Conditioning Studies in Focal Cerebral Ischemia: Model Selection, Physiological Monitoring, and Other Methodological Issues

Springer Series in Translational Stroke Research

Series Editor John Zhang

For further volumes: http://www.springer.com/series/10064 wwwwwwwwwwww Jeffrey M. Gidday Miguel A. Perez-Pinzon John H. Zhang Editors

Innate Tolerance in the CNS

Translational Neuroprotection by Pre- and Post-Conditioning Editors Jeffrey M. Gidday Miguel A. Perez-Pinzon Department of Neurosurgery Department of Neurology Washington University School of Medicine Leonard M. Miller School of Medicine St. Louis, MO, USA University of Miami, Miami, FL, USA

John H. Zhang Loma Linda University Loma Linda, CA, USA

ISBN 978-1-4419-9694-7 ISBN 978-1-4419-9695-4 (eBook) DOI 10.1007/978-1-4419-9695-4 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2012945397

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Springer is part of Springer Science+Business Media (www.springer.com) Preface

The past two decades have witnessed remarkable advances in our molecular understanding of how brain cells die after stroke, brain injury, and neurodegeneration. Yet a clinically effective neuroprotectant remains elusive. Perhaps, a gentle paradigm shift may be required. CNS disease comprises a complex mix of pathophysiologic mechanisms. Stroke is primarily an energetic insult. Traumatic brain injury is caused by biomechanical lesions. And in neurodegenerative disorders, a wide range of environmental agents and genetic perturbations has been implicated. However, in spite of this diversity in initial disease triggers, downstream molecular pathways of neuronal death and brain dysfunction show a large degree of overlap. This gives us hope. If we understand the common mechanisms of neuronal death, then we should be able to design rational and focused therapies to prevent this from happening. Thus, our current model of neuroprotection is based on the premise that curing CNS disease is best achieved by blocking deleterious pathways. But what might be missing from this conceptual formulation is the possibility that embedded within the pathophysiology of CNS disease are endogenous mechanisms of neuroprotection. As the CNS becomes damaged or diseased, it instinc- tively activates innate pathways of self-preservation. These evolutionarily conserved responses may provide a treasure trove of new “targets.” Instead of searching for exogenous agents that attempt to block brain cell death, is it possible to seek clever ways to augment innate neuroprotection? This exciting volume assembles an impressive list of leading authorities in the ﬁ eld to deﬁ ne, dissect, and debate this hypothesis. The fundamentals provided here will help us explore new frontiers of neuroprotection for many years to come.

Eng H. Lo, Ph.D. Harvard Medical School Boston, MA, USA

v wwwwwwwwwwww Contents

Part I Comparative Physiology and Historical Background

1 Tolerance, Historical Review ...... 3 Roger Simon 2 Anoxia Resistance in Lower and Higher Vertebrates ...... 19 John W. Thompson, Göran E. Nilsson, and Miguel A. Perez-Pinzon 3 Hibernation: A Natural Model of Tolerance to Cerebral Ischemia/Reperfusion ...... 37 Kelly L. Drew, Jeffrey A. Zuckerman, Phillip E. Shenk, Lori K. Bogren, Tulasi R. Jinka, and Jeanette T. Moore 4 Preconditioning in the Heart ...... 51 Derek J. Hausenloy and Derek M. Yellon

Part II Conditioning Methods

5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia, and Hyperthermia ...... 105 Ryan Kochanski, David Dornbos III, and Yuchuan Ding 6 A New Future in Brain Preconditioning Based on Nutraceuticals: A Focus on a-Linolenic Omega-3 Fatty Acid for Stroke Protection ...... 133 Nicolas Blondeau and Joseph S. Tauskela 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen, and Hydrogen Sulﬁde ...... 165 Zhiyi Zuo 8 Hypoxic Preconditioning in the CNS ...... 183 Robert D. Gilchrist and Jeffrey M. Gidday

vii viii Contents

9 Pharmacologic Preconditioning ...... 213 Jian Guan, Richard F. Keep, Ya Hua, Karin M. Muraszko, and Guohua Xi 10 Surgical Methods to Induce Brain Preconditioning ...... 225 Giuseppe Pignataro, Ornella Cuomo, and Antonio Vinciguerra

Part III Conditioning Models for Cerebral Ischemia

11 Tolerance Against Global Cerebral Ischemia: Experimental Strategies, Mechanisms, and Clinical Applications ...... 243 Kunjan R. Dave, Hung Wen Lin, and Miguel A. Perez-Pinzon 12 Preconditioning and Neuroprotection in the Immature Brain ...... 259 Nicole M. Jones and Adam A. Galle 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection, Physiological Monitoring, and Other Methodological Issues ...... 269 Thaddeus S. Nowak Jr. and Liang Zhao 14 Preconditioning for SAH ...... 291 Robert P. Ostrowski and John H. Zhang 15 Preconditioning and Intracerebral Hemorrhage ...... 309 Richard F. Keep, Ya Hua, and Guohua Xi 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke ...... 317 Heng Zhao

Part IV Mechanisms of Preconditioning

17 Synaptic Signaling in Ischemic Tolerance...... 339 Robert Meller 18 The Genomics of Preconditioning and Ischemic Tolerance ...... 363 Keri B. Vartanian, Susan L. Stevens, and Mary P. Stenzel-Poore 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred Neuroprotection? ...... 387 Peiying Li, Rehana Leak, Yu Gan, Xiaoming Hu, R. Anne Stetler, and Jun Chen 20 Ischemic Preconditioning-Mediated Signaling Pathways Leading to Tolerance Against Cerebral Ischemia ...... 429 Srinivasan Narayanan, Jake T. Neumann, Kahlilia C. Morris-Blanco, Miguel A. Perez-Pinzon, and Hung Wen Lin Contents ix

21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells...... 457 Ann M. Stowe and Jeffrey M. Gidday

Part V Other Neurological Disorders

22 Preconditioning for Surgical Brain Injury ...... 485 Cherine H. Kim, Han Chen, and John H. Zhang 23 Intrinsic Neuroprotection in Traumatic Brain Injury ...... 499 Esther Shohami and Michal Horowitz 24 Preconditioning for Epilepsy ...... 521 David C. Henshall and Eva M. Jimenez-Mateos 25 Ischemic Pre- and Post-conditioning in the Retina ...... 541 Steven Roth and John C. Dreixler

Part VI Clinical Applications

26 Clinical Cerebral Preconditioning and Postconditioning ...... 553 Cameron Dezfulian 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage, Temporary Proximal Vessel Occlusion in Carotid Revascularization, and Intracranial Aneurysm Surgery ...... 567 George Kwok Chu Wong, Matthew Tak Vai Chan, and Wai Sang Poon 28 HBO Preconditioning for TBI and Stroke Patients ...... 579 Qiang Wang and Lize Xiong 29 Electroacupuncture Preconditioning for Stroke Patients ...... 591 Qiang Wang and Lize Xiong 30 Clinical Trials of Ischemic Conditioning ...... 601 Michael Katsnelson and Sebastian Koch

Epilogue ...... 615

Author Index ...... 617

Subject Index ...... 687 wwwwwwwwwwww Part I Comparative Physiology and Historical Background Chapter 1 Tolerance, Historical Review

Roger Simon

1.1 Fever

Tolerance to fever is perhaps the fi rst thread in the history of tolerance. Induced fever therapy was a major therapeutic in the pre-antibiotic period (Bennett and Nicastri 1960 ) . Coley’s toxin, made from bacteria and bacterial toxins, produced fever and was used as an early cancer therapeutic (Patyar et al. 2010 ) . Particular ef fi cacy was noted with fever therapy for neurosyphilis and for gonorrhea, infections which are not ordinarily associated with fever. Foreign proteins were used to induce fever, fi rst with malaria and later with typhoid-paratyphoid vaccine (Albert 1999 ) . Wagner-Jauregg was awarded the Nobel Prize in medicine in 1927 for pyrotherapy for general paresis. Tolerance, which developed to the fever induction of typhoid vaccine in humans, led Paul Beeson, in 1947, to demonstrate that repeated injection of puri fi ed bacterial pyrogen (lipopolysaccharide, LPS) to rabbits resulted in resistance to the induction of fever. He termed this endotoxin tolerance. He further showed that tolerance to one bacteria resulted in tolerance to an unrelated bacteria, that tolerance occurred during the fi rst week of treatment and was lost after 3 weeks between treatments, that passive transfer could not be accomplished, and that the fever itself was not the tolerizing element (coadministration of antipyret- ics did not block the induction of tolerance) (Beeson 1947 ) . LPS is cleared more rapidly in tolerant animals, and hepatic macrophages exposed to LPS produce a pyrogen which tolerance silences (Greisman and Woodward 1970 ) . LPS has been used as an infl ammatory stimulus to produce ischemic tolerance in numerous systems. In the heart, LPS protects against reperfusion injury (Zacharowski et al. 1999 ) . In the brain (Tasaki et al. 1997 ; Dawson et al. 1999 ) , the characteristics

R. Simon (*) Departments Medicine and Neuroscience , Neuroscience Institute, Morehouse School of Medicine , Atlanta , GA , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 3 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_1, © Springer Science+Business Media New York 2013 4 R. Simon of preconditioning LPS administration are intriguingly similar to those of preconditioning ischemia. LPS-induced tolerance requires 3 days to develop (Bordet et al. 2000; Chen et al. 1996a ) and is protein synthesis dependent. Doses of LPS that produce tolerance do not result in brain injury, although they do induce inﬂ ammation. Higher doses of LPS abolish tolerance (Bordet et al. 2000 ; Ahmed et al. 2000 ) . LPS does not cause acute changes in cerebral blood ﬂ ow (Dawson et al. 1999 ; Chen et al. 1996a ; Barone et al. 1998 ) . Preconditioning with LPS also stimulates NO synthesis (Dawson et al. 1999 ) . NO has been suggested as an effector of cancer therapy with Coley’s toxin; LPS has been used experimentally and clinically in cancer therapeutics (Lundin and Checkoway 2010 ) . Fever alone produces tolerance—the heat shock response or thermotolerance. Exposure to heat stress results in tolerance to subsequent heat challenge, a response conserved throughout biology (Gilbert et al. 2010 ) . The time window of the protective effect of acquired thermotolerance is congruent with the expression of heat shock proteins (Chen et al. 1996b ) which effect protein folding as chaperones (Vabulas et al. 2010 ) but also inhibit apoptosis, produce cytoskeletal protection, and effect immune regulation (Benarroch 2011 ; Sharp et al. 1999 ) . While transient hyperthermia produces tolerance to subsequent neuronal injury, light-induced damage to the retina (Zhu et al. 2010 ; Barbe et al. 1988 ) and forebrain ischemia (Chopp et al. 1989) , even mild fever in the acute setting post-injury is associated with an increased relative risk of poor outcome from acute brain injury (stroke) (Reith et al. 1996 ) .

1.2 Trauma

R.L. Nobel carried out another early study of tolerance in 1942. He made the observation of tolerance to whole body, blunt trauma. Rats or guinea pigs were rotated in a circular drum, which was constructed with two side projections on the inner surface so that upon rotation the animal was carried a short distance on a projection and then dropped as the rotation continued (Noble and Collip 1942; Nobel 1943 ) . The cause of death was said to be “shock”; there was a fall in blood pressure and hemo- concentration. Some concussion of the nervous system was suspected although there was no macroscopic hemorrhage in or about the brain. Death was associated with mesenteric congestion. Tolerance to this “drum stress” (as it was called) occurred with mild stress preceding what would have been fatal stress. Tolerance developed over a few days and persisted for over 1 month between stresses. Tolerance was produced in awake or anesthetized animals. Transfer of blood from a resistant to a naïve animal failed to confer tolerance. In tolerant animals, trauma-induced increase in blood lactate was markedly attenuated. The release of lysosomal enzymes (acid phosphatase and beta-glucuronidase) into the circulation was attenuated in tolerance (Janoff et al. 1962 ) . Drum stress (in mice) increased brain dopamine concentration, but the fall in dopamine occurred more rapidly in tolerance, suggesting accelerated turnover (Goldberg and Salama 1972 ) . Adrenalectomized rats could be made tolerant (Janoff et al. 1962 ) . Adapted animals had a greater pressor response to catecholamines (Hruza and Zweifach 1969 ) . 1 Tolerance, Historical Review 5

1.3 Anoxia

During 1948Ð1950, Noell and Chinn used the electrocortical response to photic stimulation in rabbits to assess the survival time of the brain to anoxia. Exposure to anoxia prolonged survival time following reexposure (Noell and Chinn 1948 ; Dahl and Balfour 1964 ) . To further investigate this phenomenon, Dan and Balfour showed that in rats, a prior exposure to anoxia resulted in a marked prolongation of survival to a second anoxic exposure (Dahl and Balfour 1964 ) . The tolerant animals had brain ATP concentrations similar to naive controls but had an attenuated fall in ATP concentration during reexposure to anoxia. This was the case in the isolated head as well; thus, there is a central mechanism. The preexposed animals had increased lactate production from anaerobic glycolysis. Dan and Balfour hypothesized that increased pyruvate, from lactate oxidation, resulted in greater NADH oxidation and a slower fall in ATP and therefore prolonged anoxic survival (Dahl and Balfour 1964 ) . Increased glycogen levels and systemic acidosis have been shown to be associated with hypoxia tolerance in the absence of preconditioning (Purshottam et al. 1978 ) . Newborn mammals are resistant to anoxia. This anoxic tolerance can be extended by preexposure to hypoxia. This “adaption” persisted until day 3 (Adolph 1971 ) . That the brain’s tolerance to hypoxia is organ speciﬁ c is supported by hypoxia tolerance in the hippocampal slice (Schurr et al. 1986 ) . Hypoxic preconditioningÐ induced tolerance to oxygen deprivation occurs in C. elegans with a time window similar to that in vertebrate ischemic tolerance: onset at 16 h and duration 36 h (Dasgupta et al. 2007 ) . Curiously, tolerance in C. elegans requires the CED-4 gene, while cleavage of caspase 3 may be necessary for ischemic tolerance in mammalian systems (McLaughlin et al. 2003 ) . Attenuation of hypoxia-induced death in C. elegans has another feature of ischemic tolerance in mammals. Tolerance is inversely correlated with the rate of gene transcription, as has been shown to occur during ischemic tolerance in mice (Anderson et al. 2009 ; Stenzel-Poore et al. 2003 ) . The most recent addition to the hypoxia tolerance story is the demonstration that the tolerizing stimulus (hypoxia) can, experimentally, in a tightly controlled manner, be administered repetitively to extend the time window in which tolerance, in this case to ischemic challenge, can be induced (Stowe et al. 2011 ) .

1.4 Non-cerebral Ischemic Tolerance in the Heart

Ischemic tolerance was ﬁ rst described in the heart in 1986 (Reimer et al. 1986 ; Swain et al. 1984 ; Murry et al. 1986 ) and has since been observed in liver, intestine, lung, muscle, kidney, retina, and brain (Roth et al. 1998 ; Ishida et al. 1997 ; Tomai et al. 1999 ) . An analogous condition, termed “toughening,” has been described in the inner ear, where brief exposure to intense sound minimizes sound-induced hearing loss (Attanasio et al. 1998 ; Hamernik and Ahroon 1999 ; Yoshida and Liberman 2000) . Numerous endogenous, neuroprotective mechanisms have been characterized in the setting of ischemic tolerance (Dirnagl et al. 2003 ) . 6 R. Simon

In the heart, brief periods of ischemia render the myocardium tolerant to subsequent, normally lethal ischemia. Preconditioning reduces myocardial infarct size by as much as 70% and improves the recovery of regional myocardial function during reperfusion (Murry et al. 1986; Li et al. 1990; Lawson and Downey 1993 ) . In tolerant myocardium, the duration of ischemia needed to cause infarction is dou- bled (Lawson and Downey 1993 ) . Early tolerance starts 1Ð60 min after preconditioning but lasts less than 3 h in most studies (Alkhulaiﬁ et al. 1993 ) . The mechanisms underlying early tolerance are unknown. A second window of protection, late tolerance, develops approximately 24 h after preconditioning. Late tolerance requires new protein synthesis and persists for several days (Bolli 2000 ) . No pathway linking early tolerance to late tolerance has been identiﬁ ed, but certain effector molecules, including nitric oxide (NO), protein kinase C (PKC), and ATP-sensitive K+

(KATP ) channels, are common to both. In late tolerance, PKC potentiates the opening of mitochondrial K ATP channels. This reduces the rate of ATP hydrolysis (Garlid et al. 1997 ) and decreases the electrochemical gradient for calcium entry into the mitochondria (Holmuhamedov et al. 1999 ) , thereby attenuating the toxic effects of Ca2+ overload and ultimately preserving the integrity of the mitochondria (Rubino and Yellon 2000 ) . Bcl-2, a critical neuroprotective protein synthesized during late tolerance, may also exert its effect on mitochondria (Maulik et al. 1999 ) .

1.5 Cerebral Ischemic Tolerance

Ischemic tolerance in the brain was described by Kitagawa in 1990. Using two-vessel occlusion in gerbil, brief periods of global cerebral ischemia protected against subsequent prolonged global ischemia (Kitagawa et al. 1990 ) . The time window of effectiveness was narrow. Two minutes, but not one minute, of preconditioning produced tolerance following a 24-h, but not a 12-h, interval. Multiple brief preconditioning episodes were more protective than a single dosing. Tolerance persisted for at least 2 days. Subsequently, ischemic tolerance was described using either global or focal ischemia to induce tolerance to either global or focal ischemia (Chen and Simon 1997) . Focal-focal tolerance in rat is perhaps the best characterized model. In this model, infarct size is reduced when prolonged focal ischemia is preceded by brief, transient focal ischemia. Tolerance develops at 48 h, is maximal at 72 h, and persists for about 7 days (Chen et al. 1996a ) . Analogously, several large retrospective studies have suggested that transient ischemic attacks (TIAs) in humans are associated with improved clinical outcome after stroke, perhaps because TIAs are capable of inducing ischemic tolerance (Weih et al. 1999 ; Moncayo et al. 2000 ; Wegener et al. 2004 ; Fu et al. 2008 ) (Fig. 1.1 ). Numerous physiologic changes have been implicated as inducers of cerebral ischemic tolerance, including changes in blood ﬂ ow, glutamate concentration, and protein expression. Preconditioning with brief, transient focal ischemia produces tolerance, but does not increase blood ﬂ ow in the cortex during or after the ischemic challenge. Preconditioning with more prolonged transient focal ischemia does 1 Tolerance, Historical Review 7

Fig. 1.1 Development of an ischemic tolerance to focal ischemia model (* Simon et al. 1993 ) increase blood ﬂ ow in the challenged cortex, but does not produce tolerance (Matsushima and Hakim 1995 ) . Tolerance, therefore, is not an artifact of increased cerebral blood ﬂ ow. Glutamate may play a role. Pharmacologic blockade of NMDA, but not AMPA, subsets of postsynaptic glutamate receptors attenuates tolerance (Bond et al. 1999 ; Kato et al. 1992 ) . Extracellular glutamate concentration increases 8 R. Simon in the ipsilateral hippocampus during middle cerebral artery occlusion (MCAO) in the gerbil, and neurons in this region are tolerant to subsequent global ischemic challenge (Miyashita et al. 1994 ) . Microdialysis has shown, though, that the concentration of glutamate released during preconditioning is the same as that released during ischemia (Nakata et al. 1992 ; Nakata et al. 1993 ) . Adenosine may play a role in the brain as well as heart, acting via the A1 receptor to cause KATP channel opening. The neuroprotective effect produced by ischemic preconditioning is greater than that produced by administration of A1 agonists (Van Wylen et al. 1986 ; Heurteaux et al. 1995 ) .

1.6 Molecular Mechanisms of Tolerance

The development of tolerance requires protein synthesis. Protein synthesis inhibitors block tolerance induction (Barone et al. 1998 ) , and gene products produced during the development of tolerance have been identifi ed. Upregulation of the immediate early genes (IEG) (c-fos, c-jun, junB, and junD) occurs after preconditioning ischemia, but the genes targeted by these transcription factors in the setting of tolerance are unknown (Abe and Nowak 1996 ; An et al. 1993 ) . The transcription factor hypoxia-inducible factor-1 (HIF-1) is involved in preconditioning in neonatal rat brain (Bernaudin et al. 2002 ; Bergeron et al. 2000 ) . Akt has been implicated as well (Wick et al. 2002 ; Noshita et al. 2001 ) . Antioxidant enzymes such as MnSOD are overexpressed in tolerant brain and may play a role in protection (Ohtsuki et al. 1992 ; Kato et al. 1995 ) . The induction of heat shock proteins follows a time course compatible with tolerance (Chen et al. 1996b ) . Transgenic mice overexpressing HSP70 have attenuated ischemic injury (Rajdev et al. 2000 ) . However, tolerance of cortical neurons to subsequent global ischemia occurs in the absence of HSP mRNA expression when cortical spreading depression is used as a preconditioning stimulus. While preconditioning in the gerbil induces IEG expression, tolerance develops in the absence of an increase in HSP72 mRNA levels (Kobayashi et al. 1995 ; Abe et al. 1996 ) . The antiapoptotic Bcl-2 family proteins were early candidate mediators of tolerance (Chen et al. 1995 ) . Antisense techniques to inhibit Bcl-2 induction exacerbate ischemic injury (Chen et al. 2000 ) . Rats treated with Bcl-2 antisense oligodeoxynucleotides (ODN) prior to ischemia develop larger cerebral infarcts than untreated rats. Rats treated with Bcl-2 antisense prior to preconditioning ischemia do not upregulate Bcl-2 protein and do not become tolerant to subsequent ischemic challenge (Shimizu et al. 2001 ) . Studies of tolerance in vitro have con fi rmed observations made in vivo. When ischemia is modeled by oxygen glucose deprivation (OGD) in vitro, tolerance requires NMDA (but not AMPA or kainate) receptor activation, calcium infl ux, and new protein synthesis (Monyer et al. 1992 ; Grabb and Choi 1999 ; Marini and Paul 1992) . Tolerance is substantially attenuated by NOS inhibition during OGD and can be restored by administration of the NOS substrate L-arginine (Gonzalez-Zulueta et al. 2000 ) . These results corroborate hypoxic preconditioning experiments 1 Tolerance, Historical Review 9 performed in neonatal rats (Gidday et al. 1999 ) . In vitro studies have also expedited understanding of the signaling pathways involved in tolerance. Studying the effects of NO after OGD, V. Dawson and colleagues demonstrated that the p21RAS pathway is induced during tolerance development and that Ras, Raf, Mek, and Erk are required. They postulate that this pathway may produce tolerance through the activation of cyclic-AMP response element-binding protein (CREB), a transcription factor that induces the expression of numerous putative neuroprotective proteins (Nandagopal et al. 2001) , including Bcl-2. This agrees with in vivo fi ndings . CREB activation, phosphorylation, and nuclear translocation occurred after preconditioning ischemia, but not after severe ischemia (Meller et al. 2005 ) .

1.7 In ﬂ ammation and Tolerance

Immune and infl ammatory mechanisms have long been a focus of progressive brain injury and more recently have been studied as mediators of neuroprotection (Hallenbeck et al. 2005 ; Hallenbeck 2010 ) . Ischemia rapidly induces the expression of cytokines, chemokines, and adhesion molecules in the brain. These molecules mount an in fl ammatory response to injury which is characterized by reactive astrocytes (Balasingam et al. 1994 ) , infi ltration of peripheral leukocytes, proliferation of microglia (Morioka et al. 1993) , and further production of cytokines (Giulian et al. 1986 ; Lieberman et al. 1989 ; Sawada et al. 1989 ) . The response facilitates repair and remodeling but also initially contributes to ischemic injury (del Zoppo et al. 2000 ; Barone and Feuerstein 1999 ) . The expression of the cytokine TNF-a increases after MCAO in rats (Liu et al. 1994 ; Wang et al. 1994 ) . Injection of TNF-a exacerbates injury in fl icted by subsequent focal ischemia (Barone et al. 1997 ) . Administration of monoclonal antibodies to TNF-a blocks the effect. Similarly, IL-1 is elevated after global ischemia. Intracerebroventricular administration of recombinant IL-1 receptor antagonists (IL-1ra) and overexpression of endogenous IL-1ra by adenoviral delivery reduce ischemic brain damage (Relton et al. 1996 ; Betz et al. 1995 ) . The same in fl ammatory responses that contribute to ischemic injury may also confer protection when induced with preconditioning stimuli. IL-1 levels increase after preconditioning global ischemia, and treatment with an IL-1 receptor antagonist prior to preconditioning abolishes tolerance. Moreover, the administration of IL-1 or TNF-a produces tolerance to subsequent severe ischemia (Ohtsuki et al. 1996 ) . Injection of TNF-a prior to MCAO reduces infarct volume in mice in a protein synthesis-dependent manner (Bordet et al. 2000; Nawashiro et al. 1997 ) . TNF-a neutralizing antibodies preclude the induction of tolerance in vitro (del Zoppo et al. 2000 ) . The mechanisms by which infl ammatory cytokines establish tolerance are unknown. Pretreatment with TNF-a renders the brain unresponsive to subsequent TNF- a exposure, thereby attenuating subsequent, deleterious effects of cytokine activity in ischemia (Ginis et al. 1999 ) . Prior cytokine exposure may also contribute to the preservation of microvascular perfusion (Bordet et al. 2000 ) . In the lung, acute TNF-a and IL-1 administration increases SOD production within 72 h (White et al. 1989 ) . 10 R. Simon

It is not understood how an infl ammatory response induced prior to ischemia protects against ischemic injury. Reactive astrocytes have been suggested to play a role. Preconditioning induces astrocyte proliferation in the forebrain 3Ð4 days after subsequent ischemia (Liu et al. 2001 ) . GFAP-knockout mice have increased infarct sizes (Nawashiro et al. 2000 ) . A role for microglia has also been suggested (Liu et al. 2001 ) . Microglia/macrophages proliferate in striatum and neocortex, but not in hippocampus, after brief global ischemia that produces ischemic tolerance (Liu et al. 2001 ) . In TNF receptor-knockout mice, microglial proliferation normally induced by preconditioning (Zielasek and Hartung 1996 ) is reduced, and injury from subsequent ischemia is increased (Bruce et al. 1996 ) . Finally, peripheral leukocytes could also be a component. In fi ltrating in fl ammatory cells induce the production of cytokines, including TNF- a and IL-1. Preconditioning reduces the number of circulating polymorphonuclear leukocytes (PMN) in ischemic brain (Dawson et al. 1999 ) . During the infl ammatory response induced by preconditioning, PMNs may contribute to tolerance by producing SOD, IL-1, and TNF-a (Davenpeck et al. 1998 ) . A proposed role for interferon induction has recently been offered (Marsh et al. 2009 ) .

1.8 Cross-Tolerance

The tolerance phenomenon is not limited to ischemia. Prolonged epileptic seizure causes brain injury. In epileptic tolerance, prior brief seizure (or kindling) protects against subsequent prolonged seizure (Kelly and McIntyre 1994 ) . Seizures also protect against ischemia, and preconditioning ischemia protects against epileptic brain injury (Plamondon et al. 1999) . Adenosine A1 receptors and KATP channels have been implicated in such “cross-tolerance” (Plamondon et al. 1999 ) . Other examples of cross-tolerance include prior hypothermia protecting against subsequent ischemia (Nishio et al. 1999 ) and prior ischemia protecting against subsequent traumatic brain injury (Perez-Pinzon et al. 1999 ) .

1.9 Recent History

1.9.1 Stem Cells

Endogenous stem cells proliferate in the setting of acute brain injury, for example, following seizures (Parent et al. 1997 ) , global ischemia (Liu et al. 1998 ) , and focal ischemia (Jin et al. 2001; Zhang et al. 2001) . A protective role for these cells has been suggested (Martino and Pluchino 2006 ) . Upregulation of this proliferative response can be neuroprotective: granulocyte colony-stimulating factor increases stem cell proliferation after ischemia and is neuroprotective (Sehara et al. 2007 ; Schabitz et al. 2003 ) . Stem cell transplantation is neuroprotective as well (Ukai 1 Tolerance, Historical Review 11 et al. 2007 ) . Preconditioning ischemia also results in progenitor cell proliferation in the subgranular zone and in the subventricular zone (Maysami et al. 2008 ) . Experimental attenuation of progenitor cell proliferation blocked tolerance induction (Maysami et al. 2008) . The paracrine activity of secreted factors from these cells has been suggested as the neuroprotective mechanism of stem cell proliferation (Bakondi et al. 2009 ) .

1.9.2 Ischemic Postconditioning

Brief periods of impaired blood ﬂ ow during reperfusion can attenuate infarct volume (Zhao et al. 2006 ) . A similar result has previously been demonstrated in the heart (Yellon and Hausenloy 2005) . The effect can be demonstrated in vivo and in vitro. The degree of protection by postconditioning is compatible with that of preconditioning. The effects in the brain are not additive, however, suggesting different mechanisms mediated at least in part by Akt (Pignataro et al. 2007 ) . Ischemia is not the only postconditioner. LPS administration during reperfusion has also been reported as neuroprotective (Davis et al. 2005 ) .

1.9.3 Remote Tolerance

Studies began in the heart with the observation that brief ischemia in the circumﬂ ex artery attenuated subsequent myocardial injury from left anterior descending artery occlusion (Przyklenk et al. 1993 ) . Subsequent studies showed that transient ischemia in a number of peripheral organs protected against myocardial infarction and ischemic injury in multiple end organs, including the brain. Evolving data in the heart suggest that neural pathways in the organ receiving the remote ischemia must be intact for the preconditioning effect. A number of circulating substances and end organ receptor systems have been implicated (for review Kharbanda et al. 2009 ) . Of particular interest is that the remote preconditioning stimulus alters gene expression in the target organ (the heart) (Konstantinov et al. 2005 ) . Limb ischemia has been used prior to elective cardiac and noncardiac surgery and during prehospital transport for acute myocardial infarction. Positive effects for end-organ ischemic injury were reported (Kharbanda et al. 2009 ; Botker et al. 2010 ) . Reports with the brain as the focus of protection are evolving (Moskowitz and Waeber 2011 ; Jensen et al. 2011 ) .

References

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John W. Thompson , Göran E. Nilsson , and Miguel A. Perez-Pinzon

The fi eld of ischemic preconditioning has become highly complex, and it is now known to involve a multifaceted network of metabolic pathways. Although this fi eld was fi rst described in the ischemic myocardium by Murry et al. ( 1986 ) and followed later by studies in the brain by Schurr et al. (1986 ) , it is plausible that this fi eld emerged a few decades earlier from studies describing adaptations to extreme environmental conditions found in Mother Nature. For example, hypoxic or anoxic tolerance is found ubiquitously in nature and has been under scientifi c scrutiny since at least the 1960s (Belkin 1963, 1968a, b) if not before. Hibernation is another natural adaptation, which has been studied extensively, where extreme low-blood fl ow perfusion occurs and will be reviewed in a later chapter of this book. Almost all vertebrates die quickly when their internal oxygen stores are depleted (Lutz et al. 1996 ) . Normal cellular function and survival depends upon the continuous supply of substrates and oxygen for energy metabolism. Whenever energy metabolism is curtailed by low substrate availability or hypoxia, normal function is rapidly suppressed and irreversible injury might occur. This vulnerability is especially evident in organs such as the brain, heart, and kidney that are bioenergetically very active. The brain, which among all other body organs has the highest energy requirements, may be the most vulnerable to energy failure. In mammals, the brain is

J. W. Thompson Cerebral Vascular Disease Research Laboratories, Department of Neurology , Leonard M. Miller School of Medicine, University of Miami , Miami , FL 33136 , USA G. E. Nilsson Department of Molecular Biosciences , University of Oslo , 0316 Oslo , Norway M. A. Perez-Pinzon (*) Cerebral Vascular Disease Research Laboratories, Neuroscience Program, Department of Neurology , Leonard M. Miller School of Medicine, University of Miami , Miami , FL 33136 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 19 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_2, © Springer Science+Business Media New York 2013 20 J.W. Thompson et al. approximately 3% of the body mass, yet it receives approximately 15% of the total cardiac output and uses 20% of the body’s oxygen (Clarke and Sokoloff 1998 ) . Despite the great susceptibility of the brain to hypoxia, a few vertebrates have developed special adaptations to survive extreme energy deprivation states. In this chapter, we will review a few of these species and will describe some of the mechanisms by which anoxia resistance occurs. It will be evident at the end of the chapter that many of these mechanisms are in fact similar to those found in ischemic preconditioning, which will be described in depth in later chapters. To better understand the mechanisms by which anoxia tolerance is achieved, a better understanding of energy production and consumption is required, as it per- tains to the brain. Cellular energy production has been reviewed extensively in many classic reviews and bioenergetic books, and they stand today as fully accepted concepts (Siesjo 1978; Harold 1986 ; Erecinska and Silver 1989 ) ; the following paragraphs are summarized from these reviews.

2.1 Energy Production

2.1.1 High-Energy Intermediates

All living cells require the ongoing input of free energy in order to survive and to perform their functional activities such as mechanical, electrical, chemical, osmotic, and thermal work. In most cells, the free energy that is used for work is stored as high-energy phosphate bonds in certain key molecules. Of these molecules, ATP is the most important. Other compounds also have phosphoryl groups, which can store and subsequently provide free energy like ATP. What makes ATP important is that its hydrolysis to ADP is coupled to many other chemical reactions that are not thermodynamically spontaneous. The release of free energy from ATP will therefore allow the progress of ATP-coupled reactions. Since most cellular energy is derived from the cleavage of phosphate bonds from ATP and its intermediates (ADP and AMP), the energy state of a cell can be indicated by a formula de ﬁ ned as “energy charge” (Atkinson 1977 ) :

Energy charge=+()[] ATP 1 / 2 [] ADP /()[][][] ATP ++ ADP AMP

As shown by Atkinson ( 1977 ) and many others, in the brain and other tissues, ATP-generating pathways are inhibited when the energy charge is high; alternatively, ATP-consuming pathways are inhibited when the energy charge is low. In addition to ATP, there are other biological compounds that have high phosphate group transfer potentials, such as phosphoenolpyruvate, acetyl phosphate, and phosphocreatine (PCr). With the exception of PCr, these are not signi ﬁ cant storage molecules for energy in the brain. In the brain, PCr plays an important role in the maintenance of ATP levels (Siesjo 1978 ) . PCr possesses a phosphoryl transfer 2 Anoxia Resistance in Lower and Higher Vertebrates 21 potential that is higher than that of ATP. Thus, the reaction described above will normally proceed in the direction of ATP formation when creatine phosphokinase is activated. This activation provides an important regulation of ATP concentration in most cells. Creatine phosphokinase activity is high when the energy charge is low. As energy charge is increased, creatine phosphokinase activity is inhibited. The distribution of high-energy intermediates is diverse in the brain. The total adenine nucleotide concentration is about 3 m mol/g wet wt, while creatine is approximately 10–11 mmol/g wet wt (Erecinska and Silver 1989 ) . The ratio of PCr/ATP appears related to the intensity of cellular activity. The highly active cells in the molecular layer of the hippocampus, for example, have high PCr/ATP ratios (Lipton and Whittingham 1982 ) .

2.1.2 ATP Production

The brain is not different than other tissues with respect to the main pathways that produce high-energy intermediates. In a broad outline, brain ATP is derived from glycolysis, from tricarboxylic acid cycle (TCA) Krebs cycle reactions, and from electron transport and oxidative phosphorylation in the mitochondrial respiratory chain (Siesjo 1978 ) . Other pathways such as pentose phosphate shunt and beta- oxidation are of little importance in the brain. One difference between the brain and most other tissues is that in the brain, glucose is the primary and nearly exclusive substrate ( Balazs et al. 1970 ) . For this reason, glucose transport into the brain is carefully regulated, and systemic glucose stores are transferred to the brain whenever brain glucose levels are decreased. For example, during hypoglycemia, other tissues like the heart, renal cortex, and liver stop metabolizing glucose, making it more available for the brain. Glucose is predominantly metabolized in the glycolytic pathway and subsequently in the Krebs cycle. But it has also been observed to be metabolized in both the pentose phosphate shunt and the gamma aminobutyric acid (GABA) shunt. It is important to notice that glucose turnover via the pentose phosphate shunt does not increase with electrical stimulation (O’Neill et al. 1965 ) or high K + , whereas the TCA and aerobic glycolysis operates at higher rates under both conditions (Machiyama et al. 1970 ) . In the glycolytic pathway, pyruvate is metabolized by two related pathways: (a) it is decarboxylated and oxidized, producing acetyl CoA, a reaction catalyzed by pyruvate dehydrogenase, a mitochondrial multienzyme complex; and (b) production of lactate by lactate dehydrogenase (LDH). In the brain under resting, normoxic conditions, glucose consumption is stoichiometrically related to CO2 release with minor lactate production (Kety 1957 ). In anoxia, lactate production is increased (Kety 1957 ). During anoxia, production of acetyl CoA is halted, and the next steps of ATP production from pyruvate (i.e., the TCA or Krebs cycle reactions and electron transport in the mitochondrial respiratory chain) cease to occur. 22 J.W. Thompson et al.

The rate of lactate production in the brain is highly controlled. Lactate dehydrogenase has ﬁ ve isozymes in the brain, and the distribution, synthesis, and activity of these isozymes appear to be inversely related to brain tissue oxygen tension (Hochachka and Somero 1984 ) . In fact, tissues known to have a high tolerance to hypoxia, such as the turtle brain, have higher concentrations of the most active isozymes for lactate production (Hochachka and Somero 1984 ) .

2.2 Energy Consumption

The predominate use of energy required by the brain can be divided into that used for basal metabolism and that used for ion pumping (Michenfelder 1974 ) . Basal metabolism incorporates the energy required for maintaining cellular structure and other survival activities such as the synthesis and maintenance of metabolic and structural proteins and axoplasmic transport. While these activities may be changed slightly during anoxia, it is unlikely that such changes signiﬁ cantly contribute to anoxia tolerance. Therefore, this section will concentrate on ion transport, electrical activities, and the relationships between these activities.

2.2.1 Ion Pumps

Membrane-bound transport systems can be divided into those which are passive and those which are active and require energy from ATP. Passive transport relates to the movement of ions down transmembrane electrochemical gradients. Active transport relates to the movement of ions against transmembrane electrochemical gradients. Intracellular calcium concentration in the brain is regulated predominantly by four ATP-dependent pump systems: (a) plasma membrane Ca+2 pump; (b) endoplasmic reticulum Ca+2 pump; (c) Ca+2 uptake by mitochondria, which is driven by proton gradients (indirectly linked to ATP); and (d) Na+ /Ca +2 exchange, which is regulated by Na+ gradients and therefore indirectly controlled by the Na + /K + pump (Carafoli 1987 ) . Estimation of the energetics of these systems has been complicated by their relationship with the Na+ ,K + -ATPase. Unlike Na+ and K+ transport via the Na+ ,K+ -ATPase (see below), it has not yet been possible to de fi ne the energy cost of calcium transport in the brain. The best studied pump is the Na + , K +-ATPase system defi ned initially by Skou (1957 ) . This ATP-dependent ion pump moves 3 Na+ from inside neurons, glia, and other excitable cells to the extracellular space against 2 K + being transported in the opposite direction and utilizes an ATP molecule in the process. Since Na+ ,K + -ATPase transport of Na+ and K+ generates an electrochemical gradient, the transport system is de fi ned as an electrogenic pump. The Na+ , K +-ATPase is mainly controlled, under + + physiological conditions, by Na i (Grisar et al. 1978 ; Logan 1980 ) , although K and 2 Anoxia Resistance in Lower and Higher Vertebrates 23

ATP are regulatory components. Inhibiting the Na+ , K+ - ATPase pump with ouabain (Post et al. 1960 ) causes motor hyperactivity, convulsions, loss of ion homeostasis, electrical suppression, and death (Bignami and Palladini 1966 ; Cornog et al. 1967 ) . The relationship between Na+ , K + -ATPase pump activity and brain energetics has been reviewed extensively in classic articles (Siesjo 1978 ; Astrup et al. 1981 ; Hansen 1985 ) . For example, Hansen (1985 ) estimated that approximately 50% of brain oxygen consumption is used by this pump during “resting” conditions. This calculation was obtained with assumptions about Na+ permeability, membrane potential, cell membrane area, and Na+ concentration in both the extracellular space and cytoplasm, and that the net ﬂ ux of Na+ under resting conditions is 37 m M/g/min. Since 3 Na + are transported per ATP molecule consumed, then ATP use should be

12 mM/g/min. Assuming a P:O ratio of 3 and oxygen consumption of 10 ml O2 /g/ min (as found in the rat brain; Eklof et al. 1973 ) , then 50% of the total brain oxygen consumption is used for Na+ transport.

2.2.2 Sodium Pump and Electrical and Synaptic Activity

Na+ and K + pumping and electrical and synaptic activity are all tightly linked to the brain’s cellular metabolism. Whenever electrical activity increases (i.e., convulsions), oxygen consumption is increased. Superfusion of the brain with high K + concentrations (Ashford and Dixon 1935 ) or electrical stimulation (McIlwain 1951 ) increases oxygen consumption of the cerebral cortex approximately 100% both in vivo and in vitro. Electrical stimulation also enhances glucose consumption, unless the Na+ ,K+ -ATPase transport system was inhibited by ouabain (Mata et al. 1980 ) . Whenever electrical activity is decreased (i.e., coma or anesthesia), oxygen consumption declines (Kety 1957 ) . Electrical activity also infl uences the levels of high-energy intermediates. For example, increases in electrical activity induced transient declines in ATP levels (Heald 1954 ) . This decline was blocked when Na+ was absent. Interestingly, PCr breakdown was very high during electrical stimulation (Heald 1954 ) , but recovered afterward (Heald 1954) . It was proposed that PCr is restored through oxidative metabolism, since oxygen consumption remained elevated for a period following electrical stimulation (Mcllwain 1954 ) . This suggests that neurons rely upon PCr to maintain ATP levels during electrical activity, and that oxidative metabolism subsequently “catches up” to restore high-energy intermediates. Na + , K + ATPase pump activity must increase severalfold during electrical stimulation to balance the increased ion fl uxes. Therefore, energy expended by ion pumps must depend on three elements: (a) the intensity of electrical activity, (b) geometry of the cellular elements, and (c) myelination. During synaptic transmission, estimates of pump activity are even higher, although the electrical currents associated with postsynaptic excitatory or inhibitory potentials (approximately 10 mV) may yield ion fl ux densities only about one-tenth of those produced during an action potential. Nevertheless, these postsynaptic depo- 24 J.W. Thompson et al. larizations may last 10–1,000 times longer than action potentials and may occupy much larger membrane areas. Therefore, the net fl ux of Na + and K+ ions will be much larger, the increased pump activity will be greater, and the ATP consumption will be higher.

2.2.3 Energy Failure

Since the brain energy consumption is so large, and the activities that use the predominate fraction of energy so critical, it might be expected that energy failure would have rapidly damaging consequences. In fact, this is the case in mammals, where insults such as anoxia, ischemia, and hypoglycemia all cause the brain to become isoelectric within a few minutes (Heiss et al. 1976 ; Astrup et al. 1977 ; Branston et al. 1978 ) . If these insults persist, the loss of electrical activity is followed by depletion of high-energy intermediates, by lactate accumulation (Lowry et al. 1964) , and by the loss of ion gradients (depolarization) (Hansen 1985 ) . Although complete suppression of electrical activity takes place before complete depletion of high-energy intermediates, Lipton and Whittingham ( 1982 ) found a correlation between declines in ATP and PCr levels in synaptic terminals and depression of synaptic transmission. It was suggested that an ATP threshold exists, below which electrical suppression occurs (Lipton and Whittingham 1982 ) . Another possibility is that an ATP pool for synaptic transmission is depleted before other ATP pools (Sick and Rosenthal 1990 ) . The next event after suppression of electrical activity during energy failure is the loss of ion homeostasis (Hansen 1985 ) . As ion gradients are lost, for example, in ischemia, neurons release the excitatory neurotransmitter glutamate, which may amplify or cause irreversible pathologies during energy failure (Greenmayre 1986 ; Choi 1987; Mayer and Westbrook 1987 ; Monaghan et al. 1989; Wroblewski and Danysz 1989 ) . Studies of excitotoxicity have centered on presynaptic physiology, which regulates glutamate levels and its release, and on postsynaptic glutamate receptors. From presynaptic studies, two pools of glutamate have been recognized in brain cells: cytosolic glutamate and transmitter glutamate (Greenmayre 1986 ) . The transmitter pool is approximately 40–45% of the total cellular glutamate concentration (Young et al. 1974 ; Fonnum et al. 1981 ) . The selective uptake of glutamate into synaptic vesicles is ATP-dependent. This uptake requires a Mg-ATPase in protein I-associated vesicles and may be regulated by a proton pump ATPase (Naito and Ueda 1983 ) . Thus, glutamate uptake is dependent upon a proton gradient. This gradient decreased when cytosolic pH declined (Naito and Ueda 1983 ), which occurs during energy failure. Therefore, it is expected that cytosolic glutamate will increase during energy failure. Glutamate can be released presynaptically by both Ca +2 -dependent and Ca+2 - independent mechanisms. The Ca+2 -dependent release of glutamate occurs through synaptic vesicles, and the Ca+2 -independent release of glutamate occurs via a bidirectional electrogenic Na+ -symport mechanism (Nicholls et al. 1987 ) . This symport 2 Anoxia Resistance in Lower and Higher Vertebrates 25 also carries K+ in the opposite direction (Fonnum et al. 1981 ). Thus, the shift in ion gradients during energy failure should cause a Ca+2 -independent release of glutamate + + (i.e., high Nai will be extruded along with glutamate in exchange for K ). In fact, inhibitors of the glutamate symport prolonged and potentiated the excitatory effects of glutamate on neurons (Greenmayre 1986 ) . This was corroborated by ﬁ ndings that inhibition of the electron transport chain with cyanide provoked a Ca +2 -dependent release of glutamate during the ﬁ rst 2 min, which was followed by Ca+2 -independent glutamate release (Sanchez-Prieto and Gonzalez 1988 ) . Glutamate release following energy deprivation results in overactivation of glutamate receptors. This phenomenon is known as excitotoxicity and has been well described by many studies and will not be further elaborated in this chapter. For more details on the topic of excitotoxicity, see the following reviews (Lipton 1999 ; Obrenovitch et al. 2000 ) .

2.3 Turtles and Anoxia Tolerance

A few species of turtles such as the freshwater turtles of the genus Trachemys and Chrysemys (Ultsch 1985 ) and the marine loggerhead turtle Caretta caretta (Lutz et al. 1980 ) are known to tolerate extended periods of anoxia. This tolerance was achieved during evolution, by physiological adaptations during diving. In fact, turtles spend 86% of their time submerged (Lutcavage et al. 1989 ) . Few vertebrate species dive as a normal behavior. Therefore, these turtle species have attained special physiological adaptations to promote diving behavior. Other diving reptiles include some species of snakes, lizards, and crocodiles, which can survive 45–120 min under water (Belkin 1963 ) . Some birds are also known to dive; however, pen- guins, which are the longest documented diving birds, can only survive 18 min under water (Kooyman et al. 1971 ). Among vertebrates, turtles are probably the species that can survive longest in a dive. For example, the green turtle (Chelonia mydas ) can remain submerged for 6 h in a voluntary dive with no measurable oxygen in their trachea or carotid artery (Berkson 1966) . Marine green turtles bury themselves for at least 3 months in the ocean ﬂ oor (Felger et al. 1976 ) . The diving capacity of turtles is independent of oxygen stores, since Trachemys scripta elegans can withstand 48 h of anoxia (breathing 100% N2 ) at room temperature (Robin et al. 1964 ) or up to 3 months at low temperatures (5°C) (Jackson and Heisler 1982 ) . Since diving behavior in turtles is based on anoxia tolerance, Hochachka and Somero (1973 ) have called turtles “facultative anaerobes.”

2.3.1 Turtle Brain

Investigators in the ﬁ eld have usually compared anoxia-tolerant and anoxia-intolerant animals to deﬁ ne mechanisms of neuroprotection. With that approach in mind, it 26 J.W. Thompson et al. was clear that anoxia-tolerant species were able to maintain brain ionic gradients + and avoid anoxic depolarization. In the turtle brain, [K ]o rises only 1–2 mM during 4 h of anoxia and only reaches 3–6 mM after 48 h of anoxia (Sick et al. 1982 ) . Maintenance of ion gradients in the brain is accompanied by the ability of anoxic tolerant brains to maintain energy status. ATP levels remain relatively stable in turtle brains for at least 3–5 h of anoxia (Lutz et al. 1985 ; Kelly and Storey 1988 ; Perez-Pinzon et al. 1992a, c) . It is now accepted that the maintenance of ion gradients and ATP levels is the result of a depression in metabolic rate (discussed in the next section). In addition, anoxia-tolerant turtles appear to utilize two cooperative strategies for maintaining ATP levels – metabolic depression coupled with enhanced glycolytic ATP production.

2.3.2 Metabolic Depression

A key adaptation for anoxia survival in turtles is the ability to accomplish a drastic decrease in the rate of ATP consumption, that is, metabolic depression. In fact, anoxic turtles become comatose and unresponsive to outside stimuli (Lutz et al. 1996 ) . This is probably a sacri fi ce related to the turtle’s impressive ability to decrease their body heat production by an impressive 90% during anoxia (Jackson 1968 ) . The fi rst evidence, although circumstantial, for metabolic depression in the nervous system of turtles came from studies showing that anoxia causes a pronounced depression of electrical activity (spontaneous as well as evoked) in the central nervous system of these animals. Feng et al. ( 1988) showed a considerable suppression of EEG as well as evoked potentials in the brain of freshwater turtles ( Trachemys scripta ) exposed to anoxia. Microcalorimetric studies of heat production on the in vitro turtle brain provided direct evidence for metabolic depression. Heat output from anoxia-tolerant turtle brain obtained from the isolated cerebellum (Perez- Pinzon et al. 1992a ) or cerebral cortical slices (Doll et al. 1994 ) showed a 30–40% decrease in heat production during anoxia, and telencephalic slices also suggested a reduction in heat production of about 40% (Johansson et al. 1995 ) . A mechanism by which the brain’s metabolic rate is reduced in anoxia-tolerant vertebrates is by downregulation or depression of electrical activity (Lutz et al. 1996 ) . As described above, most of the energy consumed by the brain appears to be used by ion pumps to balance ion movements linked to the electrical and synaptic activities. Thus, a likely target for accomplishing metabolic depression in the brain would be to temporarily reduce these ion fl uxes. This could be accomplished by (1) reducing neuronal excitability through membrane hyperpolarization, (2) directly reducing ion fl ux through the closing of ion channels, and (3) suppressing synaptic transmission by increasing the release of inhibitory neurotransmitters and/or decreasing the release of excitatory neurotransmitters. There is evidence that all of these processes are involved in anoxia survival by the freshwater turtle (Lutz et al. 1996 ; Perez-Pinzon et al. 1997 ) . A strategy of reducing ion pumping as a mechanism to reduce energy consumption in neurons is termed “channel arrest” and was suggested many decades ago (Lutz et al. 1985 ; Hochachka 1986 ) , before any proof of such a process existed. 2 Anoxia Resistance in Lower and Higher Vertebrates 27

Evidence that this phenomenon is in fact occurring during anoxia tolerance in turtles was revealed by a number of studies, which determined a substantial downregulation in K+ , Na + , and Ca2+ fl uxes over neuronal membranes (Perez-Pinzon et al. 1992c ; Bickler 1992; Pek-Scott and Lutz 1998 ) . There is, for example, a 40% reduction in the density of voltage-gated Na+ channels in the anoxic turtle cerebellum (Perez-Pinzon et al. 1992b ) . A reduced density of voltage-gated Na+ channels is possibly responsible for the increased action potential threshold seen in the anoxic turtle brain (Perez-Pinzon 1992b ). Another key ion that is altered by anoxia in the turtle and is involved in electrical activity and synaptic potential is Ca+2 . There is a slow and mild increase in cytosolic Ca +2 in turtles during anoxia, which may refl ect a rise in extracellular Ca+2 derived from the breakdown of bone and shell needed to buffer the lactate load (Bickler and Hansen 1998 ) . This mild elevation in cytosolic Ca+2 may play a key role in initiating neuroprotective mechanisms in turtles (Bickler and Hansen 1998 ) . It is interesting to note that mild increases in Ca +2 also play a key role in the induction of ischemic preconditioning (as will be reviewed in future chapters) (Raval et al. 2003 ) . Other ionic changes may arise from the release of neurotransmitters or neuromodulators during anoxia in the turtle brain. For example, adenosine, which is well known to act as an inhibitory neuromodulator in mammals, is another endogenous substance that deserves attention as a possible mediator of metabolic depression in anoxia-tolerant species. Adenosine is formed from the breakdown of the high- energy purines ATP, ADP, and AMP. Consequently, the mammalian brain greatly increases its adenosine levels both intracellularly and extracellularly during energy defi ciency induced by anoxia where it acts to produce an increase in cerebral blood fl ow (Collis 1989 ) , stimulates glycogenolysis (Magistretti et al. 1986 ) , and decreases neuronal excitability as well as suppress excitatory neurotransmitter release (Prince and Stevens 1992 ) . In consequence, adenosine is thought to be an important endogenous protective agent in mammalian ischemia and anoxia (Rudolphi et al. 1992 ) . In the turtle brain, shortly after the onset of anoxia, there is a marked but temporary rise in extracellular adenosine, which is probably linked to the contemporaneous decline in ATP (Nilsson and Lutz 1992 ) . The hypothesis that adenosine plays a critical role in anoxia tolerance is supported by the fi nding that superfusing the anoxic isolated turtle cerebellum with adenosine receptor blockers theophylline or 8-cyclopentyltheophylline caused rapid depolarization (Perez-Pinzon et al. 1993 ) . The rise in adenosine is transitory and has the consequence of increasing cerebral blood fl ow (Hylland et al. 1994 ) . In addition to its effects on blood fl ow, during anoxia adenosine was shown to reduce cerebral K+ release (Pek and Lutz 1997 ) and downregulate neuronal conductance and glutamate NMDA receptor activity (Buck and Bickler 1998 ; Ghai and Buck 1999 ; Pamenter et al. 2008a ) , which are postsynaptic events, and inhibit glutamate release (Milton et al. 2002 ; Thompson et al. 2007 ) , which is primarily a presynaptic event. + + Another potential target of adenosine is the ATP-regulated K channel (K ATP ), + which also opens when ATP levels decrease. In the turtle brain, K ATP channel opening occurred via adenosine A1 receptors and appears to be involved in mediating a reduced K+ fl ux out of anoxic turtle neurons (Pek and Lutz 1998 ) . Although probably + indirectly regulated by adenosine, the mitochondrial K ATP channel is also 28 J.W. Thompson et al. activated during anoxia in turtle brains, uncoupling mitochondria and reducing mitochondrial Ca2+ uptake, thereby increasing intracellular Ca2+ which in turn acts to reduce the activity of excitatory glutamate receptors of the NMDA type (Pamenter et al. 2008b ) . The potential role of neurotransmitters in anoxia tolerance in turtles is enhanced by the fact that during anoxia/ischemia in mammalian brains, a massive release of excitatory neurotransmitters occurs that amplify the pathological events during such insults. Thus, excitotoxicity avoidance may be a crucial step in brain survival. The turtle brain appears to be protected from this excitotoxicity event that occurs in mammalian brains during anoxia/ischemia by avoiding the release of excitatory amino acids (EAA) and in turn increasing the release of inhibitory neurotransmitters (Nilsson and Lutz 1991 ; Thompson et al. 2007 ; Milton et al. 2002 ) . It has been hypothesized that anoxic survival is enhanced by a greater release of inhibitory than excitatory neurotransmitters. In the turtle brain, extracellular GABA levels increase signifi cantly after about 100 min of anoxia, while glutamate and aspartate levels were unchanged for 4 h (Nilsson and Lutz 1991 ) . In particular, the rise in extracellular GABA levels in turtles is massive (Nilsson and Lutz 1991 ) . At such high levels, GABA can be expected to function as an endogenous anesthetic, mediating the near-comatose state that characterizes the anoxic turtle. In turtles exposed to 24 h of anoxia at room temperature, the rise in extracellular GABA is accompanied by an increase in the number of GABAA receptors, which may further increase the inhibitory action of GABA (Lutz and Leone-Kabler 1995 ) .

2.4 Anoxia Tolerance in Fish

Another well-studied example of anoxia tolerance is the crucian carp (Carassius carassius ), which has the ability to survive anoxia for a day or two at room temperature and for months at close to 0°C. Similar to the anoxic turtle, the crucian carp has the remarkable ability to depress its metabolic activity to match the reduced energy production by anaerobic glycolysis, which produces an ATP yield less than one- tenth of that obtained from complete glucose oxidation (Hochachka and Somero 2002) . It appears that the only limitation to anoxia survival by the crucian carp and turtle is their glycogen stores (Nilsson 1990 ; Warren et al. 2006 ) . This is the primary reason for the temperature dependence of anoxia survival because metabolism exponentially increases with temperature, resulting in a more rapid depletion of energy reserves. Although the freshwater turtle and the crucian carp both demonstrate remarkable abilities for anoxia survival, the mechanisms by which each responds to anoxia differ greatly. Unlike mammals, which rapidly lose ATP levels during brief periods of anoxia, the anoxic crucian carp is able to maintain brain ATP levels, thereby avoiding the detrimental processes, which are initiated by the failure of ATP-driven systems such as ion pumping. The balancing of energy production to metabolism during anoxia requires either an upregulation of glycolytic ATP production (called Pasteur effect) 2 Anoxia Resistance in Lower and Higher Vertebrates 29 or a reduction in ATP-consuming pathways, a strategy often called metabolic depression. As described above, the anoxic turtle strongly depresses both neuronal and cardiac activities, thereby entering a comatose-like state. In contrast, the crucian carp depresses metabolic activity but still maintains a degree of both neuronal and swimming activity. In the laboratory, 5 h of anoxia at 9°C reduced the crucian carp’s spontaneous swimming activity by 50%, which probably corresponds to a 34–40% reduction in whole-body ATP usage (Nilsson et al. 1993 ) . The crucian carp also doubles its brain blood fl ow within the fi rst minutes of anoxia which likely allows for the maintenance of a high degree of neuronal activity (Nilsson et al. 1994) . Therefore, whole-body metabolism during anoxia in the crucian carp is higher than in the turtle. In the goldfi sh (Carassius auratus), a close relative of the crucian carp, anoxia reduces heat production (an indicator of metabolism) by one- third of normoxic levels (Van Waversveld et al. 1989 ) . In crucian carp telencephalic brain slices, lactate and heat production (using microcalorimetry) measurements suggest only a 30% reduction in ATP consumption and an upregulation of glycolysis (Johansson et al. 1995 ) . For comparison, the turtle reduces body heat production 90–95% during anoxia (Jackson 1968 ) . A key feature which allows the crucian carp to remain active during anoxia is its ability to produce ethanol as the main anaerobic end product, which easily diffuses through the gills into the surrounding water. The mechanism by which the crucian carp “senses” anoxia and thus transitions into a hypometabolic state is primarily regulated by the net breakdown products of phosphorylated adenylates (ATP, ADP, and ADP), mainly the ratio of ADP:ATP and adenosine. Numerous studies have demonstrated the importance of adenosine in anoxia survival by the turtle. However, the role of adenosine in the crucian carp survival during anoxia is not as fi rm as for the turtle. Treating the crucian carp with the adenosine receptor blocker aminophylline prevents anoxia-induced increases in brain blood fl ow and increases the rate of ethanol release into the water threefold (Nilsson et al. 1994; Nilsson 1991) . In the goldfi sh, adenosine causes a decrease in protein synthesis and Na+ /K+ ATPase activity, indicators of reduced metabolism, when added to gold fi sh hepatocytes (Krumschnabel et al. 2000 ) . Therefore, it appears that adenosine plays a signi fi cant role in anoxic metabolic depression in the crucian carp (Nilsson 1991 ) . However, microdialysis studies in the brain of crucian carp have failed to detect an increase in extracellular adenosine (P. Hylland and G.E. Nilsson, unpublished). + ATP-regulated K channels (K+ATP ) open in response to decreased ATP levels.

Although there is good evidence for a signi fi cant role of K +ATP channels in anoxic survival by the freshwater turtle, there is little evidence for such a role of K+ATP channels in the anoxic crucian carp. For example, treating crucian carp with the K+ATP channel blocker glibenclamide had no signifi cant effect on brain K + homeostasis (Johansson and Nilsson 1995 ) . Instead, it appears that AMP-activated protein kinase (AMPK) mediates key anoxic survival mechanisms in the crucian carp. AMPK, termed a metabolic master switch, is activated by phosphorylation during periods of increased AMP/ATP ratios. Activated AMPK inhibits anabolic energy-consuming pathways and stimulates energy-producing catabolic pathways. In the anoxic crucian carp, AMPK phosphorylation levels (an indicator of activation) are increased in the brain, heart, and liver (Stenslokken et al. 2008 ) . Inhibiting AMPK activation with 30 J.W. Thompson et al. compound C signi fi cantly increases ethanol production (i.e., metabolic rate) and decreases cellular energy charge. However, these changes in the presence of compound C are modest and do not lead to anoxia-induced death. Therefore, adenosine and AMPK appear to play a signi fi cant role in activating key anoxic survival mechanisms in the crucian carp, which allow for a reduction in ATP-consuming pathways. As discussed above, channel arrest is clearly part of the turtle’s anoxia tolerance leading to reduced energy demands. However, in the crucian carp, channel arrest does not seem to play an important role in anoxia tolerance. For example, neural K + and Ca 2+ permeability appears unchanged during anoxia exposure in the crucian carp (Johansson and Nilsson 1995 ; Nilsson 2001 ) . Furthermore, a recent study which examined expression of genes associated with excitatory neurotransmission demonstrates that at 1 week of anoxia (12°C), Ca2+ channels and AMPA receptor expression were maintained, whereas expression of voltage-gated Na+ channels were increased by 50% with a corresponding decrease in expression of some units of the NMDA receptor (Ellefsen et al. 2008 ) . Therefore, it appears that the crucian carp has evolved strategies other than reductions in ion channel activity to reduce the metabolic demand of the anoxic brain yet maintain a high level of neuronal and physical activity. A more dynamic and rapid control of ion channel activity may occur through the regulation of neurotransmitter release. Glutamate when released from the cell stimulates neuronal activity, which would increase ion pump activity and therefore increase energy use. As a result, the anoxic crucian carp maintains normal extracellular glutamate levels, thereby preventing excessive ATP usage (Hylland and Nilsson 1999 ) . In contrast to glutamate, GABA activates ion channels which increases the membrane − + conductance to Cl (through GABAA receptors) or K (through GABAB receptors) resulting in membrane hyperpolarization. Therefore, GABA decreases ion membrane depolarization and action potential formation, which presumably would reduce ion channel activity and thus preserve ATP levels. In the crucian carp brain, extracellular GABA levels increase approximately 50% after 6 h of anoxia (Hylland and Nilsson 1999 ) . This rise in extracellular GABA levels in the crucian carp is modest when compared to the turtle which increases extracellular GABA levels nearly 80-fold within 6 h of anoxia (Nilsson and Lutz 1991 ) . Interestingly, gene expression of several components of the GABAergic neurotransmission system reveals a slight reduction in

GABAA receptor subunit expression in the crucian carp after 1–7 days of anoxia at 8°C (Ellefsen et al. 2009 ) , suggesting a modest activation of the GABAergic system. Blocking GABA receptors or GABA synthesis increases ethanol release into the water threefold, pointing to a role of GABA in reducing whole-body metabolic depression in anoxic crucian carp brains (Nilsson 1992 ) .

2.5 Conclusions

In conclusion, as it will be obvious in latter chapters, the mechanisms of anoxia tolerance in lower vertebrates resemble many of the already well-de ﬁ ned mechanisms involved in ischemic preconditioning in the brain. Additional research in both 2 Anoxia Resistance in Lower and Higher Vertebrates 31 areas of research, anoxia tolerance in lower vertebrates and ischemic preconditioning in anoxic/ischemic susceptible species, will further shed light into potential new mechanisms that may indeed have novel therapeutic value.

References

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Kelly L. Drew , Jeffrey A. Zuckerman , Phillip E. Shenk , Lori K. Bogren , Tulasi R. Jinka , and Jeanette T. Moore

3.1 Hibernation

Hibernation, a means of systemic energy conservation, is a remarkable orchestration of behavioral, physiological, and molecular adaptations that de ﬁ es the need for most life-sustaining processes. Diverse mammalian species including one species of primate (Dausmann et al. 2004 ) hibernate. The phenomenon is well characterized in large mammals such as the black bear (Toien et al. 2011 ) , but is studied most frequently in smaller rodent species such as hamsters (Hoffman et al. 1968 ) and ground squirrels (AGS) (Carey et al. 2003 ) . When hibernating, animals may spend from a few days to several weeks at a time in a highly regulated and reversible state of torpor during which whole body metabolic rate, core body temperature (T b), heart rate, and blood ﬂ ow decrease to dramatic lows. In small hibernating mammals, T b decreases in parallel with ambient temperature down to near 0°C.

When ambient temperature is less than 30°C, T b falls below 30°C, and bouts of prolonged torpor are interrupted by brief (approx. 1 day), spontaneous arousals. Interbout arousals occur at regular intervals during which animals spontaneously return to high, euthermic Tb of 35–37°C (Geiser and Ruf 1995 ) and blood ﬂ ow returns to vital organs (Osborne et al. 2005 ) .

K. L. Drew , Ph.D. (*) • L. K. Bogren • T. R. Jinka • J. T. Moore Institute of Arctic Biology, Alaska Basic Neuroscience Program , University of Alaska Fairbanks , 902 N. Koyukuk Dr. , Fairbanks , AK 99775-7000 , USA e-mail: [email protected] J. A. Zuckerman • P.E. Shenk Radiology Consultants, Inc. , Fairbanks , AK , USA

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 37 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_3, © Springer Science+Business Media New York 2013 38 K.L. Drew et al.

3.2 Tolerance to Hypoxia, Cerebral Ischemia, and Brain Injury in Hibernating Rodents

During hibernation, cerebral blood fl ow waxes and wanes as much as tenfold in parallel with body temperature. Mass-weighted cerebral blood fl ow in hibernating animals is 7 ± 4 ml 100 g −1 min−1 compared to 62 ± 18 ml 100 g −1 min−1 in active, euthermic animals (Frerichs et al. 1994) . During interbout arousals, blood fl ow returns in a heterogeneous manner (Osborne et al. 2005 ) as illustrated in Fig. 3.1 . Despite prolonged ischemic-like levels in local cerebral blood fl ow, ground squirrels (AGS) show no evidence of ischemic injury (Frerichs et al. 1994; Ma et al. 2005 ) . Indeed, hibernating species demonstrate robust neuroprotection from which novel therapies may come to light. Hibernating species tolerate hypoxia better than nonhibernating species (D’Alecy et al. 1990 ) and experience hypoxemia regularly during emergence from hibernation (Ma et al. 2005 ) . Acute hippocampal slices from 13-lined ground squirrels (AGS) tolerate oxygen glucose deprivation (OGD) in a hibernation state and species-dependent manner (Frerichs and Hallenbeck 1998 ) , and hibernating ground squirrels (AGS) demonstrate pronounced neuroprotection in vivo following penetrating brain injury (Zhou et al. 2001 ) .

3.3 Is Resistance to Injury Due to the Hibernating State?

Evidence suggests that hibernation is associated with a variety of adaptations that may contribute to resistance to brain injury. These include hypothermia (Busto et al. 1987 ) with associated metabolic suppression, immunosuppression, leukocytopenia and lymphopenia (Bouma et al. 2011 ; Prendergast et al. 2002 ) , decreased numbers of platelets, and increased blood clotting time (Lechler and Penick 1963 ; Pivorun and Sinnamon 1981 ) as well as increased activity and concentration of antioxidants (Orr et al. 2009 ; Toien et al. 2001 ) . This combination of adaptations, collectively

Fig. 3.1 (continued) Institute of Arctic Biology animal facility at the University of Alaska Fairbanks to the MRI department at Fairbanks Memorial Hospital within 15–20 min. The chest and belly of the animals were shaved, cardiac gating leads were positioned on the skin, and rectal temperature was recorded at the beginning when animals were torpid and at the end of the imaging session. Torpid animals were placed in a plastic cylinder with a head restraint and placed into the phased-array, small extremity, 4-element surface coil and then into the bore of a General Electric 1.5 Tesla Horizon LX, and images were acquired using 11.1 software. 2D TOF magnetic resonance angiography was performed in recurrent succession as the animal aroused from torpor. Blood fl ow was calculated using the General Electric software at the following sites: carotid arteries, celiac artery, and the iliac vessels. These respective locations represented blood fl ow to the brain, liver, and lower extremities. Ten to thirteen scans were performed utilizing magnetic resonance angiography for qualitative and quantitative analysis of blood fl ow. Movement interfered with imaging once the animal reached the irritable phase of heterothermia. Movement thus de fi ned the irritable phase and was minimized by placing cotton moistened with metofane loosely over the animal’s nose. Images shown are from a representative animal at the three anatomic locations as indicated while the animal was torpid with body temperature of 3.4°C (left ), just after the animal was anesthetized with metofane (irritable phase, middle ) and again after rectal temperature reached 34°C (right ) 39

Fig. 3.1 Hibernating arctic ground squirrels (AGS) experience pronounced fl uctuations in blood fl ow during torpor and arousal. During arousal, there is preferential perfusion of the brain, followed by the vital abdominal organs (kidney, liver, spleen, and bowel), and, fi nally, the extremities. Five arctic ground squirrels (AGS) in deep torpor while housed at 2°C were transported at 4°C from the 40 K.L. Drew et al. referred to as the hibernation phenotype, provides proof of concept for combination therapies (Drew et al. 2001 ) . Understanding the contribution of individual adaptations that protect these animals from brain injury and the mechanisms that regulate adaptations expressed by hibernating species may lead to novel therapies for treatment of traumatic brain injury, stroke, cardiac arrest, and other conditions associated with ischemia and reperfusion.

3.4 Tolerance Associated with Torpor

Arctic ground squirrels (AGS) (arctic ground squirrels (AGS); Urocitellus parryii ; reclassi ﬁ ed from Spermophilus parryii ) demonstrate remarkable protection from brain injury when torpid (Zhou et al. 2001 ) . However, because hibernating ground squirrels (AGS) experience tissue temperatures that are unlikely to be tolerated by humans, it is important to separate the in ﬂ uence of tissue temperature from other neuroprotective mechanisms that may more likely translate to clinical applications. Reports dating back to the early 1800s describe hypoxia tolerance in the hibernating state in bats, marmots, and ground squirrels (AGS) and show that tolerance displayed during hibernation exceeds tolerance observed during euthermy (Bullard et al. 1960) . To distinguish effects of temperature from other neuroprotective mechanisms, Frerichs and Hellenbeck ( 1998 ) studied modeled ischemia/reperfusion (I/R) in acute hippocampal slices from 13-lined ground squirrels (AGS) (Spermophilus tridecemlineatus ) and rats. These authors found that at 36°C, slices from torpid ground squirrels (AGS) tolerated modeled I/R better than slices from active ground squirrels (AGS). In these experiments, lowering bath temperature increased duration of modeled I/R tolerated in all slices studied from 5–8 min at 36°C to 100–250 min at 7°C. The magnitude of effect of cooling was greater in slices from ground squirrels (AGS) than from rats and in slices from torpid ground squirrels (AGS) than from active ground squirrels (AGS). At 36°C, the duration of ischemia tolerated by slices prepared from active ground squirrels (AGS) did not differ from slices prepared from rats, and slices from both rats and active ground squirrels (AGS) were more vulnerable to modeled I/R than slices prepared from torpid ground squirrels (AGS). By contrast, the arctic ground squirrels (AGS) resists cerebral I/R injury regardless of the hibernating state. Although tolerance may be greater when torpid, a limit on tolerance in the euthermic state has not been determined. Studies of semi-acute hippocampal slices prepared from arctic ground squirrels (AGS) found that at 36°C, slices from hibernating and active ground squirrels (AGS) resisted cell death induced by modeled I/R equally well and better than rats (Ross et al. 2006) . When slices were cultured for an additional 24 h, in an organotypic-like culture, hippocampal slices from active ground squirrels (AGS) became more vulnerable to modeled I/R. Thus, after 48 h in culture at 36°C, slices from torpid arctic ground squirrels (AGS) tolerated modeled I/R better than slices from euthermic animals. Hippocampal slices from adult arctic ground squirrels (AGS) did not survive well in the organotypic preparation and could not be maintained beyond 3–4 days. Indeed, a decrease in ischemia tolerance in slices from euthermic arctic ground squirrels (AGS) may have been related to loss of overall viability after 48 h in culture. 3 Hibernation: A Natural Model of Tolerance to Cerebral Ischemia/Reperfusion 41

3.5 Mechanisms of OGD Tolerance Associated with Torpor

Additional mechanisms may contribute to I/R tolerance in the hibernating state. Two of these mechanisms include downregulation of Na+ /K + ATPase (Ross et al. 2006 ) and a decrease in NMDA receptor–induced increase in intracellular calcium (Zhao et al. 2006 ) . Both of these mechanisms fall under the general category of ion channel arrest. Downregulation of ion channels that contribute to loss of ion homeostasis, ATP depletion, and calcium overload may play a role in overall metabolic suppression during hibernation (Hochachka 1986 ) and be an important neuroprotective aspect of the hibernation phenotype that is expressed in other examples of ischemia tolerance (Stenzel-Poore et al. 2003) . Slower decline of ATP in brain slices taken from torpid ground squirrels (AGS) than in slices taken from euthermic ground squirrels (AGS) is also consistent with ion channel arrest during torpor (Drew et al. 2009 ) .

3.6 Tolerance Independent of Torpor

As noted above, acute hippocampal slices prepared from arctic ground squirrels (AGS) tolerate modeled I/R at 36°C better than rat hippocampal slices whether slices are prepared from arctic ground squirrels (AGS) in the torpid or euthermic state. This fi nding reported initially by Ross et al. ( 2006 ) was replicated using a standard acute hippocampal slice method (Christian et al. 2008 ) . Active arctic ground squirrels (AGS) fall into two categories: those that emerge from torpor for brief periods of euthermy during the winter hibernation season (termed interbout euthermy or interbout arousal) and those that are active (euthermic) during the summer season. Christian et al. ( 2008 ) showed that slices obtained from torpid arctic ground squirrels (AGS) or from arctic ground squirrels (AGS) during an interbout arousal resist cell death following OGD equally well, showing slight but signifi cant increases in cell death after 2 h of OGD, but not after 30 min of OGD. Chemical inhibition of glycolysis and cellular respiration with iodoacetate and NaCN during OGD fail to increase cell death beyond the slight increase seen with OGD alone. Slices obtained from summer-active arctic ground squirrels (AGS) resist cell death to a greater degree than slices obtained from arctic ground squirrels (AGS) during interbout arousal. Even 2 h of OGD fails to increase cell death in slices from this group of animals (Christian et al. 2008 ) . Arctic ground squirrels (AGS) hippocampus resists OGD-induced cell death despite signi fi cant decreases in ATP. In stroke, the mitogen-activated protein kinase (MAPK) family mediates a range of activity from metabolism, motility, and in fl ammation to cell death and survival (Sawe et al. 2008 ) . Using an acute slice model, Christian and others found that MAPK proteins are regulated in a manner consistent with promoting survival in arctic ground squirrels (AGS). However, blocking activation of the MAPKs, ERK1/2 and JNK failed to impair resistance to OGD (Christian et al. 2008 ) . Hibernation is a seasonal phenomenon. In some tissues from some species, tolerance to I/R during winter interbout euthermy is greater than during summer euthermy. Liver and intestine isolated from the 13-lined ground squirrel during interbout arousal resist I/R injury; however, tolerance is lost or decreased when tissue is obtained from animals during the summer season (Kurtz et al. 2006 ; Lindell et al. 2005 ) . By contrast, 42 K.L. Drew et al. tolerance to cerebral I/R in arctic ground squirrels (AGS) is actually greater during the summer season than during winter interbout euthermy (Christian et al. 2008 ) . Tolerance to cerebral I/R observed in vitro in euthermic arctic ground squirrels (AGS) suggested that euthermic arctic ground squirrels (AGS) would tolerate global cerebral ischemia in vivo. Dave et al. (2006 ) challenged summer-active arctic ground squirrels (AGS) and rats with 8 min of asphyxia leading to cardiac arrest. Apnea induced an immediate bradycardia followed by cardiac arrest and global cerebral ischemia in both species. Moreover, the duration of hypotension below 50 mmHg and measures of blood pressure during cardiac arrest and resuscitation were similar between the two species. Rats showed signifi cant cell death in hippocampus, striatum, and cortex 7 days after restoration of spontaneous circulation (ROSC). However, despite the apparent similarity in severity of global cerebral ischemia in the two species, arctic ground squirrels (AGS) showed no evidence of cell death in any of the brain regions studied. Moreover, rats showed signifi cant neurological impairment with evidence of hind limb paralysis while arctic ground squirrels (AGS) showed no evidence of paralysis.

3.7 Mechanisms of OGD Tolerance Independent of Torpor

While multiple adaptations contribute to ischemia tolerance during torpor, protection that does not depend on the torpid state may be more readily attainable in nonhibernating species such as humans. Moreover, mechanisms of tolerance in the euthermic state impart resistance to injury that if translated to other species would serve as a prophylactic to injury rather than as a neuroprotectant after injury. Tolerance to OGD in euthermic arctic ground squirrels (AGS) occurs downstream to ATP depletion (Christian et al. 2008 ) and involves preservation of ionic homeostasis that is dependent upon protein kinase C epsilon (e PKC) signaling. Dave et al. (2009 ) monitored ischemic depolarization (ID) in cerebral cortex during cardiac arrest in vivo and during OGD in vitro in acutely prepared hippocampal slices from arctic ground squirrels (AGS) and rat. In both the in vitro and in vivo model of global cerebral ischemia, the onset of ID was signi ﬁ cantly delayed in arctic ground squirrels (AGS) compared with rat. During cardiac arrest, ID occurred on average at 1.9 min in rats and at 3.1 min in arctic ground squirrels (AGS). During OGD in hippocampal slices, ID occurred at 2.8 min in rat and at 6.6 min in arctic ground squirrels (AGS). Blocking or delaying the ID can signi ﬁ cantly improve recovery (Anderson et al. 2005 ; Takeda et al. 2003 ) . The selective peptide inhibitor of ePKC (eV1-2) shortened the time to ID in brain slices from arctic ground squirrels (AGS) but not in rats even though e V1-2 decreased activation of e PKC in brain slices from both species. Activation of e PKC inhibits Na+ /K + ATPase and voltage-gated sodium channels (Chen et al. 2005; Nowak et al. 2004) , both of which contribute to the collapse of ion homeostasis during ischemia and may be targets of ePKC during cerebral ischemia in arctic ground squirrels (AGS) (Dave et al. 2009 ) . Other mechanisms downstream to ID may also contribute to cerebral ischemia tolerance in arctic ground squirrels (AGS). During cerebral ischemia the loss of neuronal membrane potential that leads to ID results in the massive release of neurotransmitters including the excitatory neurotransmitter glutamate (Lipton 1999 ) . 3 Hibernation: A Natural Model of Tolerance to Cerebral Ischemia/Reperfusion 43

Fig. 3.2 Arctic ground squirrels (AGS) hippocampus resists cell death following OGD despite a surge in glutamate ef fl ux. Acute hippocampal slices from rats show evidence of OGD-induced cell death (a ) and glutamate ef fl ux (c ). By contrast, OGD-induced cell death is substantially less in slices prepared from euthermic arctic ground squirrels (AGS) (eAGS) (b ), although OGD-induced glutamate ef fl ux is similar in rat (c ) and arctic ground squirrels (AGS) (d ). According to the method developed previously (Kirschner et al. 2009 ) , transverse hippocampal slices (400 m m) were prepared from adult male Sprague Dawley rats (age 3–4 months, 350–400 g) and adult euthermic arctic ground squirrels (AGS) (eAGS) of both sexes (950–1,690 g) in 2°C oxygenated HEPES- aCSF. Slices were allowed to recover 1 h in 22°C oxygenated HEPES-aCSF prior to placement in individual chambers (4–8 chambers operated in parallel). Acute slices were perfused in micro- chambers at a fl ow rate of 7 m l/min at 36°C with oxygenated aCSF to collect baseline samples.

OGD solution was a N2 -saturated aCSF with glucose omitted. After stable baseline, slices were perfused with OGD (~50 mmHg O2 ) for 30 min. Perfusate was collected at 15-min intervals during pretreatment, treatment, and reperfusion for up to 8 h. LDH analysis was carried out by Cayman Chemical LDH cytotoxicity assay kit and normalized to total protein determined by Bio-Rad protein assay. Glutamate was quantiﬁ ed in perfusate samples after derivatization with naphthalene- 2,3-dicarboxaldehyde using capillary electrophoresis with laser-induced ﬂ uorescence detection

Glutamate effl ux into the extracellular space activates N-methyl- d -aspartate (NMDA) and a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, causing excitotoxic calcium infl ux (Lipton 1999 ) . Pharmacological inhibition of NMDA receptors during early reperfusion following cerebral ischemia reduces ischemic damage in preclinical studies (Lipton 1999 ) . We have shown recently that 30-min period of exposure to OGD in vitro increases glutamate effl ux in acute hippocampal slices from summer-active arctic ground squirrels (AGS) and rat, but produces little evidence of cell death in arctic ground squirrels (AGS) (Fig. 3.2 ). Thus, glutamate released during OGD is not excitotoxic. Attenuated 44 K.L. Drew et al. excitotoxicity in arctic ground squirrels (AGS) that is independent of the hibernation state is further supported by evidence in the semi-acute slice preparation developed by Ross et al. ( 2006 ) . With this preparation, hippocampal slices from active arctic ground squirrels (AGS) resist cell death induced by 500 m M NMDA plus 20 mM KCl. The level of cell death following this excitotoxic challenge in slices from active or hibernating arctic ground squirrels (AGS) is no different from control treated slices. By contrast, the same concentrations of NMDA and KCl applied to slices from rat produce a signifi cant increase in cell death (Ross et al. 2006) . Protective mechanisms downstream to glutamate ef fl ux may be related to differences in the effects of glutamate receptor activation in arctic ground squirrels (AGS) compared to rat. Lower levels of functional NMDA receptors located in the plasma membrane and less glutamate-induced increases in intracellular calcium are associated with ischemia tolerance in euthermic arctic ground squirrels (AGS). Membrane expression of NR1, an obligatory subunit of the NMDA receptor, is lower in arctic ground squirrels (AGS) (both active and hibernating) compared to rat. Moreover, intracellular calcium increased by bath-applied glutamate does not exceed 400 nM in hippocampal slices prepared from euthermic or hibernating arctic ground squirrels (AGS). In rats, intracellular calcium exceeds 500 nM (Zhao et al. 2006 ) . 500 nM may underestimate intracellular calcium in rats. Fura-2, the calcium indicator used in these studies, has a K d of about 300 nM and may be saturated at calcium concentrations above 500 nM. In organotypic slices from rat, OGD increases intracellular calcium concentrations to almost 2,000 nM (Bickler and Fahlman 2004 ) . A Ca2+ set-point hypothesis suggests 2+ that a critical level of [Ca ]i is necessary to stimulate pro-survival and regenerative pathways (Snider et al. 2002 ) . More work is necessary to determine the impact of OGD on intracellular calcium in arctic ground squirrels (AGS) hippocampus. Other aspects of the arctic ground squirrels (AGS) phenotype may contribute to cerebral I/R tolerance. Euthermic arctic ground squirrels (AGS) appear to have a lower respiratory drive than other mammals and experience mild, chronic hypoxemia when active. Chronically low O2 saturation is associated with chronically high hypoxia-inducible factor-1a (HIF-1 a) protein levels in brain (Ma et al. 2005) . In rats, hypoxia-induced preconditioning is linked to HIF-1a -regulated gene expression and enhanced tolerance to subsequent ischemic events (Bergeron et al. 2000 ) . Repeated intermittent hypoxia extends the duration of hypoxic preconditioning from days to months in the rodent brain (Stowe et al. 2011 ) . A similar phenomenon could contribute to chronic ischemia tolerance in arctic ground squirrels (AGS).

3.8 Arctic Ground Squirrels Do Not Rely on Glycogen or Oxidative Phosphorylation for Energy Needs During OGD

Many organisms tolerant of low oxygen levels possess large stores of glycogen and pH buffering mechanisms that fuel and protect against pH shifts resulting from anaerobic glycolysis (Jackson 2004 ; Lutz and Milton 2004 ) . Indeed, arctic ground squirrels (AGS) demonstrate enhanced pH buffering capacity since blood pH remains around 7.4 despite arterial PCO2 levels of 60 mmHg (Ma et al. 2005 ) and 3 Hibernation: A Natural Model of Tolerance to Cerebral Ischemia/Reperfusion 45

− arterial HCO3 concentrations tend to be higher in arctic ground squirrels (AGS) compared with rat. However, ischemia tolerance cannot be explained by enhanced peripheral stores of glycogen. Firstly, glucose derived from glycogen is not expected to reach ischemic tissue when blood ﬂ ow is stopped during cardiac arrest. Secondly, addition of iodoacetate, an inhibitor of glycolysis, as well as the addition of NaCN, an inhibitor of cellular respiration, during OGD fails to increase cell death in an acute slice preparation (Christian et al. 2008 ) .

3.9 Tolerance to Cerebral I/R in Arctic Ground Squirrels May Be Necessary to Tolerate Transitions into and Out of Torpor

Hibernating animals may have acquired aspects of I/R tolerance as a means to tolerate transitions into and out of torpor. Hibernation per se is not a hypoxic state where, due to decreases in O2 utilization, Pa O2 can exceed normoxic values. However, as arctic ground squirrels (AGS) work to rewarm, Pa O2 falls to a minimum of 7 mmHg

(0.9 kPa) for as long as 60 min at the same time as oxygen consumption peaks. P a O2 then remains below 35 mmHg (4.7 kPa) for as long as 4 h (Ma et al. 2005 ) . Evidence suggests that arctic ground squirrels (AGS) and other hibernating mammals experience signiﬁ cant challenge to physiological homeostasis during arousal from torpor (Lee et al. 2002 ; Ma et al. 2005 ; Osborne and Hashimoto 2005 ) . We speculate that a discord between blood ﬂ ow supply and metabolic demand may occur during entrance into torpor whereas during arousal, metabolic demand must be precari- ously titrated to match the supply of nutrients and oxygen. Capacity to tolerate challenges to physiological and cellular homeostasis during transitions into and out of torpor stems, in part, from mechanisms that operate at the cellular and tissue level. Some hibernating species such as golden-mantled ground squirrels (AGS) ( Spermophilus lateralis) and hedgehogs (E. europaeus ) may become hypoxic during torpor owing to long periods of apnea. These species often breathe intermittently during hibernation, waiting up to 30 min or longer between breaths. In hedgehogs,

P a O2 is higher in torpor than in the active state (120 vs. 105·mmHg) but falls to 10 mmHg at the end of the apneic period lasting 50–70 min (Tahti and Soivio 1975 ) . Understanding the unique physiology and cellular resistance to I/R in arctic ground squirrels (AGS) holds clues for development of prophylactic treatments for patients at risk for cardiac arrest and stroke. Prophylactic treatments would impart sustained tolerance to cerebral I/R (Savitz and Fisher 2007b ) and avoid variables that may compromise benefi t of neuroprotective therapies (Savitz and Fisher 2007a ) . We discovered that arctic ground squirrels (AGS) tolerate cerebral I/R when not hibernating serendipitously while searching for mechanisms of neuroprotection in the hibernating state. Torpor remains a promising means to suppress metabolic demand, oxidative metabolism, infl ammation, and oxidative stress as well as to facilitate cooling for purposes of therapeutic hypothermia. Other aspects of torpor such as increased SUMOylation may also be benefi cial (Lee et al. 2007 ) . The question remains then, is human hibernation possible? 46 K.L. Drew et al.

3.10 Inducing Hibernation

The capacity to translate reduced metabolic demand displayed during hibernation as well as other neuroprotective aspects of the hibernation phenotype to humans could have therapeutic applications for patients suffering from disruptions in blood ﬂ ow and oxygen delivery (Drew et al. 2001 ) . Recent evidence points toward A1 adenosine receptor (A 1 AR)–dependent signaling within the central nervous system (CNS) as a means to induce hibernation. Using the seasonal characteristics of hibernation as a clue, Jinka et al. ( 2011 ) found that A 1 AR activation within the CNS meets all of the necessary requirements for an endogenous mediator of torpor. However, A1 AR agonist-induced torpor is effective only in the winter season (Jinka et al. 2011 ) . Similarly, spontaneous torpor expressed during the winter season requires A 1 AR stimulation. The A 1 AR antagonist cyclopentyltheophylline (CPT), but not the A2A AR antagonist (MSX-3), delivered into the lateral ventricle (icv) reverses spontaneous torpor. Thus, A1 AR-dependent signaling within the CNS is both necessary and sufﬁ cient to induce and sustain the onset of torpor in arctic ground squirrels (AGS), an obligate, seasonal hibernator.

A 1 AR-dependent signaling is also necessary for onset of other types of torpor.

The A1 AR antagonist CPT reverses spontaneous torpor when administered to Syrian hamsters ( Mesocricetus auratus) during torpor onset (Shiomi and Tamura 2000 ) .

Fasting-induced torpor in mice also depends on central A1 AR activation (Swoap and Lliff 2011) and not 5’-AMP as suggested previously (Swoap et al. 2007; Zhang et al. 2006) . It does not necessarily follow, however, that A 1AR activation is a common means to induce torpor. A1 AR agonists mimic the decline in T b characteristic of torpor, but in some cases, it is unclear if animals enter torpor or simply cool to torpor-like T b . In general, hypothermia is distinguished from torpor by a faster rate of cooling that precedes or parallels a decrease in metabolic rate. Although not tested in all types of torpor, a change in A 1AR signaling within the CNS during preparation for hibernation or torpor may alter the thermoregulatory response to central A1 AR agonists and transform a hypothermic response into torpor.

H2 S, a gaseous transmitter and poisonous gas, induces metabolic suppression in a manner that resembles onset of torpor when ambient temperature is decreased gradually during H2 S exposure (Blackstone et al. 2005 ) . Although not studied directly, A1 AR activation is a viable mechanism for H2 S-induced suspended animation. Hypoxia and ischemia increase adenosine release (Dunwiddie and Masino 2001) . Hypoxia suppresses metabolic rate (hypoxic metabolic response) (Tattersall and Milsom 2003 ) and Tb (hypoxia-induced anapyrexia). Hypoxia-induced decrease in T b depends on central adenosine receptors (Barros and Branco 2000 ; Steiner and Branco 2002 ) , and chemical hypoxia induced by cyanide suppresses neuronal activity via adenosine (Zhu and Krnjevic 1997 ) . Hydrogen sul fi de and IK-101, a liquid sul fide donor, have produced improvements in outcome following severe hypoxia or hemorrhage in small animals (Blackstone and Roth 2007 ; Elrod et al. 2007 ) . Improved outcome following myocardial I/R is associated with preserved myocardial mitochondrial function suggesting that the benefi t may be related to metabolic suppressing effects of systemic H2 S. IK-1001 did not, however, improve outcome in a porcine model of prolonged cardiac arrest 3 Hibernation: A Natural Model of Tolerance to Cerebral Ischemia/Reperfusion 47 and cardiopulmonary resuscitation (Derwall et al. 2010 ) . Recent developments of a method to monitor exhaled H2 S concentrations should facilitate study of the therapeutic bene fi t of H2 S (Toombs et al. 2010 ) . We have begun to explore the effects of dietary restriction (DR) on sensitivity to

A 1 AR agonists as a means to suppress metabolic demand in nonhibernating species.

Dietary restriction lowers T b and enhances longevity presumably through a decrease in oxidative metabolism (Contestabile 2009 ; Ungvari et al. 2008 ) . DR-induced modiﬁ cation of thermoregulation is associated with changes in components of the purinergic neuromodulatory system. Dietary restriction imposed by every other day feeding sensitizes rats to the cooling and metabolic depressant effects of the A1 AR agonist N 6 -cyclohexyladensoine (CHA) and is associated with increased surface expression of A1 AR in the hypothalamus (Jinka et al. 2010 ) . These data suggest that

DR sensitizes A1 AR and in this way contributes to the decline in Tb and metabolic rate during dietary restriction. In summary, small mammals that hibernate provide a natural model of tolerance to brain injury and cerebral I/R. Tolerance is signiﬁ cant whether animals are hibernating or not hibernating. Hibernation is associated with neuroprotective adaptations including cold tissue temperatures that enhance the protected phenotype. Recent discovery that adenosine mediates the onset of torpor brings us one step closer to applying aspects of torpor to human medicine.

Acknowledgments This work was supported by the US Army Research Ofﬁ ce W911NF-05-1-0280, the US Army Medical Research and Materiel Command 05178001, the National Institute of Neurological Disorders and Stroke NS041069-06 and R15NS070779, and Alaska INBRE and Alaska EPSCoR. The authors acknowledge Bianca Zuckerman for assistance with MRI angiography and data analysis. The authors also acknowledge Daniel Kirschner, Thomas Green and Kili Wetherell for assistance with microperfusion experiments and glutamate analysis.

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Derek J. Hausenloy and Derek M. Yellon

4.1 Introduction

In 1986, the possibility of harnessing the heart’s own ability to protect itself from acute ischaemia-reperfusion injury (IRI) was ﬁ rst proposed in a seminal publication by Murry and co-workers ( Murry et al. 1986 ) . In that landmark study, the intriguing discovery was made that subjecting the canine heart to four brief non-lethal episodes of ischaemia and reperfusion could protect it from a subsequent myocardial infarction ( Murry et al. 1986) . This was achieved by occluding the left anterior descending (LAD) coronary artery for 5 min and then reperfusing it for 5 min, a cycle which was repeated 4 times prior to a sustained 40-min episode of lethal myocardial ischaemia and 3 days of reperfusion, a manoeuvre which resulted in a myocardial infarct size, 25% of that observed in control hearts ( Murry et al. 1986 ) . It is interesting to explore the background as to why this somewhat paradoxical study was performed in the ﬁ rst place. At the time, several investigators had been investigating the effects of repetitive bouts of myocardial ischaemia and reperfusion on myocardial ATP content, cell death and myocardial function ( Swain et al. 1984 ; Reimer et al. 1986 ) . Surprisingly, rather than the expected cumulative detrimental effect, they found no depletion in ATP content, no cell death and impairment in function ( Swain et al. 1984; Reimer et al. 1986) . On this basis, Murry et al. ( 1986 ) hypothesized that ‘…multiple brief ischemic episodes might actually protect the myocardium during a subsequent sustained ischemic insult so that, in effect, we could exploit ischemia to protect the heart from ischemic injury’. This powerful endogenous cardioprotective phenomenon was termed ‘ischaemic preconditioning’ (IPC), and its protective effect has been shown to be ubiquitous, being effective in every species tested, including man, and in every organ, including those that

D. J. Hausenloy • D. M. Yellon (*) The Hatter Cardiovascular Institute , University College London Hospital and Medical School , 67 Chenies Mews , London , WC1E 6HX , UK e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 51 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_4, © Springer Science+Business Media New York 2013 52 D.J. Hausenloy and D.M. Yellon comprise the CNS. In both brain and heart, the ongoing challenge has been to elucidate the mechanistic pathways underlying IPC. In the fi eld of cardiac IPC, such investigations have made a major contribution to the 7,000 publications listed on PubMed over the last 25 years, considerably more than that representing the cerebral preconditioning fi eld. Ischaemic heart disease (IHD) is the leading cause of death and disability worldwide. Despite optimal therapy, the morbidity and mortality from IHD remains signifi cant. As such, novel therapeutic strategies are required to protect the heart from the detrimental effects of acute ischaemia-reperfusion injury (IRI) in order to preserve cardiac function and improve clinical outcomes in patients with IHD. Although the clinical application of IPC has not yet been realized, several proof-of- concept clinical studies have reported benefi cial effects with endogenous cardioprotective phenomena closely related to IPC, such as ischaemic postconditioning ( Staat et al. 2005 ) and remote ischaemic conditioning ( Hausenloy et al. 2007 ) . Large multicentre clinical trials are now underway to determine whether these therapeutic strategies can actually improve clinical outcomes in patients with IHD. In this chapter, an overview will be provided of the different forms of ischaemic conditioning for IHD, the underlying mechanisms and their potential clinical application. A single chapter cannot do justice to the amount of research which has already been undertaken in this fi eld, but the highlights of cardiac IPC reviewed here can hopefully serve as a reference point and provide a unique scientifi c juxtaposition for the studies of cerebral preconditioning and ischaemic tolerance discussed in other chapters in this book, given that ischaemic injury in the heart and brain shares many mechanisms. Studies of cardiac preconditioning have identi fi ed many novel targets for pharmacological manipulation, so-called pharmacological conditioning—however, for the sake of brevity, this chapter will be restricted to the mechanical forms of conditioning. For detailed comprehensive reviews on the different forms of ischaemic conditioning for IHD, the reader is referred to the following references ( Yellon and Downey 2003a ; Ovize et al. 2010; Hausenloy and Yellon 2008, 2010b ) .

4.2 The Evolution of Ischaemic Conditioning

Since its initial discovery in 1986, the concept of IPC has evolved and expanded into a variety of different forms, which can be put under the collective term, ischaemic conditioning. The fi rst major development was the discovery in 1993, by Przyklenk and co-workers ( Przyklenk et al. 1993 ) , that the IPC stimulus could be applied to an organ or tissue away from the heart—a phenomenon termed remote ischaemic preconditioning (RIPC). The introduction of RIPC has greatly facilitated the translation of ischaemic conditioning into a variety of clinical settings. It is now possible to protect the heart using a non-invasive, virtually cost-free strategy comprising the infl ation and defl ation of a blood pressure cuff placed on the upper arm or leg to induce brief episodes of ischaemia and reperfusion ( Kharbanda et al. 2002 ) . 4 Preconditioning in the Heart 53

The next major development was the discovery in 2003 by Zhao and co-workers ( Zhao et al. 2003 ) that interrupting myocardial reperfusion with several short-lived episodes of myocardial ischaemia could also reduce myocardial infarct size—a phenomenon termed ischaemic postconditioning (IPost). The introduction of an endogenous cardioprotective strategy which could be applied at the onset of myocardial reperfusion, in contrast to IPC which had to be implemented prior to the index myocardial ischaemia, has meant that IPost could be rapidly translated by Staat and co-workers ( Staat et al. 2005 ) into the clinical setting of ST-segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PPCI). The ability to ischaemic condition the upper or lower limb has made it possible to apply the remote ischaemic conditioning (RIC) stimulus after the onset of myocardial ischaemia but prior to myocardial reperfusion (termed remote ischaemic preconditioning) ( Schmidt et al. 2007 ) , a strategy which has been successfully applied to STEMI patients prior to PPCI ( Rentoukas et al. 2010 ; Botker et al. 2010 ) . Finally, the application of the RIC stimulus at the onset of myocardial reperfusion has also been described (termed remote ischaemic postconditioning) ( Kerendi et al. 2005 ; Andreka et al. 2007 ) in the experimental setting but remains to be translated into the clinical setting. In parallel to this, evolution has been the use of pharmacological agents, which target different components of the signalling pathway underlying ischaemic conditioning, to mimic its cardioprotective effects giving rise to the terms pharmacological preconditioning, preconditioning and postconditioning. This area of research does not standstill with newly introduced concepts being introduced such as repeated remote ischaemic postconditioning (remote lower limb postconditioning applied every day or every 3 days for 28 days reduced adverse LV remodelling post-MI in the rat heart) ( Wei et al. 2011 ) and remote preconditioning of trauma (a surgical incision alone preconditioned the murine heart against MI via a neurogenic mechanism) ( Jones et al. 2009 ) .

4.3 Ischaemic Preconditioning

A standard IPC stimulus induces two phases of cardioprotection: the fi rst phase (termed classical preconditioning or the fi rst window of protection) begins immediately following the IPC stimulus and lasts for 2–3 h, after which the cardioprotective effect wanes and disappears, with the second phase (termed the second window of cardioprotection) or delayed or late preconditioning reappearing 12–24 h later and lasting up to 72 h ( Marber et al. 1993 ; Kuzuya et al. 1993 ) . The memory effect of the fi rst phase of IPC is a critical feature of this cardioprotective phenomenon, and the underlying mechanism for this remains unclear although it has been attributed to the activation of signalling protein kinases such as PKC. The second phase of cardioprotection or the second window of protection (SWOP) is dependent on the transcription of new protein signalling mediators which are believed to convey the cardioprotective signal from the IPC stimulus to the next day ( Hausenloy and Yellon 2010 ) . 54 D.J. Hausenloy and D.M. Yellon

Fig. 4.1 The metabolic and biochemical changes which occur during myocardial ischaemia and 2+ + reperfusion . During myocardial ischaemia, there is an increase in intracellular Ca , Pi , Na , reactive oxygen species ( ROS ), NADH, H + and a fall in ATP levels and collapse of the mitochondrial membrane potential (D ym ). At myocardial reperfusion, there is repolarization of the D ym and res- 2+ toration of ATP production, a further increase in Ca , Pi , ROS, oxidation of NADH and restoration of physiological pH

It is also possible to maintain a preconditioned state by the repeated application of the preconditioning stimuli such as the administration of an adenosine receptor agonist (a known pharmacological preconditioning agent) ( Dana et al. 1998 ) .

4.3.1 Signalling Pathways Underlying IPC

The signal transduction pathways underlying IPC are both numerous and complex, and only a simplistic overview can be provided in this section. The current paradigm has proposed that the signalling pathway is initiated at the cell membrane surface with the activation of a variety of G-protein-coupled receptors by a number of autacoids (such as adenosine, bradykinin, endothelin, opioids) which are generated by the cardiomyocyte in response to the IPC stimulus. The activation of this receptor then recruits a wide variety of signal transduction pathways, many of which converge on the mitochondria (see Fig. 4.1 for simpli ﬁ ed scheme). 4 Preconditioning in the Heart 55

The signal transduction pathway can be conceptually classiﬁ ed into triggers (these are factors which act before the index ischaemic episode and activate downstream signalling mechanisms and display a ‘memory’ effect) or mediators/end-effectors (these are factors which act during or after the onset of the index ischaemic episode and mediate the protective effect). This separation is not rigid as certain components have been demonstrated to act as both triggers and mediators/effectors.

4.3.1.1 Triggers of IPC

In 1991, Downey’s group examined the role of adenosine, which is generated during myocardial ischaemia from the hydrolysis of ATP, as a potential trigger of preconditioning ( Liu et al. 1991 ) . They demonstrated that the protection associated with IPC could be abolished by pretreatment with a non-speciﬁ c adenosine receptor antagonist, and that a 5-min intracoronary infusion of adenosine followed by 10 min of washout could reproduce IPC ( Liu et al. 1991 ) . These ﬁ ndings had several important implications including the following: (1) IPC-induced protection could be mimicked by pharmacological agents, with these agents acting as triggers of preconditioning (so-called pharmacological preconditioning), and (2) the endogenous activation of a G-protein-coupled receptor (GPCR) was an essential component of IPC-induced protection—that is, IPC was a receptor-mediated phenomenon. Several studies followed, providing support for the involvement of other GPCR ligands as triggers for IPC such as bradykinin ( Parratt 1994 ; Goto et al. 1995) , opioids ( Schultz et al. 1995 ) , acetylcholine ( Yao and Gross 1993 ) , catecholamines ( Tsuchida et al. 1994 ) , angiotensin II ( Liu et al. 1995 ) and endothelin-1 ( Wang et al. 1996 ) . Interestingly, IPC via the GPCR appears to be a highly redundant phenomenon, with multiple cycles of IPC able to overcome the effect of antagonism at the bradykinin receptor ( Goto et al. 1995 ) . Therefore, the blockade of a single receptor type served only to raise the ischaemic threshold required to trigger protection, rather than completely blocking it ( Goto et al. 1995) . The simultaneous activation of these GPCRs during the IPC stimulus suggested that the IPC signal converged on a single downstream mediator, PKC. Other triggers of IPC which are covered in later sections include the MitoKATP channel, reactive oxygen species and nitric oxide.

4.3.1.2 Mediators of IPC

The mediators of IPC cardioprotection mainly comprise pro-survival protein kinases such as protein kinase C, tyrosine kinase, Akt, mitogen-activated protein kinases (MAPKs), the JAK-STAT pathway and so forth. In general, the mediators, which are activated by the triggers of IPC, recruit the effector mechanisms of cardioprotection. Studies by Ytrehus et al. in (1994 ) were the fi rst to demonstrate that the activation of protein kinase C (PKC) was required for IPC-induced protection, using PKC analogues to induce protection. Subsequent studies have implicated PKC as a 56 D.J. Hausenloy and D.M. Yellon downstream mediator of IPC triggers such as bradykinin ( Goto et al. 1995 ), adenosine ( Sakamoto et al. 1995 ) , opioids ( Miki et al. 1998 ) and ROS ( Baines et al. 1997 ) . Furthermore, the redundancy effect observed with the GPCR ligands in mediating IPC-induced protection could be correlated to the extent of kinase activity of PKC ( Goto et al. 1995 ) . Tyrosine kinases can be divided into (1) receptor tyrosine kinases which may act as IPC triggers by activating PKC and (2) cytosolic receptor tyrosine kinases which may act as IPC mediators by acting downstream or in parallel with PKC. Activation of receptor tyrosine kinases can take place in response to GPCR-ligand binding ( Sadoshima et al. 1995 ) . In 1996, Maulik et al. ( 1996 ) were the fi rst to demonstrate that genistein, the tyrosine kinase antagonist, could block IPC-induced protection in rat hearts. Experimental studies have implicated signalling through the pro-survival, phosphatidyl inositol 3-OH kinase (PI3K)-Akt cascade during the preconditioning phase before the index ischaemic episode ( Tong et al. 2000 ) and at the time of myocardial reperfusion as part of the reperfusion injury salvage kinase (RISK) pathway ( Hausenloy et al. 2005) , in IPC. Tong et al. ( 2000) were the fi rst to demonstrate that IPC activates the PI3K-Akt kinase cascades prior to the index ischaemic episode, and they showed that inhibiting Akt activity, using the PI3K inhibitor wortmannin, abolished IPC-induced protection using the recovery of function as the end point. Mocanu et al. (2002 ) later con fi rmed these fi ndings in the isolated perfused rat heart infarct model. The MAPKs encompass four major kinase cascades in the heart, the p38 MAPK, the c-Jun NHP2 terminal kinase (JNK), the 42- and 44-kDa extracellular signal- regulated (Erk1/2) MAPK and the big MAP kinase 1 (BMK1 or Erk5) ( Zhou et al. 1995 ; Lee et al. 1995 ; Widmann et al. 1999 ) . These four major kinase pathways exhibit the same conserved three-tier module of a MAPKKK/MKKK (MEKK) phosphorylating a MAPK kinase (MKK or MEK) which then activates the MAPK which comprises the JNK, p38, BMK1 or Erk1/2 MAP kinases. These kinase cascades are activated in response to stress such as ischaemia-reperfusion and receptor protein tyrosine kinases, GPCRs and PKC ( Widmann et al. 1999 ) . Whether Erk1/2 MAPK acts as a mediator of IPC prior to myocardial ischaemia is unclear, with studies demonstrating that it is activated prior to the index ischaemic period ( Mocanu et al. 2002 ; Strohm et al. 2000 ; Fryer et al. 2001 ; Saurin et al. 2000 ) , but only some of these studies show these MAPKs contributing to IPC protection ( Strohm et al. 2000 ; Fryer et al. 2001 ) . In contrast, there are studies reporting no change in Erk1/2 activity in the setting of IPC ( Behrends et al. 2000 ; Takeishi et al. 2001 ) . A recent study suggests that diazoxide-induced mitochondrial ROS release may activate Erk1/2 ( Samavati et al. 2002 ) . This is interesting given the fi ndings of Baines et al. demonstrating that PKC-e modules can form complexes with Erk1/2- BAD, p38 and JNK at the mitochondria ( Baines et al. 2002) . In this scenario, PKC- e appeared to phosphorylate both Erk1/2 and p38 but downregulate JNK ( Baines et al. 2002 ) . Erk1/2 can mediate cellular protection by phosphorylating recruiting several anti-apoptotic mechanisms. There is more evidence that Erk1/2 contributes to IPC at time of myocardial reperfusion as part of the RISK pathway ( Hausenloy et al. 2005 ) . 4 Preconditioning in the Heart 57

The role of p38-MAPK in IPC-induced protection is controversial with studies demonstrating increased activation of p38-MAPK during the index ischaemia in preconditioned isolated rabbit and rat hearts ( Weinbrenner et al. 1997 ; Baines et al. 1999 ; Maulik et al. 1998 ; Iliodromitis et al. 2002 ) and rabbit cardiomyocytes ( Armstrong et al. 1999 ) , as well as activation of MAPKAPK2, a downstream substrate of p38-MAPK (Nakano et al. 2000b ) . In contrast, several studies have reported that the p38-MAPK activation that occurs during the index ischaemia is transient and does not correlate with the preconditioning effect ( Behrends et al. 2000 ; Talmor et al. 2000 ) . To complicate matters further, studies have also shown that p38-MAPK activity is attenuated in preconditioned rabbit ( Gysembergh et al. 2001 ) and rat hearts ( Nagarkatti and Sha’a fi 1998; Marais et al. 2001 ) , suggesting that p38-MAPK activation is pro-injurious, a fi nding which is supported by studies demonstrating that inhibiting p38-MAPK, using SB203580, protects the heart against ischaemia- reperfusion injury ( Mackay and Mochly-Rosen 1999 ; Ma et al. 1999 ; Barancik et al. 2000 ) . In contrast, other studies demonstrate that inhibiting p38-MAPK abrogated preconditioning-induced protection ( Maulik et al. 1998 ; Nakano et al. 2000a ) , but only if p38-MAPK was present during the index ischaemic episode ( Mocanu et al. 2000 ) . The discrepancy in results may be attributed to the different p38-MAPK isoforms that exist with p38 a contributing to myocyte death and p38 b responsible for cell survival ( Saurin et al. 2000 ; Wang et al. 1998) , with Marber’s group demonstrated that IPC decreased p38 a MAPK activity during the index ischaemia ( Saurin et al. 2000 ) , suggesting that this isoform is responsible for mediating injury during ischaemia-reperfusion injury. In further studies, Marber’s group have demonstrated that the nitric oxide donor S-nitro N-acetyl penicillamine (SNAP) could delay the ischaemia-induced activation of p38a in cardiomyocytes subjected to hypoxia- reoxygenation ( Rakhit et al. 2001 ) . The JNK MAPK family comprises two isoforms, the 46-kDa JNK1 and the 54-kDa JNK2, both of which are present in the heart ( Clerk et al. 1998 ) . Clerk et al. ( 1998; Bogoyevitch et al. 1996) have demonstrated that both JNK1 and JNK2 are activated upon at reperfusion but are not affected by ischaemia, whereas a recent report has suggested that ischaemia-reperfusion may also activate JNK ( Yue et al. 2000 ; Sugden and Clerk 1998 ) . In the context of IPC, Takeishi et al. (2001 ) found that IPC resulted in a more rapid activation of JNK during the index ischaemic period, although the signifi cance of this to IPC-induced protection is unclear. Iliodromitis et al. ( 2002 ) found that IPC induced an increase in both JNK1 and JNK2, confi rming the fi ndings of Ping et al. who demonstrated a PKC-dependent activation of both JNKs during IPC in conscious rabbits (Ping et al. 1999b ) . However, Downey’s group reported no change in JNK activation when isolated rabbit hearts were subjected to IPC (Nakano et al. 2000b ) . The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is a stress-responsive mechanism that transduces signals for the cell membrane to the nucleus, where gene expression is modulated (Bolli et al. 2003 ). The JAK-STAT pathway is activated in response to ischaemia-reperfusion and has been demonstrated to mediate apoptosis (STAT1) ( Stephanou et al. 2000) and myocardial dysfunction (STAT5A, STAT6) ( Mascareno et al. 2001 ) . 58 D.J. Hausenloy and D.M. Yellon

In contrast, STAT3 was demonstrated to contribute to IPC-induced protection in a study by Hattori et al. They found that IPC produced an early activation of JAK2 and STAT3 in isolated rat hearts ( Hattori et al. 2001 ) . IPC reduced infarct size and attenuated apoptotic cell death concurrent with upregulation of the anti-apoptotic protein BCl 2 and downregulation of the pro-apoptotic protein BAX. The presence of the JAK inhibitor, AG-490, abolished JAK2 and STAT3 phosphorylation and abrogated the IPC-mediated reduction in infarct size and apoptosis, suggesting an important role for STAT 3 ( Hattori et al. 2001 ) .

4.3.1.3 Effectors of IPC

A number of different effectors have been proposed over the years, but most evidence has implicated mitochondria as the end-effectors of IPC.

4.3.2 Mitochondria and IPC

The initial experimental studies to implicate mitochondria in the signalling pathway of IPC were designed to investigate the mechanism for the preserved myocardial ATP content in preconditioned hearts as originally proposed by Murry et al. ( 1986 ) Over the years, the role of mitochondria in IPC has expanded to them being potential triggers and effectors of cardioprotection. It appears that any stimulus which mildly stresses mitochondrial function is capable of initiating IPC. This mitochondrial stress then sets into place, through the activation of various pro-survival signalling pathways, a mitochondrial phenotype which is protected against a sustained lethal insult of IRI.

4.3.3 IPC and Myocardial Energy Production

In the initial IPC, studies undertaken by Murry et al. in 1990 ( Murry et al. 1986, 1990 ) ﬁ rst proposed that IPC may protect the heart by reducing myocardial energy demand during ischaemia, thereby preventing cell death by preserving myocardial ATP content and/or reducing catabolite accumulation. This proposal has been supported by a number of experimental studies reporting preserved mitochondrial function and maintained levels of high-energy phosphates ( Kobara et al. 1996 ) .

The opening of the MitoKATP channel has been demonstrated to increase ATP synthesis ( Fryer et al. 2000 ) , preserve mitochondrial energy production ( Iwai et al. 2000 ) , decrease ATP hydrolysis ( Dos et al. 2002 ) and improve energy transfer at reperfusion ( Dos et al. 2002 ) . 4 Preconditioning in the Heart 59

Fig. 4.2 Signalling pathways linking IPC and IPost to the mPTP. This scheme provides a simpli ﬁ ed overview of two of the main signalling pathways implicated in both IPC and IPost which link the cell surface receptor to the mPTP. Of course, many other signalling pathways have been implicated in both IPC and IPost, but for the purposes of clarity, these have not been included in this scheme. IPC and IPost are both seen to activate cell surface receptor which then recruit a number of signal transduction pathways including the PI3K-Akt and MEK1/2-Erk1/2 pathways which terminate on the mitochondria with the activation of the MitoKATP channel and the inhibition of the mPTP (Adapted from ﬁ gure in reference Hausenloy et al. 2009 )

4.3.4 IPC and pH

During a sustained lethal episode of myocardial ischaemia, the reduction in oxygen favours anaerobic glycolysis leading to ATP hydrolysis and the accumulation of lactic acid and a fall in the intracellular pH to about 6.0. The increase in intracellular H+ activates the Na+ -H + exchanger which extrudes H+ ions in exchange for Na+ ions resulting in intracellular Na+ which in turn causes the Na+ -Ca 2+ exchanger to work in reverse mode extruding Na + ions in exchange for Ca 2+ ions, the end result of which is intracellular Ca 2+ overload. On reperfusing the ischaemic myocardium, the washout of lactate and the reactivation of ion pumps result in the rapid restoration of intracellular pH to pre-ischaemic physiological levels of pH 7.4 (see Fig. 4.2 for summary of biochemical events which occur during myocardial ischaemia and reperfusion). In 1990, Murry et al. ( 1990 ) fi rst proposed that IPC may protect the heart by increasing glycogen depletion and reducing the rate of anaerobic glycolysis during myocardial ischaemia. Initial experimental studies did fi nd glycogen depletion, decreased myocardial lactate and less intracellular acidosis during myocardial ischaemia in IPC-treated hearts ( Murry et al. 1990 ; Kida et al. 1991 ; Asimakis et al. 1992 ) , 60 D.J. Hausenloy and D.M. Yellon the expected result of which would be reduced activation of the Na+ -H + and Na+ -Ca 2+ exchangers and less intracellular Ca2+ accumulation ( Steenbergen et al. 1993 ) . However, subsequent experimental studies have dissociated glycogen depletion, decreased lactate accumulation and less intracellular acidosis from IPC cardioprotection ( Vander Heide et al. 1996 ; Cave and Garlick 1997 ) . Interestingly, the rapid restoration of intracellular physiological pH at the onset of myocardial reperfusion did not appear to be affected by IPC ( Headrick 1996 ) , a fi nding which differs from IPost, which has been shown to delay the restoration of intracellular physiological pH within the heart, as a cardioprotective mechanism linked to inhibition of mPTP opening ( Fujita et al. 2007 ; Cohen et al. 2007, 2008 ) .

4.3.5 IPC and Calcium

Intracellular and mitochondrial calcium overload during myocardial ischaemia and reperfusion can cause cardiomyocyte death by a number of different mechanisms including mPTP opening and cardiomyocyte hypercontracture (Shen and Jennings 1972a, b ) . IPC has been reported to protect the heart by reducing intracellular calcium and mitochondrial calcium accumulation. The underlying mechanism for this beneﬁ cial effect of IPC on calcium handling is unclear and has been attributed to the opening of the sarcolemmal K ATP channel (with action potential duration shortening) ( Auchampach and Gross 1993 ) , opening of the MitoK ATP channel (with partial mitochondrial membrane depolarization and less mitochondrial calcium accumulation) ( Holmuhamedov et al. 1998, 1999; Wang et al. 2001 ; Murata et al. 2001 ; Ishida et al. 2001 ; Riess et al. 2002 ; Crestanello et al. 2000, 2002a, b ) , and reduced intracellular acidosis during myocardial ischaemia (see previous section) ( Steenbergen et al. 1993 ) .

4.3.6 IPC and ROS

Reactive oxygen species (ROS) appear to play a dual role in the setting of IPC. On the one hand, the production of mitochondrial ROS (such as superoxide anion, hydrogen peroxide and hydroxyl radical) from the re-energization of the mitochondrial electron transport chain in the ﬁ rst few minutes of myocardial reperfusion mediates cardiomyocyte death by inducing mitochondrial permeability transition pore (mPTP) opening and causing cell membrane damage by lipid peroxidation. On the other hand, the generation of small amounts of signalling ROS prior to the index episode of myocardial ischaemia in response to a standard IPC stimulus is also required to mediate cardioprotection through the activation of pro-survival protein kinases such as PKC ( Baines et al. 1997 ) , Erk1/2 ( Samavati et al. 2002 ) and p38 MAPK ( Yue et al. 2001 ) . 4 Preconditioning in the Heart 61

4.3.6.1 IPC Attenuates Mitochondrial Production of ROS at Reperfusion

In 1994, Tosaki et al. ( Yao and Gross 1993 ) fi rst reported that IPC attenuated the production of ROS (detected indirectly by levels of malondialdehyde) at the onset of myocardial reperfusion in isolated perfused rat hearts. Using lucigenin-enhanced chemiluminescence to directly measure levels of ROS in the isolated perfused rat heart, Crestanello et al. ( Tosaki et al. 1994; Crestanello et al. 1996 ) found that IPC generated a burst of ROS immediately following the IPC stimulus and decreased the production of ROS in the fi rst 4 min of myocardial reperfusion when compared to control hearts. These fi ndings have been confi rmed in a number of subsequent experimental studies ( Vanden Hoek et al. 2000 ; Narayan et al. 2001 ; Ozcan et al. 2002; Raphael et al. 2005 ; Clarke et al. 2008) . However, the actual mechanism through which IPC attenuates mitochondrial production of ROS at the onset of myocardial reperfusion remains unclear. It has been suggested that IPC may prevent mitochondrial respiratory impairment during myocardial ischaemia, thereby resulting in less mitochondrial ROS production at time of myocardial reperfusion. Another hypothesis is that IPC somehow prevents the following process: during myocardial ischaemia, outer mitochondrial membrane permeabilization allows the release of mitochondrial cytochrome C, the effect of which is increased mitochondrial ROS production ( Pasdois et al. 2011 ) . Whether IPC can affect ROS production during myocardial ischaemia is unclear. Experimental studies have demonstrated that minimal amounts of ROS were produced during myocardial ischaemia and IPC had no effect on this ( Crestanello et al. 1996; Vanden Hoek et al. 2000) , whereas one experimental study has reported that IPC reduced the production of ROS in the last 10 min of myocardial ischaemia in the isolated guinea pig heart ( Kevin et al. 2003) . In the latter study, it was suggested that the ROS generated in the terminal phase of ischaemia may somehow prime the heart for injury during myocardial reperfusion ( Kevin et al. 2003 ) .

4.3.6.2 IPC Generates Signalling ROS Prior to Myocardial Ischaemia

When present in low concentrations, reactive oxygen species (ROS) can modify cellular activities and participate in intracellular signalling ( Droge 2002 ) . In 1988, Murry et al. ( 1988) fi rst demonstrated that antioxidants could abolish the protective effect of IPC, implicating for the fi rst time a potential role for signalling ROS as a mediator of IPC, a fi nding which was later con fi rmed by several subsequent experimental studies ( Baines et al. 1997 ; Tanaka et al. 1994 ; Chen et al. 1995 ) . In 1996, Crestanello et al. (1996 ) demonstrated that IPC generated a burst of ROS, a fi nding which has been confi rmed in several studies ( Kevin et al. 2003; Vanden Hoek et al. 1998) . In 1997, Tritto et al. ( 1997) were the fi rst to demonstrate directly that a low dose of ROS could mimic IPC-induced protection. A year later, Vanden Hoek et al. ( 1998) demonstrated using chick neonatal cardiomyocytes that the source of the preconditioning signalling ROS (mainly H2 O2 ) was complex III of the mitochondrial electron transport chain. The mechanism through which IPC generates a burst of 62 D.J. Hausenloy and D.M. Yellon mitochondrial signalling ROS has been attributed to the opening of the mitochondrial

ATP-sensitive potassium (MitoKATP ) channel (see the next section).

4.3.7 IPC and the Mitochondrial KATP Channel

The role of the KATP channel in IPC has had an interesting and often controversial history. In 1983, Noma ( 1983 ) fi rst identi fi ed a sarcolemmal ATP-sensitive potassium (KATP ) channel in isolated guinea pig cardiomyocytes, the opening of which in response to hypoxia or ischaemia could protect the heart against IRI by shortening the action potential duration, thereby reducing intracellular Ca2+ loading. In 1992, Gross et al. ( Gross and Auchampach 1992; Auchampach et al. 1992 ) were the fi rst to implicate the sarcolemmal KATP channel as a mediator of IPC, by demonstrating that IPC cardioprotection could be abolished by the sarcolemmal KATP channel blockers glibenclamide or 5-hydroxydecanoic acid (5-HD). Mice lacking the Kir6.2 component of the sarcolemmal KATP channel blockers have been reported to be resistant to IPC ( Suzuki et al. 2003 ) .

Following the discovery in 1991 of a KATP channel in the inner mitochondrial membrane ( Inoue et al. 1991 ) , Garlid et al. in ( 1997 ) and Liu et al. in ( 1998 ) demonstrated a role for the MitoKATP channel as a trigger of IPC. The current paradigm suggests that the opening of the MitoKATP channel in response to the IPC stimulus is required to generate the mitochondrial signalling ROS required for the activation of downstream mediators of IPC cardioprotection such as PKC ( Pain et al. 2000 ; Wang et al. 2001a; Forbes et al. 2001; Krenz et al. 2002 ; Oldenburg et al. 2003 ) . The mechanism through which MitoK ATP channel activation generates ROS is unclear, but the current proposition is that the opening of the mitochondrial KATP channel causes K+ in fl ux into the mitochondrial matrix coupled with H + ef fl ux out of the mitochondria. The increase in matrix pH is accompanied by the infl ux of anions such as P i, but because of the relatively low cytosolic concentration of P i, the net effect of K+ infl ux is matrix alkanization, which in turn increases mitochondrial ROS production from complex I ( Jaburek et al. 1998, 2006; Andrukhiv et al. 2006 ) .

However, the role of the MitoKATP channel as a trigger of IPC cardioprotection has been surrounded by controversy due to two main factors: (1) much of the evidence supporting the role of this channel in IPC is based upon the use of pharmacological activator and inhibitors of the MitoKATP channel such as diazoxide and 5-HD, respectively, agents which have been reported to have non-speciﬁ c effects on mitochondrial function ( Hanley et al. 2002 ) ; and (2) although the molecular structure of the sarcolemmal KATP channel is known to consist of an octomeric complex containing four Kir6.2 (an inwardly rectifying K+ channel) subunits and four SUR2 (sul- phonylurea receptor) subunits ( Inagaki et al. 1995, 1996 ) , the molecular composition of the MitoK ATP channel remains unknown. The situation has been further complicated with the discovery of a Ca 2+ -activated K+ channel in the inner mitochondrial membrane that can mediate protection against IRI ( Xu et al. 2002 ) . 4 Preconditioning in the Heart 63

4.3.8 IPC and Nitric Oxide

Whether the signalling molecule nitric oxide (NO) is a trigger for classical IPC was initially controversial (Ping et al. 1999a ; Rakhit et al. 2000 ; Lebuffe et al. 2003 ; Nakano et al. 2000c ) . However, in the current paradigm for IPC signalling, NO is a critical mediator in the IPC signalling pathways. The evidence for NO and iNOS in the setting of delayed IPC or SWOP is more persuasive ( Jones et al. 1999; Guo et al. 2005 ) .

4.3.9 IPC and the mPTP

The mPTP is a non-selective channel of the inner mitochondrial membrane which forms and opens in the fi rst few minutes of myocardial reperfusion in response to calcium, oxidative stress, phosphate and ADP ( Crompton and Costi 1988 ; Hausenloy and Yellon 2003 ) . Its opening collapses the mitochondrial membrane potential, halting mitochondrial oxidative phosphorylation, resulting in ATP depletion and cell death. Furthermore, its opening allows water and solutes to equilibrate with the mitochondrial matrix leading to mitochondrial swelling and rupture of the outer mitochondrial membrane and the translocation of pro-apoptotic factors such as cytochrome C into the cytosol inducing apoptosis. Preventing its opening at the time of myocardial reperfusion is therefore an obvious therapeutic strategy for cardioprotection. In this regard, Crompton’s research laboratory ( Crompton et al. 1988 ) made the crucial discovery that the opening of the mPTP induced by calcium, phosphate and oxidative stress could be pharmacologically inhibited by the immunosuppressant drug cyclosporine-A (CsA) which targets cyclophilin-D, an important regulatory component of the mPTP ( Baines et al. 2005 ; Nakagawa et al. 2005 ) . Since then, we and others have demonstrated that pharmacologically inhibiting mPTP opening at the onset of reperfusion can reduce MI size ( Grif fi ths and Halestrap 1993; Hausenloy et al. 2002 , 2003) , a therapeutic strategy which has been applied in the clinical setting ( Piot et al. 2008 ) . Importantly, mice de fi cient in cyclophilin-D sustained smaller MI ( Baines et al. 2005; Nakagawa et al. 2005 ) and cerebral infarcts ( Schinzel et al. 2005 ) , underscoring the importance of the mPTP as a mediator of IRI in both the heart and brain. The fi rst study to suggest that the mPTP may be a potential target for preconditioning protection was by Ashraf’s group ( Xu et al. 2001) in 2001, although no direct experimental evidence was provided to support this proposition. A year later, we postulated and demonstrated for the fi rst time that IPC elicited its cardioprotective effect by targeting and inhibiting the opening of the mPTP ( Hausenloy et al. 2002) . We found that the preconditioning mimetic, diazoxide, was able to reduce calcium-induced mPTP opening in adult rat cardiac mitochondria ( Hausenloy et al.

2002 ) , ﬁ rst suggesting a link between the MitoK ATP channel and mPTP inhibition. Subsequent studies have conﬁ rmed mPTP inhibition in several different settings of preconditioning including the preconditioned perfused rat heart ( Javadov et al. 2003 ; 64 D.J. Hausenloy and D.M. Yellon

Argaud et al. 2004 ) , the preconditioned adult rat cardiomyocyte ( Hausenloy et al. 2004; Juhaszova et al. 2004 ) , the anaesthetic-preconditioned in vivo rabbit heart ( Piriou et al. 2004 ) and the rat heart protected by delayed preconditioning ( Rajesh et al. 2003 ; Hausenloy and Yellon 2004b ) . The mechanism through which IPC actually inhibits the opening of the mPTP at the time of myocardial reperfusion is currently unresolved, but several mechanisms have been proposed and are reviewed in a recent article ( Hausenloy et al. 2009) . The current paradigm as proposed by

Garlid’s laboratory ( Costa et al. 2006 ) suggests that the opening of the MitoKATP resulted in the inhibition of mPTP opening via ROS and PKC- ε.

4.3.10 Second Window of Protection

One or more brief non-lethal episodes of myocardial ischaemia and reperfusion can precondition the myocardium to withstand a sustained episode of lethal acute ischaemia-reperfusion injury (termed classical or early ischaemic preconditioning, IPC) ( Murry et al. 1986 ) . The preconditioned state manifests immediately following the IPC stimulus and lasts for 2–3 h after which it disappears ( Lawson and Downey 1993) . In 1993, two research laboratories working independently of each other made the intriguing discovery that the cardioprotective effect actually reappeared 24 h later (termed the second window of protection or delayed or late IPC). Yellon’s group ( Marber et al. 1993 ) observed infarct-size reduction using an in vivo rabbit model 24 h following a standard IPC stimulus and termed this the second window of protection (SWOP). Kuzuya and co-workers ( Kuzuya et al. 1993 ) demonstrated myocardial infarct size reduction using an in vivo canine model either immediately or 24 h following a standard IPC stimulus but failed to observe an infarct-limiting effect at 3 or 12 h following the IPC stimulus, confi rming the presence of a biphasic cardioprotective response to IPC. Delayed preconditioning can be elicited by both non-pharmacological stimuli (ischaemia, heat stress, pacing and exercise) and pharmacological stimuli and can protect against myocardial infarction, myocardial stunning, arrhythmias and endothelial dysfunction. The studies which originally described delayed IPC used four 5-min cycles of myocardial ischaemia and reperfusion to elicit delayed ischaemic preconditioning (delayed IPC) in the rabbit heart ( Marber et al. 1993 ) and canine heart ( Kuzuya et al. 1993 ) , probably because this was the standard protocol used for eliciting early IPC. However, it has been shown that a single 5-min cycle is also suf fi cient to elicit delayed IPC ( Baxter et al. 1997 ) . One of the fi rst studies describing the phenomenon of delayed preconditioning had demonstrated that heat stress could limit myocardial infarct size 24 h later and this benefi cial cardioprotective effect was associated with increased myocardial levels of heat shock protein 72 KD (HSP72) ( Marber et al. 1993 ) . However, the original experimental study to link heat stress with cardioprotection was published several years earlier by Currie and co-workers in 1988 ( Currie et al. 1988 ) , who fi rst demonstrated that subjecting a rat to whole body hyperthermia (15 min at 42°C) 4 Preconditioning in the Heart 65 improved post-ischaemic ventricular function, reduced cardiac enzyme release in excised perfused hearts, reduced ultrastructural damage to mitochondrial and decreased oxidative stress. Subsequent studies have linked heat stress-induced delayed preconditioning with the generation of myocardial heat shock proteins and many of the mediators associated with delayed IPC.

4.3.10.1 Underlying Mechanisms

Since the original description of SWOP or delayed IPC 18 years ago, the mechanisms underlying this endogenous cardioprotective phenomenon have been the subject of intense investigation (see Fig. 4.3 for overview). The signal transduction pathway underlying delayed IPC requires ‘triggers’ (substances generated during the IPC stimulus such as adenosine, bradykinin, opioids, cytokines, nitric oxide [NO] or reactive oxygen species [ROS]) which recruit ‘early mediators’ (such as PKC, tyrosine kinase, PI3K-Akt, MEK1/2-Erk1/2 and JAK), which in turn activate transcription factors (such as STAT1/3, NFk B, AP-1, Nrf2 and HIF-1 a), resulting in the synthesis 12–24 h later of ‘distal mediators’ (such as iNOS, HSP and COX-2) which protect the heart against infarction by acting on

‘end-effectors’ or ‘targets’ (such as the mPTP or the mKATP channel). The requirement for the transcription and synthesis of de novo protein cardioprotective mediators is one of the major factors distinguishing delayed from early IPC, and it may account for the observed absence of cardioprotection in the 3–12 h following the IPC stimulus. Clearly, this classi ﬁ cation is not rigidly adhered to with some triggers/mediators/effectors dif ﬁ cult to classify under one heading, as they may overlap one or more categories.

4.4 Remote Ischaemic Conditioning

The disadvantages of both IPC and IPost are that they require an intervention being applied directly to the heart. Therefore, the discovery that the heart can be conditioned by an organ or tissue away from the heart has facilitated the translation of RIC into the clinical setting. In 1993, Przyklenk et al. (1993 ) made the intriguing discovery that inducing brief episodes of ischaemia and reperfusion in the circum ﬂ ex coronary artery territory had the capacity to reduce MI size, arising from the occlusion of the LAD coronary artery. This form of intramyocardial protection was later extended to non-cardiac organs, with the report that MI size could actually be reduced in the animal heart by inducing brief ischaemia and reperfusion in either the kidney (McClanahan et al. 1993 ) or the small intestine, ( Gho et al. 1996a ) immediately prior to the sustained coronary artery occlusion (reviewed in Przyklenk et al. 2003; Bolte et al. 2007 ) . The concept of RIPC has now been extended to different organs and tissues such that it has emerged as a true therapeutic strategy of interorgan protection against the detrimental effects of acute IRI. 66 D.J. Hausenloy and D.M. Yellon

Fig. 4.3 Signalling pathways in delayed preconditioning or SWOP. Overview of major signal transduction pathways underlying delayed ischaemic preconditioning (IPC) or the second window of protection (SWOP0). (a ) On day 1, delayed IPC generates trigger factors such as adenosine 4 Preconditioning in the Heart 67

4.4.1 Potential Mechanisms Underlying RIC

The actual mechanism through which an episode of brief ischaemia and reperfusion in an organ or tissue exerts protection against a subsequent sustained insult of IRI in a remote organ or tissue is currently unclear. Studies suggest that some of the underlying mechanistic pathways and signal transduction cascades activated within the protected organ may be similar to those recruited in the setting of IPC and IPost ( Hausenloy and Yellon 2007b ) . However, the mechanistic pathway linking the remote organ or tissue to the heart is currently unclear, although several mechanisms have been proposed including a neural and hormonal pathway. However, it is important to appreciate that these mechanistic pathways may interact with each other and may therefore not be mutually exclusive.

4.4.2 Evidence for a Potential Humoral Factor in RIC

The ﬁ nding that a period of reperfusion of the remote preconditioning organ was required in addition to the brief ischaemia suggested that the reperfusion period may be needed to ‘washout’ a substance or humoral factor generated by the preconditioning

Fig. 4.3 (continued) (acting via adenosine A1 and A3 receptors), opioids (acting via δ and μ opioid receptors) and bradykinin (acting via the bradykinin B2 receptor) which activate intracellular signalling pathways PIK3-Akt, Raf-MEK1/2-Erk1/2 and protein tyrosine kinase (PTK) which convey the cardioprotective signal to downstream pathways such as p70S6K, PKC- ε -STAT-3, eNOS-NO- ε PKG-PKC -MitoK ATP . This results at the nucleus in the activation of transcription factors such as NFκ B, STAT1/3, HIF-1α , Nrf2, which transcribe de novo proteins (distal mediators) over the next 12–24 h such as MnSOD, iNOS, COX-2, heat shock proteins (HSP), heme-oxygenase-1 (HO-1), aldose reductase (AR), which then recruit cardioprotective pathways 24–48 h later on the day of lethal insult of ischaemia-reperfusion injury (IRI). In response to the delayed IPC stimulus, the generation of cytokines such as TNF-α (acting via the TNF- α receptors I and II), IL-β and IL-6, which activate the JAK-STAT pathway, also results in the transcription of distal mediators. At the level of the mitochondria, the opening of the mitoKATP channel results in the release of signalling reactive oxygen species, which further activate protein kinases such as Erk1/2 and Akt. The opening sensitivity of the mitochondrial permeability transition pore (mPTP) is decreased by the IPC stimulus, which confers cardioprotection 24–48 h later in response to the IRI. ( b ) On day 2–3, the activated distal mediators confer cardioprotection at the time of myocardial infarction or stunning through a variety of pathways. The nitric oxide generated by iNOS mediates cardioprotection by inhibiting mPTP α opening. The activation of COX-2 generates prostaglandins PGE2 and PGF 1 which then mediate cardioprotection through an unclear mechanism. MnSOD and HO-1 exert an antioxidant effect, reducing detrimental ROS generated during IRI which may mediate cardioprotection by inhibiting mPTP opening at the time of myocardial reperfusion. The activation of AR mediates cardioprotection by decreasing cytotoxic lipid-derived aldehydes. The pro-survival kinases such as Erκ1/2 and Akt are activated at the onset of myocardial reperfusion and confer cardioprotection through mPTP inhibition β via GSK3 and the mK ATP channel. Whether autacoids such as adenosine, bradykinin or opioids activate these kinases at the time of myocardial reperfusion is unknown. The activation of HSP27 and HSP70 mediates cardioprotection by bene fi cial effects on stabilization of the actin cytoskeleton and calcium regulation, respectively (Reproduced from Hausenloy and Yellon 2010 ) 68 D.J. Hausenloy and D.M. Yellon ischaemia, which was then transported to the heart (McClanahan et al. 1993 ; Gho et al. 1996b ) . This hypothesis was further supported by a study reporting that blood taken from a rabbit which had been subjected to simultaneous ischaemic preconditioning of both the heart and kidney could reduce a subsequent myocardial infarct size by 77% when transfused to an untreated rabbit, ( Dickson et al. 1999a ) suggesting the transfer of one or more humoral cardioprotective factors. The same authors went on to demonstrate that coronary effl uent from an isolated rabbit heart treated with a standard ischaemic preconditioning protocol could reduce myocardial infarct size by 69% ( Dickson et al. 1999b) and improve recovery of left ventricular function ( Dickson et al. 2000 ) when used to perfuse an untreated isolated rabbit heart. Convincing evidence in support of a humoral mechanism for RIPC was provided in an elegant experimental study by Konstantinov et al. (2005a ) Remote limb preconditioning of a pig that had received a donor heart was able to reduce myocardial infarct size in the denervated donor heart, providing strong evidence that a humoral mediator was responsible for RIPC protection, although an afferent sensory nerve pathway from the limb could be excluded ( Konstantinov et al. 2005a ) . A similar study was conducted by Kristiansen et al. ( 2005 ) who demonstrated that hearts excised from a remote limb preconditioned rat experienced a smaller infarct size, when subjected to IRI. Other studies have investigated whether endogenous substances such as adenosine ( Pell et al. 1998 ) , bradykinin ( Schoemaker and van Heijningen 2000 ) , opioids ( Patel et al. 2002 ) , CGRP ( Tang et al. 1999 ) , and endocannabinoids ( Hajrasouliha et al. 2007 ) , are released from the remote organ during the preconditioning ischaemia and are carried to the heart in the blood stream where they then activate intracellular pathways of cardioprotection. Alternatively, the endogenous mediator may activate afferent neural pathways within the remote preconditioned organ to confer cardioprotection, as is the case with adenosine, bradykinin and CGRP. In 1998, our laboratory was the fi rst to implicate adenosine as a potential media- tory factor underlying cardioprotection in the setting of RIPC, demonstrating that the administration of the non-specifi c adenosine receptor antagonist, 8-sulphophe- nyltheophylline (8-SPT), prior to the RIPC protocol could abolish the reduction in myocardial infarct size induced by a remote preconditioning stimulus in the rabbit kidney ( Pell et al. 1998 ) . In a subsequent study by Takao et al. ( 1999 ) , it was demonstrated that 8-SPT administered after the renal RIPC stimulus also had the ability to block cardioprotection, suggesting that myocardial adenosine receptor binding was required for cardioprotection. This was supported by their fi nding of elevated plasma levels of adenosine in blood sampled from the carotid artery of rabbits subjected to RIPC compared to those treated with IPC alone ( Takaoka et al. 1999 ) . The involvement of opioid signalling in RIPC was fi rst reported by Patel et al. in ( 2002) , who demonstrated that the non-specifi c opioid receptor antagonist naloxone was capable of abolishing the myocardial infarct-limiting effects conferred by remote intestinal preconditioning in the rat ( Patel et al. 2002 ) . It has been proposed that endogenous opioids generated by the preconditioning stimulus in the remote organ enter the blood stream where they act directly on the myocardium to confer 4 Preconditioning in the Heart 69 cardioprotection ( Patel et al. 2002 ) , although further studies are required to both investigate this proposal and delineate the individual contributions of the different receptor subtypes to RIPC. Previous studies have implicated binding at the CB2 endocannabinoid receptor of the endogenous cannabinoid system in protection from myocardial ischaemia- reperfusion injury ( Di Filippo et al. 2004 ) . A recent experimental study has implicated endogenous activation of the CB2 receptor in the myocardial infarct-limiting effects of remote intestinal preconditioning, using a pharmacological CB2 antagonist to abolish RIPC protection ( Hajrasouliha et al. 2007 ) . The authors proposed that endocannabinoids generated by the intestinal ischaemia may enter the blood stream and activate CB2 receptors on the myocardium, but of course further studies are required to test this hypothesis.

4.4.3 Evidence for a Potential Neural Pathway in RIC

One of the early studies of RIPC fi rst provided potential evidence that a neural pathway may underlie the cardioprotection elicited by remote preconditioning a noncardiac organ. Gho et al. (1996b ) demonstrated that the reduction in myocardial infarct size induced by brief ischaemia and reperfusion of the anterior mesenteric artery could be reversed in the presence of the ganglion blocker hexamethonium. The hypothesis for a neural pathway was further developed with the proposition that endogenous substances such as adenosine ( Pell et al. 1998 ) , bradykinin ( Schoemaker and van Heijningen 2000 ) and calcitonin gene-related peptide (CGRP) ( Tang et al. 1999) , released by the remote preconditioned organ, stimulated afferent nerve fi bres, which then relay to efferent nerve fi bres terminating on the myocardium to confer cardioprotection. Ding et al. (2001 ) demonstrated that renal nerve section abolished the cardioprotective effect induced by a preconditioning renal ischaemia stimulus, providing strong supportive evidence of a neural pathway. They then reported that during the renal preconditioning stimulus, renal afferent nerve discharge was increased and that this enhanced neural activity could be abrogated by 8-SPT ( Ding et al. 2001 ) . Further confi rmatory evidence implicating adenosine in a neural pathway of cardioprotection was provided by Liem et al. ( 2002 ) who, after confi rming that the prior administration of hexamethonium or 8-SPT abolished the myocardial infarct size reduction induced by brief mesenteric ischaemia and reperfusion, demonstrated that the local administration of adenosine into the mesenteric vascular bed also conferred cardioprotection in a manner which was sensitive to hexamethonium ( Liem et al. 2002 ) . These fi ndings suggested that brief episodes of ischaemia of the small intestine may generate adenosine which would then activate mesenteric afferent sensory nerves. However, the investigators went on to report that 8-SPT administered after the remote preconditioning stimulus was also able to inhibit cardioprotection, suggesting that adenosine receptor binding in the heart may also be required for protection ( Liem et al. 2002 ) . 70 D.J. Hausenloy and D.M. Yellon

Schoemaker and van Heijningen ( 2000 ) demonstrated that the reduction in myocardial infarct size elicited by brief mesenteric artery occlusion and reperfusion could be abolished by prior administration of HOE140, a speci fi c bradykinin B2 receptor antagonist. Interestingly, they went on to fi nd that intra-mesenteric arterial administration of bradykinin was also able to confer cardioprotection in a manner which was sensitive to ganglion blockade by hexamethonium ( Schoemaker and van Heijningen 2000) . The authors suggested that bradykinin generated during the remote preconditioning intestinal ischaemia may stimulate mesenteric afferent sensory nerves which then mediate the cardioprotective effect ( Schoemaker and van Heijningen 2000) . These fi ndings were confi rmed in a subsequent study by Wolfrum et al. ( 2002 ) who also observed that the activation of myocardial PKC-e by brief intestinal ischaemia was blocked by HOE-140 and hexamethonium, suggesting that PKC- e was positioned downstream of bradykinin and the neural pathway. Several experimental studies have implicated calcitonin gene-related peptide (CGRP), a neurotransmitter released from capsaicin-sensitive sensory nerves, as a potential mediator of both ischaemic preconditioning ( Li et al. 1996 ) and RIPC ( Tang et al. 1999 ; Xiao et al. 2001 ) . These can be summarized as follows: remote intestinal preconditioning generates nitric oxide which stimulates capsaicin-sensitive sensory nerves in the intestinal vasculature, releasing CGRP into the bloodstream (where levels have reported to be increased by RIPC), which is then carried to the heart where it activates myocardial PKC-e ( Li et al. 1996 ; Wolfrum et al. 2005 ) .

4.4.4 Evidence for a Systemic Response in RIC

Several experimental studies have examined the effect of remote preconditioning of an organ or tissue on the myocardial gene transcription pro fi le ( Konstantinov et al. 2004 ; Konstantinov et al. 2005b ) , and the infl ammatory response ( Peralta et al. 2001 ) , and have discovered that the in fl ammatory response is suppressed and a favourable profi le of gene transcription appears to be activated that is both anti-infl ammatory and anti-apoptotic. The relevance of such a response to the cardioprotective effect elicited by RIPC is currently unclear and requires further investigation.

4.4.5 Myocardial Mechanisms of Cardioprotection in RIC

Once the cardioprotective signal has been conveyed from the remote preconditioning organ to the heart, intracellular signal transduction mechanisms are recruited within cardiomyocytes which are similar to those that participate in ischaemic preconditioning ( Yellon and Downey 2003a ) and postconditioning ( Vinten-Johansen 2007) . These include the ligand binding to G-protein cell surface-coupled receptors such as adenosine ( Pell et al. 1998 ) , bradykinin ( Schoemaker and van Heijningen 2000 ) , opioids ( Patel et al. 2002 ) , angiotensin ( Singh and Chopra 2004 ) and 4 Preconditioning in the Heart 71 endocannabinoids. ( Hajrasouliha et al. 2007 ) The binding to these cell surface receptors appears to then activate intracellular kinases such as PKC-e ( Wolfrum et al. 2002 ) and other signalling components such as reactive oxygen species ( Weinbrenner et al. 2002) , nitric oxide and the mitochondrial K ATP channel (see Fig. 4.2 ) ( Pell et al. 1998 ) . Whether RIPC also activates pro-survival kinases of the reperfusion injury salvage kinase (RISK) pathway and results in the inhibition of the mitochondrial permeability transition pore (mPTP), as in IPC and IPost, remains to be determined ( Hausenloy and Yellon 2007b ) .

4.4.6 Novel Concepts in RIC

Recent studies suggest that the heart can be protected from acute IRI by simply inducing surgical trauma elsewhere—a phenomenon termed remote preconditioning of trauma (RPCT) ( Jones et al. 2009 ; Ren et al. 2004 ) . Jones et al. (2009 ) demonstrated that an abdominal surgical incision was enough to protect the murine heart from MI. The mechanistic pathway underlying this cardioprotective effect appeared to be mediated through a neural pathway involving sensory fi bres, spinal nerves and cardiac sympathetic nerves which via bradykinin, activate myocardial intracellular mediators of cardioprotection such as PKC and the MitoKATP channel. Somewhat remarkably, the cardioprotection could be recapitulated by simply applying capsaicin cream to stimulate the C sensory fi bres in the skin ( Jones et al. 2009 ) . A recent experimental study has suggested that the heart can be repeatedly remote ischaemic postconditioned (RIPost) post-MI to elicit bene fi cial effects on LV remodelling ( Wei et al. 2011 ) . Wei et al. ( 2011 ) found that repeating the RIPost stimulus either every day or every 3 days for 28 days reduced adverse LV remodelling post-MI and improved survival at 84 days post-MI. Whether repeated RIPost every 3 days, in post-MI patients, has bene fi cial effects on LV remodelling is an intriguing possibility.

4.5 Ischaemic Postconditioning

In 2003, Zhao et al. ( 2003 ) made the surprising observation that interrupting myocardial reperfusion with several short-lived episodes of myocardial ischaemia was cardioprotective. These authors found that by applying three 30-s cycles of alternating LAD reperfusion and LAD occlusion at the onset of myocardial reperfusion could reduce MI size by 44% in the canine heart ( Zhao et al. 2003 ) . Interestingly, the term ischaemic ‘postconditioning’ had fi rst been coined in an earlier experimental study by Na et al. in 1996 ( Na et al. 1996 ) in which it was demonstrated that intermittent reperfusion achieved by ventricular ectopic beats could reduce reperfusion arrhythmias in a feline model of IRI. In fact, the concept of intermittent or gradual reperfusion as a cardioprotective strategy was fi rst investigated in the 1980s, ( Okamoto et al. 1986 ; Sato et al. 1997 ) but it is the concept of IPost which has captured the imagination of the research fi eld of cardioprotection. 72 D.J. Hausenloy and D.M. Yellon

The discovery of IPost as therapeutic cardioprotective strategy which can be applied at the onset of myocardial reperfusion has revitalized interest in myocardial reperfusion injury as a target for cardioprotection and provided solid evidence for the existence of lethal myocardial reperfusion in both animal models and man. In addition to protecting the heart from the known proponents of lethal myocardial reperfusion injury such as oxidative stress, calcium overload, in ﬂ ammation and mPTP opening, IPost is known to recruit a number of signal transduction pathways, many of which are similar to those utilized by IPC.

4.5.1 Signalling Pathways Underlying IPost

The signal transduction pathways underlying IPost are both numerous and complex, and only an overview will be given here. For more detailed accounts, the reader is kindly directed to the following reviews ( Ovize et al. 2010 ; Hausenloy 2009 ) . In common with IPC, the current paradigm is that the signalling pathway is initiated at the cell membrane surface with the activation of a variety of G-protein-coupled receptors by a number of autacoids (such as adenosine, bradykinin, opioids), the activation of which recruits a wide variety of signal transduction pathways, many of which appear to converge on the mitochondria.

4.5.1.1 Cell Surface Receptor Activation

The fi rst G-protein-coupled receptor (GPCR) to be linked to IPost was the adenosine receptor. An experimental study by Downey’s research group ( Yang et al. 2005 ) w a s the fi rst to observe that the reduction in myocardial infarct size elicited by IPost could be abolished in the presence of 8-p -(sulphophenyl) theophylline, a non-specifi c adenosine receptor blocker. Interestingly, some 14 years earlier, the same research group had been responsible for fi rst implicating adenosine receptor activation with the phenomenon of IPC ( Liu et al. 1991 ) . The actual adenosine receptor subtype which is responsible for IPost is unclear with studies implicating the adenosine A1 ( Xi et al. 2008) , A2A ( Kin et al. 2005 ) and A2B ( Philipp et al. 2006 ) receptor subtypes. A subsequent study by Penna et al. (2007 ) has implicated the endogenous activation of the GPCR, bradykinin B2, in IPost protection. These authors fi rst demonstrated that two different pharmacological antagonists of the bradykinin B2 receptor abolished IPost protection in perfused rat hearts ( Penna et al. 2007 ) . Finally, the recent fi nding that mice lacking the bradykinin B2 receptor were resistant to IPost protection provides genetic evidence for the obligatory role of endogenous bradykinin B2 receptor activation in the setting of IPost ( Xi et al. 2008) . The role for the bradykinin B1 receptor was less clear, as the mice were partially protected by the IPost stimulus ( Xi et al. 2008 ) . Recently, Zatta et al. (2008 ) have linked IPost with the endogenous activation of the opioid GPCR. After demonstrating that the non-speci fi c opioid receptor antagonist 4 Preconditioning in the Heart 73 naloxone was capable of abolishing IPost protection in the intact rat heart, they investigated the effect of IPost in the presence of pharmacological antagonists of the d -, k - and m -opioid receptors ( Zatta et al. 2008 ) . The data implicated endogenous stimulation of the m - and possibly the d -opioid receptors in the setting of IPost ( Zatta et al. 2008 ) . In addition, hearts subjected to IPost accumulated higher levels of pro-encephalin, suggesting perhaps that IPost was capable of increasing endogenous opioid content in the reperfused myocardium ( Zatta et al. 2008 ) . Other receptors which have been implicated in IPost include the protease-activated receptor 2 (PAR2) (Jiang et al. 2007 ) and particulate guanylyl cyclase, the natriuretic peptide receptor ( Burley and Baxter 2007 ) .

4.5.1.2 Signal Transduction Pathways

A number of different signalling pathways have been investigated in the setting of IPost. The fi rst of these was the reperfusion injury salvage kinase (RISK) pathway ( Hausenloy and Yellon 2004a, 2007a ) . We and others have demonstrated that the pharmacological activation of pro-survival kinases such as Akt and Erk1/2 (the RISK pathway) at the immediate onset of myocardial reperfusion using a diverse variety of agents, which include growth factors, cytokines, GPCR agonists, natriuretic peptides, adipocytokines and ‘statins’, reduces myocardial infarct size in the region of 40–50% ( Hausenloy and Yellon 2004a, 2007a ) . Our laboratory and others have demonstrated that the cardioprotective bene fi ts of IPost are dependent on the activation of Akt and Erk1/2 at the immediate onset of myocardial reperfusion ( Tsang et al. 2004 ; Yang et al. 2004 ) . Subsequent studies have con fi rmed the role for Akt and Erk1/2 in the setting of IPost in both non-diseased animal hearts and diseased ones ( Zhu et al. 2006 ; Feng et al. 2006 ) as well as human atrial muscle ( Sivaraman et al. 2007 ) . Interestingly, obese mice have been reported to be resistant to IPost protection, and this fi nding was associated with insuf fi cient activation of the RISK pathway in the hearts harvested from obese animals compared to control ones ( Bouhidel et al. 2008 ) . This fi nding underscores the importance of using relevant experimental animal models capable of simulating disease pathologies present in patients with coronary heart disease. The survival activator factor enhancement (SAFE) pathway, which comprises the TNF-a receptor and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, has also been linked to cardioprotection elicited by IPost and IPost ( Lacerda et al. 2009; Hausenloy et al. 2010a ) . Experimental studies have reported that pharmacologically inhibiting the JAK-STAT pathway at the onset of myocardial reperfusion abrogates the infarct-limiting effects of IPost (Boengler et al. 2008a; Goodman et al. 2008) . However, mice with a cardiac-specifi c STAT3 deletion were found to still be amenable to the infarct-limiting effects of IPost, providing a suitable IPost protocol was used, that is, IPost using 5 × 5-s cycles of ischaemia-reperfusion reduced myocardial infarct size but three 10-s cycles did not (Boengler et al. 2008a ) . Using mice with the same cardiac-restricted STAT-3 deletion, Goodman et al. (2008 ) were able to demonstrate improved LV function using the IPost protocol of three 10-s cycles of ischaemia-reperfusion, suggesting that 74 D.J. Hausenloy and D.M. Yellon

STAT-3 may not be an obligatory mediator of IPost. The mechanism underlying the acute form of myocardial protection mediated by the JAK-STAT pathway is unclear but may relate to as yet unidenti fi ed mitochondrial effects (Boengler et al. 2008b ) . Sphingosine kinase (SPK) is a lipid kinase which generates sphingosine 1 phosphate (S1P), which in turn regulates cell mitosis, apoptosis, cytoskeletal rearrangement and survival ( Vessey et al. 2006 ) . Jin et al. (2008 ) have recently demonstrated an obligatory role for SPhK1 as a mediator of IPost, which is potentially upstream of the RISK pathway. The authors reported that hearts isolated from mice lacking SPhK1 sustained larger myocardial infarcts, were resistant to IPost and did not demonstrate activation of the Akt and Erk1/2 components of the RISK pathway in response to IPost ( Jin et al. 2008 ) . It is well established that protein kinase C acts as a critical mediator of protection in the setting of IPC, providing for the ‘memory’ elicited by an IPC stimulus (reviewed in Yellon and Downey 2003b ) . IPost has been reported to also be dependent on PKC activation. Penna et al. (2006a ) were the fi rst to demonstrate that the non-specifi c PKC inhibitor could abolish the infarct-limiting effects of IPost in perfused rat hearts, suggesting that IPost required the activation of PKC to confer cardioprotection. A subsequent study by Zatta et al. ( 2006) has found that IPost protection could be abolished by pharmacological inhibition of the PKC-e isoform in early reperfusion. The translocation to the mitochondria of the detrimental PKC-d was reduced in postconditioned hearts. The mechanism through which IPost activates PKC is unclear. With respect to the cardioprotective effects of PKC, it has been postulated that PKC may sensitize the adenosine A2B receptor on the cell surface ( Philipp et al. 2006 ; Kuno et al. 2008 ) and that a special mitochondrial pool of PKC-e confers inhibition of mitochondrial permeability transition pore (mPTP) opening ( Jaburek et al. 2006 ) . Protein kinase G (PKG) has emerged as a critical mediator of cardioprotection in both IPC and IPost (reviewed in Burley et al. 2007 ) . Much of the experimental data suggests that in the setting of IPC, it forms the fi nal link in the signalling pathway which begins at the plasma membrane and terminates at the mitochondria ( Costa et al. 2005 ) In the context of IPost, its myocardial infarct-limiting effects have been demonstrated to be sensitive to pharmacological inhibition of the NO-sGC-cGMP-PKG pathway ( Yang et al. 2005 ; Penna et al. 2006b ) . Activated PKG at the level of the mitochondria is then believed to open the ATP-sensitive mitochondrial potassium channel through protein kinase C- e ( Costa et al. 2005) . This pathway is presumed to be activated through the Akt component of the RISK pathway via eNOS in response to IPost ( Tsang et al. 2004) . The downstream target of this pathway is believed to be PKC, resulting in sensitization of the adenosine A2B receptor ( Kuno et al. 2008 ) or the inhibition of mPTP opening ( Costa et al. 2005a, 2006 ) .

4.5.1.3 Mitochondria and IPost

Many of the signalling pathways underlying IPost appear to converge on the mitochondria, which may come as no great surprise, given the critical role this organelle plays in terms of survival and death of the cardiomyocyte. In this regard, the mitochondrial permeability transition pore (mPTP) has emerged as a critical target 4 Preconditioning in the Heart 75 for cardioprotection (reviewed in ( Hausenloy and Yellon 2003 ; Leung and Halestrap 2008) ). Preventing its opening at the onset of myocardial reperfusion using pharmacological mPTP inhibitors ( Hausenloy and Yellon 2003 ; Hausenloy et al. 2002, 2003) or genetically ablating one of its critical components ( Baines et al. 2005 ; Nakagawa et al. 2005 ; Lim et al. 2007 ) reduces myocardial infarct size by 40–50%. IPost ( Argaud et al. 2005 ) has been reported to prevent mPTP opening at the onset of myocardial reperfusion, although the mechanism underlying this effect is currently unclear. It may involve components of the RISK pathway such as Akt, Erk1/2 or GSK-3b ( Bopassa et al. 2006 ; Davidson et al. 2006 ) and/or changes in intracellular pH in the fi rst few moments of myocardial reperfusion ( Fujita et al. 2007 ; Cohen et al. 2007 ) . Argaud et al. (2005 ) found that mitochondria isolated from a perfused rabbit heart, which had been subjected to a standard IPost protocol, were more resistant to calcium-induced opening of the mPTP, suggesting that IPost was capable of inhibiting mPTP opening. A subsequent study by the same group reported that this inhibitory effect on mPTP opening was sensitive to pharmacological PI3K inhibition, suggesting that IPost mediated mPTP inhibition through the activation of the PI3K-Akt pathway ( Bopassa et al. 2006 ) . We have demonstrated that mice lacking cyclophilin-D are resistant to IPost, providing con fi rmatory evidence supporting the role of the mPTP in IPost ( Lim et al. 2007 ) . Further studies suggest that the changes in cellular pH in early reperfusion of postconditioned hearts may also contribute to the inhibition of mPTP opening ( Cohen et al. 2007 ) . In the fi rst few minutes of myocardial reperfusion, there is a rapid restoration of physiological pH within the cardiomyocyte, mediated by the washout of lactic acid and the actions of the Na + -H + exchanger (NHE) and the Na + -HCO − co-transporter. Interestingly, IPost ( Fujita et al. 2007 ; Cohen et al. 2007 ; Inserte et al. 2008 ) has been reported to delay the restoration of physiological pH in the early moments of myocardial reperfusion. This transient acidosis in the fi rst couple of minutes of reperfusion may be suf fi cient to permit the activation of the RISK pathway ( Fujita et al. 2007 ) , suppress mPTP opening ( Cohen et al. 2007 ) , inhibit cardiomyocyte hypercontracture and prevent detrimental calpain activation ( Inserte et al. 2008 ) , over this critical period of time. The mechanism through which IPost modifi es cellular pH in early reperfusion is currently unclear, but it has been attributed to delayed washout of lactate, but another potential explanation could be the inhibition of the NHE by the RISK pathway, which is activated in IPost-treated hearts. A recent study by Avkiran’s laboratory ( Snabaitis et al. 2008 ) has demonstrated that Akt is able to phosphorylate and inhibit the actions of NHE in cardiomyocytes, and so whether this process occurs in postconditioned hearts is an interesting possibility. Previous experimental studies have suggested that the ATP-sensitive mitochondrial potassium (MitoK ATP ) channel plays a pivotal role in IPost, although much of the evidence is circumstantial and based on the use of pharmacological activators and inhibitors of the channel. Studies have demonstrated that the pharmacological inhibition of the MitoKATP channel in early reperfusion abolished the infarct-limiting effects of IPost ( Yang et al. 2004 ; Donato et al. 2007 ; Mykytenko et al. 2008 ) . IPost ( Zhao et al. 2003) has been reported to reduce the amount of detrimental ROS generated at the onset of myocardial reperfusion. In contrast, ROS may also participate as critical signalling mediators in the signal transduction pathway underlying 76 D.J. Hausenloy and D.M. Yellon

IPost ( Penna et al. 2006a ) . Penna et al. (2006a ) were the fi rst to demonstrate that the administration of the non-speci fi c ROS scavenger, N-acetylcysteine (NAC), was able to abrogate the infarct-limiting effect of IPost in perfused rat hearts. Crucially, if NAC was administered after the fi rst 3 min of myocardial reperfusion had elapsed, no effect on IPost was observed, suggesting that ROS signalling during the fi rst couple of minutes of myocardial reperfusion was critical to IPost protection ( Penna et al. 2006a ) . Subsequent studies using intact rabbit ( Downey and Cohen 2006 ) and murine ( Tsutsumi et al. 2007 ) hearts have confi rmed the involvement of a signalling form of ROS in IPost protection.

4.6 Clinical Application of Ischaemic Conditioning

The translation of ischaemic conditioning as a therapeutic strategy for cardioprotection in the clinical arena has been a remarkably slow process. The major disadvantages of ischaemic preconditioning (IPC) as a cardioprotective strategy include the fact that as a therapeutic intervention, it needs to be implemented prior to the index myocardial ischaemic event, and that it requires an intervention applied directly to the heart. As a result, the application of IPC is restricted to clinical settings in which the timing of the index myocardial ischaemic episode can be anticipated and in settings in which the heart can be directly manipulated. In this regard, coronary artery bypass graft (CABG) surgery provides a controlled environment in which part of the perioperative myocardial injury is due to global IRI. Both the limiting factors to translation of ischaemic conditioning have been, in part, overcome with the discovery of ischaemic postconditioning (IPost) and remote ischaemic conditioning (RIC). IPost has allowed the therapeutic strategy to be applied at the onset of myocardial reperfusion such as in patients presenting with an acute myocardial infarction who are undergoing primary percutaneous coronary intervention (PCI). RIC has permitted the ischaemic conditioning intervention to be applied to the upper or lower limb, thereby, and allowed the intervention be applied either prior to or after the onset of myocardial ischaemia and even at the onset of myocardial reperfusion, lending itself to a number of different clinical settings. Two other major obstacles to translation which are not unique to the research ﬁ eld of cardioprotection have been the inappropriate animal models of IRI used to investigate novel cardioprotective strategies and the inadequate design of the clinical studies for testing them.

4.6.1 CABG Surgery as Setting for Cardioprotection

Perioperative myocardial injury during CABG surgery as measured by serum cardiac enzymes such as CK-MB ( Brener et al. 2002 ) , troponin-T ( Kathiresan et al. 2004) and troponin-I ( Croal et al. 2006) is linked to worse clinical outcomes. It is caused by a number of factors including coronary embolization, manual 4 Preconditioning in the Heart 77 handling of the heart, inﬂ ammation and global myocardial ischaemia-reperfusion injury (Venugopal et al. 2009a) . As such, the discovery of novel cardioprotective strategies for minimizing this form of myocardial injury during CABG surgery would be expected to preserve cardiac function and improve clinical outcomes in this clinical setting, particularly in those high patients who are most vulnerable to this form of myocardial injury.

4.6.1.1 IPC and CABG Surgery

In 1993, our research group ( Yellon et al. 1993 ) were the fi rst to demonstrate that IPC could be applied as a potential therapeutic strategy for cardioprotection in the clinical setting. We found that intermittently clamping the aorta to induce short periods of global ischaemia and reperfusion prior to cross-clamp fi brillation could preserve myocardial ATP levels ( Yellon et al. 1993 ) and reduce the release of serum troponin-T ( Jenkins et al. 1997 ) in patients undergoing CABG surgery. The obvious limitations of this approach include the invasive nature of the therapeutic intervention and the risks of thromboembolism arising from clamping an atherosclerotic aorta. Despite these disadvantages, a number of clinical studies have investigated IPC in CABG surgery patients, the clinical outcomes of which 22 studies have been reviewed in a recent meta-analysis ( Walsh et al. 2008 ) . Overall, it was suggested that IPC resulted in signi fi cant reductions in ventricular arrhythmias, inotrope score and ITU stay ( Walsh et al. 2008 ) . Given the limitations of IPC in this setting, it is diffi cult to imagine that this approach to cardioprotection will be clinically applied to adult patients undergoing CABG surgery.

4.6.1.2 RIPC and CABG Surgery

The ability to apply the preconditioning stimulus to the upper or lower limb to protect the heart has greatly facilitated its translation into the clinical setting of CABG surgery, a setting in which the global IRI can be reliably anticipated. Cheung et al. in (2006 ) published the fi rst clinical study to demonstrate bene fi cial effects of RIPC in cardiac bypass surgery. Children undergoing corrective cardiac surgery for con- genital heart disease were randomized to receive either RIPC (four 5-min cycles of lower limb ischaemia and reperfusion) or control prior to surgery ( Cheung et al. 2006) . RIPC was benefi cial in terms of reduced troponin release, less inotrope use and lower airway pressures ( Cheung et al. 2006 ) . We have shown that RIPC (three 5-min cycles of upper limb ischaemia and reperfusion) can reduce perioperative myocardial injury in patients undergoing elective CABG surgery ( Hausenloy et al. 2007) . A number of clinical studies have confi rmed these benefi cial effects, although not all the studies have been positive (see Table 4.1 ). The explanation for these discrepant fi ndings is unknown, but it may be due to concomitant medication such as volatile anaesthetics and nitrates, patient selection and the RIPC stimulus. Large multicentre clinical trials (the ERICCA and RIPHeart trials) are now under way to confi rm that RIPC is cardioprotective and that it can improve clinical outcomes in patients undergoing CABG surgery. 78 D.J. Hausenloy and D.M. Yellon cre- remote CK RIC not reported, NR glyceryl trinitrate, en fl urane or urane or fl glyceryl trinitrate, en fl urane sevo fi brillation CABG Cardioplegia Cardioplegia of RIC in CABG First investigation No diabetic patients Cardioplegia intravenous Most patients received First clinical application of delayed RIC Cardioplegia Cardioplegia No diabetic patients NR NR Cardioplegia Cardioplegia First clinical application of RIC First clinical application of RIC in not applicable, coronary artery bypass graft surgery, coronary artery bypass graft surgery, NA CABG inotrope, ↓

airway pressures airway unclamping ↓ or LVEF troponin T signi ﬁ cant) troponin T LDH 5 min after aortic 24 h AUC troponin I, 24 h AUC troponin I (by 35%) 48 h AUC Cold crystalloid cardioplegia 8 h troponin I (by 27%) Cold crystalloid cardioplegia 2 h, 4 12 24 h CK-MB and 72 h AUC troponin T (by 42%) 72 h AUC Cardioplegia 72 h troponin T (by 26%, not 6 h, 24 48 h CK-MB and 72 h AUC troponin T (by 43%) 72 h AUC and cross-clamp Cardioplegia ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ in 48 h troponin T No difference aortic cross-clamp time, AXC left ventricular ejection fraction, left ventricular LVEF CABG ± valve CABG ± valve replacement CABG ± valve CABG ± valve replacement CABG ± valve CABG ± valve replacement pump CABG surgery CABG CABG ± valve CABG ± valve replacement CABG or urgent 8 adults undergoing 8 adults undergoing 101 adults undergoing 101 adults undergoing lactate dehydrogenase, LDH (leg) (leg) 18 h prior 3 × 5 min (arm) × 3 60 children (aged <7 years) RIC protocol (site) Patients Outcome Notes ) 3 × 5 min (arm) 45 adults undergoing ) 5 min (arm) × 3 CABG 53 adults undergoing ) 5 min (arm) × 3 57 adults undergoing ) 3 or 2 min × 3 ) area under the receiver operating characteristic curve, operating characteristic curve, area under the receiver ) 5 min (arm) × 3 elective 162 adults undergoing ) 5 min (leg) × 4 37 children (aged 1–2 years) ) 3 × 5 min (arm) cardiac bypass time, 2010 2007 ) 5 min (arm) × 4 off 130 patients undergoing 2009b 2000 2010 AUC 2010 ) 5 min (arm) × 3 elective 100 adults undergoing 2006 2010 CPB 2010 2010 Major clinical studies of remote ischaemic preconditioning in CABG surgery patients Major clinical studies of remote ischaemic preconditioning in CABG surgery Zhou et al. ( Cheung et al. ( Table Table 4.1 Study Gunaydin et al. ( Rahman et al. ( et al. ( Wagner Hong et al. ( Thielmann et al. ( Wenwu et al. (2010); Wenwu Ali et al. ( Abbreviations: Hausenloy et al. ( Hausenloy ( et al. Venugopal atine kinase, ischaemic conditioning 4 Preconditioning in the Heart 79

4.6.1.3 IPost and CABG Surgery

In the clinical setting, IPost has been mainly investigated in PPCI patients. However, in 2007, Luo et al. (2007 ) reported that IPost could also be applied at the time of unclamping the aorta, as at this time the heart is subjected to global myocardial reperfusion injury—30 s after unclamping the aorta, the clamp was reapplied for 30 s, a cycle which was repeated three times in total. The same authors have reported bene ﬁ cial effects on serum cardiac biomarkers, ventilation support, inotrope requirements and ITU stay in patients undergoing cardiac surgery for Tetralogy of Fallot and aortic valve replacement ( Luo et al. 2007, 2008; Li et al. 2009 ) . However, this cardioprotective strategy is subject to the same limitations as IPC, in that it is invasive and would be associated with a thromboembolic risk. For paediatric patients undergoing corrective cardiac surgery, it has promise.

4.6.2 PPCI Patients

For patients presenting with an ST-segment elevation myocardial infarction (STEMI), timely myocardial reperfusion using PPCI remains the most effective treatment strategy for limiting MI size, preserving cardiac function and improving clinical outcomes. Ongoing improvements in both antiplatelet and antithrombotic therapy to maintain the patency of the infarct-related coronary artery have further optimized the process of myocardial reperfusion. However, there currently exists no effective therapeutic intervention for protecting the heart from the lethal myocardial reperfusion injury which occurs at the time of PPCI. Novel therapeutic interventions which can be applied prior to PPCI are required to protect the heart from lethal myocardial reperfusion injury.

4.6.2.1 IPost in PPCI Patients

A major advance in the fi eld of cardioprotection and its translation into the clinical setting was made with the discovery of ischaemic postconditioning (IPost), an intervention which could be applied at the onset of myocardial reperfusion ( Zhao et al. 2003 ) , lending itself to the setting of PPCI in patients presenting with a STEMI where it was rapidly applied. In 2005, only 2 years following the initial discovery of IPost ( Zhao et al. 2003) , Ovize’s research group ( Zhao et al. 2003 ) had demonstrated in a small proof-of-concept clinical study comprising 30 PPCI patients that IPost could reduce myocardial infarction by 43% (as measured by 72 h area under curve total serum creatinine kinase). In this landmark clinical study, IPost was applied immediately following direct stenting of the infarct-related coronary artery by interrupting myocardial reperfusion with four 1-min cycles of low-pressure in fl ations and de fl ations of the coronary angioplasty ( Zhao et al. 2003 ) . A number of clinical studies have con fi rmed these initial fi ndings showing that IPost confers long-term cardioprotection in terms of MI size reduction and preserved LV ejection fraction ( Thibault et al. 2008 ) (these clinical trials are summarized in Table 4.2 ). 80 D.J. Hausenloy and D.M. Yellon MI ↓ seru1m ↓ MI size ↓ TNF levels TNF levels ↓ hsCRP value (by hsCRP value ↓↓ EF at 1 year (by 7%) CK-MB (by 32%); ↓ MI size at 3 months by ↑ CK-MB (by 41%); ↓ ↓ improved coronary fl ow ow fl coronary improved myocardial perfusion; ↑ ↑ MI size at 7 days by SPECT (both ↓ WMSI at 8 weeks WMSI at 8 weeks ↑ 72 h AUC for CK-MB (by 26%); 72 h AUC ↓ ST-segment resolution ST-segment ↑ EF after PPCI WMSI at 1 year (3 × 1 min > 3 × 0.5 min); ↑ ↑ endothelial function; levels of malondialdehyde; levels reserve; velocity at 6 months on SPECT (by 39%); size at 7 days on SPECT (by 46%); 10%); cardiac MRI (by 18%) ↑ protocol 27%) peak levels of total CK (by 28%); peak levels 72 h AUC for total CK (by 36%) 72 h AUC 72 h AUC for total CK and 72 h AUC resolution; ST-segment coronary blood fl ow and ST-segment resolution and ST-segment ow fl coronary blood (by 19%); peak total CK level 1 min × particularly with 3 soluble Fas, Plasma apoptotic marker EF and 72 h AUC for total CK (by 40%); 72 h AUC ↓ ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↑ 30 41 43 17 94 24 38 75 74 End point 118 in troponin T or EF; No difference n de fl ation and de fl ation de fl ation de fl ation and 4–5 min de fl ation fl and 4–5 min de and de fl ation ation fl and 4–5 min de de fl ation and de fl ation and de fl ation protocol Ischaemic postconditioning protocol Anterior STEMI only TIMI 0 fl ow No collaterals TIMI 0 fl ow All-comers TIMI 0 fl ow No collaterals TIMI 0 fl ow No collaterals TIMI 0 fl ow No collaterals All-comers TIMI 0 fl ow No collaterals ) Presented within 12 h ation and fl 0.5 min in × 4 ) Presented within 6 h ation and fl 1 min in × 4 ) Presented within 12 h ation fl of 1.5 min in 2 cycles ) Presented within 12 h ation fl of 0.5 min in 3 cycles ) Presented within 12 h ation fl 0.5 min or 1 in × 3 ) Presented within 6 h ation and fl 1 min in × 4 ) Presented within 12 h ation and fl 0.5 min in × 4 2010 2008 ) Presented within 12 h ation fl 0.5 min or 1 in × 3 ) Presented within 12 h ation fl of 0.5 min in 3 cycles 2008 ) Presented within 12 h ation fl of 1.5 min in 2 cycles 2007 2009 2005 2010 2010 2006 Major clinical studies of ischaemic postconditioning in patients with PPCI Major clinical studies of ischaemic postconditioning in patients with PPCI 2005 Staat et al. ( Table Table 4.2 Study Patient population Thibault et al. ( Laskey Laskey ( Ma et al. ( Zhao et al. ( Xue et al. ( Laskey et al. ( Laskey Lin et al. ( Yang et al. ( Yang et al. ( Lonborg 4 Preconditioning in the Heart 81

SPECT plasma nitrotyrosine; CK-MB (by 20%); ↓ ↓ high-sensitivity C-reactive protein, C-reactive high-sensitivity EF after PPCI (by 9%) ↑ hsCRP , primary percutaneous coronary intervention, , primary percutaneous coronary intervention, PPCI ejection fraction, cardiac function myocardial blush grade; EF ↑ ↑ difference in overall myocardial salvage by cardiac MRI at myocardial salvage in overall difference iNOS activity in white blood cells; iNOS activity peak levels of total CK (by 11%); peak levels ↓ ↓ not reported, 50 76 no for CK-MB and troponin T; in 48 h AUCs No difference 43 NR creatine kinase, CK wall motion score index motion score index wall WMSI number of patients, n de fl ation de fl ation de fl ation myocardial infarct, myocardial infarct, -segment elevation MI, elevation -segment MI ST , STEMI TIMI 0 fl ow No collaterals TIMI 0 fl ow No collaterals TIMI 0 fl ow ) Presented within 6 h ation and fl 0.5 min in × 4 area under the receiver operating characteristic curve, operating characteristic curve, area under the receiver ) Presented within 12 h ation and fl 0.5 min in × 4 2010 ) Presented within 12 h ation and fl 0.5 min in × 3 AUC 2011 2010 inducible nitric oxide synthase, iNOS single-photon-emission CT, Abbreviations: Abbreviations: Fan et al. ( Fan Sorensson et al. ( Garcia et al. ( 82 D.J. Hausenloy and D.M. Yellon

Whether IPost can improve clinical outcomes in PPCI patients is currently being investigated in a large multicentre randomized clinical trial in Denmark.

4.6.2.2 RIPerC in PPCI Patients

In 2010, Botker et al. ( 2010) were the fi rst to demonstrate that remote ischaemic preconditioning (RIPerC) was benefi cial in STEMI patients undergoing PPCI. In the ambulance on transit to the PPCI centre, paramedics applied either RIPerC (four 5-min infl ations/de fl ations of a blood pressure cuff placed on the upper arm) or control to STEMI patients. Those patients who received RIPerC have improved myocardial salvage at 1 week, as determined by myocardial nuclear scanning. In this study, all patients were eligible for recruitment—the benefi cial effects of RIPerC were more pronounced in those presenting with TIMI 0 fl ow and LAD infarcts. One other study has reported bene fi cial effects of RIPerC in conjunction with morphine in PPCI patients ( Rentoukas et al. 2010 ) . Whether RIPerC can improve clinical outcomes in PPCI patients needs to be investigated in a large multicentre randomized clinical trial.

4.6.3 Coronary Revascularization by PCI

Peri-procedural myocardial injury during PCI as measured by serum cardiac enzymes such as CK-MB, troponin-T and troponin-I or cardiac MRI ( Selvanayagam et al. 2005 ) is linked to worse clinical outcomes (reviewed in reference 257). It can be caused by a number of factors including myocardial ischaemia-reperfusion injury arising from the angioplasty balloon in ﬂ ations, coronary embolization and distal branch occlusions ( Babu et al. 2011 ) . As such, the discovery of novel cardioprotective strategies for minimizing this form of myocardial injury during PCI may improve clinical outcomes in this clinical setting, particularly in those high patients with complex disease who are most vulnerable to this form of myocardial injury.

4.6.3.1 RIPC in PCI Patients

In 2009, Hoole et al. (2009 ) were the fi rst to demonstrate that RIPC was bene fi cial in patients undergoing elective PCI for stable chronic coronary artery disease. They randomized 242 consecutive patients to receive either RIPC (three 5-min 4 Preconditioning in the Heart 83 cycles of infl ation/de fl ation of cuff placed on upper arm) or control prior to elective PCI. Those patients who received RIPC experienced less peri-procedural myocardial injury as evidenced by a negative troponin I in 42% of RIC-treated patients compared to 24% in control patients. In addition, the median level of troponin I was greater in the control group when compared to the RIC-treated group (16 ng/ ml compared to 0.06 ng/ml). However, an earlier small clinical study failed to demonstrate any cardioprotection with RIPC ( Iliodromitis et al. 2006 ) . But in that study, only 30 patients were randomized, and they used bilateral cuff infl ation. Again, whether RIPC impacts on clinical outcomes in this patient group remains to be determined.

4.6.4 Failure to Translate Cardioprotection for Patient Bene ﬁ t

The translation of the many novel cardioprotective strategies identiﬁ ed in the preclinical animal studies into the clinical setting for patient beneﬁ t has been a huge disappointment. This failure to translate cardioprotection has been discussed in several articles ( Bolli et al. 2004 ; Kloner and Rezkalla 2004 ; Ludman et al. 2010 ; Hausenloy et al. 2010b) and has been the subject of a recent NHLBI workshop. The main causative factors, which include the design of the animal studies of IRI and the design of the clinical studies, have been extensively discussed in the literature and are summarized in Tables 4.3 and 4.4 .

4.7 Conclusions

The phenomenon of ischaemic conditioning has evolved into a number of different forms, a process which has facilitated its translation into the clinical setting of cardioprotection. Ongoing experimental studies continue to elucidate the mechanisms underlying ischaemic conditioning and over the years have made huge progress toward the understanding of adaptation of the cardiomyocyte to ischaemia-reperfusion injury and the intracellular signal transduction pathways that mediate this process. Proof-of-concept clinical studies have demonstrated benefi cial effects with ischaemic preconditioning, ischaemic postconditioning and remote ischaemic conditioning in a variety of clinical settings including cardiac bypass surgery, acute myocardial infarction and elective PCI. Large multicentre randomized clinical trials are now under way to determine whether ischaemic conditioning can improve clinical outcomes. Therefore, harnessing the powerful cardioprotection elicited by ischaemic conditioning could 1 day provide a therapeutic strategy for protecting the heart against acute IRI, preserving cardiac function and improving 84 D.J. Hausenloy and D.M. Yellon in the clinical setting, it needs to be in all animal demonstrated to be effective models of IRI tested canine models of IRI cardiac arrest, acute myocardial surgery, strategy The cardioprotective infarction. long-term to offer should be shown cardioprotection occlusion in atherosclerotic animal models. Before testing the cardioprotective strategy strategy Before testing the cardioprotective Use large animal models such as porcine and Use large c animal models of cardiac fi Use speci Use close chested internal coronary artery Use animals with co-morbid conditions Use animals on concomitant medication strated to be consistently effective in both small strated to be consistently effective IRI animal models of in vivo and large ischaemia in of IRI should ideally only be investigated PPCI patients thrombotic occlusion on a ruptured unstable atherosclerotic plaque dyslipidaemia, hypertension, previous MI, dyslipidaemia, hypertension, previous which may all interfere with cardioprotection tions which can interfere with cardioprotection The cardioprotective strategy should be demon- strategy The cardioprotective Patients often present after 3–6 h of myocardial Patients An AMI is a pro-in fl ammatory condition due to fl An AMI is a pro-in cardioprotection ischaemia (30–60 min) and reperfusion (2–72 h) coronary artery Needs to offer conclusive conclusive Needs to offer Needs to match clinical setting tested in animal models strategies Cardioprotective Short duration of myocardial Current models Non-diseased animals co-morbidities such as diabetes, age, have Patient Problem Potential solution Concomitant medication medica- can be on a number of different Patients Problems with current design of animal models of cardioprotection Problems with current design of animal models cardioprotection strategy strategy Cardioprotective Cardioprotective Animal selection animals Juvenile are middle-aged Patients Use aged animals IRI model External occlusion of a health Table Table 4.3 4 Preconditioning in the Heart 85 markers of cardioprotection such as markers by CMR or myocardial salvage myocardial nuclear scanning. Multicentre RCTs will be required on hard clinical to assess effect end points surgery, cardiac arrest, acute surgery, The cardio- myocardial infarction. should be shown strategy protective long-term cardioprotection to offer and canine models of IRI to be administered prior or at the onset of myocardial reperfusion presenting with minimal collateralization presenting with TIMI 0 coronary prior to PPCI ow fl presenting with a proximal LAD RCA infarct or large For PPCI studies, use robust surrogate PPCI studies, use robust For Use speci fi c animal models of cardiac fi Use speci Use large animal models such as porcine Use large For PPCI patients, the intervention needs PPCI patients, the intervention For For PPCI patients, only select patients For For PPCI patients, only select patients For For PPCI studies, only select patients For strategy is most likely to impact on such as MI is most likely strategy function, LV size, myocardial salvage, and CV death incidence of heart failure models of IRI should ideally only be in PPCI patients investigated ischaemia reperfusion injury occurs in the fi rst few rst few fi reperfusion injury occurs in the needs minutes of reperfusion, the intervention to be administered prior or at the onset of cial fi myocardial reperfusion to be bene area at risk may impact on cardioprotection are most likely to have not yet undergone not yet undergone to have are most likely reperfusion cardioprotective strategy are those presenting strategy cardioprotective infarcts with the larger Use clinical end point which the cardioprotective Use clinical end point which the cardioprotective Patients often present after 3–6 h of myocardial Patients For PPCI patients, where the lethal myocardial For The PPCI patients most likely to bene fi t from a fi to bene The PPCI patients most likely for cardioprotective strategies such as strategies for cardioprotective MI and revascularization non-fatal (30–60 min) and reperfusion (2–72 h) match the animal studies. there is a risk of diluting any there is a risk of diluting any effect cardioprotective Use of inappropriate clinical end points Needs to match clinical setting tested in animal strategies Cardioprotective Short duration of myocardial ischaemia The timing of the intervention needs to The timing of the intervention Collateral fl ow ow fl Collateral cant collateralization to the fi The presence of signi Coronary fl ow prior to PPCI ow fl Coronary prior to PPCI ow fl with TIMI 0 coronary Patients Current studies Problem Potential solution Problems with the current design of clinical studies of cardioprotection Problems with the current design of clinical studies cardioprotection end points intervention intervention Cardioprotection Timing Timing of Patient selection Patient By including all-comers in PPCI studies, Table Table 4.4 86 D.J. Hausenloy and D.M. Yellon clinical outcomes in patients with IHD. A similar hope is held out for the clinical application of cerebral preconditioning, for both ischaemic and other disorders in the CNS.

Acknowledgments We thank the British Heart Foundation (Program Grant RG/03/007 and FS/10/039/28270) for ongoing funding and support. This work was undertaken at University College London Hospital/University College London (UCLH/UCL) who received a proportion of funding from the Department of Health’s National Institute of Health Research (NIHR) Biomedical Research Centres funding scheme.

Con ﬂ ict of Interest None declared.

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Ryan Kochanski , David Dornbos III , and Yuchuan Ding

5.1 Introduction

Exercise, hypothermia, and hyperthermia are preconditioning methods that vastly differ in terms of the physical properties of their respective stimuli. Despite their differences, these varying physical stimuli provide different value in terms of potential clinical utility. This chapter will review the features and mechanisms underlying the cerebral protection that is conferred through the utilization of exercise, hypothermia, and hyperthermia. The neuroprotection derived from these various treatment modalities underlies their use in clinical settings, and it provides necessary background information for potential therapeutic and pharmaceutical interventions in the future.

5.2 Exercise Preconditioning

The benefi cial effects of exercise preconditioning on stroke prevention and stroke- induced brain injury reduction have been demonstrated as a function of both risk factor management and endogenous neuroprotection. Several meta-analyses have indicated that physical activity correlates with increased neuroprotection in total, ischemic, and hemorrhagic strokes (Lee et al. 2003 ; Wendel-Vos et al. 2004 ) . It is well known that much of this decrease in neurologic dysfunction following stroke is the result of exercise-induced risk factor modifi cation, which notably effects blood pressure, lipid pro fi les, weight, hypertension, and diabetes (Evenson et al.

R. Kochanski ¥ D. Dornbos III ¥ Y. Ding (*) Department of Neurological Surgery , Wayne State University School of Medicine , Detroit , MI , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 105 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_5, © Springer Science+Business Media New York 2013 106 R. Kochanski et al.

1999 ; Gillum et al. 1996 ; Hu et al. 2004 ) . In addition to amelioration of stroke risk factors, exercise preconditioning has been shown to provide endogenous neuroprotective bene fi t in the setting of stroke-induced brain injury (Endres et al. 2003 ) , resulting in decreased infarct volume and improved functional recovery (Ding et al. 2004b, c, 2005, 2006a, b, c ; Curry et al. 2010 ; Davis et al. 2007 ; Zwagerman et al. 2010b ; Chaudhry et al. 2010 ; Liebelt et al. 2010 ; Guo et al. 2008 ) . In previous human studies, exercise has been shown to enhance learning, memory, and executive function while decreasing age-related cerebral atrophy and disease-related dementia and mental impairment (Colcombe and Kramer 2003 ) . Exercise preconditioning training has also been proven bene fi cial in reducing the risk of Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Cotman et al. 2007 ) . Through amelioration of cerebral in fl ammation, strengthening of the blood-brain barrier (BBB), mediating apoptosis, and diminishing metabolic dysfunction, exercise preconditioning enhances neuronal viability through a multitude of different mechanisms.

5.2.1 Methods of Preconditioning

Previous human studies have looked at the duration and intensity of exercise and the quality of life improvements associated with these activities, and they have found that the best outcomes correlate with moderate exercise intensity and duration (Larson et al. 2006 ) . Although pre-ischemic exercise has been shown to be cardioprotective in retrospective human studies, no specifi c guidelines or methods have been established for exercise preconditioning to convey either cardioprotection or neuroprotection (Abete et al. 2001 ; Kloner 2001) . However, exercise for as little as 2 weeks and as long as 12 weeks in rats has been shown to decrease brain infarction and neuronal damage (Ang et al. 2003 ; Stummer et al. 1994 ; Wang et al. 2001 ; Curry et al. 2010 ; Davis et al. 2007 ; Ding et al. 2004b ) . Studies have also shown that this protective effect remains for at least 3 weeks after exercise has ceased (Ding et al. 2004c ) , revealing the long- lasting extent of neuroprotection conveyed from exercise training. Various types of exercise, such as treadmill, running wheel, and enriched environment, have been shown to provide neuroprotection. Studies also found that various types of exercise induced differing neurological outcomes following stroke. Forced exercise, as opposed to voluntary, conveys more potent neuroprotection in ischemic models. Forced exercise on a treadmill provides longer and slower run but more consistent intervals. Unlike these forced-exercise animals, voluntarily exer- cised rats run long total distances, but it takes place at a more rapid pace in shorter spurts (Noble et al. 1999). Even though total running distance was shorter in the forced exercise group, they showed signifi cantly smaller infarct size and decreased total neurologic de fi cit (Hayes et al. 2008 ) . This change was correlated with upregulation of heat shock proteins (Hayes et al. 2008 ) , increased neurogenesis (Leasure and Jones 2008) , and increased cerebral metabolism (Kinni et al. 2011 ) . Further studies have assessed the neuroprotection garnered from various types of exercise, notably simple and complex. Simple exercise utilizes a treadmill, requiring only 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 107

Exercise

+ + + + + + +- +

Neurotrophins Basal Lamina Astrocytosis Angiogenesis TNF-α ERK1/2 ICAM-1 Glucose (BDNF, NGF) (Collagen IV, (VEGF, Ang1, Ang2) HSP-70 Expression transport, Integrins) Glycolysis HIF-1α + + + + +- + + +- +

Neurogenesis BBB Strength Blood vessel MMP-9 Anti:Pro Leukocyte ATP density Apoptotic Invasion Production Ratio

+ + +- +- +- +

Neurovascular unit + BBB Dysfunction Apoptosis Inflammation Neuronal integrity Metabolism

+ + + + + +

Neuroprotection

Fig. 5.1 Mechanisms of neuroprotection conferred by exercise preconditioning repetitive movements, whereas complex exercise uses enriched activities which involve the use of balance and coordination (Ding et al. 2003 ). Using complex exercise in a postischemic setting yields increased synaptogenesis and better functional outcomes when compared to simple treadmill exercise (Ding et al. 2003; Jones et al. 1999 ). Nonetheless, simple exercise still carries signi fi cant bene fi t for exercise preconditioning when compared to nonexercised controls, but complex exercise may be better able to benefi t functional recovery in the poststroke individual (Ding et al. 2003 ). Future studies will be needed to both determine the duration of the neuroprotective effect of exercise and to develop a more precise training regimen in order to maximize cerebral protection in the setting of acute stroke.

5.2.2 Mechanisms of Neuroprotection

Understanding the mechanisms underlying exercise preconditioning and its enhancement of cerebral neuroprotection de fi nes its clinical importance and provides a framework for potential future therapeutic intervention. These neuroprotective effects are brought about by a number of differing mechanisms (Fig. 5.1), all of which are designed to strengthen neuronal integrity and improve cell survival. This is done through the increased release of neurotrophins and other growth factors, aimed at increasing neurogenesis and synaptic plasticity. Furthermore, the neurovascular unit is strengthened as a result of exercise training, including reinforcement of the blood-brain barrier (BBB), increased astrocytosis, and ampli fi ed angiogenesis. Chronic exercise also dampens the in flammatory effect and secondary complications 108 R. Kochanski et al. that is often seen following an ischemic insult by downregulating leukocyte invasion and fl uid permeability into the interstitial space. In addition to the previously mentioned acceleration of neurogenesis, exercise preconditioning also serves to decrease the rate of apoptosis of neurons by upregulating anti-apoptotic signaling pathways while simultaneously downregulating pro-apoptotic signaling pathways. Finally, exercise training increases the capacity for cerebral metabolism, thus ameliorating the metabolic dysfunction seen in poststroke patients. These metabolic changes have been seen in conjunction with upregulation of hypoxia-inducible factor 1 a (HIF-1a ) following exercise, also correlating with better neurologic outcomes. Altogether, these mechanisms provide increased neuroprotection against cerebral ischemic insults, equipping the brain with an ability to improve neuronal survival following a cerebrovascular accident.

5.2.2.1 Neurotrophins

Exercise increases overall brain health by facilitating increases in growth factor cascades, subsequently triggering an increase in neurogenesis. Human and animal studies have shown that brain-derived neurotrophic factor (BDNF) plays a vital role in the learning process and is essential for synaptic plasticity (Kuipers and Bramham 2006 ) . Previously shown to increase neuronal development, synaptic plasticity, and protection of the central nervous system vasculature, BDNF and nerve growth factor (NGF) have long been implicated as neuroprotective factors (Cohen-Cory et al. 2010 ; Kim et al. 2004 ) . An upregulation of the mRNA levels of these neurotrophins has also been demonstrated in the rat brain following physical exercise training, speci ﬁ cally in neuronal and astrocytic cells (Ding et al. 2004b ) . This upregulation in neurotrophin expression is believed to be the result of increased neuronal activity during exercise. Previous reports have also demonstrated an increase in BDNF mRNA and protein expression in the immediate reperfusion stage following an ischemic insult, revealing its natural function in maintaining neuronal integrity (Schabitz et al. 1997 ). Furthermore, rats which had been preconditioned with physical exercise and maintained signiﬁ cantly elevated levels of endogenous NGF were shown to have substantial neuroprotection after complete middle cerebral artery (MCA) occlusion (Ang et al. 2003 ) . These elevated levels of BDNF and NGF seen after physical exercise are able to increase neuronal health prior to injury and provide the cerebral architecture with a necessary ability to self-repair following ischemic insult. The increase in synaptic plasticity and neuronal development as a result of neurotrophin upregulation provides a fundamental component to the neuroprotective nature of exercise.

5.2.2.2 Neurovascular Unit Integrity

The integrity of the neurovascular unit, composed of capillary endothelial cells, glial cells, and neurons, provides the primary innate barrier in the setting of acute stroke, and its microvasculature and architectural framework provides the basis for 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 109 exercise-induced neuroprotection. Integrity of this structure requires robust interaction between endothelial cells, the basal lamina, astrocytic end-feet, and neurons, providing sound structural support and appropriate permeability of the blood-brain barrier (BBB). Dynamic interaction between the BBB, astrocytes, and capillaries has been previously shown in response to exercise and is also known to ameliorate dysfunction in response to ischemic insults. The integrity of the BBB is needed for both structural stability and maintenance of appropriate permeability of the cerebral vasculature. The permeability barrier is primarily composed of the endothelial wall and the basal lamina, including the extracellular matrix (ECM). This ECM, which consists of collagen type IV, laminin, fi bronectin, proteoglycan, and heparan sulfate, works as a selective barrier, making its functionality vital to neuronal health and viability. The integrity of the neurovascular unit has profound implications in acute stroke and is the fi rst structure disrupted at the onset of ischemia (del Zoppo and Hallenbeck 2000 ; del Zoppo and Mabuchi 2003 ) . Following stroke, the disruption of the BBB through alterations in collagen type IV, laminin, and fi bronectin (del Zoppo and Mabuchi 2003 ) subsequently generates vasogenic edema and disturbance of the neuronal environment. Reperfusion, the goal of the current standard of care, actually worsens this disruption, leading to increased vascular damage and edema formation (Yang and Betz 1994 ) . Because of this aggravation by treatment strategy, maintenance of BBB integrity during both ischemia and reperfusion is of essential importance. Previous studies have revealed that this necessary BBB integrity has been augmented by chronic exercise preconditioning, leading to decreased cerebral edema and brain injury (Masada et al. 2001 ) . When comparing alterations in the cerebral microvasculature to exercise preconditioned rats, nonexercised rats have shown greater parenchymal edema, swollen astrocytic end-feet, and thinner basal lamina (Ding et al. 2006a ) . Furthermore, exercise preconditioning increases the main basal laminar protein, collagen type IV, and expression and decreases its loss after stroke (Davis et al. 2007 ) . In addition to collagen IV and other basal lamina structural proteins, integrins are cell adhesion molecules which enhance the interaction between endothelial cells and astrocytes with ligands in the basal lamina. Cerebral microvasculature integrity depends on these proteins to anchor the cellular cytoskeleton to the underlying basal lamina, and it depends on integrins to serve as signaling receptors for appropriate interaction with the surrounding environment, primarily through cellular proliferation and differentiation (del Zoppo and Mabuchi 2003 ) . Following ischemia, integrins have been shown to lose their affi nity for laminin and collagen, generating BBB disruption, astrocyte swelling, and cerebral edema (Wang and Lo 2003 ; Hamann et al. 2002 ) . In response to exercise preconditioning, the disruption in integrin functionality is signifi cantly attenuated. Rats preconditioned with physical exercise were shown to upregulate expression of integrin proteins, and this upregulation was associated with a decrease in ischemic injury (Ding et al. 2006b) . The increase in integrin and collagen type IV expression provided by exercise training strengthens the BBB and the neurovascular unit, which most importantly correlates with better outcomes following acute stroke. 110 R. Kochanski et al.

In addition to increased strength, the BBB integrity is also improved by physical exercise through reduction of matrix metalloproteinase (MMP) expression. These proteins are produced by endothelial cells, microglia, and astrocytes, and they serve to degrade ECM and nonmatrix proteins. MMP expression has been shown to increase after ischemic insults in both rat and human studies (Romanic et al. 1998 ; Planas et al. 2001 ; Clark et al. 1997 ) . This increase in MMP expression correlates with tissue injury and infl ammation and leukocyte invasion (Romanic et al. 1998 ) . Furthermore, MMP defi ciency (Asahi et al. 2001 ) and inhibition (Romanic et al. 1998 ) are associated with decreased edema, infarction volume, and better overall neurologic outcomes. In a similar manner, physical exercise correlates with reduced MMP expression (Davis et al. 2007) , and this serves as the underlying factor in the observed increase in collagen type IV expression following exercise preconditioning (Guo et al. 2008 ) . These biomolecular changes in exercise preconditioned rats correlate with decreased neurologic defi cit, infarct volume, and leukocyte infi ltration following cerebral ischemia (Curry et al. 2010 ) . Furthermore, MMP-signaling triggers a caspase-mediated cascade, resulting in neuronal apoptosis (Gu et al. 2002 ) ; however, this mechanism is suppressed through tissue inhibitors of metalloproteinases (TIMPs), extracellular signal-regulated kinases (ERK1/2), and tumor necrosis factor (TNF)-a (Arai et al. 2003 ; Brew et al. 2000 ) . Elevated levels of TIMPs, ERK1/2, and TNF- a have been shown to be upregulated following physical exercise preconditioning and correlate with reduced MMP expression and neuronal apoptosis following stroke (Guo et al. 2008 ; Chaudhry et al. 2010 ) . This observed decrease in MMP, a destructive enzyme of the BBB, coupled with the increased expression of collagen IV and integrins, key components of the BBB, underlie the mechanism of increased BBB integrity and neurovascular unit strength capable of providing neuroprotection through chronic exercise preconditioning. In response to exercise preconditioning, the cerebral microvasculature is remodeled to meet the metabolic demands of the brain. Physical activity and chronic exercise increases blood vessel density (Swain et al. 2003 ) , angiogenesis (Ding et al. 2004c ) , and arteriogenesis (Lloyd et al. 2005 ) , generating more extensive collateral circulation and a more adequate response to ischemic injury. Developed in response to chronically increased energy demand during exercise, this microvasculature development has been associated with a reduction in brain damage following an ischemic insult (Ding et al. 2004b ) . The mechanism underlying the observed vasculature morphology has been found to be under the regulation of vascular endothelial growth factor (VEGF) and angiopoietin (Ang 1 and 2) (Ding et al. 2004c ) . In addition to increased angiogenesis and arteriogenesis following exercise preconditioning, another study revealed that these changes correlate with astrocytosis (Li et al. 2005 ) , further strengthening the neurovascular unit and guarding against neuronal damage in the setting of acute ischemia. Similar to the increase in the cerebral vasculature, exercise preconditioning has also been shown to preserve cerebral blood fl ow (CBF) during the reperfusion stage after stroke which correlates with a decrease in brain infarct volume (Zwagerman et al. 2010b ) . Similarly, preconditioned rats preserved signi fi cant glucose metabolism in the reperfusion stage following ischemia (Bequet et al. 2001 ) . These increases 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 111 in CBF and metabolism have also been seen during dynamic exercise in previous human studies (Ide and Secher 2000 ; Williamson et al. 1997 ) . Increased blood vessel density and CBF likely provides an enriched vascular bed which is better able to adapt to the changes of acute stroke, both in the ischemic and reperfusion stages.

5.2.2.3 In ﬂ ammatory Response

Hypoperfusion is a common problem in the reperfusion stage following acute ischemia due to incomplete return of CBF in the microvasculature. This may be due to either occlusion from cellular elements, such as leukocytes, or vascular damage due to free radicals, infl ammatory cells, or endothelial dysfunction (Hallenbeck et al. 1986 ) . Following ischemia, cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF- a), stimulate the expression of adhesion molecules, including intracellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin, on endothelial cells. These adhesion molecules are well known for increasing leukocyte in fi ltration into the brain parenchyma, further occluding microvessels and promoting neuronal damage. Chronic exercise preconditioning has been shown to decrease ICAM-1 expression, subsequently decreasing leukocyte infi ltration into the brain parenchyma following reperfusion (Ding et al. 2005 ) . By decreasing adhesion molecule expression and leukocyte migration, exercise preconditioning thus ameliorates many of the damaging secondary changes that occur during the reperfusion stage. As previously mentioned, TNF-a is a major pro-infl ammatory cytokine with profound effects on the cerebral response to hypoxic injury. Despite its known role as a deleterious cytokine, it is upregulated following exercise preconditioning (Ding et al. 2005 ) . It is known to exert both trophic and toxic effects on neuronal tissues, depending on its concentration (Rothwell and Hopkins 1995 ) . A previous study revealed that this gradual upregulation of TNF-a is associated with reduced brain injury (Ding et al. 2005 ) . It is believed that chronic low-level elevations of TNF-a , which are seen with exercise and ischemic preconditioning, may result in the development of neuronal tolerance to the cytokine (Wang et al. 2000b ; Liu et al. 2000 ) and further promote angiogenesis (Fajardo et al. 1992 ) . Chronically elevated levels of TNF- a lead to a decrease in TNF-a receptor expression, generating this neuronal tolerance. These receptors have also been shown to be downregulated following exercise preconditioning (Reyes et al. 2006 ) , promoting a model in which chronic stimulation by low levels of TNF-a leads to desensitization of the TNF-a receptor. With these receptors in decreased abundance, the elevated levels of TNF-a seen in the poststroke period no longer carry the same deleterious power seen in conditions without exercise preconditioning, leading to better overall neuronal survival. In a similar manner, toll-like receptors are thought to be involved in the immune response and may trigger cytokine cascades, leading to leukocyte in fi ltration. Recent studies have shown that exercise reduces systemic infl ammatory markers, such as toll-like receptors (McFarlin et al. 2006 ) . Furthermore, in toll-like receptor knockout mice, ischemia/reperfusion injury has been substantially decreased, resulting in enhance neuronal survival (Cao et al. 2007 ) . In rats preconditioned with physical 112 R. Kochanski et al. exercise, reduced expression of toll-like receptors correlated with decreased brain injury (Zwagerman et al. 2010a ) . Chronic exercise preconditioning decreases the overall in fl ammatory response to insults such as ischemia, resulting in improved neuronal survival and better neurologic outcomes.

5.2.2.4 Neuronal Death and Survival Signaling Pathways

Neuronal death following ischemic/reperfusion injury is mediated by a variety of genes and regulatory proteins that trigger cascades leading to either cell death or survival. Recent studies have revealed that exercise preconditioning decreases the rate of neuronal apoptosis (Chaudhry et al. 2010 ) . In addition to the roles played by TNF-a and HSP-70, anti-apoptotic genes, such as Bcl-2 and Bcl-xL, and pro-apoptotic genes, such as Bax, Bad, Bak, and AIF (apoptosis-induced factor), are responsible for the neuronal response to hypoxic conditions (Lazou et al. 2006 ) . Rats preconditioned with physical exercise have been found to have increased expression of anti-apoptotic proteins and an associated decrease in pro-apoptotic proteins following an ischemic/reperfusion insult (Chaudhry et al. 2010 ) . Furthermore, as this ratio favors anti-apoptotic markers, neuronal survival is enhanced even in the setting of lethal ischemic injury (Rybnikova et al. 2006) . Thus, chronic exercise training leads to increased levels of anti-apoptotic protein expression, correlating with superior neuroprotection. Heat shock proteins (HSP-70) are expressed in response to various types of stress, including hypoxia and ischemia, and have been shown to provide neuroprotection in the setting of acute stroke. HSP-70 mediates this neuroprotection through downregulation of pro-apoptotic proteins, such as AIF (Matsumori et al. 2005 ) , and upregulation of anti-apoptotic proteins, such as Bcl-2 (Liebelt et al. 2010 ) . Furthermore, exercise preconditioning has been shown to upregulate HSP-70 expression in neurons and the vasculature, and this upregulation has been associated with heightened neuroprotection (Masada et al. 2001 ) . Even though the neuroprotective nature of HSP-70 is dependent on very high levels of the protein (Lee et al. 2001 ) , inhibition of even mildly elevated levels of HSP-70 completely ameliorates exercise-induced neuroprotection, making it likely that HSP-70 works in conjunction with other factors to prevent apoptosis rather than working alone. A recent study has also shown that HSP-70 works with TNF-a to properly regulate the ratio between pro- and anti-apoptotic genes (Goel et al. 2010 ) . It is therefore likely that exercise training generates elevated levels of HSP-70, in correlation with elevated TNF- a , thus promoting anti-apoptosis and mediating neuronal survival.

5.2.2.5 Metabolic Alterations

Chronic exercise preconditioning has also been associated with changes in the metabolism of neuronal tissues. This has previously been shown through increases in regional utilization of oxygen and glucose (Querido and Sheel 2007 ) and increased regional cerebral blood ﬂ ow (Ogoh and Ainslie 2009 ) during exercise training. 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 113

Further studies have shown that these changes are associated with an increased capacity for ATP generation in the tissues of exercise preconditioned rats (McCloskey et al. 2001 ) , giving these rats an increased capacity for energy production in periods of hypoxia. Another recent study revealed that increased metabolism and glycolysis following treadmill exercise in rats (Kinni et al. 2011 ) correlates with decreased neurologic deﬁ cits after stroke and enhanced neuroprotection (Hayes et al. 2008 ) . Another element of the metabolic changes occurring after exercise training relates to hypoxia-inducible factor 1a (HIF-1a ). Known to be neuroprotective following ischemia (Bernaudin et al. 2002 ; Schubert 2005 ), HIF-1a has been shown to be upregulated following exercise preconditioning and induces both angiogenesis and glycolysis (Bernaudin et al. 2002; Schubert 2005 ; Bergeron et al. 1999 ). These metabolic changes seen following upregulation of HIF-1a promote neuronal survival (Kinni et al. 2011) and serve as another neuroprotective mechanism of exercise preconditioning. This ability of exercise preconditioning to increase the metabolic rate and energy generating capacity in cerebral tissues allows for a more rapid and ef ﬁ cient response to ischemic injury, thus providing another mechanism of neuroprotection.

5.2.3 Poststroke Exercise

While exercise preconditioning provides neuroprotection prior to the onset of acute ischemic stroke, the role of exercise is a core component to many rehabilitation programs. The use of poststroke exercise provides substantial beneﬁ t to patients and correlates with better outcomes in rehabilitation (Rabadi 2007 ) . Previous studies have shown that the brain undergoes morphologic changes after stroke in an attempt to restore functionality (Kleim et al. 2002 ) . Poststroke exercise has also been shown to increase neurotrophin expression (Vaynman and Gomez-Pinilla 2005 ) and neurogenesis (Leasure and Grider 2010 ) , and these changes are correlated with improved outcomes and more rapid recovery. Multiple studies have also shown that exercise training in the poststroke period correlates with increased synaptogenesis at the infarct location (Stranahan et al. 2007 ; Ding et al. 2003 ; Jones et al. 1999 ). Complex exercise, involving balance and coordination, is able to increase this synaptogenesis, more so than simple treadmill exercise, and correlates with better functional outcomes (Ding et al. 2003 ; Jones et al. 1999 ). These changes seen in postischemic conditioning through exercise may provide possible avenues for therapeutic intervention.

5.2.4 Clinical Application

The neuroprotective nature of exercise is not only useful in prevention and amelioration of stroke-induced brain damage but has therapeutic implications as well. The various neuroprotective changes that occur in relation to neurotrophic factors, the neurovascular unit, the inﬂ ammatory environment, and cerebral metabolism provide potential 114 R. Kochanski et al. mechanisms for future drug targets. Pharmaceutical intervention that increases the innate neuroprotection seen in association with exercise could potentially strengthen the BBB, decrease cerebral edema, or work through a variety of other pathways to confer enhanced neurologic outcomes. This would be particularly useful as a preconditioning method in a patient with a history of stroke, transient ischemic attack, or any other cerebrovascular event. Furthermore, using exercise preconditioning in advance of neurologic surgery could utilize the known neuroprotective effects of exercise to achieve better outcomes after surgery. Clearly, more research needs to be done, especially in the development of exercise guidelines for ideal neuroprotection.

5.3 Hypothermia

Hypothermia as a treatment modality both during and after the onset of ischemic stroke has been studied extensively; however, much less is known about its utility as a preconditioning stimulus. It has been shown to reduce cerebral ischemic injury in animals, but presently no clinical therapies have been implemented to reduce the neurological complications resulting from cerebral ischemia. The following section will outline the current knowledge of hypothermic preconditioning and assess its utility as a potentially viable clinical treatment.

5.3.1 Experimental Models

Hypothermic preconditioning is performed by administering hypothermia of a specifi c depth and duration at a time point prior to the onset of ischemia. It has been shown in animal models to confer protection against cerebral hypoxia/ischemia both in vivo and in vitro (Nishio et al. 1999, 2000 ; Yunoki et al. 2002, 2003 ; Mitchell et al. 2010 ; Yuan et al. 2004 ) . In vivo studies, through manipulation of several key parameters, have optimized the ability of the preconditioning stimulus to confer protection in response to 1 h of transient focal ischemia induced by a three-vessel occlusion model (Nishio et al. 2000; Yunoki et al. 2003) . This model consists of occlusion of both carotid arteries and unilateral occlusion of the middle cerebral artery (MCA). The above-mentioned studies have established that the depth and duration as well as the specifi c onset of the hypothermic stimulus prior to ischemia are all crucial parameters in conferring optimal cerebral protection. Like ischemic preconditioning, hypothermia is capable of inducing either rapid or delayed tolerance depending on the specifi c timing of the stimulus in relation to the onset of cerebral ischemia (Yunoki et al. 2003 ; Stetler et al. 2008 ) . In vitro studies have further characterized the underlying mechanisms involved in both the rapid and delayed phases induced by hypothermia (Yuan et al. 2004 ; Mitchell et al. 2010 ) . The key aspects of the hypothermic stimulus that render it capable of providing optimal cerebral protection are outlined below. 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 115

Induction of Rapid Tolerance

Begin hypothermia End hypothermia Ischemic onset

20 minPost-hypothermic interval of 20 – 60 min Ischemia/Reperfusion

Fig. 5.2 Rapid tolerance confers maximal neuroprotection when induced 20Ð60 min prior to the onset of ischemia. Hypothermia applied for 20 min duration is sufﬁ cient to induce rapid tolerance

5.3.1.1 Rapid vs. Delayed Tolerance

The rapid tolerance phase induced by hypothermic preconditioning was fi rst reported in rats subjected to 1 h of transient focal ischemia by three-vessel occlusion (Yunoki et al. 2003) . Signifi cant reductions in infarct volume were seen when the hypothermic preconditioning stimulus was applied between 20 and 60 min prior to the onset of ischemia; however, neuroprotection was nearly reversed by 120 min. Figure 5.2 demonstrates the general time interval capable of inducing rapid tolerance. Rapid tolerance confers maximal neuroprotection when induced 20Ð60 min prior to the onset of ischemia, and a hypothermic stimulus applied for 20 min duration is suf fi cient to induce rapid tolerance. The administration of protein synthesis inhibitors prior to preconditioning does not ameliorate the neuroprotective response, indicating that this form of tolerance does not depend on de novo protein synthesis (Yunoki et al. 2003 ) . An in vitro study subjecting Purkinje cells in rat cerebellar slices also evaluated the ef fi cacy of a brief hypothermic stimulus at time points immediately to 3 h prior to the onset of oxygen glucose deprivation (Yuan et al. 2004) . This stimulus was shown to elicit rapid tolerance that decreased Purkinje cell death when administered at time points up to 3 h after the onset of the stimulus. The delayed tolerance phase was fi rst demonstrated in a similar three-vessel occlusion model as used to elicit rapid tolerance (Nishio et al. 1999 ) . Delayed tolerance elicits signifi cant neuroprotection when the hypothermic stimulus is administered between 6 and 48 h prior to the onset of cerebral ischemia. This protective response peaks in the vicinity of 24 h and is completely abolished by 7 days after the initial stimulus. Figure 5.3 demonstrates the ideal time interval for inducing delayed tolerance. Maximal neuroprotection is conferred when delayed tolerance is induced 24 h prior to the onset of ischemia, and a hypothermic stimulus duration of at least 20 min can effectively induce delayed tolerance. Besides the difference in the onset of protection conferred by delayed tolerance in comparison to rapid tolerance, another important defi ning feature of delayed tolerance is its dependence on de novo protein synthesis (Nishio et al. 2000 ) . Treatment with protein synthesis inhibitors prior to the preconditioning stimulus was shown to completely reverse its protective effect suggesting dependence on gene product expression. 116 R. Kochanski et al.

Induction of Delayed Tolerance

Begin hypothermia End hypothermia Ischemic onset

20 –120 minPost-hypothermic interval of 24 hrs Ischemia/Reperfusion

Fig. 5.3 Delayed tolerance confers maximal neuroprotection when induced 24 h prior to the onset of ischemia. A hypothermic stimulus of at least 20 min is effective in inducing delayed tolerance

5.3.1.2 Depth and Duration

A series of experiments evaluating the ef fi cacy of hypothermic preconditioning has shown that the induction of delayed tolerance is dependent on both the depth and duration of the hypothermic stimulus (Yunoki et al. 2002 ) . Whole body cooling using ice packs to temperatures of 31.5, 28.5, or 25.5¡C showed signi fi cant temperature dependent reduction in brain infarct volume after focal cerebral ischemia in rats. In this experiment, the duration of hypothermia at the desired temperature was 20 min, although the total amount of time below normal body temperature was around 120 min (due to the time required to reach the target body temperature and to rewarm back to normal body temperature). Another experiment showed that the greatest reduction in infarct volume occurred when milder hypothermia (33¡C) was administered for 120 min (180 min total) compared to only 20 and 60 min. It is important to note however, that hypothermic preconditioning at a temperature of only 1.5¡C greater (34.5¡C) for 120 min did not show a signifi cant reduction in infarct volume indicating the dependence of stimulus duration on stimulus depth. Rapid tolerance has been shown to be dependent only upon the depth of the hypothermic stimulus (Yunoki et al. 2003 ) . In these experiments, a greater stimulus was required to elicit rapid tolerance in comparison to the levels of hypothermia capable of inducing delayed tolerance. Signi fi cant reductions in infarct volume were only seen in 28.5 and 25.5¡C and not 31.5¡C. No signi fi cant difference was observed in animals exposed to hypothermia for a period of 20 min in comparison to those exposed for 60 min, suggesting that rapid tolerance is not dependent on stimulus duration.

5.3.2 Mechanism

The underlying mechanisms inducing delayed or rapid tolerance in response to hypothermic preconditioning are not well characterized; however, the differing characteristics between rapid and delayed tolerance, especially in regard to de novo protein synthesis, suggest that hypothermic preconditioning may induce protection through contrasting mechanisms (Yunoki et al. 2003 ) . The current mechanistic understanding of hypothermic preconditioning is outlined in Fig. 5.4 . 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 117

Hypothermic Preconditioning

+ + -

Adenosine A1 HMG I(Y) TNF-α receptors protein

- + -

COX-2, NO synthase, MMP-9 K channels ATP cytokines

- + -

BBB dysfunction Hyperpolarization Inflammation

+ + +

Neuroprotection

Fig. 5.4 Mechanisms of neuroprotection conferred by hypothermic preconditioning

5.3.2.1 Adenosine Receptors, KATP Channels, and High-Mobility Group I (Y) Protein

Rapid tolerance in Purkinje cells induced by 20 min of 33¡C hypothermia prior to oxygen glucose deprivation demonstrated decreased expression of high-mobility group I (Y) protein and dependence on activation of adenosine A1 receptors and

K ATP channels (Yuan et al. 2004 ) . High-mobility group I (Y) protein (HMG I(Y)) is a nuclear transcription factor known to induce expression of deleterious proteins including inducible nitric oxide (NO) synthase, cyclooxygenase-2 (COX-2), and cytokines which may be elevated during ischemia (Perella et al. 1999 ; Pellacani et al. 2001; Yuan et al. 2004 ) . A1 receptor activation is associated with a decrease in synaptic activity which may decrease the spread of excitotoxicity (Pugliese et al.

2003) and indirectly activate KATP channels leading to cell hyperpolarization (Heurteaux et al. 1995 ; Yamada and Inagaki 2005 ) . Ischemic preconditioning involves a similar protective mechanism involving A1 receptors and KATP channels suggesting a potentially shared common pathway (Stetler et al. 2008 ) .

5.3.2.2 TNF- a

As mentioned earlier in this chapter, exercise induces chronic elevation of TNF-a which is capable of downregulating expression of TNF-a receptors I and II during ischemia and correlates with reduced MMP expression and increased BBB integrity 118 R. Kochanski et al. following stroke (Reyes et al. 2006 ; Guo et al. 2008 ; Chaudhry et al. 2010 ) . Induction of tolerance by hypothermic preconditioning may also involve a similar mechanism. Hypothermic preconditioning for 90 min at 30¡C demonstrated an increase in TNF-a expression in rat hippocampus cultures 24 h after the initial stimulus (Mitchell et al. 2010 ) . The protective effects of this pro-in fl ammatory cytokine were attenuated by pretreatment with IL-11, an anti-in fl ammatory cytokine, and blockage of IL-11 lead to an increase in protection conferred by TNF-a . This tolerance inducing stimulus provided by TNF- a has also been seen in ischemic preconditioning (Romera et al. 2004 ) . Pretreatment with TNF-a in the absence of ischemic preconditioning has even been shown to induce tolerance, further demonstrating the crucial role of this pro-infl ammatory cytokine in conferring protection when administered prior to ischemia (Nawashiro et al. 1997 ) .

5.3.3 Intra- and Postischemic Hypothermia

Clinical studies have shown that decreases in body temperature are associated with decreased mortality following stroke (Jorgensen et al. 1999 ; Wang et al. 2000a ; Kammersgaard et al. 2002 ) . Numerous experimental models have been created to assess the viability of hypothermia as a treatment method for ischemic stroke (Tang and Yenari 2010 ) . Intra-ischemic hypothermia has been deemed the “gold standard” due to its ability to signifi cantly reduce brain injury in a number of in vivo animal studies (Barone et al. 1997 ; Corbett and Thornhill 2000 ; Miyazawa et al. 2003 ) . Postischemic hypothermia has also been shown to be effective depending on the duration of hypothermia (Miyazawa et al. 2003 ) . Some clinical studies have suggested that the neuroprotective properties of mild or moderate hypothermia in acute ischemic stroke can only be achieved by either earlier initiation of brain cooling after onset of stroke or by prolonged hypothermia for up to 48Ð72 h (Schwab et al. 1998, 2001; Kammersgaard et al. 2000 ; Steiner et al. 2000 ) . Many studies in animal models with global and focal transient cerebral ischemia have demonstrated the effectiveness of postischemic hypothermia (Coulborne et al. 1997; Kawai et al. 2000; Maier et al. 2001 ) , but prolonged application of postischemic hypothermia seems to be necessary to achieve signi fi cant and persistent neuroprotection (Coulborne et al. 1997 ; Inamasu et al. 2000 ; Kollmar et al. 2002 ) . Furthermore, it is likely that mild postischemic hypothermia simply delays neuronal damage caused by ischemia (Dietrich et al. 1993 ; Inamasu et al. 2000 ) . A number of mechanisms have been proposed for hypothermia induced neuroprotection both during and after stroke. These include reductions in cerebral metabolism (Polderman 2009 ) , decreased free radical formation (Karibe et al. 1994 ) , preservation of blood-brain barrier integrity (Huang et al. 1999 ; Chi et al. 2001 ) , and suppression of infl ammation (Aibiki et al. 1999 ; Kimura et al. 2002 ; Luan et al. 2004; Suehiro et al. 2004 ) . Interestingly, hypothermia seems to induce contrasting responses depending on whether it is administered as a preconditioning stimulus or as a treatment during an ischemic event. When administered during ischemia, hypothermia has been shown to decrease pro-in fl ammatory cytokine release includ- 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 119 ing TNF-a (Aibiki et al. 1999 ; Kimura et al. 2002 ; Suehiro et al. 2004 ) . However, when administered as a preconditioning stimulus prior to ischemia, hypothermia induces TNF-a expression in a similar manner as described earlier with exercise preconditioning which appears to be a signifi cant factor in conferring tolerance (Mitchell et al. 2010 ) . This dual role may be a reason why hypothermia, unlike most other preconditioning modalities, is effective both as a preconditioning stimulus and as a treatment for stroke. Further studies are necessary to elucidate any potential relationship between preconditioning, intra-, and postischemic mechanisms of neuroprotection conferred by hypothermia.

5.3.4 Clinical Applications

Hypothermia is unlike other preconditioning stimuli in that it serves a dual protective role both as a tolerance conferring stimulus and as an intra- and postischemic neuroprotectant. Unlike ischemic or hyperthermic preconditioning, a hypothermic stimulus could theoretically be induced close to the onset of ischemia without exacerbating the ischemic event. This unique characteristic could make it a clinically bene ﬁ cial treatment modality in situations like cardiac or neurological surgery where cerebral ischemia may transiently occur (Nishio et al. 1999 ; Yunoki et al. 2003 ) . It also makes it one of the more suitable options for inducing rapid tolerance. There are, however, some important factors that must be considered should any clinical treatment strategies be implemented.

5.3.4.1 Safety

Hypothermia has been well studied in relation to its effect on humans which further substantiates its ability to be of clinical bene fi t compared to other less studied therapies (Nishio et al. 1999 ) . There are, however, serious side effects that can occur, the most signi fi cant being cardiac arrhythmia which can occur at temperatures below 28¡C (Polderman 2009 ) . This could present as an obstacle because the highest level of neuroprotection was shown to be conferred by temperatures as low as 25.5¡C when eliciting delayed tolerance (Yunoki et al. 2002 ) . Also, rapid tolerance conferred cerebral protection only at temperatures of 28.5 and 25.5¡C and not 31.5¡C, indicating the need for signi fi cant hypothermia. Parameters of the hypothermic stimulus may have to be manipulated in order to avoid these side effects. An increased risk of infection can occur in the context of prolonged hypothermia (Polderman 2009 ) . Even though hypothermia for periods as brief as 20 min has been shown to confer protection, the risks associated with prolonged hypothermia may still be signi fi cant because of the time it takes to cool to target temperature and rewarm to normal body temperature. It is also important to note that the only current studies involving hypothermic preconditioning have been performed in small animals and any benefi t cannot be readily extrapolated to humans. Despite the potential side effects of hypothermia, continued investigation into its use as a clinical preconditioning therapy seems worthwhile. 120 R. Kochanski et al.

5.3.4.2 Cooling Methods

Induction of therapeutic hypothermia has been classically achieved through whole body surface cooling using, such as externally applied ice packs, cooling blankets, alcohol applied to exposed skin, and can take as long as 3Ð7 h to reach target body temperatures of 32Ð34¡C (Kammersgaard et al. 2000 ; Schwab et al. 2001) . Focal cooling of the head using externally applied ice packs as the preconditioning stimulus has been shown to be similarly effective in conferring cerebral protection compared to whole body cooling while also eliminating potential side effects by keeping core body temperature close to normal (Yunoki et al. 2002) . Externally selective cooling, however, may only be effective in reducing temperatures of the superﬁ cial cerebrum and not deeper brain structures like the basal ganglia (Mellergard 1992 ; Nelson and Nunneley 1998 ; Diao et al. 2003 ) . The ideal method of hypothermic preconditioning should be regionally selective with the capability to include either deep or super ﬁ cial brain structures and should achieve rapid cooling to the desired brain temperature in order to provide optimal neuroprotection and mitigate systemic complications. Selective cooling using intracarotid saline infusion has been shown to provide both intra- and postischemic neuroprotection (Ding et al. 2004a, b, c ; Luan et al. 2004 ; Li et al. 2004 ) . This therapy is particularly desirable after ischemia because it provides extremely fast and effective heat exchange leading to rapid tissue cooling because the cold liquid is transported through the microvasculature and distributed throughout the parenchyma. Theoretically, cooling of a localized cerebral infarction having a mass of 300 g is 30 times faster than classic surface cooling (Konstas 2007 ) . When compared to both systemic and external head cooling, regionally selective vascular cooling has been shown to be more effective at reducing brain injury and improving functional outcome in rats (Wang et al. 2010 ) . It has also been shown that local brain hypothermia induced by autocirculating cold arterial blood (13Ð15¡C) into ischemic territory reduces infarct volume by decreasing the ischemic territory to 32Ð34¡C within 5Ð10 min in rats (Cheng et al. 2009 ) . Most importantly, a recent pilot study has demonstrated the safety and feasibility of selective brain cooling with endovascular intracarotid infusion of cold saline in humans (Choi et al. 2010 ) . Hypothermic preconditioning prior to instances of predicted ischemia could theoretically be induced in a similar manner.

5.3.4.3 Timing

As previously mentioned, the speci ﬁ c timing of the stimulus in relation to ischemia is crucial in conferring either rapid or delayed tolerance. Rapid tolerance would be most ideal in regards to initiating prior to surgery because it could be induced after the patient has been placed under anesthesia. Eliciting delayed tolerance would be far less practical in that it would require cooling the patient at a period of 24 h prior to the onset of the surgical procedure. This would most likely require sedative anesthesia because a conscious patient is unlikely to tolerate being cooled and compensatory shivering may prevent body cooling to the desired temperature (Sessler 2009 ; Moore et al. 2011 ) . 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 121

5.4 Hyperthermia

Hyperthermia, when administered at speci ﬁ c time intervals prior to the onset of cerebral stress, has been shown to provide neuroprotection. Like hypothermic and ischemic preconditioning, the speci ﬁ c time at which the hyperthermic stimulus is administered prior to the onset of ischemia is a key parameter. The following section will outline the effects of hyperthermia both as a preconditioning method and when administered during times cerebral ischemia.

5.4.1 Experimental Models

Hyperthermic preconditioning has been shown to confer neuroprotection when administered at speci fi c time points prior to ischemia both in vivo and in vitro (Chopp et al. 1989 ; Zhang et al. 2000; Xu et al. 2002; Kelty et al. 2002; Du et al. 2010 ) . In a transient forebrain ischemia model, rats were heated within a high humidity container and maintained at rectal temperatures of 41.5¡C for 15 min (Chopp et al. 1989 ) . Rats that underwent hyperthermia showed a signi fi cantly attenuated loss of neuronal cells after ischemia. Similar neuroprotection was noted in a 2-h middle cerebral artery (MCA) occlusion model which subjected rats to 42¡C by immersion in a water bath for 15 min (Xu et al. 2002 ) . Signi fi cant decreases in infarct volume were noted only between 18 and 24 h after the preconditioning, and the protective effect of the stimulus was abolished by 48 h. Hyperthermic preconditioning, when administered for as little as 15 min, effectively induces tolerance and confers optimal neuroprotection between 18 and 24 h prior to the onset of ischemia (Chopp et al. 1989; Zhang et al. 2000; Xu et al. 2002) . This protective time interval is consistent with delayed tolerance and is similar to hypothermic preconditioning. Figure 5.5 demonstrates the general time interval for effective hyperthermic preconditioning. It is important to note, however, that the ability of a hyperthermic preconditioning stimulus to induce rapid tolerance has not been effectively demonstrated. No signifi cant neuroprotection was conferred when ischemia occurred at 30 min after the stimulus was applied which would normally be consistent with rapid tolerance (Xu et al. 2002) . Rapid tolerance induced by hypothermia was conferred between 20 and 60 min prior to the onset of ischemia (Yunoki et al. 2003 ) , and the lack of protection conferred by hyperthermia between 20 and 60 min prior to ischemia may be indicative of its inability to induce rapid tolerance.

5.4.2 Mechanism

Like hypothermic preconditioning, the mechanism involving hyperthermic preconditioning is not well characterized. The majority of studies involve the expression of heat shock proteins (HSPs) in response to hyperthermia. More recent studies have demonstrated involvement of adenosine receptors and hypoxia-inducible factor 1 a 122 R. Kochanski et al.

Hyperthermic Preconditioning

Begin hyperthermiaEnd hyperthermia Ischemic onset

15 min Post-hyperthermic interval of 18-24 hrs Ischemia/Reperfusion

Fig. 5.5 Hyperthermic preconditioning confers optimal neuroprotection when administered between 18 and 24 h prior to the onset of ischemia. Hyperthermia for as little as 15 min effectively induces tolerance

Fig. 5.6 Mechanisms Hyperthermic Preconditioning of neuroprotection conferred by hyperthermic + + preconditioning + +

Adenosine A1 α HIF-1 receptors

+ +

HSP-72 KATP channels

+- +

Apoptosis Hyperpolarization

+ +

Neuroprotection

(HIF-1 a ). The following section will touch brie ﬂ y on the current mechanisms believed to be involved in hyperthermic preconditioning. The current understanding of the mechanism is outlined in Fig. 5.6 .

5.4.2.1 Heat Shock Proteins

HSPs are molecular chaperones believed to play a vital role in the cellular response to stressful stimuli by decreasing denatured protein accumulation and improving cell survival through a reduction in pro-apoptotic proteins (Chen and Simon 1997 ; 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 123

Kirino 2002 ; Stetler et al. 2008 ; Liebelt et al. 2010 ) . Several studies have demonstrated the ability of a hyperthermic stimulus to induce HSP expression prior to cerebral stress. Preconditioning at 41.5¡C for 15 min induced cerebral HSP72 expression in glial and endothelial cells as well as neuroendocrine areas when assessed at 24 h after the stimulus (Li et al. 1992 ) . Rats preconditioned at 42¡C for 15 min have also demonstrated increased levels of HSP72 as early as 16 h after the initial stimulus (Yang and Lin 1999 ) . This increase in HSP72 decreased back to baseline at 48 h after the initial stimulus which is consistent with delayed tolerance. Hyperthermic preconditioning at 40¡C for 15 min was also shown to protect synaptic function in mouse medullary slices subjected to stressful stimuli (Kelty et al. 2002 ) . When slices were treated with exogenous HSP72 in the absence of a hyperthermic stimulus, similar synaptic protection was conferred, indicating a key protective role by HSP72.

5.4.2.2 Adenosine Receptors

Rats subjected to 15 min of hyperthermic preconditioning at 42¡C at both 18 and 24 h prior to MCAO showed a reduction in infarct volume compared to control (Xu et al. 2002 ) . This protective response was partially attenuated in animals receiving a central adenosine receptor antagonist, indicating at least partial dependence on adenosine receptors during hyperthermic preconditioning. It has also been proposed that HSPs may be involved; however this has yet to be demonstrated (Xu et al. 2002 ) . Adenosine receptor involvement, as previously mentioned, has been shown to play a role in the protective response conferred through rapid tolerance induced by hypothermia (Yuan et al. 2004 ). This may be suggestive that adenosine receptors play a crucial role in both rapid and delayed tolerance and can be induced through various modes of preconditioning.

5.4.2.3 HIF-1 a

HIF-1 a plays a key role in maintaining oxygen homeostasis and is also involved in tolerance conferred by ischemic preconditioning (Zhu et al. 2007, 2008; Du et al. 2010 ) and exercise preconditioning (Kinni et al. 2011 ) . Hyperthermic preconditioning of mouse astrocytes at either 38 or 40¡C for 6 h was shown to increase both expression of HIF-1a and overall cell survival after a 24-h period of ischemia and reperfusion (Du et al. 2010 ) . HIF-1 a has also been shown to upregulate transcription of heat shock proteins (Huang et al. 2009 ) . This evidence further indicates that the link between hyperthermic preconditioning stimulus and heat shock protein expression may be controlled by the induction of HIF-1a .

5.4.3 Intra- and Postischemic Hyperthermia

The major discrepancy between hyperthermia and hypothermia lies in their opposite effects on outcome when administered immediately before, during, or after isch- 124 R. Kochanski et al. emia. Elevated body temperatures, especially within the ﬁ rst week in patients suffering from an ischemic stroke have been shown to have a negative effect on outcome (Greer et al. 2008 ; Saini et al. 2009 ) . Several in vivo studies have demonstrated increases in infarct volume when hyperthermia of 39Ð40¡C is administered either immediately prior to or during ischemia (Busto et al. 1987 ; Dietrich et al. 1990a ; Chen et al. 1991 ) . A hyperthermic temperature of 40¡C administered 1 day after cerebral ischemia has also been shown to exacerbate the injury (Kim et al. 1996) . Several of the mechanisms by which hyperthermia exacerbates preexisting ischemic injury include an increase in metabolic rate in the absence of effective blood ﬂ ow (Holtzclaw 1992; Corbett and Thornhill 2000 ) , increased glutamate release (Sternau et al. 1992 ; Takagi et al. 1994 ) , increased cytoskeleton degradation (Morimoto et al. 1997 ) , and BBB breakdown (Dietrich et al. 1990b , 1991 ) .

5.4.4 Clinical Implications

It seems unlikely that hyperthermia could be utilized clinically to confer cerebral protection due to its ability to exacerbate any concurrent cerebral stress. Not only has an increased cerebral temperature been shown to be detrimental during stroke, but it is also unlikely that human subjects could tolerate the degree of hyperthermia administered during preconditioning. Temperatures between 40 and 42¡C have been shown in the majority of experiments to induce the most robust protective responses. Heat stroke occurs in humans at temperatures above 40¡C and is often fatal (Bouchama and Knochel 2002 ) . The inability to elicit rapid tolerance also makes hyperthermia a less enticing mode of preconditioning. Even if hyperthermia can elicit rapid tolerance, it would be unsafe due to the potential for exacerbating ischemia if body temperatures remain elevated prior to onset of the ischemic insult. Nevertheless, the elucidation of the mechanism behind hyperthermia’s protective response as a preconditioning method is still important in discovering new therapeutic strategies to prevent cerebral damage.

5.5 Conclusion

Despite their differences, exercise, hypothermia, and hyperthermia are all effective physical preconditioning methods, and studies suggest that they may share certain mechanisms. Evidence that exercise confers cerebral protection both through preconditioning and after brain injury further substantiates its encouragement as a preventive measure of future disease and supports its use in rehabilitation after brain injury. Hypothermic preconditioning may eventually be utilized to induce cerebral protection prior to surgery. Besides the protection conferred by the physical properties of the above-mentioned preconditioning stimuli, substantial beneﬁ t lies in the elucidation of the underlying mechanism of tolerance which may eventually be 5 Neuroprotection and Physical Preconditioning: Exercise, Hypothermia… 125 exploited through the development of targeted pharmaceutical therapies. Ideally, these targeted therapeutics should be capable of inducing tolerance without the risks associated with the physical preconditioning methods described in this chapter. Further research is therefore necessary in order to confer clinically optimal neuroprotection through the use of preconditioning.

References

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Nicolas Blondeau and Joseph S. Tauskela

6.1 Introduction

Prevention and treatment of stroke remain major challenges in modern medicine. Stroke represents a leading cause of death in developed (3rd) and Third World countries due to its high incidence, the severity of the insult, and grave defi ciencies in treatment options. Ischemic stroke is not so much a disease – although once early treatment options are exhausted, clinically stroke is treated as such – but is more akin to a traumatic event, caused by a disruption in blood fl ow to part of the brain, commonly due to occlusion of a blood vessel(s) feeding the brain (in approximately 85% of the stroke patient population). The only therapeutic is restoration of cerebral blood fl ow, which is achieved by the disruption of the blood clot by recombinant tissue plasminogen activator (tPA) treatment. It is somewhat ironic that blood is considered the best “neuroprotectant,” given that tPA mediates its effect through the vasculature, as opposed to exerting any direct protection in neurons. Unfortunately, “clot busters” are ultimately provided to only approximately 5% of stroke patients, although this percentage varies across different clinical stroke units. Numerous neuroprotective drugs and therapies identi fi ed in a multitude of preclinical studies block the neurotoxic ischemic signaling transduction cascade, but, of those tested in clinical trials, all have failed, leaving clinicians without any repertoire of therapeutic

N. Blondeau , Ph.D. (*) Université de Nice Sophia Antipolis , 28 Avenue de Valrose , 06103 Nice Cedex 2 , France Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, UMR 6097 , 660 Route des Lucioles , 06560 Valbonne , France e-mail: [email protected] J. S. Tauskela , Ph.D. National Research Council of Canada, Human Health Therapeutics, Neurotherapeutics Program , Ottawa , ON , K1A 0R6 , Canada

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 133 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_6, © Springer Science+Business Media New York 2013 134 N. Blondeau and J.S. Tauskela opportunities and causing a major exodus of pharmaceutical companies (O’Collins et al. 2006 ) . On the positive side, this failure in translation from experimental models to clinical trials led our community to reevaluate the science of ischemic stroke, resulting in a proposal of a set of drug development criteria, collectively known as the Stroke Therapy Academic Industry Roundtable (STAIR) recommendations (Fisher 2003 ; Fisher et al. 2009 ) . A rami fi cation of clinical failures of conventional neuroprotective drug therapies has been acceleration in the study of unconventional therapies, with one such effort being to interrogate how the brain protects itself. This endogenous mechanism of protection of the brain is known as brain preconditioning. Preconditioning of the brain elicits complex endogenous neuroprotective responses that act by pleiotropic mechanisms to block death pathways, promote survival pathways, and increase resistance. The preconditioning approach represents a major departure from most conventional neuroprotective approaches since tolerance to cerebral ischemia is brought about primarily through the activation of endogenous cellular protective signaling pathways rather than through inhibition of a speci fi c neurotoxic receptor or signaling molecule. As a result, and of crucial importance, the adverse effects that are so often encountered with conventional neuroprotection should be averted with preconditioning. Preconditioning is multicellular in nature, with a capability of targeting not only neurons but the entire neurovascular unit, which is so important in preserving neuronal networks. Preconditioning research should help in the identifi cation of cellular signaling involved in inducing cerebral ischemic tolerance, perhaps ultimately shaping the design of successful stroke treatments. Given that neuroprotection appears ineffective per se, an emerging direction to identify the “best-in-class” therapeutics is to seek drugs combinatorial in nature, which could protect the whole neurovascular unit and target time-dependent neurotoxic mechanisms. This is the major focus of preconditioning research with the discovery of novel preconditioning molecules that activate complex cellular signaling cascades to render the brain resistant to subsequent ischemia. Despite the relative novelty of the preconditioning approach, in order to not repeat past mistakes in neuroprotection evaluations, it is mandatory that agents selected as preconditioners should be considered as any other drug or therapeutic candidate, as early in their development as possible (Tauskela and Blondeau 2009 ) . Considered from this perspective, it is immediately obvious that a crucial consideration is how a preconditioning agent is to be delivered, and how much risk is involved in its delivery. Preconditioning achieved by classical means – ischemia or neurotoxins – represents substantial stressors to neurons, often causing brain tissue to approach the “brink of death” in order to successfully precondition, and therefore represents an unacceptable risk (Dirnagl et al. 2003; Kirino 2002) . Moreover, the blood-brain barrier often limits penetration of drugs beyond the brain vasculature. As a result of these challenges, in addition to considering neuroprotective properties, as has classically been done, it is important to consider delivery and risk as early as possible in the drug/ therapy discovery process. To address these and other issues raised later in this chapter, in parallel to chemical preconditioners, momentum is building in this fi eld with convincing demonstrations that natural/endogenous compounds, 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 135 such as adenosine, lysophospholipids, and omega-3 polyunsaturated fatty acids that are found in our daily diet, could be excellent preconditioners against stroke. Consequently, these natural molecules may overcome major diffi culties associated with more traditional therapies, introducing a major new concept in preconditioning to combat stroke, which is preconditioning achieved through supplementation of an essential item in diet or as a nutraceutical. In this spirit, this book chapter addresses this new idea that selected nutraceutical may act in fact as preconditioners of the brain and may therefore represent a paradigm shift in the management of ischemic stroke. We begin with an overview of hard lessons learned from the failure of neuroprotective agent development and then attempt to de fi ne how this should alter pursuit of brain preconditioning research. This is followed by a review and critical insight into how nutrition might be of relevance in the prevention of stroke as well as in identifying new preconditioners. The goal of this chapter is to introduce a unique perspective, as inspired by preconditioning achieved by omega-3 polyunsaturated fatty acids, especially a -linolenic acid (ALA) as a nutraceutical, thereby presenting the fi eld with new opportunities and avenues of investigation in developing stroke prevention and therapy.

6.2 Lessons from the Failure of Neuroprotective Agent Development in Stroke

About 780,000 Americans each year suffer a new or recurrent stroke, meaning that on average, a stroke occurs every 40 s in developed countries. Stroke kills patients on an average of every 4 min, with an estimated 25% death rate within the fi rst week and 50% of the patient population within 5 years after the brain attack (Rosamond et al. 2008 ) . Work capacity is compromised in 70% of patients surviving a stroke, among which a third would need assistance with self-care. The most common consequences of stroke are functional control impairment, paralysis, speech and sensory problems, and memory and reasoning defi cits, often leading to long-term disability, dementia, and poststroke depression. In the United States alone, its eco- nomic cost has been estimated to be greater than 70 billion dollars (Lloyd-Jones et al. 2010; Rosamond et al. 2008) . Altogether with its substantial social and psychological costs, stroke is a global burden of the developed nations affecting all racial or ethnic groups indiscriminately. Stroke is a neurovascular event caused by a disruption in blood fl ow to part of the brain. In approximately 85% of the cases, stroke is an ischemic syndrome, commonly due to occlusion of a blood vessel. Interruption of blood fl ow by occlusion deprives the surrounding brain tissue of nutrients and oxygen and allows toxic metabolic products to build up. It elicits a complex interplay of multiple signaling pathways in neuronal, glial, and endothelial cells, leading to irreversible damage of the neurovascular unit within the ischemic territory (Iadecola 2004 ) . Within the core of the affected brain tissue, blood fl ow is most severely restricted, and excitotoxic and necrotic cell death occur within minutes. Between the core and the brain tissue that 136 N. Blondeau and J.S. Tauskela is still normally perfused, collateral arteries may temporarily buffer the reduction of blood fl ow and therefore delay the effect of stroke. In this area, termed ischemic penumbra, the tissue is ischemic but still momentarily preserved as apoptosis leads to a slower rate of cell death (Endres and Dirnagl 2002 ) . In any case, the core will spread if reperfusion is not reestablished rapidly. This raises an issue of paramount importance in stroke, which is the limited time window of intervention required for neuroprotection, and represents perhaps the major reason of failure to develop stroke therapeutics. Echoing a concern raised throughout the clinical trial process, in a recent seminal review, Ginsberg notes that failure to implement a neuroprotective treatment within a 4–6-h window following stroke is suffi cient to account for the abundance of nonsuccessful clinical trials (Ginsberg 2008 ) . Timing represents the major restriction for clinical treatment with the only therapeutic available, recombinant tissue plasminogen activator, to restore cerebral blood fl ow by disrupting the blood clot causing vessel occlusion to a maximum temporal window extending up to 4.5 h window post stroke. It is important to recognize that timing is an issue even within this 4.5-h window: every 15-min reduction in door-to-needle time was associated with a 5% lower odd of in-hospital mortality. Moreover, treatment time is not likely to substantially change since fewer than one-third of patients treated with intravenous tPA have door-to-needle times £60 min, with only modest improvement noted over the past 6 years (Fonarow et al. 2011) . Another recent major study notes that the odds of a favorable outcome decrease by a factor of 2 for each 90-min period of intravenous tPA treatment after symptom onset (Lees et al. 2010 ) . This pessimistic view of stroke therapy has only been strengthened by the continued failure of drugs aimed at directly inhibiting the ischemic cascade by targeting primarily neurons. In 2006, O’Collins reviewed 1,026 neuroprotective treatments identi fi ed for acute stroke between 1957 and 2003, among which 114 were tested in clinical trials and failed (O’Collins et al. 2006 ) . As further underlined in a recent viewpoint expressed by Moskowitz, a growing community of scientists and clinicians question the merits of the translational process taking a neuroprotective drug or therapy from experimental models to clinical trials in stroke (Moskowitz 2010 ) . Perhaps even more troubling has been the exodus of neuroprotection stroke programs in big pharma. Retrospective analyses raise the possibility that the preclinical, translational, and clinical trial process was inadequate to select the “best-in-class” agents. An undeniable absence of clarity in choos- ing a drug candidate for translation to clinical trials was pointed out by O’Collins, since no clinical drug candidate distinguished itself as being better when compared against all other drugs preclinically identi fi ed in experimental models of cerebral ischemia (O’Collins et al. 2006 ) . In addition, preclinical studies examining the clinically tested drugs displayed poor adherence to a series of criteria proposed by the Stroke Therapy Academic Industry Roundtable (STAIR) designed to increase the stringency of preclinical evaluation of prospective neuroprotective agents. The O’Collins study raised the question of whether the best drugs were being chosen to proceed to clinical trial. However, STAIR was not initially designed to identify leading candidates but only on how to test candidates once identi fi ed. We recently proposed new ideas and recommendations for the development of neuroprotective 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 137 strategies in combating stroke, with particular emphasis on how these strategies could be implemented to brain preconditioning studies (Tauskela and Blondeau 2009) . The basic tenet proposed was to devote some effort beyond mechanistic studies and to consider preconditioning candidates much like any other neuroprotective drug under investigation so that progress made in identifying major issues in preclinical studies of neuroprotective drugs and therapies could be extended to the preconditioning fi eld. Retrospectively, much critique has focused on inadequate timing and dosing of drugs used in clinical trials. Several reasons may account for this unsuccessful translation. The ineffective management came in part from the failure to incorpo- rate preclinical information into clinical trials, as well as major concerns that animal data do not adequately refl ect the complexity encountered in clinical settings (Jonas et al. 1999) . A better knowledge of animal model limitations in mimicking the multifactorial aspects of human stroke, and of their value in speci fi c pathophysiological features of human stroke, may reduce the failure rate. Improvements can be anticipated by testing the effi ciency of the drug in several experimental models of stroke, as well as in mammals, which include primates. The clinical failures also lead to redefi ning the view of the pathology of cerebral ischemia as a cerebrovascular – and not exclusively neuronal – disease. The era of considering only neurobiological aspects of stroke is over, and it is increasingly acknowledged that stroke triggers vascular, cerebral blood fl ow (CBF), and multicellular alterations (neuronal, astroglial, and oligodendroglial) and associated microglial activation. This integrative view of stroke intuitively necessitates taking into consideration ef fi cacy, or at least the effect of the treatment being investigated, on all cell types within the entire neurovascular unit (fundamentally comprising endothelium, astrocyte, and neuron). Ideally, the “best-in-class” drug for a clinical trial would have clear and demonstrable effi cacy on all the neurovascular unit cells supporting neuronal protection as well as vascular functions. Thus, an experimental drug should be tested for safety and ef fi cacy not only on neurons but also on cerebral endothelial cells, astrocytes, oligodendrocytes, microglia, and so on (Lo and Rosenberg 2009) . The ischemic response not only occurs within different cell types but is also temporally and mechanistically multimodal. Stroke induces cellular damage through multiple pathways, which include mechanisms like excitotoxicity, oxidative stress, and programmed cell death. The failure of stroke trials, with each usually targeting only one of these or other pathways, suggests that targeting a single element of a single pathway may not yield suffi cient neuroprotection. Consequently, an emerging view is that combination or “multi” therapy, or discovering drugs exhibiting multimodal actions at multiple cell types, is required to address the multifactorial nature of stroke. Finally, even with or without acute neuroprotective therapy, molecules that trigger regenerative mechanisms following cerebral ischemia are also being increasingly considered as particularly attractive therapeutic agents (Lo 2008 ; Minnerup and Schabitz 2009 ; Zaleska et al. 2009 ) . A pertinent and effective experimental approach to identify such candidates would be to seek understanding of how the brain protects itself against ischemia, through the study of brain preconditioning. 138 N. Blondeau and J.S. Tauskela

6.3 Brain Preconditioners May Lead to Innovative Therapies Against Ischemic Stroke

Ischemic preconditioning is a cellular and adaptive biological process that confers resistance to ischemia. Ischemic preconditioning of the brain, heart, and other organs refers to an endogenous protective process that is induced by a sublethal ischemia and which increases the tissue tolerance to a subsequent, normally lethal ischemia. Since its original description in the brain by Kitagawa in 1990 (Kitagawa et al. 1990 ) , nonischemic preconditioners including various sublethal insults like epilepsy, endotoxins, anoxia, hyperthermia, and spreading depression were shown to also induce delayed tolerance to normally lethal forms of themselves. An interesting point to note is that the nonischemic preconditioners promote additional tolerance to ischemia, a phenomenon known as “cross-tolerance” (Gidday 2006 ; Plamondon et al. 1999 ; Tauskela and Blondeau 2009 ) . The large body of evidence showing that preconditioning promotes both protection and regeneration against stroke through direct and/or indirect mechanisms establishes that the preconditioning response is multicellular and, as a consequence, that preconditioners are considered pleiotropic in nature. Regarding the translation of brain preconditioning, the stroke research community expressed initial concerns that its potential clinical applications may be very limited, due to the requirement of bringing neurons to the “brink of death” during the sublethal preconditioning challenge (Dirnagl et al. 2003 ) . The discoveries that brain preconditioning and its tolerance to ischemia may be pharmacologically promoted by drugs like adenosine and K ATP channel agonists (Blondeau et al. 2000) offered new perspectives in the drug discovery ﬁ eld. Several other compounds including 3-nitropropionic acid and the glutamate receptor agonist, N -methyl- d -aspartate (NMDA), have been investigated in both in vivo and in vitro models of preconditioning (Tauskela and Blondeau 2009) . Moreover, retrospective case-control studies showed a clinical correlate of the experimental preconditioning paradigm, suggesting that patients with a history of transient ischemic attack (TIA) exhibit a decreased morbidity after stroke, so preconditioning may also be taking place in the human brain (O’Duffy et al. 2007 ; Weih et al. 1999 ) . Finally, the recent demonstration that a variety of natural products like polyunsaturated fatty acids and lysophospholipids may precondition the brain against stroke circumvents this initial concern of its potential applications (Blondeau et al. 2002a, b ) . With this encouraging insight, studies on preconditioning provide an innovative approach for the discovery of novel cerebroprotective compounds. Regarding the relevance of the time course and dose of preconditioning for translation, preconditioning protection against ischemia occurs within two different temporal windows. The ﬁ rst window is known as “rapid preconditioning” and occurs within minutes after the stimulus used for induction of preconditioning, and the ischemic- tolerant state persists for only ~1 h. The so-called delayed window that produces a more robust state of protection develops ~24 h after preconditioning induction, peaks at 72 h, and fades 7 days later in general. A 2- to 4-day interval between preconditioning and lethal ischemia provides the greatest protection, at least in the rodent brain. 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 139

Several high-impact reviews have already elegantly addressed the molecular sensors, transducers, and effectors of preconditioning and underline key conceptual differences between preconditioning and more conventional neuroprotection strategies, notably the time frame and the protection paradigms (Dirnagl et al. 2003; Gidday 2006 ; Kirino 2002 ; Obrenovitch 2008 ) . Indeed, most drugs aim to directly block toxic signaling pathways activated post stroke, while preconditioning indirectly interferes with the toxic signaling effects by activating complex endogenous pro-survival responses. Since the preconditioning response is strongly associated with gene remodeling, development of an ischemic-tolerant phenotype requires a certain delay in order to attain subsequent maximal protection. Because of this requirement for a delay, the scientiﬁ c and clinical communities sometimes incorrectly perceive the potential applications of preconditioning as a preventive strategy. Instead, preconditioning protects the brain against an ischemic insult, but virtually nothing is known regarding the ability of preconditioning to reduce the incidence of stroke. Since preventive strategies against stroke are mainly designed to limit occurrence of the stroke itself, often by reducing the risk factors, this view of preconditioning as preventive strategy or pretreatment has generated considerable confusion in the ﬁ eld.

6.4 Focus on Omega-3 PUFAs in Stroke: From Insuf ﬁ ciency as a Risk Factor to Supplementation as a Preventative Strategy, Culminating in Discovery as a Preconditioner

6.4.1 Nutrition as a Risk Factor in Stroke

The poor translation from experimental models to clinical trials has led to adoption of additional strategies in combating stroke, most notably drawing attention to the importance of prevention. By defi nition, prevention or/and treatment of the risk factors reduce the occurrence of stroke, and such strategies continue as the highest priority in reducing the burden of stroke. Some risk factors are not modifi able, such as a family history of cerebrovascular diseases, aging, male sex, and Hispanic or Black race (Allen and Bayraktutan 2008 ) . But other risk factors including cardiovascular complications, hypertension, diabetes, hypercholesterolemia, cigarette smoking, increased in fl ammatory markers, dyslipidemia, and obesity may be addressed by pharmacological and lifestyle changes, thereby preventing or minimizing the possibility of having a stroke. Modi fi able risk factors often coexist and have been estimated to account for 60–80% of stroke incidence in the general population (Allen and Bayraktutan 2008 ; Moskowitz et al. 2010 ) . Interestingly, the modifi able risk factors of stroke often coexist with improper lifestyle and nutrition, causing imbalances in essential vitamins and nutriments, suggesting that these imbalances may be amenable to lifestyle changes. Many clinical and epidemiologic studies have shown that de fi ciencies in vitamins, nutrients, and essential omega-3 polyunsaturated fatty acids (PUFAs) may be risk factors of stroke per se. 140 N. Blondeau and J.S. Tauskela

Insuf fi cient dietary intake of fruits and vegetables or of food containing omega-3, in the form of a -linolenic acid (ALA) and the long-chain derivatives (LC-n-3), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is a risk factor for cardiovascular and cerebral diseases, including coronary heart disease and stroke (Dauchet et al. 2005; de Goede et al. 2011; Riediger et al. 2009; Simopoulos 2008). Encouragingly, supplementation with such factors in the diet may exhibit a preventative role in reducing the incidence of the pathologies. Intriguingly, several studies described below in cerebral disease fi elds identifi ed benefi cial effects of diets rich in omega-3 PUFAs, achieved by consumption of seafood (rich in LC-n-3: EPA and DHA) and/or vegetable oils rich in precursor (ALA), suggesting that omega-3 PUFAs may have a neuroprotective role.

6.4.2 A Balanced PUFA Intake Is Crucial for Health

The primary role of the PUFAs is to act as structural components due, in part, to their presence within the bilayer of cell membranes. PUFAs thus contribute to the fl uidity and permeability properties of cellular membranes, as well as being involved in a myriad of intracellular signaling pathways (for reviews, see Jump 2002 ; Spector 1999; Uauy et al. 2001 ) . Omega-3 de fi ciencies have been observed in humans and certain symptoms share those with panhypovitaminose. This was probably due to the fact that vitamins such as vitamin B regulate many enzymatic activities using an omega-3 derivates as a substrate. For example, 32 clinical cases presenting symptoms of beriberi responded to a diet enriched in omega-3 (60% omega-3/AG essential (Rudin 1982 ) ). The risk of an omega-3 defi ciency was fi rst identifi ed with the use of total parenteral nutrition (TPN) since original preparations were devoid of lipids or contained high omega-6 and low omega-3 fatty acids (Holman et al. 1982 ) . A 6-year-old girl undergoing 5 months of TPN exhibited episodes of numbness, tingling, weakness, inability to walk, leg pain, and psychological and visual disturbances. Administration of a TPN solution enriched in omega-3 primarily from soy- bean oil sources (EPA + DHA) reversed the neuropathy and restored omega-3 toward normal levels. The syndrome of omega-3 defi ciency may be associated with neurological and psychological disorders (Rudin 1981 ) . These types of studies ultimately convinced the scienti fi c and medical communities to investigate the necessity of omega-3 PUFAs.

6.4.3 The In ﬂ uence of Nutritional PUFAs on Brain Biochemistry

Omega-3 and omega-6 fatty acids exhibit substantial differences in location and uptake in the brain. Neuronal membranes are enriched in DHA and AA, despite lacking the desaturase and elongase enzymes required for their synthesis; however, astrocytes and endothelial cells produce and release DHA and AA (Moore et al. 1991 ) . EPA is found in trace amounts in the brain, mostly in phosphatidylinositol (PI) form, but 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 141

DHA is concentrated in synaptic regions in gray matter and is especially abundant in phosphatidylethanolamine (PE) and phosphatidylserine (PS). Treatments, which raise blood DHA levels rapidly, increase DHA uptake into and retention by brain cells. In contrast, AA is widespread throughout the brain and is abundant in PI and phosphatidylcholine (PC). Daily treatment (4 weeks with 300 mg/kg gavage in gerbils) with DHA and EPA, but not AA, increases brain phospholipid content. This increase is associated with an increase in PSD-95, the GluR1 subunit of AMPA receptors and synapsin-1 (a presynaptic vesicular protein), and spine density in CA1 pyramidal neurons, suggesting that EPA/DHA supplementation increases neuroplasticity and neurotransmitter release and thus impacts animal behavior. Integration of omega-3 within neuronal matter achieved through supplementation might “set the stage” for investigation of potential bene fi ts to be explored for patients with neurodegenerative diseases or stroke or brain injury (Cansev et al. 2008 ) . In rodents, in order to observe maximal improvements in cognition, learning, epilepsy, and hormonal regulation, omega-3 supplementation should strive to achieve an ideal omega-6/omega-3 ratio of 4:1 (Yehuda and Carasso 1993; Yehuda et al. 1998). Since humans must also strive for a diet allowing a similar ratio of omega-6/omega-3 to be achieved, the experimental studies were extended to clinical studies examining different neuropathies, including Alzheimer disease for which bene fi cial effects, as well as improvements in appetite, autonomy, and short-term memory, were described (Morris et al. 2003a; Soderberg et al. 1991 ) . In many animal models of disease, treatment with omega-3 injection reduces the incidence of epileptic seizures, as well as displaying other anticonvulsive properties (Blondeau et al. 2002b; Lauritzen et al. 2000, Yehuda et al. 1994). Extension to initial small-scale studies in humans seems to suggest the same type of benefi cial effects, but this remains to be confi rmed by epidemiologic studies of larger cohorts of patients (Schlanger et al. 2002 ; Spirer et al. 1994 ; Yuen and Sander 2004 ; Yuen et al. 2005 ) . The more widely described and accepted benefi ts concern cardiovascular diseases, particularly coronary diseases. Omega-3 intake decreases levels of circulating trig- lycerides and the incidence of thrombosis associated with atherosclerosis, and is associated with antiarrhythmic properties (Dirnagl et al. 2003 , Leaf and Kang 1996; Nair et al. 1997; Nordoy 1999). Omega-3 PUFAs are involved in many physiological processes, such as the regulation of the plasmatic lipid rate (Harris 1997 ) and a variety of immune functions (Hwang 2000 ) , probably underlying why omega-3 has a signifi cant impact on health and chronic diseases like diabetes and obesity (Storlien et al. 1998 ) . However, in many domains, aspects of the consumption of omega-3, as well as detailed mechanisms of action, remain undefi ned and controversial. Much insight has been gained into the importance of EPA and DHA in neuro- and cardio- logical diseases, but very little is known regarding their precursor ALA.

6.4.4 Bene ﬁ cial Effects of EPA in Neurological Diseases

Although EPA is not a component of neuronal membranes, EPA acts as an eico- sanoid precursor and modulates cytokines level, and is involved in the regulation of 142 N. Blondeau and J.S. Tauskela

Table 6.1 EPA supplementation for the treatment of schizophrenia (add-on = added to existing medication) Diet Outcome References Schizophrenia EPA 2 g/day, add-on EPA > DHA = placebo Peet et al. (2001 ) EPA 2 g/day EPA > placebo Peet et al. (2001 ) EPA 1–4 g/day, add-on EPA > placebo Peet and Horrobin (2002a ) EPA 3 g/day, add-on EPA > placebo Emsley et al. (2002 ) EPA 3 g/day, add-on EPA = placebo Fenton et al. (2001 ) Depression DHA 2 g/day DHA = placebo Marangell et al. (2003 ) EPA 3 g/day, add-on EPA > placebo Nemets et al. (2002 ) EPA 1–4 g/day, add-on 1 g/day EPA > placebo Peet and Horrobin (2002a ) Fish oil 9.6 g/day, Fish oil > placebo Su et al. (2003 ) add-on EPA 1 or 2 g/day EPA > placebo Frangou et al. (2006 ) EPA 1,050 mg/day and Only a trend toward Lesperance et al. (2011 ) DHA 150 mg/day superiority of omega-3 over placebo Modi ﬁ ed from M. Peet and C. Stokes (2005 )

various cerebral functions (Fenton et al. 2000 ) . In approximate chronological order, effects of supplementation of omega-3, using fi sh oils or their derivatives as sources, were fi rst studied in schizophrenia, then in mood disorders and depression. Several studies showed that a diet enriched with EPA, administered either alone or combined with conventional medications, improves the symptoms (Table 6.1 ). One of the fi rst clinical studies on patients treated for schizophrenia reported that a diet which included 2 g/day EPA for 3 months, when combined with drug treatment, improved symptoms, with no benefi cial effect being reported for DHA alone or placebo (Peet and Horrobin 2002b ) . Further studies largely focused on EPA-enriched diets (Table 6.1 ). If preliminary results continue to be promising, larger studies will be necessary to establish the potential of EPA in the treatment of the schizophrenia. Three leading studies examining the effect of diet-enriched EPA on depression appear to have produced more homogenous results, showing additional bene fi cial effects compared to the effect of drug treatment alone (Nemets et al. 2002; Peet and Horrobin 2002a ) . Several studies showed that the combination of EPA/DHA also reduces the symptom of depression (Rocha Araujo et al. 2010 ) , although a recent major randomized controlled trial for major depression did not show any effi cacy of omega-3 supplementation (Lesperance et al. 2011 ) . EPA, but not DHA, appears to be responsible for the effi cacy of omega-3 long-chain PUFA supplementation in depression (Martins 2009 ) . Bene fi cial outcomes of EPA may derive from indirect effects, such as in eico- sanoid synthesis or by acting as an agonist on the nuclear receptors of peroxisome (peroxisome proliferator-activated receptors, PPARs) (Xu et al. 1999). Similar to DHA, EPA modi fi es the expression of certain genes; perhaps of most relevance to brain functions, EPA modi fi es genes involved in synaptic plasticity, cell cytoskeleton, and signal transmission (Barcelo-Coblijn et al. 2003; Kitajka et al. 2002). EPA 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 143 likely inhibits phospholipase A2 (which exhibits higher levels of activity in schizophrenia patients (for review, see Bennett and Horrobin 2000) ), an enzyme which is a major participant in phospholipid metabolism and arachidonic acid generation. In addition, schizophrenia is often associated with higher levels of proin fl ammatory cytokines (Muller and al. 2000; Gaughran 2002); inhibitors of cyclooxygenase-2 activity have shown benefi cial effects in this pathology (Muller and al. 2002), suggesting that EPA could act by its anti-in fl ammatory properties. In mood disorders, EPA appears to mimic protein kinase C inhibitors such as lithium or valproate, which are currently used for the treatment of these symptoms (Mirnikjoo et al. 2001; Seung Kim et al. 2001). Although the mechanisms of action remain still to be de fi ned, EPA seems pleiotropic in nature by stimulating endogenous pro-survival responses and genetic and cellular remodeling, features that have been strongly associated with preconditioning.

6.4.5 Importance of DHA in CNS Development and Maintenance

In vivo research has shown that omega-3 defi ciency leads to a decrease in DHA concentration in the brain and retina (Innis 1991; Okuyama et al. 1996; Tinoco 1982), resulting in marked functional consequences. It has been known since the 1970s that rats de fi cient in omega-3 exhibit abnormalities in electroretinograms (Wheeler et al. 1975) . Such rats display a decrease in motor, olfactory, and spatial performances such as in light discrimination and in learning and cognition (Frances et al. 1996; Greiner et al. 1999; Moriguchi et al. 2000; Nakashima et al. 1993; Spirer et al. 1994 ) . Similar results were observed in cats and primates, with visual acuity strongly impaired (Connor et al. 1984, 1991; Neuringer and Connor 1986). The same type of symptoms has been reported in humans. Brains of children fed with milk formulas de fi cient in omega-3 have lower levels of DHA in the brain, which is compensated for by an increase in docosapentaenoic acid (Farquharson et al. 1992; Jamieson et al. 1999; Makrides et al. 1994). A defi ciency of DHA in children’s diet depletes DHA at the level of the cerebral nervous system, causing functional declines in brain and retina. DHA is crucial for proper cerebral development, primarily from a fetal stage up to 5–6 years of age (Clandinin et al. 1980 ) . During fetal development, the mother furnishes the requirements for DHA. DHA supplementation of milk formula shows benefi cial effects in children, based on various functional parameters, such as vision and cognition (Helland et al. 2003; Hoffman et al. 2000; Williams et al. 2001). The lack of DHA also affects the elderly, since aging results in a decrease of DHA in neural membranes (Horrocks and Yeo 1999; Lauritzen et al. 2001). This DHA reduction was proposed to be correlated to memory losses and learning de fi cit in elderly. DHA declines could contribute to cholinergic malfunction of the hippocampus, a privileged zone of the brain responsible for memory (Favreliere et al. 2003; Jones et al. 1997). Cognitive de fi ciencies are also one of the major symptoms of Alzheimer disease, and, interestingly, the brains of patients who have this neurodegenerative disorder are severely de fi cient in DHA (Soderberg et al. 1991 ) . Therefore, many neurological disorders seem linked to de fi ciencies in DHA (Table 6.2 ). 144 N. Blondeau and J.S. Tauskela

Table 6.2 Relationship between the alterations in DHA levels and the bene fi cial effects of DHA supplementation in Alzheimer disease and neuropsychiatric disorders Modi fi cation Effect Of DHA of DHA levels supplementation References Alzheimer disease ⇓ Bene fi cial Morris et al. (2003 ) , Soderberg et al. (1991 ) Depression ⇓ Bene fi cial Hibbeln and Salem (1995 ) Schizophrenia ⇓ Bene fi cial Mahadik et al. (1996 ) Hyperactivity ⇓ Bene fi cial Burgess et al. (2000 ) , Stevens et al. (2003 ) Multiple sclerosis ⇓ Bene fi cial Nordvik et al. (2000 ) Ischemia ⇓ Bene fi cial Gamoh et al. (1999 ) , Okada et al. (1996 ) Modi fi ed from L. Horrocks and A. Farooqui (2004 )

6.5 Nutr ition As the Principal Cause of the Biochemical Imbalance Between Omega-6 and Omega-3

The human body is not able to synthesize the omega-6 and omega-3 PUFA precursors, linoleic acid (LA, C18:2 n-6) or a -linolenic acid (ALA, C18:3 n-3), respectively. These PUFAs – and their products (see Fig. 6.1 ) – are essential to humans, but humans do not possess the capability for their syntheses. The conversion rate to long-chain derivatives via sources other than LA or ALA is very slow, so to meet these demands, these PUFAs must be present in the diet (Burr 1981 ) . The chemical structures of omega-3 and omega-6 differ in the position of the fi rst double bond (Fig. 6.1 ). In the omega-3 family, the double bond is located at the third carbon from the methyl-terminal extremity of the PUFA and for the omega-6 at the sixth one. This is the basis for the “n-3 or w-3” designation for omega-3 (Holman 1964 ) . In mammals, LA and ALA serve as precursors for omega-6 and omega-3 products, respectively, and no transformation or functional substitution can occur between these two families (see Fig. 6.1 ). In contrast, LA can be metabolized to ALA in plants, serving as an important source for humans. The different metabolite products of LA and ALA are identifi ed as long-chain PUFA. LA metabolism leads to arachidonic acid (AA, C20:4 n-6) and docosapentaenoic acid (C22:6 n-6). ALA is successively transformed to eicosapentaenoic acid (EPA, C 20:5 n-3) which con- verts to docosahexaenoic acid (DHA, C22:6 n-3) (Klenk and Mohrhauer 1960 ; Marcel et al. 1968 ) . The same enzymes (elongase and desaturase) are involved in both omega-3 and omega-6 synthesis (Fig. 6.1 ). Thus, a competition exists, at the level of these enzymes, for the development of long-chain derivatives (Mohrhauer and Holman 1963; Rahm and Holman 1964). An important rami fi cation of this competition for enzymes is that omega-6 intake could negatively compromise EPA and DHA production. Recent epidemiologic studies have shown that intake of the w -3 PUFA family is well below the Dietary Reference Intake (or DRI, known formally as Reference 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 145

Fig. 6.1 Metabolism of omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) . The omega-6 family precursor, linolenic acid (LA), and the omega-3 precursor, a -Linolenic acid (ALA) is essential for humans. Humans cannot synthesize either of these PUFA precursors, but plants can process ALA from LA. Humans posses the enzymes to increase the degree of saturation (desaturases) and length of the carbon chain (elongases), allowing formation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from ALA (n-3), or arachidonic acid (AA) and docosapentaenoic acid (DA) from LA (n-6). In theory, these PUFAs are not essential, but the synthesis rate is so slow that most long-chain PUFAs are supplied by the diet, thereby satisfying their biological functions (basically making them essential). The enzymes that mediate omega-3 and omega-6 synthesis are identical, consequently generating a competition between the two families for the development of their metabolites

Daily Intake or Recommended Daily Intake, RDI) in the majority of the adult population in developed countries. The majority of individuals, ostensibly in good health, do not conform to the DRI concerning omega-3 fatty acids. As well, the average intake in LA is more than 10 times higher than for ALA. Consequently, the reported omega-6/omega-3 ratio is higher than 10, compared to an optimal ratio of approximately 5 (Vazquez Martinez et al. 1998; World Health Organization 1995). This elevated ratio represents strong evidence of an imbalance in the intake of these two families of essential fatty acids. Omega-3 PUFAs are not currently provided at adequate levels in many of the foods common to diet, raising concerns of omega-3 levels being too low to support protection or resistance against disease development. In industrialized countries, such as within Europe and the United States, the large availability and the low cost 146 N. Blondeau and J.S. Tauskela

Table 6.3 Recommendations for average population intakes of unsaturated fatty acids (% total energy) Omega-6 Omega-3 w -6/ w -3 Department of Health (1994) >1 >0.2 5 British Nutrition Foundation (1992) 3–10 0.5–2.5 4–6 Scienti fi c Committee for Food (1993) 2 0.5 4 FAO/World Health Organization (1998) 4–10 0.4–2 5–10 Modi fi ed from H. Roche (1999 ) of food sources containing omega-6 facilitate their consumption in excess. The imbalance between omega-3 and omega-6 intake is further augmented by the increasing prevalence of poor food habits since “junk food” snacks are rich in lipids and trans-fatty acids. Consumption of trans-fatty acids is now widely recognized as being detrimental to health, resulting in increases in body mass and in levels of low-density lipoprotein (LDL), a heightened risk of platelet aggregation, and reductions in levels of high-density lipoprotein (HDL). At least in vitro, trans-fatty acids inhibit delta-6-desaturase, an enzyme that plays a dominant role in omega-3 metabolism (Mahfouz 1981 ) . In modern times, our major food sources of omega-3 PUFAs have been exchanged for omega-6-rich and omega-3-poor sources. Animal products that are primarily derived from herbivores represent the main constituents of current human diet. Nowadays, herbivores are a weak source of omega-3 relative to omega-6 because of the “industrially controlled” diet of herbivores such as corn rich in omega-6, resulting in animals that are de fi cient in omega-3; compounding this problem is the lower preference of humans for fi sh and other sources high in omega-3. Foodstuffs richer in omega-3 are mainly sea products and vegetables, which contain a high content in ALA; examples include citrus fruits (lemon, orange, grapefruit), green vegetables (cabbage, watercress, spinach), seeds and their oils (sesame, linen, rape, nuts), and basil, soya, and olive oils. The fl esh of fi sh, particularly fatty fi sh of the cold seas such as salmon and sardine, are rich in EPA and DHA. Shell fi sh consume great quantities of phytoplankton and are therefore also very rich in ALA. Nevertheless, it is important to note that sources of ALA are also sources of omega-6, although in a weaker proportion, harmonizing the omega-6/omega-3 ratio toward the desired target ratio of 5:1. At present, >75% of the insuffi ciency in ALA intake appears attributable to consumption of meat and animal products since such practices cause a parallel rise in intake of omega-6 fatty acids. Thus, changes in the development of consumer habits and available sources of food over the past half century have contributed to insuf fi cient ALA intake and LA consumption far exceeding recommended levels, resulting in the imbalance in levels between omega-6 and omega-3 families. Many scientifi c or medical authorities, including the World Health Organization, have provided recommendations relating to omega-3 consumption (Table 6.3). However, a wide range of qualities and quantities are recommended, refl ecting different methods used to assess diet, as well as differing concepts of nutrition, within institutions in different countries. 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 147

The use of omega-3 in a therapeutic capacity may be quite advantageous when considered from the adverse event or “side effect” perspective, since often the effective dose/duration required by drugs or preconditioners approaches the threshold which can cause deleterious effects or toxicity. The potential toxicity of PUFAs has been evaluated, primarily within the Eskimo population, which consumes 15–20 g/day of omega-3. The consequences of chronic and massive omega-3 ingestion seem restricted to symptoms of nausea associated with a raw fi sh taste characteristic of the oxidized FA. Certain studies have raised the possibility of an increased delay of erythrocyte sedimentation when intake in EPA and DHA exceeds 9 g/day. Nevertheless, the risk of hemorrhagic events resulting from any ability to inhibit platelet aggregation remains rather theoretical. To our knowledge, no recommendations have been made for ALA intake, while DHA should not exceed more than 15 times the DRI (Martin 2001 ) . In the United States, the statute of Generally Recognized As Safe (GRAS) defi nes a maximal limit of 3 g/day of long-chain omega-3 in nutritional complements. Therefore, the interest in omega-3 as a neuroprotectant, potentially without encountering issues which raise safety concerns, suggests the possibility of more rapid translation to the clinic. On the one hand, such a scenario is bene fi cial if regulatory issues can be largely bypassed; indeed, there is currently a strong emphasis in exploring ways to circumvent this issue, such as in a recent evaluation of >1,000 FDA-approved compounds and natural products – which are currently approved for humans – as potential neuroprotectants (Li et al. 2011 ) . On the other hand, the fi nancial issues that are particularly acute with phase III clinical trials may persist since a “big” pharmaceutical partner may not be found for nutraceuticals (a similar problem exists in pursuing hypothermia as a neuroprotectant).

6.6 Omega-3 and Excitotoxicity, the Primary Driving Force of Damage in Stroke

Before considering the relevance of omega-3 to cerebral ischemia, it is useful to examine the effect of omega-3 on epileptic seizures and acute spinal cord injury (SCI), due to similarities in neurotoxic mechanisms. In stroke, epilepsy, and SCI – as well as many other acute and chronic neurodegenerative diseases – glutamate- mediated excitotoxicity is considered as a major cause of initial substantial neuronal damage. Moreover, as might be expected from this shared susceptibility, epileptic and ischemic preconditioning share common mechanisms of neuronal protection and the cross-tolerance phenomenon was initially described between ischemia and epilepsy (Plamondon et al. 1999 ) . In 1999, Plamandon et al. showed that sublethal epileptic seizures induced by kainic acid (KA) administration also promotes tolerance to global ischemia, and conversely ischemic preconditioning protects against neuronal damage induced by KA-induced seizures (cross-tolerance). The following year, omega-3 PUFAs were reported to provide strong neuroprotection in epilepsy models: ALA and, to a lesser extent, DHA prevented neurotoxicity in in vivo and in vitro model of seizure-like activity. A dose-response relationship was observed 148 N. Blondeau and J.S. Tauskela between ALA concentration and neuroprotection (Lauritzen et al. 2000 ) . Earlier work had shown that i.v. infusion of EPA or DHA, but not LA or oleic acid (OA, C18:n-9; a control FA displaying a similar degree of saturation), resulted in a modest anticonvulsant effect in rats (Voskuyl et al. 1998 ) . Post-insult acute or chronic treatment with ALA or DHA in animals subjected to SCI models ranging in severity provided improvements in function, neuronal/glial survival, and biochemical markers (reviewed in (Michael-Titus 2007 ) ). In hippocampal slices, application of EPA or DHA, but not the controls OA or stearic acid (SA, C18:0), raised the stimulatory thresholds (Xiao and Li 1999). DHA has been reported to suppress baseline neurotransmission and to interfere with LTD (Young et al. 2000), while others found no effect of DHA on LTP or LD; however, DHA or AA was required for the induction (but not maintenance) of LD in hippocampal CA1 neurons in rat brain slices (Fujita et al. 2001). In other electrophysiological investigations, intracerebroventricular injection of DHA, but not ALA, decreased the slope of fi eld excitatory postsynaptic potentials (fEPSPs) in the CA1 region and decreased the fEPSPs’ slope in the dentate gyrus; regional differences were also identi fi ed since DHA inhibited LTP in the CA1 but not the dentate gyrus (Itokazu et al. 2000). DHA inhibited neuronal excitability only at higher frequencies of long-duration depolarizing current pulses in hippocampal slices exposed to bicuculline (a GABAA receptor antagonist) or Mg 2+-free medium used to induce epileptiform activity (Young et al. 2000). Hence, the overall effect of DHA appears to be suppressive of neuronal excitability, thereby justifying continued investigation in epilepsy, SCI, and neurodegenerative diseases and probably other neurodegenerative diseases or insults with a strong excitotoxicity component. With regard to potential incorporation of natural brain preconditioners in the diet or functional foods, a diet of LA and ALA administered in a 4:1 ratio for 3 weeks (termed the “SR-3 compound”) at 200 mg/kg, but not 40 mg/kg (which represents an increase of only 1% in daily intake for rats), increased resistance to pentylenetetrazol-induced seizures (Taha et al. 2006, 2009), although others report increased resistance at the lower dose (Rabinovitz et al. 2004; Yehuda et al. 1994). Chronic intake of omega-3 fatty acids also promoted neuroprotection in the hippocampus of rats subjected to pilocarpine-induced status epilepsy (Ferrari et al. 2008). In contrast to this encouraging preclinical data suggesting a neuroprotective role for omega-3 in epilepsy, clinical trials with omega-3 dietary supplementation have been somewhat disappointing. However, concerns exist regarding limited patient enrolments and the duration and type of diet. The fi rst randomized clinical trial was performed in 2005 using EPA (1 g) and DHA (0.7 g) daily over 12 weeks, resulting in a 6-week transient decrease in seizure frequency (Yuen et al. 2005 ) . In another small randomized trial, dietary supplementation with EPA and DHA in a 3:2 ratio at 3.2 mg/day made no signi fi cant improvement (Brom fi eld et al. 2008 ) . Nonetheless, in a clinical trial which enrolled epileptic patients, a 6-month diet enriched in omega-3 PUFAs decreased the strength and frequency of epileptic seizures (Schlanger et al. 2002 ) . Further clinical trials are required with higher patient enrollment and with improvements in design based on knowledge gained in previous trials and preclinical studies. 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 149

The benefi cial effects of EPA and DHA in humans are promising, with the mechanisms of action seemingly pleiotropic in nature, but require further defi nition. In most of the cases where bene fi ts were observed, patients presented marked imbalances in omega-3 intake. Preconditioning can be achieved by long low-intensity (nonischemic) stimuli, so conceivably, if a preconditioner was naturally present in the diet, its de fi ciency may actually weaken the brain. This concept could be extended to vitamins (with the exception of vitamins D and PP, minerals, and certain amino acids) as well as some fatty acids (FA), which are characterized as being “essential” because their synthesis is not performed by the human organism and must therefore be provided by diet. Epidemiologic studies that identify nutritional defi ciencies in crucial elements may support the discovery of new preconditioners, the bene fi ts of which could be proven by exerting a protective effect due to supplementation.

6.7 Omega-3 “Rapid Preconditioning” as an Acute Treatment in an Experimental Model of Cerebral Ischemia and Stroke

During the acute phase of ischemic injury, glutamate excitotoxicity and hyperactivation of its receptors represent a major source of neurotoxicity within the core of an ischemic infarct and eventually in the surrounding penumbra. Most excitotoxic- mediated neurotoxicity occurs quite rapidly, within minutes to hours following onset cerebral ischemia, so therapeutics aiming to inhibit this acute phase must be administered very shortly after the ischemic insult. Excitotoxicity may represent the major mechanistic underpinning of why the success clot-buster reperfusion is also so highly time-dependent (“time lost is brain lost”). With regard to neuroprotection, the fi rst ischemic tolerance window – occurring within minutes to hours after the induction of “rapid preconditioning” – may be useful to counteract this acute temporal phase, even if the time constraints imposed by acute neuroprotection may be diffi cult (if not impossible) to achieve clinically. Nevertheless, it is important to test prospective drugs both as acute neuroprotectants and as rapid preconditioning agents, in order to estimate neuroprotective capacity. ALA and DHA provided acute neuroprotection in an in vivo transient model of global ischemia, a model for which neuronal death of CA1 hippocampal pyramidal cells is mainly driven by glutamate excitotoxicity (Pulsinelli and Brierley 1979 ) . Seven days after a 20-min global ischemia, only 15% of the CA1 neurons survived such an ischemic challenge, while the injection of ALA (i.c.v., 10 m M/5 m l) within 30 min post ischemia preserved 80% of the CA1 neurons. ALA was also protective when administered intravenously (i.v., 500 nmol/kg) 30 min before or after the ischemic challenge (Fig. 6.2a ). However, in this ischemia model, EPA and DHA had less pronounced and reproducible protective effects (Lauritzen et al. 2000 ) . Contrasting with these positive fi ndings, intraperitoneal injection of AA or DHA 1 h after transient ischemia aggravated injury (Yang et al. 2007). While recognizing that experimental optimization may yield more promising results, we nonetheless pursued 150 N. Blondeau and J.S. Tauskela

Fig. 6.2 Acutely administered omega-3 a -linolenic acid reduces ischemic stroke damage . (a ) Intravenous injection of ALA (i.v., 500 nmol/kg) 30 min prior to an ischemic challenge (20-min global ischemia) prevents glutamate excitotoxicity-driven CA1 hippocampal neuronal death. ( b ) Left panel: ALA is vasoactive, triggering a 30% release of the basilar artery diameter ex vivo. Right panel: ALA injection in vivo (administered i.v. at a neuroprotective dose of 500 nmol/kg) induces a 20% increase in the cerebral blood fl ow (CBF) within 30 min. ( c ) ALA signifi cantly reduces the infarct volume when injected up to 6 h after reperfusion post-MCAO. The maximal protection was achieved with a dose of 500 nmol/kg injected 2 h postischemia, with the window of successful intervention maintained for up to 6 h, but fading by 12 h after the onset of reperfusion further investigation using the omega-3 precursor rather than long-chain derivatives. To clearly evaluate if ALA could inhibit glutamate excitotoxicity, we tested its protective potential in an in vivo model of seizure induced by the administration of the glutamate analog kainic acid, as well as in an in vitro model of glutamate excitotoxicity. In vivo, using the same dosage paradigms as for ischemia, ALA was neuroprotective when injected i.v. either 30 min before or after KA treatment (Lauritzen et al. 2000) . In vitro, the same range of concentrations known to be protective both in vivo with i.c.v. injection and in vitro on granule cells prevented hippocampal neuronal death triggered by the addition of an excitotoxic concentration of glutamate (50 m M) for 24 h (Blondeau et al. 2009 ) . This was an important fi nding mechanistically since it demonstrated postsynaptic-mediated neuroprotection. In all these models a saturated fatty acid, palmitic acid, failed to induce any benefi cial effects, underlying the importance of the omega-3 polyunsaturated class of fatty acids. 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 151

Antiexcitotoxic activity by a drug is important but, using the failed clinical trials as a guide, may not be suf fi cient to achieve adequate neuroprotection. Rather, therapeutics able to target the entire neurovascular unit are likely more desirable (Barone 2009 ) , given the complex etiology of cerebral ischemia and the success demonstrated by timely restoration of blood fl ow. As previously discussed, the failures of therapeutics targeting only neurons and the hyperacute nature of glutamate release-driven neuronal death in ischemia lead the scientifi c community to identify drugs that could also help in restoring and/or preserving CBF (del Zoppo 2009 ) . Given their involvement as a cellular structural component and participation in cellular signaling, it is perhaps not surprising that bene fi ts provided by omega-3 against ischemia involve the neurovascular unit. The effect of omega-3 PUFA on the vascular tone and CBF was investigated as early as 1984, when it was shown that intravenous administration of EPA in gerbils improved CBF after ischemia and reperfusion, compared to LA-treated animals (Black et al. 1984 ) . The in vivo and in vitro neuroprotective 10 and 100 m M concentrations of ALA, respectively, increased the diameter of the basilar but not carotid artery (Blondeau et al. 2007 ) . In both mice and rats (Fig. 6.2b ), ALA acted as a vasoactive drug, leading to an approximately 30% increase of the diameter of the basilar artery ex vivo, which could account for a 20% increase of the in vivo CBF observed within 30 min postin- jection (i.v. neuroprotective dose of 500 nmol/kg). Since ALA did not dilate carotid arteries with elastic properties, the omega-3-induced relaxation appears to be speci fi c to cerebral resistance arteries such as those of the cerebral vascular bed, without affecting the systemic blood pressure. In post-insult treatment paradigms, treating rats by gavage on a daily basis for 4 weeks starting 1 day following ischemia had no effect on cerebral blood fl ow or glucose utilization in the ischemic core; however, in peri-infarcted areas (i.e., the frontal cortex and medial caudoputamen), CBF and glucose utilization were signifi cantly improved in the ethyl eicosapentate- treated group, compared to the saline-treated group (Katsumata et al. 1999). Taken together, the ability of ALA injections to provide resistance to glutamate excitotoxicity and a capacity to induce vasodilation of brain arteries in the penumbra – which in turn may increase collateral fl ow and help preserve CBF – potentially contribute to protection against ischemic stroke. Preclinical investigations into the utility of postischemic treatment of ALA, based on two clinically relevant parameters, are suggestive of clinical potential. The window of intervention to effi ciently reduce the infarct volume measured 24 h post- MCAO was characterized, and a delivery protocol was designed to improve the long-term survival rate. ALA signi fi cantly reduced the infarct volume when injected up to 6 h after reperfusion onset (Heurteaux et al. 2006 ) . Maximal protection was achieved using 500 nmol/kg injected 2 h post ischemia, with the window of intervention dissipated 12 h after reperfusion (Fig. 6.2 ). In contrast, the saturated palmitic fatty acid failed to reduce the infarct volume when injected 2 h after reperfusion. ALA-induced neuroprotection correlated with a decrease in cytopathological features of cell injury, DNA fragmentation, and proapoptotic Bax protein upregulation. For all parameters used as measures of neuroprotection, the natural omega-3 precursor provided values similar to riluzole, a drug currently in clinical use for amyotrophic 152 N. Blondeau and J.S. Tauskela lateral sclerosis. Interestingly, a single ALA injection that provides the best cerebral protection when measured 24 h post stroke did not display any benefi cial effect on the long-term survival rate. To achieve a threefold improvement in the survival rate at 10 days and 1 month post ischemia, repeated injections were required (Blondeau et al. 2009 ; Heurteaux et al. 2006 ) . This data suggested that the reduction of glutamate excitotoxicity within the acute phase after stroke was insuffi cient to reduce long-term mortality rates. Indeed, excitotoxicity suppression may instead only delay neuronal death, by failing to prevent later stages of cell death including apoptosis and associated infl ammatory events, events known to cause progressive tissue damage hours to days or weeks later. The benefi cial effects caused by repeated injections spaced several days apart implied multiple temporal and mechanistic benefi ts provided by ALA. A growing body of preclinical data suggests that pharmacological therapies able to enhance brain-repair processes substantially improve functional recovery when delivery is delayed until the later recovery stage of stroke. This represents a paradigm shift from focusing solely on neuroprotection to facilitating spontaneous recovery of function following stroke (Barone 2010 ) . The concept underlying these restorative therapies is the pleiotropic targeting of many parenchymal cell types including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, and neurons, leading to enhancement of neurotrophic factor production, endogenous neurogenesis, angiogenesis, and synaptogenesis in ischemic brain tissue (Zhang and Chopp 2009) . These events collectively improve outcome after stroke, including the all-important neurological function. Subchronic ALA administration provides long-term benefi ts on neuronal plasticity. ALA increased neurogenesis following three sequential injections using the same time course and dose that improved long-term survival rate post stroke (Blondeau et al. 2009 ) . Subchronic ALA treatment signi fi cantly increased the number of proliferating immature neurons, as identi fi ed by the colocalization of incorporated BrdU, a DNA synthesis marker, in dividing progenitor cells and DCX, a microtubule-associated protein specifi cally expressed in all migrating neuronal precursors. These immature neurons identifi ed 3 days after the last injection survived and matured by 21 days after the fi nal ALA injection (Fig. 6.3a ), suggesting that the repeated ALA injections triggered the induction of neurogenesis. Upregulation of key proteins involved in synaptic functions – synaptophysin-1, VAMP-2, and SNAP-25 – as well as proteins supporting glutamatergic neurotransmission – V-GLUT1 and V-GLUT2 – also indicated that ALA subchronic treatment promoted synaptogenesis (Fig. 6.3b). These changes correlated with an increase in BDNF protein levels, both in vivo, following subchronic ALA treatment, as well as in vitro using neural stem cells and hippocampal cultures (Blondeau et al. 2009 ) . Altogether these results imply a pleiotropic effect of ALA in protecting the brain from stroke, due to a capability to target multiple cell types, and by providing combined acute neuroprotection, CBF regulation, and long-term repair/ compensatory plasticity. This pleiotropism parallels the wide-ranging abilities of brain preconditioning to provide combined neuroprotective, regenerative, and anti-in fl ammatory effects. 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 153

Fig. 6.3 Omega-3 a -linolenic acid increases neuroplasticity . ( a ) Subchronic ALA treatment signifi cantly increases cell proliferation in the dentate gyrus 3 and 21 days later (left panel ). The proliferating cells were identifi ed as immature neurons (middle panel; using BrdU and DCX) 3 days following the fi nal ALA injection but were mature neurons (right panel ; using BRdU and NeuN) 21 days later. (b ) Subchronic ALA treatment signifi cantly increases the levels of different markers of synaptogenesis (synaptophysin, VAMP-2, SNAP-25, VGLUT-1, and VGLUT-2), measure in cortex and hippocampus at day 10. The inset images were immunostained for SNAP- 25, a synaptosome-associated exocytosis protein, in the cortex of ALA-treated animals at day 10, showing the increased in synaptogenesis by ALA treatment

6.8 Omega-3 a -Linolenic Acid as a Complete Preconditioner in Experimental Models of Cerebral Ischemia and Stroke

ALA has been convincingly demonstrated to be a “natural” preconditioner able to induce delayed cerebral ischemic tolerance: ALA preconditioning triggered the late phase of tolerance against two models of excitotoxicity-driven neuronal death 154 N. Blondeau and J.S. Tauskela induced by global ischemia and kainic acid injection. ALA injection (i.v., 500 nmol/ kg) 3 days before 6-min global ischemia or kainic acid-induced epileptic seizure almost fully prevented the CA1 neurodegeneration (Blondeau et al. 2002b ) . This data suggests that ALA acts as a nonischemic preconditioner capable of inducing delayed cross-tolerance to ischemia, which is usually de fi ned as a nonlethal insult inducing tolerance to an otherwise lethal, but dissimilar, insult (Tauskela et al. 2004 ) . The narrow temporal window of cerebral protection conferred by ALA preconditioning paralleled fi ndings observed using preconditioning by ischemia or epileptic activity. Similarities in signal transduction pathways activated with ischemic preconditioning and other well-established chemical preconditioners con fi rmed that ALA may be studied as a new natural preconditioner of the brain, as discussed in a subsequent section. ALA preconditioning induced the neuroprotective HSP70 heat shock protein within a similar time frame and neuronal localization shared by ischemic, epileptic preconditioning, and adenosine and K ATP channel opener preconditioning (Blondeau et al. 2000, 2002b) . In addition, preconditioning by ALA, ischemic, and kainic acid-induced epileptiform activity rapidly increased the expression of the transcription factor nuclear factor-kB. NFkB is a ubiquitously expressed inducible regulator of a broad range of genes that play pivotal roles in cell death and survival pathways. The three different preconditioning paradigms increased NFkB DNA-binding activity and nuclear translocation of p65 and p50 subunits of NFkB in similar manners and cellular compartmentalization (Blondeau et al. 2001 ) . Pretreatment with the NFkB inhibitor diethyldithiocarbamate or kB- decoy DNA suppressed the increased DNA-binding activity and the nuclear translocation of NFkB, leading to the loss of the preconditioning-induced neuroprotection against an otherwise lethal ischemic or epileptic challenge. Thus, the signaling pathways established by ALA preconditioning that are required for the development of brain tolerance match with other preconditioners. ALA preconditioning was also demonstrated in mice since ALA injection 3 days before a transient focal ischemia signi fi cantly reduced the infarct volume (Blondeau et al. 2009 ) . The similarities in the narrow window of cerebral protection, as well as overlap in signal transduction pathways, activated by ischemia and other well-established chemical preconditioners, clearly establish ALA as a compound that can trigger the ubiquitous pleiotropic protective signaling pathways important to brain preconditioning. In vitro ischemia data suggest the possibility of preconditioning by omega-3 fatty acids at the neuronal level. A 24-h preincubation, but not coincubation, with DHA in hippocampal slices induced tolerance to OGD (Strokin et al. 2006). In hippocampal neuron cultures, a 24-h preincubation combined with a 24-h coincubation of DHA with glutamate prevented a glutamate-induced increase in Ca 2+ in fl ux and neurotoxicity (Wang et al. 2003 ) . In organotypic hippocampal slice cultures, a 24-h preincubation, followed by a 24-h coincubation of DHA, but not with EPA or AA, resulted in protection of CA1 neurons against AMPA, but not NMDA or kainate, which was attributed to a decrease in plasma membrane AMPA receptors (Menard et al. 2009). 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 155

In addition to employing similar effectors of protection, omega-3 preconditioning could also use the same triggers used by other forms of preconditioning. Emerging results suggest that n-3 PUFAs might exert their neuroprotective effects in part through direct modulation of activity of channels such as K+ channels. More than 77 genes encoding K+ channels have been identi ﬁ ed in the human genome. A newly + + discovered family of K channels is tandem pore domain K channels (K 2 P), also called background K+ channels, which possess four transmembrane-spanning domains and two pore regions for each protein subunit. K2 P channels are important in tuning the neuronal resting membrane potential, action potential duration, and membrane input resistance and consequently in the regulation of transmitter release.

K 2P channels can be regulated by membrane-receptor-coupled second messengers and pharmacological agents (reviewed by Lesage and Lazdunski 2000 ) . The class of mammalian K2P channel subunits now comprises 17 members, one of which is the TREK-1 channel (Fink et al. 1998). Regulation of this channel may be particularly relevant to neuronal disease states. Mechano-gated and arachidonic acid-activated TREK-1 channels are highly expressed in the brain, at both pre- and postsynaptic locations (Kim et al. 1995; Lauritzen et al. 2000 ) , and are regarded as signal integra- tors capable of responding to a wide range of physiological and pathological inputs. These channels can be activated by physical stimuli such as membrane stretching, depolarization, intracellular acidosis, and warm temperature. Recent studies indicate a central role for TREK-1 in diverse CNS settings, such as in general anesthesia, pain perception, depression, and neuroprotection (Alloui et al. 2006; Heurteaux and Blondeau 2005; Heurteaux et al. 2004, 2006 ) . TREK-1 KO mice develop seizures of progressive severity when subjected to transient bilateral occlusion of common carotid arteries (CCA) concomitant with systemic hypotension (mean arterial blood pressure, MABP 30 ± 3 mmHg), leading to a 40% increase in the number of deaths for Trek1−/− mice compared to the Trek1+/+ mice. Trek1−/− mice were also much more sensitive to epileptic seizures induced by kainate (a glutamate agonist; 22 mg/ kg) or pentylenetetrazole (a GABA antagonist; 40–55 mg/kg) (Heurteaux et al. 2004). Activation of c-fos, routinely used as a biochemical marker of neuronal excitability, was drastically enhanced in Trek1−/− mice compared to Trek1+/+ mice, particularly in the CA3 subﬁ eld, 120 min after kainate injection. Interestingly, the protection afforded by omega-3 PUFA pretreatment against cerebral ischemia and epileptic seizure is eliminated in TREK-1 KO animals, suggesting a strong link between omega-3, TREK-1 channels, and acute neuroprotection against cerebral ischemia and epilepsy, insults dominated by glutamatergic excitotoxicity. Several lines of evidence are also consistent with involvement of TREK-1 channels in preconditioning, at least in vivo. Palmitic acid, which does not activate TREK-1 channels, fails to induce rapid and delayed preconditioning protection of the brain against both epilepsy and ischemia (Blondeau et al. 2001, 2002b ; Lauritzen et al. 2000 ) . TREK-1 activity is known to be upregulated or activated by lysophospholipids, omega-3 fatty acids such as ALA, volatile anesthetics, and riluzole, which are also able to induce ischemic tolerance (Blondeau et al. 2002a, b ; Heurteaux et al. 2006 ; Honore 2007; Lang-Lazdunski et al. 2003; Lesage and Lazdunski 2000 ) . 156 N. Blondeau and J.S. Tauskela

6.9 Omega-3 Preconditioning by Chronic Administration

In view of the strong link between preconditioning and omega-3 fatty acids, improved outcomes after brain injury by stroke in vivo suggest that chronic pretreatment administration of omega-3 fatty acids may occur as the result of preconditioning. Gerbils fed a diet supplemented with menhaden fi sh oil (which contains 17% EPA) for 2 months after weaning displayed a reduction in postischemic cerebral edema and hypoperfusion (Wang et al. 2003 ) . Daily oral administration of ethyl DHA in gerbils for 4–10 weeks suppressed neuronal injury due to subsequent transient ischemia (Cao et al. 2004, 2005, 2006, 2007), and provision of a fi sh-oil- supplemented diet for 6 weeks in rats prior to ischemia reduced infarct volumes by ~30% (Choi-Kwon et al. 2004). Rats fed a diet with a decreased omega-6/omega-3 ratio immediately after birth, and then subjected to 2-vessel occlusion at 3 months, displayed improved BBB function and behavior measured at 7 months (de Wilde et al. 2002). Rats fed a diet supplemented with fi sh n-3 fatty acids for 2 weeks prior to global ischemia displayed decreased apoptotic death and improved biochemical and histopathological parameters (Bas et al. 2007 ; Ozen et al. 2008 ) . In contrast, fi sh-oil-supplemented diets (2 weeks) prior to ischemia resulted in an improvement in spatial memory de fi cits despite no improvement in surviving CA1 hippocampal neurons (Plamondon and Roberge 2008 ) . Overall, neuronal protection obtained with long-chain omega-3 by both parenteral and enteral supplementation seems quite positive, while the overall properties exerted by ALA seems to ful fi ll criteria necessary to rank this omega-3 PUFA as a good neuroprotectant, or at least as a crucial molecule for brain resistance.

6.10 From Omega-3 a -Linolenic Acid Preconditioning Toward Nutraceuticals: A Natural Preconditioner in the Diet to Prevent Stroke-Induced Damage

ALA is a fatty acid characterized as “essential” because its synthesis is not performed by the human organism and must therefore be provided by diet. This could explain in part why the severe de ﬁ ciency in omega-3 intake that has been reported in numerous epidemiologic studies may weaken the brain, representing an important risk factor in the development and/or deterioration of certain neuropathologies and neurovascular diseases, like stroke. In the context of stroke treatment, the rationale of supplementation with a nonischemic preconditioner like ALA has seemed promising to investigate, especially since no detrimental effects of ALA have been documented as far as we are aware. An unobtrusive method of increasing ALA intake would be to increase levels in the daily diet. We addressed this important hypothesis by investigating whether ALA supplementation achieved using a diet enriched in rapeseed oil, a rich source of ALA as the only source of lipids, decreased infarct volumes in stroked mice. A 6-week diet enriched with ALA ( 0.75% by weight) signi ﬁ cantly 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 157

Fig. 6.4 ALA-enriched diet decreases the infarct volume induced by 60-min MCAO. The upper panel summarizes the protocol followed for a 6-week diet of regular or supplemented with rapeseed in rodents, as well as the ALA composition and how this affected the w -6/w -3 ratio. The bottom panel shows that the cerebroprotection occurs mainly at the cortical level and not subcortical regions reduced mortality rate and infarct size induced by 60 min of middle carotid artery occlusion and increased the probability of spontaneous reperfusion in the postischemic period (Nguemeni et al. 2010 ) . Figure 6.4 shows that the ALA-enriched diet decreased the total infarct volume by 35–45% (total infarct, cortical infarct). This reduction of the focal ischemic lesion matched that observed in rats supplemented with high dietary levels of long-chain omega-3 (1.75% by weight) over 6 weeks (Relton et al. 1993 ) . This result implies that lower levels of supplementation may be required when using precursors (ALA, 0.75% by weight over a 6-week diet) compared to long-chain derivatives. In addition, supplementation of 0.4 g/kg/day of EPA/DHA during 14 days before surgery may reduce apoptosis of hippocampal neurons in ischemic rat (Bas et al. 2007 ; Ozen et al. 2008 ) but fails to prevent ischemia-induced hippocampal damage despite improving memory and behavioral outcome, when given once daily 3 days before ischemia and continued until day 41 (Fernandes et al. 2008 ) . Besides supplementation by force-feeding, fi sh oils incorporated to the diet have also been tested. Again, a positive effect on memory recovery was induced by a fi sh-oil-enriched diet (11.5% w/w fi sh oil from menhaden fi sh and 3.5% corn oil to standard rat chow), while a 17-week diet (13 pre- and 4 postischemia) had no effect on hippocampal damage in rats subjected to transient global ischemia (Plamondon and Roberge 2008 ) . This study suggests that the 158 N. Blondeau and J.S. Tauskela absence of neuroprotection by the long-chain omega-3 cannot be attributed to the length of the diet duration, nor to the quantity provided in the diet. Since the neuronal protection obtained with supplementation with long-chain omega-3 appears inconsistent at the present time, ALA provided by vegetable rapeseed oil should be considered as an interesting alternative to long-chain omega-3 derivate from fi sh oil. Finally, a drastic reduction of lipid peroxidation levels was also observed in the ischemic brain of animals fed the ALA-enriched diet, suggesting that benefi cial effects of ALA have not yet been exhaustively categorized (Nguemeni et al. 2010 ) .

6.11 Conclusion

In this chapter, we present the novel concept of nutraceutical preconditioning, one which largely circumvents the administration and timing issues which thus far has plagued the preconditioning and neuroprotectant fi elds. We propose that certain nutraceuticals can act as “natural preconditioners” to increase brain resistance against devastating insults such as stroke. We have highlighted how preconditioning studies led the group of Blondeau to discover that a natural molecule, a -linolenic acid ALA, an omega-3 polyunsaturated fatty acid precursor, protects the brain from in vivo and in vitro models of stroke (Blondeau 2011 ) . Overall, ALA emerged as the “best-in-class” natural preconditioner therapeutic; although recommendations made by the Stroke Therapy Academic Industry Roundtable (STAIR) do not directly address drug discovery, the best chance for success in following the STAIR protocol is to move forward with an optimum candidate. The surprising pleiotropic properties of ALA to trigger responses that are multicellular, mechanistically diverse, and with a wide temporal range mirror those responses typically elicited by preconditioning, resulting in neuronal protection and brain artery vasodilation and neuroplasticity stimulation. The potential of ALA as preconditioner supports a novel and extremely relevant concept in the context of nutraceutical and functional food development against stroke. The fi eld of brain preconditioning is more than 2 decades old, yet little progress has been made in issues faced by more conventional neuroprotectants, issues that must ultimately be confronted if preconditioning is ever to enter the clinical arena. Indeed, ALA supplementation achieved through modi fi cation of the daily diet prevented MCAO-induced mortality and cerebral damage, essentially evading the problem of delivery to the brain, which has normally to be addressed for chemical drugs. If omega-3 FAs such as ALA are taken prophylactically in the diet, as is being increasingly recommended to prevent/diminish various affl ictions, the possibility of concomitant preconditioning occurring circumvents what is probably the major issue in the fi eld, which is timing. Currently – and probably for the foreseeable future – the notion of preconditioning is contemplated only for patients at imminent risk of cerebral infarction (i.e., surgery in the brain). Ultimately, the future of preconditioning may largely depend upon its successful translation not only to the clinical arena but also to daily life. This novel concept of nutraceutical preconditioning is not 6 A New Future in Brain Preconditioning Based on Nutraceuticals... 159 restricted to omega-3 PUFAs such as ALA but may in fact extend to other existing or novel nutraceuticals (in fact, it is hoped that this is the case). Different classes of nutraceuticals contain molecules which, implemented in the laboratory setting, are implicated in preconditioning (as well as by other neuroprotective mechanisms which may include antioxidant capability) in in vivo or in vitro models of cerebral ischemia including epigallocatechin 3-gallate (green tea), resveratrol (red grapes), quercetin (apples), the organosulfur compounds allicin (garlic) and l -sulforaphane (broccoli), phenolic acids such as rosmarinic and carnosic acid (rosemary), and gin- seng (Kelsey et al. 2010 ; Wang et al. 2008 ) . Determination of whether compounds such as these actually function as preconditioners in a nutraceutical capacity will be required, and further mechanistic enquiry is urgent for any nutraceutical being considered as a preconditioner or even as neuroprotectant. Development of “functional” foods by integrating knowledge drawn from preconditioning research may represent an important avenue to achieve this goal.

Acknowledgments The authors are grateful to G.L.N, ONIDOL, the “Fondation de la Recherche Médicale,” and CNRS for their support. Nicolas Blondeau is also grateful to Pr Michel Lazdunski and Dr Catherine Heurteaux for providing the opportunity and their continued support since 1997 to work on omega-3 PUFAs and brain protection. N. Blondeau also wishes to thank Pr Bernadette Delplanque for many helpful discussions. Finally, we thank all our past and present team members who have contributed to the data discussed in this chapter.

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Zhiyi Zuo

7.1 Introduction

Ischemic preconditioning has drawn a signi fi cant attention from the scienti fi c community since it was introduced in 1986 (Murry et al. 1986 ; Dirnagl et al. 2003 ) . Part of the reasons for drawing a signi fi cant attention is that ischemic preconditioning has been considered to induce endogenous mechanisms to protect cells. Thus, research on ischemic preconditioning can identify novel targets and design interventions for organ and cell protection. There are two temporal phases for ischemic preconditioning-induced protection. The differentiation of phases is based on when the detrimental insult occurs after the preconditioning stimuli. The acute phase starts a few minutes after the preconditioning stimuli and lasts for a few hours. The mechanisms for this phase of protection generally involve modi fi cations of existing proteins. The delayed phase begins a few hours after the preconditioning stimuli and can persist for a few days or longer. This phase of protection is mediated usually by synthesis of protective proteins (Dirnagl et al. 2003 ; Barone et al. 1998 ) . Ischemic preconditioning involves application of short episodes of ischemia prior to a detrimental insult. This form of protection induction may be applicable under certain clinical situations, such as during cardiovascular surgeries or procedures. However, application of this method carries risks because of the nature of the preconditioning stimuli. Its application may not be practical in many organs including brain and spinal cord because of the diffi culty to apply well-controlled short episodes of ischemia to those organs. Subsequent studies show that many stimuli, in addition to ischemia, also can induce the preconditioning phenomenon (Gidday 2006 ) . Among them are medical gases including volatile anesthetics and hyperbaric

Z. Zuo (*) Departments of Anesthesiology, Neuroscience, Neurological Surgery , University of Virginia , 1 Hospital Drive , PO Box 800710, Charlottesville , VA 22908-0710 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 165 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_7, © Springer Science+Business Media New York 2013 166 Z. Zuo oxygen (HBO) (Zheng and Zuo 2004 ; Ostrowski et al. 2008 ) . These gases are relatively safe and have been used in clinical practice for many years. They usually readily cross the blood-brain barrier and, therefore, can be applied systemically to induce neuroprotection.

7.2 Volatile Anesthetics

7.2.1 General Description

Volatile anesthetics are the most commonly used general anesthetics in clinical practice. It is estimated that ~80% patients who have surgeries under general anesthesia will receive volatile anesthetics as their primary anesthetics (Clergue et al. 1999 ) . This is because these anesthetics are easy to use and make patients unconscious, unaware, insensate, amnesic, and muscle relaxed, effects that are often required for general anesthesia. Many volatile anesthetics have been used in the anesthesia practice over the years since 1842. Newer and better anesthetics are developed to replace the previous ones. Currently, three volatile anesthetics are commonly used in the anesthesia practice in the USA: isoﬂ urane (CHF 2 ÐOÐ

CHClÐCF3 ), sevo ﬂ urane (CH 2 FÐOÐCHÐ(CF3 )2 ), and des ﬂ urane (CHF2 ÐOÐ

CHFÐCF3 ). Halothane (CF3 ÐCHBrCl) was introduced into clinical practice in 1956 and was phased out in late 1990s in the USA. In addition to volatile anesthetics, two gas anesthetics are worth mentioning in the context of preconditioning. One is xenon (Xe), a noble gas. Although it is perceived as a good anesthetic, its wide use as an anesthetic in clinical practice may not be possible due to the limited resource and the signi ﬁ cant cost to produce it.

The other gas anesthetic is nitrous oxide (N2 O). Nitrous oxide was ﬁ rst used as an anesthetic/analgesic in 1835 and has been used since then in the clinical practice. Thus, nitrous oxide may be one of the longest used drugs in modern medicine.

7.2.2 Iso ﬂ urane

Most studies on anesthetic preconditioning-induced neuroprotection involve using iso fl urane. All in vivo studies published so far on anesthetic preconditioning in the brain are performed in rodents. Two studies have shown isofl urane preconditioning effects in the spinal cord of rabbits (Park et al. 2005 ; Sang et al. 2006 ) . One study showed neuroprotective effects of iso fl urane pretreatment in a global brain ischemia model of dogs (Blanck et al. 2000 ) . Since there was no interval between the application of iso fl urane and the onset of brain ischemia, it is dif fi cult to know whether the neuroprotection shown in the study is purely from the preconditioning effect. 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen... 167

7.2.2.1 Iso ﬂ urane Preconditioning-Induced Neuroprotection

Very few studies have examined the acute phase of iso fl urane preconditioning effects in the brain. An initial study using rat cerebral slices showed that isofl urane preconditioning induced an acute phase of protection against a -amino-3-hydroxy- 5-methyl-4-isoxazol propionic acid-induced neurotoxicity (Li et al. 2002 ) . A subsequent study showed that isofl urane applied 15 min before oxygen-glucose deprivation (OGD) induced an iso fl urane dose- and exposure duration-dependent protection in rat cerebral slices. This protection was maximized with the exposure to 2% isofl urane for 20 min (Zheng and Zuo 2003 ) . Interestingly, the potency of currently used volatile anesthetics including isofl urane to induce this preconditioning effect correlates well with the potency of these anesthetics to induce anesthesia. In fact, the EC50 for these two effects are very similar (Wang et al. 2007a ) . This concentration for iso fl urane is ~1.2% (Zheng and Zuo 2003 ; Wang et al. 2007a ) . Of note, the EC50 for volatile anesthetics to induce anesthesia/analgesia is called one minimum alveolar concentration (MAC) that is de fi ned to be the alveolar concentration at which 50% subjects will not move in response to painful stimulation. The iso fl urane preconditioning-induced acute phase of neuroprotection also has been shown in the adult rats after focal brain ischemia (Liu et al. 2006 ) and neonatal rats after hypoxic- ischemic (HI) insult (Sasaoka et al. 2009 ) . However, it is suggested that this acute phase of neuroprotection may not improve the long-term neurological outcome after HI insult in the neonatal rats (Sasaoka et al. 2009 ) . Numerous studies have shown that isofl urane preconditioning induces a delayed phase of neuroprotection. This protection has been shown in neuronal cultures and hippocampal slice cultures of rodents (Kapinya et al. 2002a ; Bickler et al. 2005 ; McMurtrey and Zuo 2010 ) . However, detailed dose-response and time-course studies that allow calculation of the EC50 and optimal exposure duration have not been performed yet under in vitro or in vivo conditions. The neuroprotection has been shown consistently in both rats and mice when the interval between iso fl urane exposure and brain ischemia is 24 h or shorter and the neuroprotection disappears if the interval is 48 h (Kapinya et al. 2002a, b ) . Thus, most of in vivo studies use 24 h as the interval. One exposure to 1Ð2% isofl urane for 30 min to 4 h is commonly used to precondition the animals, although multiple exposures to iso fl urane for 5 consecutive days also have been used (Kitano et al. 2007a ) . The neuroprotection has been observed in adult rodents after permanent or transient focal brain ischemia, global brain ischemia, or intracerebral hemorrhage, and neonatal rats after HI insult (Zheng and Zuo 2004 ; Kapinya et al. 2002a, b; Kitano et al. 2007a ; Gigante et al. 2011; Xiong et al. 2003; Zhang et al. 2010; Zhao and Zuo 2004 ) . This neuroprotection also is iso fl urane dose-dependent in both adult and neonatal brains of rodents (Zhao and Zuo 2004; Xiong et al. 2002; Nasu et al. 2006) . In addition, this neuroprotection translates into long-term neurological outcome improvement (Zheng and Zuo 2004; Zhao et al. 2007 ; Li and Zuo 2009) . Of note, the delayed phase of iso fl urane preconditioning-induced neuroprotection has been shown in the human neuroblastoma SH-SY5y cells (Zuo et al. 2006 ) , suggesting that iso fl urane preconditioning-induced neuroprotection may not just occur in rodents. 168 Z. Zuo

Although iso fl urane preconditioning-induced delayed phase of brain protection is shown in the neonatal rats of both genders (Zhao and Zuo 2004 ; Zhao et al. 2007 ) , this neuroprotection is easily induced in adult male rodents but may not be induced in young and middle-aged female mice after focal brain ischemia (Kitano et al. 2007b) . These fi ndings suggest a gender difference in this isofl urane preconditioning effect. Consistent with this suggestion, the iso fl urane preconditioning effects are shown to be androgen-dependent in male mice (Zhu et al. 2010 ) and attenuated by estradiol in ovariectomized female mice (Wang et al. 2008a ) . In addition to the brain, isofl urane preconditioning-induced acute and delayed phases of protection have been shown in the spinal cord of rabbits (Park et al. 2005 ; Sang et al. 2006 ) . The acute phase of this protection is iso fl urane concentration- dependent (Park et al. 2005) . It is yet to determine the dose-dependency in the delayed phase of the protection. Rabbits exposed to 1 MAC isofl urane for 40 min each day for 5 consecutive days have improved neurological and histopathological outcome after a 20-min spinal cord ischemia if the interval between the last exposure to isofl urane and spinal cord ischemia is 24 or 48 h but not 72 h (Sang et al. 2006) . This fi nding suggests that the effective time window for this isofl urane effect is within 48 h after the preconditioning stimuli.

7.2.2.2 Possible Mechanisms for Iso ﬂ urane Preconditioning-Induced Neuroprotection

Iso fl urane preconditioning-induced acute phase of protection in rat cerebral slices is inhibited by glutamate transporter inhibitors, suggesting a role of glutamate transporters in this effect (Zheng and Zuo 2003; Wang et al. 2008b) . One of the major functions of glutamate transporters is to transport glutamate from extracellular space to intracellular compartments (Danbolt 2001 ) . The events downstream of glutamate transporters, such as maintaining extracellular glutamate homeostasis, to mediate the iso fl urane preconditioning effects have not been determined yet. The isofl urane preconditioning effects also may involve protein kinase C (PKC) and nitric oxide synthase (NOS) (Zheng and Zuo 2005 ) , important intracellular signaling molecules. While the ATP-sensitive potassium (KATP ) channels may not play a role in this iso fl urane preconditioning effect in rat cerebral slices (Zheng and Zuo

2003 ) , KATP channels may mediate isofl urane preconditioning-induced acute phase of protection in the rabbit spinal cord (Park et al. 2005 ) . Also, the role of adenosine A1 receptor is implicated in the iso fl urane preconditioning-induced acute phase of protection in rats after a transient focal brain ischemia (Liu et al. 2006 ) but not in rat cerebral slices stimulated by glutamate (Zheng and Zuo 2005 ) . These results suggest that different molecular mechanisms may be involved in the iso fl urane preconditioning effects in different neural tissues suffering from different detrimental insults. Since the potency for volatile anesthetics to induce a preconditioning effect and anesthesia is correlated, it is suggested that similar mechanisms may be involved in these two effects caused by volatile anesthetics (Wang et al. 2007a ) . Multiple molecules have been indicated to contribute to iso fl urane preconditioning-induced delayed phase of protection. The list of molecules includes signaling 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen... 169 molecules, such as free radicals (Sang et al. 2006 ) , intracellular Ca ++ (Bickler et al. 2005) , calcium/calmodulin-dependent protein kinase II (CaMKII) (McMurtrey and Zuo 2010 ) , mitogen-activated protein (MAP) kinase (Zheng and Zuo 2004 ) , protein kinase B/Akt (Bickler et al. 2005 ) , hypoxia-inducible factor (HIF)-1a (Li et al. 2008a ) , and inducible NOS (Kapinya et al. 2002a ; Zhao and Zuo 2004 ) . These signaling molecules often are called transducers because they transduce the signals from the triggers to the effectors in the signaling transduction for the preconditioning effects (Dirnagl et al. 2003 ) . KATP channels can be considered as a trigger (regulating free radical production), transducer (participating in signaling transduction by being sensitive to regulation induced by signaling molecules, such as PKC), and effector (attenuating ischemia-induced calcium accumulation in the mitochondria and facilitating energy production in the mitochondria after ischemia) (Dirnagl et al.

2003; O’Rourke 2004 ) . The role of KATP channels in isofl urane preconditioning- induced delayed phase of neuroprotection has been suggested (Xiong et al. 2003 ; Kaneko et al. 2005 ) . A series of studies performed by Zuo’s group have established the role of B-cell lymphoma-2 (Bcl-2), a protective protein, in the isofl urane preconditioning-induced delayed phase of neuroprotection in rats (Zhao et al. 2007 ; Li and Zuo 2009 ; Zuo et al. 2006 ; Li et al. 2008b ; Gwak et al. 2011 ) . Bcl-2 should be an effector for this preconditioning effect. A recent study has added metallothioneins I/II as a potential effector for this effect in mouse neuronal-glial cocultures (Edmands and Hall 2009 ) . Metallothioneins are a family of proteins with low molecular weights. These molecules can bind heavy metals and protect against oxidative damage. In human SH-SY5Y cells, the extracellular signal-regulated kinase (ERK, one type of MAP kinase)-early growth response gene 1 (a transcription factor)-Bcl-2 pathway is indicated for the iso fl urane preconditioning-induced delayed phase of protection (Zuo et al. 2006 ) . Finally, iso fl urane preconditioning reduces ischemia-induced ubiquitin-conjugated protein aggregation (Zhang et al. 2010 ) . However, it is yet to be determined how iso fl urane can induce this effect. In addition to the intracellular changes in the neural tissues, a recent study shows that isofl urane preconditioning at 24 h before focal brain ischemia improves the regional cerebral blood fl ow and oxygen consumption in the ischemic brain tissues (Chi et al. 2010 ) . This fi nding suggests a novel mechanism for isofl urane preconditioning-induced delayed phase of neuroprotection. However, isofl urane-induced intracellular changes in the neural tissues must be important for the isofl urane preconditioning effects because these effects are apparent in neuronal cultures and brain slices under in vitro conditions (Zheng and Zuo 2003; Kapinya et al. 2002a ; Bickler et al. 2005 ) , in which the in fl uence of blood fl ow does not exist.

7.2.3 Halothane

Two studies have examined the preconditioning effects of halothane in the brain. One study showed that halothane preconditioning induced an acute phase of protection in rat cerebellar slices and that this effect was halothane dose-dependent. Similar to isoﬂ urane, the halothane preconditioning effect also was inhibited 170 Z. Zuo by a glutamate transporter inhibitor, suggesting the involvement of glutamate transporters in the effect (Wang et al. 2007a ) . The second study showed that halothane preconditioning induced a delayed phase of protection in rats after permanent focal brain ischemia. However, the mechanisms for this effect were not investigated (Kapinya et al. 2002a ) . Since halothane is no longer used in the USA, China, and many other countries, it is unlikely that additional studies on halothane preconditioning will be performed in the future.

7.2.4 Sevo ﬂ urane

In recent years, many studies have been performed on sevofl urane preconditioning- induced neuroprotection, possibly due to its increased popularity in the clinical practice, especially in pediatric anesthesia. Sevo fl urane preconditioning-induced acute phase of neuroprotection was fi rst shown in rat hippocampal slices (Kehl et al. 2004 ) . This effect was inhibited by a mitochondrial K ATP channel inhibitor (Kehl et al. 2004 ) , suggesting a role of KATP channels in this protection. This role is supported by fi ndings in a recent study using neuronal-glial cocultures (Velly et al. 2009) . An early study using rat cerebellar slices showed that this protection was sevofl urane dose-dependent with an EC50 similar to its MAC. This neuroprotection was inhibited by a glutamate transporter inhibitor in the cerebellar slices (Wang et al. 2007a ) . The involvement of ERK in this sevo fl urane preconditioning-induced acute phase of protection is suggested by a study using rat hippocampal slices (Wang et al. 2010 ) . All of these in vitro studies expose cells or brain slices to 0.5Ð3 MAC sevofl urane for 30 min and have an interval of 15 min between the sevo fl urane exposure and OGD. Three in vivo studies have shown sevofl urane preconditioning-induced acute phase of neuroprotection. The fi rst study exposed rats to 1 MAC sevo fl urane for 30 min at 15 min before a 7-min global brain ischemia. These rats had better neuropathological results in the hippocampus than those without preconditioning at 7 days after the brain ischemia (Payne et al. 2005 ) . In a similar experimental paradigm, the second study showed that exposure of rats to 1 or 2 MAC sevofl urane for 1 h increased the number of intact neurons in the hippocampus at 6 weeks after a 10-min global ischemia, suggesting that sevo fl urane preconditioning-induced acute phase of protection can translate into an improved long-term neurological outcome (Wang et al. 2007b) . However, a recent study showed that exposure of rats to 2.7% sevofl urane (~1 MAC) for 45 min at 1 h before a 60-min focal brain ischemia reduced brain infarct size and improved neurological functions at 3 days, but not at 7 and 14 days, after the brain ischemia (Codaccioni et al. 2009 ) . These results suggest that sevofl urane preconditioning only temporally improves neurological outcome after focal brain ischemia. Of note, these three in vivo studies did not examine the molecular mechanisms for the sevofl urane preconditioning-induced acute phase of neuroprotection. Exposure to 0.1 mM sevo fl urane for 1 h at 3 h before OGD signi fi cantly reduced OGD-induced cell injury in rat hippocampal slices (Sigaut et al. 2009 ) . 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen... 171

This may represent a delayed phase of neuroprotection. A recent study showed the delayed phase of sevofl urane preconditioning-induced neuroprotection in mouse neuronal-glial cocultures (Bantel et al. 2009 ) . Multiple in vivo studies have shown this neuroprotective effect (Payne et al. 2005 ; Wang et al. 2007b ; Codaccioni et al. 2009; Luo et al. 2008; McAuliffe et al. 2007; Adamczyk et al. 2010; Wang et al. 2011 ; Yang et al. 2011 ; Ye et al. 2012 ) . Most studies exposed rodents to ~1 MAC sevofl urane for 30Ð60 min once only. Exposure to sevofl urane once per day for several days also has been used in several studies (Payne et al. 2005 ; Wang et al. 2011 ; Yang et al. 2011 ) . Exposure to 1 MAC sevofl urane for 15 min at 72 h before a 1-h focal brain ischemia was used in one study (Adamczyk et al. 2010 ) . Global and transient focal brain ischemia and HI insult were used in these studies. Improvement of long-term neurological function by sevofl urane preconditioning has been suggested by two studies in neonatal rats and mice after HI insult (Luo et al. 2008 ; McAuliffe et al. 2007 ) . Molecules that are suggested to be involved in this protection by these in vivo studies include K ATP channels (Adamczyk et al. 2010 ; Ye et al. 2012 ) , free radicals (Yang et al. 2011 ) , cAMP response element-binding (CREB) (Luo et al. 2008 ) , and antioxidant enzymes (Yang et al. 2011 ) . Interestingly, one study suggests that inhibition of nuclear factor (NF)- kB, p38 MAP kinase, and the subsequent neuroin fl ammation contributes to the sevo fl urane preconditioning-induced neuroprotection (Wang et al. 2011 ) . Another study indicates that activation of p38 kinase may be involved in the neuroprotective effect (Ye et al. 2012 ) . Another interesting note is that KATP channels are not activated by sevofl urane and may not be involved in sevofl urane preconditioning-induced delayed phase of protection in the mouse neuronal-glial cocultures (Bantel et al. 2009 ) . However, the role of K ATP channels in this sevofl urane effects has been indicated by two in vivo studies in rats using transient focal brain ischemia models (Adamczyk et al. 2010 ; Ye et al. 2012 ) . Finally, sevo fl urane preconditioning has been shown to protect the blood-brain barrier after transient focal brain ischemia in rats (Yu et al. 2011 ) . Two studies have examined the sevo fl urane preconditioning effects in the spinal cord. The fi rst study exposed rats to 3.5% sevo fl urane (~1.3 MAC) for 1 h and subjected these rats to a 12-min spinal cord ischemia at 1 h or 24 h after the exposure. No neuroprotective effects were observed with this protocol when neurological outcome was evaluated at 7 days after the ischemia (Zvara et al. 2006 ) . The second study exposed rabbits to 3.7% sevo fl urane (~1 MAC) for 30 min at 1 h before the spinal cord ischemia. Signi fi cant improvement in neurological outcome was observed at 48 h after the ischemia. This protection was inhibited by an ERK inhibitor, suggesting the role of ERK in this protection (Ding et al. 2009 ) .

7.2.5 Desﬂ urane

Des fl urane preconditioning-induced acute phase of protection is desfl urane dose- dependent in rat cerebellar brain slices. This protection is inhibited by a glutamate transporter inhibitor, indicating that glutamate transporters are involved in this 172 Z. Zuo protection (Wang et al. 2007a ) . The delayed phase of protection induced by desfl urane preconditioning is relatively mild in neonatal mice after HI insult. This limited protection presents with improved performance in some (novel object recognition) but not other (cued maze) cognitive tests and no improvement in histopathology (McAuliffe et al. 2007 ) . Interestingly, des fl urane pretreatment (6 or 12% that is ~1 and 2 MAC for 30 min) does not appear to induce a delayed phase of neuroprotection in adult rats after transient focal brain ischemia (Li and Zuo 2009 ) . Consistent with this failure of protection, des fl urane pretreatment does not increase Bcl-2 expression in rat brain, a mechanism that is involved in iso fl urane preconditioning-induced delayed phase of protection in rats (Zhao et al. 2007 ; Li and Zuo 2009 ; Zuo et al. 2006 ; Li et al. 2008b ; Gwak et al. 2011 ) . These results suggest that volatile anesthetics behave differently in inducing the delayed phase of neuroprotection, unlike the situation with the acute phase of preconditioning-induced neuroprotection that can be provoked by all volatile anesthetics tested at clinically relevant concentrations (Wang et al. 2007a ) .

7.2.6 Xeon and Nitrous Oxide

It is yet to determine whether xenon induces an acute phase of preconditioning effect. However, the delayed phase of xenon preconditioning-induced neuroprotection has been shown in neuronal-glial cocultures, hippocampal slices, and intact brains of rodents (Bantel et al. 2009 ; Luo et al. 2008 ; Ma et al. 2006 ; Limatola et al. 2010 ) . This neuroprotection is xenon dose- and exposure time-dependent and is maximized with the exposure to 75% xenon (~1 MAC) for 2 h (Ma et al. 2006 ) . This neuroprotection also improves long-term neurological outcome (Luo et al. 2008 ) . Interestingly, xenon preconditioning induces protection against focal brain ischemia in young adult mice of either gender (Limatola et al. 2010 ) , unlike the situation of gender-dependency of isoﬂ urane preconditioning-induced protection in mouse brains. The neuroprotection of xenon preconditioning may be mediated by activation of mitochondrial KATP channels (Bantel et al. 2009 ) . Consistent with this possibility, xenon activates K ATP channels as shown by electrophysiological studies in neuronal-glial cell cultures. The role of CREB, Bcl-2, Akt, and HIF-1a in the xenon preconditioning-induced neuroprotection has been suggested (Bantel et al. 2009 ; Luo et al. 2008 ; Ma et al. 2006 ; Limatola et al. 2010 ) . Nitrous oxide has been shown to induce a preconditioning effect in the heart (Weber et al. 2005 ) . However, it is yet to be shown whether it will induce a preconditioning effect in the central nervous system.

7.3 Hyperbaric Oxygen

HBO preconditioning-induced protection in the brain was fi rst reported in 2000 (Xiong et al. 2000 ) , and the following studies did not appear in the English literature until 2006 (Freiberger et al. 2006 ; Hirata et al. 2007 ) . Many studies on this subject 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen... 173 are published in recent years. Of note, all studies investigated the delayed phase of HBO preconditioning-induced neuroprotection. It is yet to determine whether HBO preconditioning can induce an acute phase of protection. Also, it is not known whether HBO preconditioning-induced neuroprotection can be translated into a long-lasting improvement of neurological outcome. In the fi rst study on the subject, HBO preconditioning was shown to improve the neurological outcome after transient focal brain ischemia in rats. HBO at 2.5 atmosphere absolute (ATA) in 100% oxygen for 1 h every day for 5 days appeared to provide better neuroprotection than 2.5 ATA for 1 h per day for 3 days (Xiong et al. 2000) . This 5-day preconditioning protocol is very popular in the subsequent studies. However, one exposure to HBO at 2.5 ATA for 2.5 h was used in two studies investigating HBO preconditioning-induced neuroprotection in the neonatal rats after HI insult (Freiberger et al. 2006 ; Li et al. 2008c) . The last HBO preconditioning exposure is applied at 24 h before a detrimental insult in all studies published so far. HBO has been shown to provide protection against HI insult (Freiberger et al. 2006 ; Li et al. 2008c ) , transient focal and global brain ischemia (Ostrowski et al. 2008 ; Xiong et al. 2000 ; Hirata et al. 2007 ; Yan 2011 ; Cheng et al. 2011 ; Ostrowski et al. 2010; Yamashita et al. 2009; Li et al. 2008d; Gu et al. 2008 ) , traumatic brain injury (Hu et al. 2008, 2010) , decompression sickness (Fan et al. 2010 ) , surgical brain injury (Jadhav et al. 2009, 2010) , hypoxic injury (Peng et al. 2008 ) , intracerebral hemorrhage (Qin et al. 2007, 2008 ) , and posttraumatic syndrome (Peng et al. 2010) in rodents. However, HBO preconditioning does not appear to improve neurological outcome after permanent focal brain ischemia (Xiong et al. 2000 ) . The degree of neuroprotection induced by HBO preconditioning depends on the number of HBO episodes (5 episodes may be better than 3 episodes) (Xiong et al. 2000 ) and the degree of ATA (3.5 ATA appears to be better than 2 ATA) used as preconditioning stimuli (Yamashita et al. 2009 ) . The effective time window for HBO preconditioning-induced neuroprotection against an 8-min forebrain ischemia is less than 72 h after the last episode of 3.5 ATA HBO in rats (Hirata et al. 2007 ) . Various molecular mechanisms have been suggested for HBO preconditioning to protect against different neurological insults. The role of activated ERK in this protection is suggested in the intracerebral hemorrhage (Qin et al. 2007 ) . HBO increases the expression of ribosomal protein S6 kinase, an enzyme that is involved in protein synthesis, in the brain (Qin et al. 2008 ) . However, there are no additional data to suggest the involvement of this enzyme in HBO preconditioning effects. HBO preconditioning reduces apoptosis pathway in rats after transient focal and global brain ischemia as well as HI insult (Ostrowski et al. 2008 ; Li et al. 2008c ) . Cyclooxygenase 2 may play a role in the HBO effects against transient global brain ischemia and surgical brain injury (Jadhav et al. 2009, 2010) . Inhibition of p38 MAP kinase has been indicated in the HBO preconditioning-induced protection against forebrain ischemia (Yamashita et al. 2009 ) . Nitric oxide (NO) may be involved in the HBO effect against decompression sickness (Fan et al. 2010 ) . Autophagy activation may mediate the HBO preconditioning-induced neuroprotection after transient focal brain ischemia (Yan 2011 ) . Increased expressions of HIF-1a (Gu et al. 2008 ) , erythropoietin (Gu et al. 2008) , and antioxidant enzymes (Li et al. 2008d ) as well as 174 Z. Zuo inhibition of matrix metalloproteinase 9 expression (Ostrowski et al. 2010 ) are associated with HBO preconditioning-induced protection. Additional evidence to suggest the role of the changed expression of these proteins in the HBO preconditioning effect has not been provided. In addition to those molecular mechanisms discussed above, HBO preconditioning has been shown to preserve regional cerebral blood fl ow and partial oxygen pressure in rat brain tissues with traumatic injury (Hu et al. 2010 ) . In a rabbit permanent middle cerebral arterial occlusion model, HBO preconditioning attenuated the ischemia-increased lactate and glutamate in the ischemic penumbral region (Gao-Yu et al. 2011) . This is the only study in which HBO preconditioning effects in the brain was performed on an animal species other than rodents. Unfortunately, this study did not examine the neurological outcome. Thus, it is yet to determine whether HBO preconditioning occurs in other species including humans. There are four studies on HBO preconditioning-induced protection in the spinal cord. The fi rst study showed that 5-day HBO preconditioning (100% oxygen at 2.5 ATA for 1 h each day for 5 days), not a 3-day protocol, provided signifi cant protection against a 20-min spinal cord ischemia in rabbits (Dong et al. 2002 ) . Hyperbaric air did not induce ischemic tolerance (Dong et al. 2002 ) , suggesting that the HBO preconditioning-induced neuroprotection is not simply due to hyperbaricity. In the second study, the HBO preconditioning-induced protection against spinal cord injury was inhibited by a free radical scavenger inhibitor in rabbits. The inhibitor also reduced HBO-induced expression of antioxidant enzymes (Nie et al. 2006 ) . These results establish the role of free radicals as a trigger in inducing HBO preconditioning effects. The third study showed that HBO preconditioning-induced protection against oxidative stress may be mediated by increased expression of heme oxygenase 1 in the primary spinal cord neuronal cultures (Li et al. 2007 ) . The most recent study showed in rats that the HBO preconditioning improved neurological functions after spinal cord injury, increased antioxidant enzymes and Bcl-2, inhibited mitochondria-mediated apoptosis process, and NO may be a trigger for these benefi cial effects (Wang et al. 2009 ) . Similar to the situation in HBO preconditioning-induced protection in the brain, it is yet to determine whether HBO preconditioning induces an acute phase of protection and whether HBO preconditioning-induced protection translates into a long-term improvement of neurological outcome after spinal cord ischemia/injury.

7.4 Hydrogen Sul ﬁ de

Hydrogen sul ﬁ de (H2 S) has been considered as a toxic gas. It is realized now that

H 2 S is a gaseous messenger, just like NO in humans and animals (Wang 2003 ) .

Endogenous H2 S can be synthesized from cysteine by cystathionine b synthase, an enzyme that is found in the central nervous system (Lowicka and Beltowski 2007 ) . Multiple functions, including regulation of neurotransmission, smooth muscle relaxation, and neuroprotection, have been found for H2 S (Kimura et al. 2005 ) . 7 Medical Gases for Conditioning: Volatile Anesthetics, Hyperbaric Oxygen... 175

Injection of an H2 S donor, NaHS, at 25 m mol/kg at 30 min before a 15-min global brain ischemia reduces the brain ischemia-induced neuronal injury in rats (Ren et al. 2010 ) . Application of NaHS prior to hypoxia reduces the human neuroblastoma

SH-SY5Y cell injury. This protection may involve KATP channels, PKC, ERK, and heat shock protein 90 (Tay et al. 2010 ) . However, these NaHS-induced neuroprotection may not fall into the category of preconditioning effects because NaHS is very likely still releasing H2 S during the detrimental insults and no interval between the application of H2 S and the detrimental insults exists in these studies.

A recent study suggests that H2 S can induce a preconditioning effect in neural tissues. Rats were breathing 80 ppm H2 S for 1 h. They were subjected to retinal ischemia for 60 min at ~10 min after the H2 S exposure. Rats exposed to H2 S had reduced retinal ganglion cell loss and activated caspase 3 expression. H 2 S exposure increased the heat shock protein 90 expression (Biermann et al. 2011 ) .

7.5 Prospective

Preconditioning is a unique and naturally evolved protective mechanism. It involves induction/activation of endogenous strategies prior to a detrimental insult to reduce the insult-induced injury. Pharmacological preconditioning with relatively safe drugs has been considered as a promising approach to provide protection in various organs including brain. Although there is a need to predict the occurrence of a detrimental insult for the effective use of preconditioning mechanism, there are many clinical scenarios where such a predication is not too dif fi cult. For example, patients for coronary artery bypass grafting will often require being on cardiopulmonary bypass while the heart stops beating during the surgery. This arti fi cial bypass and procedures associated with establishing this bypass can be a signifi cant insult to many organs including the brain. In fact, perioperative stroke incidence for this type of surgery ranges from 1.6 to 5.2% (Arrowsmith et al. 2000 ; Dacey et al. 2005 ; Hogue et al. 1999 ) . Another example is carotid endarterectomy (CEA). Perioperative stroke incidence for CEA ranges from 0.25 to 7% (Allain et al. 2005 ; Wilson and Ammar 2005) . About 35% perioperative strokes occur during the surgery and the rest of them after the surgery. About 56% of those occurred after surgery happen within 24 h (Ferguson et al. 1999 ) . Thus, most strokes in patients having CEA occur within the effective time window of preconditioning-induced neuroprotection. Preconditioning with medical gases including volatile anesthetics would seem to be suitable for these clinical scenarios. However, almost all research on preconditioning induced by medical gases in the brain has been performed in rodents so far. Although evidence has suggested volatile anesthetics-induced cardioprotection in the humans (De Hert et al. 2002 ; Belhomme et al. 1999 ; Julier et al. 2003 ) , no study has been performed to determine the preconditioning effects of medical gases in human brains. Future studies should include multiple species, especially the higher- order animals, in the studies to determine whether medical gases can induce a preconditioning effect in the brains of species other than rodents. 176 Z. Zuo

Future studies should also focus on determining whether preconditioning by medical gases can improve long-term neurological outcome. This determination is especially important because clinical outcome is often evaluated weeks or months after the brain ischemia or other detrimental insults. There is emerging evidence of gender difference in volatile anesthetics-induced neuroprotection (Kitano et al. 2007b ) . This issue needs attention because better use of the strategy to provide protection can be designed for different groups of patients. Similar to this issue is aging effect. There is a lack of data on whether a preconditioning effect can be induced by medical gases in aged brains. Whether various clinical conditions that are often associated with stroke or other detrimental insults can modify the preconditioning effects induced by medical gases is unknown. All of this information is critically important for designing clinical trials to determine whether medical gases can induce preconditioning effects in human central nervous system.

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Zheng S, Zuo Z (2003) Isofl urane preconditioning reduces Purkinje cell death in an in vitro model of rat cerebellar ischemia. Neuroscience 118:99Ð106 Zheng S, Zuo Z (2004) Isofl urane preconditioning induces neuroprotection against ischemia via activation of p38 mitogen-activated protein kinase. Mol Pharmacol 65:1172Ð1180 Zheng S, Zuo Z (2005) Iso fl urane preconditioning decreases glutamate receptor overactivation- induced Purkinje neuronal injury in rat cerebellar slices. Brain Res 1054:143Ð151 Zhu W, Wang L, Zhang L, Palmateer JM, Libal NL, Hurn PD et al (2010) Iso flurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice. Neuroscience 169:758Ð769 Zuo Z, Wang Y, Huang Y (2006) Isofl urane preconditioning protects human neuroblastoma SH-SY5Y cells against in vitro simulated ischemia-reperfusion through the activation of extracellular signal-regulated kinases pathway. Eur J Pharmacol 542:84Ð91 Zvara DA, Bryant AJ, Deal DD, DeMarco MP, Campos KM, Mansfi eld CM et al (2006) Anesthetic preconditioning with sevofl urane does not protect the spinal cord after an ischemic-reperfusion injury in the rat. Anesth Analg 102:1341Ð1347 Chapter 8 Hypoxic Preconditioning in the CNS

Robert D. Gilchrist and Jeffrey M. Gidday

8.1 Introduction/Overview

Hypoxia represents an interesting stressor, given that, evolutionarily, alterations in environmental oxygen have represented an ongoing challenge for adaptation at the species level, particularly when life moved from sea to land, but also continuing today with the world’s temporary and permanent high-altitude-dwelling species (including humans). Of course, at the organ and cellular level, the source of this fundamental metabolic substrate can, for any number of pathological reasons, become limited, but we now realize that adaptive responses have evolved to low- oxygen conditions that help the organ or cell better resist such threats. Consider also the many anoxia- or hypoxia-tolerant vertebrates whose brains have adapted to survive long periods of time with little or no oxygen, not to mention the protracted periods of hypoxia that hibernating animals successfully deal with. Moreover, recall that, for mammals, the intrauterine environment is hypoxic; while some of the adaptive responses that permit fetal development in this environment are also “carried forward” and exhibited by neonates of most species, adults appear to have “lost” such a capacity. Or have they? The emergence of the entire fi eld of ischemic tolerance would suggest that many of these innate, evolutionarily conserved responses may just lie dormant and can be reactivated again with the appropriate preconditioning stimulus. Indeed, we now recognize that the mammalian brain reacts to noninjurious hypoxic challenges with a host of molecular and genetic changes that increase its resistance to ischemic and other life-threatening injuries. This chapter reviews this fi eld. Some of the fi ndings and mechanisms described herein have been validated to be applicable to other preconditioning stimuli, while it would appear that many others are unique to hypoxic preconditioning (HPC) itself.

R.D. Gilchrist • J. M. Gidday (*) Department of Neurosurgery , Washington University School of Medicine , St. Louis , MO 63110 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 183 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_8, © Springer Science+Business Media New York 2013 184 R.D. Gilchrist and J.M. Gidday

8.2 Models of HPC-Induced Protection for Cerebral Ischemia

HPC has been proven ef fi cacious for inducing tolerance in a number of diverse models of experimental cerebral ischemia. The ability of hypoxia to precondition the brain was fi rst documented in a slice model by Schurr and Rigor in 1987 (Schurr et al. 1986 ) and in vivo in a neonatal model of hypoxia-ischemia (Gidday et al. 1994 ) . Protection of the neonatal brain by HPC and other preconditioning stimuli is covered brie fl y here (Alkan et al. 2008 ; Bernaudin et al. 2002b ; Feng et al. 2010 ; Jones and Bergeron 2001 ; Laudenbach et al. 2007 ; Shu et al. 2010; Vannucci et al. 1998; Yin et al. 2007 ) , but details about its mechanistic basis and their relation to those operative in the adult brain are covered in detail elsewhere in this book (see Chap. 12 by Jones and Galle). The vast majority of in vivo HPC work to date has been performed in adult or newborn rodents (mice, rats, and gerbils), but recently, HPC ef fi cacy was also demonstrated in newborn piglets (Ara et al. 2011 ) . Protection from brain injury following global ischemia or cardiac arrest (Duszczyk et al. 2009 ; Geocadin et al. 2005; Nalivaeva et al. 2004 ; Wu et al. 2010b ; Zhan et al. 2010, 2011) or severe global hypoxia (Chang et al. 2006 ; Gorgias et al. 1996 ; Kalpana et al. 2008 ; Omata et al. 2002 ; Rybnikova et al. 2002, 2005, 2006, 2008, 2011; Samoilov et al. 2003; Stroev et al. 2004a, b, 2005, 2009 ) by HPC in adult models is often evidenced by histological assessments of the highly ischemia-sensitive CA1 hippocampal pyramidal cells (Churilova et al. 2010 ; Duszczyk et al. 2009 ; Gorgias et al. 1996 ; Nalivaeva et al. 2004 ; Rybnikova et al. 2005, 2006 ; Stroev et al. 2004a, b ; Taie et al. 2009 ; Zhan et al. 2010, 2011 ) and/or other endpoints (Chang et al. 2006 ; Duszczyk et al. 2009 ; Geocadin et al. 2005 ; Gorgias et al. 1996 ; Kalpana et al. 2008 ; Nalivaeva et al. 2004 ; Omata et al. 2002 ; Rybnikova et al. 2002, 2008, 2009, 2011; Samoilov et al. 2003 ; Stroev et al. 2004a, b, 2005, 2009; Wu et al. 2010b) . HPC effi cacy has also been documented in rodent models of transient (Baranova et al. 2007 ; Fan et al. 2011 ; Leconte et al. 2009 ; Lin et al. 2003, 2008 ; Miller et al. 2001 ; Prass et al. 2002, 2003; Stowe et al. 2011 ; Wacker et al. 2009 ) and permanent (Bernaudin et al. 2002a ; Prass et al. 2003 ; Stowe et al. 2011 ; Tang et al. 2006 ; Zhang et al. 2011 ) focal ischemia. HPC serves as an effective preconditioning stimulus in slice and organotypic culture models of ischemic tolerance (Bickler and Fahlman 2009, 2010 ; Bickler et al. 2009, 2010; Gage and Stanton 1996 ; Levin and Godukhin 2009 ; Levin et al. 2010; Omata et al. 2002 ; Semenov et al. 2008) , which provide unique opportunities for dissecting the mechanistic basis of HPC-induced tolerance. Cell death in pure or glial-mixed cultures of neurons or neuronal cell lines secondary to simulated ischemia (oxygen and glucose deprivation [OGD]) or excitatory amino acids is also attenuated when HPC precedes the OGD stimulus within minutes or several hours/ days (Arthur et al. 2004 ; Bruer et al. 1997 ; He et al. 2008 ; Liu et al. 2000 ; Ruscher et al. 1998 ; Wick et al. 2002 ; Wu et al. 2004, 2005, 2010a; Yu et al. 2008 ; Zhang et al. 2006 ) . Cell culture studies also document the HPC-induced protection of nonneuronal, resident brain cells from simulated ischemia, including, so far to date, cerebral endothelial cells (Zhang et al. 2007b ) and astrocytes (Chu et al. 2010 ; Yu et al. 2008 ) . Finally, preconditioning of human and other stem cell populations with hypoxia (Francis and Wei 2010 ; Hu et al. 2011 ; Jaderstad et al. 2010 ; Oh et al. 2010 ; 8 Hypoxic Preconditioning in the CNS 185

Theus et al. 2008 ) or the hypoxia-mimetic and hypoxia-inducible factor-1a stabilizer deferoxamine (Chu et al. 2008 ) prior to their implantation into ischemic foci improved their survival and differentiation, enhanced graft-host communication, reduced infarction volumes, and augmented poststroke recovery of function. Preconditioning with hypoxia, for both rapid and delayed CNS tolerance, has been achieved in a number of ways. For in vivo studies, some labs have exposed animals to hypobaric hypoxia (Chang et al. 2005, 2006; Duszczyk et al. 2009 ; Gutsaeva et al. 2008 ; Rybnikova et al. 2002, 2005, 2006, 2008, 2009, 2011 ; Samoilov et al. 2003 ; Semenov et al. 2008 ; Stroev et al. 2004a, b, 2005, 2009 ) , but most commonly, various durations of normobaric hypoxia are employed (Alkan et al. 2008 ; Ara et al. 2011 ; Bernaudin et al. 2002a, b ; Dale-Nagle et al. 2010 ; Dhodda et al. 2004; Fan et al. 2011 ; Feng et al. 2010; Gagnon et al. 2007; Garnier et al. 2001 ; Geocadin et al. 2005 ; Gidday et al. 1994, 1999; Gorgias et al. 1996; Grimm et al. 2002; Laudenbach et al. 2007; Miller et al. 2001 ; Nalivaeva et al. 2004 ; Prass et al. 2003; Shu et al. 2010; Stowe et al. 2011; Taie et al. 2009; Tang et al. 2006 ; Wacker et al. 2009, 2012 ; Wu et al. 2010b ; Zhu et al. 2002, 2007, 2010, 2011 ). Typically, the stimulus involves exposure of the entire animal to a few hours of mild hypoxia (8–12% hypoxia), administered a day or two before the lethal cerebral ischemic insult; this approach raises the interesting question of whether a circulating mediator, released from a hypoxic tissue other than that in which protection is measured, is really the molecular trigger of cerebral protection in a “remote preconditioning” kind of way (see below). Attempts to simulate the adaptation-inducing effects of high-altitude hypoxia, by exposing rodents to chronic or near-chronic hypoxia for extended periods of time, have been effective in preclinical models with respect to rendering the brain more resistant to ischemia (Chang et al. 2005, 2006 , Lin et al. 2003; Wei et al. 2008) . But, in fact, the jury is still out on whether living at moderate altitude confers an advantage with respect to stroke incidence (Faeh et al. 2009 ) . Therefore, such epigenetic changes, while sometimes similar to those induced by single or repetitive hypoxia, do not really constitute examples of changes induced by more brief and/or short-term hypoxic challenges and thus will not be discussed in much detail in this chapter; moreover, such considerations probably fall more into the category of stroke prophylaxis than stroke preconditioning. Some labs use intermittent hypoxia as the preconditioning stimulus, underpinning both an acute auto-hypoxia model (Bu et al. 2007, 2011; Duan et al. 1999 ; Lu et al. 1999 ; Shao et al. 2009 ; Xie et al. 1999 ; Zhang et al. 2007b, 2011 ; Zhang and Lu 1999 ) and a more standard delayed preconditioning approach (Churilova et al. 2010; Duszczyk et al. 2009 ; Gorgias et al. 1996; Lin et al. 2002, 2003; Omata et al. 2002 ; Peng et al. 2009 ; Rybnikova et al. 2002, 2005, 2006, 2008, 2011 ; Samoilov et al. 2003 ; Semenov et al. 2008 ; Stowe et al. 2011 ; Stroev et al. 2004a, b, 2005, 2009; Zhu et al. 2007 ) . Although it is unclear in most of these latter reports whether more than one hypoxic challenge was required to provide any unique ischemia- tolerant phenotypes since cerebral ischemia was induced within a day or so of the last hypoxic exposure. However, recent work in our laboratory revealed that 2 weeks of repetitive hypoxic preconditioning of adult mice (nine hypoxic challenges) can actually promote a sustained period of tolerance to both permanent and transient focal stroke, such that neurovascular protection is present months after the last HPC 186 R.D. Gilchrist and J.M. Gidday stimulus (Stowe et al. 2011 ) . We also demonstrated a similar extension of the duration of ischemic tolerance in the retina using either repetitive preconditioning with hypoxia (Zhu et al. 2007 ) or repeated administration of deferoxamine (Zhu et al. 2008) . Such ﬁ ndings may reﬂ ect a unique kind of long-lasting plasticity that can be induced by intermittently presented, but not single, preconditioning stimuli; if demonstrated to occur in humans as well, such a discovery is sure to raise excitement about the translational potential of preconditioning for patients at risk for CNS ischemia and perhaps other injuries. In most in vivo tolerance models promoted by HPC, cardiac arrest or focal stroke follows 24–48 h after the HPC stimulus. However, HPC has also been used for “rapid preconditioning,” wherein various neuroprotection endpoints are studied within minutes to perhaps an hour or two after preconditioning (Bu et al. 2007, 2011; Duan et al. 1999 ; Geocadin et al. 2005 ; Gorgias et al. 1996 ; Levin and Godukhin 2009 ; Levin et al. 2010 ; Lu et al. 1999 ; Shao et al. 2009 ; Xie et al. 1999 ; Zhang et al. 2006, 2007a, 2011) . Another variation on this latter theme is repeatedly exposing mice to hypoxia until gasping is evidenced and then examining the biochemical and molecular basis of the acutely enhanced tolerance to each successive hypoxic challenge (Bu et al. 2007, 2011 ; Duan et al. 1999 ; Lu et al. 1999 ; Shao et al. 2009 ; Xie et al. 1999 ; Zhang et al. 2007a, 2011; Zhang and Lu 1999 ) or to a subsequent stroke (Bu et al. 2011 ; Zhang et al. 2011 ) . An intermittent hypoxia stimulus paradigm has also been leveraged for inducing rapid preconditioning in in vitro models (Levin and Godukhin 2009 ; Levin et al. 2010 ; Wu et al. 2004, 2005, 2010a ; Zhang et al. 2006 ) . It is worthy of mention that some investigators have used “hypoxia mimetics” for both in vivo and in vitro preconditioning; this typically involves the administration of agents such as deferoxamine (Bergeron et al. 2000 ; Chu et al. 2010 ; Hamrick et al. 2005 ; Li et al. 2008b ; Prass et al. 2002 ; Yao et al. 2008 ) , cobalt chloride (Bergeron et al. 2000 ; Jones and Bergeron 2001 ; Kalpana et al. 2008 ; Valsecchi et al. 2011; Whitlock et al. 2005; Yao et al. 2008 ) , or ethyl-3,4-dihydroxybenzoate (EDHB) (Chu et al. 2010 ) that stabilize the transcription factor known as hypoxia- inducible factor (see below). Accumulating evidence suggests that other pharmacologic approaches for inducing tolerance may work by mechanisms quite similar to those induced by hypoxia (e.g., ginkgolides (He et al. 2008 ) ).

8.3 Molecular-Genetic Basis for HPC-Mediated Ischemic Tolerance

8.3.1 Induction Mechanisms

8.3.1.1 Hypoxia “Receptor”?

As outlined below in more detail, oxygen-dependent enzymes such as those in the prolyl hydroxylase family can serve as “receptors” or sensors for hypoxia-induced posttranslational modi ﬁ cations and changes in gene expression. Another candidate is 8 Hypoxic Preconditioning in the CNS 187 the mitochondria, wherein free radical and associated oxygen-nitrogen species formed in response to hypoxia serve as subsequent signals (Bailey et al. 2011 ) . The activity of AMPK may be another (Li and McCullough 2010 ) . Adenosine and/or other metabolites formed during metabolic stress or oxygen supply/demand imbalances may also act as intra- or intercellular signals for the induction of a genomic response on the part of any of the brain’s resident cells (Lin et al. 2008 ; Zhang and Lu 1999 ) . Whether one particular cell (i.e., the endothelial cell) serves as the master responder and through such a hypoxic response initiates in a paracrine fashion adaptive responses on the part of neighboring glia and neurons is a possibility not yet investigated, despite the general acceptance of the neurovascular unit concept. But the fact that endothelial cells are the ﬁ rst cell exposed to reductions in PaO 2 secondary to systemic hypoxia (as well as circulating mediators released into blood in remote preconditioning paradigms) warrants further investigations of such a hypothesis.

8.3.1.2 Signal Transduction Pathways

While rapid preconditioning, by defi nition, involves posttranslational modifi cations of structural and functional proteins to afford a quickly inducible, ischemia-resistant phenotype, hypoxic and other delayed preconditioning stimuli lead to genomic reprogramming, both before and after the actual ischemic insult. This, in turn, implicates the involvement of one or more signaling pathways proximal to the activation of transcription factors and their engagement with promoters, enhancer RNAs, and other regulators. Some of the key mediators involved in the proximal signaling of delayed (classic) ischemic tolerance are reviewed below. HPC does not seem to require membrane-localized NMDA receptor activation, given that blockade of this glutamate receptor subtype, as well as the AMPA receptor subtype, did not block HPC-induced tolerance in a hippocampal slice model (Gage and Stanton 1996 ) . Other “negative” fi ndings include the lack of microglial activation in the gerbil brain 48 h or 7 days following HPC (Garnier et al. 2001 ) . Whether Toll- like receptors are involved in HPC has not been studied directly, but the involvement of “infl ammatory” signaling (e.g., TNF-a (Liu et al. 2000 ) ) secondary to the HPC stimulus and the reoxygenation that follows is not unlikely. Considerable evidence supports the notion that the phosphorylation and activation of Akt is promoted by HPC, and that such increases occur both before (Bickler et al. 2010 ; Wick et al. 2002 ; Zhang et al. 2007b, 2010) and after ischemia (Feng et al. 2010 ; Li et al. 2008a; Yin et al. 2007 ) ; these fi ndings suggest the activation of the upstream source of Akt, membrane-bound PI3-kinase, by hypoxia, with further modulation of Akt signaling by nitric oxide and other mediators. As a kinase, phosphorylated Akt phosphorylates other pro-survival targets, including bad (Rybnikova et al. 2006 ; Wang et al. 2010 ) , NF k B (Rybnikova et al. 2008 ) , eNOS (Hashiguchi et al. 2004 ) , and the antiapoptotic, IAP-family protein member called survivin (Zhang et al. 2007b ) . pAkt also inactivates, by phosphorylation, proapoptotic factors like GSK-3b . Phosphorylated levels of another kinase, p38 MAPK, increase acutely in response to repetitive HPC in mouse cortex, hippocampus, and hypothalamus, and immunostaining revealed this increase to be localized to microglia (Bu et al. 2007 ) ; this acti- 188 R.D. Gilchrist and J.M. Gidday vation of p38 could be secondary to HPC-induced increases in infl ammatory cytokines and growth factors. There is also evidence for the membrane activation/ translocation of protein kinase C (PKC) in HPC-induced rapid tolerance (Bu et al. 2011 ; Zhang et al. 2011 ) . mRNA levels of the calcium and phosphate homeostasis- regulating stanniocalcin-1 are increased in an IL-6-dependent manner in response to HPC in the mouse brain (Westberg et al. 2007 ) , consistent with a signaling and/or effector role for this peptide. Using a genetic and immunoneutralizing approach, we recently found that, in a mouse model of HPC-induced tolerance to transient focal stroke, the proin fl ammatory chemokine CCL2 is essential (Stowe et al. 2012 ) . Finally, bioactive lipid signaling is also involved in HPC-induced tolerance, based on the fi nding that HPC increases the expression level and activity of sphingosine kinase 2, one isoform of the enzyme responsible for generating the pleiotropic lipid signaling intermediate sphingosine-1-phosphate (S1P), and that pharmacologic inhibition of this enzyme, or its genetic deletion, blocks HPC-induced tolerance to transient focal stroke in mice (Wacker et al. 2009, 2012 ) . It is not unlikely that the restoration of normoxia following HPC is associated with the generation of reactive oxygen species (ROS). Even hypoxia itself is associated with increases in mitochondrial superoxide production secondary to mitochondrial “uncoupling.” Thus, the signaling of HPC-induced tolerance may also involve the activation of many redox-sensitive signaling and transcriptional regulatory systems. However, direct evidence for HPC-induced ROS production is not widespread (Bickler et al. 2009 ; Rauca et al. 2000 ) . Although there is evidence for the involvement of nitric oxide (NO) in HPC- induced tolerance in neonate and adult brain and retina (Chen et al. 2010 ; Gidday et al. 1999 ; Zhu et al. 2006 ) , as with other “mediators,” it is often diffi cult to determine from studies using NO synthase (NOS) knockouts or NOS inhibitors whether NO is playing a signaling role proximal to gene expression changes or is acting as an effector or both. NO exerts many modulatory functions with respect to intra- and extracellular signaling and organelle function among cells of the neurovascular unit, so its role in preconditioning-induced tolerance is broad. NO production by the inducible NOS (iNOS) isoform, a target of the transcription factor hypoxia- inducible factor-1 a (HIF-1a ), has been linked mechanistically with HPC-induced protection (Taie et al. 2009 ) , but other studies support a more prominent role for endothelial NOS (eNOS) (Gidday et al. 1999 ; Vellimana et al. 2011 ) . NO appears to recipro- cally regulate the production of EPO in some cell types; in a neuronal cell culture study, it infl uenced at a transcriptional level the HPC-induced elevation in EPO receptor expression on neurons (Chen et al. 2010 ) .

8.3.2 Transcriptional Activation/Genetic Reprogramming

Early investigations of HPC in the hippocampal slice documented that the tolerance to ischemia required the synthesis of both new mRNA and proteins (Gage and Stanton 1996 ) , a fundamental dependence that we now know applies to all models 8 Hypoxic Preconditioning in the CNS 189 of “delayed” stroke tolerance independent of the preconditioning stimulus. Shortly after this report, microarray-based studies began to be published that refl ected the shared goal of understanding the genomic basis of ischemic tolerance. One of the fi rst was conducted using the hypoxic-ischemic neonatal rat model of HPC that our lab advanced (Gidday et al. 1994 ) in which temporal expression changes were identi fi ed for a host of genes in response to HPC (Bernaudin et al. 2002b ) . Others followed in HPC-induced adult stroke tolerance models (Bickler and Fahlman 2009, 2010 ; Tang et al. 2006 ) , including a recent paper on regional changes in cerebral gene expression in response to HPC (Xu et al. 2011 ) . Although measured at distinct timepoints experimentally, gene expression is obviously a fl uid process, with ongoing, dynamic changes in both gene activation and repression occurring in response to the HPC stimulus and in response to the ischemic insult in a preconditioned brain. The former suggests that changes in phenotype may “prime the pump” for an impending ischemic event, with “maximal” expression changes correlating temporally with the ideal therapeutic time window for preconditioning-induced tolerance (Dhodda et al. 2004 ; Tang et al. 2006 ) ; in other models, however, the transcriptional response has waned, and yet the brain remains tolerant (Stowe and Gidday unpublished observations; Marsh et al. 2009 ) . The postischemic transcriptional changes, in turn, have come to form the notion that preconditioning “reprograms” the brain’s response to stroke (Stenzel-Poore et al. 2003 ) (for more on preconditioning genomics, see Chap. 18 by Vartanian et al.). Although the speci fi cs are unique depending on the exact nature of the HPC stimulus (i.e., its frequency, magnitude, and duration), genes that regulate signal transduction, transcriptional activation, ionic homeostasis, metabolism, infl ammation, apoptosis, and cellular plasticity are often upregulated by hypoxia (Bernaudin et al. 2002b ; Bickler and Fahlman 2009 ; Tang et al. 2006 ) ; it is expected that many other genes would be downregulated in response to HPC, but, surprisingly, HPC-related array studies reporting downregulated genes are rare, except in the neonate (Bernaudin et al. 2002b ; Gustavsson et al. 2005 ) . Given the parallels between the hypometabolic effects of hypoxia (see below) and the cerebral metabolic suppression that occurs in hibernating and hypoxia-tolerant animals, even in invertebrate models of HPC (Mao and Crowder 2010 ) , a rather robust repression of gene expression by HPC would be expected. As a brief aside, the transcriptional repression that occurs among many metabolism-, transport-, synthesis-, and cell cycle-related genes following ischemia in preconditioned animals, recently discovered in a model of preconditioning with brief ischemia to be regulated by polycomb group proteins (Stapels et al. 2010 ) , may not necessarily defi ne the ischemia-tolerant phenotype induced by other preconditioning stimuli, despite their relatively equal ability to induce tolerance (Bickler and Fahlman 2009 ; Stevens et al. 2011 ) . Epigenetic transcriptional regulation can occur by many ways, including DNA methylation, chromatin remodeling, noncoding RNAs (including microRNAs), and RNA/DNA editing; thus, the adaptive responses a cell can mount at the genomic level to environmental stressors are impressive. As it turns out, a large number of genes are regulated by hypoxia, and hypoxia-response elements (HREs) on promoter sequences of genes continue to be identi fi ed. This, in turn, implies that 190 R.D. Gilchrist and J.M. Gidday hypoxia-sensitive transcription factors participate in directing the genomic response to HPC. Below, we address the intensively investigated transcription factor called hypoxia-inducible factor in some detail and also include a brief discussion of other hypoxia-sensitive transcription factors that are likely responsible for these aforementioned changes in gene expression induced by HPC.

8.3.2.1 Hypoxia-Inducible Factor

Hypoxia-inducible factor (HIF)-1a , and to a lesser extent its isoforms HIF-2 a and HIF-3 a, represents evolutionarily conserved transcription factors that have garnered considerable experimental attention as prominent players in hypoxia-inducible gene expression, secondary to their stabilization by hypoxic stimuli and subsequent binding to HREs on many pro-survival genes across many cell types and species (for reviews, see Majmundar et al. 2010; Ratan et al. 2004; Semenza 2009; Sharp and Bernaudin 2004 ; Wenger 2002 ) . In a nutshell, during normoxia, HIF-1a is post- translationally modi fi ed by the oxygen-dependent HIF prolyl hydroxylases (PHDs), essentially tagging it for proteasomal degradation. With the reduction in oxygen availability that de fi nes hypoxia, the oxygen-dependent PHDs become functionally inhibited, thereby stabilizing the alpha subunit and allowing it to transport to the nucleus; there, after dimerizing with HIF-1b , it becomes transcriptionally active and associates with other coactivators (e.g., CBP/p300), as well as with another oxygen-dependent hydroxylase called factor-inhibiting HIF-1 (FIH-1) which regulates HIF-1 DNA binding and transcriptional activity. Additional negative feedback loops operate to limit HIF-1 a activity during hypoxia as well as during the return to normoxia, including complex pathways (microRNAs, others) controlling PHD transcription. As expected, many other metabolites also regulate PHDs and/or the stability of HIF-1a , including nitric oxide (Chen et al. 2010 ; Martinez-Romero et al. 2009 ) , insulin-like growth factor 1 (IGF-1) (Chavez and LaManna 2002 ; Wang et al. 2004) , reactive oxygen species (ROS) (Martinez-Romero et al. 2009 ; Semenza 2007) , PI3K/Akt (Jiang et al. 2001 ; Li et al. 2008a) , p38 MAPK, and CREB, and thus may also participate in the adaptive response to hypoxia. In turn, these regulators may serve as indirect therapeutic targets for HIF-1a modulation (Majmundar et al. 2010 ) . Dose-dependent increases in DNA binding and transcriptional activity of HIF- 1 a occur during hypoxia, as well as immediately following reoxygenation (Tzeng et al. 2010 ) . HIF-1 a is now known to be responsible for activating the transcription of over 200 genes, including glycolysis-promoting enzymes, structural membrane proteins, ion-gated channels, antiapoptotic factors, and angiogenesis-promoting species (Majmundar et al. 2010 ; Semenza 2009 ; Wenger 2002 ) , many of which can be linked to the promotion of cell survival. Moreover, cellular metabolism is also regulated directly by HIF-1a at several levels (Aragones et al. 2008, 2009 ; Majmundar et al. 2010 ) . The evidence linking HIF-1a to HPC-induced tolerance continues to accumulate. In neonates, HPC-induced elevations in HIF-1 a protein expression were documented 8 Hypoxic Preconditioning in the CNS 191 throughout the brain immediately after HPC (Bernaudin et al. 2000 ) ; interestingly, following the ischemic insult, HIF-1a expression was evidenced largely in microvessels of the preconditioned animals (Bergeron et al. 2000 ) , perhaps playing a role in postischemic endothelial cell survival. In this same model, a microarray analysis identi fi ed several HIF gene targets (e.g., adrenomedullin) whose expression was changed by the HPC stimulus in a time-dependent manner (Bernaudin et al. 2002b ) , including some that were already upregulated or downregulated by the end of the 3-h hypoxic stimulus (Bernaudin et al. 2002b ) . Further examination of the genomic and proteomic basis of tolerance in this model revealed an upregulation of the glucose transporter protein GLUT-1 and other proteins involved in glycolysis (Jones and Bergeron 2001) . (For more on neonatal ischemic tolerance, see Chap. 12 by Jones and Galle). In adults, HIF-1a expression increases in response to hypoxia (Bernaudin et al. 2002a ; Prass et al. 2003 ) and the hypoxia associated with global ischemia (Chavez and LaManna 2002 ) . In fact, nuclear HIF-1a levels and HIF-1 DNA binding activity increase in response to HPC, along with the protein levels of some of its prototypical gene targets (i.e., EPO, VEGF, the sodium-calcium exchanger-1 [NCX1]) at timepoints coincident with ischemic tolerance (Bernaudin et al. 2002a ; Prass et al. 2003 ; Valsecchi et al. 2011 ) . HPC elevates HIF-1a and cell survival in C6 glioma cells (He et al. 2008 ) . HIF-1 a upregulation also occurs in response to HPC in neurally differentiating embryonic stem cell cultures (Theus et al. 2008 ) and in deferoxamine-preconditioned human neural stem cells (Chu et al. 2008 ) ; such a stem cell-priming strategy improves both morphological and functional outcome endpoints following ischemia (Chu et al. 2008 ; Theus et al. 2008 ) . In the mouse retina, repetitive HPC (Peng et al. 2009; Zhu et al. 2007 ) and repetitive deferoxamine treatments (Zhu et al. 2008 ) elevated HIF-1 a in a dose- and time- dependent fashion and protected against acute retinal ischemic injury. In the rat retina, cobalt preconditioning augmented heat shock protein 27 expression secondary to HIF-1a activation (Whitlock et al. 2005 ) , suggesting one mechanism whereby HPC may protect this tissue. HPC also protects the photoreceptors of the mouse retina from phototoxic injury (Grimm et al. 2002 ) , although a causal role for HIF- 1 a in mediating this response has not been easy to document (Thiersch et al. 2009 ) . (For more on retinal preconditioning and tolerance, see Chap. 25 by Roth and Dreixler). Employing HIF-1 a knockouts to con fi rm the role of this transcription factor in HPC-induced cerebral ischemic tolerance has led to some controversial results. In neuron-speci fi c HIF-1a -null mice, the increase in brain tissue pO2 during a severe hypoxic challenge that was measured in wild-type mice with prior HPC was no longer observed in HIF-1a -de fi cient mice, suggesting a causal role for HIF-1a - driven gene expression in establishing a phenotype consistent with neuroprotection (Taie et al. 2009 ) ; of note, this study did not actually assess lesion volumes or other focal stroke outcome endpoints. In another Cre-Lox mouse model, the neuron-speci fi c deletion of HIF-1a did not lead to the abolishment of HPC-induced ischemic tolerance (Baranova et al. 2007 ) , suggesting either that neuronally derived HIF-1a is not necessary for the overall cerebroprotection that de fi nes the ischemia-tolerant brain 192 R.D. Gilchrist and J.M. Gidday or that other resident brain cells upregulate HIF-1a , and the resulting upregulation in the synthesis of survival-promoting mediators acts in a paracrine manner to afford protection to ischemic neurons. The latter hypothesis is supported by the fi nding that HPC in Cre-Lox mice with a photoreceptor-selective knockout of HIF-1a still results in photoreceptor protection (Thiersch et al. 2009 ) . While these surprising study outcomes could be the result of differences in preconditioning and CNS injury models, the Cre-linked promoter used in the generation of the respective Cre-Lox mice (e.g., nestin vs. CaMKII a ) may also contribute. Approaching the testing of the hypothesis that HIF-1a is critical to preconditioning-induced ischemic tolerance in a different way, we found that HPC-induced tolerance to focal stroke in adult mice was lost when the generation of HIF in response to HPC was prevented with the diacylglycerol kinase inhibitor R59949 (Wacker et al. 2012 ) , and another lab reported that HIF-1 a silencing in adult rats blocked the establishment of ischemic tolerance in response to brief ischemic preconditioning (Valsecchi et al. 2011 ) . Because of differences in its transactivation domain, the HIF-2a isoform is believed to have unique target genes relative to HIF-1a (Hu et al. 2003 ; Majmundar et al. 2010 ; Sowter et al. 2003 ) that may include several glycolytic enzymes, the endothelial-speci fi c angiopoietin receptor tie-2, the VEGF-R2 receptor ( fl k-1) (Elvert et al. 2003 ) , eNOS (Coulet et al. 2003 ) , and EPO (Chavez et al. 2006 ) . Although direct evidence for HIF-2a being causal to ischemic tolerance is lacking to date, its expression increases in a time-dependent manner in response to hypoxia (Wiesener et al. 2003 ) , it regulates neuronal vulnerability to oxidative stress-induced injury (Siddiq et al. 2009 ) , and its lentiviral-mediated overexpression activates a number of known and putative neuroprotective genes (Ralph et al. 2004 ) ; thus, some kind of participatory role is likely. Its discovery and hypoxia-sensitive expression in endothelial cells (Takahashi et al. 2004 ; Wiesener et al. 2003 ) , and its delayed increase in endothelial cells in hypoxic border zones of the ischemic infarct where poststroke neovascularization is initiated (Thiersch et al. 2009 ) , would also suggest that the HIF-2a isoform could contribute to one or more of the cerebrovascular- protective phenotypes that defi ne the ischemia-tolerant brain (see Stowe and Gidday, Chap. 21 ). That being said, there is also elegant in vitro evidence that HIF-2 a expressed in astrocytes accounts for a paracrine-mediated survival of cocultured neurons in a preconditioning-like model (Chavez et al. 2006 ) . The bottom line seems to be that the regulation of HIF-1a and HIF-2 a is exquisitely regulated in a time-, cell-type-, and stimulus-dependent manner (Majmundar et al. 2010 ) , and that more convincing elucidation of their respective roles in CNS tolerance in response to HPC will require considerable further study.

8.3.2.2 Non-HIF Transcription Factors

Other transcription factors, including Mtf-1, Egr-1, Nrf-2, ATF-2, JNK and other MAPKs (ERK1/2), c-fos, Jun B, NGFI-A (Egr-1), STAT3, mTOR, PGC-1a , CREB, and NF k B, also directly or indirectly regulate hypoxia-responsive gene expression in important ways (Kenneth and Rocha 2008 ; Majmundar et al. 2010 ; Ran et al. 2005 ; Rybnikova et al. 2009 ; 8 Hypoxic Preconditioning in the CNS 193

Seta et al. 2002 ; Trachootham et al. 2008 ) . Thus, the signaling and transcription factors responsible for HPC-mediated changes in gene expression are certainly more extensive than just those which activate HIF. Several examples of hypoxia-induced “activation” of these transcription factors in ischemia tolerance models are available (Bickler et al. 2009, 2010; Bu et al. 2007 ; Churilova et al. 2010 ; Gutsaeva et al. 2008 ; Rybnikova et al. 2008, 2009; Zhang et al. 2007a) , and, at a more general level, the pro-survival functions of many of these regulators have been extensively documented.

8.3.3 Effector Mechanisms

While transcriptional changes in gene expression are critical to understanding HPC- induced tolerance, ultimately microarray data must be integrated and causally linked with posttranslational protein expression patterns, as not all mRNA changes will result in proportional and physiologically signi fi cant changes in the levels of proteins a cell expresses nor reveal posttranslational changes that signi fi cantly affect phenotype. In fact, it has been estimated that posttranslational modifi cations provide for more than 200,000 different proteins in a given mammalian cell, even though the total number of genes is roughly a tenth of that. Below, we focus at a more integrative, phenotypic level on specifi c endpoints often used to defi ne the ischemically injured brain and how these are changed in ischemic tolerance. Of course, many more examples exist in the literature regarding cytoprotective neural, glial, and vascular phenotypes induced by a variety of different preconditioning stimuli; here, we only summarize those that have been documented to occur in HPC models.

8.3.3.1 Attenuated Excitotoxicity

Given the central role of acute excitotoxicity in cerebral ischemic injury, it would be expected that HPC-treated animals/cells would exhibit phenotypes indicative of a reduction in excitotoxic tone, thereby lowering calcium in ﬂ ux. Indeed, in a mouse model of repetitive HPC for acute tolerance, the concentration of the excitatory amino acids aspartate and glutamate decreased, which was purported to contribute to the enhanced anoxia tolerance in this model (Xie et al. 1999 ) . In addition to reductions in presynaptic release, neuronal culture studies of glutamate toxicity suggest that HPC also reduces the sensitivity of neurons to glutamate and/or downregulates one or more glutamate receptors (Turovskaya et al. 2011 ; Zhang et al. 2006) . The increased survival time in severe hypoxia of rat hippocampal slices treated with the AMPA receptor antagonist kynurenic acid (with NMDA receptor antagonism by MK801 having no effect) was attenuated in slices from rats treated with a chronic hypobaric hypoxia stimulus, indicating that the protective effects of this HPC-like stimulus may also be downregulation in, or reduced activity of, AMPA receptors (Chang et al. 2006 ) . Consonant with this, HPC blocked hippocampal injury in rats caused by kainate injection (Chang et al. 2005 ) . 194 R.D. Gilchrist and J.M. Gidday

8.3.3.2 Enhanced Antioxidant Capacity

Reactive oxygen and nitrogen species play many direct and indirect roles in ischemic brain injury, at all levels of cellular organization, including DNA. Early investigations of HPC recorded an accumulation of the sulfur-containing compounds cystine, cysteine, and cystamine, all of which are capable of scavenging oxygen free radicals and thereby preventing oxidative damage (Schurr et al. 1986 ) . In other studies, HPC-induced increases in manganese and copper-zinc superoxide dismutases (SOD1 and SOD2), heme oxygenase-1 (HO-1), glutathione peroxidase, glutathione reductase, and thioredoxin-2 (Txn2) occur, often in proportion to the magnitude of the ischemic tolerance so invoked, both rapid and delayed, and in inverse proportion to the quantity of radical species and/or lipid peroxidation levels measured (Alkan et al. 2008 ; Arthur et al. 2004 ; Duan et al. 1999 ; Garnier et al. 2001 ; Gorgias et al. 1996 ; Lin et al. 2003 ; Stroev et al. 2004a, b ; Zhu et al. 2007 ) . HO-1 presents itself as an interesting candidate for establishing long-term ischemic tolerance to stroke (Stowe et al. 2011 ) , given that, at least in retina, much longer durations of elevated expression were achieved with repetitive HPC relative to a single HPC stimulus (Zhu et al. 2007) . This may also result from the observation that beneﬁ cial or detrimental effects of HO-1 upregulation exhibit some dependence on the frequency and magnitude of the inducing stimulus, the cell type under consideration, the existing redox environment, and many other interdependent factors (Schipper 2004 ) . As a result of the reaction products HO-1 catalyzes, which include carbon monoxide, biliverdin, and iron, it is often said that HO-1 exerts pleiotropic cytoprotective effects beyond free radical scavenging, including vasodilation, angiogenesis, anti-in ﬂ ammation, and antiapoptosis.

8.3.3.3 Metabolic Downregulation

Generally speaking, tissue and cellular metabolism runs lower in hypoxic environments – a manifestation of the well-known Pasteur effect, as well as a reduction in protein synthesis, and other adaptive responses (Buck and Pamenter 2006 ) . As mentioned above, this response is now known to be coordinated at several levels by glucose transporters (Yu et al. 2008 ) , glycolytic enzymes (Jones and Bergeron 2001) , calcium infl ux (Bickler et al. 2009 ; Semenov et al. 2008 ) , mitochondrial respiration (Buck and Pamenter 2006 ) , the endoplasmic reticulum (Bickler et al. 2009 ; Lin et al. 2007) , HIF (Aragones et al. 2008, 2009; Majmundar et al. 2010 ) , and mRNA translation (Liu et al. 2008 ) . However, few intentional measures of alterations in cerebral metabolic rate (for oxygen or glucose) in response to established HPC stimuli are available. In brain slices obtained from normal rats but subsequently exposed to a sublethal hypoxic stimulus that promotes tolerance, CMR glc did not change (Omata et al. 2002 ) , whereas in slices obtained from rats already exposed to tolerance-inducing chronic hypobaric hypoxia, the spontaneous fi ring rate of locus coeruleus neurons was reduced by a third (Chang et al. 2006 ) . HPC led to increases in the expression of the mRNA for the glucose transporter GLUT-1 in 8 Hypoxic Preconditioning in the CNS 195 cultured astrocytes and both GLUT-1 and GLUT-3 mRNA in cultured neurons, as well as to an increase in metabolism as measured by 2-deoxyglucose uptake; interestingly, blocking neuronal glucose uptake blocked their anoxia tolerance (Yu et al. 2008 ) . Several large questions still require experimental inquiry with respect to metabolism. First, it must be determined whether the HPC “reprograms” the metabolic response of the cell/tissue to ischemia, as has been hypothesized for preconditioning’s effect on the genome. Second, it would be helpful to identify components of the ischemia-tolerant “metabolome” at any given postischemic time and how they contribute to increased ischemic resistance. For example, the enhanced IGF-1 synthesis in the neonate brain secondary to HPC (Wang et al. 2004 ) may not only augment metabolic growth processes and the cell’s response to growth-promoting signals critical to neuronal survival, but its role as a global anabolic factor in most cell types – including neurons, endothelial cells, and hematopoietic cells – may also prepare the tissue through autocrine/paracrine signaling for enhanced postischemic recovery. HPC-induced tolerance is also characterized by preservation of postischemic Na + /K +-ATPase levels (Zhan et al. 2011) ; the participation of large conduc- + tance, calcium-activated potassium (BKCa ) channels (Levin and Godukhin 2009 ) ; a preservation of the astrocyte gap junction protein connexin 43 (Lin et al. 2008 ) ; and a consequent increase in protective levels of extracellular adenosine (Lin et al. 2008 ; Zhang and Lu 1999) , all of which refl ect metabolic phenotypes likely to be critical to the overall cytoprotection. In the neonatal rat, HPC-induced neuroprotection was not the result of a change in the extent of anaerobic glycolysis, tissue acidosis, or depletion of high-energy phosphate reserves following ischemia but did improve the rate of restoration of high-energy phosphates in the early hours of postischemic reperfusion (Vannucci et al. 1998 ) . In cultured PC12 cells, HPC increased mRNA levels of aldose reductase, suggesting metabolic consequences secondary to increases in sorbitol production, but this effect may relate more to adaptive changes in cellular volume regulation (Wu et al. 2010b ) . Accumulating evidence from a number of preconditioning models indicates that mitochondria are likely to be critical effectors of the ischemia-tolerant state (Correia et al. 2010; Dirnagl and Meisel 2008 ; Zhan et al. 2002 ) and may be the instrumental organelle responsive to hypoxia (Buck and Pamenter 2006 ) . In addition, transient hypoxia stimulates mitochondrial biogenesis in a CREB- and PGC-1 a -dependent fashion (Gutsaeva et al. 2008 ) . HPC of cultured hypothalamic (Wu et al. 2004 ) or hippocampal (Wu et al. 2005 ) neurons results in better preservation of mitochondrial membrane potential in response to anoxia. With better preservation of mitochondrial function, ATP production by aerobic respiration and the necessary catalytic activity of mitochondrial Mn-SOD (Arthur et al. 2004 ; Stroev et al. 2005 ) and mitochondrial thioredoxin-2 (Stroev et al. 2004a ) are optimized, with concomitant reduction of superoxide secondary to fewer electrons leaked to molecular oxygen from the electron transport chain. Finally, the activation of ATP-sensitive potassium channels (KATP ) located directly on the mitochondrial inner membrane is also likely to be involved in preconditioning signaling (Busija et al. 2008 ; Correia et al. 2010 ) . 196 R.D. Gilchrist and J.M. Gidday

8.3.3.4 In ﬂ ammatory and Immunological Changes

Neurovascular and glial infl ammation, as well as alterations in immune cell traffi cking and Toll-like receptor function, are hallmark characteristics of the postischemic brain. While changes in these endpoints have been documented in response to preconditioning with other non-hypoxic stimuli (see Chap. 18 by Vartanian et al.), relatively little has been forthcoming in this regard from HPC- based models of tolerance. Repeated HPC reduces postischemic leukocyte adherence and diapedesis in cortical venules in vivo secondary to downregulation of adhesion molecule mRNA expression (Stowe et al. 2011 ) , consistent with similar anti-infl ammatory phenotypes established by other preconditioning stimuli with respect to interactions between infl ammatory cells and the cerebrovascular endothelium. Following hypoxia-ischemia in the neonatal rat, the expression of several proin fl ammatory cytokines and other proin fl ammatory markers was reduced in HPC-treated animals, coincident with reductions in lesion size (Yin et al. 2007 ) . We are not aware of any studies to date that have reported HPC-induced reductions in the microglial activation aspects of the postischemic in fl ammatory response.

8.3.3.5 Reduced Apoptosis

It is well established that many preconditioning stimuli attenuate apoptotic ischemic cell death, and HPC is no exception. HPC upregulates Bcl-2 and Bcl-xL levels in hippocampus and cortex of rats and reduces the extent of Bax upregulation caused by the subsequent severe hypoxic injury (Rybnikova et al. 2006 ) . In an in vitro model of cerebral endothelial cell preconditioning, we found that the phosphorylation of the inhibitor-of-apoptosis protein survivin occurs secondary to an HPC- mediated enhancement of phosphorylated Akt expression, and that blocking this Akt-mediated survivin upregulation attenuated HPC-induced tolerance to simulated ischemia in these cells (Zhang et al. 2007b ) . Subjecting embryonic stem cell-derived neural progenitor cells and other stem cells to HPC leads to augmented expression of the antiapoptotic protein Bcl-2, which was surmised, in turn, to contribute to their improved survival and the enhancement of other benefi cial cell-based therapy effects following their transplantation into the ischemic rat brain (Theus et al. 2008 ) . Enhanced neuronal differentiation and a number of other survival-enhancing phenotypic signatures (elevated HIF, VEGF, EPO, Bcl-2) were also identifi ed in these cells in a separate study from the same laboratory (Francis and Wei 2010 ) . These fi ndings suggest that HPC’s antiapoptotic effects may be initiated in the very earliest stages of cellular development. HPC can protect the nematode C. elegans from death due to severe hypoxia (Dasgupta et al. 2007 ; Mao and Crowder 2010 ) , re fl ecting the strength of evolutionarily conserved mechanisms of HPC-induced tolerance. Interestingly, in this invertebrate model of tolerance, expression of the Apaf-1 homologue ced-4 actually 8 Hypoxic Preconditioning in the CNS 197 increases following HPC, and by mutational and RNAi silencing studies, the authors showed ced-4 to be necessary for HPC-induced protection (Dasgupta et al. 2007 ) . Assuming the homology between the apoptosis-related nematode and mammalian genes that largely drove apoptotic cell death research forward a couple decades ago, it would appear that small, hypoxia-induced changes in Apaf-1 expression may play important signaling roles in promoting either the induction and/or expression of cerebral ischemic tolerance. This hypothesis remains to be tested.

8.3.3.6 Improved Blood Flow/Tissue Perfusion (Short-Term Vascular Adaptations)

As likely with all preconditioning stimuli, HPC promotes adaptive changes not only in neurons but in vascular (and glial) cells of the brain as well. This, in turn, implies that an assortment of vascular ischemic injury phenotypes is mitigated in the ischemia-tolerant brain. With respect to perfusion, vessel diameters of leptomeningeal anastomoses (which subserve perfusion in the penumbra in the setting of occlusions of the middle cerebral artery) are increased after HPC (Woitzik et al.

2006 ) . In addition, tissue oxygenation (pO 2) of the mouse brain is improved during a subsequent (24 h later) bout of severe hypoxia (Taie et al. 2009 ) ; the extent to which the latter resulted from an increase in neuroglobin, the mRNA and protein expression levels, which were documented to increase acutely in mouse cortex after repeated HPC (Shao et al. 2009) , will require further testing. Both of the aforementioned fi ndings imply an HPC-induced improvement in tissue perfusion. Actual improvements in blood fl ow in the ischemic periphery as a result of prior HPC were measured in mice in response to transient focal ischemia (Fan et al. 2011 ) . Similarly, an increase in intraischemic fl ow characterized HPC-treated neonatal rats (Gustavsson et al. 2005 ) .

8.3.3.7 Angiogenesis (Long-Term Vascular Adaptations)

An enhancement of the postischemic cerebrovascular angiogenic response is expected to accompany reductions in infarction volumes in HPC-treated animals, but not many studies to date have examined this possibility in any model of preconditioning. We do know that vascular endothelial growth factor (VEGF) is upregulated in response to HPC (Bernaudin et al. 2002a ; Fan et al. 2011 ; Feng et al. 2010 ; Gustavsson et al. 2005 ; Laudenbach et al. 2007 ) , consistent with the promotion of a proangiogenic phenotype. In fact, in the neonatal rat, HPC led to remarkably acute increases in cerebral vascular density (Gustavsson et al. 2005 ) , but again, systematic examinations of longer postischemic recovery time periods are needed. 198 R.D. Gilchrist and J.M. Gidday

8.3.3.8 Reductions in Overt Vascular Injury

In addition to perfusion-related changes, the cerebral vasculature of the ischemia- tolerant brain exhibits less blood-brain barrier (BBB) dysfunction after focal ischemia. In our experience, HPC in adult mice leads to reduced immunoglobulin G permeability through the BBB in response to transient focal stroke (Stowe et al. 2011 ; Wacker et al. 2012 ) , as a result, in part, of the structural preservation of tight junction and adherens junction proteins secondary to an HPC-induced elaboration of sphingosine-1-phosphate (Wacker et al. 2012 ) . It would not be surprising to ﬁ nd that HPC also resulted in improvements in basal lamina integrity as well and perhaps effects on pericyte structure/function too. As mentioned earlier, HPC of cultured human cerebral endothelial cells protects them from oxygen/glucose deprivation-induced apoptosis (Zhang et al. 2007b ) , suggesting that, in addition to effects on perfusion, vascular reactivity, and barrier phenotypes, HPC also has direct cytoprotective effects on cerebral endothelium.

8.3.3.9 Miscellaneous Cytoprotective Effectors

Erythropoietin

While originally thought to function exclusively as a “hormone” stimulating red cell production from bone marrow, it is now clear that erythropoietin (EPO) acts more like a circulating cytokine and exhibits a number of cytoprotective and growth factor-like actions in the brain that are completely independent of its erythropoietic effect (van der Kooij et al. 2008 ) . HPC, as well as treatment with the hypoxia mimetics cobalt chloride and deferoxamine, increase erythropoietin (EPO) mRNA expression in neuron-astrocyte cocultures (Bernaudin et al. 2000) and in rodent brain cortex/hippocampus (Bernaudin et al. 2000, 2002a ; Li et al. 2008b ; Westberg et al. 2007 ) . Following HPC, EPO transcription increases secondary to HIF-1a promoting EPO transcription (Prass et al. 2002, 2003 ) , resulting in upregulation of the protein 24 h after reoxygenation (Bernaudin et al. 2002a ) . That pretreatment with the PHD inhibitor and HIF-stabilizing agent deferoxamine mimicked this response indicates that induction of EPO transcription occurs through the activation of HIF- 1 a (Prass et al. 2002 ) . Increases in EPO expression, secondary to p42/p44-MAPK and PI3K/Akt/GSK-3b activation, occur in C6 glioma cells in response to HPC and may prevent the apoptosis that follows an ischemic insult to these cells (He et al. 2008) . EPO and its receptor were also upregulated in neurally differentiating embryonic stem cells and primary bone marrow cultured cells in response to HPC, and this upregulation promoted survival of the cells after transplantation into ischemic rat brain (Theus et al. 2008 ) . Causal evidence supporting a role for EPO in HPC- induced tolerance was forthcoming from a study in adult mice showing that infusion of a soluble receptor for EPO, which effectively reduces EPO signaling, attenuated HPC’s neuroprotective effect by ~40% (Prass et al. 2003 ) . In a novel in vitro model of hypoxic post-conditioning-induced protection of pure neuronal cultures, elevations 8 Hypoxic Preconditioning in the CNS 199 in EPO protein expression were documented in response to hypoxia even when the hypoxic challenge followed OGD by 14 h (Leconte et al. 2009 ) .

VEGF

In the mouse, VEGF mRNA and protein are increased following a variety of HPC stimuli (Bernaudin et al. 2002a ; Fan et al. 2011 ; Shao et al. 2009 ) . The role of VEGF in establishing tolerance has received support in culture and animal models. In particular, in cerebellar granule cells, HPC increased VEGF and VEGF receptor-1 (VEGF-R1) and VEGF-R2 levels, and a monoclonal antibody to VEGF-R2 blocked HPC-induced tolerance to glutamate and other cytotoxic treatments; pretreatment with VEGF in the absence of HPC was also protective (Wick et al. 2002 ) . In adult mice, antagonism of the VEGF-R2 receptor blocked HPC-induced tolerance to transient focal stroke in adult mice (Fan et al. 2011 ) and HPC-induced tolerance to hypoxic-ischemic injury in neonatal mice (Laudenbach et al. 2007 ) . Moreover, VEGF mRNA and protein increases in the neonatal rat brain following hypoxia-ischemia were prolonged and ampli ﬁ ed in those animals receiving HPC (Feng et al. 2010 ) . Finally, the release of VEGF in response to preconditioning primary astrocyte cultures with the hypoxia mimetic deferoxamine is integral to protection of these cells from oxidative stress, secondary to HIF-1a -mediated transcription (Chu et al. 2010 ) .

Humoral Mediators

Adrenomedullin is a circulating pleiotropic cytokine/hormone that exerts autocrine and paracrine vasodilatory, angiogenic, anti-infl ammatory, and antiapoptotic effects. Increases in its message and protein levels following HPC in neonates (Bernaudin et al. 2002b) , hypoxic post-conditioning in adult mice (Leconte et al. 2009 ) , and OGD in HPC-treated neuronal cultures (Tixier et al. 2008 ) have been reported; in the latter model, its antagonism after OGD blocked tolerance, indicative of a phenotype-de fi ning role for this peptide (Tixier et al. 2008 ) . While the mechanism was not identi fi ed, HPC-treated rats subjected to global ischemia/cardiac arrest exhibited improvements in cardiovascular sympathovagal balance (as assessed by barorefl ex sensitivity) that correlated with reductions in neurodefi cit scores (Wu et al. 2010b ) . How changes in glucocorticoid and mineralocorticoid receptor expression in response to HPC (Rybnikova et al. 2011 ) comprise part of the body’s overall neuroendocrine response to systemic hypoxia, and the expected concomitant changes in the hypothalamic-pituitary-adrenal axis, remains to be elucidated.

Opioids

Delta-opioid receptor activation protects cortical neurons from glutamate-induced injury with ligand binding density, but not expression, increasing following HPC 200 R.D. Gilchrist and J.M. Gidday and documented as necessary for evocation of rapid tolerance after HPC (Zhang et al. 2006 ) . Delta opioid receptors are also upregulated in retina in response to intermittent HPC through HIF-1 a-mediated transcriptional pathways (Peng et al. 2009 ) . These ﬁ ndings suggest that opioids may participate in mediating the ischemia- tolerant phenotype, perhaps through the regulation of MAPK, Bcl-2 expression, and cytochrome c release.

Heat Shock Proteins (HSPs)

HSP’s are chaperone proteins known to exhibit ubiquitous protective effects in the cell, most notably by reducing protein misfolding, preventing the ER stress response, scavenging ROS, and blocking caspase-mediated apoptosis. While seeming “natural” candidates for effectors of CNS tolerance, due to temporal dissociations between their expression pro ﬁ les following brief ischemic preconditioning and the actual period of ischemic tolerance so induced, several early studies were unable to provide support for a role for HSP-70 and HSP-72. HSP-72 was not induced at all in gerbil hippocampus following HPC and thus is not likely to underlie the antioxidant and antiapoptotic phenotypes so noted (Garnier et al. 2001 ) , nor was HSP-70 or HSP-90 elevated after HPC in an in vitro model of cerebellar neuronal tolerance (Wick et al. 2002 ) . However, in retina, expression of the HIF-1a gene target HSP-32 (heme oxygenase-1) increased in a dose-dependent manner after single or repetitive preconditioning of the adult mouse (Zhu et al. 2007 ) . Similarly, the HPC-mimetic cobalt chloride upregulated retinal HSP-27 through HIF-1a activation (Whitlock et al. 2005 ) , suggesting that, generally speaking, some but not all HSPs may participate in HPC-induced protection of retina and brain.

Amyloid-Related Proteins

HPC restored the reduction in the expression levels of the putative cytoprotective molecule soluble amyloid precursor protein (sAPP) following global ischemia in rats (Nalivaeva et al. 2004 ) , suggesting the activation of an a -secretase-like metalloproteinase by HPC.

8.4 HPC-Mediated Cytoprotection in Nonischemic Conditions

HPC protects the CNS not only against ischemic injury but against a host of other injury paradigms as well. Recently, we showed that HPC exhibited vascular and neuronal protective effects in mouse subarachnoid hemorrhage (SAH), reducing vasospasm and improving functional outcome metrics; the increase in eNOS expression and activity following HPC, and the loss of the neurovascular SAH protection afforded by HPC in eNOS-null mice, strongly implicated eNOS-derived NO in 8 Hypoxic Preconditioning in the CNS 201 these protective effects (Vellimana et al. 2011 ) . HPC also lessened the susceptibility to PTZ-induced clonic seizures in both rat and mouse epilepsy models (Rauca et al. 2000; Rubaj et al. 2000 ) . In animal and cell culture models of excitotoxic injury (Chang et al. 2005 ; Laudenbach et al. 2007 ; Zhang et al. 2006 ) , iron- and oxidative stress-induced oxidative injury (Chu et al. 2010 ; Lin et al. 2002 ) , MPP+ neurotoxicity (Tzeng et al. 2010 ) , sPLA2 III-induced apoptosis (Wang et al. 2010 ) , and cold cryolesions (Westberg et al. 2007 ) , HPC exhibited signi fi cant protective effects. Injury-induced changes in NADPH and nNOS immunostaining in crushed hypo- glossal and vagus nerves were also attenuated in proportion to survival time by prior HPC (Wei et al. 2008) . In addition, HPC is protective in a mouse model of noise- induced hearing loss (Gagnon et al. 2007 ) , implying protective effects on the cochlea, cochlear nerve, and/or related auditory pathways, as well as in a model of light-induced phototoxicity of retinal photoreceptors (Grimm et al. 2002 ) . Preconditioning cultured oligodendrocytes with the hypoxia mimetics cobalt chloride or deferoxamine prevented TNF-a -mediated apoptosis (Yao et al. 2008 ) . Acute intermittent hypoxia also gives rise to long-lasting phrenic motor facilitation via spinal mechanisms, refl ecting the possibility of restoring respiratory motor neuron function in conditions causing respiratory insuf fi ciency (Dale-Nagle et al. 2010 ) . Finally, HPC treatment of mesenchymal stem cells prior to implantation improved their survival in a rat model of spinal cord compression injury (Oh et al. 2010 ) . Thus, it is likely that HPC triggers survival-enhancing adaptive changes in the CNS that afford a level of cytoprotection to a wide variety of pathological conditions, consistent with the notion that bene fi cial responses of neurons to mild hypoxic stress are broad based and, not surprisingly, phylogenetically conserved (Dasgupta et al. 2007 ; Mao and Crowder 2010 ) .

8.5 Translational Potential for HPC?

Several reviews have been written about translational preconditioning in general, but the use of hypoxia as a preconditioning stimulus does not get a lot of attention, despite intentional hypoxic breathing being a common intervention for enhancing human athletic performance (e.g., see, www.go2altitude.com ). Achieving the ideal titration of the stressor will be a challenge for any preconditioning stimulus employed clinically, including hypoxia. Too little hypoxia (be it the intensity, duration, and/or frequency of the challenge) and no adaptive responses will be induced; too much hypoxia and at risk is cellular injury to the most vulnerable of brain neurons (e.g., CA1 hippocampal pyramidal cells). As illustration, we originally found that 2 h of 11% oxygen breathing in adult mice was only neuroprotective against brain damage by transient, but not permanent, focal stroke (Miller et al. 2001 ) , and others have found HPC to be ineffective in inducing cerebral tolerance in other in vitro (Meloni et al. 2002 ) and in vivo (Prass et al. 2003 ) ischemia models, raising the question of whether the hypoxic challenge was severe enough in the latter models to trigger the necessary adaptive responses. However, increasing the duration and magnitude of 202 R.D. Gilchrist and J.M. Gidday the hypoxic stimulus was ef ﬁ cacious in the adult mouse for inducing tolerance to permanent focal stroke in another laboratory (Bernaudin et al. 2002a ) and, subsequently, in our hands as well (Stowe et al. 2011 ) . Nevertheless, con ﬁ rming the risk for preconditioning-induced morphologic or functional injury to be nonexistent, even after what is presumed to be a mild or nonlethal hypoxic challenge, will always be a key criterion to meet, both in animal models (Sommer 2008 ) and of course in the clinic. As further examples of each, in what could be an age-dependent and/or injury-dependent effect, HPC was found to exacerbate volatile anesthetic-induced apoptosis in the neonatal brain (Shu et al. 2010 ) . In turn, 9 h of 12.9% oxygen breathing in humans – while promoting some degree of ROS formation and, presumably, ROS-dependent signaling – was apparently without any adverse effects on the volunteers (Bailey et al. 2011 ) .

8.6 Future Studies

While our mechanistic knowledge about the ability of the CNS and its resident cells to adapt to hypoxia by presenting robust, ischemia-resistant phenotypes has grown quickly over the last decade or so, much of the preclinical work to date has focused on fairly simple models that do not accurately re fl ect the clinical scenarios and the hope we place in successful translational preconditioning. For example, as with other preconditioning stimuli, it is possible that the ef fi cacy of HPC for promoting ischemic tolerance will be lost with advancing age (Bickler et al. 2010 ) , in females, and/ or in the presence of other comorbidities like hypertension, diabetes, hypercholesterolemia, etc. Thus, new dose-response relationships will have to be elucidated in preclinical models for these subpopulations to support preconditioning’s translational potential. It is also not unlikely that combinations of preconditioning stimuli will be required to establish any signifi cant degree of tolerance, with HPC being part of a combinatorial treatment strategy. In the hippocampal slice, for example, isofl urane can enhance the protective effect of HPC (Bickler and Fahlman 2010 ) , so ultimately identifying synergistic preconditioning stimuli will be important, at least for some of the aforementioned patient subgroups. The previous fi nding is somewhat inconsistent with the assumption held for many years that different preconditioning stimuli ultimately converge on the same transcription factors and induce the same “quality” of tolerance. However, the preclinical fi ndings that the genomic response to different preconditioning stimuli can be distinct, despite producing equivalent levels of stroke tolerance (Bickler and Fahlman 2009 ; Stevens et al. 2011 ) , would suggest that endogenous neuroprotection may be achieved in redundant ways; some of these may be “additive,” as with iso fl urane and HPC (Bickler and Fahlman 2010 ) , and some may not. In the evolving era of personalized medicine, it is not unlikely that distinct preconditioning cocktails will be selected for a given patient subgroup; whether HPC is part of any of those regimens remains to be seen. Another translational roadblock that must be circumvented relates to preconditioning the patient populations at highest risk for impending stroke (e.g., those with 8 Hypoxic Preconditioning in the CNS 203 a history of TIAs, prior stroke, carotid endarterectomy, coronary bypass) or cerebral ischemic injury (e.g., SAH). An ideal preconditioning “therapy” for such individuals may be a protracted state of tolerance to cover their respective windows of highest stroke risk. However, current preclinical models that exhibit only a couple of days of protection are not likely to translate well to these ideal preconditioning treatment groups. Thus, our recent fi nding that repetitive presentations of HPC can extend the period of stroke tolerance in the mouse from days to months (Stowe et al. 2011 ) is an exciting development in the preclinical world with tangible clinical ramifi cations. An intentional melding of preconditioning treatment strategies with stroke prophylaxis provided by exercise, diet (including, possibly, dietary restriction), statins, etc., may also provide clinical bene fi ts, given our current understanding that preconditioning- induced tolerance only implies a reduction in the extent of injury should a stroke occur, but not necessarily a reduction in stroke incidence. Finally, post-conditioning with hypoxia (or other stimuli) to engage innate neurovascular protective responses that can improve poststroke recovery and restoration of function is a relatively nascent fi eld deserving of more experimental attention, since this approach could conceivably be administered to many more stroke victims than those in the aforementioned high-risk groups. In support of this contention is a recent study in mice in which repetitive delayed hypoxic post-conditioning (starting 5 days after stroke) reduced thalamic atrophy measured many weeks later (Leconte et al. 2009 ) . In closing, despite the potency of HPC for promoting CNS tolerance in our animal and culture models against not only ischemic but other injuries, and despite our growing understanding of the mechanistic basis of the protection so achieved, a considerable number of issues still require investigation for this and most other preconditioning stimuli before we can translate the potential of innate protection from bench to bedside.

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Stroev SA, Tjulkova EI, Gluschenko TS, Rybnikova EA, Samoilov MO, Pelto-Huikko M (2004b) The augmentation of brain thioredoxin-1 expression after severe hypobaric hypoxia by the preconditioning in rats. Neurosci Lett 370:224–229 Stroev SA, Gluschenko TS, Tjulkova EI, Rybnikova EA, Samoilov MO, Pelto-Huikko M (2005) The effect of preconditioning on the Cu, Zn superoxide dismutase expression and enzyme activity in rat brain at the early period after severe hypobaric hypoxia. Neurosci Res 53:39–47 Stroev SA, Tyul’kova EI, Glushchenko TS, Tugoi IA, Samoilov MO, Pelto-Huikko M (2009) Thioredoxin-1 expression levels in rat hippocampal neurons in moderate hypobaric hypoxia. Neurosci Behav Physiol 39:1–5 Taie S, Ono J, Iwanaga Y, Tomita S, Asaga T, Chujo K, Ueki M (2009) Hypoxia-inducible factor-1 alpha has a key role in hypoxic preconditioning. J Clin Neurosci 16:1056–1060 Takahashi R, Kobayashi C, Kondo Y, Nakatani Y, Kudo I, Kunimoto M, Imura N, Hara S (2004) Subcellular localization and regulation of hypoxia-inducible factor-2alpha in vascular endothelial cells. Biochem Biophys Res Commun 317:84–91 Tang Y, Pacary E, Freret T, Divoux D, Petit E, Schumann-Bard P, Bernaudin M (2006) Effect of hypoxic preconditioning on brain genomic response before and following ischemia in the adult mouse: identifi cation of potential neuroprotective candidates for stroke. Neurobiol Dis 21:18–28 Theus MH, Wei L, Cui L, Francis K, Hu X, Keogh C, Yu SP (2008) In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefi ts after transplantation into the ischemic rat brain. Exp Neurol 210:656–670 Thiersch M, Lange C, Joly S, Heynen S, Le YZ, Samardzija M, Grimm C (2009) Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors. Eur J Neurosci 29:2291–2302 Tixier E, Leconte C, Touzani O, Roussel S, Petit E, Bernaudin M (2008) Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner. J Neurochem 106:1388–1403 Trachootham D, Lu W, Ogasawara MA, Nilsa R-DV, Huang P (2008) Redox regulation of cell survival. Antioxid Redox Signal 10:1343–1374 Turovskaya MV, Turovsky EA, Zinchenko VP, Levin SG, Shamsutdinova AA, Godukhin OV (2011) Repeated brief episodes of hypoxia modulate the calcium responses of ionotropic glutamate receptors in hippocampal neurons. Neurosci Lett 496:11–14 Tzeng YW, Lee LY, Chao PL, Lee HC, Wu RT, Lin AMY (2010) Role of autophagy in protection afforded by hypoxic preconditioning against MPP+ -induced neurotoxicity in SH-SY5Y cells. Free Radic Biol Med 49:839–846 Valsecchi V, Pignataro G, Del Prete A, Sirabella R, Matrone C, Boscia F, Scorziello A, Sisalli MJ, Esposito E, Zambrano N, Di Renzo G, Annunziato L (2011) NCX1 is a novel target gene for hypoxia-inducible factor-1 in ischemic brain preconditioning. Stroke 42:754–763 van der Kooij MA, Groenendaal F, Kavelaars A, Heijnen CJ, van Bel F (2008) Neuroprotective properties and mechanisms of erythropoietin in in vitro and in vivo experimental models for hypoxia/ischemia. Brain Res Rev 59:22–33 Vannucci RC, Tow fi ghi J, Vannucci SJ (1998) Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates. J Neurochem 71:1215–1220 Vellimana AK, Milner E, Azad TD, Harries MD, Zhou M-L, Gidday JM, Han BH, Zipfel GJ (2011) Endothelial nitric oxide synthase mediates endogenous protection against subarachnoid hemorrhage-induced cerebral vasospasm. Stroke 42:776–782 Wacker BK, Park TS, Gidday JM (2009) Hypoxic preconditioning-induced cerebral ischemic tolerance: role of microvascular sphingosine kinase 2. Stroke 40:3342–3348 Wacker BK, Freie AB, Perfater JL, Gidday JM (2012) Junctional protein regulation by sphingosine kinase 2 contributes to blood-brain barrier protection in hypoxic preconditioning-induced cerebral ischemic tolerance. J Cereb Blood Flow Metab 32(6):1014–1023 Wang X, Deng J, Boyle DW, Zhong J, Lee WH (2004) Potential role of IGF-I in hypoxia tolerance using a rat hypoxic-ischemic model: activation of hypoxia-inducible factor 1alpha. Pediatr Res 55:385–394 8 Hypoxic Preconditioning in the CNS 211

Wang G, Zhou D, Wang C, Gao Y, Zhou Q, Qian G, DeCoster MA (2010) Hypoxic preconditioning suppresses group III secreted phospholipase A2-induced apoptosis via JAK2-STAT3 activation in cortical neurons. J Neurochem 114:1039–1048 Wei IH, Huang C-C, Tseng C-Y, Chang H-M, Tu H-C, Tsai M-H, Wen C-Y, Shieh J-Y (2008) Mild hypoxic preconditioning attenuates injury-induced NADPH-d/nNOS expression in brainstem motor neurons of adult rats. J Chem Neuroanat 35:123–132 Wenger RH (2002) Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia- inducible transcription factors, and O2-regulated gene expression. FASEB J 16:1151–1162 Westberg JA, Serlachius M, Lankila P, Penkowa M, Hidalgo J, Andersson LC (2007) Hypoxic preconditioning induces neuroprotective stanniocalcin-1 in brain via IL-6 signaling. Stroke 38:1025–1030 Whitlock NA, Agarwal N, Ma JX, Crosson CE (2005) Hsp27 upregulation by HIF-1 signaling offers protection against retinal ischemia in rats. Invest Ophthalmol Vis Sci 46:1092–1098 Wick A, Wick W, Waltenberger J, Weller M, Dichgans J, Schulz JB (2002) Neuroprotection by hypoxic preconditioning requires sequential activation of vascular endothelial growth factor receptor and Akt. J Neurosci 22:6401–6407 Wiesener MS, Jurgensen JS, Rosenberger C, Scholze CK, Horstrup JH, Warnecke C, Mandriota S, Bechmann I, Frei UA, Pugh CW, Ratcliffe PJ, Bachmann S, Maxwell PH, Eckardt KU (2003) Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs. FASEB J 17:271–273 Woitzik J, Hecht N, Schneider UC, Pena-Tapia PG, Vajkoczy P (2006) Increased vessel diameter of leptomeningeal anastomoses after hypoxic preconditioning. Brain Res 1115:209–212 Wu L-Y, Ding A-S, Zhao T, Ma Z-M, Wang F-Z, Fan M (2004) Involvement of increased stability of mitochondrial membrane potential and overexpression of Bcl-2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons. Brain Res 999:149–154 Wu LY, Ding AS, Zhao T, Ma ZM, Wang FZ, Fan M (2005) Underlying mechanism of hypoxic preconditioning decreasing apoptosis induced by anoxia in cultured hippocampal neurons. Neurosignals 14:109–116 Wu L-Y, Ma Z-M, Fan X-L, Zhao T, Liu Z-H, Huang X, Li M-M, Xiong L, Zhang K, Zhu L-L, Fan M (2010a) The anti-necrosis role of hypoxic preconditioning after acute anoxia is mediated by aldose reductase and sorbitol pathway in PC12 cells. Cell Stress Chaperones 15:387–394 Wu W, Guo X, Jiang D, Sun J, Qiu Y, Zhu Y, Thakor NV, Tong S (2010b) Inﬂ uence of hypoxic- preconditioning on autonomic regulation following global ischemic brain injury in rats. Neurosci Lett 480:191–195 Xie J, Lu GW, Hou YZ (1999) Role of excitatory amino acids in hypoxic preconditioning. Biol Signals Recept 8:267–274 Xu H, Lu A, Sharp FR (2011) Regional genome transcriptional response of adult mouse brain to hypoxia. BMC Genomics 12:499 Yao S-y, Soutto M, Sriram S (2008) Preconditioning with cobalt chloride or desferrioxamine protects oligodendrocyte cell line (MO3.13) from tumor necrosis factor-alpha-mediated cell death. J Neurosci Res 86:2403–2413 Yin W, Signore AP, Iwai M, Cao G, Gao Y, Johnnides MJ, Hickey RW, Chen J (2007) Preconditioning suppresses in ﬂ ammation in neonatal hypoxic ischemia via Akt activation. Stroke 38:1017–1024 Yu S, Zhao T, Guo M, Fang H, Ma J, Ding A, Wang F, Chan P, Fan M (2008) Hypoxic preconditioning up-regulates glucose transport activity and glucose transporter (GLUT1 and GLUT3) gene expression after acute anoxic exposure in the cultured rat hippocampal neurons and astrocytes. Brain Res 1211:22–29 Zhan R-Z, Fujihara H, Baba H, Yamakura T, Shimoji K (2002) Ischemic preconditioning is capable of inducing mitochondrial tolerance in the rat brain. Anesthesiology 97:896–901 Zhan L, Wang T, Li W, Xu ZC, Sun W, Xu E (2010) Activation of Akt/FoxO signaling pathway contributes to induction of neuroprotection against transient global cerebral ischemia by hypoxic pre-conditioning in adult rats. J Neurochem 114:897–908 212 R.D. Gilchrist and J.M. Gidday

Zhan L, Peng W, Sun W, Xu E (2011) Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase. Neurochem Int 59:65–72 Zhang WL, Lu GW (1999) Changes of adenosine and its A₁ receptor in hypoxic preconditioning. Biol Signals Recept 8:275–280 Zhang J, Qian H, Zhao P, Hong SS, Xia Y (2006) Rapid hypoxia preconditioning protects cortical neurons from glutamate toxicity through delta-opioid receptor. Stroke 37:1094–1099 Zhang N, Gao G, Bu X, Han S, Fang L, Li J (2007a) Neuron-speci ﬁ c phosphorylation of c-Jun N-terminal kinase increased in the brain of hypoxic preconditioned mice. Neurosci Lett 423:219–224 Zhang Y, Park TS, Gidday JM (2007b) Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent survivin activation. Am J Physiol Heart Circ Physiol 292:H2573–H2581 Zhang N, Yin Y, Han S, Jiang J, Yang W, Bu X, Li J (2011) Hypoxic preconditioning induced neuroprotection against cerebral ischemic injuries and its cPKC g-mediated molecular mechanism. Neurochem Int 58:684–692 Zhu Y, Ohlemiller KK, McMahan BK, Gidday JM (2002) Mouse models of retinal ischemic tolerance. Invest Ophthalmol Vis Sci 43:1903–1911 Zhu Y, Ohlemiller KK, McMahan BK, Park TS, Gidday JM (2006) Constitutive nitric oxide synthase activity is required to trigger ischemic tolerance in mouse retina. Exp Eye Res 82:153–163 Zhu Y, Zhang Y, Ojwang BA, Brantley MA Jr, Gidday JM (2007) Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1. Invest Ophthalmol Vis Sci 48:1735–1743 Zhu Y, Zhang L, Gidday JM (2008) Deferroxamine preconditioning promotes long-lasting retinal ischemic tolerance. J Ocul Pharmacol Ther 24:527–535 Chapter 9 Pharmacologic Preconditioning

Jian Guan , Richard F. Keep , Ya Hua , Karin M. Muraszko , and Guohua Xi

9.1 Background

Pharmacological preconditioning (PPC) is a rapidly growing area of investigation in the larger realm of cerebral preconditioning for the attenuation of stroke-induced brain injury. The appeal of PPC is multifactorial. From a basic science perspective, the study of a wide array of pharmacologic agents with a broad range of actions could allow for a clearer picture of the complex mechanisms of injury following ischemic and hemorrhagic stroke. From a translational perspective, the well-studied and proven safety proﬁ les of many of these agents, which include such staples as opioids (Lim et al. 2004 ) and macrolide antibiotics (Koerner et al. 2007 ) , carry the promise of more direct clinical applicability. On a practical level, the possibility of the rapid administration of a drug to induce a preconditioned state is very attractive for situations such as traumatic injury where other forms of preconditioning such as hyperbaric oxygen or transient ischemia would be unwieldy. In terms of effectiveness, animal studies of various pharmacological agents have shown neuroprotective effects similar to that of the more extensively studied ischemic preconditioning (Gidday 2010 ) . A large range of agents can induce PPC (see the recent review by Gidday 2010 ) . Some, such as volatile anesthetics and dietary factors, are covered by other chapters in this book. This chapter focuses on just six agents that can induce PPC: thrombin, erythropoietin (EPO), deferoxamine, erythromycin, opioids, and lipopolysaccharide (LPS). These have been chosen for several reasons. While they all act to upregulate

J. Guan ¥ R. F. Keep ¥ Y. Hua ¥ K. M. Muraszko Department of Neurosurgery , University of Michigan , Ann Arbor , MI , USA G. Xi , M.D. (*) Department of Neurosurgery , University of Michigan , Ann Arbor , MI , USA R5018 Biomedical Science Research Building , University of Michigan , 109 Zina Pitcher Place , 48109-2200 Ann Arbor , MI , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 213 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_9, © Springer Science+Business Media New York 2013 214 J. Guan et al. defense mechanisms in the brain, EPO and thrombin are endogenous compounds that may, therefore, act naturally to modify stroke (e.g., the release of thrombin in a cerebral hemorrhage may upregulate iron handling proteins affecting iron toxicity during hematoma lysis). LPS, a component of the wall of gram-negative bacteria, is an example of a potentially injurious exogenous natural compound that induces an upregulation of endogenous defense mechanisms. Deferoxamine, erythromycin, opioids, and EPO are currently used therapeutics which may facilitate their use in the clinic for preconditioning. Thrombin and LPS were also chosen because they represent the often close divide between a preconditioning agent being bene ﬁ cial and harmful. While both of these agents can induce PPC, both can enhance brain injury at high concentrations. Finally, although these PPC agents have some common ﬁ nal protective pathways, they differ in upstream events. They also appear to differ in whether they can induce acute (classical) rather than chronic (delayed) preconditioning. This is an important clinical consideration. While the role of PPC in the clinic is still uncertain, one potential role relates to surgery-induced ischemic events where acute induction of protection would have advantages. In contrast, the use of PPC to prevent the recurrence of stroke or reduce injury from the second stroke would not require acute effects of the PPC agent.

9.2 Individual Agents

9.2.1 Thrombin

Thrombin is an endogenous serine protease best known for the central role it plays in the coagulation cascade by cleaving fi brinogen to fi brin (Coughlin 2000 ) . Thrombin is produced from the cleavage of prothrombin by the activation of factor X through the coagulation cascade. Though the majority of prothrombin is synthesized in the liver, prothrombin mRNA is also expressed in cells of the central nervous system, and this expression has been shown to increase following ischemic events in animal models (Riek-Burchardt et al. 2002 ) . More recently, thrombin has also been shown to play an important part in the infl ammatory response following brain injury (Nishino et al. 1993 ) . In hemorrhagic stroke models, infusion of thrombin or prothrombin results in signi fi cant brain edema, a consequence attenuated by concomitant treatment with thrombin inhibitors (Lee et al. 1996 ) . Similarly, in models of ischemic stroke, intra-arterial administration of thrombin led to greater vascular disruption and cell death (Chen et al. 2010 ) . Despite these deleterious effects, the administration of low-dose intracerebral thrombin has been demonstrated to have substantial neuroprotective effects. Thrombin preconditioning (TPC) has been shown to mitigate brain edema following hemorrhage (Xi et al. 2000 ) and to protect from subsequent injections of higher doses of thrombin (Xi et al. 2000 ) . TPC has also demonstrated effectiveness in 9 Pharmacologic Preconditioning 215

Fig. 9.1 TTC-stained coronal brain sections obtained 24 h after permanent middle cerebral artery occlusion in rats receiving an intracerebral injection of either saline (a ), thrombin (b ), or thrombin/ hirudin, a thrombin inhibitor (c ) 7 days prior to the onset of ischemia. Note, in particular, that in the thrombin-injected animal, there appear to be areas of both caudate and cortex close to the site of injection that are protected compared to the other two groups (Masada et al. 2000 )

reducing infarction volume and behavioral defi cits in murine models of cerebral ischemia (Fig. 9.1 ) (Masada et al. 2000 ) and reducing neuronal death after oxygen- glucose deprivation (Fig. 9.2 ). In addition, recent animal studies also suggest that TPC may have a protective role in Parkinson’s disease, with signifi cant TPC- associated behavioral benefi ts seen in a rat 6-hydroxydopamine model of the disease (Cannon et al. 2005 ) . The mechanisms of thrombin’s harmful and protective effects are complex and likely involve multiple pathways. Many of thrombin’s actions are mediated by protease-activated receptors (PARs). These G-protein-coupled receptors are activated by proteolytic cleavage, with subtypes 1, 3, and 4 being activated by thrombin (Coughlin 2000) , and are expressed by neurons and astrocytes (Wang et al. 2002 ) . Through these PARs, thrombin can impact a wide variety of intracellular signaling pathways with effects on cell shape, secretion, mobility, and metabolism (Coughlin 2000 ) . One of the pathways thought to contribute to thrombin-induced brain injury is the potentiation of NMDA receptors through activation of PAR1, leading to a neurotoxic state of hyperexcitability (Hamill et al. 2009 ) . Thrombin may also play a role in compromise of the bloodÐbrain barrier by inducing endothelial hyperpermeability through Rho kinase and protein tyrosine kinase (van Nieuw Amerongen et al. 2000 ) . In addition, thrombin infusion has been associated with large increases in substances such as complement C9, tumor necrosis factor- a, and matrix metalloproteinase, all of which may be important mediators of brain injury following hemorrhagic stroke (Hua et al. 2007 ) . TPC is also likely dependent on multiple mechanisms. Infusion of low-dose thrombin causes upregulation of heat shock protein 27 (HSP27), the peak of which has been shown to closely correlate with the window of maximum TPC effectiveness 216 J. Guan et al.

a 160

120

80 * # # 40

LDH (% of control) 0 0 0.25 0.5 1.0 2.0 Thrombin (U/ml)

b 100

80 # # # # 60

Dead Cell (%) 20

0 0 0.25 0.5 1.0 2.0 Thrombin (U/ml) c

Control TPC

Fig. 9.2 Primary cultured neurons were pretreated with different doses of thrombin for 24 h and then exposed to oxygen-glucose deprivation (OGD ) for 2 h. The levels of lactate dehydrogenase (LDH ) released into the medium (a ) and the percent of dead cells by live/dead cell staining ( b and c ) were measured after 22 h of reoxygenation. Values are expressed as means ± SD. *p < 0.05, # p < 0.01 versus control (0 U/ml thrombin). In (c ), neurons stained red indicate dead neurons, and those stained green are live. TPC thrombin preconditioning with 1 U/ml. Scale bar = 20 m M (Hu et al. 2010 )

(Xi et al. 1999 ) . HSP27 has been demonstrated to have a variety of protective actions including chaperoning damaged proteins and suppression of cell death signaling (Stetler et al. 2009 ) . Thrombin-receptor activation also likely plays a central role in TPC. This is supported by animal studies demonstrating TPC-like effects with 9 Pharmacologic Preconditioning 217

1 2 3 4 5 6 7 8 9 Activated p44/42 MAPK

Activated p70S6K

β−actin

3000 Activated Vehicle p44/42 MAPK * Thrombin PD98059+Thrombin 2000

Pixels * 1000

0 42 kDa 44 kDa

3000 Activated Vehicle * p70S6K Thrombin PD98059+Thrombin

2000

Pixels * 1000

0 85 kDa 70 kDa

Fig. 9.3 Activated p44/42 MAPK and p70S6K (Thr421/Ser424) protein levels in neurons after vehicle (lanes 1Ð3), thrombin (1 U/ml, lanes 4Ð6), or thrombin (1 U/ml) plus PD098059, a MEKI inhibitor (20 mM, lanes 7Ð9) treatment for 24 h. Values are mean ± SD, *p < 0.05 versus the other groups (Hu et al. 2010 ) administration of a thrombin-receptor agonist and inhibition of TPC with simultaneous administration of a thrombin-receptor antagonist (Jiang et al. 2002 ) . These effects may be secondary to downstream activation of p44/42 mitogen-activated protein kinases, which could play a role in synthesis of neuroprotective proteins (Hu et al. 2010 ) (Fig. 9.3 ). One of these proteins, hypoxia-inducible factor-1a , has been 218 J. Guan et al.

-120 kDa

123456789

b 10000 #,##

7500

# (pixels) 5000

HIF-1 2500

0 137 Time (days)

Fig. 9.4 ( a ) Western blot analysis showing the time course of HIF-1a protein levels in the ipsilateral basal ganglia 1 (lanes 1Ð3), 3 (lanes 4Ð6), and 7 days (lanes 7Ð9) after intracerebral injection of thrombin (1 U). ( b ) HIF-1a protein levels were semiquantitated by Western blot analysis. Values are mean ± SD, n = 3. # p < 0.01 versus day 7; ##p < 0.05 versus day 1 (Hua et al. 2003 ) shown to have protective functions following infusion of lysed red blood cells or iron Ð two mediators of neurotoxicity following intracerebral hemorrhage (Lee et al. 1996 ) Ð into the basal ganglia of rats (Hua et al. 2003 ) (Fig. 9.4 ). The apparent dose-dependent effects of thrombin administration, with injurious effects at high concentrations and protective effects at low concentrations, further complicate the picture. It is currently unclear whether the dichotomous natures of thrombin’s actions are the result of activation of disparate pathways or the alternate expressions of a single mechanism. With further investigation into the mechanisms of thrombin-induced neurological damage and TPC, important therapeutic modalities could be discovered.

9.2.2 Erythropoietin

Erythropoietin (EPO), an endogenous cytokine produced primarily in the peritubu- lar cells of the kidney, functions as an important component of erythropoiesis. By inhibiting apoptosis of erythrocyte precursors, EPO increases red blood cell production, leading to its current usage in treating severe anemia (Eid and Brines 2002 ) . Studies have also demonstrated the expression of EPO and EPO receptor genes in the brain (Marti et al. 1996) , expression that is upregulated following ischemic injury (Sirén et al. 2001 ) . 9 Pharmacologic Preconditioning 219

Endogenous expression of EPO is thought to play a major role in the neuroprotective effects of ischemic preconditioning (IPC). In vitro studies demonstrated that astrocytes, when subjected to ischemic stress, release EPO as a form of paracrine signaling, leading to increased neuronal resistance to subsequent ischemia (Prass et al. 2003 ) . Similar to its role in erythropoiesis, EPO is thought to mediate neuroprotection through its anti-apoptotic actions. This is mediated through EPO receptors and the Janus tyrosine kinase 2 pathway, eventually leading to phosphorylation of BAD and inhibition of apoptosis (Ruscher et al. 2002 ) . The protective effect of EPO is supported by in vivo studies demonstrating increased brain injury following infusion of exogenous soluble EPO receptors that act to “scavenge” endogenous EPO and attenuate its neuroprotective actions (Eid and Brines 2002 ) . In addition to its direct action on neurons, EPO has been shown to have numerous other protective effects on the central nervous system. Exposure to EPO signifi cantly increases levels of glutathione peroxidase, an important scavenger of harmful free radicals (Kumral et al. 2005) . EPO acts to inhibit recruitment of infl ammatory cells and decrease production of varying proinfl ammatory cytokines (Villa et al. 2003) . Administration of EPO has also been shown to have a protective effect on endothelial cells in ischemic injury and appears to improve hemodynamics through stimulation of angiogenesis and prevention of bloodÐbrain barrier compromise (Hasselblatt et al. 2006 ) . EPO is attractive as a therapeutic agent for multiple reasons. When given for short periods, EPO is relatively safe and well tolerated (Dawson 2001 ) . Neuroprotective effects are seen within minutes (Ruscher et al. 2002 ) and can last for up to 3 days even without continuous dosing (Dawson 2001 ) . The ability of EPO to penetrate the bloodÐbrain barrier (Eid and Brines 2002 ) , thus allowing for systemic administration, is also extremely appealing. Though initial clinical trials of EPO in ischemic stroke were promising (Ehrenreich et al. 2002 ) , more recent phase III studies demonstrated no therapeutic bene fi t and increased mortality in the treatment arm (Ehrenreich et al. 2009) Ð possibly secondary to concomitant administration of tissue plasminogen activator.

9.2.3 Deferoxamine

Deferoxamine, a compound originally derived from the bacterium Streptomyces pilosus, is a potent iron chelator (Keberle 1964 ) . When deferoxamine comes into contact with ferric iron, the two molecules form an extremely stable complex, thus facilitating iron excretion (Keberle 1964 ) . Used for decades in the treatment of iron overload secondary to poisoning, inherited storage diseases, and transfusion-dependent anemia, the drug is well tolerated with a relatively benign side effect pro fi le (Selim 2009 ) . Iron has been shown to play a signi fi cant role in brain injury following both ischemic and hemorrhagic stroke (Hua et al. 2003, 2006; Palmer et al. 1994 ) . Much of this is thought to be secondary to the generation of free radicals, with iron 220 J. Guan et al. catalyzing the conversion of peroxide and superoxide to reactive hydroxyl radicals through the Haber-Weiss reaction (Hua et al. 2006 ) . These free radicals cause extensive oxidative stress, leading to lipid peroxidation and increased excitotoxicity (Selim 2009 ) . The role of iron in free radical-induced damage has been demonstrated by studies showing a strong correlation between regional iron levels in the brain and oxidative damage (Palmer et al. 1994 ) . Iron may also play a part in long-term de fi cits following hemorrhagic stroke, with elevated levels seen in animal models of intracerebral hemorrhage months after the initial event (Hua et al. 2006 ) . The neuroprotective effects of deferoxamine are likely accomplished through a variety of mechanisms. At a basic level, deferoxamine administered subcutaneously can penetrate the bloodÐbrain barrier and act to inhibit the iron-mediated free radical formation described above (Nakamura et al. 2003 ) . Deferoxamine reduces acute edema formation, neurological de fi cits, and levels of APE/Ref-1 Ð a marker of oxidative DNA damage Ð in rat models of intracerebral hemorrhage (Nakamura et al. 2003) . It is also a direct scavenger of free radicals, reacting with hydroxyl radicals and possibly reducing their harmful effects (Halliwell 1989 ) . Administration of deferoxamine has also been shown to signifi cantly increase binding of hypoxia-inducible transcription factor 1 (HIF-1) to DNA (Prass et al. 2003 ) . HIF-1 has been shown to play a large role in cellular oxygen homeostasis and vascularization (Semenza 2000) . In addition, deferoxamine can also inhibit prolyl 4-hydroxylase activity which may lead to protection from oxidative-stress-induced cell death (Siddiq et al. 2008 ) .

9.2.4 Erythromycin

Erythromycin, an antibiotic of the macrolide family fi rst isolated in 1952 from Streptomyces erythreus , is regularly used to treat a wide variety of gram-positive infections (Mc Kendrick 1979 ) . Erythromycin’s antibiotic function derives from its ability to disrupt bacterial protein synthesis by binding to the 50S subunit of bacterial ribosomes (Straughan 1978 ) . Due to its relatively benign side effect pro fi le and its spectrum of coverage, erythromycin is commonly used in the treatment of respiratory infections and in the treatment of patients with penicillin allergy (Straughan 1978 ) . The ubiquitous nature of antibiotic use in current clinical practice, along with their well-described safety profi les, makes erythromycin and other antibiotics extremely attractive as preconditioning agents. In vivo studies in rats have demonstrated myriad bene fi ts of preconditioning using erythromycin prior to ischemic insults. These include reductions in hippocampal and parietal cortex neuronal loss, and improved scores on tests of neurologic function (Brambrink et al. 2006 ) . Signi fi cant improvements were seen following single doses of erythromycin prior to ischemia and benefi ts peaked with administration 12 h prior to injury (Brambrink et al. 2006 ) . The bene fi cial effects of preconditioning with erythromycin are thought to be secondary to downregulation of gene expression following ischemia. Specifi cally, pretreatment with erythromycin appears to signi fi cantly reduce the induction of cytokines, chemokines, and iNOS Ð potent mediators of infl ammatory neuronal damage (Koerner et al. 2007 ) . 9 Pharmacologic Preconditioning 221

9.2.5 Opioids

With a history of usage predating written recording, opioids are one of the oldest drug classes in use today (Lehmann 1997 ) . Most commonly prescribed for analgesia, opioids function by interacting with a variety of opioid receptors scattered throughout the central and peripheral nervous system, resulting in a range of effects from inhibition of nociceptive signal transmission to respiratory depression (Lehmann 1997 ) . A variety of studies have shown signifi cant neuroprotective effects with morphine preconditioning. Lim et al. demonstrated decreased levels of Purkinje cell death in rat cerebellar slices exposed to ischemic conditions 1 h after pretreatment with morphine (Lim et al. 2004 ) . In vivo studies in rats also suggested that opioids may have a delayed preconditioning effect Ð infarct volume was reduced following middle cerebral artery occlusion in rats treated with morphine 24 h prior to the ischemic event (Lehmann 1997 ) . The mechanism of opioid preconditioning likely involves a number of pathways and has not yet been fully elucidated. Studies with various opioid antagonists suggest that the receptors activated in acute and delayed preconditioning may be different. Naloxone, an d -opioid receptor antagonist, appears to abolish acute preconditioning, while b -FNA, an m -opioid receptor antagonist, abolishes delayed neuroprotection (Zhao et al. 2006 ) . Opioid preconditioning also appears to have protective effects on important non-neuronal components of the CNS. Preconditioning with morphine reduces lipopolysaccharide- and interferon-mediated injury to microglial cells, important immune mediators in the brain (Gwaks et al. 2010 ) . Another factor in opioid-induced neuroprotection may be an improvement in blood fl ow to ischemic regions Ð rats preconditioned with fentanyl had signifi cantly increased cerebral blood fl ow in ischemic areas compared to controls (Chi et al. 2010 ) .

9.2.6 Lipopolysaccharide

Lipopolysaccharide (LPS) is an integral component in the cell wall of gram-negative bacteria. A potent endotoxin, the body’s violent immune reaction to LPS Ð involving cytokine production, complement activation, and coagulation Ð plays a central role in the development of gram-negative sepsis (Davies and Cohen 2011 ) . Even low doses of LPS can lead to dramatic symptoms (Davies and Cohen 2011 ) , and a higher concentration of the toxin in septic patients correlates with higher mortality rates (Davies and Cohen 2011) . Due to the deleterious effects of LPS and its potent in fl ammatory effects, studies were undertaken seeking to quantify the contribution of LPS to brain injury following stroke (Ahmed et al. 2000 ) . Surprisingly, the results of these experiments suggested that LPS preconditioning in fact decreased subsequent brain injury in animal models of ischemic stroke (Ahmed et al. 2000 ) . One of the possible mediators of LPS-induced neuroprotection is thought to be tumor necrosis factor alpha (TNF-a ). A key player in the in fl ammatory response, TNF-a is expressed in the brain following a wide variety of insults including infection 222 J. Guan et al. and infarction and has been shown to be produced by astrocytes following LPS exposure (Tuttolomondo et al. 2008 ) . Though high concentrations of TNF-a have been demonstrated to be correlated with worse outcomes following stroke (Tuttolomondo et al. 2008 ) , more recent data suggests that it can also induce neuroprotection, possibly by reducing free radical-mediated damage via induction of manganese superoxide dismutase and inhibition of apoptosis (Mallard and Hagberg 2007 ) . LPS may also play a role in improving blood fl ow following infarction, with one study showing improved blood fl ow in the preinfarct area minutes and days after occlusion (Furuya et al. 2005 ) . This is thought to be mediated through increased expression of endothelial NOS, an important player in endothelial regulation (Furuya et al. 2005 ) .

9.3 Future Directions

PPC holds great promise for reducing the morbidity and mortality associated with ischemic and hemorrhagic stroke. Though research is ongoing, many barriers remain to implementation of PPC in the clinical setting. Many potential preconditioning agents, including some discussed above, have very narrow therapeutic indices. This is further complicated by the bloodÐbrain barrier compromise often seen in stroke, a factor that can make precise systemic dosing of agents diffi cult, and may inherently alter the toxicity of some agents (Chen et al. 2010 ) . Though many PPC agents have shown signifi cant benefi ts in animal models, most have not yet been studied in clinical settings and some, like erythropoietin, have had mixed results in limited trials (Ehrenreich et al. 2009 ) . Finally, the fact that neuroprotection derived from PPC can often take hours to days to develop due to the need for new protein synthesis is also problematic, as long- term maintenance on many of these substances is unrealistic and potentially dangerous (Gidday 2010 ) . For these reasons, PPC remains an area in which further research is needed. The bene fi ts of these agents, both for patient care and scienti fi c inquiry, are enormous, but to realize this potential, many vital questions must fi rst be answered.

References

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Giuseppe Pignataro , Ornella Cuomo , and Antonio Vinciguerra

10.1 Introduction

Surgical methods to induce preconditioning are among the most common techniques used to experimentally induce ischemic preconditioning. In effect, when someone approaches this ﬁ eld of research, the most intuitive method to induce preconditioning is to use a short subliminal occlusion of the artery that will undergo subsequent harmful occlusion. Despite the relatively simpleness of the idea, the deﬁ nition of the surgical techniques that can be used in animals to induce brain ischemic preconditioning is not that easy. In this chapter, we will review the most relevant surgical methods used to induce brain preconditioning. The authors desire to apologize from now on for all the missing citations that, merely for inattention, will not appear in this chapter.

10.2 Surgical Methods to Induce Brain Preconditioning

10.2.1 Ischemic Preconditioning

The ability to withstand, to respond to, and to cope with ongoing stress is a fundamental property of all living organisms. The fate of the brain tissue after cerebral ischemia is determined by the degree and duration of ischemia, and even without preconditioning, resident brain cells naturally respond to brain ischemia by mobiliz- ing a series of defensive mechanism and organize responses to mitigate cell injury and death. If the subthreshold noxious stimulus is too mild or negligibly mild, it

G. Pignataro, M.D., Ph.D. () ¥ O. Cuomo, Ph.D. ¥ A. Vinciguerra, Ph.D. student Division of Pharmacology, Department of Neuroscience, School of Medicine , “Federico II” University of Naples , Via Pansini, 5 , 80131 Naples , Italy

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 225 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_10, © Springer Science+Business Media New York 2013 226 G. Pignataro et al. may not induce any response, whereas if it is suf ﬁ cient enough, it may serve as a preconditioning trigger, or if it is too severe, over the threshold, it may permanently injure tissues. The peculiarity of preconditioning (PC) stimulus is that it is sublethal. In the scenario of ischemic tolerance, PC stimulus prepares the brain for subsequent injurious ischemic injury. Danger signal evoked in the brain by the stressing preconditioning stimulus induces complex endogenous protective mechanisms resulting in a latent protective phenotype. When the lethal ischemic insult is applied within this latent protective phenotype, a separate set of responses are triggered that constitute ischemia-tolerant phenotype, which strikingly differs from the unprimed or unpreconditioned brain’s phenotype. Therefore, thanks to preconditioning induction, the negative outcome of the brain cells induced by focal or global ischemia is shifted from death to survival.

10.2.2 Focal Ischemia Versus Global Ischemia

Focal ischemia, which accounts for a majority of strokes, occurs when an artery supplying a region of the brain is occluded, either by an embolus, which is generally material broken off from a plaque in a large artery or a thrombus in the heart, or by a thrombus or platelet plug which is generated directly in the affected artery. While focal ischemic insults generally refl ect the distribution of vascular supply to a region, the area of infarction is typically less than the entire distribution of the occluded artery due to the presence of collateral circulation at the borders of the region supplied by the occluded vessel. The ultimate area of infarction will depend on the duration and degree of vascular occlusion and on the availability of collateral blood supply. The region of the brain supplied uniquely by the occluded artery develops the most severe injury, termed the ischemic core, while the rim of tissue surrounding the core, termed the penumbra, which has the bene fi t of some maintained blood fl ow supplied by collateral circulation, sustains less severe injury (Lo 2008 ) . Focal ischemia may also accompany other acute brain insults, such as intracerebral hemorrhage or trauma. Reversible global ischemia, such as occurs during cardiac arrest and resuscitation, re fl ects a transient loss of blood fl ow to the entire brain and generally results in the death of certain selectively vulnerable neuronal populations. Hypoxia accompanies ischemic insults but may also occur without loss of blood fl ow, for example, during drowning or carbon monoxide poisoning. Hypoglycemia produces brain injury that has several features in common with ischemic injury. Neurons are more susceptible than glial cells to ischemia, hypoxia, or hypoglycemia, and the phylogenetically newer regions of the brain, including the cortex and cerebellum, are affected to a greater extent of damage than the brainstem. Several other cell types, such as astrocytes, oligodendroglias, and endothelial cells, show a phenotype that is more resistant to ischemic conditions, compared to neurons (Calabresi et al. 2000 ) . However, the selective vulnerability of certain neurons cannot be explained by simply considering the vascular distribution, in fact, the juxtaposition of relatively vulnerable and relatively resistant neuronal populations 10 Surgical Methods to Induce Brain Preconditioning 227 within a single vascular distribution suggests that intrinsic tissue factors contribute heavily to ischemic neuronal vulnerability. For example, pyramidal neurons in the CA1 sub fi eld of the hippocampus die after 5Ð10 min of global ischemia, while neurons in the nearby CA3 region are preserved. Populations of neurons that are selectively vulnerable to ischemia include cortical pyramidal neurons, cerebellar Purkinje cells, hippocampal CA1 pyramidal neurons, and subpopulations in the amygdala, striatum, thalamus, and brainstem nuclei (Calabresi et al. 2000 ) .

10.2.3 Experimental Induction of Focal and Global Ischemia

Several questions come suddenly into the mind of researchers that are going to plan an experimental protocol to study neuroprotection induced by ischemic preconditioning; the ﬁ rst ones to be answered are the following: (1) How to induce surgically ischemia and preconditioning? (2) Which species of animal should be used? Several animal models are currently used to experimentally reproduce cerebral ischemia. Although cats, rabbits, dogs, and nonhuman primates have been used for animal experiments on cerebral ischemia, most of these experiments are usually performed with rodents. The fact that the anatomy of the cerebral vasculature does not differ grossly between rodents and higher species, including humans, supports this choice. Additionally, there exist some pragmatic reasons for giving the preference to rodents: 1. Higher acceptance from social and ethical perspective 2. Lower costs 3. A rather high homogeneity due to inbreeding The focal ischemia can be induced either by transient occlusion or by permanent occlusion. The global ischemia can be induced by occluding the two vertebral arteries (VA) and the two common carotid arteries (CCA), so-called four-vessel occlusion (4-VO), or by occluding the two CCA (2-VO).

10.2.3.1 Focal Ischemia

Focal transient ischemia is generally modeled in mice and rats by occluding the middle cerebral artery (MCA) at its origin using the protocol fi rst described by Longa (Longa et al. 1989 ) . Brie fl y, after isolating the external carotid artery (ECA), a stump is created by cutting it, and a 5.0 (for rats) or a 6.0 (for mice) nylon fi lament is introduced through the ECA stump into the internal carotid artery (ICA) and gently advanced into the circle of Willis until reaching the branching point of the MCA, thereby occluding the MCA. Achievement of ischemia is usually con fi rmed by monitoring regional cerebral blood fl ow in the area of the right MCA by means of a disposable microtip fi ber optic probe (diameter 0.5 mm) connected through a Master Probe to a laser Doppler computerized main unit. The fi lament is 228 G. Pignataro et al.

Fig. 10.1 Transient middle cerebral occlusion procedure. After a midline ventral incision on the neck, the experimental surgical practice to induce tMCAO proceeds through the following steps: ( a ) isolation of the common carotid artery (CCA ) from the vagus nerve, (b ) isolation of the external carotid artery ( ECA) that is cut in order to create a stump, (c ) formation of a micro-hole on the ECA stump, and ( d ) insertion of a nylon fi lament trough the hole created on the ECA into the internal carotid artery (ICA ). The fi lament is gently advanced into the circle of Willis until reaching the branching point of the MCA, thereby occluding the MCA. Achievement of ischemia is usually confi rmed by monitoring regional cerebral blood fl ow in the area of the right MCA by means of a disposable microtip fi ber optic probe (diameter 0.5 mm) connected through a Master Probe to a laser Doppler computerized main unit kept in situ for a time interval of 60Ð180 min. After this time interval, the fi lament is withdrawn and reperfusion allowed (Fig. 10.1 ). Focal permanent ischemia is modeled on the basis of the original protocol fi rstly described by Tamura et al. ( 1981 ) . Briefl y, a 2-cm incision is made verti- cally between the orbit and the ear. Under an operating stereomicroscope, an incision is made to divide the temporal muscle. The left lateral surface of the skull is then exposed by re fl ecting the temporal muscle surrounding the soft tissue. A small window is made just over the visibly identi fi ed middle cerebral artery. Saline solution is applied to the area throughout this procedure to prevent heat injury. The left middle cerebral artery occlusion is performed by electrocoagulation of the artery with a bipolar electrocauterizer. In order to achieve a consistent brain damage, it is important to perform the MCAO as close as possible to its origin, near the circle of Willis. 10 Surgical Methods to Induce Brain Preconditioning 229

Alternatively, permanent focal ischemia can be obtained by introducing a nylon ﬁ lament in the ICA and by advancing it to the origin of middle cerebral artery as described for the induction of transient focal cerebral ischemia model. However, in the permanent model, the ﬁ lament is kept in situ until the animal is euthanized.

10.2.3.2 Global Ischemia

As regards global ischemia models, in the beginning of the 1980s, three models of global ischemia in rodents were described which are mainly in use today: 1. The four-vessel occlusion (4VO) model in the rat where the vertebral arteries are permanently occluded and the carotid arteries are clamped transiently for 10Ð30 min (Pulsinelli et al. 1982a ) . 2. The two-vessel occlusion plus hypotension (2VO + hypo) model in the rat which is produced by transient occlusion of both common carotid arteries and simultaneous arterial hypotension (ca 50 mm HG) due to withdrawal of blood (Smith et al. 1984 ) . 3. In gerbils, a transient occlusion of both common carotid arteries (2VO) is suf fi cient to induce a global cerebral ischemia as these animals display an incomplete circle of Willis (Kirino 1982 ) . In addition to these three models of global ischemia, two other less commonly used models have been established which are used less often: (1) the so-called three- vessel occlusion that consists in the occlusion of the basilar artery and of the two CCA (Thal et al. 2010) and (2) the cardiac resuscitation model, obtained by IV injection of KCl. After 10-min cardiac standstill, cardiopulmonary resuscitation is initiated by administration of epinephrine, ventilation with 100% oxygen, and chest compressions (Ko fl er et al. 2004 ) . The production of a global cerebral ischemia mouse model has been diffi cult. Attempts have been made to apply the rat global ischemia models to the mouse, but the success of these attempts appears to be limited. The resulting high level of mortality, inconsistency of brain damage, and frequent complications such as seizures have limited the success of mouse global ischemia. With respect to histopathology, blood fl ow, and metabolism, the three main models of global ischemia have been thoroughly characterized (Pulsinelli et al. 1982a, b ; Smith et al. 1984 ; Kirino et al. 1991 ) . Selective neuronal damage can be observed in the CA1 sector of the hippocampus (Pulsinelli et al. 1982a ; Kirino et al. 1991 ) . It develops within 48Ð72 h following ischemia and is thus termed delayed neuronal death (Kirino et al. 1991 ) . A duration of 3 min of vessel occlusion in gerbils and of 10 min in rats is suffi cient to induce this hippocampal damage (Pulsinelli et al. 1982a; Kirino et al. 1991) . Increase up to 20 min of ischemia in rats results in an increase of neuronal damage in the CA1 sector which then is almost complete (Pulsinelli et al. 1982a) . Furthermore, following an ischemia duration of 20 or 30 min, neuronal damage can also be observed in the striatum and cortex (Pulsinelli et al. 1982a ) . Striatal neuronal damage is mainly located in the dorsolateral part and 230 G. Pignataro et al. affects medium-sized spiny striatal projection neurons (Chesselet et al. 1990 ) . Within the cortex, mainly the layers 3, 5, and 6 display neuronal damage (Pulsinelli et al. 1982a ) . In these regions of selective neuronal damage, activation of microglia and astrocytes has been observed (Morioka et al. 1992 ) . Within the hippocampus, this glial activation precedes the neuronal damage and persists up to 4 weeks. In addition to these neuropathological changes, functional alterations at the level of glucose metabolism, second messenger systems, and neurotransmitter receptors have been demonstrated (Kozuka et al. 1993 ) . These functional changes are observed in the regions of selective neuronal damage where they partially precede the neuronal degeneration. Furthermore, several other regions which are devoid of ischemic neuronal damage display functional changes. These changes seem to be due either to synaptic connections between the affected region and a region with ischemic neuronal damage or to a lower degree of reduced cerebral blood fl ow which does not result in neuronal degeneration but induces these functional alterations.

10.2.4 Ischemic Preconditioning

It does not matter whether the dangerous ischemia is focal or global; the subliminal stimulus able to shift brain cells to a tolerant state can be represented either by a focal ischemia or by a global ischemia. Beside the kind and the duration of the preconditioning stimulus, in the de fi nition of the surgical protocol used to induce ischemic preconditioning, it is very important also to de fi ne the duration of the time interval between the preconditioning stimulus and the subsequent ischemic insult. Overall, the parameters that must be taken into consideration in setting a surgical strategy to induce ischemic preconditioning are: 1. The typology of ischemic insult, focal versus global 2. The duration of the preconditioning stimulus and the possible number of ischemic cycles 3. The time interval between preconditioning and injurious stimulus 4. The typology and the duration of the injurious stimulus, transient versus permanent focal ischemia or global ischemia Therefore, it is possible to classify the different preconditioning/harmful ischemia situations as focal/focal (Table 10.1 ), focal/global, global/focal, or global/global (Table 10.2 ), where the fi rst term indicates the typology of preconditioning stimulus and the second term indicates the typology of harmful ischemia (Fig. 10.2 and Fig. 10.3 ) .

10.2.4.1 Focal Preconditioning/Focal Permanent Harmful Ischemia

Transient focal-permanent focal transient occlusion of the middle cerebral artery (MCA) by intraluminal insertion of a nylon monoﬁ lament is the most common 10 Surgical Methods to Induce Brain Preconditioning 231

) 2009 ) , 1996 ) , ) ) 2002 ) 2008, ) ) and ) ) , 2003 2011 2007 2011 1998 2005 2011 2008 2003 al. ( and Chen et al. ( ( and Chen et al. Lee et al. ( ( Lee et al. Alkayed et al. ( ( Alkayed et al. Pignataro et al. Naylor et al. ( ( Naylor et al. Rats Mice Gesuete et al. ( Rats Barone et al. ( (pMCAO) (pMCAO) (pMCAO) (pMCAO) Typology of the duration Typology of the injurious stimulus Species References 3 days 120 min tMCAO 30 min Rats 60 min tMCAO Hao et Mice ( Atochin et al. Time Time interval between PC and injurious stimulus MCAO MCAO separated 5 min MCAO by 10 min reperfusion 15 min tMCAO 15 min tMCAO 3 days 60 min tMCAO Mice ( Lusardi et al. 7 min tMCAO 7 min tMCAO 2 periods of 5 min 4 days 3 days Permanent focal ischemia 30 min tMCAO 90 min tMCAO Mice 3 days ( Zhang et al. 100-min tMCAO Rats Lusardi et al. 10 min tMCAO 10 min tMCAO 1Ð3 periods of 10 min 3 days 1 h tMCAO of 5 min or three cycles SHR, Duration of PC stimulus and the possible number of ischemic cycles; 10 min tMCAO 1, 2, and 7 days Permanent focal ischemia

Review of the most common surgical strategies to induce focal ischemic preconditioning to induce focal ischemic preconditioning strategies of the most common surgical Review focal focal focal focal focal focal focal focal Transient Transient focal-transient Transient Transient focal-permanent Transient focal-transient Transient focal-transient Table Table 10.1 of Ischemic Typology Insult Transient Transient focal-transient Transient focal-transient Transient focal-transient Focal-Focal Transient focal-permanent 232 G. Pignataro et al. ) ) ) ) 2005 ) 1994 2009 2010 2004

Rats Glazier et al. ( P7 rats ( Lee et al. (4-VO) (4-VO) hypoxia Typology of the Typology duration of the injurious stimulus Species References 1 min or day 17 min BCCAO Mice ( Rehni et al. 8 min Ð 2 days BCCAO Rats ( Mishima et al. Time interval between interval Time PC and injurious stimulus 24 h 2 h of 8% oxygen BCCAO followed by followed BCCAO 1 min of reperfusion electroconvulsive shock electroconvulsive (ECS) application artery ligation 20 min tMCAO 20 min tMCAO 24 h 10 min Global ischemia Duration of PC stimulus and the possible number of ischemic cycles; 1 min Three cycles 5 min BCCAO 5 min BCCAO 24 h 20 min tMCAO Mice ( et al. Faraco

Review of the most common surgical strategies to induce focal ischemic preconditioning with the use of stroke global model global model to induce focal ischemic preconditioning with the use of stroke strategies of the most common surgical Review global (BCCAO) (BCCAO) focal Focal – Global Focal Transient focal-transient ECS-Global ischemia ECS-Global ischemia Single or repetitive Typology of Ischemic Insult Typology Global – Two-vessel occlusion Neonatal Hypoxia Neonatal Hypoxia Unilateral common carotid Table Table 10.2 Global-Focal Transient global-transient 10 Surgical Methods to Induce Brain Preconditioning 233

Typology of Ischemic Experimental Procedure Insult

Focal-Focal

10’ 1-2-7 days Transient focal-Permanent pMCAO focal Rats tMCAO REP

7’ 4 days Transient focal-Permanent pMCAO tMCAO REP focal Mice

5’ 5’ 5’ 3 days 90 ‘ tMCAO Transient focal-Transient focal tMCAO REP tMCAO REP Mice

Transient focal-Transient focal 10’ 3 days Adult male spontaneously tMCAO REP 1h tMCAO hypertensive Rats

10’ 10’ 10’ 10’ 10’ 3 days Transient focal-Transient focal 120 ‘ tMCAO tMCAO REP tMCAO REP tMCAO REP Rats

30’ 3 days 100’ tMCAO Transient focal-Transient focal tMCAO REP Rats

10’ 3 days 60’ tMCAO Transient focal-Transient focal tMCAO REP Mice

5’ 10’ 5’ 10’ 5’ 30’ 60 ‘ tMCAO Transient focal-Transient focal tMCAO REP tMCAO REP tMCAO REP Mice

Fig. 10.2 Schematic illustration of the experimental procedures used to induce ischemic preconditioning in the focal/focal models described in this chapter

Typology of Ischemic Experimental Procedure Insult

Global -Global 1’ 1’ 1’ 1’ 1’ 1’ 17 ‘ BCCAO Two-vessel occlusion (BCCAO) BCCAO REP BCCAO REP BCCAO REP

Focal-Global Transient focal-Transient 20’ 24h 10’ Global Ischemia (4-VO) Global Rats tMCAO REP Global-Focal Transient global-Transient 5’ 24h 20’ tMCAO focal Mice BCCAO Neonatal Hypoxia

Neonatal Hypoxia P7 Rat Pups Unilateral 24h 2h 8% Oxygen Hypoxia CCAO ECS-Global ischemia ECS-Global ischemia Single 8 min or 2 days BCCAO or repetiive ECS

Fig. 10.3 Schematic illustration of the experimental procedures used to induce ischemic preconditioning in the models using global ischemia described in this chapter 234 G. Pignataro et al. model to induce focal cerebral ischemic preconditioning in rats (Takano et al. 1997 ; Durukan et al. 2008 ) , and it is also available in mice (Huang et al. 1994 ) . This method was introduced fi rst time in a rat ischemic tolerance experiment, applying 10 min of transient MCA occlusion (tMCAO) as the PC stimulus and permanent MCAO as the fi nal lethal ischemia (Barone et al. 1998 ) . Authors provided a systematic evaluation on the effect of focal PC on permanent focal ischemia. These researchers carried out a series of studies to characterize the temporal pattern of a PC paradigm, to systematically evaluate the importance of protein synthesis in PC-induced IT, and to explore candidate gene expression changes associated with IT. Temporary middle cerebral artery occlusion (MCAO) (10 min) was used as stimulus for PC. Various periods of reperfusion (i.e., 2, 6, and 12 h and 1, 2, 7, 14, and 21 days) were allowed after PC and before permanent MCAO (PMCAO) to establish IT compared with non-PC (sham-operated) rats. Infarct size, forelimb and hind limb motor function, and cortical perfusion have been measured after PMCAO. Hemispheric infarct was signi fi cantly reduced only if PC was performed 1 day (decreased 58.4%), 2 days (decreased 58.1%), or 7 days (decreased 59.4%) before PMCAO. PC signifi cantly reduced neurological defi cits in a manner similar to the reduction in infarct size. The PC stimulus did not produce any signi fi cant brain injury, alter cortical blood fl ow after PMCAO, or produce contralateral cortical neuroprotection. PC stimulus was able to protect the brain in a time window ranging between 2 and 7 days (Barone et al. 1998 ) . This model was applied successfully by others to obtain delayed ischemic tolerance (IT) (Masada et al. 2001; Pradillo et al. 2005) . Repeated brief transient ischemia regimen was also proved as a preconditioning paradigm inducing early IT in mice subjected to permanent focal ischemia (Stagliano et al. 1999 ) . Recently, a successful protection was achieved by subjecting mice to 7-min occlusion of the right middle cerebral artery 4 days before permanent occlusion of the right middle cerebral artery (Gesuete et al. 2011 ) . A modi fi ed model of transient focal PC/transient focal MCAO has been recently described (Naylor et al. 2005 ) . The surgical procedure was performed following the scheme here described: the MCA of adult male spontaneously hypertensive rats was occluded for 10 min for preconditioning and 1 h for ischemia. In some experiments, a 1-h occlusion was induced 3 days after a 10-min occlusion. In brief, the rat is anesthetized with halothane, placed in a stereotaxic frame fi tted with a nose cone with 2% halothane anesthesia. A craniectomy (4 mm in diameter, 2Ð4 mm lateral, and 1Ð2 mm caudal to bregma) is performed with extreme care over the MCA territory using a trephine. The dura is left intact and a laser Doppler fl owmeter probe is placed on the surface of the ipsilateral cortex and fi xed to the periosteum with a 4Ð0 silk suture. The probe is connected to a laser fl owmeter device for continuous monitoring of regional cerebral blood fl ow (rCBF). The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) are exposed through a ventral midline incision. A 3Ð0 monofi lament nylon suture with a rounded tip is introduced into ECA lumen and gently advanced to ICA until rCBF is reduced to 15Ð20% of the baseline. After the desired period of occlusion (10 min or 1 h), the suture is withdrawn to restore the blood fl ow. The wound is sutured and the rat is allowed to recover from anesthesia (Naylor et al. 2005 ) . 10 Surgical Methods to Induce Brain Preconditioning 235

10.2.4.2 Transient Focal Preconditioning/Transient Focal Harmful Ischemia

In the transient focal preconditioning/transient focal harmful ischemia model, both stimuli are induced by transiently occluding the MCA. One (Hao et al. 2003 ; Lee et al. 2007 ) or three times of 10-min transient focal cerebral ischemia protects from subsequent 120 min of tMCAO in rats (Alkayed et al. 2002 ; Chen et al. 1996 ) . Shorter durations (2 and 3 min) of tMCAO were severe enough to induce delayed ischemic tolerance but did not provide early tolerance to transient ischemia (Glantz et al. 2005 ; Puisieux et al. 2004 ) . Transient focal/ focal IT paradigm induced IT also in mice and spontaneously hypertensive rats (Hoyte et al. 2006 ) . In a recent mouse model, delayed tolerance is achieved by inducing two periods of 5-min tMCAO as the PC method against 90-min tMCAO applied in 3 days, but not in 2 or 4 days (Zhang et al. 2008 ) . According to another recent protocol to induce tolerance, rats are subjected to 30 min of tMCAO followed, 3 days later, by 100 min of injurious tMCAO. The protection is already evident if the time interval between the preconditioning stimulus and the injurious stimulus is of 1 day, but it reaches the maximum extent of bene ﬁ t when the harmful stimulus is delivered 3 days after preconditioning. The duration of the protection is lost in 7 days (Lusardi et al. 2011 ; Pignataro et al. 2008, 2009 ) . A similar protocol can be applied to mice, but in that case, the duration of the PC stimulus is 15 min, whereas the duration of the injurious middle cerebral artery occlusion is 60 min. Once again, in order to reach the best protection, the time interval between preconditioning and harmful ischemia must be 3 days (Lusardi et al. 2011 ) . The rapid preconditioning has also been induced in mice. In the preconditioned groups, the ﬁ lament was advanced into the ICA until the origin of the MCA for either 5 min or three cycles of 5 min separated by 10-min reperfusion. Thirty minutes separated the preconditioning from the severe ischemia, induced by 60 min occlusion of the MCA (Atochin et al. 2003 ) .

10.2.4.3 Focal Preconditioning/Global Harmful Ischemia

Brief unilateral occlusion of the MCA induced signiﬁ cant protection from global ischemia in both gerbils (Miyashita et al. 1994 ) and rats (Belayev et al. 1996 ) . Interestingly, transient (20 min) occlusion of the distal MCA protected only ipsilateral parietal cortex of the rat from global ischemia (10 min 4-VO) (Glazier et al. 1994 ) .

10.2.4.4 Global Preconditioning/Focal Harmful Ischemia

Brief global ischemia can protect from both subsequent transient and permanent focal ischemia (Simon et al. 1993 ) . Chiarugi et al. report that 5-min bilateral common carotid artery occlusion (BCCAO) in the mouse prompted reduction of infarct volume triggered 24 h later by 20-min middle cerebral artery occlusion (Faraco et al. 2009 ). 236 G. Pignataro et al.

10.2.4.5 Global Preconditioning/Global Harmful Ischemia

Kitagawa and coworkers ﬁ rstly described the phenomenon of ischemic tolerance in the brain (Kitagawa et al. 1990 ) . Cerebral ischemia was produced in gerbils by occlusion of both common carotids for 5 min; this experimental approach consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. An ischemia duration less than 2 min is able to deplete high-energy phosphate compounds and to perturb the protein synthesis, but never causes neuronal necrosis, and therefore has been chosen as mild ischemic treatment. Single 2-min ischemia performed 1 or 2 days before 5-min ischemia exhibited only partial protective effects against delayed neuronal death. However, two 2-min ischemic treatments at 1 day intervals 2 days before 5 min of ischemia exhibited drastically complete protection against neuronal death. The duration and intervals of ischemic treatment, enough to perturb cellular metabolism and cause protein synthesis, were needed respectively, because neither 1-min ischemia nor 2-min ischemia exhibited protective effects. In a recent paper, a new model of global PC/global ischemia has been described in mice. In this model, adult Swiss albino mice of either sex were anesthetized using chloral hydrate (400 mg/kg, i.p.). A midline ventral incision was made in the neck to expose right and left common carotid arteries, which are isolated from surrounding tissue and vagus nerve. A cotton thread was passed below each of the carotid artery. Global cerebral ischemia was induced by occluding the carotid arteries. After 17 min of global cerebral ischemia, reperfusion was allowed for 24 h. The incision was sutured back in layers. The sutured area was cleaned with 70% ethanol and sprayed with antiseptic dusting powder. The animals were shifted individually to their home cage and were allowed to recover overnight. Behavioral assessment of the animals was done both immediately before as well as 24 h after the surgery. During surgery, the animals were kept on a heating pad in order to maintain the body temperature so as to avoid the effect of temperature variations on the ﬁ nal results. In the prior ischemic preconditioning episode, the carotid arteries are occluded for a period of 1 min followed by 1 min of reperfusion time. Three such cycles of ischemia and reperfusion are allowed immediately before (for acute preconditioning) and 24 h before (for delayed preconditioning) the bilateral carotid artery occlusion, performed for 17 min (Rehni et al. 2010 ) . The protection achieved with this method is very relevant as both rapid and delayed preconditioning are able to produce a reduction in the infarct volume of more than 50% (Rehni et al. 2010 ) .

10.2.5 Other Surgical Methods to Induce Preconditioning

In recent years, a growing interest in the preconditioning fi eld has given the input to several studies and generated numerous papers and new models of PC/ischemia. Beside the myriad of new models proposed, it is interesting to underline the PC in neonatal animals. To settle up this model in P7 rat pups, unilateral common carotid artery ligation followed 1 h later by 8% oxygen hypoxia for 2 h was performed. This 10 Surgical Methods to Induce Brain Preconditioning 237 hypoxic/ischemic stimulus produces selective damage in the hemisphere ipsilateral to the occluded artery that resembles HI damage to the human neonatal brain. To examine whether the carotid artery ligation preconditioning could be established in a time-dependent manner, the carotid artery ligation was performed in postnatal day 7 rat (P7) 24 h (24-h ligation group), 6 h (6-h ligation group), or 1 h (1-h ligation group) before exposing them to 2 h of 8% oxygen hypoxia. The animals were anesthetized with 2.5% halothane (balance, room air), and the right common carotid artery was surgically exposed and permanently ligated with 5Ð0 surgical silk. After surgery, the pups were returned to the dam for a 1-, 6-, or 24-h recovery before hypoxia. On P7, the three groups were placed in airtight 500-ml containers partially submerged in a 37¡C water bath through which humidifi ed 8% oxygen (balance, nitrogen) was maintained at a fl ow rate of 3 L/min for 2 h. After completion of hypoxia, the rat pups were returned to their cage. When the carotid artery ligation was performed 24 h before hypoxia, a remarkable neuroprotection was achieved, thus showing that the preconditioning phenomenon can be induced in neonatal mice by modifying the time interval between preconditioning stimulus and harmful stimulus. Notably, the same stimulus represented by the ligation of the carotid artery applied 1 h before subjecting neonatal rats to hypoxia was not able to induce any protection (Lee et al. 2004 ) . Another model has been proposed in 2005 by Mishima and coworkers (Mishima et al. 2005 ) . They demonstrated that electroconvulsive shock (ECS) can be used as a preconditioning stimulus in what is commonly called cross-protection. In particular, both single and repetitive ECS application confers neuroprotection from subsequent global ischemia induced in rats by bilateral common carotid artery occlusion (BCCAO) (Winston et al. J Neurochem 1990 ). Single ECS received ECS only 2 days before 8-min global ischemia, whereas repetitive ECS group received ECS once a day for 9 consecutive days until 2 days before global ischemia induction. No differences were found in the neuroprotection induced by the two experimental procedures.

10.2.6 Conclusions

In conclusion, the past two decades have provided interesting insights into the mechanisms and potential applications of ischemic tolerance in the brain. However, in order to have consistent results and valid indications on the directions to follow for developing new effective treatment in stroke therapy, it is important to choose the right experimental model. Up until now, the ischemic preconditioning fi eld is populated by a myriad of animal models. From what appeared in the scienti fi c literature, it is now well established that delayed preconditioning is more effective than rapid preconditioning in terms of neuroprotection. In addition, the best time interval between preconditioning induction and harmful stimulus ranges between 3 and 7 days. As regards the duration of the preconditioning stimulus, it is usually one quarter of the harmful ischemia. In fact, for the focal/ 238 G. Pignataro et al. focal model in rats, PC is represented by 30-min MCAO whereas harmful ischemia is around 120 min, while in mice, 15 min of MCAO represents the PC and 60 min of MCAO the harmful ischemia. The correct execution of the experimental protocol is of fundamental importance to achieve the best results and to compare the fi ndings of different laboratories; therefore, an accurate examination of the different preconditioning models is mandatory before starting any new project.

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Kunjan R. Dave , Hung Wen Lin , and Miguel A. Perez-Pinzon

11.1 Introduction

Cardiopulmonary arrest (CA) is one of the major causes of global cerebral ischemia. Most patients that are resuscitated after CA die from brain injuries, while relatively small proportion of patients resume their former lifestyles (Krause et al. 1986 ) . Brain injury following global cerebral ischemia induced by CA remains a key problem. To circumvent the detrimental effects of CA, ischemic preconditioning (IPC) mimetics have the potential to activate endogenous neuroprotective pathways, which can protect the brain against CA-induced global cerebral ischemia. IPC refers to the ability of a brief (“sublethal”) ischemic episode, followed by a period of reperfusion, to increase an organ’s resistance to injury (also known as ischemic tolerance). Induction of ischemic tolerance has gained wide acceptance as a robust neuroprotective mechanism against conditions of metabolic stress in many organs, such as the brain, heart, and kidney among others (Alkhulaiﬁ et al. 1993 ; Kato et al. 1992; Kitagawa et al. 1990 ; Lin et al. 1992, 1993; Murry et al. 1986 ; Walker et al. 1993; Peralta et al. 1996; Pudupakkam et al. 1998 ) . In this review, we discuss how the induction of IPC may offer novel therapeutic approaches to protect the human brain against global cerebral ischemic damage.

K. R. Dave (*) • H. W. Lin Cerebral Vascular Disease Research Laboratories, Department of Neurology, Leonard M. Miller School of Medicine , University of Miami , Miami , FL 33136 , USA e-mail: [email protected] M. A. Perez-Pinzon Cerebral Vascular Disease Research, Laboratories, Neuroscience Program, Department of Neurology , Leonard M. Miller School of Medicine, University of Miami , Miami , FL 33136 , USA

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 243 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_11, © Springer Science+Business Media New York 2013 244 K.R. Dave et al.

11.2 Experimental Models

There are several in vivo and in vitro models in which tolerance can be induced against global cerebral ischemia. For studying the effect of ischemic tolerance, it is essential to establish reliable and reproducible in vivo and in vitro models. There are two components in establishing an IPC model. The fi rst is to induce “sublethal” ischemia and second to implement “lethal” ischemia at an appropriate time after induction of “sublethal” ischemia. The goal of “sublethal” ischemia should be to activate neuroprotective pathways without inducing detectable deleterious effects on the target tissue. The duration of sublethal ischemia must be long enough to induce cellular metabolism stress activating neuroprotective pathways. This careful balance must be met as longer durations of ischemia will promote neuronal death. Guidelines for establishing the minimum ischemic duration to induce neuronal death are described previously (Ljunggren et al. 1974 ; Nowak et al. 1985 ) . The duration of sublethal ischemia varies between animal models. For example, 2 min of global cerebral ischemia induced by occlusion of both common carotid arteries plus hypotension is enough to induce ischemic tolerance in the rat brain (Dave et al. 2005 ) . However, just an additional minute turns a sublethal ischemic insult into a lethal one (Kato et al. 1992 ; Nakata et al. 1992 ) . Thus, careful titration of the duration of sublethal ischemia is highly recommended while developing an IPC paradigm. Some investigators use more than one sublethal ischemic insult for induction of ischemic tolerance (Kitagawa et al. 1990) , but repeated sublethal ischemic insults can also be irreversibly damaging depending on their number, intensity, and frequency (Kato and Kogure 1990 ; Kato et al. 1989; Tomida et al. 1987 ) . Similarly, the intensity of lethal cerebral ischemia is also important, as protective pathways induced following a sublethal ischemic insult may not be suf fi cient to induce tolerance against lethal ischemia of any arbitrary intensity (duration). For example, IPC was protective against a 6- or 8-, but not a 10-min, period of lethal ischemia (Liu et al. 1993 ) . The time interval between sublethal and lethal ischemia is also of importance. Neuroprotection against cerebral ischemia is short-lived when the interval between the two insults is minutes to hours (Perez-Pinzon et al. 1997 ) . In contrast, long-lasting neuroprotection can be induced by a relatively longer duration between two insults (Corbett and Crooks 1997 ) . If IPC is administered via global cerebral ischemia, it is common to use a sublethal ischemia dose of 2–3 and 6–10 min of ischemia as a lethal episode. In this case, the duration between the two insults would range from minutes to days to achieve neuroprotection (Kitagawa et al. 1990 ; Perez-Pinzon et al. 1997 ) . Tolerance against global cerebral ischemia can also be induced by other means. For example, repeated exposure of Mongolian gerbils to hyperbaric oxygen has been shown to induce ischemic tolerance against global cerebral ischemia (Wada et al. 1996 ) . Mild inhibition of mitochondrial electron transport chain complex II by 3-nitropropionate lowered cell death in the CA1 region of the hippocampus following 11 Tolerance Against Global Cerebral Ischemia: Experimental Strategies... 245 global cerebral ischemia (Sugino et al. 1999 ) . Other factors such as activation of adenosine receptors A1 (A1AR) also induced hippocampal neuroprotection against global cerebral ischemia (Blondeau et al. 2000 ) . Apart from classical IPC, a short period of sublethal ischemia and reperfusion in various organs/parts such as the kidney, intestines, limbs, and liver can induce ischemic tolerance in the organ/part itself or in another organ/part as well (i.e., the heart) (Ates et al. 2002; Birnbaum et al. 1997; Brzozowski et al. 2004; Gho et al. 1996; Harkin et al. 2002; Kuntscher et al. 2002; Oxman et al. 1997; Przyklenk et al. 1993 ; Waldow et al. 2005 ; Xia et al. 2003 ) . This is now referred to as “remote ischemic preconditioning.” For example, sublethal ischemia in a remote organ/ part, such as intestines and hind limbs, can induce ischemic tolerance against global cerebral ischemia (Dave et al. 2006a ; Rehni et al. 2007a, b; Sun et al. 2006 ; Zhao et al. 2004 ) . Another modality for the induction of ischemic tolerance is referred to as ischemic post-conditioning, which can afford protection to the brain against cerebral ischemia. Ischemic post-conditioning can be induced by controlled reperfusion (intermittent or slow reperfusion) following cerebral ischemia preventing ischemic injury in the brain (Zhao 2009 ) . Ischemic post-conditioning can mitigate the development of cerebral ischemic damage induced by global cerebral ischemia when post-conditioning is conducted immediately or at 2 days after ischemia (Zhao 2009 ; Rehni and Singh 2007 ; Wang et al. 2008 ; Burda et al. 2006 ) . Primary neuronal or organotypic slice cultures are commonly used to mimic IPC in vitro (Dave et al. 2005; Arthur et al. 2004; Badaut et al. 2005; Bickler et al. 2005; Bruer et al. 1997 ; Grabb et al. 2002 ; Hassen et al. 2004 ; Khaspekov et al. 1998; Kim et al. 2007 ; Lange-Asschenfeldt et al. 2005 ; Xu et al. 2002 ) . In vitro models are preferred over in vivo models to study molecular mechanisms of action because they help de fi ne intrinsic parenchymal mechanisms of neuroprotection. In vitro models lack cerebral blood fl ow that might otherwise have autocrine/ paracrine effects on the tissue. Another advantage of in vitro cultures is the direct drug access to the tissue rendering ease of pharmacological or environmental manipulations (Xu et al. 2002 ; Raval et al. 2006a ) . The duration of sublethal as well as lethal ischemia is longer in in vitro models. For example, 15 min of oxygen-glucose deprivation (OGD) is necessary to induce ischemic tolerance in rat hippocampal organotypic slices, while 1 h of OGD is required for the induction of ischemic tolerance in mixed cortical neuron/astrocyte cell cultures (Kim et al. 2007 ) . Similarly, the duration of lethal OGD in rat hippocampal organotypic slices and mixed neuron/astrocyte cell culture is 40 and 240 min, respectively. There are certainly advantages and disadvantages to both in vivo and in vitro models of global cerebral ischemia. In vitro models of global cerebral ischemia have been developed to target drug delivery and control environmental conditions useful for mechanistic studies, while in vivo models may more accurately depict actual physiology and function of a particular ischemia paradigm. 246 K.R. Dave et al.

11.3 Mechanisms of Ischemia Tolerance

11.3.1 Triggering Pathways

Several signaling events are activated immediately following an IPC stimulus, which in turn activates a cascade of signaling pathways leading to a state of ischemic tolerance in the tissue. Sublethal activation of N-methyl-D-aspartic acid (NMDA) receptors by IPC-induced release of glutamate is one of the triggering events (Pringle et al. 1999; Raval et al. 2003; Pringle et al. 1997) . Activation of NMDA receptors build-up a critical level of intracellular calcium, which in turn, mediates activation of phospholipase C and subsequently generates diacylglycerol (Nishizuka 1992 ) . In parallel, increased extracellular adenosine levels owing to breakdown of ATP during preconditioning insult activate adenosine A1 receptors (A1ARs) (Heurteaux et al. 1995 ) . Activation of NMDA and A1AR leads to activation of the epsilon protein kinase C (e PKC) pathway via activation of phospholipase C (Lange-Asschenfeldt et al. 2004 ) . We previously showed that e PKC is activated following IPC. We also demonstrated that activation of e PKC is a key to the induction of ischemia tolerance as ePKC inhibition also ameliorated IPC-induced ischemia tolerance (Raval et al. 2003 ; Lange- Asschenfeldt et al. 2004 ; Dave et al. 2001 ; Raval et al. 2007 ) .

11.3.2 Suppressed Excitotoxicity

The role of excitotoxicity in cerebral ischemic damage is well-established. IPC can reduce cerebral ischemia-induced excitotoxicity. IPC-induced presynaptic alterations results in enhanced g -aminobutyric acid (GABA) release together with reduced glutamate release during a subsequent period of lethal ischemia (Dave et al. 2005 ; Grabb et al. 2002 ) . This enhanced release of GABA after IPC was correlated with an increase in glutamic acid decarboxylase (GAD) activity: the predominant pathway of GABA biosynthesis in the brain (Dave et al. 2005 ) . Earlier, using an in vitro model, we observed that IPC signiﬁ cantly enhanced the frequency of GABA A min- iature postsynaptic currents in CA1 pyramidal neurons, suggesting an increase in the number of presynaptic GABA release sites or an increase in release probability (DeFazio et al. 2009 ) . In different models of cerebral ischemia, speciﬁ c activation of either GABAA or GABAB receptors during an otherwise lethal period of ischemia resulted in less ischemic damage (Dave et al. 2005 ; Grabb et al. 2002 ) . These studies suggest that IPC suppresses lethal cerebral ischemia-induced excitotoxicity.

11.3.3 Suppressed In ﬂ ammation

Cerebrovascular infl ammation plays a central role in the pathogenesis of cerebral ischemia. Post-ischemic activation of infl ammation contributes to blood–brain barrier damage, cerebral edema, increased intracranial pressure, and possibly brain 11 Tolerance Against Global Cerebral Ischemia: Experimental Strategies... 247 herniation (del Zoppo et al. 2000 ; de Vries et al. 1997 ) . The important process in cerebrovascular in fl ammation is the induction of proin fl ammatory mediators such as cyclooxygenase-2 (COX-2) (McKay and Cidlowski 1999 ; Nogawa et al. 1998 ) . These infl ammatory molecules also play important roles in ischemia tolerance. Earlier, we observed that IPC resulted in delayed activation of COX-2. Inhibition of COX-2 following IPC abolished IPC-mediated neuroprotection (Kim et al. 2007 ) . Surprisingly, while various other effectors of preconditioning were demonstrated to be activated shortly after IPC, our study demonstrated delayed activation of COX-2 (Kim et al. 2007) . Consistent with our observations, COX-2 expression has been shown to occur 24 h after IPC induction in the heart (Shinmura et al. 2000 ) . The delayed COX-2 expression after IPC emphasizes the need for de novo protein synthesis. IPC, as well as hyperbaric oxygen preconditioning, suppressed global cerebral ischemia-induced COX-2 expression (Cheng et al. 2011; Choi et al. 2006a ) . These studies suggest that COX-2 activity or COX-2-derived prostaglandins may be required for induction of the late protective phase of IPC.

11.3.4 Elevated Neurotrophic Factors

Neurotrophic factors have been implicated in protection of cerebral ischemia- vulnerable regions following IPC. Earlier, we observed that brain-derived neurotrophic factor (BDNF) mRNA was elevated in hippocampal CA1, CA3, and dentate gyrus neurons early after IPC. Besides BDNF, nerve growth factor (NGF) was found to be upregulated transiently after IPC (Truettner et al. 2002 ) . The exact role of these neurotrophic factors in the induction of ischemic tolerance remains to be deﬁ ned. However, considering the diverse and crucial neuronal functions of neurotrophic factors including neuronal survival and neuroplasticity, it is plausible that these factors suppress neuronal death and preserves neuroplasticity following lethal cerebral ischemia (Jiang et al. 2003 ; Castren 2004 ; Semkova and Krieglstein 1999 ) .

11.3.5 Anti-cell Death Pathways

Suppression of cell death pathways appears to hold the most promise in the development of a novel drug to prevent cerebral ischemic damage. The mechanisms of cell death after brain ischemia are complex. It has been postulated that delayed cell death after brain ischemia may result from apoptosis and/or necrosis (Lo et al. 2003 ; Martin et al. 1998 ; Moskowitz and Lo 2003 ) . In both pathways, however, mitochondrial dysfunction appears to play a central role (Fiskum et al. 1999 ; Friberg and Wieloch 2002 ) . Generation of reactive oxygen species, release of cytochrome c , and their consequent effects on mitochondrial dysfunction are considered key factors for the induction of cerebral ischemic injury (Fiskum et al. 1999 ) . Mitochondrial dysfunction such as release of cytochrome c into the cytosol leads to leakage of reactive oxygen species (ROS) from mitochondrial electron transport chain (ETC), 248 K.R. Dave et al. which results in damage not only to mitochondrial DNA, proteins, and lipids but also to nuclear DNA and non-mitochondrial proteins and lipids (Heron et al. 1993 ; Dirnagl et al. 1995 ; Perez-Pinzon et al. 1999 ) . The ROS and activation of other apoptotic pathways are responsible for oxidative DNA damage and fragmentation following lethal cerebral ischemia (Chen et al. 1998 ; Cui et al. 2000 ; Krajewski et al. 1999 ; Liu et al. 1996 ) . IPC prevents ROS-induced cerebral ischemic damage (Frassetto et al. 1999 ) . Upregulation of DNA repair mechanisms such as increased expression of Ku70 (a multifunctional DNA repair protein) is observed following IPC (Sugawara et al. 2001 ) . Lethal ischemia also results in excessive PARP-1 (poly (ADP-ribose) polymerase – 1) activation that functions normally in DNA repair. Overactivation of PARP-1 is considered one of the major causes of ischemic cell death as it depletes stores of nicotinamide adenine dinucleotide (NAD+ ). Using an in vitro model, Garnier and colleagues demonstrate that cleavage of PARP-1 owing to sublethal activation of caspase-3 following IPC prevented excessive PARP-1 activation and leads to neuroprotection (Garnier et al. 2003) . Earlier, we reported that the late, but not early, IPC was able to protect mitochondrial function following lethal cerebral ischemia (Dave et al. 2001 ; Perez-Pinzon et al. 2002 ) . IPC-induced activation of ePKC may play a key role in preserved mitochondrial functions following lethal cerebral ischemia (Dave et al. 2008 ) . IPC also leads to activation of cell survival pathways, such as Akt/protein kinase B (a serine/threonine protein kinase) and extracellular receptor kinase 1/2 (ERK1/2), and inhibition of cell death pathways mediated by c-Jun N-terminal kinases (JNKs), p53 (a tumor suppressor transcription protein), and caspase-9 (Lange-Asschenfeldt et al. 2004 ; Choi et al. 2006b; Garcia et al. 2004 ; Liu et al. 2007; Tomasevic et al. 1999; Zhang et al. 2006, 2007 ) . These mechanisms are summarized in Fig. 11.1 .

11.4 Natural Solutions for Ischemia Tolerance

Some species are naturally resistant to severe hypoxia (low oxygen), anoxia (zero oxygen), or ischemia (zero glucose and oxygen). A variety of coordinated adaptations contribute to the tolerance of cerebral ischemia in these animals. These adaptations are not only limited to their ability to survive these insults, but also protect highly susceptible organs such as the heart and brain from ischemia/hypoxia. Our laboratory has a special interest in hypoxia/anoxia tolerance in turtles and in hibernating arctic ground squirrels (AGS). Anoxia-tolerant turtles avoid hypoxic/anoxic brain injury by delaying anoxic depolarization by either silencing/downregulating NMDA receptor activity by dephosphorylation of the receptor, or by altering potassium homeostasis (Bickler et al. 2000 ; Sick et al. 1982 ) . Turtle brains also have a relatively larger brain glycogen storage capacity leading to continued anaerobic ATP production, resulting in normal metabolic activities during hypoxia (Lutz et al. 1996 ) . Reduced brain energy consumption during metabolic depression is also responsible for extended anoxia survival in turtles (Lutz et al. 1996 ) . Besides anaerobiosis, several groups of genes that are upregulated under anoxia have been identiﬁ ed in turtles which may be 11 Tolerance Against Global Cerebral Ischemia: Experimental Strategies... 249

Fig. 11.1 Neuroprotective mechanisms activated following ischemic preconditioning. These mechanisms include pathways that trigger other neuroprotective signaling, anti-excitotoxic mechanisms, anti-infl ammatory pathways, induction of neurotrophic factors, and anti-cell death pathways. These mechanisms are compartmentalized depending on their cellular/subcellular localization. For detailed description, see Sect. 11.3 . A1AR adenosine receptors, AA arachidonic acid, BDNF brain- derived neurotrophic factor, COX-2 cyclooxygenase-2, CREB transcription factor cAMP response element-binding, Cyt c cytochrome c , ePKC epsilon protein kinase c, ERK1/2 extracellular receptor kinase 1/2, ETC electron transport chain, GABA g -aminobutyric acid, GAD glutamic acid decarboxylase, Ku70 a multifunctional DNA repair protein, NF- k B transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells, NMDA N-methyl-D-aspartic acid, PARP-1 poly (ADP-ribose) polymerase-1, PGE2 prostaglandin E2, PG receptor prostaglandin receptors, PLA2 phospholipase A2, ROS reactive oxygen species, TrkB tyrosine kinases B receptor responsible for anoxia/hypoxia tolerance (Storey 2007 ) . These include mitochondrial DNA-encoded electron transport chain proteins, iron storage proteins, antioxidant enzymes, and neurotransmitter receptors and transporters, among others. Hibernating mammals such as arctic ground squirrels (AGS) also tolerate up to 90% reduction of cerebral blood fl ow during hibernation when the core body temperature is near 0°C (Drew et al. 2002, 2009 ) . During euthermia, AGS can also tolerate up to at least 10 min of cardiac arrest (CA) without any visible brain damage (Dave et al. 2006b, 2009) . Thus, tolerance to acute challenge at warmer body temperatures suggests that AGS tolerate CA even when the infl uence of temperature is excluded. The pathways that are activated during hibernation are described earlier (see review Drew et al. 2007 ) . Understanding the mechanism of natural tolerance to global cerebral ischemia/hypoxia in ischemia-/anoxia-/hypoxia-tolerant animals could aid in the development of novel therapeutics against cerebral ischemia- induced brain damage (Zhou et al. 2001 ) .

11.5 Clinical Applications

CA and stroke are the leading causes of death and disability in the USA. Out of 1.5 million cases of myocardial infarction annually, half of these patients experience sustained CA. Despite a quick response to such emergencies and better defi brillation 250 K.R. Dave et al. procedures, 83% of resuscitated patients die within the fi rst 6 months and only 3–13% of patients survive with adequate brain function thereafter (Krause et al. 1986; Brain-Resuscitation-Clinical-Trial-II-Study-Group 1991 ) . Brain damage following CA appears to be a major limitation in patient survival. Epidemiological studies suggest that patients with previous myocardial infarction, lower left ventricular ejection fraction, hypertension, heavy coffee consumption, diabetes mellitus, and cigarette smoking have increased risk for CA (de Vreede-Swagemakers et al. 1999 ; Khot et al. 2003 ; Diabetes Statistics 2011 . American Diabetes Association ) . IPC offers novel therapeutic targets to protect the brain in patients with high risk for CA. Cardiopulmonary bypass is another condition where global cerebral ischemia is expected. With improved techniques, mortality rates in pediatric cardiopulmonary bypass are decreasing while incidence of adverse neurological outcomes, such as compromised cognitive and speech impairments, behavioral abnormalities, and motor defi cits, are increasing in these patients (Su and Undar 2010 ) . In another study, Barber and colleagues evaluated cognitive functions after cardiac valve surgery in 40 patients (Barber et al. 2008 ) . They observed that cognitive impairment was associated with perioperative ischemia. The cognitive impairment was more severe in patients with greater ischemic burden. Overall, the already mentioned studies indicate neurological and cognitive impairments in patients undergoing cardiac surgery. Induction of ischemia tolerance in these patients will help lower brain damage following in patients undergoing cardiac surgery.

11.6 Ischemic Preconditioning Mimetics in Preclinical Studies

Investigators in the fi eld of preconditioning are attempting to dissect out signaling pathways involved in induction of tolerance against ischemia so that preconditioning can be mimicked pharmacologically. Such successful pharmacological intervention might provide a prophylactic strategy against serious brain ischemic insult in patients. As of now, several pharmacological agents emulate IPC. In recent years, erythropoietin (EPO, the primary humoral regulator of erythropoiesis) is emerging as a promising IPC mimetic. EPO is synthesized in renal peri- tubular interstitial cells in an oxygen concentration-dependent manner and is a potent neuroprotectant against several cerebral insults by direct activation of cell survival pathways (Baker 2005 ; Dawson 2002) . Several studies have also shown the potential of EPO to induce ischemic tolerance in in vivo and in vitro models of ischemia (Bernaudin et al. 2002 ; Grimm et al. 2002, 2004; Malhotra et al. 2006 ; Prass et al. 2003 ; Ruscher et al. 2002 ) . A clinical trial has also the bene fi cial effects of administering erythropoietin to patients with acute ischemic stroke (Coleman and Brines 2004) . The use of EPO as a prophylactic agent to induce ischemic tolerance needs extensive characterization, as less than 1% of systemically administered EPO crosses the blood–brain barrier, thus large volumes and multiple doses of EPO are required to achieve benefi cial effects in the brain (Coleman and Brines 2004 ) . Careful consideration should be used due to the fact that EPO use is also associated 11 Tolerance Against Global Cerebral Ischemia: Experimental Strategies... 251 with undesirable side-effects (Kennedy and Buchan 2005 ) such as increased hema- tocrit, blood viscosity, and increased platelet activation (Velly et al. 2010 ) . Nonerythropoietic, non-platelet activating, and tissue-protective EPO analogues are available. Of these, non-sialic EPO (asialoEPO) and carbamylated EPO (CEPO) are neuroprotective in animal models of cerebral ischemia (Price et al. 2010 ; Leist et al. 2004) . Overall, EPO derivatives have a potential to be used in cerebral ischemia patients. Another potential IPC mimetic is resveratrol. Resveratrol is a natural polyphenol found in grapes and wine and has been associated with protective effects against cardiovascular diseases. Earlier, we demonstrated that IPC activates sirtuin 1 (SIRT1), a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases, by altering reduced to oxidized nicotinamide adenine dinucleotide (NADH/NAD+ ) ratio (Lin et al. 2004 ; Raval et al. 2006b ) . Resveratrol also activates SIRT1. Pretreatment with resveratrol mimics IPC in in vivo and ex vivo models of cerebral ischemia (Raval et al. 2006b ; Della-Morte et al. 2009 ) . Based on these fi ndings, resveratrol could be used as a prophylactic preconditioning agent. The translational value of resveratrol is supported by the fact that phase I safety trials have already been conducted in healthy volunteers (Boocock et al. 2007) . However, before it can be used as a preventative agent against cerebral ischemia, a better understanding of the mechanisms by which resveratrol induces protection in the brain is essential. This can be potentially challenging due to the ubiquitous nature of resveratrol-activating multiple signaling pathways resulting in unwarranted side-effects. Erythromycin is a broad-spectrum antibiotic used originally to treat a wide variety of bacterial infections. Recently, it has been shown to have ef fi cacy to induce ischemic tolerance using a rat model of transient global cerebral ischemia. Intramuscular injection of erythromycin improved postischemic neuronal survival as well as a reduction in ischemia-induced functional de fi cits when injected up to 24 h prior to exposure to cerebral ischemia (Brambrink et al. 2006 ) . Currently, the mechanism of action of erythromycin to induce ischemic tolerance is still unknown, but other family of macrolides may possess similar or more effective tolerance characteristics. This may prove highly benefi cial since erythromycin and other macrolides are used in humans frequently. Presently, IPC mimetics are available to induce transient ischemia tolerance. However, the effect of chronic treatment with these agents on induction of sustained ischemia tolerance is not known.

11.7 Summary

Overall, preclinical studies demonstrate that IPC or IPC mimetics have potential to inhibit or mitigate brain damage following global cerebral ischemia. Inducing the proper ischemic tolerance resulting in neuroprotection remains a challenge. More effective pathological models (in vivo and in vitro) are needed for better understanding of ischemic tolerance which may provide novel therapeutic targets applicable to 252 K.R. Dave et al. a wide-range of cerebral ischemic scenarios. More innovative approaches in IPC mimetic development are greatly needed as induction of persistent cerebral ischemia tolerance remains an obstacle.

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Nicole M. Jones and Adam A. Galle

12.1 Preconditioning and Tolerance to Brain Injury

In spite of the many preclinical animal studies and clinical trials, the current state of clinical neuroprotection against brain injury is essentially non-existent. One interesting and rapidly evolving approach in the fi eld of neuroprotection is that of development of tolerance to an injury, produced by cerebral preconditioning. Whereby preconditioning treatments enhance ‘endogenous’ neuroprotective adaptations that can be induced by mild stressful but non-injurious stimuli presented to cells, tissues or organisms, and there will be a reduction in the damage received from an exposure to a subsequent injury (Gidday 2006 ) . Tolerance to injury produced via ischemic preconditioning was fi rst demonstrated by Murry et al. ( 1986 ) , whereby it was shown that brief ischemic episodes reduced the size of myocardial infarcts, following a sustained ischemic insult in dogs. This phenomenon was confi rmed to exist in the brain, where hippocampal CA1 pyramidal neurons were protected, if carotid artery blood fl ow was transiently interrupted 2 days prior to a prolonged global ischemia in gerbils (Kitagawa et al. 1991 ) . Furthermore, a clinical correlate was discovered for this preconditioning phenomenon in the brain, as patients with a history of transient ischemic attacks have decreased morbidity following stroke (Weih et al. 1999 ) . Many types of mild noxious stimuli are capable of preconditioning, including temporary ischemia, hyperthermia, isofl urane, physical exercise, lipopolysaccharide (LPS) and glutamate. These preconditioning stimuli are effective in a range of tissue types including heart, liver, kidney, lung and brain (Wang et al. 2002 ; Shpargel et al. 2008 ) in a variety of mammalian species (Wang et al. 2002 ; Shpargel et al. 2008 ; Obrenovitch 2008 ) . It should be noted, however, that a comparison of the genetic pro fi les of preconditioned brains in response to subsequent ischemia differs contingent on the preconditioning stimuli (Stenzel-Poore et al. 2007 ) . One particular modality

N. M. Jones (*) ¥ A. A. Galle Department of Pharmacology , University of New South Wales , Sydney 2052 , Australia e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 259 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_12, © Springer Science+Business Media New York 2013 260 N.M. Jones and A.A. Galle of cerebral preconditioning successfully and repeatedly demonstrated to reduce injury following an ischemic episode is the use of sublethal hypoxia (Bergeron et al. 2000a ; Sharp et al. 2004 ) . Even without preconditioning, the mammalian brain is able to mobilise a range of potential endogenous defence mechanisms in an attempt to reduce the likelihood of cell death following a brain injury. Defence mechanisms designed to decrease neuronal excitability, extracellular glutamate concentration and intracellular Ca2+ concentration and to enhance neuronal energetics are some of the processes activated (Sapolsky 2001 ) . Cerebral preconditioning, however, elicits a fundamentally different response. In contrast to the reactive mechanisms activated following cerebral ischemia, preconditioning initiates a series of adaptive responses aimed at priming the brain to cope with and withstand the subsequent injurious episode (Gidday 2006 ) . This adaptive response is characterised by a biphasic pattern of ischemic tolerance; a short-term protective phenotype can be induced almost immediately following exposure to a preconditioning stimulus (Perez-Pinzon et al. 1997 ; Stagliano et al. 1999) . This is followed by a delayed phase of tolerance, which only comes into effect following an interval of multiple hours or even days following exposure to preconditioning stimuli and can remain active for days or even weeks (Hagberg et al. 2004 ) . The ‘rapid’ form of preconditioning results from changes in ion-channel permeabilities, reduction in neuronal energy demands, protein phosphorylation and post-translational protein modiﬁ cations and is active for approximately 1 h (Gidday 2006; Obrenovitch 2008; Lu et al. 2005; Meller et al. 2006 ) . In contrast, delayed preconditioning is dependent on gene activation and de novo protein synthesis (Gidday 2006 ) .

12.2 Molecular and Cellular Mechanisms Involved in Preconditioning

Studies have shown that decreased protein expression is at least as important as protein up-regulation in the preconditioned brain (Stenzel-Poore et al. 2003 ; Kawahara et al. 2004 ) . Preconditioning appears to modify the brain’s response to ischemic stimuli with a distinct set of improved injury response mechanisms, suggesting a general reprogramming of the brain’s innate response to injury (Stenzel- Poore et al. 2003 ; Tang et al. 2006 ) . The physiological effects of cerebral preconditioning are extensive, modulating a wide range of cellular functions, and many molecular mechanisms have been proposed. In adult mice subjected to preconditioning, the changes in gene expression suggested metabolic downturn combined with a reduction in membrane ion-channel activity (Obrenovitch 2008 ) . When cellular adaptations are taken into account with the altered genetic proﬁ les presented in preconditioning microarray studies, it becomes clear that this phenomenon of preconditioning/ischemic tolerance is highly complex and multifaceted (Bernaudin et al. 2002a ) . Numerous molecular mechanisms have been proposed as putative effectors of cerebral preconditioning, including various protein kinases, transcription factors and immediate early genes, in addition to adenosine A1 receptors, nuclear factor- k B (NF-k B), vascular endothelial growth factor (VEGF), erythropoi- 12 Preconditioning and Neuroprotection in the Immature Brain 261 etin (EPO), tumour necrosis factor-a (TNF-a ), cAMP response element-binding protein (CREB) (Lin et al. 2009 ) and hypoxia-inducible factor (HIF-1) to name but a few (Obrenovitch 2008 ; Sharp et al. 2004 ; Hagberg et al. 2004 ) .

12.2.1 Glial Response to Preconditioning

It is interesting to note that resident glial cells, akin to their neuronal counterparts, also respond uniquely to preconditioning. In response to preconditioning stimuli, both astrocytes and microglia become transiently activated. Neuronal support processes mediated by astrocytes, such as glycogen storage, extracellular ion buffering, scavenging of reactive oxygen species, EPO production and blood-brain barrier modulation through VEGF and adrenomedullin, are augmented following exposure to a preconditioning stimulus (Gidday 2006 ; Lin et al. 2008 ; Trendelenburg and Dirnagl 2005 ) . In addition, the generation of anti-inﬂ ammatory cytokines, heat- shock proteins (HSP32) and neurotrophic factors (brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF)), by preconditioned astrocytes contribute to the phenotype of ischemic resistance (Trendelenburg and Dirnagl 2005 ) .

12.2.2 Vascular Response to Preconditioning

Whilst the focus of cerebral preconditioning rests primarily with the effects it has directly on neurons, and to a lesser extent glia, it is important to also appreciate the neuroprotective effects that preconditioning can derive from the cerebral vasculature. Like neurons and glia, the functioning of the vascular endothelium is altered following exposure to a preconditioning stimulus. In a mouse model of middle cerebral artery occlusion (MCAO), the administration of LPS resulted in a reduction in cerebral infarct by 68% accompanied by a 114% improvement in cerebral blood (Kunz et al. 2007 ) . Moreover, these beneﬁ cial effects were conditional on the presence of NO, as neuroprotection was not observed in mice lacking the gene for iNOS (Kunz et al. 2007 ) . In addition, studies in a rat model of permanent focal stroke demonstrated an increase in the number of patent microvessels coinciding with a decrease in the reduction of CBF post-injury (Dawson et al. 1999 ) .

12.3 Preconditioning and Neuroprotection in the Immature Brain

Whilst still in utero, a foetus, and then subsequently a newly born baby, may inherently be more able to adapt to hypoxic-ischemic (HI) conditions, having been previously being exposed to the hypoxic intrauterine environment. Preconditioning of 262 N.M. Jones and A.A. Galle the neonatal brain can also result in resistance to injury, and in some cases the mechanisms mimic those described for tolerance to injury in the mature brain, but there are also some con fi ned to the immature brain which are likely to be related to distinct developmental gene expression profi les. Over the years, there have been a number of different types of preconditioning models that have been shown to protect against brain injury in the immature brain, and these include hyperthermia (Wada et al. 1999) , anaesthetics (Zhao et al. 2007 ) , hypoxia (Gidday et al. 1994 ) and LPS (Eklind et al. 2005 ) exposure. Whilst these preconditioning paradigms have proven useful in animal models, to date, the mechanisms and long-term effects of preconditioning in the immature brain are still under investigation, and neuroprotective (Cheung et al. 2006) effi cacy of preconditioning has not yet been examined in human infants. The potential clinical application of ischemic preconditioning has been examined in a clinical trial in children undergoing heart surgery, and the study reported myocardial protection in patients that received remote ischemic preconditioning in the lower limb (Cheung et al. 2006 ) . Understanding some of the common endogenous protective mechanisms involved in preconditioning in the immature brain could lead to major advances in the treatment of brain injuries occurring in newborns as well as adults.

12.3.1 Ligation and Ischemic Preconditioning

Ischemic preconditioning produced by unilateral carotid artery ligation performed at differing times prior to hypoxia can reduce lesion size and long-term memory de ﬁ cits after HI injury in the neonatal rat (postnatal day 7 = p7) (Lee et al. 2004 ) , and this protection appears to be mediated by CREB. Ligation preconditioning alone results in a sustained increase in CREB phosphorylation, and the protection is reduced when CREB antisense treatment is used. Similarly, ischemic preconditioning using reversible occlusion of carotid artery at various times (between 2 and 22 h) prior to HI can also reduce injury severity, as indicated by a reduction in TUNEL and caspase-3 staining and improved recovery of memory deﬁ cits. There was an increase in CREB and BCl2 levels produced by ischemic preconditioning, suggesting an induction of anti-apoptotic pathways may promote neuronal survival (Lin et al. 2009 ) .

12.3.2 Anaesthetic Preconditioning

Iso fl urane preconditioning (1.5% for 30 min) performed on p6 rats can result in neuroprotection in cortex and hippocampus after HI performed on p7, which is accompanied by an improvement in motor function, but not memory de fi cits after HI (Zhao et al. 2007 ) . Preconditioning with isofl urane increased expression of BCl2 protein, and protection was prevented by using an inhibitor of iNOS, indicating a 12 Preconditioning and Neuroprotection in the Immature Brain 263 vital role for NO production in isofl urane preconditioning (Zhao et al. 2007 ) . However, short-term exposure to 2% isofl urane preconditioning, at 60 or 90 min prior to HI injury, can reduce hippocampal damage up to 7 days after HI, but 49 days after HI the protection is not sustained, indicating a more rapid preconditioning effect (Sasaoka et al. 2009 ) . Sevo fl urane and xenon are both inhalational anaesthetic agents that have the potential to be used during birth. Preconditioning with xenon alone (7.5%) or combination (20% xenon, 0.75% sevo fl urane), but not sevo fl urane (1.5%), can reduce infarct size and reduce long-term behavioural defi cits after HI in p7, when given 4 h prior to HI. This effect was associated with an increase in pCREB expression, and the neuroprotective effects of preconditioning were prevented by using wortmannin, indicating that PI3K signalling is important for protection produced by anaesthetic agents (Luo et al. 2008 ) . In p9 mice, isofl urane preconditioning (1.8%) was able to improve mortality after HI as measured by improved striatal function (apomorphine-induced circling); however, there was no improvement on the spatial learning (McAuliffe et al. 2006 ) . Iso fl urane was shown to be only slightly protective in the striatum of mice, and the protection may have occurred as a result of hypothermia.

12.3.3 Lipopolysaccharide Preconditioning

A number of studies have examined the effects of preconditioning with LPS prior to neonatal brain injury which appears to have diverse effects, dependent upon the timing of LPS administration and also the dose. When administered 4 h prior to HI injury, LPS can exacerbate or sensitise the p7 rat brain to an HI injury, whereas if given 24 h before HI, LPS can reduce the extent of brain injury in cortex, striatum and hippocampus (Ikeda et al. 2006 ) , suggesting that LPS can have both protective and degenerative properties. LPS preconditioning can also improve some of the long-term memory defi cits that occur after HI (Lin et al. 2010 ) . There also appears to be an age-dependent effect of LPS preconditioning, which may relate to differential expression of Toll-like receptor-4 (TLR4) on microglia and astrocytes during the postnatal period. In rat pups exposed to LPS preconditioning at different ages (between p3 and p14) and subsequently exposed to HI, LPS had no effect when administered on p3 or p5; however, when used on p7, p9 and p14, it was able to prevent HI injury (Hickey et al. 2011 ) . When a higher dose of LPS (0.3 mg/kg) was administered, there was an increase in microglial activation and TNFa and iNOS expression, and an increase in mortality after HI was observed. However, when 0.05 mg/kg of LPS is used, there was no change in in fl ammatory mediators, and after an HI injury, there was a reduction in HI-induced infl ammation, indicating that preconditioning with low-dose LPS may reprogram the microglia and prevent the subsequent activation after HI insult (Hickey et al. 2011 ) . Corticosterone may also be involved in LPS preconditioning as protection can be reversed by using the glucocorticoid antagonist RU486, indicating a role for corticosteroids in neuroprotection (Ikeda et al. 2006 ) . LPS preconditioning can also increase levels of eNOS 264 N.M. Jones and A.A. Galle and pAkt (Lin et al. 2010 ) . Decreasing NOS activity using antisense treatment and inhibiting eNOS expression were able to reverse the protective actions of LPS-induced preconditioning, and similarly, inhibition of PI3K was able to prevent the protective actions of LPS preconditioning (Lin et al. 2010 ) .

12.3.4 Hypoxic Preconditioning

Hypoxic preconditioning in the immature rat brain was fi rst demonstrated by Gidday et al. 1994 in a study where rat pups that were exposed to 8% oxygen for 3 h, followed 24 h later by HI injury displayed signifi cant neuroprotection (Gidday et al. 1994 ) . This hypoxic preconditioning afforded very robust neuroprotection, and these fi ndings have been subsequently con fi rmed, and the molecular and cellular mechanisms have been further examined by many other groups (Vannucci et al. 1998 ; Jones and Bergeron 2001, 2004 ; Gustavsson et al. 2007a ) . Importantly, preconditioning with hypoxia is able to improve some of the behavioural de fi cits that occur following an HI injury (Gustavsson et al. 2005 ; Jones et al. 2008 ) . Tests for sensorimotor function, spatial learning and cognition are all improved up to 5 weeks after the initial HI injury on p7 and are accompanied by sustained histological protection. The robust neuroprotection afforded by hypoxic preconditioning is the product of gene activation and de novo protein synthesis, and many of the neuroprotective molecules implicated in other preconditioning paradigms are induced by hypoxic preconditioning or hypoxia alone (Sharp et al. 2004 ) . Genes shown to be up-regulated following 3 h of mild hypoxic exposure have diverse functions including transcription factors, signal transduction molecules and receptors, cell structure, transporters, ion channels, proteases, ROS detoxifi cation, apoptosis, molecular chaperones and in fl ammation (Tang et al. 2006 ; Bernaudin et al. 2002a ) . Conversely, many gene products were shown to be down-regulated following hypoxic preconditioning, including genes involved in cell adhesion, signal transduction and DNA synthesis (Bernaudin et al. 2002a ) . Gene array studies have shown that in the neonatal rat, hypoxic preconditioning caused an increase in the expression of many anti-apoptotic genes which is accompanied by a reduction in some anti-apoptotic genes. Members of the BCl2 family, MAPK and PI3K/AKT signalling pathways, were shown to be increased after hypoxic preconditioning (Gustavsson et al. 2007b ) . ERK activity was found to be important for the protection conferred by hypoxic preconditioning in the p6 rat (Jones and Bergeron 2004 ) ; as an inhibitor of ERK activity, U0126 was able to reduce the protective action of hypoxia. A similar key role for PI3K/AKT signalling in hypoxia-induced tolerance has been reported; with PI3K inhibition, the protective effects of hypoxic preconditioning were reduced. These effects are accompanied by a reduced infl ammatory response to HI in preconditioned brains, which are also dependent upon PI3K/AKT signalling (Yin et al. 2007 ) . Additional mechanisms involved in hypoxic preconditioning include reduction in oxidative stress by increasing endogenous antioxidant activity (Alkan et al. 2008 ; Sheldon et al. 2007 ) and potential changes in expression of glutamate transporters (Cimarosti et al. 2005 ) . 12 Preconditioning and Neuroprotection in the Immature Brain 265

Preconditioning with hypoxia increases levels of the hypoxia-inducible transcription factor HIF-1 (Bergeron et al. 2000b ) and its downstream target gene expression. HIF-1 modulates the expression of many target genes including VEGF, transforming growth factor b (TGFb ), glycolytic enzymes (aldolase, lactate dehydrogenase, phosphofructokinase), glucose transporter 1 (GLUT1) and EPO (Jones and Bergeron 2001 ; Bernaudin et al. 2002b ) . Increases in expression of GLUT1 and glycolytic enzymes observed after hypoxic preconditioning (Jones and Bergeron 2001) indicate that modulation of glucose transport and glycolysis by hypoxia contributes to the development of hypoxia-induced tolerance. It also appears that brain glycogen is increased following hypoxic preconditioning and that residual ATP levels are markedly higher following HI injury in preconditioned animals (Brucklacher et al. 2002) . Promoting glucose transport and glycolysis may thus play a role in hypoxia-induced tolerance. Indeed, hypoxic preconditioning has been shown to prevent the secondary depletion of phosphocreatine and adenosine triphosphate that is generally associated with HI in newborn rats (Vannucci 1998 ) , suggesting a greater preservation of metabolic capacity in preconditioned animals. Several other HIF-1 target genes are increased after hypoxia preconditioning in the neonatal rat (Jones and Bergeron 2001 ; Jones et al. 2006 ) . Differential expression patterns of HIF-1 and one of its regulatory enzymes (HIF-1 prolyl hydroxylase 2 (PHD2)) have been shown to change after hypoxic preconditioning (Jones et al. 2006) . Recent studies examining conditional knockout of the HIF-1 a gene in different types of cells have highlighted the complex roles that HIF-1/PHD signalling may play in injury and repair. Surprisingly, a neuroprotective effect is observed in adult mice following ischemia, where HIF-1a was speciﬁ cally knocked out in neurons (Helton et al. 2005 ) . Additional studies have found that HIF-1a does have roles in apoptosis, via the induction of pro-apoptotic proteins (BNIP3 and p53) after brain injury (Althaus et al. 2006 ; Halterman and Federoff 1999 ) . However, it is also clear that HIF-1 is involved in endogenous repair mechanisms because if HIF-1a and HIF-2 a are knocked out in astrocytes, the protective effect of preconditioning is lost (Chavez et al. 2006 ) . Adding to this controversy, selective knockout of HIF-1a in neurons is required for survival after hypoxia, whilst in astrocytes it is involved in cell death caused by hypoxia (Vangeison et al. 2008 ) . Similar to LPS preconditioning, hypoxia also has protective actions on the brain vasculature (Gustavsson et al. 2007a ) . Preconditioning with hypoxia can prevent the decrease in cerebral blood ﬂ ow observed after HI insult in p7 rats and causes an increase in the expression of a number of vascular associated genes. This is accompanied by an increase in the number of brain microvessels, indicating that hypoxia may be causing an increase in brain angiogenesis. One important regulator of angiogenesis is VEGF, which has been found to increase after hypoxic preconditioning and HI, and a number of VEGF splice variants (VEGF120 , VEGF164 ) are increased for a sustained period of time subsequent to HI (Feng et al. 2010 ) . In addition, hypoxic preconditioning can reduce injury caused by excitotoxic lesions in immature rats which predominantly result in white matter damage (Laudenbach et al. 2007 ) , and when VEGF activity is blocked, the protective effect of hypoxia is reduced. Further vascular effects of hypoxia in the neonatal rat brain have been shown via 266 N.M. Jones and A.A. Galle preconditioning being prevented by non-selective inhibition of NOS suggesting that eNOS is likely to be important for mediating protection produced by hypoxic preconditioning (Gidday et al. 1999 ) .

12.4 Conclusions

Since it was ﬁ rst described, there has been a huge increase in our knowledge regarding the mechanisms involved in mediating the endogenous neuroprotection resulting from preconditioning in the neonatal brain. These preconditioning strategies may eventually be useful as preventative strategies for neuroprotection in high-risk patients (in pregnancy or surgery situations). Clearly, there are many common cellular and molecular pathways which are likely to mediate protection produced by preconditioning, and these pathways may themselves be the target of future drug therapies that could prove useful for minimising damage as a result of brain injuries in human neonates.

References

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Lu G-W et al (2005) Hypoxic preconditioning. Mol Neurobiol 31(1):255Ð271 Luo Y et al (2008) Xenon and sevo fl urane protect against brain injury in a neonatal asphyxia model. Anesthesiology 109:782Ð789 McAuliffe J, Miles L, Vorhees C (2006) Adult neurological function following neonatal hypoxia- ischemia in a mouse model of the term neonate: water maze performance is dependent upon separable cognitive and motor components. Brain Res 1118(1):208Ð221 Meller R et al (2006) Rapid degradation of Bim by the ubiquitin-proteasome pathway mediates short-term ischemic tolerance in cultured neurons. J Biol Chem 281(11):7429Ð7436 Murry CE, Jennings RB, Reimer KA (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74(5):1124Ð1136 Obrenovitch TP (2008) Molecular physiology of preconditioning-induced brain tolerance to ischemia. Physiol Rev 88(1):211Ð247 Perez-Pinzon MA et al (1997) Rapid preconditioning protects rats against ischemic neuronal damage after 3 but not 7 days of reperfusion following global cerebral ischemia. J Cereb Blood Flow Metab 17(2):175Ð182 Sapolsky RM (2001) Cellular defenses against excitotoxic insults. J Neurochem 76(6):1601Ð1611 Sasaoka N et al (2009) Isofl urane exerts a short-term but not a long-term preconditioning effect in neonatal rats exposed to a hypoxic-ischaemic neuronal injury. Acta Anaesthesiol Scand 53(1):46Ð54 Sharp FR et al (2004) Hypoxic preconditioning protects against ischemic brain injury. NeuroRx 1(1):26Ð35 Sheldon RA et al (2007) Hypoxic preconditioning reverses protection after neonatal hypoxia-ischemia in glutathione peroxidase transgenic murine brain. Pediatr Res 61(6):666Ð670 Shpargel KB et al (2008) Preconditioning paradigms and pathways in the brain. Cleve Clin J Med 75(Suppl 2):S77 Stagliano NE et al (1999) Focal Ischemic preconditioning induces rapid tolerance to middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab 19(7):757Ð761 Stenzel-Poore MP et al (2003) Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states. Lancet 362(9389):1028Ð1037 Stenzel-Poore MP et al (2007) Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis. Stroke 38(2):680Ð685 Tang Y et al (2006) Effect of hypoxic preconditioning on brain genomic response before and following ischemia in the adult mouse: identifi cation of potential neuroprotective candidates for stroke. Neurobiol Dis 21(1):18Ð28 Trendelenburg G, Dirnagl U (2005) Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia 50(4):307Ð320 Vangeison G et al (2008) The good, the bad, and the cell type-speci fi c roles of hypoxia inducible factor-1 a in neurons and astrocytes. J Neurosci 28(8):1988Ð1993 Vannucci R (1998) Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates. J Neurochem 71:1215Ð1220 Vannucci RC, Towfi ghi J, Vannucci SJ (1998) Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates. J Neurochem 71(3):1215Ð1220 Wada T, Kondoh T, Tamaki N (1999) Ischemic “cross” tolerance in hypoxia ischemia of immature rat brain. Brain Res 847:299Ð307 Wang YP et al (2002) Lipopolysaccharide triggers late preconditioning against myocardial infarction via inducible nitric oxide synthase. Cardiovasc Res 56(1):33Ð42 Weih M et al (1999) Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain? Stroke 30(9):1851Ð1854 Yin W et al (2007) Preconditioning suppresses in fl ammation in neonatal hypoxic ischemia via Akt activation. Stroke 38:1017Ð1024 Zhao P et al (2007) Isofl uorane preconditioning improves long-term neurologic outcome after hypoxic-ischemic brain injury in neonatal rats. Anesthesiology 107:963Ð970 Chapter 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection, Physiological Monitoring, and Other Methodological Issues

Thaddeus S. Nowak Jr. and Liang Zhao

13.1 Introduction

The earliest studies of preconditioning in experimental stroke examined the potential impact of such interventions on the key variable of cerebral perfusion. Based on limited regional or temporal sampling, these were interpreted to suggest that protection could be dissociated from overt CBF changes, and the majority of conditioning studies in experimental stroke have since focused on cellular protection mechanisms. However, evolving results indicate a strong association between delayed improvements in penumbral perfusion and subsequent tissue protection. This presentation will briefl y consider the strengths and limitations of commonly used focal brain ischemia models and address the requirements for adequately monitoring those physiological parameters that are recognized to in fl uence outcome in experimental stroke. It will then summarize observations regarding changes in blood fl ow and relevant physiological variables in conditioning models.

13.2 Stroke Models Used in Conditioning Studies

Essentially, all established methods to model focal ischemic stroke have been used to evaluate conditioning effects, usually targeting with varying speci ﬁ city the middle cerebral artery (MCA) territory of rats or mice. The choice of models signiﬁ cantly impacts infarct distribution and the time course of its evolution, as well as its

T. S. Nowak Jr. (*) • L. Zhao Department of Neurology , University of Tennessee Health Science Center , 855 Monroe Ave., Link 415 , Memphis , TN 38163 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 269 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_13, © Springer Science+Business Media New York 2013 270 T.S. Nowak and L. Zhao sensitivity to a number of physiological variables. Of primary importance is the extent of collateral perfusion, which is determined by the interacting factors of occlusion method and species/strain selection.

13.2.1 Surgical MCA Occlusion in Rodents

Initial approaches to surgical occlusion of the MCA in rats, targeting relatively accessible distal occlusion sites, produced small and variable lesions restricted to cortex (Robinson et al. 1975 ) , and this proved to be a consistent fi nding in commonly used outbred strains (Coyle 1986 ; Coyle et al. 1984 ) . Later studies involved more proximal occlusion sites and comparatively invasive surgical procedures to produce ischemia in MCA cortex as well as the striatal territory served by lenticulostriate arteries (Bederson et al. 1986 ; Tamura et al. 1981a ) . The same subtemporal route could also be used to produce somewhat more distal occlusions that spared striatum (Shigeno et al. 1985 ) . The spontaneously hypertensive rat (SHR), which possesses reduced collateral perfusion, had been found to exhibit reasonably consistent infarcts even after distal transtemporal occlusions, dorsal to the rhinal fi ssure (Coyle 1986; Coyle et al. 1984) . Subsequent surgical occlusion studies in other strains therefore returned to this technically simpler approach but with additional procedures to reduce collateral perfusion and improve reliability. The most straight- forward of these involves tandem occlusion of the ipsilateral common carotid artery (Brint et al. 1988 ; Kaplan et al. 1991 ) , which can be applied most effectively to SHR but also has proven applicable to certain other strains (Hashimoto et al. 2008 ; Ma et al. 2006 ; Ren et al. 2004 ) . However, commonly used strains, such as Sprague– Dawley and Wistar rats from some suppliers, can have suffi cient collateral perfusion that they may fail to produce infarcts even when these two vessels are permanently occluded (Ma et al. 2006 ; Ren et al. 2004 ) . For such strains, the contralateral common carotid artery (CCA) can be targeted as well, although this is not compatible with permanent occlusion. Perfusion can be restored to all of the occluded vessels to produce an unambiguously transient ischemia model (Buchan et al. 1992; Hiramatsu et al. 1993; Yip et al. 1991) . Alternatively, MCA occlusion is maintained, but reperfusion is allowed via one or both CCAs after a comparatively short interval (Chen et al. 1986 ; Markgraf et al. 1993 ) , resulting in a hybrid model with a component of persistent partial fl ow attenuation. Although not initially recognized, this use of bilateral CCA occlusion can be confounded by prolonged hyperthermia, particularly if rats are allowed to recover from anesthesia prior to reperfusion (Yanamoto et al. 1996, 2001) , somewhat analogous to the hyperthermia produced in the gerbil global ischemia model (Kuroiwa et al. 1990 ) . This presumably arises due to the involvement of hypothalamus, as considered below in the context of fi lament occlusion methods. Conversely, a technical concern common to all approaches that involve exposure of the brain surface is the need to avoid incidental cooling at the time of surgery (Barone et al. 1997 ) . This becomes increasingly important in strains with robust collateral perfusion. For example, an interval 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 271 of cooling at the onset of permanent occlusion is protective in Wistar rats but not in SHR (Baker et al. 1992 ; Kader et al. 1992 ; Ren et al. 2004 ; Ridenour et al. 1992 ) . It is also important to consider the heterogeneity intrinsic in outbred rats. It has long been appreciated that, in addition to yielding larger infarcts, the SHR exhibits less variability than some commonly used normotensive strains (Brint et al. 1988 ; Duverger and MacKenzie 1988 ) . MCA territory distribution and/or extent of collateralization clearly vary among outbred individuals, requiring larger numbers of animals to establish statistically signi fi cant effects. There can also be drifts in population characteristics, resulting in marked differences in outcome following surgical MCA occlusion in animals of the same strain obtained from different vendors, a result reported for both Sprague–Dawley (Oliff et al. 1995) and Wistar rats (Ma et al. 2006 ) . Since animal sources are not always speci fi ed in publications, this can complicate the comparison of results among studies using seemingly comparable models and may lead to the selection of methods requiring different measures to reduce collateral perfusion according to the characteristics of animals available to a given laboratory. This issue will be considered again in the context of fi lament occlusions. The same spectrum of surgical approaches has also been used to produce MCA occlusion in mice, with a corresponding range of outcomes. Distal MCA occlusion alone has produced reliable infarcts in some strains (Barone et al. 1993 ; Keum and Marchuk 2009; Kuraoka et al. 2009; Majid et al. 2000; Xi et al. 2004) . As in the rat, coincident ipsilateral CCA occlusion increases infarct volume but also increases mortality in those strains that already exhibit large infarcts after MCA occlusion alone (Barone et al. 1993 ) . Possible variations in outcome for the same strain obtained from different suppliers have not been systematically investigated, but this would be expected to be less of an issue for inbred mice. In several studies, the largest infarcts consistently have been observed in BALB/c mice (Barone et al. 1993 ; Keum and Marchuk 2009 ; Majid et al. 2000 ) . However, varied responses to distal MCA occlusion have been reported for C57BL/6 mice, with infarcts ranging in size from negligible (Keum and Marchuk 2009 ) to moderate (Kuraoka et al. 2009 ; Majid et al. 2000 ) . Differences in anesthesia, temperature control, and other procedural variables also would be expected to contribute to such heterogeneity.

13.2.2 Intraluminal Filament Occlusions

Although nominally the most common approach now used to produce focal brain ischemia in rodents, the implementation of intraluminal fi lament occlusion actually varies widely among laboratories. Details of procedure optimization by individual investigators are beyond the scope of consideration here, but there are several features common to all such methods. It is essential to realize that the term “MCA occlusion” does not apply to these approaches, either as originally described for rats (Koizumi et al. 1986 ; Zea Longa et al. 1989 ) or as currently performed in most laboratories. The devices intended to obstruct the origin of the MCA also attenuate 272 T.S. Nowak and L. Zhao perfusion via the anterior cerebral artery (ACA) and often occupy a considerable extent of the internal carotid artery (ICA). In animals that lack adequate communi- cating vessels in the posterior circle of Willis, perfusion of the posterior cerebral artery territory also may be reduced, resulting in hippocampal damage (Özdemir et al. 1999 ) . Perhaps most importantly, hypothalamic ischemia secondary to ICA obstruction can produce profound hyperthermia in rats that are allowed to recover from anesthesia during the occlusion (Ábrahám et al. 2002 ; He et al. 1999 ; Li et al. 1999; Zhao et al. 1994 ) . Even when devices are scaled to permit introduction of a small tethered embolus directly into the MCA, heparin is required to avoid hyperthermia due to occasional clotting along the delivering fi lament (Ma et al. 2006 ) . Temperature elevation markedly exacerbates brain vulnerability (Chen et al. 1991 ; Meden et al. 1994; Morikawa et al. 1992 ; Sick et al. 1999) . Together with the larger volume of ischemic tissue that results due to involvement of other vascular territories (Kanemitsu et al. 2002 ) , this contributes to the high mortality often observed after permanent fi lament placement (Nagasawa and Kogure 1989 ) . Transient occlusions are therefore the norm for intraluminal fi lament models, and in rats these almost always involve Sprague–Dawley and Wistar strains in which the ischemic territory distribution may be limited by more robust collateral perfusion. Given the rapid increase in temperature that can occur during hypothalamic ischemia, hyperthermia is expected in most models in which anesthesia is discontinued during an interval of fi lament occlusion. When anesthesia and normothermia were maintained during occlusion, rats showed normal temperature control following reperfusion at 90 min, but persistent hyperthermia occurred after 2-h occlusions, refl ecting the progression to overt hypothalamic infarction after the longer insult (Li et al. 1999 ) . This illustrates the potential for complex interactions between occlusion duration, infarct distribution, and temperature and emphasizes the need for comprehensive postischemic temperature monitoring in any study involving fi lament occlusions. Another important factor to be considered with such endovascular occlusion methods is the potential for endothelial damage and its consequences. Microemboli have been detected in brain after just a few minutes of fi lament insertion and removal (Zhan et al. 2008) . Details of fi lament fabrication and dimensions will determine the risk of such incidental damage and will clearly vary among laboratories. For example, polylysine coating has been used to improve occlusion ef fi cacy in some studies (Belayev et al. 1996) but, in our hands, was completely incompatible with maintaining ICA patency when attempting selective MCA occlusion by the intraluminal route (J. Ma and T. S. Nowak, Jr., unpublished observation 2005). To our knowledge, the potential impact of delayed thrombosis or embolism secondary to vascular injury has not been systematically studied in fi lament models. However, it should be noted that cortical infarcts are sometimes observed after fi lament occlusions as short as 1 h (Belayev et al. 1996) , which is below the temporal threshold for infarction in surgical MCA occlusion models (Kaplan et al. 1991 ; Ren et al. 2004 ) , or with fi laments that selectively occlude the MCA (Ma et al. 2006 ) . This may re fl ect in part a more effective reduction of cortical CBF, or interacting effects of coincident striatal involvement, but the possible contribution of prolonged perfusion de ficits that extend the effective duration of the ischemic insult remains a potential consideration. 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 273

As a practical matter, this also prolongs the interval during which variations in systemic physiological parameters can impact infarct progression. In this context, it is particularly important to consider the distinction between overt infarction and selective neuronal injury, speci fi cally in the evaluation of striatal vulnerability. Proximal MCA or fi lament occlusions can sometimes produce more profound CBF defi cits in striatum than in cortex (Belayev et al. 1997 ; Kametsu et al. 2003; Tamura et al. 1981b ) and together with the greater vulnerability has led to the frequent reference to striatum as “core” ischemic territory in such models. However, it must be recognized that the abundant projection neurons of dorsolateral striatum are among the intrinsically vulnerable cell populations in brain, showing marked loss after longer durations of global ischemia (Chesselet et al. 1990 ; Meade et al. 2000 ; Pulsinelli et al. 1982 ; Smith et al. 1984 ) . This explicitly confounds the interpretation of striatal pathology after fi lament occlusions (Katchanov et al. 2003 ; Korematsu et al. 1993 ) , since such lesions must be considered mechanistically distinct from infarction. This type of striatal damage is often possible after the intervals of fi lament occlusions used as preconditioning insults in many studies and may contribute to the protection mechanism, as considered further below. The above has dealt largely with fi lament occlusions in rats. Parallel issues arise in mice with respect to the extension of the ischemic insult beyond MCA territory (McColl et al. 2004; Özdemir et al. 1999) . However, although delayed temperature increases have been described in apparent association with immunosuppression and secondary infection (Meisel et al. 2004 ) , heat loss is by far the more critical problem during acute anesthesia recovery in mice (Barber et al. 2004 ; Connolly et al. 1996 ; Herrmann et al. 2003 ) . This is not simply due to the effects of surgical anesthesia, since cooling is more pronounced with increasing insult duration (Barber et al. 2004) . Sustained temperature control through 90 min after permanent fi lament occlusion in C57BL/6 mice resulted in larger infarcts than if only 10 min of postoperative warming were provided (Connolly et al. 1996 ) , core temperature of the latter group falling below 32°C during that interval. This is comparable to the protective effect of early cooling during permanent MCA occlusion in Wistar rats noted above (Baker et al. 1992 ; Kader et al. 1992 ; Ren et al. 2004 ) . Incidental hypothermia can persist for several hours after transient occlusions in mice, resulting in reduced infarct volume even when temperature control is maintained during the interval of fi lament insertion (Barber et al. 2004 ) . Long-term postischemic temperature monitoring is therefore essential in the context of any protection study.

13.2.3 Other Models

Several alternative occlusion methods and non-rodent species have been used in conditioning studies. Endothelin-induced vasoconstriction of the MCA (Sharkey et al. 1993 ) was applied to produce stroke in an evaluation of thrombin preconditioning (Henrich-Noack et al. 2006 ) . Photothrombotic microvascular occlusion in rats (Watson et al. 1985 ) was found sensitive to acute melatonin pretreatment, but 274 T.S. Nowak and L. Zhao not to hypoxic preconditioning (Matějovská et al. 2008 ) . Surgical MCA occlusion has been applied to hyperbaric oxygen preconditioning studies in rabbits (Gao-Yu et al. 2011 ) . Preconditioning effects have been recently studied in the rhesus monkey, Macaca mulatta (Bahjat et al. 2011 ) , using a retro-orbital approach to transiently occlude both ACAs and the MCA at a comparatively distal site (West et al. 2009 ) . The 60-min occlusion interval applied in this study was below the threshold for overt infarction defi ned in earlier work using more proximal MCA occlusion alone in the same species (Crowell et al. 1970 ; Gao et al. 2006 ) or in Macaca fascicularis (Marcoux et al. 1982) . In part, the effi cacy of this model may refl ect the attenuation of collateral perfusion achieved by simultaneous occlusion of the ACAs, although the methods applied to in vivo imaging and histopathological evaluation also are likely to have included regions of selective neuron loss in the measured lesions. Additional models and re fi nements of established methods are continuously under development, and it is likely that focal brain ischemia models not mentioned here have been or will be applied to the study of conditioning effects. For any large vessel occlusion method, collateral perfusion will remain the primary variable impacting model design and evolution of pathology.

13.3 Physiological Variables and Anesthesia in Conditioning Studies

13.3.1 Impact of Conditioning on Cerebral Perfusion

Initial preconditioning studies had suggested that protection was not associated with overt improvements in perfusion, but these involved measurements at either discrete loci or at comparatively early time points. Hydrogen clearance measurements of absolute cortical CBF during fi lament occlusion found equivalent local fl ow defi cits in sham-operated animals and in rats that exhibited protection after a previous global ischemic insult (Matsushima and Hakim 1995 ) , and comparable results were obtained for preconditioning induced by cortical spreading depression (CSD) in two laboratories using distinctly different models (Matsushima et al. 1996 ; Yanamoto et al. 1998 ) . Although providing reliable quantitative measurement during prolonged intervals, the method permitted monitoring at only one site. Laser Doppler fl owmetry at a single recording location also identi fi ed no signi fi cant difference in relative perfusion between rats preconditioned by a prior interval of occlusion and a corresponding sham group (Barone et al. 1998 ) . Interpretation of the above results is limited by the concern that the regions evaluated may not have corresponded to the penumbral territory that is protected. However, using quantitative autoradiography to assess absolute regional CBF, it was shown that rats preconditioned by a series of 10-min fi lament occlusions showed perfusion reduction comparable to that of sham animals at a 30-min time point during the subsequent 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 275 test occlusion (Chen et al. 1996) . A subsequent autoradiographic study identifi ed no signifi cant effect of a similar preconditioning regimen on the distribution of perfusion de fi cit at a somewhat later 2-h time point (Alkayed et al. 2002 ) . Likewise, there was no effect of LPS-induced preconditioning on regional CBF at 15 min after a permanent surgical MCA occlusion (Dawson et al. 1999 ) . These independent results established that the initial distribution of CBF reduction remained unaltered by effective preconditioning insults. Importantly, although the acute fl ow de fi cit was not changed, LPS-preconditioned SHR maintained better microvascular fi lling in spared cortex at 24 h (Dawson et al. 1999) . Subsequent work explicitly established the delayed nature of preconditioning-associated CBF improvements. Using magnetic resonance imaging to identify the rim of spared penumbral cortex, repeated laser Doppler measurements specifi cally targeting this region demonstrated increased relative perfusion in LPS- preconditioned SHR at intervals 6 h or longer after occlusion (Furuya et al. 2005 ) . In agreement with previous studies using the same preconditioning methods and occlusion model (Dawson et al. 1999) , no difference was observed at 15- or 30-min time points. Corresponding results were also obtained in SHR preconditioned by a brief transient occlusion interval, using quantitative autoradiographic CBF measurements in awake animals (Zhao and Nowak 2006b) . The volume of cortex falling below the perfusion threshold for infarction was initially identical in naïve and preconditioned rats but declined heterogeneously in the latter group between 90 min and 3 h after occlusion (Fig. 13.1 ), with an apparent bimodal distribution. Ischemic territory volume after 3 h accurately predicted the distribution of fi nal infarct volumes in naïve, sham, and preconditioned groups (Zhao and Nowak 2006b ) . This interval corresponds closely to the temporal threshold for maximal infarction in the SHR (Jacewicz et al. 1992 ) . Results from several laboratories therefore indicate that CBF improvement within a critical time window contributes to a protection mechanism common to LPS and ischemic preconditioning in the surgically occluded SHR. Some studies in other stroke models have identifi ed more acute perfusion differences in preconditioned animals. An early hydrogen clearance study of ischemic preconditioning in Wistar rats noted somewhat better maintained CBF within the ischemic territory during 2 h of intraluminal fi lament occlusion (Matsushima and Hakim 1995) , although the infarcts produced were too small and variable to document a preconditioning effect. More recently, improved perfusion was reported throughout the duration of comparatively short 45- or 60-min fi lament occlusions in a mouse ischemic preconditioning model, using both laser Doppler and perfusion- weighted magnetic resonance imaging (Hoyte et al. 2006 ) . In the latter study, perfusion was determined relative to baseline or as a percentage of the contralateral side, respectively, and could have been infl uenced by factors that might alter either ipsilateral baseline CBF or global brain perfusion. In fact, a number of results suggest that reductions in ipsilateral CBF can occur secondary to tissue injury, whereas global CBF can be in fl uenced by prior anesthesia exposure. Some preconditioning manipulations clearly impact baseline CBF. In a study of CSD-induced preconditioning that involved topical KCl application to cortex, it was 276 T.S. Nowak and L. Zhao

Fig. 13.1 Preconditioning-associated CBF changes in the spontaneously hypertensive rat. Quantitative autoradiography establishes that the fl ow de fi cit at 3 h following permanent MCA occlusion is markedly attenuated after prior ischemic preconditioning (PC), with a modest effect of sham treatment ( upper panel). Arrowheads identify the margin of severely ischemic territory (~30 ml/100 g/min) in representative animals of the respective treatment groups. The volume of cortex below this threshold for infarction is initially identical in naïve and PC groups and remains constant in naïve animals, but penumbral perfusion improves by 3 h in the preconditioned animals, with an apparently bimodal distribution (lower panel ) (*, P < 0.05 vs. corresponding Naïve rats) (Adapted from data of Zhao and Nowak 2006b ) noted that perfusion relative to baseline, assessed by laser Doppler fl owmetry, remained signifi cantly higher than that of sham-operated rats during 2 h of fi lament occlusion 3 days later (Otori et al. 2003 ) . However, autoradiographic measurements of absolute regional perfusion in comparably treated animals actually showed a reduction in absolute ipsilateral baseline CBF, and the fl ow defi cit during occlusion was calculated to have been unaltered in preconditioned animals. A following study showed that prior application of 5 M NaCl, which produced cortical lesions but did not induce CSD, comparably reduced infarct volume and impacted fl owmetry during occlusion (Muramatsu et al. 2004 ) . It was suggested that reductions in baseline perfusion re fl ected the decreased metabolic demand (glucose utilization) also reported following such lesions (Kawahara et al. 1999 ; Pappius 1981 ) , which could directly contribute to their protective ef fi cacy. Variable decreases in ipsilateral CBF were 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 277 noted following preconditioning by brief intervals of focal ischemia (Zhao and Nowak 2006b ) and were subsequently associated with the presence of incidental lesions (Zhao and Nowak 2006a ) . However, as noted above, CSD induced by more discrete KCl microinjection, with correspondingly less tissue damage, led to robust preconditioning without apparent impact on pre-occlusion CBF (Matsushima et al. 1996 ; Yanamoto et al. 1998 ) . Changes in baseline perfusion and metabolism therefore re fl ect the extent of prior tissue injury, but do not appear to be required for conditioning effects. This will be considered in greater detail below in the context of recent studies speci fi cally investigating preconditioning effects of cortical lesions. Global perfusion has not been extensively examined in preconditioning models, largely because this requires quantitative monitoring methods that are not widely in use, but some evidence suggests that generalized increases in absolute CBF may contribute to the preconditioning effects of prior anesthesia. For example, contralateral perfusion remained low for several hours in naïve SHR awake after halothane anesthesia following MCA occlusion surgery but recovered more rapidly in rats that had experienced either an interval of preconditioning ischemia or a sham procedure, which was itself partially protective (Zhao and Nowak 2006b ) . Higher global CBF (measured in contralateral cortex) correlated with reduced ipsilateral ischemic territory volumes. Both of the previously manipulated groups also showed slight decreases in blood pressure relative to naïve animals, indicative of generalized vasodilation. One other study speci fi cally examining perfusion effects of iso fl urane preconditioning also noted improved ipsilateral CBF, measured autoradiographi- cally 1 and 3 h after permanent distal MCA occlusion in Wistar rats (Chi et al. 2010b ) . Although contralateral CBF was unchanged relative to controls, the animals of this study had been maintained under iso fl urane anesthesia throughout the time of perfusion measurement. A similar result was obtained in a model of opioid preconditioning by the same laboratory (Chi et al. 2010a ) . Given the myriad confounds in the evaluation of CBF under anesthesia, considered further below, it is clear that more comprehensive studies are required to adequately assess the potential contribution of perfusion changes to conditioning effects in a broad range of models.

13.3.2 Tissue Damage and Preconditioning Effects

In the idealized scenario, a preconditioning treatment is intended to be below an insult threshold that would itself be associated with detectable injury. However, it is increasingly recognized that many such manipulations can have substantial impacts (Sommer 2008 ) . As noted above, varying degrees of cortical injury can be produced in the course of inducing CSD for preconditioning studies (Otori et al. 2003 ) . Similarly, striatal neurons are vulnerable to the durations of fi lament occlusions used to produce some priming insults (Kametsu et al. 2003 ; Li et al. 1995 ) . Striatal hyperemia had been noted at 24 h following preconditioning occlusions in an early rat study (Chen et al. 1996) , progressing to regions of focal stri- 278 T.S. Nowak and L. Zhao atal injury and selective cortical neuron loss at later intervals. In another study, striatal damage was indicated by magnetic resonance imaging 24 h after a preconditioning fi lament occlusion (Mullins et al. 2001 ) . Occasional injury to striatal neurons was also mentioned in a mouse model (Stagliano et al. 1999 ) . A comprehensive optimization of preconditioning by repeated intraluminal fi lament occlusions in mice was successful in identifying a series of manipulations after which no neuronal damage could be detected but also confi rmed the presence of lesions under many commonly used experimental conditions (Zhang et al. 2008 ) . Furthermore, although short occlusion durations may remain below the threshold for producing consistent pathology (Pedrono et al. 2010 ) , the risk of microembo- lism after even very brief fi lament insertion, noted above (Zhan et al. 2008 ) , demonstrates the potential for diffuse local injury after preconditioning insults. No acute lesions were reported following preconditioning ischemia produced by prior surgical occlusions that selectively target MCA territory cortex (Purcell et al. 2003 ) . However, local neuronal apoptosis at the site of vascular manipulation is common in such models (Currie et al. 2000 ) . Moreover, preconditioning insults can induce neuronal expression of the 70-kDa stress protein, hsp70, throughout MCA territory, with even more widespread glial activation that was accompanied by astrocytic hsp27 expression (Currie et al. 2000 ) . Although such responses may not refl ect overt neuron loss, they seem to occur at an insult threshold associated with subtle dendritic pathology (Nishino and Nowak 2004 ) . Finally, although it had been our impression that overt damage was absent from such a model (Zhao and Nowak 2006b ) , a retrospective analysis of the histological sections used for CBF autoradiography in that study subsequently identi fi ed small foci of injury in the subset of both preconditioned and sham-operated animals that also exhibited the most robust reduction in ischemic territory (Zhao and Nowak 2006a ) . This would appear to explain the bimodal preconditioning observed in the model as the combination of a uniform anesthesia effect with sporadic additional contributions of focal injury. The latter observation has provided the basis for development of a preconditioning model based on the production of small cortical freeze lesions (cold lesions, CL) (Zhao and Nowak 2011b ; L. Zhao and T. S. Nowak, Jr., manuscript in preparation). In brief, (1) prior lesions reduce infarct volume after subsequent permanent focal ischemia in the SHR; (2) protection is associated with delayed recovery of penumbral perfusion after awakening from anesthesia, as described above for ischemic preconditioning in this model; (3) the magnitude of protection is essentially independent of lesion size, which can be made small enough to avoid overt injury-induced decreases in CBF and glucose utilization; and (4) the anesthesia interval required for production of preconditioning lesions is suf fi ciently short that no signi fi cant sham preconditioning effect is observed under routine modeling conditions. As described below, several issues were encountered in the course of model development that emphasize the critical importance of physiological monitoring in studies that involve repeated surgical procedures and illustrate the particular challenges associated with anesthesia selection in experimental stroke. 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 279

13.3.3 Anesthesia Confounds in the Study of Acute Stroke Pathophysiology

Effective anesthesia and analgesia are essential elements of the surgical procedures required to produce focal brain ischemia under most experimental conditions. Anesthetics are well recognized to impact cerebral blood fl ow and metabolism and can therefore alter stroke pathophysiology. In general, sustained exposure to volatile anesthetics during transient occlusions is considered protective (Kawaguchi et al. 2000 ; Saito et al. 1997 ; Warner et al. 1995 ) , although not under all conditions (Hu et al. 2011 ) . Procedural details must be considered since, for example, rats ventilated under halothane anesthesia during transient fi lament occlusion better maintained blood pressure and blood gases and showed smaller infarcts and better survival than those breathing spontaneously under halothane or choral hydrate anesthesia (Zausinger et al. 2002 ) . Halothane and isofl urane have been reported to differentially impact the ef fi cacy of ischemic preconditioning (Zhang et al. 2008 ) . Concerns may be reduced in permanent occlusion models, but anesthesia duration can still be a critical variable, particularly in strains characterized by appreciable collateral perfusion. Thus, occlusion site and anesthesia duration interact to in fl uence infarct size in Long-Evans rats (Hashimoto et al. 2008 ) . Tandem MCA/CCA occlusions targeting the MCA at the level of the rhinal fi ssure yielded larger infarcts than those at slightly more distal occlusion sites when halothane anesthesia was discontinued immediately after occlusion surgery, consistent with better preservation of collateral perfusion during distal occlusion. When anesthesia duration was prolonged, infarct volumes after distal occlusions increased to equal those after more proximal occlusions. Likewise, transient 3-h ischemia under sustained anesthesia resulted in similarly large infarcts after occlusion at either location. If anesthesia intervals had not been kept comparable, this would have been interpreted as an apparent increase in infarct volume after transient occlusion at the distal site. Such a phenomenon would appear to account for previous reports of “reperfusion injury” in this rat strain (Aronowski et al. 1997 ) . A likely contributor to such effects is the extended interval of reduced blood pressure under sustained anesthesia (Hashimoto et al. 2008 ) . Hypotension is well recognized to exacerbate injury during focal ischemia (Zhu and Auer 1995 ) , and hypertension can be protective (Chileuitt et al. 1996 ; Drummond et al. 1989 ; Smrcka et al. 1998 ) . Adequate blood pressure during early reperfusion is critical to achieve robust re fl ow after prolonged global ischemia (Böttiger et al. 1997 ; Hossmann 1988 ) , and acute tissue injury after prolonged focal ischemia can be reduced by brief hypertension during initial reperfusion (Cole et al. 1992 ) . As noted previously, modest blood pressure reductions indicative of generalized vasodilation can sometimes be associated with increased CBF in awake animals (Zhao and Nowak 2006b ) , as well as under some anesthesia conditions (Takeda et al. 2011 ) . The interaction between blood pressure and perfusion is therefore a critical variable in the context of any conditioning intervention, including postconditioning models. Blood glucose is another physiological variable critically impacting stroke outcome, particularly in the context of increased collateral blood fl ow or reperfusion. 280 T.S. Nowak and L. Zhao

As reviewed previously (Kent et al. 2001 ) , hyperglycemia more markedly worsens damage in transient occlusion models (Kamada et al. 2007 ) , or in brain regions with more robust collateral perfusion (Prado et al. 1988 ) , and a component of this effect may be mediated by more severe CBF defi cits (Kawai et al. 1997 ; Quast et al. 1997 ) . Conversely, elevated glucose can reduce the incidence of peri-infarct depolarizations (PIDs) during occlusion (Nedergaard and Astrup 1986 ) . PIDs are increasingly recognized as likely contributors to the expansion of tissue injury and functional de fi cits in both experimental and clinical stroke (Lauritzen et al. 2011 ; Takeda et al. 2011) . Apparent protective effects of hyperglycemia in a model of microvascular coagulation (Ginsberg et al. 1987 ) could represent the special case of a model with a negligible perfusion penumbra in which the benefi ts of PID reduction may predominate (Prado et al. 1988) . Complex anesthesia effects on blood glucose were encountered in recent studies to develop a preconditioning model based on prior cortical freeze injury (Zhao and Nowak 2011c ) . Iso fl urane is known to have hyperglycemic effects both clinically (Lattermann et al. 2001 ) and in the experimental setting (Constantinides et al. 2011 ; Tanaka et al. 2009 ) . Glucose levels can be reasonably well controlled by an overnight fast in naïve animals. However, a modest but signi fi cant increase in blood glucose levels occurred even in fasted rats if they had experienced prior anesthesia and surgery to produce preconditioning lesions, and a comparable effect was seen after a sham procedure (Fig. 13.2 , upper panel). The highest levels reached in these studies were associated with reduced PID incidence following MCA occlusion (Fig. 13.2, lower panel), requiring that animals with glucose levels exceeding 200 mg/dl be excluded from the study to avoid confounds in comparing the groups. It is unclear whether such variability is suf fi cient to impact infarct volume in the model, but it clearly interferes with the study of stroke pathophysiology under prolonged anesthesia. It should be noted that iso fl urane effects on blood glucose may not be of general relevance in preconditioning models. Glucose levels did not appear to be systematically altered in an early study of anesthesia preconditioning in Wistar rats (Kapinya et al. 2002) or after isofl urane or ischemic preconditioning in C57BL/6 mice (Wang et al. 2008; Zhang et al. 2008 ) . In another mouse study, glucose levels were high and variable during fi lament occlusion under iso fl urane anesthesia, but no signi fi cant differences were seen between sham and isofl urane preconditioned groups (Zhu et al. 2010 ) . It remains good practice to evaluate glucose status in the context of any manipulation, but its susceptibility to change would appear to vary considerably among models. Additional anesthesia effects emerged in the course of evaluating PID incidence in the lesion-induced preconditioning model, which appear to provide some insight into anesthesia preconditioning mechanisms. It is well recognized that PID incidence and propagation are attenuated by volatile anesthetics (Patel et al. 1998 ; Saito et al. 1997 ; Takeda et al. 2011 ) , and a -chloralose has become the alternative anesthetic of choice for studies of such events (Hashemi et al. 2009 ; Luckl et al. 2008 ; Takeda et al. 2011 ) . This agent decreases blood fl ow relative to volatile anesthetics but also maintains more stable perfusion (Takeda et al. 2011 ) . As expected, PID incidence (measured as peri-infarct CBF increases by speckle contrast perfusion imaging) was higher in rats maintained under a -chloralose vs. isofl urane (Fig. 13.3 , 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 281

Fig. 13.2 Hyperglycemia after repeated iso fl urane exposure and its impact on peri-infarct depolarization. Whereas blood glucose can be adequately controlled by an overnight fast in the naïve spontaneously hypertensive rat, animals that experienced prior anesthesia and skull exposure to produce a cortical cold lesion (CL), or the corresponding sham surgery, exhibit signifi cant hyperglycemia when re-anesthetized with iso fl urane for MCA occlusion surgery the following day ( upper panel ). Glucose levels above 200 mg/dl are associated with a decrease in the incidence of peri-infarct depolarization (PID), as assessed by speckle contrast perfusion imaging of the associated CBF transients in these groups ( lower panel). Although even normoglycemic rats exhibit fewer PIDs after preconditioning, failure to exclude hyperglycemic animals would overestimate the impact of preconditioning on PID incidence (Adapted from data of Zhao and Nowak 2011c ) upper panel). However, there was an important anesthesia-dependent difference in the relative effect of sham treatment vs. CL preconditioning. Under a -chloralose, there was no effect of sham surgery, and only the CL group exhibited a reduction in PIDs, corresponding to the absence of a sham effect on fi nal 24-h infarct volume in the model, noted above. A statistically signifi cant reduction in infarct size was already evident in the CL group at the end of the 4-h recording session (Fig. 13.3 , lower panel), re fl ecting the rapid time course of infarct evolution under a -chloralose anesthesia (Saito et al. 1997 ; Takeda et al. 2011 ) . However, under sustained isofl urane anesthesia, rats that had experienced sham surgery showed a reduction in PID incidence identical to that of the CL group, as well as a trend toward reduced infarct volume. Therefore, a prominent sham effect emerges when animals are maintained under isofl urane anesthesia for prolonged monitoring, suggesting that 282 T.S. Nowak and L. Zhao

Fig. 13.3 Anesthesia regimen impacts acute infarct evolution and peri-infarct depolarization in the spontaneously hypertensive rat. Animals maintained under continuous isofl urane anesthesia exhibit reduced PID incidence relative to those switched to a-chloralose at the time of MCA occlusion (upper panel ). Only rats preconditioned by cortical cold lesions (CL) have reduced PID number relative to the naïve group under a -chloralose anesthesia, whereas both sham and CL animals show signifi cant reductions under isofl urane. Infarct volumes at the end of recording (4 h after MCA occlusion) followed similar trends (lower panel ), although a statistically signi fi cant reduction in infarct volume was observed only for the CL group under a -chloralose anesthesia (*, P < 0.05 vs. corresponding Naïve rats) (Adapted from data of Zhao and Nowak 2011a ) isofl urane preconditioning in this case refl ects a change in the impact of the anesthetic itself on stroke pathophysiology (Zhao and Nowak 2011a ) . Given the complex in fl uence of volatile anesthetics on CBF, differences in perfusion would seem a plausible candidate mechanism, but this remains to be investigated. Such an effect would be predicted to particularly impact those transient occlusion models in which anesthesia is maintained throughout the ischemic insult.

13.4 Conclusions

CBF remains the central variable in ischemic stroke and warrants renewed attention to its role in brain protection by conditioning treatments as well as other interventions (Sutherland et al. 2011 ) . Available evidence clearly indicates the relevance of perfusion effects in at least a subset of conditioning models. Many studies addressing the role of changes in physiological variables in conditioning have involved surgi- 13 Conditioning Studies in Focal Cerebral Ischemia: Model Selection... 283 cal occlusion methods. In part, this refl ects a greater emphasis on routine physiological monitoring and control in such models, since they require longer anesthesia intervals than typically required for intraluminal fi lament occlusions. The generally lower variability of MCA occlusion in the SHR also facilitates acquisition of inter- pretable data in a study of manageable scale. However, fi lament occlusion models are sensitive to the same physiological variables, which may in fact be even more relevant in view of the intrinsically greater collateral perfusion and larger volume of salvageable penumbral cortex in the strains used for such studies. Prolonged or repeated anesthesia is usually required for transient occlusions, further extending the interval during which attention to physiological variables is required. Comprehensive evaluation of potential perfusion effects would appear to be warranted in all conditioning models. More generally, not all manipulations that attenuate pathology in a tissue injury model can be expected to prove clinically useful as protective preconditioning or postconditioning treatments. It is essential to understand the critical variables that in fl uence outcome in the model under study, to determine whether these are impacted by the conditioning intervention, and if so, to critically examine the relevance of the affected parameter to the clinical condition. This must be a primary consideration in assessing the translational relevance of conditioning effects in stroke.

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Robert P. Ostrowski and John H. Zhang

14.1 Introduction

Subarachnoid hemorrhage (SAH) is a bleeding into the space situated between the middle (arachnoid) and the innermost (pia mater) meninges covering the brain (MacDonald 1989 ) . Aneurysmal SAH most often occurs spontaneously and cannot be predicted. Therefore, a question arises to what extent conditioning modalities are of use for this clinical entity. Are all these means primarily serving to increase brain tolerance to injury in case of hemorrhage or ischemia complicating aneurysm surgery? Although several strategies can be used for prophylactic neuroprotection, including hypothermia, barbiturates, and free radical scavengers, the intraoperative rupture of aneurysm is still associated with signi fi cant morbidity (Keedy 2006 ) . It is hoped that conditioning could improve overall clinical outcomes including survival rates, neurological status, and quality of life after SAH. Even in the treated SAH patients, the mortality up to 30% is sustained (Cahill and Zhang 2009 ) . Although it remains uncertain whether the conditioning can be applied to target acute brain injury in SAH, it surely can be considered when cerebral vasospasm is anticipated. Principally because of cerebral vasospasm (CVS), SAH cases have high risk of ischemic stroke in the 2 weeks following the initial bleed (Fergusen and Macdonald 2007 ) . Since the effectiveness of conditioning for SAH remains largely unexplored, its therapeutic pro fi le and mechanisms should be fi rst characterized in the preclinical studies.

R. P. Ostrowski Department of Physiology & Pharmacology , Loma Linda University School of Medicine , Loma Linda , CA , USA J. H. Zhang (*) Loma Linda University, Loma Linda , CA , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 291 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_14, © Springer Science+Business Media New York 2013 292 R.P. Ostrowski and J.H. Zhang

Due to a clinical complexity of SAH and CVS, the terms denoting conditioning in SAH need to be carefully selected. The conditioning after SAH and before the development of vasospasm cannot be classically termed preconditioning (PC) because it is implemented after the initial bleeding, i.e., during early brain injury phase, which is an integral component or SAH pathology. This opens several possibilities in terminology of conditioning procedures (e.g., postconditioning or preconditioning) depending upon type of stimulus, conditioning regimen, and with respect to phase of SAH injury.

14.2 Types of Conditioning for SAH

Multiple conditioning modalities and corresponding stages of SAH-related injury have been summarized in Fig. 14.1 . Preconditioning is conducted before anticipated bleed into the subarachnoid space. It can be done by a brief occlusion of cerebral arteries. Also, hyperbaric oxygen (HBO) for PC in SAH seems to be a reasonable option although it has not been studied so far in this aspect. In contrast, the classical hypoxic preconditioning prior to SAH has been recently carried out in one experimental series of mice and resulted in the attenuation of cerebral vasospasm and neurological outcomes (Vellimana et al. 2011 ) . Postconditioning strategies involve

Preconditioning Perconditioning Postconditioning Perconditioning

SAH CVS

CONDITIONING Time OF THE BRAIN

REMOTE CONDITIONING

Fig. 14.1 Types of conditioning for SAH and intracranial aneurysms. Preconditioning is done before hemorrhagic brain insult or neurovascular surgery. Postconditioning may be carried out after the initial bleeding. Perconditioning denotes conditioning during bleeding or developing vasospasm 14 Preconditioning for SAH 293 exposure to the conditioning stimulus after SAH occurs. Remote conditioning is the modality of neuroprotection in which brief subinjurious insult to one organ will result in the protection of the brain against the subsequent major harmful stimuli (Ren et al. 2009 ; Tapuria et al. 2008 ) . This is usually achieved by the placement of the pressure cuff on the upper or lower extremity in order to constrict peripheral arteries temporarily to the point of transient limb ischemia (Ren et al. 2008 ) . Remote postconditioning induced by transient limb ischemia after SAH could protect against progression of brain injury and prevent from detrimental SAH sequelae including cerebral vasospasm. Special position in this classiﬁ cation is occupied by remote perconditioning (e.g., four cycles of 5-min limb ischemia intertwined with 5-min periods of reperfusion) (Saxena et al. 2010) . In case of SAH, perconditioning would signify the remote conditioning induced during subarachnoid bleed and ultra-early vascular narrowing or applied upon evolving cerebral vasospasm. As such, perconditioning, when feasible, could become an immediate intervention targeting critical stages of SAH-induced brain injury (Ostrowski et al. 2006 ) .

14.3 Conditioning Modalities

Established conditioning modalities produce the state of endogenous brain protection that lasts several days after induction (Dirnagl et al. 2009 ; Obrenovitch 2008) . All strategies must take this into consideration as the effect of procedures done too early will dwindle before the insult occurs. With high likelihood, there are different therapeutic windows of opportunity for pre- and postconditioning in SAH, which however has not been researched in detail so far. From experimental studies of cerebral ischemia comes an indication that the range for postconditioning therapeutic windows stretches from a few seconds to 2 days after reperfusion (Zhao 2009 ) . In contrast, recently developed 2-week-long protocol of repetitive hypoxic preconditioning induces strong epigenetic anti-in fl ammatory phenotype and maintains neuroprotection for 8 weeks after completion (Stowe et al. 2011 ) . Viable translation of long preconditioning regimens with regard to SAH is however uncertain. In the setting of repair surgery for ruptured intracranial aneurysms, brief occlusion of the proximal artery has been proven effective as a preconditioning technique (Chan et al. 2005 ) . It seems though that iso fl urane could be also used as a safe and effective PC modality before aneurysm surgery. A high concentration of iso fl urane (to induce EEG isoelectricity) has been found to offer brain protection during aneurysm repair surgeries as reported by Dr. Mayer’s team at the Mayo Clinic (Meyer and Muzzi 1992) . Breathing hypoxic air before experimental SAH (8% oxygen for 4 h at 24 h prior to SAH) offers neurological benefi ts and prevents vasospasm from developing (Vellimana et al. 2011 ) . Likewise, statins have been tested as the preconditioning agents against cerebral vasospasm, and this treatment resulted in signi fi cant level of protection (Chang et al. 2010 ) . This approach, however, according to a classical view on the preconditioning produces rather preconditioning-like effect, because statins do not seem to impose a stressful stimulus on the brain or other 294 R.P. Ostrowski and J.H. Zhang organs (Domoki et al. 2009 ; Keep et al. 2010 ) . As shown by Godman and collaborators, the protective molecular phenotype can be also induced by HBO-PC, and one may consider it as the candidate means of brain protection against consequences of subarachnoid bleeding (Godman et al. 2010 ) . HBO-PC appears especially suitable when the risk of traumatic SAH exists. Interestingly, without much of elaboration on the basic science grounds, clinical trials with remote conditioning after SAH are currently being carried out (Koch et al. 2011 ) . The usefulness of other means has not been speci fi cally veri fi ed with regard to SAH, however. Thrombin PC (1 U intracerebrally) provides neuroprotection in experimental ICH via the p44/42 MAPK pathways, which seems to offer therapeutic implication for SAH as well (Hua et al. 2003 ) . Thrombin has been demonstrated to play a role in the mechanism of injury in both entities, ICH and SAH (Gong et al. 2008; Sugawara et al. 2009 ) . Therefore, it is reasonable to expect that preconditioning with a low dose of thrombin may produce clinical bene fi t in SAH.

14.4 Clinical Applications and Strategies

In the recent years, early surgery after aneurysmal SAH has been advocated especially for the anterior circulation aneurysms. One approach relies on placing a metal clip on the aneurysm (clipping) through craniotomy (van der Jagt et al. 2009 ) . Coiling is a vial alternative although for certain locations clipping remains a dominant option such as for the aneurysms of middle cerebral artery (Regli et al. 2002 ) . Despite advancements in clipping and coiling techniques, the percentage of dead or dependent patients after treatment of ruptured aneurysms remains high (23.5Ð30.9% after 1 year) (Molyneux et al. 2005 ) . For these early surgeries, which due to acute edema and infl ammation require more brain retraction, preconditioning would be justifi able (Fig. 14.2). In addition, certain procedures used during aneurysm surgery create the risk of hypoxic injury, e.g., systemic hypotension used sometimes in the fi nal surgery phase in order to facilitate placement of the clip on the aneurysm neck (Han et al. 2004 ) . For the same reason, focal hypotension is applied intraoperatively, by transiently occluding the artery with aneurysm (Lavine et al. 1997 ) . Even though this maneuver helps to clip the aneurysm, focal ischemia may ensue. It is also known that occasionally small arteries need to be sacri fi ced in order to open the access to the aneurysm that needs clipping (Ahn et al. 2007 ) . Moreover, unruptured aneurysm may rupture intraoperatively, which enables conditioning. Notably, coiling carries a higher risk of intraprocedural rupture than clipping (Guan and Wang 2008) . Suitable conditioning may be provided by limb occlusion, hypoxic air inhalation, or even brief occlusion of cerebral arteries; however, the applicability of other modalities is debatable. The use of HBO for this purpose awaits experimental verifi cation. Generally, HBO should be used after aneurysm is secured (ruptured or not). There is a concern that after aneurysm rupture, HBO might induce clot dislodgement and rebleeding (Ostrowski and Zhang 2011 ) . One experimental study, however, demonstrated functional bene fi t and 14 Preconditioning for SAH 295

Intraprocedural Intraprocedural Cerebral bleeding ischemia SAH tSAH vasospasm

Hypoxic PC ++++ + HBO PC +/? +/? +/? + + Remote PC + + + + + Postconditionig + + + + +/?

Perconditionig + + +/? +/? +

Fig. 14.2 Conditioning modalities and proposed clinical applications. Hypoxic/ischemic preconditioning of the brain or nonvital remote organ can be done in patients faced with increased risk of intraprocedural bleed or brain hypoxia or when the risk of SAH is imminent. Postconditioning, including limb ischemia and HBO exposure, is suitable for patients with SAH or with neurosurgical complications. Perconditioning is applicable intraoperatively or upon vasospasm although it can be hardly doable for spontaneous or traumatic bleeds. Postconditioning for CVS may be considered although it appears as too late intervention, and, therefore, its role is uncertain

reduced mortality with HBO-based therapeutic approach (Ostrowski et al. 2005 ) . Without further experiments utilizing models of aneurysm, this issue will remain somewhat unresolved. In patients with unruptured aneurysms and especially in those treated with endovascular approach, postconditioning may not be required because patients with coiled aneurysms recover rather rapidly and oftentimes without complications (Brilstra et al. 2004 ) . The combination of both pre- and postconditioning is not justi fi able as the bene fi ts from these modalities may not be additive (Pignataro et al. 2008) . In principle, it seems more benefi cial to stimulate endogenous defenses even before injurious stimuli are received. While increasing resilience of cells with postconditioning still makes a lot of sense clinically, only boosting resistance to injury will prevent certain population of neurons from dying on impact and through early apoptosis (Ostrowski et al. 2008 ) . The major rationale of conditioning for SAH is however the increased risk of ischemic stroke due to cerebral vasospasm after SAH (Pluta et al. 2009 ) . Early perconditioning could be considered in case of subangiographic and angiographic vasospasm where it could prevent clinical deterioration. This could be brief general hypoxia or brief limb ischemia alternating with reperfusion. As long as the 296 R.P. Ostrowski and J.H. Zhang

aneurysm has been secured, hyperbaric oxygenation might as well be benefi cial. Furthermore clinical vasospasm would also require conditioning of some sort; preferably utilizing remote organ ischemia. All these modalities need further evaluation of clinical safety and risk of complications in so treated patients, however (Keep et al. 2010; Koch et al. 2011) . It is the severe condition of many patients with SAH that precludes intense prophylactic therapy before vasospasm, such as preventive triple H. This approach has been oftentimes advised against due to a high risk of complications in the clinical setting (Hunt and Bhardwaj 2007; Meyer et al. 2011 ) . There are also some other clinical considerations. Notably the clinical grade and characteristics of patients selected for postconditioning matter. It of concern that severely compromised patients may have only little benefi t from conditioning. This stays in line with the caliber of the ruptured vessel, which may inversely affect the effectiveness of any conditioning modality. On the other hand, SAH usually creates severe injuries, and, therefore, devising conditioning procedures in hope for mild brain insult would not adhere to reality. Some authors have argued that when treated aggressively, poor-grade SAH patients do improve (Taylor et al. 2011 ; Wartenberg 2011) . The existing literature may suggest that some modalities such as HBO can ameliorate the extent of neurological defi cits in severe experimental SAH (Ostrowski et al. 2005 ; Walid and Zaytseva 2009 ) . To what extent the benefi cial effect is derived from conditioning with elevated reactive oxygen species or whether HBO principally ameliorates tissue oxygenation is not known. Although it is believed that oxidative stress accompanying HBO is crucial for conditioning effect, it is also a matter of concern (Frietsch and Kirsch 2004 ; Thom 2009 ) . Study by Alken and colleagues, however, has demonstrated the benefi cial effect of HBO in the acute phase of cerebral vasospasm, which due to a signi fi cant hyperbaria (3 ATA) could also have a preconditioning component. If so, a signifi cant reduction in neurologic defi cits in rats with vasospasm may indirectly point toward robustness of such conditioning effect of HBO (Kocaogullar et al. 2004 ) . Remote conditioning is a powerful and fl exible clinical approach. One of the cerebral blood vessels that entail dif fi culties in the course of aneurysm surgery is the choroidal artery. In addition, patients with choroidal artery aneurysms have higher risk for postoperative stroke (Piotin et al. 2004 ) . It would be diffi cult to propose that this artery can be selected for preconditioning by a brief occlusion so that tissue damage in its territory could have been reduced had the bleed actually occurred. Remote preconditioning paradigm, however, surpasses this obstacle, by spreading beyond any given territory of vascular supply. This is furthermore important as closing and opening the artery harboring aneurysm may induce intraluminal pressure changes and add to aneurysm manipulations increasing the risk of rupture (Kheireddin et al. 2007 ) . Although the hypoxic or ischemic brain conditioning offers a noteworthy option it may not be optimal also because the conditioning stimulus is injurious in nature therefore dif fi cult to titrate (Hua et al. 2005 ) . It seems reasonable to imply that conditioning procedures carried out remotely from the brain offer the greatest level of clinical safety. 14 Preconditioning for SAH 297

14.5 Conditioning for Traumatic SAH

Traumatic subarachnoid hemorrhage (tSAH) is most frequently the result of severe blunt head trauma and is the most common type of SAH in humans (van Gijn and Rinkel 2001) . There are far going differences in the blood distribution and clinical distinctions in cerebral vasospasm following aneurysmal versus traumatic SAH reviewed elsewhere (Armin et al. 2006 ) . Preconditioning can be carried out in face of the imminent risk of blunt head trauma with tSAH. The risk for blunt head trauma is increased in adolescents, single and low-income individuals, racial and ethnic minorities, men, and individuals with a history of substance abuse (Da Dalt et al. 2006; Pickett et al. 2004 ) . Therefore, the feasibility of preconditioning in most of tSAH risk groups is limited (trafﬁ c accidents, falls, narcomaniacs, alcoholics, etc.) (Okten et al. 2006 ; Wang et al. 2011 ) . Presumably, it would be more reasonable to prescribe preconditioning for individuals practicing challenging sports and for service members participating in direct combat operations. Hyperbaric oxygen appears especially well suited as a conditioning modality for tSAH as it can increase cellular resistance against other coexisting sequelae of brain trauma and non-neurological injuries (Hu et al. 2008 ; Harch et al. 2009 ) .

14.6 Mechanisms of Conditioning

The mechanisms of preconditioning in SAH are centered around the action of endothelial nitric oxide synthase (eNOS) and NO signaling which is unsurprising considering that the disturbances of this pathway play a central role in the early brain injury and cerebral vasospasm after SAH (Pluta and Oldﬁ eld 2007 ) . Indeed, a recent pioneering study has shown that eNOS is induced by hypoxic preconditioning to prevent vasospasm after SAH (Vellimana et al. 2011 ) . Since the used means of preconditioning was hypoxia, it is also plausible that the modi ﬁ cation of hypoxic signaling was involved. It is well established that the induction of hypoxia inducible factor 1 a (HIF-1) a solely in the conditioning phase translates into the enhanced expression of protective genes upon brain injury, including erythropoietin (EPO), glucose transporters, glycolytic enzymes, and vascular endothelial growth factor (Bergeron et al. 2000; Semenza 2001; Sharp et al. 2001) (Fig. 14.3). Several studies pointed out, however, that also after major brain hypoxia, the induction of HIF-1a can be enhanced in the preconditioned cells in association with increased levels of adaptive genes such as EPO (Liu et al. 2005; Ratan et al. 2004 ) . While HIF-1 controls hypoxic signaling and its moderate activation can induce adaptive target genes, its excessive stimulation can lead to the activation of proapoptotic downstream targets such as BNIP3, NOXA, and PUMA in cerebral tissues after SAH (Ostrowski et al. 2005 ; Schmidt-Kastner et al. 2004 ) . It appears that the prevention of this excessive signaling offers neuroprotection (Ostrowski et al. 2005 ; Halterman et al. 1999 ) . Similar to hypoxic PC, the mechanism of 298 R.P. Ostrowski and J.H. Zhang

Hypoxic PC

HIF-1 EPO Vasospasm

eNOS

Statins Blood degradation Thrombin PC Neuronal injury products

Nrf2 HO-1, Trx-1 Inflammation Neurotoxicity SOD, CAT Oxidative stress

SAH HBO Stem cells Neuroregeneration PC Growth factors

Fig. 14.3 Candidate mechanisms of conditioning for SAH. As discussed in the text, only the involvement of endothelial nitric oxide synthase has been experimentally proven. Indirectly, the induction of protective molecules such as bcl-2 and HSP27 in preconditioning tissues may be considered as playing a protective role against SAH-related morbidity. With almost certainty, however, the preconditioning effect cannot be exerted without the involvement of major transcriptional factors that control protective transcriptome. The most plausible candidates include HIF-1 and Nrf2, considering these transcriptional factors have been found upregulated with common conditioning modalities; Trx-1: thioredoxin 1. See text for other abbreviations

pharmacological preconditioning with statins appears to rely on the upregulation of eNOS in cerebral microvasculature, which may contribute to the observed reduction of neurological de fi cits (Chang et al. 2010 ) . The inducible NOS seems also involved in the mechanism of anesthetic preconditioning, being released 6 h after the beginning of isofl urane exposure (Kapinya et al. 2002 ) . The results of many experiments seem to suggest that the mechanism of iso fl urane PC further includes the activation of Akt, bcl2, and KATP channels (Zhou et al. 2010 ; Xiong et al. 2003 ; Li et al. 2008 ) . In turn, the mechanism of remote pre- and postconditioning has been postulated to depend upon activation of opioid receptors (Zhou et al. 2011 ; Rehni et al. 2007 ) . In this scenario, remotely released opioids can travel to the brain and exert protective effects (Zhou et al. 2011 ; Peart et al. 2005 ) . There are many modalities of conditioning that have not been speci fi cally tested for SAH however capable of activating innate resistance to injury in the healthy rats. In the en masse population endangered by stroke, the effect of preconditioning may be limited because chronic cerebrovascular diseases by itself tend to maximally upregulate innate neuroprotective mechanisms. Whereas in SAH patients, such preexisting upregulation seldom occurs as SAH usually strikes amid good health and in the relatively young patients. Conditioning stimuli such as brief hypoxia or hyperbaric oxygen are able to induce antioxidant endogenous defenses in cerebral tissues, including superoxide dismutase (SOD), catalase (CAT), and heme oxygenase-1 (HO-1), which could protect against the anticipated major brain insult (Bigdeli et al. 14 Preconditioning for SAH 299

2009 ; Park et al. 2008 ; Wada et al. 2001 ) . The induction of HO-1 might be especially bene fi cial in dealing with blood deposits in the subarachnoid space (Li et al. 2007 ) . In this setting, HO-1 could provide neuroprotection by facilitating the sequestration of blood-derived catalytically active iron (Matz et al. 1997 ) . Another upstream of HO-1 that may be activated upon preconditioning is Nrf2 (nuclear factor E2-related factor 2). Nrf2 is a major transcription factor for the genes involved with reducing oxidative stress, infl ammation, and accumulation of toxic metabolites (Al fi eri et al. 2011 ; Zhao et al. 2007 ) . One-hour-long course of HBO at 2.4 atm induced Nrf2 mRNA in human microvascular endothelial cells acutely after exposure (Godman et al. 2010 ) . Interestingly, Nrf2 activation was recently implicated to play a bene fi cial role in ameliorating early brain injury after SAH (Chen et al. 2011 ) . Question, however, arises if HBO-PC in vivo can induce genes that protect against blood-brain barrier disruption and brain edema formation in SAH. In the series of our recent experiments, HBO-PC by itself potently induced tissue inhibitor of metalloproteinases 1 (TIMP-1) in the cerebral tissues (Dr. Yoshiteru Soejima, unpublished observation) (Ostrowski et al. 2010 ) . Further studies are required to fi nd out whether the observed TIMP-1 upregulation can lead to a reduction of vasogenic edema after subarachnoid bleeding. As indicated by several studies, endothelial apoptosis plays an important role in the mechanism of vasospasm (Yan et al. 2008 ; Zhou et al. 2005 ; Yatsushige et al. 2008 ) . Of note, preconditioning modalities alone may activate anti-apoptotic molecules such as bcl2 and PI2K/Akt in cerebral tissues, leading to increased cell survivability (Wada et al. 2001 ; Miyawaki et al. 2008 ) . However, the impact of conditioning on vasoconstriction mechanisms is less known. A recent study has shown that the pretreatment with atorvastatin, termed “atorvastatin preconditioning,” reduced the level of endothelin 1 in CSF on the fi fth day after experimental SAH (Chang et al. 2010 ) . Moreover, vascular preconditioning may carry a promise of applicability to SAH patients (Harada et al. 2001 ) . In addition, several studies have indicated that ischemic preconditioning can target the infl ammatory response after SAH (Bowen et al. 2006 ; Sercombe et al. 2002 ) . The induction of anti-infl ammatory mechanisms occur through Akt activation and has a potential to target infl ammatory component of cerebral vasospasm (Dumont et al. 2003 ; Yin et al. 2007 ) . Beside these few con fi rmed molecular pathways for conditioning in SAH, the involvement of other mechanisms remains hypothetical at this point. Promising set of mechanisms for conditioning in SAH appears to be triggered by thrombin, well-established preconditioning stimulus against intracerebral hemorrhage (Xi et al. 1999 ) . By causing activation of heat shock proteins (HSP), protease-activated receptors, and 70-kDa ribosomal protein S6 kinase (p70S6K), thrombin appears to act in concert with other established inducers of preconditioning, such as brief hypoxia (Hu et al. 2011 ; Xi et al. 2000 ) . An interesting phenomenon that also has been found induced with conditioning is mobilization of stem cells, demonstrated in radiotherapy patients subjected to HBO (Thom et al. 2006 ) . The activation of multipotent and self-renewable cells for angiogenesis and neuroregeneration appears to be a desirable effect of conditioning for SAH (Ali et al. 2011 ; Sgubin et al. 2007 ) . 300 R.P. Ostrowski and J.H. Zhang

14.7 Outcome Measures of Conditioning for SAH

Quite naturally, there will be diversed endpoints of evaluation in the experimental studies (Fig. 14.4 ). Some of these methods produce results that can be easily extrapolated into the clinical arena. Other, such as histological analysis, cannot directly serve this purpose. However, histological protection still needs to be accompanied by improved functional performance (cognitive and sensory motor function) in order to consider conditioning effective. In the experimental setting, cerebrovascular casting can be done for evaluation of vasospasm in mice (Vellimana et al. 2011 ) , while in bigger rodents (rats and rabbits) as well as in dogs, catheter angiogram can be performed (Yatsushige et al. 2005; Suzuki et al. 1999; Sancak et al. 2002 ) . Finally, favorable transcriptome should be included in the conditioning-induced changes which will further speak in favor of the lasting bene ﬁ cial effect; however, this approach awaits further exploration (Stenzel-Poore et al. 2004 ) . Different SAH animal models for testing preconditioning strategies are available. However, a model that would combine early brain injury and vasospasm is

SAH Neurobehavior Neurological tests assessment histological analysis MRI, CT

Catheter angiogram TCD

morphometry

microarray

CONDITIONING OUTCOMES

Fig. 14.4 Outcome measures of conditioning for SAH. Different clinical and experimental measures used to evaluate endpoints of conditioning for SAH may create a hurdle for translation. However, subsets of methods can be used in both scenarios (e.g., catheter angiogram). It seems that the research on conditioning for SAH would bene ﬁ t from using animal models with truly aneurysmal SAH, from broad implementation of clinically relevant methods in basic research, and, conversely, from adapting basic science-derived tools in the clinical arena (microarrays). This requires closer collaboration between clinical and basic science researchers. Bold arrows indicate methods with increased translational signi ﬁ cance 14 Preconditioning for SAH 301 lacking, and, therefore, the results of animal experiments should be interpreted cau- tiously (Titova et al. 2009 ; Megyesi et al. 2000 ) . For example, endovascular perfora- tion model will accurately test brain tolerance for early injury, while blood injection models are better suited for evaluation of conditioning for vasospasm. The most important domain, however, are clinical measures for the evaluation of conditioning effects. First, clinicians should be able to observe reduction of vascular spasm; while catheter angiography remains a gold standard, transcranial Doppler (TCD) ultrasound examination should be administered as the noninvasive means (Rigamonti et al. 2008 ) . In parallel to reduced mortality, the improved neurological condition and cognitive performance of patients should be noted. Clinical trials typically use Rankin score and cognitive status evaluation done at prolonged period of time after SAH, e.g., 1 year which is in contrast with the majority of experimental, short-term studies (Scharbrodt et al. 2009 ; Jeon et al. 2010 ) .

14.8 Ongoing Clinical Trials

Clinical trials testing different strategies of remote PC for SAH are under way (Fig. 14.5 ). Several research teams in China and USA are investigating the effect of remote ischemic preconditioning (RIPC) and the applicability of PC before CVS or neurosurgery procedures. The remote preconditioning for neurosurgery has been tested by a team by Dr. Dong from Xijing Hospital in China. The study was completed in 2009.

Fig. 14.5 Ongoing clinical trials. Several clinical trials are evaluating protection Remote against brain injury following conditioning SAH or sustained during craniotomy. Remote conditioning procedure has been conducted by in ﬂ ating the pressure cuff on the upper thigh of SAH patients. However, the pressure cuff was applied on the arm to precondition patients before elective craniotomies. All craniotomy SAH CVS these procedures were carried out to cause brief limb ischemia alternating with reperfusion, and it has been hypothesized that subsequent release of endogenous opioids is responsible for brain protective effect 302 R.P. Ostrowski and J.H. Zhang

The investigators evaluated the extent of cerebral injury in preconditioned patients undergoing craniotomy. Investigators placed the cuff on patients’ arm and infl ated it three times to 200 mmHg separated by 5-min periods of reperfusion before major neurosurgical procedure. Using S-100b and NSE as primary markers they found reduced incidence of postoperative cerebral injury in the RIPC group of patients ( http://clinicaltrialsfeeds.org/ ). The investigators led by Dr. Koch propose to study patients with clipped or coiled aneurysms after subarachnoid hemorrhage, who generally have an elevated risk of ischemic stroke in the 2 weeks following the initial bleed. The investigators will apply a blood pressure cuff around thigh and use it to interrupt the circulation for 5Ð10 min. The investigators will repeat this procedure for a total of three times every 24Ð48 h up to 14 days. The cuff will be infl ated to 200 mmHg. Possible side effects (e.g., tolerability, local tissue trauma, or deep vein thrombosis) will be evaluated initially in small group of patients (Koch et al. 2011) ( http://clinicaltrials.gov ). Dr. Nestor Gonzales has initiated another trial at UCLA that aims to determine the effect of RIPC on SAH patients. The patients will receive four rounds of lower limb remote ischemic preconditioning on days 2, 3, 6, and 9 following surgery that secured ruptured aneurysm. Each RIPC procedure will include four cycles of 5-min lower limb ischemia with 5-min reperfusion. Pressure cuff will be placed on patients tight and in fl ated to a pressure 20 mm greater than systolic arterial blood pressure. Investigators will check for neurological events or vasospasm (on TCD) on days 3, 7, 15, and 30 ( http://clinicaltrials.gov ).

14.9 Future Directions

It is believed that identifying mediators of preconditioning will allow to develop their pharmacological analogs. Thereby, ischemic tolerance status could be prolonged as long the maintenance dose is being administered (Gidday 2006 ) . However, experimental treatments that are proven effective against vasospasm and neurological injury may not necessarily be effective in the clinical setting. Therefore, basic science researchers should take into account clinical speci fi cs that oftentimes are not addressed by the design of laboratory investigations. It seems that basic scientists would benefi t from short courses of clinical rotations in the neurosurgery/ neurology wards. Their main effort, however, should focus on mechanistic studies because there is still a paucity of data on preconditioning mechanisms for SAH. This research area would benefi t from studying PC with models of experimental aneurysms as well (Short et al. 2001 ) . Clinical studies will further explore remote organ ischemia to precondition the brain. There, patient selection criteria are utmostly important as is the selection of clinical outcomes to demonstrate protective effects. New outcome measures should be clinically implemented including gene expression pro fi ling. To this end, blood of preconditioned patients can be tested in order to verify the induction of protective phenotype at the transcriptome level (Tang et al. 2006 ) . That having said, it seems there is still a need for testing 14 Preconditioning for SAH 303 different novel modalities, e.g., preconditioning with spider venom-derived peptide modulators of ion channel gating or with bradykinin (Ping et al. 2005; Redaelli et al. 2010 ) . New modalities can be proposed based on observations of molecular changes associated with SAH, as for example, high mobility group box 1 (HMGB1) preconditioning. HMGB1, being an important mediator of infl ammation following brain injury, has been found elevated in plasma of patients at 3, 7, and 14 days after SAH (Nakahara et al. 2009 ) . Experimental studies done so far have shown that HMGB1 preconditioning is an effectible modality combating hepatic injury (Klune et al. 2008 ) .

14.10 Conclusions

The activation of innate defenses by conditioning modalities appears highly suitable for SAH and aneurysm patients who relatively seldom harbor preexisting conditions. It has been postulated that the remote modes of PC are the most safe as these do not further challenge the brain that is already at risk of serious injury. However, different novel models of conditioning should be further developed to better address the complexity of SAH and to study the underlying molecular mechanisms which together with implementing clinically relevant measures of assessment should enable successful translation.

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Richard F. Keep , Ya Hua , and Guohua Xi

15.1 Introduction

Intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype with more than 30,000 patients dying from spontaneous ICH in the USA each year. The causes of ICH are multiple. While spontaneous ICH is often associated with hypertension, other major causes include amyloid angiopathy, aneurysms, arterio- venous malformations, cavernous angiomas, and brain tumors (Xi et al. 2006 ) . In addition, secondary cerebral hemorrhage can occur after an ischemic stroke (hemorrhagic transformation) and traumatic brain injury. In both cases, hemorrhage is associated with worse outcome. The brain injury following an ICH has multiple components including primary injury, related to the physical trauma and the mass effect caused by the hematoma, and a variety of secondary injury mechanisms (see below; Xi et al. 2006 ) . While a number of approaches/agents have proved efﬁ cacious in preclinical studies, as yet no therapy has translated to the clinic. Preconditioning (PC) describes a phenomenon where an event (such as ischemia) or pharmacologic agent upregulates defense mechanisms in a tissue that protect against subsequent injury. Thus, for example, a short duration of cerebral ischemia can protect against a subsequent more severe ischemic event (ischemic PC; Gidday 2006 ; Obrenovitch 2008 ) . PC-induced protection may occur either early after the initial PC stimulus (acute, classical PC) or only appear after several hours (delayed PC) as it relies on new protein synthesis (Gidday 2006 ; Obrenovitch 2008) . PC, which can impact a variety of ICH-related injury mechanisms, may be a method of reducing ICH occurrence or ICH-induced brain injury. However, while

R. F. Keep , Ph.D. (*) ¥ Y. Hua ¥ G. Xi Department of Neurosurgery, R5018 Biomedical Science Research Building , University of Michigan , 109 Zina Pitcher Place , 48109-2200 Ann Arbor , MI , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 309 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_15, © Springer Science+Business Media New York 2013 310 R.F. Keep et al. there is a wealth of data on the effects of PC on models of cerebral ischemia, there have been relatively few studies examining the effects on ICH. This chapter highlights those studies and discusses the potential uses of PC for ICH.

15.2 Mechanisms of Brain Injury After ICH

Before discussing PC and ICH, it is important to understand the injury mechanisms that are thought to play a role in ICH-induced brain injury. Upon rupture of a blood vessel, there is an extravasation of blood which can physically disrupt the surrounding parenchyma. This may involve disruption of the spatial relationship between cells (e.g., synaptic junctions) and damage to individual cells (e.g., axons). In addition, the mass of blood may cause (depending on the size of the ICH and compensatory mechanisms) increased intracranial pressure which in turn may impact cerebral blood ﬂ ow (CBF). The effects of ICH on CBF have been contentious (Xi et al. 2006 ) . A number of animal studies have shown little or no reductions in CBF after blood injection into the brain parenchyma (Xi et al. 2006 ) . Human studies have sometimes seen reductions in CBF, but they have not usually reached levels that would be expected to cause ischemic brain damage (Carhuapoma et al. 2000 ; Tanaka et al. 1996; Zazulia et al. 2001 ) . In addition, ICH can reduce energy metabolism in the perihematomal zone, and reductions in CBF may be a result, rather than a cause, of those changes (Zazulia et al. 2001 ) . Very large hemorrhages will overwhelm the ability of the brain to cope with increased volume (e.g., CSF displacement) and cause very high intracranial pressures. Such changes can cause profound reductions in CBF and brain herniation, and such patients generally die very soon after the ICH. Physical trauma and mass effect are primary forms of brain injury after ICH. In addition, there are secondary forms of brain injury which are delayed to varying degrees. These are induced by clot-derived factors and the response of the brain to the clot. Of the clot-derived factors, the most intensely studied are thrombin and hemoglobin/iron. Thrombin generation by the coagulation cascade is central in limiting bleeding in ICH. However, extravascular thrombin has many effects other than hemostasis such as direct actions on astrocytes, neurons, endothelial cells, and the in ﬂ ammatory system (Xi et al. 2003 ) . High concentrations of thrombin can cause brain injury, and inhibiting thrombin reduces the brain injury induced by intracerebral blood injection (Kitaoka et al. 2002; Lee et al. 1995, 1996 ) . Another cause of brain injury is erythrocyte lysis during clot resolution with the resultant release of hemoglobin. Such hemoglobin is degraded, with one product being iron which is potentially neurotoxic, particularly through the generation of free radicals (Xi et al. 2006 ; Wagner et al. 2003 ) . Intracerebral injection of lysed erythrocytes, hemoglobin, or iron will all cause brain injury (Huang et al. 2002 ; Xi et al. 2001a ) , and inhibiting hemoglobin breakdown and chelating iron both reduce ICH-induced brain injury in animals (Gong et al. 2006 ; Nakamura et al. 2004 ) . 15 Preconditioning and Intracerebral Hemorrhage 311

Fig. 15.1 Bar graph showing brain water content 72 h after ICH in rats that received ﬁ ve sessions of hyperbaric oxygen pretreatment (HBOP5 + ICH, n = 10) or sham pretreatment (control + ICH, n = 10). Values are means ±SD.* p < 0.05 vs. control + ICH (Reprinted with permission from Neurosurgical Focus , Qin et al. 2007 )

In an ICH, there is activation of the infl ammatory and complement systems (Xi et al. 2006 ; Wang and Dore 2007 ) . Both of these are important in clot resolution. Thus, in fi ltration of leukocytes into the brain and activation of resident microglial within the brain are important for phagocytosis of the hematoma. Similarly, the complement system is involved in cell lysis and the in fl ammatory response. However, both the in fl ammatory and complement systems can have effects on parenchyma tissue resulting in injury (so-called bystander effects). Thus, a variety of inhibitors and knockouts affecting these systems result in reduced ICH- induced injury (Xi et al. 2006 ; Wang and Dore 2007 ) .

15.3 Preconditioning and ICH-Induced Injury

Direct examinations of the effects of PC stimuli on ICH are limited. PC with hyperbaric oxygen reduced brain injury after ICH (intracerebral injection of blood) in rats (Qin et al. 2007 ) (Fig. 15.1 ). This protection was associated with activation of p44/42 mitogen-activated protein kinases (p44/42 MAPK) and ribosomal protein S6 kinases (p70S6K) (Qin et al. 2007, 2008 ) . Blocking p44/42 MAPK abolished PC-induced protection (Qin et al. 2007 ) (Fig. 15.2 ). Prior intracerebral injection of a low dose of thrombin (thrombin PC) reduces ICH-induced brain injury in rats (Xi et al. 2000 ) . Thrombin PC also protects against brain injury induced by intracerebral injection of lysed red blood cells which may be linked to an upregulation of iron-handling proteins in the brain (Hua et al. 2003 ) . There have, as yet, been no published studies on remote PC and ICH. In remote PC, an ischemic event in one organ induces protection in another organ. Thus, for example, Ren et al. ( 2008 ) found that a period of limb ischemia protected against a stroke induced by middle cerebral artery occlusion (MCAO) in rats. 312 R.F. Keep et al.

Fig. 15.2 Bar graph showing brain water content 24 h after ICH. Rats were pretreated with an intracaudate injection of a MAPK p44/p42 inhibitor PD098059 (HBOP5 + PD098059 + ICH, n = 5) or vehicle (4% DMSO, HBOP5 + vehicle + ICH) plus ﬁ ve sessions of hyperbaric oxygen pretreatment (HBOP5) before ICH. Values are means ±SD. * p < 0.05 vs. HBOP5 + PD098059 + ICH (Reprinted with permission from Neurosurgical Focus , Qin et al. 2007 )

15.4 Preconditioning and Mechanisms That Are Involved in ICH-Induced Injury

While evidence on the direct effects of PC on ICH is scarce, there is much more data on the effects of PC on individual injury mechanisms that are thought to be involved in overall ICH-induced brain injury. As discussed above, an ICH may cause primary or secondary brain injury. The primary injury is related to the physical trauma caused by the in ﬂ ux of blood into the brain parenchyma and the mass effect of the blood which may impact intracranial pressure and blood ﬂ ow, with potential reductions in CBF. There is evidence that a variety of PC stimuli can protect against both traumatic and ischemic brain injury. For ischemia, a wide variety of PC stimuli protect against global ischemia, as may occur in response to increased intracranial pressure, and focal cerebral ischemia (see reviews: Gidday 2006 ; Obrenovitch 2008 ) . Although traumatic brain injury has not been as well studied as ischemia, several studies have shown that PC can protect against brain trauma (Perez-Pinzon et al. 1999 ; Shein et al. 2007 ) . Similarly, with respect to the secondary injury mechanisms discussed above, there is evidence that at least some PC stimuli can protect against each of them. The brain injury induced by high concentrations of thrombin is reduced by PC with an intracerebral injection of a low dose of thrombin (Xi et al. 1999 ) . This PC stimulus is associated with activation of p44/42 MAPK and kinase inhibition abolished the protection (Xi et al. 2001b ) . The effects of thrombin PC appear to be mediated by thrombin receptor activation (Jiang et al. 2002 ) . Iron-induced brain injury is reduced by PC with a low intracerebral dose of thrombin (Hua et al. 2003 ) . Iron is thought to induce brain injury after ICH through oxidative stress (Nakamura et al. 2005 ) , and a variety of PC stimuli increase antioxidant defense mechanisms within the brain (Gidday 2006 ; Obrenovitch 2008 ) . 15 Preconditioning and Intracerebral Hemorrhage 313

While the effects of PC stimuli on in fl ammation have not been examined in models of ICH, they have been extensively studied in cerebral ischemia models. Such studies have shown reduced leukocyte in fi ltration into brain and less microglial activation within the brain of preconditioned animals (Gidday 2006 ; Obrenovitch 2008 ; Rosenzweig et al. 2004) . Some of the reduced infl ammation may be a result of the reduced brain injury (infarct size) with PC. However, in vitro experiments have shown direct effects of PC on endothelial adhesion molecule expression which will impact leukocyte in fi ltration (Andjelkovic et al. 2003 ) .

15.5 Preconditioning and Bleeding

Hemorrhage size is a major determinant of ICH-induced brain injury and outcome (Broderick et al. 1993 ) . In human ICH, there continues to be hematoma expansion for several hours after ictus in ~30% of patients (Xi et al. 2006 ) . Platelet plug formation and the coagulation cascade together regulate the extent of hemorrhage after a vessel rupture. Studies on the effects of PC stimuli on these hemostatic systems have mostly been limited to ischemic PC. Stenzel-Poore et al. (2003 ) used transient MCAO for ischemic PC and found a marked ﬁ vefold increase in bleeding time. In general, a similar suppression of hemostasis has been found with ischemic PC of different tissues. For example, Linden et al. (2006 ) found that ischemic PC (transient coronary artery occlusion) decreased platelet activation-aggregation after a subsequent coronary injury, and Warzecha et al. ( 2007 ) found that transient clamping of the celiac artery prolongs the activated partial thromboplastin time (APTT). In addition to platelets and the coagulation system, the size of an ICH will depend on the nature of the vascular rupture (i.e., the endothelial and vascular wall injury) underlying the hemorrhage. There is evidence that ischemic PC (transient MCAO) reduces bloodÐbrain barrier injury from a subsequent prolonged MCAO (Masada et al. 2001 ) and “ischemic” PC also protects brain endothelial cells from oxygen glucose deprivation (in vitro ischemia; Andjelkovic et al. 2003 ) . These endothelial effects may offset some of the effects of PC on hemostasis. The above discussion raises fundamental questions for the use of PC in ICH. Data are needed on the effects of PC on hemorrhage size in animal models with vascular disruption as the basis of the ICH. In addition, data are needed on whether the effects of ischemic PC on the hemostatic system also occur with other PC stimuli.

15.6 Post-conditioning and ICH

For ischemic stroke, there has been considerable interest in post-conditioning stimuli which upregulate defense mechanisms and protect when given after ischemic onset (Zhao 2009 ) . There have, as yet, been no such studies for ICH. As some of the injury following ICH is related to the hematoma lysis and the release of hemoglobin and iron (Xi et al. 2006 ) , this may be a fruitful area of research. However, there is a caveat 314 R.F. Keep et al. to this suggestion. As noted about, thrombin PC can protect against ICH-induced brain injury, perhaps by upregulating iron-handling proteins (Xi et al. 2000; Hua et al. 2003 ) . As thrombin is produced very early after an ICH, it is possible that this endogenous thrombin triggers a PC response that protects against the later release of hemoglobin and iron. There is a large increase in the expression of iron-handling proteins within the brain after an ICH (Wu et al. 2003 ) . Another potential use of post-conditioning may be in tissue plasminogen activator (tPA)-induced hemorrhagic transformation. Hemorrhage after tPA-induced reperfusion of ischemic stroke is still a major concern for this therapy. If an agent could be given prior to or at the time of tPA administration that would reduce the impact of any hemorrhage should it occur, this might increase the frequency that tPA is used by physicians.

15.7 Clinical Considerations

There have started to be clinical trials using PC stimuli to reduce ischemic brain injury related to surgery, for example, coronary artery bypass graft (CABG) surgery, carotid artery endarterectomy, and elective neurosurgical procedures (Keep et al. 2010 ) . As yet, there are no trials directed at ICH or hemorrhage secondary to ischemia or trauma. The scarcity of the preclinical data on the potential effects of different types of PC on the occurrence and size of ICH as well as ICH-induced brain injury is a caveat for the current ischemia-related trials where, for example, bene fi ts of PC on ischemia-induced injury might be offset if there was increased occurrence of ICH. Ideally, a PC stimulus should protect against both ischemic and hemorrhagic brain injury. Although there are differences in the mechanisms of injury with ischemia and hemorrhage, there are also some similarities (e.g., role of free radicals and in fl ammation), and there is evidence that both hyperbaric oxygen and thrombin PC can protect against both types of injury (Qin et al. 2007 ; Xi et al. 2000 ; Masada et al. 2000 ; Matchett et al. 2009 ) . More studies are needed to determine the extent to which this is the case with other forms of PC. If a PC stimulus can be found that reduces ICH-induced injury and does not adversely affect the risk of bleeding, would such an agent have a clinical use? There are cases with known risk of hemorrhage such as neurosurgical procedures and patients with cerebrovascular disease (e.g., cavernous malformations) that might benefi t from PC. For the latter, however, studies would be needed on how long the PC stimulus would provide protection and whether repeating the stimulus would prolong the effects.

15.8 Future Directions

There has been a real scarcity of studies on the effects of PC on ICH. This is needed both to examine potential utility in the treatment of ICH and because of impact on current clinical trials of PC for cerebral ischemia. With respect to the latter, there are 15 Preconditioning and Intracerebral Hemorrhage 315 now a very wide range of PC paradigms that have shown protection in preclinical models of ischemic stroke. Data on their relative effects on hemostasis and ICH occurrence are needed. In addition, there is a great need to examine the effects of post-conditioning and remote PC on ICH-induced injury. The former could greatly extend the potential clinical usefulness of the conditioning phenomenon (i.e., not needing to know when a patient will undergo an ICH), while the relative ease and safety of inducing remote PC have led to it being used frequently in current clinical trials (Keep et al. 2010 ) .

References

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Nakamura T, Keep RF, Hua Y, Hoff JT, Xi G (2005) Oxidative DNA injury after experimental intracerebral hemorrhage. Brain Res 1039(1Ð2):30Ð36 Obrenovitch TP (2008) Molecular physiology of preconditioning-induced brain tolerance to ischemia. Physiol Rev 88(1):211Ð247 Perez-Pinzon MA, Alonso O, Kraydieh S, Dietrich WD (1999) Induction of tolerance against traumatic brain injury by ischemic preconditioning. Neuroreport 10(14):2951Ð2954 Qin Z, Song S, Xi G, Silbergleit R, Keep RF, Hoff JT et al (2007) Preconditioning with hyperbaric oxygen attenuates brain edema after experimental intracerebral hemorrhage. Neurosurg Focus 22(5):E13 Qin Z, Hua Y, Liu W, Silbergleit R, He Y, Keep RF et al (2008) Hyperbaric oxygen preconditioning activates ribosomal protein S6 kinases and reduces brain swelling after intracerebral hemorrhage. Acta Neurochir Suppl 102:317Ð320 Ren C, Gao X, Steinberg GK, Zhao H (2008) Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning. Neuroscience 151(4):1099Ð1103 Rosenzweig HL, Lessov NS, Henshall DC, Minami M, Simon RP, Stenzel-Poore MP (2004) Endotoxin preconditioning prevents cellular in fl ammatory response during ischemic neuroprotection in mice. Stroke 35(11):2576Ð2581 Shein NA, Horowitz M, Shohami E (2007) Heat acclimation: a unique model of physiologically mediated global preconditioning against traumatic brain injury. Prog Brain Res 161:353Ð363 Stenzel-Poore MP, Stevens SL, Xiong Z, Lessov NS, Harrington CA, Mori M et al (2003) Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states. Lancet 362(9389):1028Ð1037 Tanaka A, Yoshinaga S, Nakayama Y, Kimura M, Tomonaga M (1996) Cerebral blood fl ow and clinical outcome in patients with thalamic hemorrhages: a comparison with putaminal hemorrhages. J Neurol Sci 144(1Ð2):191Ð197 Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF (2003) Heme and iron metabolism: role in cerebral hemorrhage. J Cereb Blood Flow Metab 23(6):629Ð652 Wang J, Dore S (2007) In fl ammation after intracerebral hemorrhage. J Cereb Blood Flow Metab 27(5):894Ð908 Warzecha Z, Dembinski A, Ceranowicz P, Dembinski M, Cieszkowski J, Kusnierz-Cabala B et al (2007) Infl uence of ischemic preconditioning on blood coagulation, fi brinolytic activity and pancreatic repair in the course of caerulein-induced acute pancreatitis in rats. J Physiol Pharmacol 58(2):303Ð319 Wu J, Hua Y, Keep RF, Nakamura T, Hoff JT, Xi G (2003) Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke 34(12):2964Ð2969 Xi G, Keep RF, Hua Y, Xiang J, Hoff JT (1999) Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning. Stroke 30(6):1247Ð1255 Xi G, Hua Y, Keep RF, Hoff JT (2000) Induction of collagen may attenuate brain edema following intracerebral hemorrhage. Acta Neurochir Suppl 76:501Ð505 Xi G, Hua Y, Bhasin RR, Ennis SR, Keep RF, Hoff JT (2001a) Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood fl ow and bloodÐbrain barrier integrity. Stroke 32(12):2932Ð2938 Xi G, Hua Y, Keep RF, Duong HK, Hoff JT (2001b) Activation of p44/42 mitogen activated protein kinases in thrombin-induced brain tolerance. Brain Res 895(1Ð2):153Ð159 Xi G, Reiser G, Keep RF (2003) The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective? J Neurochem 84(1):3Ð9 Xi G, Keep RF, Hoff JT (2006) Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 5(1):53Ð63 Zazulia AR, Diringer MN, Videen TO, Adams RE, Yundt K, Aiyagari V et al (2001) Hypoperfusion without ischemia surrounding acute intracerebral hemorrhage. J Cereb Blood Flow Metab 21(7):804Ð810 Zhao H (2009) Ischemic postconditioning as a novel avenue to protect against brain injury after stroke. J Cereb Blood Flow Metab 29(5):873Ð885 Chapter 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke

Heng Zhao

In contrast to ischemic preconditioning, which is sublethal ischemia and reperfusion performed before stroke onset to mitigate its effects, ischemic postconditioning refers to the protective effects of reperfusion interruption performed after the restoration of blood fl ow following stroke. This chapter focuses on the protective effects of ischemic postconditioning in various ischemic models, including in vivo focal and global ischemia models as well as in vitro oxygen glucose deprivation (OGD) models. Different triggers used to mimic the effects of ischemic postconditioning are discussed, including brief, repetitive reperfusion/occlusion of blood vessels, a single period of brief ischemia or hypoxia, and application of neurotoxic or anesthetic agents. In addition, the chronic protective effects of ischemic postconditioning and the therapeutic time windows are summarized. Furthermore, the potential for a combination of ischemic pre- and postconditioning to generate synergistic effects is explored. Last, the underlying protective mechanisms of postconditioning, such as the Akt cell survival pathway, MAPK pathways, and PKC pathways, among others, are discussed. This chapter therefore offers a comprehensive review on the progress of research in ischemic postconditioning for stroke. Ischemic postconditioning is a relatively innovative technique derived from the concept of preconditioning. Classical ischemic postconditioning refers to the protective effects of interrupting early reperfusion following ischemia (Zhao et al. 2003b ) , whereas ischemic preconditioning is conducted hours to days before ischemia. Postconditioning and preconditioning are so closely related, they must each be discussed in context of the other to be fully appreciated. In this chapter, I fi rst brie fl y compare pre- and postconditioning. I then review postconditioning models used in stroke research and their short- and long-term effects. I also discuss possible synergies between pre- and postconditioning. Last, I discuss the potential protective mechanisms of postconditioning.

H. Zhao , Ph.D. (*) Department of Neurosurgery and Stanford Stroke Center , Stanford University School of Medicine , MSLS Building, 1201 Welch Road, P306 , 94305-5327 Stanford , CA , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 317 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_16, © Springer Science+Business Media New York 2013 318 H. Zhao

16.1 Preconditioning Versus Postconditioning

As previously detailed in other chapters of this book, preconditioning in an organ, such as the brain or heart, refers to a phenomenon in which a brief sublethal ischemic insult to the organ protects it against future injury induced by a prolonged ischemia. Investigators from diverse backgrounds have used different approaches to show that stimuli, such as hypothermia/hyperthermia (Chap. 5), anesthetic agents (Chap. 7 ), hypoxia (Chap. 8 ), ischemia (Chap. 10 ), as well as low doses of certain toxins (Chap. 9), can activate endogenous protective mechanisms and inhibit the impact of a subsequent and more severe attack. Preconditioning has been extensively studied over several decades (Chap. 3). In contrast, ischemic postconditioning is a relatively novel concept (Zhao et al. 2003b ) . Similar to ischemic preconditioning, ischemic postconditioning was fi rst defi ned in the research fi eld of myocardial ischemia as a series of mechanical interruptions of reperfusion performed at immediate reperfusion (Zhao et al. 2003b ) . The term “ischemic postconditioning” was adopted to contrast with ischemic preconditioning because temporally it occurs after and not before the prolonged ischemia, but both preconditioning and postconditioning consist of one or a series of short ischemic events (Zhao et al. 2003b) . Extensive research on postconditioning in the heart has led to promising human clinical trials (Staat et al. 2005 ; Garcia et al. 2011 ; Lonborg et al. 2011 ) . It is not surprising that ischemic postconditioning is also protective against cerebral ischemia (Zhao et al. 2006 ) , as ischemic brain injury and myocardial ischemic injury share many similar mechanisms. For example, reperfusion injury in both has been linked to free radical products, apoptosis, and necrosis, as well as various other similar cell signaling pathways (Zhao 2009 ) . The protective effects of both rapid and delayed ischemic postconditioning have been established in brain ischemia (Zhao 2009 ) . Here, we use the established defi nition of rapid postconditioning as postconditioning performed immediately after reperfusion. In contrast, we arbitrarily defi ne delayed postconditioning as postconditioning performed from 3 h to a few days after stroke onset.

16.2 Rapid Postconditioning in Brain Ischemia

16.2.1 The Effects of Rapid Postconditioning in Focal Ischemia

16.2.1.1 Distal Middle Cerebral Artery (MCA) Occlusion

To study the effects of ischemic postconditioning, my laboratory employed a focal ischemic model generated by transient bilateral common carotid artery (CCA) occlusion in combination with permanent occlusion of the distal middle cerebral artery (dMCA) (Zhao et al. 2006 ; Gao et al. 2008a ; Ren et al. 2008b ; Zhao 2009 ) . We prefer this model because of its clinical relevance to ischemic postconditioning. First, it generally mimics frequent clinical cases in which partial reperfusion occurs. 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 319

Previous studies have reported that most spontaneous recanalization after stroke results in partial reperfusion (Neumann-Haefelin et al. 2004 ) , and further, t-PA treatment leads to partial reperfusion in most stroke patients (Alexandrov et al. 2001) . Second, this ischemic model produces a well-defi ned infarction in the cortex with a highly reproducible infarct region (Chen et al. 1986 ; Zhao et al. 2003a, 2004, 2005, 2006) and reliable experimental data related to the mechanisms involved in postconditioning (Zhao et al. 2006 ; Gao et al. 2008a, b ) . Third, performing postconditioning by occluding and releasing the CCAs is clinically relevant. We chose this method because the cervical carotid arteries are accessible. In fact, physicians often occlude the carotid artery during insertion of guiding catheters (Yoshimura et al. 2006) or for temporary balloon occlusion in the treatment of intracranial aneurysms and tumors (Sakakibara et al. 1998 ) . In addition, carotid endarterectomy is widely used for stroke prevention; briefl y occluding the carotid artery has been proven safe (Deriu et al. 1994 ) . In our initial study, focal ischemia was induced by transient occlusion of the bilateral common carotid artery (bCCA) for 15, 30, or 60 min combined with permanent occlusion of the dMCA (Zhao et al. 2006 ) . At the end of the bCCA occlusion (bCCAo), postconditioning following stroke was performed with three cycles of 30-s bCCA release and 10-s bCCAo (Zhao et al. 2006 ) (Fig. 16.1 ). We found that rapid postconditioning reduced infarct size as a function of ischemic severity Ð that is, it is less effective with longer periods of ischemia (Fig. 16.1). In subsequent studies, we selected the 30-min bCCAo combined with permanent dMCAo ischemic model (Gao et al. 2008a) because it generates a prominent cortical infarction against which postconditioning offers reliable and reproducible protection. Our studies showed that bilateral CCA release after stroke led to reperfusion in the penumbra as detected by laser Doppler fl owmetry (LDF) probe (Fig. 16.2a ). In the penumbra, cerebral blood fl ow (CBF) recovered to above 100% of pre- ischemic levels after 30 min of bilateral CCAo combined with permanent MCAo (Fig. 16.2a ). Rapid postconditioning disrupted the early reperfusion but improved CBF thereafter. We further con fi rmed this data using 14 C-iodoantipyrine (IAP) autoradiography. Our experiment showed that CBF was reduced in the ipsilateral cortex during ischemia, and a hyperemic response was found in the peri-infarct region at 30 s after CCA release. CBF subsided at 30 min after reperfusion, and rapid postconditioning appeared to improve CBF in the penumbra (Fig. 16.2b ).

16.2.1.2 MCA Suture Occlusion Model

The MCA intraluminal MCAo model is one of the most frequently used stroke models. Pignataro and colleagues used this model to demonstrate very strong protection with postconditioning using a 100-min MCAo (Pignataro et al. 2008 ) . They found that postconditioning with three cycles of 5-min reperfusion/5-min occlusion reduced infarction by 38%, and that one 10-min occlusion initiated after a 10-min reperfusion reduced infarct size by approximately 70% compared to rats subjected to control ischemia. However, postconditioning with a 10-min occlusion started after a 30-min reperfusion offered no protection. Xing and colleagues also found using the MCA 320 H. Zhao

Fig. 16.1 Revised from Zhao et al. ( 2006 ) . Rapid postconditioning reduces infarct size. ( a ) Surgery protocols for cerebral ischemia with and without rapid postconditioning. Rats were divided into six groups. All rats were subjected to permanent MCA occlusion followed by bilateral CCA occlusion (represented by two black bars ) 2 min later. The duration of CCA occlusion after 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 321

Fig. 16.2 Revised from Gao et al. ( 2008a ) . Effects of ischemic postconditioning on CBF after stroke. ( a ) Rapid postconditioning improves CBF after stroke. Using an LDF probe, CBF was detected in the penumbra from 2 min before bCCA occlusion (baseline) to 30 min after bCCA release. Rapid postconditioning was induced by three cycles of 30 s/10 s of bCCA. Bilateral CCA occlusion plus dMCA occlusion decreased CBF to about 25 %. After bCCA release, CBF fully recovered and was interrupted by postconditioning of three cycles of 30 s/10 s of bCCA. Postconditioning improved overall CBF levels from 5 min to 30 min after bCCA release (P = 0.034, two-way ANOVA). *vs. control, P = 0.031. n = 5Ð7/group. CCAo, CCA occlusion; MCAo, MCA occlusion; CCAr, CCA release; RPC, rapid postconditioning. Penumbra is de ﬁ ned by the cortical region saved by postconditioning (Zhao et al. 2006 ) . ( b ) Representative images of spatial changes in CBF measured by 14 C-iodoantipyrine (IAP) autoradiography (unpublished). From left to right: Blood ﬂ ow was evenly distributed in both hemispheres in rat brain without ischemia. CBF was severely reduced in the ischemic hemisphere at 30 min after ischemia. Hyperemic response was measured 30 s in both the peri-infarct region and contralateral hemisphere after bilateral CCA release, as indicated by arrows . The penumbral region was poorly perfused 30 min after CCA release in rat brain without rapid postconditioning ( see asterisks ). However, CBF in the peri-infarct region or penumbra appears to be improved by ischemic postconditioning 30 min after reperfusion compared with control ischemia (indicated by arrows in the right column). Rats were euthanized 60 s after 14 C-IAP injection; 12 blood samples were collected during 60 s 14 C-IAP infusion. Rat brains were cut, and seven coronal slices were harvested, exposed for 1 month

Fig. 16.1 (continued) MCA occlusion was 15, 30, or 60 min. Rats in groups 1, 3, and 5 were subjected to ischemia without postconditioning, and animals in groups 2, 4, and 6 were subjected to both ischemia and postconditioning. Rapid postconditioning with three cycles of 30-s reperfusion/10-s occlusion was performed within 2 min after the initial CCAs reperfusion. RPC, rapid postconditioning. (b ) Typical infarcts stained by cresyl violet in four coronal sections after ischemia with and without postconditioning. Ischemia caused cortical injury ( outlined pale region ) without postconditioning in groups 1, 3, and 5. Minimal infarct was detected in group 2; postconditioning reduces infarct size in all four coronal levels. Rapid postconditioning reduces infarct size from levels 3Ð4 in group 4 compared to group 3. In group 6, infarct in level 4 was decreased by postconditioning. ( c ) Rapid postconditioning reduces overall infarct size after focal ischemia. Infarct size was measured 2 days after ischemia. A mean infarct size for each group was calculated by summing all four levels for each animal and dividing by the number of animals in each group. *vs. groups 3 and 5 (P < 0.05); **vs. groups 3, 5, and 6 ( P < 0.001), vs. group 1 (P = 0.029); # vs. groups 3 and 5 ( P < 0.001); ## vs. all the rest of the groups ( P < 0.001). Two-way ANOVA (factor A: with or without postconditioning, factor B: various brain sections) was used to compare infarct size among six groups. C control group without postconditioning; P rapid postconditioning 322 H. Zhao suture occlusion model that rapid postconditioning reduced infarction by only 16 and 12% at 1 and 3 days after stroke, respectively (Xing et al. 2008 ) . The protective effects of ischemic postconditioning using this model were conﬁ rmed recently in several other studies (Abas et al. 2010 ; Robin et al. 2010 ; Yuan et al. 2010 ) .

16.2.2 Global Ischemia

Whether ischemic postconditioning offers protection in transient forebrain ischemia has also been studied. Rapid postconditioning applied immediately after reperfusion attenuated neuronal death in both the hippocampus and the parietal cortex after a 10-min transient global ischemia as measured days after reperfusion (Wang et al. 2008) . Rapid postconditioning was also shown to attenuate behavioral deﬁ cits after global ischemia in mice (Rehni and Singh 2007 ) , although this study did not report how rapid postconditioning affected neuronal loss. More recently, Zhang and colleagues demonstrated in a 10-min global ischemia model in rats that postconditioning increased the numbers of surviving pyramidal cells in the hippocampal CA1 region from approximately 23.5% in the control ischemic group to approximately 62.8% in the postconditioning group (Zhang et al. 2010 ) .

16.2.3 Rapid Postconditioning in In Vitro Ischemia

Tissue culture models of postconditioning have also been established. Postconditioning with oxygen glucose deprivation (OGD) reduced neuronal death in cortical cultures (Pignataro et al. 2008 ) . Following a 120-min OGD insult, postconditioning with 10 min of OGD initiated 10 min after reperfusion robustly blocked cell death; however, postconditioning with 30 min of OGD at 10, 30, or 60 min after reperfusion did not reduce cell death (Pignataro et al. 2008 ) . This study agrees with the in vivo ﬁ nding that the onset time and duration of postconditioning are critical to generate neuroprotection. In rat organotypic hippocampal slice culture, rapid postconditioning with brief OGD also blocked ischemic injury (Scartabelli et al. 2008 ) . In this study, postconditioning with 3 min of OGD started at 5 min after reperfusion reduced cell injury by approximately 40%.

16.3 Delayed Postconditioning

As discussed above, the protective effects of rapid postconditioning against cerebral ischemia have been confi rmed by a number of independent groups. However, the extremely narrow therapeutic time window of rapid postconditioning may preclude its clinical translation in some stroke patients. When ischemic stroke patients receive tissue plasminogen activator (t-PA) treatment, the exact reperfusion time cannot be defi ned 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 323 because a few minutes to a few hours are necessary to dissolve the blood clot after t-PA bolus (Christou et al. 2000 ; Alexandrov et al. 2001 ) . Therefore, it is essential to explore whether delayed postconditioning still offers neuroprotection. At least three independent laboratories (Burda et al. 2006b ; Ren et al. 2008b ; Leconte et al. 2009 ) , including our own group, have studied the protective effects of delayed postconditioning against brain ischemia. Burda et al. reported that delayed postconditioning conducted 2 days after transient global brain ischemia in gerbils blocked selective neuronal death in the hippocampus (Burda et al. 2006a ) . Delayed postconditioning not only reduced acute infarction but also attenuated secondary chronic brain degeneration. Leconte and colleagues reported that delayed hypoxic postconditioning performed 5 days after stroke in mice and 14 h after OGD in culture protected against secondary thalamic degeneration measured 43 days post-stroke (Leconte et al. 2009 ) . In our laboratory, we demonstrated that delayed postconditioning conducted as late as 6 h after stroke onset (5.5 h after reperfusion) reduced infarct size, improved metabolism, attenuated BBB leakage and edema formation, and offered long-term protection for up to 2 months (Ren et al. 2008b ) . Our studies on rapid postconditioning showed that its protective effects depend on the cycle number and duration of each reperfusion or occlusion as well as the onset time of postconditioning (Gao et al. 2008a ) . We hypothesized that delayed postconditioning using various parameters would protect against ischemia and tested up to 41 conditions to determine the optimal parameters to reduce infarct size (Ren et al. 2008b ) . We found that delayed postconditioning performed at 3 and 6 h reduced infarct size and that postconditioning with six cycles of 15 min/15 min performed at 6 h in the ipsilateral CCA provided the strongest and most reproducible protection (Fig. 16.3 ). In our studies, delayed postconditioning improves F-18-fl uorodeoxyglucose (FDG) uptake as measured by positron-emission tomography (PET), attenuates edema, and inhibits BBB permeability (Ren et al. 2008b ) . Because postconditioning improves CBF after stroke, and CBF is coupled with glucose uptake, we were interested in determining whether postconditioning improves glucose uptake. In our study, we detected glucose uptake by FDG-PET and verifi ed that delayed postconditioning improved FDG uptake in the penumbra but not in the core. In addition, we demonstrated that delayed postconditioning inhibited edema at 2 days and BBB permeability at 2 days but not 1 day after stroke.

16.4 Long-Term Protective Effects of Postconditioning

Some neuroprotectants, such as postischemic hypothermia (Dietrich et al. 1993) and rapid ischemic preconditioning (Perez-Pinzon 2004 ) , offer only transient protection for a few days after ischemia. Whether postconditioning provides lasting protection and preserves brain function has not been studied. In addition, it is unknown if postconditioning will improve behavioral deﬁ cits as reduction in brain tissue injury may not translate into preserved neurological function (Dumas and Sapolsky 2001 ) . 324 H. Zhao

Fig. 16.3 Revised from Ren et al. (2008b ) . Delayed postconditioning reduced infarction. (a ) Comparative timelines for experimental cerebral ischemia and delayed postconditioning. We compared the protective effects of rapid and delayed postconditioning after varying protocol parameters. Focal ischemia was induced by 30-min bCCA occlusion plus permanent dMCA occlusion. Rapid postconditioning was induced immediately after bCCA reperfusion (30 min after stroke onset) and delayed postconditioning at 3 and 6 h after stroke onset. Rats were divided into 11 groups. Group 1, control ischemia without postconditioning. Group 2 was designed to detect if iso ﬂ urane alone after stroke has neuroprotection; isoﬂ urane was applied from 6Ð9 h. Groups 3Ð11 were designed for rapid and delayed postconditioning; either was performed by occluding and releasing the bilateral CCA (bCCA) in groups 3, 4, 5, 6, 8, and 9 for the period indicated, while delayed postconditioning was performed by occluding (15 min) and releasing (15 min) the ipsilateral CCA (iCCA) only in groups 7, 10, and 11. 10Ð10 s/10 s, ten 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 325

To address these two issues, we performed two long-term studies of both rapid postconditioning and delayed postconditioning. Our results showed that rapid postconditioning reduced lesion size approximately 40% in rats subjected to ischemia at 30 days post-ischemia, and it improved neurological function as measured by the vibrissae test, which detects asymmetrical forelimb usage (Gao et al. 2008a ) . Rapid postconditioning also improved subject performance on spatial learning and memory in a water maze test 3 weeks after reperfusion in global ischemia (Wang et al. 2008 ) . We also found that delayed postconditioning conducted at 6 h post-stroke attenuated brain injury and improved behavioral test outcomes for up to 2 months using multiple methods, including the vibrissae, postural re ﬂ ex, tail hang, and home cage tests (Ren et al. 2008b ) .

16.5 Triggers for Postconditioning

In cerebral ischemia research, the classic, narrowly de ﬁ ned concept of ischemic postconditioning induced by brief, repetitive occlusions and reperfusions of blood vessels has evolved similarly to that of ischemic preconditioning: it now refers to a broad range of stimuli or triggers, including serial interruptions of reperfusion, a brief focal cerebral ischemia, metabolic inhibition or oxidative stress, and some anesthetics. “Remote postconditioning” is also performed in a distal organ to generate protective effects against a previous severe ischemia induced in another vital organ (Ren et al. 2008a, 2009) . The protective effects of remote postconditioning are reviewed in Chap. 17 . In our rapid ischemic postconditioning models (Gao et al. 2008a ; Ren et al. 2008b) , we compared different occlusion/reperfusions combinations of 30 or 10 s, repeated for three to ten cycles, and found that ten cycles of 10-s occlusion/10-s reperfusion of the bilateral CCA starting at 10 s after reperfusion offered the strongest protection. For delayed postconditioning, six cycles of 15-min occlusion/15- min reperfusion of the ipsilateral CCA at 6 h post-stroke provided the largest reduction in infarct size (Ren et al. 2008b) . We concluded that the protective effects of ischemic postconditioning depend on the number and duration of cycles as well as the onset time. However, there are no de ﬁ nitive rules for inducing ischemic postconditioning. Pignataro et al. reported that three cycles of 5-min occlusion/5-min

Fig. 16.3 (continued) cycles of 10-s release and 10-s occlusion; 3Ð30 s/30 s, three cycles of 30-s release and 30-s occlusion; 6Ð15 min/15 min, six cycles of 15-min release and 15-min occlusion; 1Ð5 min, 5-min occlusion once; 1Ð15 min, 15-min occlusion once. (b ) Average infarct size in rats treated with delayed postconditioning. Infarct size was measured 2 days after ischemia by TTC staining. Rat brains were cut into fi ve slices; a mean infarct size for each group was calculated by summing all fi ve levels for each rat and dividing by the number of rats in each group. Conditions for each group are indicated below the bar. bCCA, bilateral CCA; postcon, postconditioning; iCCA, ipsilateral CCA; N/A, not applicable. ***, vs. ischemic control (group 1), P < 0.001; #, ### vs. iso fl urane control (group 2), P < 0,05, 0.001; †, vs. each of the other groups, P is at least smaller than 0.05. n = 6Ð8/group 326 H. Zhao reperfusion of the MCA were also protective (Pignataro et al. 2008 ) . In addition, they showed that even a single period of MCA occlusion for 10 min starting 10 min after occlusion robustly reduced infarction in focal ischemia. In an in vitro OGD model, they further showed that a single 10-min hypoxia reduced neuronal injury (Pignataro et al. 2008 ) . The application of pharmacological neurotoxic agents can also trigger postconditioning (Burda et al. 2006a ) . In a delayed postconditioning study, global ischemia was induced by 4-vessel occlusion in rats, and postconditioning was performed using four techniques: short ischemia, injection of 3-nitropropionic acid (3-NP), norepinephrine, or bradykinin (Burda et al. 2006b ; Danielisova et al. 2006 ) . Delayed postconditioning with 5Ð6 min of ischemia or with intraperitoneal injection of norepinephrine or 3-NP at 2 days resulted in neuronal survival of 80Ð100% after global ischemia (Burda et al. 2006a) . Postconditioning could also be induced by adding a low dose of the pharmacological agent 3,5-dihydroxyphenylglycine (DHPG), a group 1 mGlu receptor agonist. Scartabelli et al. found that giving a high dose of DHPG at 5 min after reperfusion and incubating for 30 min exacerbated neuronal injury. However, a low dose of DHPG given under the same parameters was protective and inhibited neuronal injury (Scartabelli et al. 2008 ) . Volatile anesthetic agents such as isofl urane and sevofl urane have been used to trigger postconditioning. Rapid postconditioning can also be induced by iso fl urane in slice organ cultures (Lee et al. 2008 ) . Rats were exposed to OGD for 15 min to stimulate ischemia, and corticostriatal slices were prepared and subjected to iso fl urane. The protective effect of iso fl urane postconditioning was dependent on the duration and concentration of iso fl urane exposure. This study suggests a similar therapeutic time window to ischemic postconditioning in vivo. Isofl urane postconditioning started at 0 or 10 min, but not greater than 30 min post-reperfusion, reduced cell damage (Lee et al. 2008 ) . Most recently, postconditioning with sevofl urane reduced neurological defi cits, infarct size, and brain edema in focal ischemia in rats (Wang et al. 2010 ) .

16.6 Combination of Rapid Conditioning with Ischemic Preconditioning

Because pre- and postconditioning share some common mechanisms, we were very interested in determining whether the combination of rapid pre- and postconditioning acted synergistically in the ischemic brain. In the heart, both pre- and postconditioning protect the ischemic organ by enhancing adenosine activity, reducing the products of reactive oxygen species (ROS) and lipid peroxidation (Zhao et al. 2003b ; Halkos et al. 2004 ) , inhibiting JNK/P38 activity (Sun et al. 2006 ) , and promoting ERK1/2 activity (Yang et al. 2004 ) . The Akt pathways are also involved in both pre- and postconditioning. Some of these mechanisms are also present in the ischemic brain receiving pre- and postconditioning. 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 327

We ﬁ rst compared the protective effects of postconditioning combined with either rapid or delayed preconditioning (Gao et al. 2008a ) Ð the two therapeutic time windows well deﬁ ned for preconditioning. Rapid preconditioning was induced with transient left dMCA occlusion for 15 min at 60 min before stroke (Gao et al. 2008a ) . Delayed preconditioning was induced with transient left dMCA occlusion for 5 or 15 min 3 days before stroke onset. Postconditioning by ten cycles of 10 s occlusion/10 s reperfusion was performed immediately after reperfusion. We found the protective effects of postconditioning to be comparable to those of rapid preconditioning but less effective than delayed preconditioning. We further addressed whether postconditioning plus preconditioning can act synergistically (Gao et al. 2008a ) . We found that postconditioning combined with either rapid or delayed preconditioning does not further reduce infarction. Our results are consistent with a previous study where Pignataro and colleagues (2008 ) reported that the protective effects of postconditioning were comparable to delayed preconditioning in a suture MCA occlusion model in rats, while a combination of pre- and postconditioning provided no greater protection.

16.7 The Underlying Protective Mechanisms

As discussed previously (Zhao 2009 ) , postconditioning primarily targets early reperfusion, reduces free radical generation, and attenuates edema formation and BBB leakage. The strategy to interrupt early reperfusion is consistent with in vivo evidence that partial or gradual reperfusion leads to reduced infarct size (Zhao et al. 2003a ; Gao et al. 2008a ) and in vitro evidence that gradual reoxygenation after ischemia by OGD produces less neuronal death in cell culture (Burda et al. 1995) . In addition, the hyperemic response shortly after reperfusion is suspected to be detrimental to the ischemic brain (Frerichs et al. 1992 ) , but rapid postconditioning attenuates the hyperemic response and mitigates hypotension thereafter. Furthermore, sudden, abrupt reperfusion results in the overproduction of ROS, which leads to apoptosis (Chan 1996 ) . Therefore, it is reasonable for rapid postconditioning to attenuate ROS production and apoptosis (Zhao et al. 2006) . It is known that apoptosis is determined by a balance between neuronal survival and death signaling pathways. The next section reviews how postconditioning affects these cell signaling pathways.

16.7.1 The Akt Pathway

The Akt/PKB cell signaling pathway is critical for neuronal survival. In the Akt pathway, growth factors activate Akt through a receptor tyrosine kinase (RTK) (Franke et al. 2003 ) . Akt activity is regulated by upstream molecules, including phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and phosphatase and tensin homologue deleted on chromosome 10 (PTEN). 328 H. Zhao

Akt is activated by phosphorylation of Ser-473 (P-Akt (Ser473)) and Thr-308 (Franke et al. 2003 ; Fresno Vara et al. 2004 ) . Activated Akt translocates from the membrane to the cytosol, mitochondria, or the nuclei to block caspase-3-mediated apoptosis by phosphorylating its substrates, such as Bcl-2-associated death protein (Bad), forkhead transcription factor (FKHR), PRAS, and glycogen synthase kinase 3 b (GSK3 b ) (Franke et al. 2003 ; Hanada et al. 2004 ) . Akt downregulates GSK3b activity by phosphorylating it at Ser-9 (Bhat et al. 2000 ) . Dephosphorylation of GSK3 b leads to its activation and phosphorylation of b -catenin, thus marking b -catenin for degradation. Akt dysfunction is a direct outcome of ROS activity. Previous studies have shown that ROS induce a conformational change in Akt and Akt dephosphorylation(Cao et al. 2009 ) , and overexpression of SOD 1, a free radical scavenger, enhances Akt phosphorylation (Noshita et al. 2003 ) . We found that moderate hypothermia, which is known to inhibit ROS activity, maintains Akt function (Zhao et al. 2005 ) . Therefore, we were interested in determining whether ischemic postconditioning mediates Akt activity via the inhibition of ROS production. We dissected ischemic penumbra and core for Western blot and kinase assay analysis. We demonstrated that rapid postconditioning increased Akt phosphorylation and activity (Fig. 16.4 ) (Gao et al. 2008b ) . In addition, the PI3K inhibitor, LY294002, enlarged the infarct in postconditioned rats, suggesting that Akt activity contributes to the protective effects of rapid postconditioning. Nevertheless, rapid postconditioning did not affect protein levels of phosphorylated PTEN or PDK1 but did inhibit an increase in phosphorylated GSK3b (Gao et al. 2008b ) . In addition, rapid postconditioning blocked

Fig. 16.4 Revised from Gao et al. (2008b ) . Akt activity contributed to the protective effect of rapid postconditioning (RPC). Focal ischemia was induced by 30-min bCCA occlusion combined with permanent dMCA occlusion; RPC was conducted immediately after bCCA release by three cycles of 30 s/10 s of bCCA. ( a ) Changes in P-Akt and total Akt after stroke in rats with and without RPC. Representative protein bands for P-Akt (Ser473), total Akt, and b -actin in both the ischemic penumbra and the core. Relative optical densities of protein bands in ischemic rats were normalized to those in sham rats and calibrated with b -actin. RPC with three cycles of 30 s/10 s upregulated the overall protein level of P-Akt across all time points compared with control ischemia (two-way ANOVA: P = 0.005 in the penumbra, P = 0.027 in the ischemic core). *, **P < 0.05, 0.01 vs. sham, respectively; #, ##, P < 0.05 and 0.01 vs. control at the corresponding time point, respectively. Total Akt decreased after ischemia in both the ischemic core and penumbra; postconditioning had no statistically signi ﬁ cant effect. *P < 0.05, * P < 0.01 vs. sham. n = 5Ð7/group. c control; p postconditioning. (b ) RPC increased in vitro Akt activity at 5 h . Akt activity as assessed by protein levels of phosphorylated Akt substrate, a fusion protein of GSK3, is expressed as percentages of P-GSK3 from sham animals. * P < 0.05 vs. sham; # P < 0.05 vs. 5 h con. n = 5/group. ( c ) The PI3K inhibitor LY294002 inhibited Akt activity at 5 h in rapid postconditioned animals. Antibody directed against phosphorylated GSK3 was used for detection. *P < 0.05 vs. sham; # P < 0.05 vs. PI3KI. PI3KI, PI3K inhibitor; Post, RPC. (d ) The PI3K inhibitor LY294002 partially blocked the protective effect of RPC. Infarct size was detected by TTC staining 2 days after stroke. The left columns show representative TTC staining from rat brains that received RPC or control ischemia, treated with vehicle (DMSO) or with the PI3K inhibitor LY294002. Bar graph (right panel) shows quantitation of the data. *P < 0.001 vs. other groups; # P < 0.05 vs. DMSO/con, and inhib/con. RPC, rapid postconditioning; con, control ischemia; inhib, PI3K inhibitor 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 329 330 H. Zhao

b -catenin phosphorylation subsequent to inhibiting GSK3b but had no effect on total or non-phosphorylated active b -catenin protein levels (data not shown). Taken together, changes in Akt phosphorylation and activity do not appear to synchronize with its upstream and downstream signals. Therefore, studying PTEN, PDK 1, and GSK-3b may not provide insights into understanding the protective mechanisms of ischemic postconditioning. Our ﬁ ndings are consistent with other reports. Pignataro and colleagues showed that rapid postconditioning increases Akt phosphorylation (as measured by Western blot) (Gao et al. 2008b; Pignataro et al. 2008) , and Akt inhibition partially blocked the protective effects of rapid postconditioning in a MCA suture occlusion model (Gao et al. 2008b ; Pignataro et al. 2008 ) . We concluded that the Akt pathway plays a critical role in the protective effects of postconditioning. This is further supported by a recent in vitro experiment showing that Akt inhibition abolished the protective effect of OGD and DHPG postconditioning in hippocampal slice cultures, a model previously discussed in this article (Scartabelli et al. 2008 ) .

16.7.2 The MAPK Pathways

The MAPK pathways include extracellular signal-regulated kinase 1/2 (ERK1/2), P38, and c-Jun N-terminal kinase (JNK) pathways, which are also closely associated with ischemic injury and neuronal survival (Sawe et al. 2008 ) . While JNK and p38 are detrimental to brain injury induced by stroke, whether ERK1/2’s activity is protective or detrimental is controversial (Sawe et al. 2008 ) . We demonstrated that both JNK and ERK are involved in the protective effects of postconditioning. In our study, stroke resulted in increases in ERK1/2 phosphorylation (P-ERK1/2) from 1 to 24 h after stroke, and such increases were inhibited by rapid postconditioning in the penumbra (Gao et al. 2008b ) , suggesting that P-ERK1/2 might be detrimental after ischemia; therefore, its inhibition may contribute to the protective effects of rapid postconditioning. Our observation differs from Pignataro et al. who demonstrated that rapid postconditioning enhanced ERK1/2 phosphorylation (Pignataro et al. 2008 ) . However, it is possible that changes in P-ERK1/2 are unrelated to the protective effects of rapid postconditioning, since U0126, an antagonist of ERK1/2, did not block its protective effects (Pignataro et al. 2008 ) . More studies are needed to clarify the role of ERK1/2 in rapid postconditioning.

16.7.3 The PKC Pathways

Our studies demonstrated that rapid postconditioning inhibits d PKC cleavage and improves e PKC phosphorylation, two well-known modulators of brain injury after stroke. Both d PKC and e PKC belong to the PKC family, which includes at least 11 isozymes (Casabona 1997 ) . The functions of these isozymes are determined by their 16 The Protective Effects of Ischemic Postconditioning in Experimental Stroke 331 intracellular location, cleavage form, and phosphorylation. While d PKC activity usually leads to cell death (Shimohata et al. 2007b ) , e PKC promotes neuronal survival (Shimohata et al. 2007a ) . In our study, protein levels of total d PKC were unchanged after rapid postconditioning, but less of its cleaved form was observed in the penumbra at 1 h after stroke (Gao et al. 2008b ) . In addition, rapid postconditioning did not affect the levels of phosphorylated d PK C (thr 505) after stroke, which decreased by 24 h after stroke onset. In contrast, levels of phosphorylated e PKC increased. Therefore, it is likely that rapid postconditioning reduced brain injury by inhibiting d PKC cleavage and promoting e PKC phosphorylation (Gao et al. 2008b ) .

16.7.4 The KATP Channels

ATP depletion after ischemia causes KATP channels to open. Previous studies have shown this is critical to induce the protective effects of ischemic pre- and postconditioning in the ischemic heart and for preconditioning in the ischemic brain. KATP channels include sarcolemmal and mitochondrial types. When mitochondrial KATP channels open, an outward current is generated that stabilizes the mitochondrial membrane, which generates protective effects. Lee and colleagues reported that both a general channel blocker, glibenclamide, and a mitochondrial channel blocker, 5-HD, abolished the protective effect of isoﬂ urane postconditioning, suggesting that

K ATP channels may be involved. This fi nding was confi rmed by a recent study that diazoxide, a mitochondrial KATP opener, mimics ischemic postconditioning while the addition of 5-HD blocked its bene fi cial effects (Robin et al. 2010 ) .

16.7.5 Other Cell Signaling Pathways

In addition to the cell signaling pathways and proteins discussed above, emerging evidence implicates the involvement of additional proteins in postconditioning. For instance, glutamate transporter-1 is increased 3, 6, 24, and 72 h post-reperfusion in rats with global ischemia receiving ischemic postconditioning (Zhang et al. 2010 ) . Postconditioning also attenuated endoplasmic reticulum (ER) stress-related protein expression, including C/EBP-homologous protein (CHOP), caspase-12, and glucose- regulated protein 78 (GRP78) (Yuan et al. 2010 ) . Postconditioning selectively upregulated protein and mRNA levels of Na(+)/Ca(2+) exchanger (NCX) 3 but not NCX 1 and 2 in focal ischemia (Pignataro et al. 2011 ) . NCX are plasma membrane ionic transporters involved in the control of Na(+) and Ca(2+) homeostasis and in the progression of stroke damage. Akt regulates NCX3; therefore, NCX3 is a potential target for stroke treatment (Pignataro et al. 2011) . In global ischemia, ischemic postconditioning promoted glutamine synthetase, an enzyme predominately expressed on glia that catalyzes glutamine synthesis and leads to reduced glutamate toxicity (Zhang et al. 2011 ) . 332 H. Zhao

Rapid postconditioning also inhibits inﬂ ammation after stroke. Xing et al. showed that rapid postconditioning inhibits myeloperoxidase (MPO) activity, an indicator of leukocyte accumulation, in the cortex 24 h after stroke (Xing et al. 2008) . In addition, rapid postconditioning attenuates the expression of IL-1b and TNF- a mRNA, and ICAM-1 protein expression in the ischemic cortex 24 h after stroke (Xing et al. 2008 ) . Recently, rapid ischemic postconditioning was shown to inhibit the expression of toll-like receptor 4. This receptor has been shown to be a critical regulator of the innate immune response in the hippocampus using a thrombotic cerebral ischemia model in rats (Feng et al. 2011 ) . These results suggest that rapid postconditioning may produce an anti-in ﬂ ammatory effect.

16.8 Concluding Remarks

Many studies have demonstrated that rapid postconditioning reduces infarction, attenuates neurological de ﬁ cits, and blocks apoptosis in the immediate aftermath of stroke; the long-term protection of postconditioning has also been proven. In addition, delayed postconditioning induced a few hours to days after ischemia can offer protection depending on the ischemic models used. The protective effects of postconditioning are related to its ability to block the overproduction of ROS and lipid peroxidation, improve Akt activity, and open K ATP channels, as well as change MAPK pathways and d PKC and e PKC activities. Despite impressive progress, many of the studies spotlighting underlying protective mechanisms merely reveal correlations between changes in cell signaling pathways and postconditioning. Whether these factors are causative for postconditioning requires further study.

Acknowledgments The author wishes to thank Ms. Cindy H. Samos for assistance with the manuscript. This work was supported by NIH and AHA grants.

References

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Wang JK, Yu LN, Zhang FJ, Yang MJ, Yu J, Yan M, Chen G (2010) Postconditioning with sevo ﬂ urane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway. Brain Res 1357:142Ð151 Xing B, Chen H, Zhang M, Zhao D, Jiang R, Liu X, Zhang S (2008) Ischemic post-conditioning protects brain and reduces in ﬂ ammation in a rat model of focal cerebral ischemia/reperfusion. J Neurochem 105(5):1737Ð1745 Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV (2004) Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44:1103Ð1110 Yoshimura S, Enomoto Y, Kitajima H, Yamada J, Kaku Y, Iwama T (2006) Carotid-compression technique for the insertion of guiding catheters. AJNR Am J Neuroradiol 27:1710Ð1711 Yuan Y, Guo Q, Ye Z, Pingping X, Wang N, Song Z (2010) Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway. Brain Res 1367:85Ð93 Zhang W, Miao Y, Zhou S, Wang B, Luo Q, Qiu Y (2010) Involvement of glutamate transporter-1 in neuroprotection against global brain ischemia-reperfusion injury induced by postconditioning in rats. Int J Mol Sci 11:4407Ð4416 Zhang W, Miao Y, Zhou S, Jiang J, Luo Q, Qiu Y (2011) Neuroprotective effects of ischemic postconditioning on global brain ischemia in rats through upregulation of hippocampal glutamine synthetase. J Clin Neurosci 18:685Ð689 Zhao H (2009) Ischemic postconditioning as a novel avenue to protect against brain injury after stroke. J Cereb Blood Flow Metab 29:873Ð885 Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK (2003a) Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity. J Neurochem 85:1026Ð1036 Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J (2003b) Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285:H579ÐH588 Zhao H, Yenari MA, Cheng D, Barreto-Chang OL, Sapolsky RM, Steinberg GK (2004) Bcl-2 transfection via herpes simplex virus blocks apoptosis-inducing factor translocation after focal ischemia in the rat. J Cereb Blood Flow Metab 24:681Ð692 Zhao H, Shimohata T, Wang JQ, Sun G, Schaal DW, Sapolsky RM, Steinberg GK (2005) Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats. J Neurosci 25:9794Ð9806 Zhao H, Sapolsky RM, Steinberg GK (2006) Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cereb Blood Flow Metab 26:1114Ð1121 Part IV Mechanisms of Preconditioning Chapter 17 Synaptic Signaling in Ischemic Tolerance

Robert Meller

17.1 Introduction : Neurotransmitters in Ischemic Tolerance and Preconditioning

Numerous studies have attempted to identify the initiators of tolerance and the end target effectors for tolerance. From the pioneering work of Olney and others which leads to the glutamate hypothesis of ischemia and excitotoxicity, it is clear that ischemia, either brief or prolonged and injurious, can liberate multiple neurotransmitters. Logically, these were then pursued as potential therapeutic targets for anti-stroke strategies. Hence, we shall start off with the usual suspects and progress through the evidence for neurotransmitters as inducers and effectors of tolerance. Clearly, the ultimate goal of such studies is to identify synaptic signaling processes affected by preconditioning-induced tolerance as a means to develop neuroprotective therapies for stroke. While such studies have revealed a high degree of complexity in terms of synaptic signaling that is rich in potential therapeutic targets for intervention, it is a sobering fact that, to date, these approaches have yielded disappointing results in clinical stroke trials.

17.2 Excitatory Transmitters: NMDA Receptors and Stroke

Lucas and Newhouse ﬁ rst showed that retinal neurons were damaged by exposure to the amino acid glutamate in 1957 , and Olney showed that peripheral administration of glutamate injured the brain of both rodents and primates (Olney 1969, 1971 ;

R. Meller , D.Phil. (*) Department of Neurobiology/Pharmacology, Neuroscience Institute, Morehouse School of Medicine , Moorehouse University , 720 Westview Drive SW , Atlanta , GA 30310-1495 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 339 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_17, © Springer Science+Business Media New York 2013 340 R. Meller

Olney and Sharpe 1969 ) . Subsequent in vitro and in vivo experiments showed that blocking NMDA (N -methyl- d -aspartate) glutamate receptors attenuated ischemia- induced neuronal damage, clearly implicating a role of NMDA receptors in the pathological mechanism of stroke (Rothman 1984 ; Simon et al. 1984 ; Simon and Shiraishi 1990 ; Gonzalez-Zulueta et al. 2000 ) . The following is a brief overview of NMDA receptors (for more details, see Lau and Tymianski 2010 ; Groc et al. 2009 ) . NMDA receptors are hetero-tetramers, consisting of two NR1 subunits and two NR2 subunits. Glutamate binds to the NR2 subunit, whereas the co-agonist glycine binds to the NR1 subunit. The NR1 has eight splice variants, the NR2 contains four different subunit variants (A–D), and there are two NR3 subunits. The role of NR3 is not as well understood. Developmentally, NR2B subunits are expressed ﬁ rst, followed by the NR2A subunit following birth. The location of the NR2A vs. 2B subunit containing receptors is a little more controversial; typically, NR2A subunits are deemed to be synaptically located, and NR2B are extrasynaptic in mature neurons, but structural studies show both NR2B subunits in the synapse and NR2A subunits in extrasynaptic sites (Thomas et al. 2006 ; Harris and Pettit 2007 ) . It is thought that the developmental increase in NR2A expression may coincide with the localization of NMDA receptors to synaptic sites (Thomas et al. 2006 ; Tovar and Westbrook 1999 ) . One interesting difference between the subunits is their relative speed of diffusion in the membrane. NR2A subunit containing NMDARs are static, whereas NR2B subunit containing receptors are more mobile in the synapse (Groc et al. 2006 ) . In addition NMDA receptors are regulated by phosphorylation (Chen and Roche 2007 ) and their anchoring to the cytoskeleton scaffold (Rosenmund and Westbrook 1993 ) . Blocking NMDA receptors with pharmacological antagonists reduces brain injury associated with ischemia, seizure, and trauma, showing that these insults to the brain induce cell death via similar excitotoxic biochemistry (Rothman 1984 ; Simon et al. 1984 ; Simon and Shiraishi 1990 ; Meldrum 1993 ; Tanaka et al. 1996 ; Palmer 2001 ; Berg et al. 1993 ) . However, 20 years of research later, we are left with the disappointing fact that not a single pharmacological agent aimed at blocking NMDA receptor activation has been successful in blocking stroke-induced infarction in clinical trials, and many reviews have covered this topic. Multiple reasons have been suggested to account for these disappointing results, including pharmacological side effects (such as neuronal vacularization and CNS-related side effects (Fix et al. 1993 ) and inappropriate animal models (Hoyte et al. 2004 ) ). Perhaps one of the largest reasons behind their failure is that the therapeutic window available for a glutamate-blocking therapy is narrow (approximately 1 h) (Hoyte et al. 2004 ) , but recent experiment suggests this could be enhanced with a combination approach (Pignataro et al. 2007) . Furthermore, many of these trials were performed with antagonists based on an older knowledge of these receptors, and recent studies suggest a highly complex network of NMDA receptor-mediated signaling in neurons. Indeed, contrary to initial hypotheses, blocking physiological NMDA receptors may be detrimental to neurons due to the blockade of survival signaling (Biegon et al. 2004 ) (see later). Hence, newer strategies are being devised (Fig . 17.1 ). 17 Synaptic Signaling in Ischemic Tolerance 341

Fig. 17.1 AMPA receptors as effectors of ischemic tolerance. Multiple studies have shown a potential role of AMPA receptors in regulating excitotoxic cell death following ischemia. Preconditioning has been shown to reduce expression of the GluR2 subunit. A loss of GluR2 results in enhanced calcium permeability of the AMPA receptor. The reduction of GluR2 expression may be mediated by REST. In addition, editing of the AMPA GluR2 subunit has been reported following preconditioning, which would slow the desensitization of the AMPA receptor

17.2.1 Evidence for Altered NMDA Receptor Expression or Function in Innate Neuroprotection

Altered NMDA receptor function has been described in hibernating arctic ground squirrel, an animal model of innate protection. Hippocampi from hibernating squirrels show remarkable resistance to ischemia and NMDA (Ross et al. 2006 ) . Furthermore, NR1 phosphorylation is decreased in hibernating squirrel hippocampi cells, which translates to a decrease in NMDA-mediated calcium increases. The fraction of NR1 in the membrane pool was reduced in hibernating ground squirrel, suggesting that surface expression of NMDA receptors decreases in hibernation, but no change in brain distribution of NR1 was observed (Zhao et al. 2006 ) . These data suggest that when brain is in a neuroprotective state, reduced NMDA function can occur. Another model of tolerance requires the use of spreading depression as the tolerance inducer. Spreading depression is the phenomenon whereby the ionic composition of the extracellular milieu is compromised, such that the cells undergo excitation followed by a depression of electrical activity, which spreads as wave across the brain surface. This phenomenon is modeled using high local potassium and mimics the ionic imbalance in the parenchyma following ischemia. Interestingly, while spreading depression affects the expression of AMPA receptors and other transmitter receptor levels both in cortex and subcortical structures, no change was noted in NMDA receptors when receptor levels were assessed 24 h later by autoradiography (Haghir et al. 2009 ) . 342 R. Meller

Evidence for altered NMDA function in preconditioning-induced ischemic tolerance comes from multiple sources. Following global cerebral ischemia to gerbils, NMDA regulates a long-term potentiation (LTP)-like enhancement of neurotransmission. However, this NMDA-induced LTP response is diminished in animals subjected to prior preconditioning (Kawai et al. 1998 ) . NR2 expression increases in non-preconditioned brain following ischemia in both synaptosomal and whole brain extracts (Shamloo and Wieloch 1999 ) , and these effects were blocked by preconditioning, suggesting that preconditioning modiﬁ ed the NMDA receptor response to harmful ischemia. The changes in NMDA receptor expression were also accompanied by changes in protein tyrosine phosphorylation. Interestingly, although this was a global model of cerebral ischemia that tends to have stronger hippocampal effects (two-vessel occlusion model of global cerebral ischemia), most changes in receptors were reported in the neocortex. In rapid ischemic tolerance, no change in total NMDA receptor protein levels were observed using Western blotting (Meller et al. 2008 ) . However, NR2B receptor subunits were released from their anchoring to the actin cytoskeleton, and NMDA excitotoxicity is reduced in rapid ischemic tolerance (Meller et al. 2008 ) . This suggests that rapid tolerance can uncouple NMDA receptors from their intracellular signaling components that promote cell death (more on this later).

17.2.2 NMDA Receptors as Inducers of Tolerance

Multiple studies suggest a role for NMDA receptors in initiating tolerance (i.e., preconditioning). NMDA receptor antagonists block preconditioning-induced ischemic tolerance in multiple in vitro and in vivo models (Gonzalez-Zulueta et al. 2000 ; Tauskela et al. 2001, 2008; Chen et al. 2008) . These studies suggest that many preconditioning stimuli liberate glutamate to activate NMDA receptors resulting in tolerance to ischemia. NMDA receptor activation following preconditioning can activate the pro-survival protein kinases AKT1 and Erk5 (Miao et al. 2005 ; Wang et al. 2006 ) . Activating NMDA receptors, using NMDA or synaptic stimulation protocols, can induce tolerance against excessive ischemia, glutamate, or the cell death inducer staurosporine (a general protein kinase inhibitor which induces apoptosis at high concentrations in a caspase-dependent manner) (Tauskela et al. 2008 ; Hardingham et al. 2002 ; Grabb and Choi 1999 ) . In a series of experiments by Hardingham and Bader, it was shown that stimulation of synaptic NMDA receptors (using bicuculline to release neurons of inhibition) results in tolerance to glutamate and ischemia. This preconditioning-like effect was mediated by the pro-survival transcription factor cyclic AMP response element-binding protein, aka CREB. Synaptic activity increases CREB phosphorylation and drives the transcription of a subset of pro-survival genes (Atf3, Btg2, GADD45beta, GADD45gamma, inhibin beta-A, interferon-activated gene 202B, Npas4, Nr4a1, and Serpinb2), which strongly promote survival of cultured hip- 17 Synaptic Signaling in Ischemic Tolerance 343 pocampal neurons (Zhang et al. 2009 ) . Mimicking the effect of CREB, overexpression of Atf3 reduces infarct volume following permanent middle cerebral artery occlusion via coagulation (Zhang et al. 2011 ) . Interestingly, Atf3 is a transcriptional repressor, and gene repressor mechanisms may mediate, at least in part, the protective phenotype of ischemic tolerance (Stapels et al. 2010 ; Stenzel-Poore et al. 2003 ) . Hence, a synaptic event associated with preconditioning can couple to a genomic response. However, little overlap was observed between this in vitro synaptic protective model and an in vivo genomics study of preconditioning and ischemic tolerance (Stenzel-Poore et al. 2003 ) . The concept that synaptic NMDA receptors are the promoters of neuroprotection has been strongly promoted by Hardingham, Bading, and others (Hardingham and Bading 2010 ) . They have shown a robust series of experiments that suggest enhanced electrical activity of neurons, via bicuculline or low NMDA concentrations, promotes neuroprotection (Soriano et al. 2006 ) . If bicuculline is administered in the presence of the noncompetitive NMDA antagonist MK801, synaptic NMDA receptors are blocked and further stimulation of NMDA receptors (extrasynaptic) promotes cell death (Hardingham and Bading 2002 ) . This has been shown to be mediated by the repression of CREB, possibly by the activation protein phosphatases (Hardingham et al. 2002) . The repression of CREB was ifenprodil sensitive, suggesting a role of extrasynaptic NR2B-containing receptors (Hardingham et al. 2002 ) . Indeed, many studies have also promoted that the concept that synaptic NR2A receptors are protective, while extrasynaptic NMDA2B receptors promote cell death (Chen et al. 2008 ; Liu et al. 2007) . However, both NR2A and 2B receptors have been shown to be located in the synapse (Thomas et al. 2006 ; Harris and Pettit 2007 ; Liu et al. 2007 ) . In addition, NR2A and NR2B receptor expression is differentially regulated in development, which may account for some of these studies (von Engelhardt et al. 2007 ) . While there is evidence that extrasynaptic NR2B receptors mediate excitotoxic cell death following exposure to high glutamate or NMDA concentrations, there is plenty of confl icting evidence that ischemia-induced cell death evolves through a synaptic NMDA receptor-dependent mechanism (see later). For example, loss of postsynaptic dendritic spines (using the actin destabilizer a-latrunculin) blocks ischemia-induced cell death, but not exogenous glutamate-induced cell death (Sattler et al. 2000 ) . This suggests that additional components of ischemia result in the synaptic-mediated mechanism of cell death. Clearly, the identi fi cation of a switch between physiological and pathological functions of NMDA receptors (be they synaptic or extrasynaptic) could have great therapeutic potential. However, the biggest detractor for the extrasynaptic NR2B hypothesis of excitotoxicity is the fact that the NR2B glutamate receptor subunit selective antagonist Eliprodil was withdrawn from clinical stroke trials in 1997, reducing the enthusiasm of NR2B subunit selective NMDA receptor antagonists as a therapeutic option for stroke (see Wood and Hawkinson 1997 ) . So how to reconcile all of these models of NMDA receptors and synaptic vs. extrasynaptic receptors? One potential difference is the use of the models. Treating cells with NMDA or glutamate may activate extrasynaptic receptors rather than 344 R. Meller synaptic receptors, and careful consideration of the various models used to promote any hypothesis must be duly taken. In addition, a number of models include additional compounds to promote neuronal activity. The designation of extrasynaptic receptors is usually performed using the model of Westbrook and Tovar, whereby neurons are activated, but in the presence of MK801, an open-channel noncompetitive antagonist (Tovar and Westbrook 1999 ) . Ischemia would appear to initially promote glutamate release from synapse, but under extreme conditions, extrasynaptic release may also occur. Extrasynaptic glutamate release may also be enhanced by release from glia, either via reversal of uptake (Rossi et al. 2000 ) or via glia vesicular release (Parpura et al. 1994, 1995 ) . Under conditions of brief ischemia, i.e., preconditioning, then the glutamate release is likely to be more synaptic and act at postsynaptic sites. The initial ischemic event may also deplete presynaptic terminals of additional transmitter (Hogins et al. 2011 ) , but presynaptic depletion is only likely to be a transient event (see below). The toxic effect of the released glutamate seems to predominantly work via NR2B subunits, but this may account for only 30–40% of total NMDA-mediated current in mature neurons (Liu et al. 2007 ) . Since blocking all NMDA receptors at the time of harmful ischemia is protective, this would suggest that synaptic NR2A receptor may contribute to initial toxicity, but once the harmful event has passed, synaptic signaling is essential for maintaining trophic support to the neurons, as suggested following TBI and also shown in stroke models (Biegon et al. 2004 ; Liu et al. 2007 ) . While one would logically hypothesize that blocking NR2B receptors post-stroke may therefore be bene fi cial, such a hypothesis still has caveats, most essentially the failure of NR2B receptor antagonists in clinical trials. Perhaps newer antagonists may prove to be more ef fi cacious; however, such studies highlight the inherent dif fi culties of targeting transmitter receptors whose function may be essential for “normal” neurotransmission (Miwa et al. 2008 ) . As an alternative approach, perhaps we can target signaling pathways that connect receptors to cell death. Indeed, this is proposed to occur in rapid ischemic tolerance and has been therapeutically investigated by Tymianski’s group (see below).

17.3 AMPA and Other Glutamate Receptors

17.3.1 AMPA Receptors

While most attention has been focused on the role of NMDA receptors in ischemic tolerance, less attention has focused on AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid) and other glutamate receptors. The AMPA receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[ f ]quinoxaline-2,3-dione) prevents ischemia-induced cell damage in brain (Graham et al. 1996 ) ; however, the AMPA receptor antagonists ((3S ,4aR ,6 R ,8a R )-6-[2-(1(2)H -tetrazole-5-yl)]decahy- droisoquinoline-3-carboxylic acid, LY293558) and a noncompetitive AMPA receptor antagonist ((−)-1-(4-amino-phenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3- 17 Synaptic Signaling in Ischemic Tolerance 345

Fig. 17.2 NMDA receptors as initiators of ischemic tolerance . NMDA receptor activation has been shown to mediate tolerance following precondition neurons using ischemia and low or brief doses of glutamate/NMDA. Stimulating activity in neuronal cultures using bicuculline increases synaptic excitatory transmission, which also activates NMDA receptors. Downstream of the receptor protein, multiple intracellular signaling mechanisms have been implicated in regulating NMDA-mediated protection, including various protein kinases, and nitric oxide. The exact molecular targets for these signaling events are not yet fully understood, but CREB is one focal region whereby multiple intracellular protein kinases can converge to promote the expression of neuroprotective proteins such as Bc-2 or to activate the expression of additional factors which may play a role in gene repression responses in tolerance

acetyl-2,3-benzodiazepine, LY300164) both failed to block tolerance in a gerbil global tolerance model (Bond et al. 1999 ) . The dual AMPA/kainate receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) failed to block tolerance in an in vitro model of tolerance following ischemic preconditioning, but did block chemical/pharmacological preconditioning (Werner et al. 2007 ) . However, CNQX does have antagonist properties at the glycine site of the NMDA receptor (Lester et al. 1989) . Taken together, it is unlikely that AMPA receptors are initiators of tolerance (Fig. 17.2 ). Some studies suggest that AMPA receptor editing may be an effector of tolerance. To understand these, we must quickly review AMPA receptor biology. There are four AMPA receptor subunits GluR1–4 (other nomenclatures exist for these receptors as well). The receptor consists of a tetramer consisting of two dimers of GluR2 and either GluR1, 3, or 4. The carboxyl termini of the subunits determine their binding to intracellular scaffold proteins. A lot of focus on AMPA receptors centers on the GluR2 subunit which regulates calcium permeability. A lack of a GluR2 subunit will enable the channel to conduct calcium. The permeability of the GluR2 subunit to calcium is determined by Q/R editing of the mRNA. If the subunit is an R isoform, the pore is positively charged and unfavorable for Ca 2+ conductance. An additional site, the R/G site, is posttranscriptionally edited, and this regulates desensitization rates. 346 R. Meller

Furthermore, fl ip/ fl op editing of the subunits encodes a 38-amino acid sequence which regulates the rate of desensitization of the channel. The fl ip isoform results in prolonged AMPA currents, whereas the fl op isoform will desensitize quicker, preventing overactivation of the cell. Of note, AMPA receptors can be activated by AMPA, glutamate, or kainic acid. Kainic acid results in lower desensitization of the channel than glutamate. Interestingly, targeting calcium-permeable GluR2 lacking AMPA receptors may help reduce delayed ischemic cell death following global ischemia. One of the interesting features of global ischemia is the delay for harmful ischemia to cell death, typically 3 days. Following harmful global ischemia, the hippocampal cells undergo changes that ultimately lead to their death. This includes the expression of GluR2 lacking AMPA receptors and their traffi cking to synaptic sites (Liu et al. 2006 ; Noh et al. 2005 ) . The GluR2 subunit contains a REST silencing element in its promoter which may be dere- pressed following harmful ischemia (Calderone et al. 2003 ) . Hence, there is much focus on elucidating the role of AMPA receptors and speci fi cally GluR2 subunits in stroke. Total GluR2 mRNA levels were not affected following preconditioning in gerbil hippocampi, but GluR2 levels decrease following harmful ischemia, an effect reversed in tolerance (Yamaguchi et al. 1999 ) . Interestingly, R/G editing decreased in animals subjected to preconditioning, at the time when tolerance is observed (2 days following 2-min global ischemia) (Yamaguchi et al. 1999 ) . The decrease in editing (G) was observed predominantly in the CA1 region of the hippocampus. In contrast, the Q/R site was not changed by ischemic preconditioning, suggesting that the AMPA receptors were predominantly impermeable to calcium. Other global ischemia studies of tolerance show an increase in the GluR2 fl op isoform (Alsbo et al. 2000a ) and increases in polyadenylation of GluR2 (Alsbo et al. 2000b ) . Therefore, ischemic preconditioning may regulate GluR2 transcription and editing. When animals are subjected to harmful ischemia, there is a more pronounced decrease in GluR2 expression, which may relate to enhanced calcium-mediated toxicity in neurons. The large decrease in GluR2 mRNA following ischemia is prevented by preconditioning. However, reducing GluR2 expression using antisense is not protective; indeed, it enhances cell death when combined with preconditioning ischemia. Hence, preconditioning ischemia may work at upstream targets to the GluR2 mRNA (Tanaka et al. 2002) . While mRNA editing and changes may occur, it is interesting to note that GluR2 protein levels may increase in the hippocampus following preconditioning (Kjoller and Diemer 2000 ) , but not all studies show this (Tanaka et al. 2002 ; Sommer and Kiessling 2002 ) .

17.3.2 Metabotropic Glutamate Receptors

The results of studies of metabotropic glutamate receptors are quite mixed with regard to their role in ischemic tolerance. mGluRs are subdivided into three major groups: group 1 (mGluR1,5), group 2 (mGluR2,3), and group 3 (mGluR4,6,7,8). 17 Synaptic Signaling in Ischemic Tolerance 347

Confounding the issue of identifying their role in tolerance is the relative selectivity of compounds used in investigations. Metabotropic receptor expression has been investigated following ischemic tolerance; both mGluR1b and mGluR5 receptor levels decreased 8 h following harmful ischemia in animals subjected to prior preconditioning (Sommer et al. 2000 ) . Preconditioning has been reported when cells are pretreated with DHPG (dihydroxyphenylglycine), an mGluR1 agonist. In the same study, blocking mGluR1 receptors with LY367385 ((+)-2-methyl-4-carboxy- phenylglycine) and 3-MATIDA ( a -amino-5-carboxy-3-methyl-2-thiopheneacetic acid) blocked ischemic preconditioning (Werner et al. 2007 ) . This suggests that activation of mGluR1 may be an initiating mechanism of tolerance. Ischemia- induced preconditioning was not blocked by the mGluR5 receptor antagonist MPEP (2-methyl-6-(phenylethynyl)pyridine hydrochloride); however, the protective effect of DHPG was blocked by MPEP (Werner et al. 2007 ) . Other studies show that the nonselective mGluR agonist 1-aminocyclopentane-1,3-dicarboxylic acid is ineffective at preconditioning neurons (Grabb and Choi 1999 ) , and that mGluR1/5 antagonists do not block ischemic tolerance (Duszczyk et al. 2006 ) . Hence, the current understanding of the role of mGluR receptors in ischemic tolerance is not clear.

17.4 Inhibitory Transmission: GABA and Ischemic Tolerance

Some of the data surrounding the role of the inhibitory amino acid GABA as an inducer or mechanism of tolerance is also conﬂ icting. No change in hippocampal GABA release in tolerant brain, compared to non-preconditioned brain, was reported in a global ischemic tolerance model using gerbils (Nakata et al. 1994 ) . While an increase in GABA is not observed in the brain of hibernating ground squirrels compared to nonhibernating squirrels, in other anoxic tolerant species, turtles and epau- lette shark (Hemiscyllium ocellatum), GABA and expression of GABA receptors during tolerance are increased (Renshaw et al. 2010 ; Nilsson and Lutz 1991 ) .

GABAA receptors show a transient increase in expression in gerbil hippocampus CA1 regions in tolerance, as shown by [3 H] muscimol binding. GABA release from cortical neuronal cultures following normally injurious ischemia (OGD) is increased in cells that are subjected to prior ischemic preconditioning (Grabb et al. 2002 ) . Enhanced GABA release following preconditioning was also shown in an in vivo microdialysis study (Dave et al. 2005 ) . Consistent with this concept, it was recently shown that GABA-induced inhibitory postsynaptic potentials (IPSPs) were enhanced by preconditioning ischemia (DeFazio et al. 2009 ) . Hence, enhanced GABA-ergic neurotransmission may exert protective effects via dampening down excitatory neurotransmission in response to subsequent ischemia. Blocking GABAA receptors with bicuculline may serve as a preconditioning agent, but this effect is thought to be mediated by enhanced electrical activity of neurons (Tauskela et al. 2008 ) . Neuroprotection with GABA agonists has had mixed results. 348 R. Meller

17.4.1 Adenosine, Purines, and Ischemic Tolerance

The neuromodulator adenosine is released via a carrier-based mechanism in the

CNS. It acts at various receptors, including A 1 , A 2 , and A 3 receptors. A1 receptors are inhibitory, coupling with Gi /Go , whereas A2 receptors are excitatory coupling via

G s and G q . The biological action of adenosine is curtailed by its uptake into a cell and phosphorylation by adenosine kinase. Adenosine has inhibitory and neuroprotective effects (see Williams-Karnesky and Stenzel-Poore 2009 ) , and as such, its involvement in ischemic tolerance has been studied. Indeed, multiple studies suggest that part of the initiator mechanism of ischemic preconditioning-induced tolerance is mediated by the liberation of adenosine.

A 1 receptors are initiators of ischemic tolerance given that the A1 antagonists theophylline and DPCPX (8-cyclopentyl-1, 3-dipropylxanthine) inhibit delayed ischemic tolerance in both global and focal models (Heurteaux et al. 1995 ) . DPCPX also blocks rapid ischemic tolerance in vivo and in vitro (Ordonez et al. 2010 ; Nakamura et al.

2002) . However, it should be noted that not all studies have shown a role for A1 receptors (Tauskela et al. 2003 ; Sorimachi and Nowak 2004 ) , and, furthermore, hypothermic effects of adenosine active compounds may not have been discounted in all in vivo studies (Minakina et al. 2007 ) . Adenosine A1 agonists act as chemical preconditioning agents and induce neuroprotection against harmful ischemia and chemical ischemia, including adenosine, RPIA, and 2-chloroadenosine (Ordonez et al. 2010 ; Reshef et al.

2000a ; Perez-Pinzon et al. 1996 ) . Adenosine A1 receptors have also been implicated in seizure cross-tolerance, whereby prior exposure to kainic acid-induced seizures resulted in tolerance to ischemia (Blondeau et al. 2000 ) .

Some studies have shown adenosine A1 receptor upregulation following preconditioning stimuli, as well as rapid upregulation of A3 mRNA (von Arnim et al. 2000 ) .

Interestingly, although A1 receptors show higher expression in young animals, adenosine becomes more effective against ischemia in older animals compared to younger animals (Kulinskii et al. 1996 ) . Repeated (four) hypoxic preconditioning stimuli resulted in a lower level of A1 receptor expression as determined by autoradiography, but higher levels of adenosine, compared to a single preconditioning stimulus (Zhang and Lu 1999 ) . However, the af ﬁ nity of the A 1 receptor was higher following repeated preconditioning doses (Zhang and Lu 1999 ) . Propentofylline, an adenosine uptake inhibitor, potentiates the effect of ischemic preconditioning in gerbil model of delayed ischemic tolerance (global ischemia) (Kawahara et al. 1998 ) . The protective effect of preconditioning was blocked by theophylline (Kawahara et al. 1998 ) . One role of adenosine kinase in terminating adenosine’s metabolic effect has recently been described in LPS-induced tolerance; LPS preconditioning was abolished in ADK overexpressing animals (Shen et al. 2011) . Indeed, cortical knockdown of adenosine kinase is neuroprotective against focal ischemia (Shen et al. 2011 ) . While many studies have implicated adenosine in ischemic tolerance, few have focused on the molecular mechanisms by which adenosine exerts its effects. A number of reports suggest that ATP-sensitive potassium ion channels are a molecular target of adenosine receptor activation (Reshef et al. 2000a, b ; Perez-Pinzon and Born 1999 ) as 17 Synaptic Signaling in Ischemic Tolerance 349

the K ATP blocker glibenclamide has been shown to block A 1 -mediated ischemic tolerance.

Adenosine can activate protein kinase C Epsilon and p42/p44 MAPK (Erk1/2 ) (Ordonez et al. 2010 ; Lange-Asschenfeldt et al. 2004 ) which may promote degradation of the pro-apoptotic protein Bim by the proteasome (Ordonez et al. 2010 ) . Proteasome inhibitors block the neuroprotective effect of adenosine in a rapid tolerance model (Ordonez et al. 2010 ) . The activation of PKC epsilon following adenosine or ischemic preconditioning was blocked by a phospholipase C inhibitor (Lange-Asschenfeldt et al. 2004 ) .

Adenosine agonists acting via KATP channels have been shown to suppress p53 signaling following harmful ischemia (Huang et al. 2006 ) . ATP is also released following neurotransmission and in ischemia, and recent studies implicate a role of ATP-activated P2Y receptors in ischemic tolerance (Schock et al. 2007 ) . ATP can be released as either a co-transmitter or via connexin containing hemichannels following ischemia (Schock et al. 2007 ) . The protective effect of ATP was blocked by suramin (a nonselective P2 antagonist) and reactive blue (a P2Y selective antagonist). The P2 receptor may also tolerize glial cells to ischemia, as OGD can induce tolerance in glial cells to lethal ischemia (8 h OGD) in a suramin-dependent manner (Iwabuchi and Kawahara 2009 ) . ATP-dependent neuroprotection following cortical-spreading depression was mediated by protein kinase A and phospholipase C (Schock et al. 2007 ) . Delayed tolerance induced by ATP was cycloheximide sensitive, suggesting that like ischemic preconditioning (Barone et al. 1998 ) , ATP-mediated protection requires new gene expression. While clear similarities exist between this mechanism of ATP-induced tolerance and ischemic preconditioning-induced tolerance (and that mediated by adenosine), it should be noted that tolerance induced by ATP occurred 8 h following preconditioning/ exposure of cells to ATP rather than 24–72 h following preconditioning, as commonly reported in ischemic tolerance in vivo (Schock et al. 2007 ) .

17.5 Beyond Receptors: A Role for Synapses in Ischemic Tolerance

17.5.1 Presynaptic Mechanisms of Tolerance

Multiple studies support the view that glutamate is released from the synapse following ischemia and mediates excitotoxicity. Cutting the synaptic input to the hippocampus blocks global ischemia-induced cell injury to the hippocampal CA1 region (Buchan and Pulsinelli 1990 ) . Blocking synaptic release of glutamate using tetanus toxin blocks OGD-induced cell death in cortical cultures (Monyer et al. 1992) . Removing the synaptic heads from dendrites, using a -latrunculin A, blocks ischemia-induced cell death in vitro (Sattler et al. 2000 ) . Finally, following the preconditioning of neuronal cells with a depolarizing KCl stimuli, glutamate release in response to subsequent OGD is reduced (Grabb et al. 2002 ) . Hence, these data are consistent with synaptic glutamate being important for ischemia-induced cell death, and blocking the synaptic release of glutamate provides tolerance (Fig. 17.3 ). 350 R. Meller

Fig. 17.3 Syna ptic mechanism in ischemia-induced cell death. Multiple studies show synaptic glutamate release is critical for ischemia-induced cell death. Cutting synaptic inputs to hippocampus reduces cell death in global models of ischemia. Depleting synaptic boutons of vesicles or preventing their excitotoxic release with tetanus toxin reduces ischemia-induced cell death. Loss of postsynaptic sites, either via a -latrunculin or in rapid tolerance, also reduces ischemia-induced cell death. It should be noted that under prolonged periods of ischemia, glutamate may leak from the synapse to extrasynaptic sites and promote additional damage via glial release of glutamate

While presynaptic morphology is not thought to be permanently affected by ischemia, in a recent study it has been shown that synaptic silencing can mediate tolerance to hypoxia and ischemia in vitro (Hogins et al. 2011 ) . Following prolonged depolarization of the cultures with KCl, neurons are immediately subjected to hypoxia or ischemia. Cells preconditioned with the KCl depolarization are protected against subsequent ischemia or hypoxia (Hogins et al. 2011 ) . The authors suggest that the protection is due to silencing of synapses as shown by a reduction in uptake of FM1–43fx (a dye taken up into vesicles). This would suggest that ischemia induces the release of synaptic glutamate, and disrupting the presynaptic glutamate content is protective. Indeed, this observation is consistent with the fact that blocking presynaptic release of glutamate with tetanus toxin is protective (Monyer et al. 1992 ) . Interestingly, in the same study, the authors show that the preconditioning effect of synaptic silencing with KCl was blocked by the proteasome inhibitors MG132 (Z-Leu-Leu-Leu-CHO), but not calcium chelation (no extracellular calcium and EGTA). A postsynaptic role of the ubiquitin-proteasome system has been discussed in relation to rapid ischemic tolerance (see below Meller et al. 2008 ) . The calcium effect is more puzzling, because doctrine dictates that action potential arriving at the synapse depolarizes the cell, enabling infl ux of calcium and vesicle release. A recent study by Rossi’s group may explain this interesting observation (Andrade and Rossi 2010) . They show that glutamate release following ischemia was not calcium dependent but related to actin depolymerization in the presynaptic terminal. Actin stabilization by phalloidin prevented ischemia-induced glutamate release. Interestingly, 17 Synaptic Signaling in Ischemic Tolerance 351 actin depolymerization also prevents ischemia-induced cell death, but a postsynaptic site of action was suggested (Sattler et al. 2000 ) . A presynaptic effect of ischemia has been reported, but it tends to be transient. Ischemia can induce temporary swelling to presynaptic boutons, which is reversible within 1 h (Ekstrom von Lubitz and Diemer 1982 ; Hills 1964 ) . Interestingly, such studies show that ischemia increases vesicle aggregation in presynaptic terminals and reduces the vesicle content of presynaptic bouton (Ekstrom von Lubitz and Diemer 1982 ; Williams and Grossman 1970 ) . Presynaptic terminals appear resilient to the increases in glutamate that accompany ischemia, GAP43 staining of presynaptic sites is unaffected by NMDA. In contrast, postsynaptic sites show a remarkable and rapid remodeling of postsynaptic morphology (Hasbani et al. 2001a ) , and the dendritic spines shrink following NMDA receptor activation, but reappear following washout of NMDA. Two points must be raised about the presynaptic hypothesis. First, the depletion of the presynaptic terminals was performed in the presence of APV (a competitive NMDA antagonist) and NBQX (an AMPA antagonist). Therefore, whether this presynaptic effect is only observed in this tightly manipulated model, vs. a relevant model to explain ischemic tolerance mechanisms, is not clear. Ischemic preconditioning-induced tolerance to ischemia in vitro and in vivo is blocked by glutamate receptor antagonism and speci fi cally NMDA receptor blockade in most models (Tauskela et al. 2008; Grabb and Choi 1999; Bond et al. 1999; Mabuchi et al. 2001 ; Kato et al. 1992 , but also see Duszczyk et al. 2005 ; Wrang and Diemer 2004 ) . A second point is whether prior ischemia depletes the presynaptic terminal suffi cient enough to reduce further release, within a temporally relevant profi le. The synaptic silencing studies investigated the effect of ischemia immediately following the KCl depolarization, whereas tolerance is usually described approximately 1 h (rapid) and 24–72 h (delayed) following the preconditioning event. Transmitter release studies show a modest decrease in glutamate release following preconditioning, but this study investigated the glutamate response 24 h following the preconditioning event (Grabb et al. 2002 ) . Therefore, it is not yet clear whether such rapid silencing of synapses represents a relevant mechanism of endogenous tolerance.

17.5.2 Postsynaptic Mechanisms of Tolerance

The synapse is functionally organized, in that the form of the synapse is inﬂ uenced by its function. For example, the morphology of a synapse is determined by its transmitter, and excitatory transmitters form asymmetric synapses, whereas inhibitory transmitters form symmetric synapses. Dendritic spine volume is directly correlated with the number of vesicles in the adjacent presynaptic site. The dendrite is also plastic, and the morphology of a synapse can be rapidly changed in response to multiple pathological and physiological stimuli. 352 R. Meller

Previous studies have shown that ischemia can modify synapses. Dendritic spines are retracted within 10 min in response to ischemia or glutamate exposure, but last for 1 h and recover by 4 h post-ischemia (Meller et al. 2008 ; Hasbani et al. 2001a ; Zhang et al. 2005 ) . Spine remodeling is mediated by glutamate receptors, given that MK801 blocks spine retraction (Hasbani et al. 2001b; Waataja et al. 2008 ) . Spine loss is transient when neurons are exposed to non-harmful ischemia (Meller et al. 2008; Hasbani et al. 2001a ; Zhang et al. 2005 ; Park et al. 1996 ) . The retraction of the spine is a postsynaptic event, as the location of presynaptic GAP-43 was not affected by ischemia; however, following temporary ischemia, the spines re-emerge at the same point (Hasbani et al. 2001a ; Zhang et al. 2005 ) . In delayed ischemic tolerance, spine density was shown to be increased in the hippocampal Ca1 following preconditioning, at the time when tolerance was observed (Corbett et al. 2006 ) . In contrast, a decrease in dendritic spine density is associated with neuroprotection in hibernating ground squirrels and gerbils (von der Ohe et al. 2006 ; Popov and Bocharova 1992 ; Magarinos et al. 2006 ) . Spine remodeling has been shown to be an effector mechanism of rapid ischemic tolerance (Meller et al. 2008 ) . The loss of spines on a dendrite following preconditioning ischemia is uniform, with respect to the location of the dendrite to the cell body. Spine loss was observed on primary, secondary, and tertiary neurons. This was consistent with other studies reporting that brief glutamate receptor activation remodels synapses (Waataja et al. 2008 ; Graber et al. 2004 ) . Ischemic preconditioning-induced rapid ischemic tolerance and spine loss are blocked by the actin stabilizer jasplakinolide (Meller et al. 2008 ) . Thus changes in the actin cytoskeleton following ischemia result in spine loss and neuroprotection. Interestingly, the distribution of actin in neurons changes following a brief preconditioning dose of ischemia (Meller et al. 2008 ) . The punctuate distribution of actin associate with spines is lost and relocates into the dendritic shaft and the cell soma. The reorganization is associated with a solubilization of fi lamentous actin (f-actin) micro fi bers into free glomerulus actin (G-actin). Interestingly, in addition to spine loss, it has been shown that neuronal vascularization in response to glutamate or ischemia may be a protective phenomenon. Vacuoles or varicosities are localized swellings that occur on neuronal dendrites. A number of hypotheses have been promoted for their function, one being to reduce the localized and presumable high toxic concentration of calcium in dendrites, following prolonged activation of NMDA receptors or calcium infl ux via calcium channels. Varicosity formation is mediated by Na 2+ fl ux and AMPA internalization (Ikegaya et al. 2001) . NMDA-mediated varicosity formation induces transient, reversible neuroprotection by attenuating excitatory neurotransmission (Ikegaya et al. 2001 ) . Vacuoles are formed following preconditioning ischemia, but in contrast to dendrite, spine loss shows a bias toward tertiary dendrites rather than primary dendrites (Meller et al. 2008 ) . A number of hypotheses have been promoted for the function of varicosities, one being to reduce the localized and presumable high toxic concentration of calcium in dendrites following prolonged activation of NMDA receptors or calcium in fl ux via calcium channels. 17 Synaptic Signaling in Ischemic Tolerance 353

17.5.3 The Ubiquitin System: A Regulator of Synaptic Function and Tolerance

Rapid morphological events at the synapse following ischemia were shown to be regulated by targeted protein degradation by the ubiquitin-proteasome system (Meller et al. 2008 ) . The ubiquitin-proteasome system is the major regulated protein degradation system in a cell; however, a recent role in regulating cell signaling has been described (for more reviews, see Ikeda and Dikic 2008 ; Meller 2009 ; Hunter 2007 ) . Ubiquitin is ligated to its target protein by an E3 ligase. Usually, the target protein is fi rst phosphorylated by a kinase. Only polyubiquitinated proteins (fi ve or more ubiquitin molecules linked on residue lysine 48) is targeted for degradation. Monoubiquitination has been shown to regulate AMPA receptor internalization and p53 protein traf fi cking in the cell (interestingly both via MDM2) (Colledge et al. 2003) . Lysine 63-linked ubiquitin has been shown to act as a scaffold for signaling complexes associated with NFKB signaling (Ordureau et al. 2008 ) . Many proteins in the synapse are regulated by ubiquitination, and this has been shown to regulate synapse function (Dong et al. 2008 ; Ehlers 2003 ; Fonseca et al. 2006) . Protein ubiquitination has been described following preconditioning ischemia in rapid ischemic tolerance. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors (MG132, MG115, and lactacystin) (Meller et al. 2006, 2008 ) . It is not yet clear whether the E3-ligase TRIM2 which regulates rapid ischemic tolerance is involved in the synaptic remodeling effect of preconditioning ischemia (Thompson et al. 2011) , but interestingly TRIM2 regulates neuronal polarization and neuro fi lament degradation (Khazaei et al. 2011 ) . In addition, in vivo models of rapid and delayed tolerance have been shown to be blocked by multiple proteasome inhibitors (Rehni et al. 2010 ) , but proteasome inhibitors can also block ischemia- induced cell damage in vivo (Wojcik and Di Napoli 2004 ; Williams et al. 2003, 2004, 2006) . Previous studies have reported an increase in the accumulation of ubiquitinated proteins following harmful ischemia, an effect reduced by preconditioning (Liu et al. 2004, 2005) . Hence, in these studies, protein ubiquitination was a result of metabolic stress to cells, but briefer ischemic stress induces protection via enhanced and selective protein degradation. Ischemia-induced synaptic remodeling via the proteasomal degradation of actin- stabilizing proteins, specifi cally MARCKS and fascin (Meller et al. 2008 ) . Indeed, NMDA can increase MARCKS degradation by calpain as well (Graber et al. 2004 ) . Actin-binding proteins such as MARCKS and fascin regulate synaptic and dendritic morphology. Actin requires intermediary proteins such as MARCKS to anchor to the plasma membrane (Hartwig et al. 1992 ; Sundaram et al. 2004 ) . Given the loss of the rigidity of the cytoskeleton, as well as the loss of the multiple proteins – protein signaling cascades regulated by the cytoskeleton scaffolds following ischemia – this may be a fertile ground for the discovery of new therapeutic approaches for stroke (Fig. 17.4 ). The loss of spines and actin depolymerization observed in rapid ischemic tolerance induces protection against ischemia and excitotoxic NMDA exposure 354 R. Meller

Fig. 17.4 Synaptic remodeling in rapid ischemic tolerance and following glutamate receptor activation. Glutamate receptor activation or precondition with brief ischemia induces rapid morphological changes in the appearance of dendritic spines. Spines are lost all over the neuron, and this spine loss corresponds with the time course of rapid tolerance. The loss of spines is mediated by the reorganization of the actin cytoskeleton due to the proteasomal degradation of key actin stabilizing anchor proteins (MARCKS and fascin). Such dramatic changes in the dendritic spines are reversible, but result in reduced NMDA-mediated cell death and protection against ischemia

(Meller et al. 2008 ) , suggesting that ischemia-induced remodeling of synapses can mediate changes in postsynaptic signaling. In contrast, latrunculin is protected against ischemia-induced cell death, but not exposure to toxic glutamate. Identifying these critical proteins which mediate excitotoxic vs. essential physiological signaling from receptors is currently being investigated as a therapeutic strategy for ischemia. While the uncoupling of NMDA receptors from the cytoskeleton and subsequent protection in ischemic tolerance was recently reported, it should be duly noted that this concept was discovered via an alternative series of investigation by Tymianski’s group (Sattler et al. 1999 ) . The postsynaptic density (PDZ)-mediated interaction of NOS to the NMDA 2B receptor subunit, via PSD95, could be disrupted by a PDZ-containing TAT peptide. This peptide was effective at reducing cell death following ischemia (Sattler et al. 1999 ; Aarts et al. 2002 ) . Disrupting the NR2B-PSD interaction blocked excitotoxic signaling, but apparently spared normal neuronal function (Martel et al. 2009 ) . This strategy is currently in clinical trials for brain ischemia.

17.6 Summary and Future Directions

While a role for the synapse in transducing excitotoxic signaling following ischemia has been known for many years, translating this observation into a clinical anti- stroke strategy has proven problematic. Experiments to explain such failures of these trials have revealed a high degree of complexity in our understanding of physiological and pathological synaptic signaling. Preconditioning and recent peptide 17 Synaptic Signaling in Ischemic Tolerance 355 studies suggest that physiological and pathological synaptic signaling can be separated, and, as such, this may provide the target area for future investigations into novel anti- stroke strategies.

Acknowledgments Robert Meller is currently funded by the NIH (NS59588). Institutional support at the Neuroscience Institute at Morehouse School of Medicine was provided by NIH/NCRR/ RCMI grants G12-RR03034, U54 NS060659, and S21MD000101–10. Previous funding support provided by NIH grants NS050669 and NS054023 (Meller), NS024728 (Roger Simon), and the American Heart Association 0465430Z (Meller). Dr. Meller would like to thank collaborators and colleagues both past and present.

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Keri B. Vartanian , Susan L. Stevens , and Mary P. Stenzel-Poore

18.1 Introduction

Preconditioning is a phenomenon whereby a small dose of an otherwise harmful stimulus confers resistance or tolerance to a subsequent challenge with an injurious event. Several preconditioning stimuli have been described that provide signifi cant protection against ischemic brain injury, including exposure to brief ischemia, small seizures, immunologic activation, exposure to hypo- and hyperthermia, and inhalation of volatile anesthetics [reviewed in Dirnagl et al. 2009 ] . Protection is manifest acutely (referred to as rapid preconditioning) or delayed where protection occurs over a time period of 1Ð3 days. The neuroprotective state characteristic of delayed preconditioning lasts from 1 to 5 days and depends on new gene transcription and de novo protein synthesis. Gene regulation studies performed in models of delayed type of preconditioning have provided important clues about the brain’s endogenous mechanisms that lead to the profound neuroprotective phenotype that accompanies ischemic tolerance. High-throughput transcriptomic analysis of gene expression provides an unbiased means of capturing a global snapshot of the genomic response at a given point in time. Here, we will provide an overview of progress toward the elucidation of the genomic profi le with key pathways identifi ed as potential mediators of preconditioning and ischemic tolerance. In addition, we will discuss new approaches to transcriptomic analyses that identify overlapping molecular mechanisms of ischemic tolerance, the discovery of potential key effectors of protection, and technological advancements that hold great promise to increase our understanding of the complex interactions that regulate ischemic tolerance.

K. B. Vartanian ¥ S. L. Stevens ¥ M. P. Stenzel-Poore (*) Department of Molecular Microbiology & Immunology, Oregon Health & Sciences University , Portland , OR , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 363 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_18, © Springer Science+Business Media New York 2013 364 K.B. Vartanian et al.

18.2 Current Status: The Genomics of Preconditioning and Tolerance

Multiple stimuli have been reported as preconditioning agents that protect the brain, as well as other organs, from subsequent ischemic injury. Transcriptomic analysis of many of these preconditioning paradigms using microarrays demonstrates dramatic alterations to the genomic profi le in the brain (Fig. 18.1). Gene regulation prior to the ischemic event sets the stage for the protective phenotype, while gene changes manifested following the ischemic event represent the endogenous neuroprotective genomic response. Determination of the major genes involved in these processes has begun to provide important information in deciphering the mechanisms of preconditioning and ischemic tolerance. The genes that have been identi fi ed as major players in preconditioning-induced protection can be categorized generally into families based on function: defense/ stress, cell survival, cellular homeostasis, and signaling mediators. Genes found in these families have demonstrated roles in the setup of protection (prior to ischemia) and/or execution of the tolerant state (following exposure to ischemia) (Table 18.1 ). Together, these critical alterations comprise the genomic profi le responsible for the neuroprotective phenotype.

18.2.1 Defense/Stress Response

Ischemic injury induces a multitude of endogenous danger signals that activate the organism’s defense/stress responses to mitigate damage. These responses are designed to assist in the restoration of the system, for instance, through induction of chaperone proteins such as heat shock proteins or through clearance of cellular debris. However, these processes, left unchecked, can exacerbate injury. As defense/ stress responses are key mediators of restorative and damaging effects, their regulation in preconditioning and ischemic tolerance is critically important.

18.2.1.1 Heat Shock Proteins

Heat shock proteins (HSPs) are a family of ubiquitously expressed molecules that function in response to stress to prevent apoptosis, modulate in ﬂ ammation, and act as protein chaperones to alleviate protein aggregation (Kalmar and Greensmith 2009 ) . Multiple studies have shown that the low-level stress signaling initiated by preconditioning upregulates many HSPs prior to stroke (Dhodda et al. 2004 ; Stenzel- Poore et al. 2003 ) . Some HSPs identi ﬁ ed from these studies include HSP70, HSP27, HSP90, HSP60/10, HSP32/HO-1, HSPB2, HSP 110, HSPa1a, HSPa1b, and HSPa8 (Dhodda et al. 2004 ; Stenzel-Poore et al. 2003 ) . Following stroke, several HSPs are upregulated in the tolerant brain. In a model of ischemic preconditioning followed 18 The Genomics of Preconditioning and Ischemic Tolerance 365

Fig. 18.1 Transcriptional studies of preconditioning and tolerance . ( a) Time line of preconditioning and ischemic tolerance. ( b ) Left panel: GeneSifter-derived heat map of regulated genes. Multiple time points examined during preconditioning and tolerance denoted at the top. Genes and hierarchical-generated clustering dendrogram along right-hand side . Red denotes increased signal intensity and green denotes decreased signal intensity compared to control group. Right panel : Expanded view of a particular cluster arm of the dendrogram (Color fi gure online) by transient middle cerebral artery occlusion (MCAO), the genes HSP70, HSP27, and HSP 32 are all induced in the tolerant brain 6 h following stroke (Dhodda et al. 2004) . The most deeply studied of these HSPs are HSP70 and HSP27, both of which display functional effects that lead to the protective phenotype. Investigations into the overexpression of HSP70 in transgenic mice have demonstrated con fl icting results in response to stroke. Some studies indicate that overexpression of HSP70 Table 18.1 Summary of genomic responses in preconditioning and ischemic tolerance Regulated genesa Category Subcategory Preconditioning Tolerance References Defense response HSPs HSP70, HSP27, HSP90, HSP60/10, HSP70, HSP27, HSP32 Dhodda et al. (2004 ) and Kawahara et al. HSP32/HO-1, HSPB2, HSP100, (2004 ) HSPa1a, HSPa8 TLRs and TLR1, TLR2, Cd14, MyD88 TLR7, IL1b, MIP1 a , IL6, MCP1, Dhodda et al. (2004 ) , Stevens et al. (2011 ) , in fl ammatory ICAM1, COX2, IL12, IFIT1, and Marsh et al. (2009 ) cytokines Usp18, Oasl2, IRF7 Cell survival Apoptosis Caspase-2, caspase-3, Bcl-2, Bcl-2, POSH Dhodda et al. (2004 ) , Kawahara et al. GADD153, DRAL, BID, LICE (2004 ) , and Bernaudin et al. (2002 ) Cell cycle CyclinD1, cyclin ania-6a, NDR1, CyclinB, LINE3, ORF2, putative Kawahara et al. (2004 ) and Tang et al. CDKN1 p21, BTG3 DNA-binding protein (2006 ) Neurogenesis TGFa , TGFb , VEGF, IGF-1, TGFb, VEGF, IGF-1, FGF-2 Dhodda et al. (2004 ) and Tang et al. FGF-2, stem cell growth factor (2001 ) Cellular Glucose GLUT1, GLUT3 GLUT1, GLUT2, GLUT3 Stenzel-Poore et al. ( 2003 ) and Tang et al. homeostasis (2006 ) Metabolism Carbonyl reductase, translin, N-acetyl galactosaminidase, Stenzel-Poore et al. (2003 ) phospholipase D1, ADP- alcohol dehydrogenase, ribosyltransferase 1 carboxyl aster lipase, steroid 3- a dehydrogenase Ion K+ channels, ion transport CaMKIIa, inositol triphosphate Tang et al. (2006 ) regulator 7, cation transport receptor 2, Scn1a ATPase, Scn6a, CBGA3 Glutamate GluR2, GluR3, EAAT4 GluR2 Kawahara et al. (2004 ) and Bernaudin et al. (2002 ) Signaling mediators Signaling p38 MAPK, MKP-1, MKP-3, Rab16, Ralb, GIT-1, ADCY5, Kawahara et al. (2004 ) , Tang et al. (2006 ) , Smad1/7, guanylyl cyclase, ADCY7, ARPP-19 and Bernaudin et al. (2002 ) ARPP-21, CAP1 Transcription Silence factor B, C/EBPd , NF k B, IRFs, C/EBPd Dhodda et al. (2004 ) , Stevens et al. (2011 ) , factors EGR1, EGR2, KLF4, Kawahara et al. (2004 ) , Tang et al. NF k B p105, HIF1 a (2006 ) , and Bernaudin et al. (2002 ) The major gene families categorized by function are included with speci fi c gene subcategories and regulated genes of interest a For complete gene lists, see references 18 The Genomics of Preconditioning and Ischemic Tolerance 367 has no affect on infarct size (Olsson et al. 2004 ) , while others, including a rodent MRI study, indicate that HSP70 overexpression leads to smaller infarcts (Yenari et al. 2005 ; van der Weerd et al. 2005 ) . Despite this con fl ict, mechanistic studies of HSP70 demonstrate several protective pathways induced by HSP70. HSP70 plays a critical role in the removal of damaging proteins by targeting speci fi c proteins for degradation (Kalmar and Greensmith 2009) . HSP70 suppresses apoptosis through multiple avenues, such as inhibition of the activation of apoptosis protease factor-1 (Pandey et al. 2000 ) and upregulation of the anti-apoptotic factor Bcl-2 (Yenari et al. 2005 ) . The small HSP, HSP27, also plays an important role in suppression of proapoptotic pathways. In a study utilizing HSP27 transgenic mice, the overexpression of HSP27 suppressed the activation of the neuronal death pathway mediated by c-Jun N-terminal kinase (JNK) (Stetler et al. 2008 ) . Finally, HSP70 can also induce anti-infl ammatory effects by inhibiting nuclear translocation of NF k B, preventing the induction of COX-2, and blocking the release of NO (Kalmar and Greensmith 2009 ) . Taken together, HSPs functionally have a wide scope of protective mechanisms that contribute to the protective phenotype induced by preconditioning.

18.2.1.2 Toll-Like Receptors and In ﬂ ammatory Cytokines

Toll-like receptors (TLRs) are mediators of infl ammation that can signal upon binding of endogenous ligands released during ischemic injury (i.e., heat shock proteins, fi bronectin, fi brinogen, mRNA). TLRs initiate common signaling cascades via the adaptor molecules MyD88 and TRIF that result in downstream activation of NFk B with the induction of pro-in fl ammatory mediators (i.e., TNF, IL1b , IL6, COX-2, NOS) or the activation of interferon regulatory factors (IRFs) with the induction of interferon-associated genes. TLRs have recently been implicated in the exacerbation of ischemic injury, as mice lacking either TLR2 or TLR4 have reduced brain damage following ischemia (Cao et al. 2007 ; Lehnardt et al. 2007 ) . Although TLR activation and cytokine release play a damaging role in ischemic injury, it has also become clear that low-level TLR engagement with ligands for TLR2, TLR4, or TLR9 can result in preconditioning and provide neuroprotection against subsequent ischemic injury (Hua et al. 2008; Tasaki et al. 1997; Hickey et al. 2007 ; Rosenzweig et al. 2004 ; Stevens et al. 2008 ) . In addition, certain cytokines activated downstream of TLR signaling (e.g., TNFa and IL1b ) can induce neuroprotection against an ischemic insult when provided as a preconditioning stimulus on their own (Nawashiro et al. 1997 ; Ohtsuki et al. 1996 ) , highlighting the role of TLR-induced in fl ammatory cytokines in modulating preconditioning. Self-regulation of the in fl ammatory response may be the primary means by which TLR preconditioning provides protection from subsequent ischemic injury. As a receptor family, TLRs are known to self-regulate through redirection of TLR signaling. The genomic analysis of the tolerant brain provides strong evidence that preconditioning reprograms the TLR signaling cascade. Feng et al. identi fi ed TLR pathways as signi fi cantly modulated in rat brains of ischemic preconditioned animals exposed to global cerebral ischemia and reported a downregulation of infl ammatory 368 K.B. Vartanian et al. associated genes (i.e., COX-2, IL12) (Feng et al. 2007 ) . Suppression of in fl ammatory mediators, such as IL1 b , Mip1 a, IL6, MCP1, and ICAM1, was also reported in response to focal ischemia in rats preconditioned with brief ischemia (Dhodda et al. 2004 ) . These results suggest an inhibition of the TLR pro-in fl ammatory cascade in response to stroke in preconditioned mice. There is also evidence for redirection of TLR signaling following stroke in a manner that enhances the IRF cascade (Stevens et al. 2011; Marsh et al. 2009 ) . Research shows that a large number of genes associated with IRF signaling are induced following stroke in preconditioned animals including IRF7, Usp18, IFIT1, Oasl2, and PYCARD (Stevens et al. 2011 ; Marsh et al. 2009) . Notably, IRF-regulated genes, such as type I IFNs, have been associated with neuroprotection (Marsh et al. 2009 ; Liu et al. 2002 ; Veldhuis et al. 2003a, b ) . Thus, following stroke, in fl ammatory responses and TLR signaling are altered to suppress critical infl ammatory mediators that exacerbate damage while enhancing potential IRF-associated neuroprotective components.

18.2.2 Cell Survival

Improved cell survival is a necessary part of preconditioning that is mediated by multiple molecular pathways. Not surprisingly, genomic evidence shows that many of the genes that are regulated in preconditioning are involved in cell survival including apoptosis, cell cycle, and neurogenesis.

18.2.2.1 Apoptosis

Many genes involved in apoptosis are upregulated following preconditioning including the proapoptotic genes GADD153, DRAL, BID, LICE, caspase-2, and caspase-3 and the anti-apoptotic gene Bcl-2 (Dhodda et al. 2004 ; Kawahara et al. 2004 ) . The functions of the proapoptotic genes include induction of growth arrest and DNA damage, downstream effectors of the major cell-death transcription factor p53, or execution of the classical caspase-dependent apoptotic cascade. Caspase-3 is the most prevalent proapoptotic gene upregulated following preconditioning. Interestingly, low-level activation of caspase-3, in a manner that did not promote widespread cell death, was found to be essential for effective ischemic preconditioning in rats (McLaughlin et al. 2003 ) . In this model, blockade of caspase-3 activation rendered ischemic preconditioning ineffective in protecting against subsequent ischemic injury (McLaughlin et al. 2003 ) . Thus, activation of proapoptotic pathways following preconditioning is a necessary response for the induction or setup of the protective phenotype and suggests that low-level injury is required for the delayed protection induced by preconditioning. Additionally, the anti-apoptotic gene Bcl-2 is upregulated following preconditioning and in ischemic tolerance (Dhodda et al. 2004 ; Meller et al. 2005 ) . Bcl-2 is a member of the Bcl family of anti-apoptotic genes that function as inhibitors of caspase-dependent apoptosis by 18 The Genomics of Preconditioning and Ischemic Tolerance 369 blocking the release of cytochrome c from the mitochondria, which is the major trigger of the caspase cascade. Functional evaluation of Bcl-2 in ischemic tolerance has revealed a major role for Bcl-2 in preconditioned animals following ischemia. Inhibition of Bcl-2 following stroke using antisense Bcl-2 in preconditioned rats blocked protection (Shimizu et al. 2001 ) , indicating that the upregulation of Bcl-2 is necessary for tolerance. Taken together, the induction of low levels of proapoptotic genes during preconditioning appear beneﬁ cial to the setup of ischemic tolerance, while the functions of anti-apoptotic genes are critical in providing neuroprotection in the tolerant state following stroke.

18.2.2.2 Cell Cycle

Mature neurons are in a state of cell arrest, and forced reentry into the cell cycle is often lethal. Following stroke, many of the cell cycle genes are upregulated contributing to neuronal apoptosis. Preconditioning also upregulates cell cycle genes including cyclin D1, cyclin ania-6a, and NDR1 (Kawahara et al. 2004 ; Tang et al. 2006) . This induction likely leads to low-level injury following preconditioning and, as with low-level increase of caspase-3 discussed above, may be involved in the subsequent neuroprotection. However, inhibitors of the cell cycle are also upregulated following preconditioning. The cyclin-dependent kinase inhibitor gene p21 and the anti-proliferative gene BTG3 are upregulated after ischemic preconditioning (Kawahara et al. 2004 ; Tang et al. 2006) . The upregulation of cyclin inhibitors may balance the upregulation of the cyclin genes following preconditioning. Treatment with cyclin inhibitors has been successful in reducing infarct size in multiple models of rodent stroke (Menn et al. 2010 ) . Importantly, following stroke, preconditioned animals downregulate many cell cycle and DNA synthesis genes including cyclin B, LINE3, ORF2, and putative DNA-binding protein (Bernaudin et al. 2002 ) . This suggests that a state of cell arrest is maintained following stroke in preconditioned animals, thereby preventing neurons from entering the lethal process of cell division.

18.2.2.3 Neurogenesis

Adult neurogenesis induces the maturation of neuronal precursors that reside in the subgranular zone of the hippocampal dentate gyrus or the subventricular zone into new functional neurons (Lichtenwalner and Parent 2006 ) . Cerebral ischemia accelerates the neurogenic maturation process for several days following injury (Lichtenwalner and Parent 2006 ) . Importantly, in a rodent model, precursor cells originating in the subventricular zone migrate to the damaged striatum following stroke where they could possibly replace the necrotic neurons lost during injury (Lichtenwalner and Parent 2006 ) . Evidence suggests that preconditioning may jump-start some of these mechanisms to enhance adult neurogenesis and cell survival to ultimately promote repair. Preconditioning upregulates several growth factors 370 K.B. Vartanian et al. that are involved in neurogenesis including TGFa , TGFb , TGFb receptors, VEGF, and stem cell growth factor (Dhodda et al. 2004 ; Stenzel-Poore et al. 2003 ; Kawahara et al. 2004; Bernaudin et al. 2002 ) . TGF a expression is known to promote the survival of neuronal and glial precursor cells, and both TGFa and TGF b are neuroprotectants (Junier 2000 ; Justicia et al. 2001 ; Zhu et al. 2002 ) . VEGF, the robust angiogenesis stimulant, is also associated with promoting the survival of precursor neurons in the process of neurogenesis (Thau-Zuchman et al. 2010 ; Udo et al. 2008 ) . Interestingly, Naylor et al. demonstrated that ischemic preconditioning signiﬁ cantly enhances the proliferation of progenitor neuronal cells prior to stroke in the dentate gyrus (Naylor et al. 2005) . The induction of many growth factors in the brain correlated with this enhanced proliferative state including TGFb , insulin growth factor-1 (IGF-1), and FGF-2 (Naylor et al. 2005 ) . Taken together, this suggests that preconditioning initiates adult neurogenesis prior to ischemic injury, which likely is a contributing factor to the improved cell survival and neurological outcome following stroke in preconditioned animals.

18.2.3 Cellular Homeostasis

Neuronal homeostasis includes regulation of standard processes such as metabolism and glucose transport along with the regulation of more unique processes such as cell polarization and excitotoxic neurotransmission. Dysregulation of these cellular maintenance processes occurs following stroke and exacerbates injury. Thus, tight regulation of those processes that contribute to neuronal homeostasis would be important in promoting a neuroprotective environment.

18.2.3.1 Glucose

Glucose metabolism, a key source of energy in the brain, relies in part on the glucose transport proteins GLUT1 and GLUT3. Although these proteins are not the rate-limiting step in glucose metabolism under basal conditions, they play a critical role in facilitating glucose transport to the brain during times of minimal glucose, such as during ischemia. In fact, transcriptional upregulation of these transporters with subsequent increase in glucose transport activity has been reported in neurons, glial, and endothelial cells exposed to hypoxia (Bruckner et al. 1999 ; Takagi et al. 1998; Zovein et al. 2004 ) . Genomic analysis of neonatal rat brain exposed to hypoxia indicated that GLUT1 was upregulated following preconditioning (Bernaudin et al. 2002 ) . In addition, Stenzel-Poore et al. found that the expression of GLUT3 was induced in mouse brain following ischemic preconditioning (Stenzel-Poore et al. 2003) . Interestingly, virus-mediated overexpression of GLUT1 in cultured neurons provided protection against subsequent exposure to glucose deprivation (Ho et al. 1995 ) . Therefore, increasing the capacity to transport glucose to neurons in preparation of an ischemic event may be an effective means of protection for the brain in the setting of nutrient deprivation. 18 The Genomics of Preconditioning and Ischemic Tolerance 371

18.2.3.2 Metabolism

The metabolic demands of cells exposed to ischemia are not met due to the lack of oxygen and nutrients. A neuroprotective strategy would be to enable cells of the CNS to adapt to these conditions of deprivation leading to improved survival. Interestingly, a substantial number of genes involved in metabolic pathways (e.g., N -acetyl galactosaminidase, alcohol dehydrogenase, and carboxyl ester lipase, steroid 3-a dehydrogenase) are downregulated in animals preconditioned with exposure to brief ischemia, which predicts a suppressed metabolic state in the tolerant response to stroke (Stenzel-Poore et al. 2003 ; Bernaudin et al. 2002 ) . The functions of the identi ﬁ ed genes involve regulation of cellular energetics including energy metabolism and protein utilization. The suppression of these functions parallel those that are described in controlled cellular arrest during hibernation, a time when animals are metabolically inactive (Stenzel-Poore et al. 2003 ) . This suggests that the brain made tolerant to ischemia may function similarly to the brain in a state of hibernation where a genomic program is utilized to suppress metabolic activity, which allows cell survival in a limited environment.

18.2.3.3 Ion

A key component of the pathogenesis of stroke is dysregulation and accumulation of ions such as Ca2+ , Na2+ , K+ , and H+ , leading to plasma membrane depolarization, which culminates in cell death. Ionic homeostasis is maintained through the constant exchange of ions through channels located in the mitochondrial and plasma membranes. Under hypoxic conditions, energy required to maintain these channels is depleted and ions accumulate in the mitochondria and cytoplasm, increasing cellular depolarization. During periods of low oxygen availability, hypoxia-tolerant species have developed an endogenous mechanism to circumvent ion accumulation through modulation of ion channels (Buck and Hochachka 1993 ; Frerichs and Hallenbeck 1998 ; Ghai and Buck 1999 ) . We found that following ischemic preconditioning, several genes that encode receptors or channels associated with ionic transport (multiple potassium channels, ion transport regulator 7, a cation transport ATPase family member) were suppressed, leading us to postulate that ischemic preconditioning may induce a tolerant state through the modulation of ion channels (Stenzel-Poore et al. 2003 ) . Other genomic pro fi les have identi fi ed ion channel regulation as well; however, most channel regulation appears to be upregulated following preconditioning (Dhodda et al. 2004 ; Tang et al. 2006 ; Bernaudin et al. 2002 ) . These fi ndings, on fi rst glance, may appear to be contradictory. However, the maintenance of ionic homeostasis is a complicated balancing act that likely involves increased as well as decreased expression of specifi c receptors. Further research is needed to understand the exact correlation between the modulation of these channels and protection. Several lines of evidence support a role for ionic homeostasis in preconditioning and ischemic tolerance: (1) Horiguchi et al. showed that opening of a mitochondrial ATP-sensitive potassium channel was involved in 3-nitropropionic acid-induced ischemic tolerance in a rat model of focal cerebral ischemia (Horiguchi et al. 2003 ) ; (2) cultured rat 372 K.B. Vartanian et al. neurons exposed to a preconditioning dose of oxygen-glucose deprivation showed decreased voltage-gated potassium currents across the plasma membrane (Stenzel- Poore et al. 2003 ) ; and (3) imaging of intracellular calcium in gerbil hippocampal CA1 neurons showed that the subsequent increase in calcium levels following an anoxic-aglycemic episode was inhibited in gerbils that had been preconditioned with brief ischemia (Shimazaki et al. 1998) . Although these studies, with the exception of Horiguchi’s, do not address specifi c ion channels, the potential for an ionic homeostasis component to preconditioning is well founded.

18.2.3.4 Neurotransmitters

Following stroke, the accumulation of the excitotoxic neurotransmitter glutamate in the extracellular environment results in hyperactivation of glutamate receptors and further destabilizes the ionic balance in the cell. During ischemia, low levels of ATP result in the uncontrolled release of glutamate into the extracellular environment. The excessive glutamate results in subsequent hyperactivation of receptors, enhanced in ﬂ ux of cations (i.e., Na+ , K+ , Ca 2+ ), and excitotoxic injury, thus perpetuating the ionic imbalance. Both Kawahara and Stenzel-Poore report that ischemic preconditioning resulted in the downregulation of glutamate receptors (GluR2, GluR3, mGluR3) (Stenzel-Poore et al. 2003 ; Kawahara et al. 2004 ) . This downregulation potentially creates a cellular setting where the density of glutamate receptors is minimized at the time of the ischemic insult, dampening the excitotoxic response to the overload of glutamate in the extracellular compartment (Stenzel-Poore et al. 2003 ; Kawahara et al. 2004 ) . Other key regulators of glutamate in the CNS are excitatory amino acid transporters (EAAT), also known as glutamate transporters. During basal conditions, EAATs remove glutamate from the synapse following a synaptic transmission, thus maintaining the level of glutamate in the extracellular environment at nontoxic levels. Bernaudin et al. found that the transcript for EAAT4 was increased by 14-fold over basal levels in neonatal rats exposed to hypoxic preconditioning (Bernaudin et al. 2002 ) ; thus, direct removal of glutamate from the extracellular environment may be another route of reducing the impact of excessive glutamate during an ischemic event. These genomic studies suggest that modulation of neurotransmitter responses may be involved in the mechanism of preconditioning and ischemic tolerance.

18.2.4 Signaling Mediators

Many of the genes involved in critical cell functions that contribute to preconditioning and neuroprotection depend on the coordinated regulation of signaling cascades and transcription factor expression and activity. Thus, the differential regulation of signaling cascades and transcription factors in preconditioning and ischemic tolerance serve as the important messengers that promote the protective genomic phenotype. 18 The Genomics of Preconditioning and Ischemic Tolerance 373

18.2.4.1 Signaling Cascades

Alterations in gene expression ultimately affect the activation of downstream signaling cascades. In fact, many of the genes that are altered by preconditioning are involved in a variety of signaling cascades such as mitogen-activated protein kinase (p38 MAPK, MKP-1, MKP-3, MKK2), TGFb (Smad1, Smad7), cyclic AMP (PK1, ARPP-21, CAP1, ADCY5, ADCY7), and small GTPase (Rho activation protein 30, Rab16, Ralb, GIT-1) (Dhodda et al. 2004; Marsh et al. 2009; Kawahara et al. 2004 ; Tang et al. 2006 ; Bernaudin et al. 2002 ) . The regulation of these pathways likely has diverse functional effects that lead to the neuroprotective phenotype. For example, the MAPK family member p38 has neuroprotective properties in response to ischemic injury. Inhibition of p38 in rats caused increased infarcts and vascular leakage in response to stroke (Lennmyr et al. 2003 ) , suggesting that p38 MAPK signaling functions to protect the integrity of the brain and vasculature following stroke. Interestingly, ischemic preconditioning induces the upregulation of p38 prior to stroke (Dhodda et al. 2004 ) and preconditioning with anesthetics increased p38 phosphorylation, indicating increased activity, following stroke (Ye et al. 2012 ; Zheng and Zuo 2004 ) . Additionally, the gene MAPK phosphatase-1 (MKP-1) that is upregulated following ischemic preconditioning promotes protection against TNF a -induced apoptosis in rat mesangial cells. MKP-1 inhibits apoptosis by preventing the sustained activation of JNK (Guo et al. 1998a, b ) . Thus, MKP-1 may serve to promote protection against ischemia by preventing JNK activation following stroke. These are just two examples of the potential protective effects of the selectively regulated signaling pathways associated with preconditioning and ischemic tolerance. As many of these pathways interact with each other, it is likely they coordinate regulation of multiple signaling cascades to promote the protective functions that lead to smaller infarcts in preconditioned animals.

18.2.4.2 Transcription Factors

A variety of transcription factors are regulated in preconditioned animals prior to and following stroke. Prior to stroke, ischemic preconditioning upregulates the expression of transcription factors such as silence factor B, C/EBP d, EGR1, EGR2, KLF4, and NF kB p105 (Dhodda et al. 2004; Kawahara et al. 2004; Tang et al. 2006 ) . The upregulation of some of these transcription factors is maintained following stroke including CCAAT/enhancer binding protein delta (C/EBPd ). Many of these transcription factors play detrimental roles in ischemic injury. EGR-1 (early growth response-1) contributes to ischemic injury and infl ammation as EGR-1-defi cient animals had smaller infarcts and decreased infl ammation in response to cerebral ischemia (Tureyen et al. 2008 ) . C/EBP d and the related transcription factor C/EBPb also exacerbate brain injury in response to ischemia through the upregulation of infl ammatory cytokines and neurotoxic factors such as iNOS (Won et al. 2003 ; Yi et al. 2007 ) . However, C/EBP d also induces potential neuroprotective mediators such as IGF-1 (Ji et al. 2003 ) . The upregulation of these transcription factors associated 374 K.B. Vartanian et al. with damage may be important for the modest damage required to create the preconditioned environment that leads to neuroprotection. In addition, although the transcription factor hypoxia-inducible factor-1 (HIF-1) is not transcriptionally upregulated using microarray analysis, several of the neuroprotective genes induced following hypoxic preconditioning are associated with HIF-1 such as VEGF and GLUT-1, suggesting that HIF-1 activation may be an important mediator of multiple pathways to protection (Tang et al. 2006 ) . Several transcription factors are also regulated following stroke, and many of these changes are considered to be associated with neuroprotection. The in fl ammatory transcription factor NFk B plays a damaging role following ischemia, and inhibition of NFk B promotes neuroprotection. Preconditioning with a low dose of the TLR4 agonist, LPS, resulted in suppression of NFk B activity following ischemia (Vartanian et al. 2011 ) . Furthermore, genomic analysis of the neuroprotective environment following preconditioning shows upregulation of many genes associated with IRF transcription factors (Stevens et al. 2011; Marsh et al. 2009 ) . Additionally, IRF3 activity is enhanced following stroke in LPS-preconditioned mice (Vartanian et al. 2011 ) . The importance of IRF transcription is demonstrated by the absence of neuroprotection in IRF3- and IRF7- de fi cient mice subjected to preconditioning (Stevens et al. 2011 ; Marsh et al. 2009 ) . These changes in transcription factors indicate that there is a major shift in the transcriptional programs produced following stroke in the neuroprotective environment.

18.2.5 Summary of Genomic Changes in Preconditioning and Tolerance

The use of microarrays to evaluate the genomic environment in preconditioning and ischemic tolerance has provided a solid foundation for our understanding of neuroprotection. These studies have yielded extensive amounts of data on gene expression at a wide variety of time points allowing researchers to begin to piece together the dynamic responses induced by preconditioning that culminate in ischemic tolerance and neuroprotection. Several major themes have begun to emerge. First, preconditioning induces a modest response that is associated with cellular damage, which appears to be important to the setup for subsequent neuroprotection. This is demonstrated through the damaging cascades induced following preconditioning such as apoptosis (caspase-3), forced reentry into the cell cycle, induction of in ﬂ ammatory cytokines, and upregulation of transcription factors known to exacerbate neuronal damage. Second, several protective mechanisms are initiated following preconditioning. These include the regulation of ion transport, suppressed glutamate responsiveness, and initiation of adult neurogenesis. Many of these protective mechanisms are seen again during the tolerant response such as anti-apoptotic pathways (Bcl-2) and the promotion of cell survival and neurogenesis (growth factors). Third, following stroke, certain pathways in the tolerant brain have been reprogrammed, thereby engaging new genomic responses to combat injury and promote neuroprotection. This is demonstrated through new gene regulation resulting in 18 The Genomics of Preconditioning and Ischemic Tolerance 375 suppression of the cell cycle, decreased metabolic activity, and the redirected in ﬂ ammatory response to decrease damaging NFk B activity and increase neuroprotective interferon responses. Through these transcriptional studies, great progress has been made in understanding the endogenous programs engaged in the brain that provide protection from ischemic injury.

18.3 Emergent Trends and New Directions

Over the past decade, the focus of transcriptomic research for preconditioning has centered on identiﬁ cation of key elements of gene regulation. As we begin to decipher the large amounts of accumulated transcriptomic data, new questions about the data have developed. Researchers seek to describe the preconditioned and tolerant state and, perhaps more importantly, to understand the underlying molecular mechanisms that promote neuroprotection. As the ﬁ eld moves forward, it is aided by rapidly advancing technology to address new hypotheses about gene regulation in preconditioning and ischemic tolerance.

18.3.1 Converging Mechanisms

To date, most transcriptional studies have analyzed the effects of a single preconditioning stimulus on gene expression. However, comparison of the transcriptional pro fi les from multiple preconditioning stimuli in one model system has the potential to identify key mechanisms that are common or shared. Stevens et al. performed transcriptional profi ling of three different preconditioning paradigms in a mouse model of transient focal ischemia (Stevens et al. 2011 ) . In addition to analyzing the effect of preconditioning on the brain, the blood genomic response was also examined to explore the systemic contribution to ischemic tolerance. Although most studies have focused on the genomics of the brain, many of the stimuli that induce ischemic tolerance originate outside the central nervous system, including systemic administration of infl ammatory mediators (Rosenzweig et al. 2004 ; Stevens et al. 2008) and remote ischemic preconditioning of a limb to provide brain tolerance (Dave et al. 2006 ; Ren et al. 2008) . In addition, Sharp and colleagues have shown a distinct genomic pro fi le in the blood following stroke suggesting that there is an intimate relationship between these compartments (Stamova et al. 2010 ) . In a study by Stevens et al., mice were preconditioned with LPS (TLR4 agonist), CpG-ODN (TLR9 agonist), or brief ischemia and samples from brain tissue and blood were collected at multiple time points prior to and following ischemic challenge. Pathway analysis of the genes regulated during the preconditioning phase identi fi ed the TLR signaling cascade (i.e., Ik Ba , MyD88, IL1b , CD14, LPSbp, TLR2) and cytokine-cytokine receptor interactions (i.e., Mip1a , Mip2a , IL1r1, IL1 b , IL6) as the two most signifi cantly affected pathways associated with 376 K.B. Vartanian et al.

Fig. 18.2 Preconditioning induces shared gene regulation that maps to common pathways. Population analysis of pathways associated with the regulated genes following preconditioning in the brain (a ) and blood (b ) (3, 24 and 72h) using the Kyoto Encyclopedia of Genes and Genomes ( KEGG) database. Z -scores greater than two were considered signifi cant ( black line ). Top panel (Brain) modi fi ed from Stevens et al. (2011 ) preconditioning in all paradigms in the brain and the systemic circulation (Stevens et al. 2011; Fig. 18.2 ). Thus, TLRs may play a pivotal role not only in exogenous TLR ligand-mediated preconditioning but also in ischemic preconditioning, a fi nding further validated by Pradillo and colleagues wherein mice de fi cient in TLR4 exhibited attenuated protection when preconditioned with brief ischemia (Pradillo et al. 2009) . In addition, the brain showed a shared apoptotic-associated gene regulation (Fig. 18.2a), as was described for hypoxic and ischemic preconditioning above, while the blood shared gene regulation associated with hematopoietic cell lineage pathways (i.e., CD11b, CD5, CD14, CD34, CD55, CR2; Fig. 18.2b ). These studies suggest that TLR signaling may be a common mechanism engaged by distinct preconditioning paradigms in the setup of neuroprotection. Examination of the transcriptional pro fi le following stroke in these studies indicates that all three preconditioning stimuli induce new brain and blood gene regulation in response to the injury that was not evident in animals receiving stroke alone. As the changes in gene regulation were seen as early as 3 h following stroke, they are 18 The Genomics of Preconditioning and Ischemic Tolerance 377

Fig. 18.3 Shared gene regulation reveals common functional categories associated with ischemic tolerance . ( a) Venn diagram representation highlighting the shared functional categories of gene regulation following stroke. (b ) Promoter region analysis of the genes in the brain, common to all preconditioning paradigms 24 h following stroke (a blue area in Venn diagram). Promoter analysis and interaction network toolset (PAINT) ( http://www.dbi.tju.edu/dbi/tools/paint/; Vadigepalli et al. 2003) generated hypothesis gene-transcriptional regulatory element (TRE) network showing the relationship of identifi ed TREs to the regulated genes. Genes are depicted in blue , TREs represented in red (Modi fi ed from Stevens et al. 2011 ) (Color fi gure online) unlikely due to extent of injury, since damage is just beginning to develop at this point. Thus, this new gene regulation represents a reprogrammed response to the ischemic event (Stevens et al. 2011 ) in both the brain and in the blood cell response. The brain’s reprogrammed genomic response, shared between these preconditioning paradigms, revealed a common set of IFN-associated genes regulated following stroke that were not evident in non-preconditioned mice (Stevens et al. 2011 ; Fig. 18.3a, blue area). These genes represent a shared response to injury that suggests a common mechanism involved in the induction of ischemic tolerance. Identifi cation of potential upstream mediators of this shared transcriptional profi le 378 K.B. Vartanian et al. could provide insight into actual effectors of ischemic tolerance. Regulatory sequences for interferon regulatory factors (IRFs) have been identifi ed that are overrepresented in the commonly regulated genes (Fig. 18.3b), implying that IRFs modulate a common neuroprotective response to stroke in preconditioned mice. As discussed above, this IRF response may also re fl ect redirection of TLR signaling in the setting of stroke, which supports the idea that reprogramming of the damaging TLR pro-in fl ammatory response to stroke leads to a more neuroprotective signaling cascade. Transcriptional profi ling of the blood cell response in these animals suggests there may be overlapping mechanisms shared between two of the preconditioning stimuli that play a role in tolerance. CpG-ODN and brief ischemia both induce genes associated with the TGF family (Fig. 18.3a , orange area), which, as discussed above, may have neuroprotective effects in relation to stroke. Also, following stroke, the blood from LPS and CpG-ODN preconditioned animals shared the induction of erythropoiesis-associated genes (i.e., ankyrin 1, erythrocyte protein band 4.2 and 4.9, Rh blood group, D antigen) (Fig. 18.3a, red area). These genes are not evident prior to the ischemic challenge. These genes may play a role in increasing oxygen availability to the brain through the increase of circulating red blood cells during reperfusion. The transcriptional comparison of these three preconditioning paradigms has highlighted potential shared mechanisms of neuroprotection, which may prove to be relevant in other forms of preconditioning as well. These studies identi fi ed TLR signaling and the innate immune response (i.e., cytokine signaling) as key mediators of the preconditioning stimuli. In addition, a common IRF signaling event is identi fi ed in the brain response to stroke only in preconditioned mice and supports the notion that reprogramming of the stroke-induced TLR response may represent a shared mechanism of ischemic tolerance.

18.3.2 Effectors of Neuroprotection

The transcriptional analysis performed following preconditioning and during ischemic tolerance has created a new view of the genomic environment during these two important phases of neuroprotection. The initial period of preconditioning can be seen as the setup or priming phase, and the end stage is marked by the neuroprotective phenotype (Fig. 18.4). The precise mechanism by which the priming phase translates into neuroprotection is unclear. However, the 1Ð5-day time window of protection against subsequent injury suggests that a refractive state exists in which effectors are in place to alter the response to stroke, culminating in ischemic tolerance and protection. It is intriguing to speculate about the speci ﬁ c effectors that translate the conditions of priming into neuroprotection. Gene regulation has already peaked and dropped back to baseline at the time of ischemia making it unlikely that expression of a speci ﬁ c gene or set of genes following preconditioning choreo- graphs the tolerance phenotype. Translation from priming to protection may require 18 The Genomics of Preconditioning and Ischemic Tolerance 379

Fig. 18.4 Model of the genomics of preconditioning and ischemic tolerance. Schematic representation of the genomic progression of ischemic tolerance. The preconditioning stimulus induces genomic changes associated with modest injury. As the response evolves, a refractive phase develops wherein the response to a stroke results in reprogrammed gene expression and the development of a neuroprotective phenotype. Thus, the primary features of this process include (a ) initial TLR activation which reprograms the subsequent TLR response to stroke and thereby initiates neuroprotective gene transcription, (b ) modest activation of apoptosis that is required for subsequent inhibition of apoptotic pathways, (c ) neurogenesis initiated for potential replacement of damaged neurons, (d ) neurotransmitters and ion channels regulated to maintain ion homeostasis, (e ) glucose transporters increased to optimize glucose availability, and (f ) HSPs induced to suppress apoptosis and reduce protein aggregation. The postulated effectors of the refractive phase include epigenetic modi fi cations and miRNAs 380 K.B. Vartanian et al. specifi c executors of neuroprotection, perhaps in the form of master transcriptomic regulators. Identifi cation of recent candidates for this important role stems from new discoveries in our understanding of genomic regulation. Genome-wide regulation has recently been linked to newly discovered regulatory mechanisms including post-transcriptional effects on gene expression by miRNAs and epigenetic changes that occur at the chromosomal level. These types of gene regulation have the ability to have widespread effects on entire genomic programs and ultimately may serve as the necessary “molecular switch” that executes the tolerant genomic response. The roles of these regulatory processes are being explored in the context of preconditioning and stroke, and the results suggest that they may play key roles in modulating neuroprotection.

18.3.2.1 miRNA

The recent discovery of miRNAs has altered our understanding of gene regulation. Functional miRNAs are short sequences of nucleotides approximately 22Ð25 nucleotides long that modulate gene expression posttranscriptionally [reviewed in Bartel 2009 ] . Each individual miRNA has many select targets; thus, gene expression is greatly affected by the presence or absence of particular miRNAs. Researchers have begun to investigate the role of miRNAs in stroke and preconditioning. Several studies using specially designed miRNA microarrays have revealed robust regulation of miRNAs following ischemic preconditioning (Dharap and Vemuganti 2010 ; Lee et al. 2010 ; Lusardi et al. 2010 ) . Lee et al. compared miRNA expression in mice 3 h following ischemic preconditioning (15 min) to miRNAs expressed 3 h following ischemia (120 min). The results identi fi ed several miRNAs that were selectively upregulated following preconditioning compared to ischemia including miR200a, miR200b, miR200c, miR141, miR429, miR182, and miR96. Functionally, many of these upregulated miRNAs (miR200b, c, and miR429) targeted prolyl hydroxylase 2 for degradation. Importantly, the downregulation of prolyl hydroxylase 2 contributed to neuroprotection in an in vitro model of ischemia (Lee et al. 2010 ) . Lusardi et al. investigated changes in miRNA expression following ischemic preconditioning compared to animals receiving a sham surgery (Lusardi et al. 2010 ) . From their miRNA analysis, they identi fi ed 33 miRNAs that were downregulated in ischemic preconditioned animals compared to sham. Interestingly, all of these miRNAs converged on the target mRNA of MeCP2, an epigenetic regulator that functions as a global transcriptional repressor (Lusardi et al. 2010 ) . The downregulation of these miRNAs, in particular miR132, led to the increased expression of MeCP2 in the mouse brain following preconditioning, suggesting a possible role for MeCP2 in the priming phase for neuroprotection. Additional study has also identi fi ed dramatic regulation of miRNAs following ischemic preconditioning whereby 26 miRNAs were upregulated and 25 miRNAs were downregulated following ischemic preconditioning (Dharap and Vemuganti 2010) . Computational analysis of these regulated miRNAs suggested potential targets that include a variety of signaling cascades such as TGFb , MAPK, Wnt, p53, and JAK-STAT (Dharap and Vemuganti 2010 ) . 18 The Genomics of Preconditioning and Ischemic Tolerance 381

Taken together, the evidence strongly implicates miRNAs as regulators of gene expression in the preconditioned brain. The roles of miRNAs in this setting will likely prove to be vital to our understanding of the gene programs initiated during preconditioning and ischemic tolerance and may identify key regulators of neuroprotection.

18.3.2.2 Epigenetics

Epigenetic changes occur at the chromosomal level in a manner that broadly affects gene expression. A major mechanism of epigenetic change involves chromatin remodeling and histone modifi cation, such as methylation or acetylation, which causes the chromatin structure to reorganize, ultimately changing the availability of specifi c gene promoter regions. Furthermore, histone modifi cation can affect when speci fi c genes are regulated. Through these dramatic long-lasting alterations, entire gene programs can be affected. Research into the possible role of epigenetic changes brought on by preconditioning is in its infancy. An investigation into tolerance induced by ischemic preconditioning revealed that several gene repressor proteins were upregulated following stroke including histone H2A, histone H2b, and polycomb group protein (PcG) SCMHI (Stapels et al. 2010 ) , each of which are associated with chromatin remodeling. In particular, the PcG SCMHI protein contains histone H2A monoubiquitination activity and is known to target genes encoding electron transport, glucose transport, endopeptidases, oxidoreductases, G-coupled protein receptors, and potassium channels (Stapels et al. 2010 ) , indicating the wide breadth of effects of epigenetic mediators. Interestingly, overexpression of PcG SCMHI induced ischemic tolerance without the need for preconditioning in an in vitro model of ischemia (Stapels et al. 2010 ) . Consistent with this fi nding, knockdown of PcG SCMHI in vivo reversed the preconditioning-induced neuroprotection against ischemia. These data highlight the importance of epigenetic mediators in neuroprotection and demonstrate the signi fi cance of this new avenue of genomic research.

18.3.3 Technological Advances

Great advancement and understanding in the fi eld of preconditioning and ischemic tolerance has been achieved in recent years through the implementation of microarray technology. The applications of microarray technology are evolving with the fi eld to be able to address novel questions about transcriptional analysis. Microarray platforms have been developed and modifi ed to examine small miRNA content, single nucleotide polymorphisms (SNPs), and DNA-protein interactions (ChIP-chip arrays). This new technology will complement the current transcriptomic data obtained through microarrays with the advantage of using a similar hybridization- based system that yields easily quantifi able data. However, microarray technology 382 K.B. Vartanian et al. still requires signifi cant sample preparation, adjustments for background hybridization levels, and can only accommodate known targets that are available as probes on the individual chips. Rapid advances in DNA sequencing technology have overcome some of these challenges. Next-generation sequencing (NGS) has fundamentally altered the accessibility of DNA sequencing for biological research. Newly developed NGS technology has dramatically improved since the classical Sanger-based sequencing method developed in the 1970s. Several different platforms of NGS technology have been created which utilize miniaturized sequencing reactions to allow for the simultaneous generation of millions of DNA sequences using clonal detection of single nucleotides [reviewed in Su et al. 2011 ] . This is performed without the need for prior DNA clon- ing at a fraction of the cost of traditional DNA sequencing. NGS yields detailed sequencing of entire genomes with many applications including identifi cation of miRNAs, high-resolution analysis of protein binding sites on DNA (ChIP-seq), and SNP discovery. This technology has several advantages over microarray as it is not limited by hybridization to known sequences and therefore provides an unbiased, quantitative view of the entire transcriptome; it is more sensitive to transcripts present in low abundance and requires less sample and data processing (t’Hoen et al. 2008 ) . This type of analysis allows for discovery of low-level or unexpected transcriptomic alterations or potentially important changes outside the known exons. However, it is important to consider that microarray technology has been around for 15 years, and we have come to understand the limitations of this system while improving the technology and creating a large public database of genomic data. As NGS technology continues to develop, our understanding of the new challenges introduced by deep DNA sequencing will improve, as will our tools to analyze new data. Already, third- generation sequencing has eliminated the bias of DNA library generation necessary for some of the earlier NGS platforms. As NGS becomes more widely used, it will likely surpass microarray in many regards but also will serve to complement microarray to lead to new discoveries in genomics research.

18.4 Perspective

Transcriptomic research has established and described a wealth of information about genomic responses to preconditioning stimuli and ischemic tolerance following stroke. As the ﬁ eld of transcriptional research progresses, biological research will take on the new role of understanding how the preconditioned environment leads to ischemic tolerance. This will require the integration of current microarray data sets with new technologies that provide more detailed information about the genome including SNPs, miRNA, and DNA-protein binding. To unearth the relevant genomic alterations buried in these massive data sets, biologists will need to collaborate with computational scientists and mathematicians to apply and create new complex computer-aided data mining programs. Additionally, robust validation criteria must be implemented to con ﬁ rm the roles of newly discovered regulators of 18 The Genomics of Preconditioning and Ischemic Tolerance 383 preconditioning and ischemic tolerance. A cohesive approach among scientists will allow data to traverse from lab-to-lab and lead to testing new discoveries in multiple stroke models to gain relevant preclinical data that moves us closer to our common goal of designing a neuroprotective therapy for the treatment of stroke.

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Peiying Li , Rehana Leak , Yu Gan , Xiaoming Hu , R. Anne Stetler , and Jun Chen

19.1 Introduction

Preconditioning or tolerance is a natural phenomenon of endogenous adaptation, whereby subtoxic stress protects against subsequent higher dose stress. Although preconditioning is likely to be a ubiquitous stress response relevant to many disease conditions, it has been most successfully employed in ischemia research. Enormous efforts have focused on identifying the intrinsic mechanisms so that they can be translated into pharmacological interventions aimed at counteracting neurodegeneration following stroke. A ﬁ rm grasp of the molecular events at the level of each subcellular organelle will aid researchers to move closer toward this elusive clinical goal. Fortunately, technological innovations such as the confocal and transmission electron microscopes have transported preconditioning research into the subcellular organelle level. This has deepened our knowledge of how preconditioning affects these organelles and triggers subcellular signaling pathways that eventually lead to neuroprotective processes and cellular survival. In this chapter, the participation of mitochondria, the endoplasmic reticulum, proteasomes, lysosomes, the Golgi apparatus, the peroxisome proliferator-activated receptors on the nuclear membrane, and gene expression regulating factors in the nucleus will thus be considered.

P . L i ( *) ¥ Y. Gan ¥ X. Hu ¥ R. A. Stetler ¥ J. Chen Department of Neurology and the Center for Cerebrovascular Disease Research , University of Pittsburgh School of Medicine , Bioscience Tower S-507, 3550 Terrace St , Pittsburgh , PA 15213 , USA e-mail: [email protected] R. Leak Division of Pharmaceutical Sciences, Mylan School of Pharmacy , Duquesne University , Pittsburgh , PA 15282 , USA

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 387 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_19, © Springer Science+Business Media New York 2013 388 P. Li et al.

19.2 Mitochondria Preconditioning

Over the last few decades, many attempts have been made to identify the molecular mechanisms involved in preconditioning-induced protective responses. Recent data suggest that many of these mechanisms converge on one of the most important subcellular organelles—the mitochondria. A better understanding of the mitochondrial mechanisms underlying preconditioning-conferred neuroprotection will help in the development of novel therapeutic approaches with the primary goal of modulating mitochondria to enhance brain tolerance against neurodegenerative events. In this section, the following parts of the mitochondrial preconditioning will be discussed: mitochondrial reactive oxygen species (ROS), mitochondrial ATP-sensitive potas- + sium (mtKATP ) channels and mitochondrial permeability transition pore, mitochondrial biogenesis, mitochondrial dynamics, and mitophagy (Fig . 19.1 ).

19.2.1 Mitochondrial ROS as Mediators of Signal Transduction in Ischemic Preconditioning

Reactive oxygen species are chemically reactive molecules containing oxygen. By virtue of an unpaired electron, ROS are typically unstable and highly reactive and have the potential to cause damage to DNA, RNA, and proteins. The mitochondrion is the site at which most of the ROS are produced. ROS are a contributing factor to aging but also have a bene ﬁ cial side as they are potentially protective when mild activation of ROS is induced by preconditioning. Preconditioning with ischemic pretreatments (Hausenloy et al. 2010 ) , volatile + anesthetics (Hirata et al. 2011; Zu et al. 2011 ) , or mtKATP channel openers was reported to increase ROS generation and protect against myocardial infarction, while ROS scavengers were able to abolish the preconditioning-conferred protection (Tanaka et al. 2002 ; Kevin et al. 2003 ; Novalija et al. 2003a, b ) . In neuronal preconditioning, ROS generation was also observed as one of the earliest requisite signals. In addition, ROS scavengers also abolish the neuroprotection of preconditioning. Taken together, these data all indicate that modest ROS generation is required for both cardiac and neuronal preconditioning to be protective. With regard to the downstream events that allow ROS generation to mediate protection against ischemic and reperfusion injury, many studies have helped identify the signal transduction components involved. So far, the downstream cell signaling events of ROS generation in neuronal preconditioning include upregulation of the neuroprotective protein heat shock protein 70 (HSP70), activation of PKC, and activation of survival kinases PI3K and ERK1/2.

19.2.1.1 Upregulation of HSP70

Since the identi ﬁ cation of the heat shock response in fruit ﬂ ies by Ritossa ( 1996 ) , heat shock proteins have been widely studied as chaperones that help refold misfolded, 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 389

Fig. 19.1 Signaling pathways of mitochondrial preconditioning. Preconditioning insults stimulate the production of reactive oxygen species (ROS ). Moderate amounts of ROS exert neuroprotective effect via several signaling mechanisms. (1) ROS mediates the opening of potassium channels, such as mtK ATP and BK Ca channels, either directly or indirectly through the activation of PKC. The opening of mtKATP channels will decrease the mitochondrial transmembrane potential ( Δ Ψm) and promote ATP production. Meanwhile, the opened mtKATP channels could further enhance ROS production, developing a positive feedback cycle for neuroprotection. (2) ROS or activated PKC leads to low-conductance transient opening of mitochondrial permeability transition pore (mPTP ) and increases its threshold for reopening. (3) ROS upregulates the expression of neuroprotective protein HSP70 and activates the pro-survival signaling pathways, such as PI3K and MARK/ERK. In addition to the ROS-dependent mechanisms, preconditioning also directly causes PKC activation, calcium overload, and translocation of Cx43 through mtK ATP channels, all of which induce transient mPTP opening and increase the threshold of mPTP opening upon subsequent lethal ischemic injury

aggregated proteins. They can either be constitutively expressed or induced upon cellular stress. The generation of ROS during preconditioning was able to induce the expression of the most widely examined heat shock protein, HSP70, while the application of antioxidants or free radical spin traps during the preconditioning period blocks both the tolerance and upregulation of HSP70. Here, ROS acts as a potent signaling molecule capable of eliciting discrete posttranslational modiﬁ cation of proteins. ROS phosphorylate the adaptor protein p66Shc at serine 36, and loss of p66Shc abolishes the resistance to a subsequent lethal insult. Moreover, myocytes overexpressing HSP70 exhibited a marked resistance to ROS (Chong et al. 1998 ) , suggesting that HSP70 also induces cardioprotection upstream to ROS-mediated damage. 390 P. Li et al.

19.2.1.2 ROS Production Occurs Upstream of PKC Activation in Preconditioned Cardiomyocytes

PKC is a ubiquitous intracellular kinase consisting of approximately ten isoforms. The Ca2+ -independent isoforms PKC-d and -e have been considered the most important to preconditioning-related signaling cascades (Bouwman et al. 2007 ) . Studies have shown that activation of PKC-d , PKC-e , and PKC-a occurred downstream of ROS production in acute preconditioning by sevofl urane (Novalija et al. 2003a, b; Bouwman et al. 2004 ; Liu et al. 2008 ) . Supporting evidence came from studies showing that the ROS formation during sevo fl urane preconditioning was not altered by any of the PKC inhibitors, indicating that PKC activation occurred downstream of ROS generation (Novalija et al. 2003a, b ) . Upon acute preconditioning by sevofl urane, PKC- d translocated to the sarcolemmal membrane. This translocation could be inhibited by ROS scavenging, suggesting ROS release precedes PKC-d activation (Bouwman et al. 2004 ) . Once activated, the PKC-e translocated into the mitochondria and formed a complex or “signaling module” with MAPKs, resulting in the inhibition of mitochondrial-dependent apoptosis (Baines et al. 2002 ) . PKC activator AD198 was able to generate ROS, activate PKC-e , and induce cardioprotective signaling in cardiomyocytes (Hofmann et al. 2007 ) . Nevertheless, the activation of the Ca2+ -dependent PKC-a isoform was also involved in sevofl urane-induced cardioprotection, and this activation occurred downstream of ROS generation (Bouwman et al. 2007 ; Liu et al. 2008 ) . PKC- a inhibition attenuated cardioprotection by ischemic preconditioning, but did not affect ROS production during preconditioning (Vanden Hoek et al. 2000 ) . In a sevo fl urane preconditioning study, it was further shown that PKC- a was activated by sevofl urane via the formation of ROS (Bouwman et al. 2007 ) . In summary, these data suggest that preconditioning induces ROS production and that this, in turn, activates PKC.

19.2.1.3 Activation of the PI3K Pathway

The PI3K-Akt-Gsk3b pathway is an important pro-survival signaling cascade that has been suggested to be involved in preconditioning for many years (Tong et al. 2000 ) . Recently, this pathway was shown to be activated as the consequence of ROS generation when a potent activator of the BKCa channel, NS1619, was applied to induce preconditioning (Gaspar et al. 2008a ) . However, this is the only evidence so far to support that ROS generation in response to preconditioning activates the pro-survival PI3K pathway.

19.2.1.4 Activation of MAPK/ERK Pathway

Extracellular signal-regulated kinase (ERK), a prototypical and ubiquitous member of the mitogen-activated protein kinase (MAPK) family, can also be upregulated by reactive oxygen species production in response to numerous stimuli (Ruiz-Ramos et al. 2005 ; Yang et al. 2007) . In PC12 cells, low-level ROS production was shown to modulate the 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 391 redox condition of the cell and act as a messenger to trigger ERK1/2 activation in 35%

O2 -induced hypoxia preconditioning. The activation of the ERK pathway could promote Bcl-2 gene expression through a variety of transcription factors. Bcl-2 is a critical anti- apoptotic regulator of programmed cell death (Cao et al. 2009 ) .

19.2.2 Mitochondrial Potassium Channels

Two potassium channels located on the inner mitochondrial membrane are thought to + initiate neuronal preconditioning: the ATP-sensitive potassium (mtKATP ) and the large conductance calcium-activated potassium (BKCa ) channels. The KATP has been extensively studied and demonstrated to mediate neuronal preconditioning. The latter one, however, may not be involved in neuronal preconditioning (Gaspar et al. 2008a, 2009 ) .

+ 19.2.2.1 The Opening of mtKATP

The KATP is of particular importance in the brain because brain mitochondria contain + many more mtKATP channels than other tissues (Bajgar et al. 2001 ) . Emerging + ﬁ ndings suggest a key role for the mtKATP channels in neuroprotection afforded by preconditioning (Busija et al. 2008 ; Dirnagl and Meisel 2008 ) . It was shown that + mtK ATP blockers, such as 5-hydroxydecanoate (5-HD), not only abolished neuroprotection conferred by ischemic preconditioning (Kaneko et al. 2005; Raval et al. 2007; Watanabe et al. 2008 ) but also the protection from pharmacological preconditioning (Kaneko et al. 2005 ; Adamczyk et al. 2010 ) . Conversely, activation of + mtK ATP channels with pharmacological agents mimics preconditioning. Selective + mtKATP channel openers, such as diazoxide and BSM-191095, have been shown to induce immediate and delayed preconditioning in in vitro and in vivo models of ischemia (Kis et al. 2003; Mayanagi et al. 2007a, b; Watanabe et al. 2008 ) . Moreover, pretreatment with diazoxide was effective in protecting cultured hippocampal neurons (Goodman and Mattson 1996 ) , as well as vascular endothelial cells (Chi et al. 2000 ) against amyloid-b (A b)-induced cytotoxicity. Recently, it was shown that pretreatment with diazoxide also exerted a neuroprotective effect against MPP + - induced cytotoxicity in in vitro and in vivo models of Parkinson’s disease (Xie et al. 2010) . In another model of Parkinson’s disease, diazoxide induced protection against the neurotoxic effects of rotenone in PC12 cells (Tai and Truong 2002 ; Tai et al. 2003 ) and improved both parkinsonian symptoms and neurochemical alterations in rats treated with rotenone (Yang et al. 2006 ) . These results suggest that + mtK ATP channel activation may be involved in preconditioning against neurodegeneration and provide a new therapeutic strategy for the treatment of Alzheimer’s and Parkinson’s disease neurodegeneration. + The mechanisms underlying the role of mtKATP channels in preconditioning-mediated protection involve a decrease in the mitochondrial transmembrane potential + (D Y m) and an increase in ATP production (Inoue et al. 1991 ) . The mtKATP channel 392 P. Li et al. opener diazoxide could suppress the D Y m increase and intracellular ROS overproduction induced by Ab (Ma and Chen 2004 ) and MPP + (Xie et al. 2010 ) in + neurons. However, another mtKATP channel opener BSM-191095 could depolarize mitochondria without affecting ROS production (Mayanagi et al. 2007a, b ; Gaspar et al. 2008b ) . BSM-191095 exerted neuroprotective effects via activating the PI3K signaling pathway, increasing ATP content and catalase expression (Gaspar et al. + 2008b) . In addition, the mtKATP channel opener exerts a direct effect on endothelial mitochondria. Pretreatment with diazoxide was shown to protect the endothelium against ischemic stress and ameliorate disruption of the blood-brain barrier (Lenzser + et al. 2005 ) . Such mechanisms undoubtedly contribute to mtKATP -mediated neuro- + protection. Recently, the brain mtKATP channel was shown to be a target of epsilon protein kinase C (PKC e), which mediated preconditioning-induced neuroprotection + through the activation of mtKATP channels (Raval et al. 2007 ) . + There is potentially a positive feedback loop between ROS generation and mtKATP + opening. ROS generation can also open mtKATP channels and contribute to preconditioning-conferred neuroprotection (Forbes et al. 2001 ) . Conversely, it has been shown that diazoxide could induce a modest increase of ROS in adult cardiomyocytes, and the increase in ROS could be abrogated by the addition of antioxidants + (Forbes et al. 2001 ) . MtKATP channels do not appear to be the end effectors of protection; rather, their opening before ischemia generated free radicals that triggered entrance into a preconditioned state and activation of kinases (Pain et al. 2000 ) .

19.2.2.2 A Second Potassium Channel on the Mitochondria BKCa Channel

+ 2+ + Besides the mtKATP channel, neurons also express the Ca -activated K (BKCa ) channel that may mediate neuroprotective effects. The BKCa channel is composed of a pore-forming a-subunit (BKCa a) and an auxiliary BKCa b subunit. BK Ca channels have been localized to the inner membrane of mitochondria (Xu et al. 2002 ; Douglas et al. 2006 ) , and emerging studies have reported immediate and delayed preconditioning-induced cardioprotection (Shintani et al. 2004 ; Wang et al. 2004; Sato et al. 2005) and neuroprotection (Cheney et al. 2001; Runden-Pran et al. 2002; Hepp et al.

2005) via the activation of BK Ca channels. Recently, the neuroprotective effect of the

NS1619, a potent activator of the BKCa channel, was shown to be the consequence of ROS generation and activation of the PI3K pathway (Gaspar et al. 2008a ) .

19.2.3 Mitochondrial Permeability Transition Pores

19.2.3.1 What Is the Mitochondrial Permeability Transition Pore (mPTP)?

The mPTP is a protein pore in the inner membrane of the mitochondrion. The opening of mPTP reveals the mitochondrion’s role in cell death in addition to its normal physiological role in ATP production and metabolism. Pathological changes during 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 393 cardiac or brain ischemia and reperfusion injury, such as Ca2+ overload, especially when accompanied by oxidative stress, elevated phosphate concentration, and adenine nucleotide depletion can all induce the opening of mPTP. This causes the mitochondrion to break down and, if unrestrained, leads to necrotic cell death.

19.2.3.2 Preconditioning-Conferred Cardioprotection May Be Associated with the Inhibition of mPTP

In cardiac ischemia, there is general agreement that the end effect of acute preconditioning is the inhibition of mPTP opening. Ischemic preconditioning not only reduces mPTP opening during the early phase (1Ð2 h after preconditioning) of reperfusion but also increases subsequent pore closure as reperfusion continues (Halestrap et al. 2007 ; Lim et al. 2007 ) . In acute myocardial infarction studies, it is well established that the mitochondrial dysfunction resulting from the irreversible pathological opening of the mPTP at the onset of myocardial reperfusion is a major determinant of the death of cardiomyocytes (Hausenloy et al. 2010 ) . In the brain, there is no evidence that preconditioning might alter the opening of mPTP and subsequently impact the neurological outcome of ischemic injury. However, there are studies demonstrating that inhibition of mPTP opening by N -methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclosporin A (CsA) analog, or cyclosporine, an immunosuppressant drug after traumatic brain injury, is neuroprotective and improves cognitive function in rats (Readnower et al. 2011 ; Sullivan et al. 2011 ) . Therefore, it is reasonable to speculate that preconditioning may also inhibit the mPTP opening during the upcoming noxious insult and provide protection in the brain as it does in the heart.

19.2.3.3 How Is Preconditioning Linked to Inhibition of the mPTP?

Despite a large body of literature, there is no consensus on which signaling pathways are involved in the inhibition of mPTP in response to ischemic preconditioning and how they interact. As discussed below, there are some hypotheses trying to explain how preconditioning can induce the inhibition of mPTP. Although all the data arise from cardiac preconditioning, the mechanisms in the heart may also help us to understand those in the brain.

Phosphorylation by PKC e May Be Responsible for the Inhibition of mPTP in Response to Preconditioning

In response to preconditioning, PKC e translocates into the mitochondria (Mitchell et al. 1995; Ping et al. 1997 ; Baines et al. 2002) . In isolated heart mitochondria, it was reported that the mPTP was inhibited following 15 min of preconditioning with 394 P. Li et al.

PKC e and phorbol ester (Baines et al. 2003 ) , suggesting that PKCe might directly inhibit the mPTP by phosphorylating certain components of the mPTP, such as the voltage-dependent anion channel (VDAC) (Ping et al. 1997 ; Baines et al. 2002, 2003 ; Halestrap et al. 2007 ) .

The Contribution of ROS Production and Calcium Loading to the mPTP Inhibition Following Preconditioning

Elevated ROS production in and around mitochondria induced by ischemia and reperfusion was reported to trigger the opening of mPTP. Calcium, although not as potent as ROS, is another trigger of mPTP opening in excitable cardiomyocytes and neurons (Juhaszova et al. 2004, 2008) . Preconditioning with diazoxide was shown to limit mPTP induction by increasing the mPTP-ROS threshold and reducing calcium overload (Juhaszova et al. 2009) . In this manner, diazoxide provides cellular protection against ischemia-reperfusion injury, suggesting that both ROS production and calcium load could mediate mPTP inhibition-induced protection in preconditioning (Juhaszova et al. 2009 ) .

Translocation of Connexin 43 (Cx43) into the Mitochondria May In ﬂ uence the Opening of the mPTP

Connexin 43 is a 43-kD peptide belonging to the connexin family of gap junctions. It has been proposed that Cx43 may play a pivotal role in linking preconditioning to the mitochondria via inﬂ uencing the opening of the mPTP (Boengler et al. 2005 ; Halestrap 2006 ; Rodriguez-Sinovas et al. 2006 ) . There is also evidence that Cx43 is able to translocate to the mitochondria following preconditioning. The translocation + of Cx43 to the mitochondria was proposed to be required for opening of the mtK ATP channel, which leads to ROS production and triggers preconditioning (Boengler et al. 2005 ; Halestrap 2006 ; Rodriguez-Sinovas et al. 2006 ) .

19.2.4 Mitochondrial Biogenesis

19.2.4.1 What Is Mitochondrial Biogenesis?

Mitochondrial biogenesis is the process of generating new mitochondria by growth and division of preexisting organelles. It requires the expression of several hundred gene products for proteins that make up the functional and structural organelle. The majority of mitochondrial proteins come from the nuclear genome, whereas the mitochondrial genome encodes for only a few proteins involved in the electron transport chain. Transcriptional regulators enabling 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 395 communication between the nuclear and mitochondrial genomes include the nuclear respiratory factors (NRF) 1 and 2, which mediate expression of multiple nuclear genes for mitochondrial proteins; mitochondrial transcription factor A (TFAM, previously mtTF-1 and mtTFA), which stimulates mitochondrial DNA (mtDNA) replication and transcription; and peroxisome proliferator-activated receptor coactivator-1 (PGC-1), a stimulator of mitochondrial biogenesis in mammals (Scarpulla 2006 ) .

19.2.4.2 Modulation of Mitochondrial Biogenesis May Be a Potential Therapeutic Strategy

Mitochondrial biogenesis is a highly regulated process and has been well deﬁ ned in skeletal and heart muscle. It appears to involve adaptive remodeling in cardiac tolerance to ischemia-reperfusion injury and may be a putative target for therapeutic intervention (McLeod et al. 2005 ) . In brain, disruption of mitochondrial function plays a central role in the pathophysiology of many neurological diseases (Mandemakers et al. 2007) and places the brain at risk for compromised energy production. Therefore, generation of new mitochondria is a helpful strategy to minimize damage attributable to lost energy resources.

19.2.4.3 Mitochondrial Biogenesis and Preconditioning-Conferred Neuroprotection

Mitochondrial biogenesis has been observed in the brain in different pathophysiological conditions, and it has been suggested that alteration of mtDNA and increased expression of nuclear genes encoding mitochondrial proteins could be triggered by mitochondrial dysfunction or high energy demands (Chen et al. 2001 ; St-Pierre et al. 2006 ) . There is evidence showing that transient cerebral ischemia or hypoxia and ischemia was able to trigger mitochondrial biogenesis as an adaptive or compensatory response to stress. Transient global ischemia was shown to increase mitochondrial elongation, a well-known step in the process of mitochondrial biogenesis, in the hippocampal CA1 region in adult rats (Bertoni-Freddari et al. 2006 ; Liang and Wong-Riley 2006 ) . Similarly, another group demonstrated that transient cerebral hypoxia stimulated mitochondrial biogenesis in subcortical regions of the brain through an NO-dependent mechanism (Gutsaeva et al. 2008 ) . Hypoxia and ischemia was also demonstrated to rapidly increase mitochondrial biogenesis de ﬁ ned by alterations of mtDNA and mitochondrial-speciﬁ c transcriptional factors (Yin et al. 2008 ) . Although these studies did not directly prove that preconditioning is mediated by mitochondrial biogenesis, the consistent increase of mitochondrial biogenesis after cerebral ischemia or hypoxia may be one of the underlying mechanisms of ischemic or hypoxic preconditioning-conferred neuroprotection. 396 P. Li et al.

19.2.5 Mitophagy

Mitophagy is the process by which cells eliminate dysfunctional mitochondria via autophagy. The degradation of mitochondria is selective to those that are damaged; however, the mechanism underlying this selectivity is still under investigation.

19.2.5.1 The Association Between Mitophagy and Preconditioning-Conferred Protection

Recent studies have found that ischemic preconditioning in isolated rat hearts was able to mediate cardioprotection against ischemic and reperfusion injury in an autophagy-dependent manner. Furthermore, it was indicated that the autophagic process involves recruitment of p62 to mitochondria (Huang et al. 2010 ) . Another line of evidence comes from studies showing that ischemic preconditioning could trigger the opening of mPTP (Hausenloy et al. 2009 ) , and that mPTP plays a role in mediating mitophagy in starved HL-1 cells, a cardiac muscle cell line (Carreira et al. 2010) . Taken together, these data suggest that mitophagy might also be an important element of preconditioning-conferred cardioprotection. In nondividing neurons, mitophagy is of particular importance for the maintenance of homeostasis, because neurons are postmitotic cells and highly dependent on the endolysosomal pathway for the degradation of toxic aggregates (Lee 2009 ) . Mitochondria are one of the major sources of energy but also the major producers of ROS, cytochrome C , and apoptosis-inducing factor, all of which can facilitate apoptosis (Twig and Shirihai 2011 ) . Therefore, the clearance of damaged mitochondria by mitophagy is crucial to neuronal survival. However, the evidence showing that preconditioning-afforded neuroprotection can be mediated by mitophagy is not well established.

19.2.5.2 Parkin-Mediated Mitophagy and Preconditioning

Parkin is a ubiquitin ligase in the cytosol and was recently associated with mitophagy. It can selectively and rapidly translocate from the cytosol to depolarized mitochondria and subsequently induce mitochondrial autophagic removal. The clearance of impaired mitochondria plays a pivotal role in protecting neurons against the release of oxidative substances and apoptosis-inducing factors from dying mitochondria (Narendra et al. 2008 ; Geisler et al. 2010 ; Matsuda et al. 2010 ) . However, direct evidence that parkin mediates preconditioning-induced mitophagy is still lacking. One study of parkin knockout mice shows that they exhibited attenuated ischemic preconditioning-induced p62 translocation to the mitochondria (Huang et al. 2011 ) . The p62 translocation was previously demonstrated to be required for parkin-mediated mitophagy (Geisler et al. 2010 ) . Therefore, 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 397 further studies are still warranted to demonstrate whether preconditioning can directly induce parkin translocation into mitochondria and to elucidate the signaling pathway by which the translocation is triggered.

19.3 Endoplasmic Reticulum (ER)

19.3.1 The Endoplasmic Reticulum and ER-Associated Stress

The ER is the organelle responsible for folding and processing of newly synthesized secretory and membrane proteins (Paschen 2004 ) . It also operates as a major intracellular Ca2+ store and plays a critical role in reactions which are strictly Ca2+ dependent (Paschen and Doutheil 1999 ) . Increasing evidence shows that the ER acts as an important organelle in the transduction of various cellular stresses into cell defense/ survival or death decisions (Lin et al. 2007 ) . ER stress has been linked to various pathological conditions in the brain, such as stroke, traumatic brain injury, and neurodegenerative diseases (Paschen 2004 ; Matus et al. 2011 ) . To combat ER stress, cells use an integrated signaling response, unfolded protein response (UPR). When unfolded or misfolded proteins accumulate, the UPR will activate signaling pathways which increase the production of molecular chaperones to help with the protein folding. If ER stress persists, the UPR will activate signaling pathways which lead to apoptosis (Li et al. 2006a ) . When a particular protein is continuously misfolded, it can be selectively recognized as a potential threat to the proper functioning of ER. In this case, the cells can have these misfolded proteins aggregate to one another and accumulate and subsequently guide them to retrotrans- locate back to the proteasome in the cytosol for degradation. This is called ER-associated degradation (ERAD) and will be discussed below (Ron and Walter 2007 ) (Fig. 19.2 ). It has been recently documented that enhancing the UPR, including inducing ER stress proteins and activating ERAD, underlies brain tolerance against ischemia. Additionally, a moderate Ca 2+ response of ER appears to be implicated in the induction of ischemic tolerance following preconditioning stimuli. These studies with an emphasis on ER-associated mechanisms underlying brain preconditioning will be discussed below.

19.3.2 ER Stress Proteins

Activating the signaling pathways that lead to increased production of molecular chaperones is one of the main jobs of the UPR. Increased chaperones are expected to refold the unfolded proteins accumulated under stress. Many chaperones are heat shock proteins, which are expressed in response to elevated temperatures or other 398 P. Li et al.

Fig. 19.2 Protein degradation triggered by preconditioning contributes to neuroprotection. Multiple subcellular organelles involved in protein degradation have been shown to contribute to preconditioning-conferred neuroprotection. (1) Degradation of pro-death proteins and actin-binding proteins by the ubiquitin-proteasome system ( UPS) underlies preconditioning-conferred neuroprotection. (2) Preconditioning promotes the function of ER-associated degradation (ERAD ) of misfolded proteins, which is important for degradation of protein aggregates through the ubiquitin- proteasomal pathway under stressful conditions. (3) Preconditioning also triggers autophagy in the lysosome via the PI3K pathway or HIF-1 pathway, both of which are activated by ROS generation following preconditioning. All these processes are essential components of preconditioning- afforded neuroprotection

cellular stresses, including stroke and other neurodegenerative diseases (Stetler et al. 2009 ; Turturici et al. 2011 ) . The major evidence of the involvement of UPR in brain tolerance against ischemia comes from observations that multiple preconditioning paradigms may induce the expression of ER chaperones.

19.3.2.1 Heat Shock Proteins

Heat shock proteins are a class of functionally related proteins responsible for folding or unfolding other proteins. Most of the stress-inducible HSPs are constitutively expressed in all living cells and having a plethora of cellular functions in normal or in stressed cells to promote cell survival (Stetler et al. 2010 ) . The induction of HSPs has been acknowledged to contribute to the neuroprotection against cerebral ischemia and 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 399 neurodegenerative diseases. Recently, HSPs have also been associated with brain preconditioning. HSP70 is the most abundant HSP in cells. Its expression was found to be elevated by different paradigms of preconditioning in various in vivo or in vitro models of brain tolerance (Chen et al. 1996, 2004 ; Ikeda et al. 2000 ; Liebelt et al. 2010 ) . In the context of cerebral ischemic tolerance, preconditioning-induced HSP70 expression was found to be primarily expressed in neurons (Currie et al. 2000 ) and was correlated with the preconditioning-conferred neuroprotection. On the other hand, inhibition of HSP70 activity by antisense oligodeoxynucleotides (Chen et al. 2004 ) or by neutralizing antibody (Liebelt et al. 2010 ) or HSP70 inhibitor (Sun et al. 2010 ) was shown to abolish the protective effect of preconditioning. HSP70 may mediate preconditioning by inhibiting cytotoxic protein aggregations (Ge et al. 2008) , as a result of its chaperone activity. In addition, increased HSP70 expression was shown to reduce the release of cytochrome C and the nuclear translocation of apoptosis-inducing factor (AIF) in several ischemia models (Stetler et al. 2010 ) . Furthermore, the activation of p38 MAPK was shown to contribute to the upregulation of HSP70 by remote ischemic preconditioning in brain ischemic tolerance (Sun et al. 2010 ) . It is still unknown whether HSP70 expression reduces unfolded proteins and how this contributes to the activation of these signaling pathways in preconditioning-afforded neuroprotection. HSP27 is a member of the small HSP family. An increase in HSP27 was observed in various rodent models of ischemic tolerance (Kato et al. 1995 ; Currie et al. 2000 ; Valentim et al. 2001 ) . The expression of HSP27 was also seen in activated astrocytes (Kato et al. 1995 ; Currie et al. 2000 ) . This differs from HSP70, which is typically induced in neurons. Distinct biochemical activities have been reported for the phosphorylation status of HSP27. Dephosphorylated HSP27 acts as a chaperone and protects other proteins from denaturation. A decrease in phosphorylation was seen in the preconditioned brain after lethal global ischemia (Valentim et al. 2001 ) . However, in in vitro models of neuronal tolerance, ischemic preconditioning or preconditioning with NDMA or ethanol led to an increase in HSP27 phosphorylation, which might be involved in preconditioning by regulating actin dynamics (Valentim et al. 2003 ; Sivaswamy et al. 2010 ) .

19.3.2.2 Glucose-Regulated Proteins (GRPs)

Glucose-regulated protein 78 (GRP78) is one of the well-studied ER chaperones. GRP78 is a member of the 70-kDa heat shock protein family. The suppression of its expression by antisense treatment enhanced apoptosis in hippocampal neurons exposed to excitotoxic and oxidative insults (Yu et al. 1999 ) . On the contrary, an increased level of GRP78 renders neurons more resistant to the stressful conditions in vitro (Yu et al. 1999 ; Aoki et al. 2001 ) . Similar results were also obtained in the rodent models of cerebral ischemia (Morimoto et al. 2007; Oida et al. 2008 ) . Both ischemic (Hayashi et al. 2003; Lehotsky et al. 2009b) and resveratrol (Saleh et al. 2010) preconditioning were reported to lead to signiﬁ cantly increased levels of GRP78 400 P. Li et al. expression. GRP78 expression peaked at 2 days after preconditioning, corresponding with the permissive time window of ischemic tolerance (Hayashi et al. 2003 ) . Activation of the NMDA receptor was shown to be responsible for the upregulation of GRP78 in ischemic tolerance (Saleh et al. 2010) . Increased GRP78 expression led to decrease in eIF2a phosphorylation via a PERK-dependent manner, which may contribute to the preconditioning-induced neuroprotection (Hayashi et al. 2003 ) . Similarly, the expression of GRP94, another resident ER chaperone, was also increased in preconditioned brain (Hayashi et al. 2003 ; Saleh et al. 2009 ) . These studies indicate that the development of brain tolerance against ischemia may include an enhanced ER ability to deal with unfolded proteins under stress conditions.

19.3.3 ER-Associated Degradation

Once the misfolded or unfolded protein aggregations accumulate to the extent that exceeds the clearance ability of UPR, activation of ERAD is important for degradation of the protein aggregates through the ubiquitin-proteasomal pathway under stressful conditions. However, the ubiquitin-proteasome system (UPS) is impaired in various neuronal pathologies including acute disorders and degenerative diseases (Paschen 2003, 2004) . Following ischemic injury, the insoluble ubiquitin conjugates form aggregates in neurons may amplify neuronal damage to the neurons, although the activation of UPS appears also to be implicated in secondary brain injury after ischemia (Meller 2009 ) . Preconditioning can promote ERAD through the UPS, as evidenced by the observations that the accumulation of ubiquitin-conjugated proteins induced by harmful ischemic injury was reversed in brain with delayed ischemic or iso ﬂ urane preconditioning (Liu et al. 2005b ; Zhang et al. 2010) . Proteasome inhibition with an aldehyde inhibitor attenuated the neuroprotective effect of both the acute and delayed ischemic preconditioning (Rehni et al. 2010 ) . These studies suggest that enhancing UPS-mediated ERAD may contribute to preconditioning-induced neuroprotection. However, promoting degradation of unfolded proteins is not the only proteasome-associated mechanism underlying preconditioning-induced protection. Preconditioning stimuli may trigger the rapid degradation of multiple proteins, such as pro-apoptotic proteins, by the UPS to establish acute ischemic tolerance. These UPS-associated mechanisms will be discussed in the proteasome section.

19.3.4 ER Ca2+ Release

In addition to folding and processing newly synthesized proteins, the ER also acts as the primary source of releasable intracellular Ca 2+ in neurons and plays an important role in the maintenance of intracellular Ca2+ homeostasis (Wei and Xie 2009) . An altered intracellular Ca2+ homeostasis may play a role in ischemic damage/protection. Although extreme increases in intracellular Ca 2+ concentrations 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 401 play a role in neurodegenerative processes associated with brain ischemia (Mattson 2007 ) , moderate increases in intracellular Ca2+ concentrations (increases of 50Ð200 nM) by Ca2+ -selective ionophore treatment may activate cell survival programs and promote resistance to hypoxia or ischemia in neurons (Bickler and Fahlman 2004 ) . Similar moderate increase in intracellular Ca2+ concentrations was observed in hippocampal neurons with hypoxic preconditioning and appeared to be mostly derived from IP3 receptor-mediated Ca2+ release from the ER (Bickler et al. 2009 ) . This moderate Ca2+ release may stimulate multiple pro-survival signaling pathways including ERK1/2, Akt, and CREB (Bickler et al. 2009 ) and promote cell survival responses mediated by the Bcl-2 family of proteins (White et al. 2005) . Although we cannot rule out Ca 2+ from other sources, e.g., NMDA receptor-mediated exogenous Ca2+ in ﬂ ux (Wang et al. 2006 ) to induce ischemic tolerance, a moderate increase of intracellular Ca2+ from the ER may be one of the mechanisms of preconditioning-induced neuroprotection against ischemia.

19.4 Lysosome

19.4.1 Autophagy and Its Role in Preconditioning

Autophagy is a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. The selective autophagy for degradation of mitochondria, mitophagy, was discussed in the section on mitochondria. Autophagy plays a key role in cellular homeostasis between biosynthesis and catabolism. The autophagic process can be rapidly activated in neurons after exposure to various stimuli including hypoxia, ischemia, and excitotoxicity (Balduini et al. 2009 ) . Massive autophagy may result in the total collapse of cellular functions through excessive self-digestion and degradation of cellular constitutions and is therefore implicated in cell death. Inhibition of autophagy can attenuate neuronal cell death in in vitro and in vivo models (Sadasivan et al. 2006 ; He et al. 2008 ; Lai et al. 2008; Park et al. 2009) . On the other hand, autophagy can also work in conjunction with the proteasome to degrade misfolded proteins and reduce cell stress (Hara et al. 2006 ; Komatsu et al. 2006a, b ; Carloni et al. 2008 ) . Autophagy has also been implicated to play an important role in preconditioning-afforded neuroprotection. Ischemic preconditioning increased generation of autophagosomes, as well as autophagy-related proteins, either in the in vitro PC12 cell model (Park et al. 2009 ) or in the rat model of ischemic tolerance (Sheng et al. 2010) . Inhibiting the formation of autophagosomes attenuated the neuroprotective effect induced by preconditioning (Park et al. 2009 ; Sheng et al. 2010 ) . Furthermore, inducing autophagy by rapamycin mimicked the protective effect of ischemic (Sheng et al. 2010 ) or hyperbaric oxygen preconditioning (Yan et al. 2011 ) and elicited a neuroprotective effect against ischemic injury in the brain. These data support the pro-survival function of autophagy in the context of preconditioning. 402 P. Li et al.

19.4.2 How Does Preconditioning-Induced Autophagy Provide Protection in the Brain?

To address the question of how autophagy mediates the neuroprotection provided by preconditioning, several studies have implicated the increase of LC3-II and Beclin-1, two pro-autophagic proteins, following preconditioning (Yan et al. 2011 ) . It was proposed in an MPP + -induced neurotoxic model that autophagy induced by hypoxic preconditioning appears to be mediated by oxidative stress, which increased the nuclear HIF-1 level (Wu et al. 2010) . In addition, the class III PI3K pathway was also indicated to regulate the autophagosome formation. Beclin-1, also known as autophagy-related gene (Atg) 6, and its binding partner class III PI3K, also named Vps34, are required for the initiation of the formation of the autophagosome (Furuya et al. 2005 ) . Other mechanisms beyond HIF-1 and the PI3K pathway underlying the protective effect of preconditioning-induced autophagy still require further study.

19.5 Proteasome

19.5.1 What Are the Proteasome and the Ubiquitin-Proteasome System (UPS)?

The UPS plays a key role in various cellular processes including protein turnover under normal and pathologic conditions. The UPS also acts as a protein quality controller by removal of damaged, oxidized, misfolded, or mislocalized proteins, which requires participation of heat shock proteins (Ciechanover 2006 ; Goldberg 2007 ; Lanneau et al. 2010 ) . Proteasomes are enormous barrel-shaped protein complexes that are the major non-lysosomal structures for intracellular degradation of proteins by UPS proteolysis. In order to be targeted to this complex, a protein requires polyubiquitin modiﬁ cation by sequential addition of ubiquitins to the lysine residue, resulting in a high afﬁ nity for the proteasome and subsequent processing into peptides, which are then further processed by peptidases into amino acids for recycling by the cell. Substrates for the proteasome are usually less bulky and have shorter half-lives than the autophagic system, but there is cross talk between the two cellular incinerators, and they are not fully independent systems of proteolysis (Korolchuk et al. 2009 ) . The most common form of the proteasome is known as the 26S proteasome complex, which is about 2,000 kDa in molecular mass and contains one 20S core particle structure and two 19S regulatory caps. The 20S core exhibits little protease activity on its own and requires the 19S caps for full catalytic function (Pickart and Cohen 2004) . The 19S cap proteins stimulate proteolytic activity by regulating entry of the protein substrate into the catalytic chamber (Pickart and Cohen 2004 ) . 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 403

As well as unfolding the proteins, the 19S caps remove the ubiquitin chain prior to degradation of the target proteins. The impaired UPS is implicated in various neurological disorders. Ubiquitin- rich inclusions are a common feature of certain neurodegenerative diseases, such as Alzheimer’s disease (Oddo 2008 ) and Parkinson’s disease (Lim 2007 ) . Protein aggregates formed by polyubiquitinated proteins following global ischemia have also been reported (Liu et al. 2005b ) . Harmful ischemia can result in cap disassem- bly and thus lead to a reduction in proteasome function, which may contribute to cell stress following ischemia and amplify the neuronal damage in the acute phase (Meller 2009) . However, the UPS function may also be activated in secondary brain injury after ischemia. Proteasome inhibitors were reported to reduce the brain infarction following ischemia in animal models via reducing neuroin ﬂ ammation (Meller 2009 ) .

19.5.2 The Contribution of UPS to Ischemic Tolerance

Studies have suggested beneﬁ cial effects of the UPS in the development of ischemic tolerance. Most direct evidence comes from the observation that proteasome inhibition with an aldehyde inhibitor attenuated the neuroprotective effect of both the acute and delayed ischemic preconditioning (Rehni et al. 2010 ) . As we discussed in the ER section, the accumulation of ubiquitin-conjugated proteins in the brain induced by ischemic injury was reversed by ischemic or iso ﬂ urane preconditioning (Liu et al. 2005a; Zhang et al. 2010) , suggesting that preconditioning stimuli may enhance proteasome activity and thereby promote the UPR to deal with ischemia- induced protein aggregation and ER stress. In addition, a preconditioning-induced increase in heat shock protein expression may also reduce protein aggregation in neurons following ischemia (Ouyang et al. 2005 ) .

19.5.3 Degradation of Speci ﬁ ed Proteins by the UPS Underlies the Neuroprotection of Rapid Ischemic Tolerance

Recently, studies revealed that the rapid degradation of selective proteins by the UPS underlies the mechanism of rapid ischemic tolerance. The ﬁ rst protein shown to be degraded by the proteasome in models of rapid ischemic tolerance was Bim, a pro-death member belonging to the BH3 only subgroup of the Bcl-2 family (O’Connor et al. 1998 ) . It was shown that preconditioning with ischemia (Meller et al. 2006 ) or adenosine (Ordonez et al. 2010 ) resulted in the rapid degradation of Bim by the proteasome and that this renders neurons protected against harmful ischemia. The preconditioning-stimulated Bim degradation appears to be dependent on activation of the ERK1/2 pathway (Ordonez et al. 2010 ) . The actin-binding 404 P. Li et al. protein has been identiﬁ ed as another target in the model of rapid ischemic tolerance (Meller et al. 2008 ) . Following ischemic preconditioning, there is proteasome- dependent degradation of two actin-binding proteins: fascin and myristoylated, alanine-rich C-kinase substrate (MARCKS). The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density, leading to a reduced NMDA- mediated electrophysiological response and rendering neurons more resistant to NMDA excitotoxicity (Meller et al. 2008 ) . These data suggest the critical role of the UPS in mediating rapid ischemic brain tolerance.

19.6 Golgi Apparatus

19.6.1 The Golgi Apparatus (GA) and Its Physiological Function

The Golgi apparatus is composed of stacks of membrane-coated smooth cisternae, which are responsible for modifying, sorting, and packaging macromolecules, particularly for cell secretion. The cisternae stack has four functional regions: the cis- Golgi, medial-Golgi, endo-Golgi, and trans-Golgi network. The GA contains a great number of vesicles that are used to transport molecules. Vesicles from the ER fuse with the cis-Golgi network and travel through the stacks to the trans-Golgi network, where they are sent to their destination such as the cellular membrane for protein secretion. Each cisterna contains special Golgi enzymes, which may be different depending on the functional region, to selectively modify cargo proteins that travel through it, as well as proteins involved in vesicle formation and protein sorting (Munro 1998 ) . These modi fi cations include glycosylation and phosphorylation and may provide a signal sequence which determines the fi nal destination of the cargo protein. Apart from the role in posttranslational modi fi cation and sorting, the GA also serves as an intracellular Ca2+ store and is involved in Ca2+ -mediated signaling (Pizzo et al. 2010 ) .

19.6.2 Ischemic Preconditioning Modi ﬁ es the GA-Associated Ca2+ Signaling to Tolerate the Subsequent Lethal Injury

The role of GA in ischemic tolerance is rarely studied. Most recently, ischemic preconditioning was shown to have protective effect on the GA function via maintaining its Ca2+ homeostasis (Lehotsky et al. 2009a) . As a part of secretory pathways, the GA in neural cells represents a dynamic Ca2+ store and is involved in Ca2+ signaling (Michelangeli et al. 2005 ) . Ca 2+ accumulates within the GA lumen in an ATP- dependent manner catalyzed by the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and the secretory pathway Ca2+ ATPase1 (SPCA1) (Pizzo et al. 2010 ) . Ca 2+ was then released to the cytoplasm in response to the activation of the IP3 receptor 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 405

Fig. 19.3 Preconditioning modi ﬁ es the Ca2+ signaling in endoplasmic reticulum and Golgi apparatus to tolerate the subsequent lethal injury. Preconditioning induces moderate increase in intracellular Ca 2+ , which is mostly derived from IP3 receptor-mediated Ca 2+ release from the endoplasmic reticulum (ER ). In Golgi apparatus, preconditioning increases the activity of the secretory pathway Ca2+ ATPase1 ( SPCA1), which is responsible for Ca2+ release from the GA to the cytoplasm through the activation of the IP3 receptors on the GA. This moderate Ca 2+ release may stimulate multiple pro-survival signaling pathways including ERK1/2, Akt, and CREB and lead to neuroprotection

on the GA (Pizzo et al. 2010 ) . The SPCA1 exhibits a particularly high activity within the brain (Wootton et al. 2004 ) and plays a potential role in normal neural development, neural migration, and morphogenesis (Sepulveda et al. 2007, 2008) . Ischemic/reperfusion injury causes signiﬁ cant drops in the SPCA1 activity although it induces its gene expression in later reperfusion periods (Lehotsky et al. 2009a ; Pavlikova et al. 2009) . Ischemic preconditioning initiates an earlier GA response to the injury by signiﬁ cant elevation of SPCA1 expression and protects SPCA1 activity (Lehotsky et al. 2009a ; Pavlikova et al. 2009) . Therefore, ischemic preconditioning appears to modify the GA-associated Ca 2+ signaling in response to lethal ischemic injury. These studies shed light on the role of GA in ischemic tolerance, but further studies are still required to clarify the underlying mechanisms (Fig. 19.3 ).

19.7 Nuclear Membrane

19.7.1 Peroxisome Proliferator-Activated Receptors (PPARs)

Peroxisome proliferator-activated receptors are a group of nuclear receptor proteins that function as ligand-activated transcription factors regulating gene expression. There are three isoforms of PPARs: a, b/ d, and g, encoded by distinct genes. 406 P. Li et al.

All PPAR isoforms heterodimerize with the retinoid X receptor (RXR), another nuclear receptor, to form a transcriptionally competent complex that binds to speciﬁ c DNA elements within promoter regions of target genes (Di Paola and Cuzzocrea 2007 ) . Once activated by ligand binding, this heterodimer recruits transcription coactivators and regulates the expression of genes involved in lipid or glucose metabolism (Berger et al. 2005) . Importantly, the activation of PPAR- a and PPAR-g also shows anti-in ﬂ ammatory and antioxidant effects in peripheral organs beyond their metabolic effects (Bordet et al. 2006 ) .

19.7.2 Activation of PPARs in Cerebral Ischemic Injury

Recently, the activation of PPARs, especially of PPAR-a and PPAR-g , has been shown to induce neuroprotection in various acute and chronic CNS disorders including ischemic stroke (Bordet et al. 2006; Heneka and Landreth 2007) . This is evidenced by the observation that pretreatment with either PPAR-a or PPAR-g agonists could induce both preventative and acute neuroprotection involving both cerebral and vascular mechanisms (Bordet et al. 2006 ) . For example, pretreatment with the selective PPAR- a agonist WY14643 suppressed oxidative stress and the expression of infl ammatory mediators such as iNOS and ICAM-1 induced by transient cerebral ischemia/reperfusion (Collino et al. 2006a, b) . Also, pretreatment with the PPAR-g agonists protects the brain against ischemic/reperfusion injury by inhibiting oxidative stress and excessive infl ammation (Collino et al. 2006a, b ) . This PPAR-g -mediated anti-infl ammatory effect was shown to be associated with its suppressive effect on COX-2 expression (Collino et al. 2006a, b; Zhao et al. 2006) . Together, these studies suggest that PPARs may serve as potential pharmacological targets for preconditioning as well as the treatment of cerebral ischemia/ reperfusion injury.

19.7.3 How Does Preconditioning Induce the Activation of PPARs?

The activation of PPAR-g is involved in the cardioprotective effects of multiple preconditioning stimuli (Sivarajah et al. 2005; Lotz et al. 2011a, b ) . For example, inhibition of PPAR-g with its antagonist signi ﬁ cantly reversed the reduction in myocardial infarct size elicited by remote ischemic preconditioning (Lotz et al. 2011b ) . Increases in both iNOS and NO appear to contribute to the PPAR-g -afforded cardioprotection (Lotz et al. 2011a, b ) . Similarly, both PPAR- a and PPAR-d play a role in remote ischemic preconditioning against myocardial infarction (Li et al. 2011a ; Lotz et al. 2011b ) . 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 407

The role of PPARs in brain ischemic tolerance, however, is not well studied. One study found that ischemic preconditioning caused a signi ﬁ cant increase in nuclear PPAR-g transcriptional activity in both neurons and astrocytes and a PPAR- g antagonist markedly inhibited ischemic preconditioning-induced brain tolerance (Romera et al. 2007 ) . Moreover, this study identiﬁ ed the GLT1/EAAT2 glutamate transporter as a target gene of PPAR- g leading to neuroprotection by increasing glutamate uptake (Romera et al. 2007 ) .

19.8 Nucleus

19.8.1 Genomic Reprogramming

It is well accepted that preconditioning stimuli have the ability to alter the brain’s transcriptional response to subsequent ischemic injury via multiple signaling pathways, effectively shifting the overall outcome from cell death to cell survival (Stenzel-Poore et al. 2007 ; Dirnagl et al. 2009 ) . This ischemic tolerance-associated alteration in gene expression was fi rst apparent by gene expression profi ling of ischemic preconditioned animals (Stenzel-Poore et al. 2003 ) . Microarray analysis revealed that exposure to a brief period of ischemic insult prior to harmful ischemia resulted in transcriptional suppression of a number of genes that control metabolism, cell cycle regulation, immune response modulation, ion-channel activity, and blood coagulation (Stenzel-Poore et al. 2003, 2004 ) , indicating a complete genomic reprogramming (Stenzel-Poore et al. 2003 ) . The signi fi cant modifi cation of genomic response to ischemia was also observed in the brains preconditioned by other paradigms such as hypoxia (Bernaudin et al. 2002b ; Tang et al. 2006 ) or LPS (Stenzel-Poore et al. 2007 ) . Although the patterns of gene regulation are similar, the identity of the genes regulated is unique to different preconditioning stimuli (Stenzel-Poore et al. 2007 ) . For example, hypoxic preconditioning increases the expression of multiple HIF-1-dependent genes such as VEGF and GLUT-1 (Bernaudin et al. 2002b; Tang et al. 2006 ) . In contrast, LPS preconditioning causes a robust neuroprotective cytokine response as well as a marked absence of deleterious in fl ammatory mediators to achieve protection (Stenzel-Poore et al. 2007 ) (Fig. 19.4 ).

19.8.2 Epigenetics

Gene regulation in ischemic tolerance may also depend on epigenetic modi ﬁ cation such as DNA methylation and histone acetylation. Suppression of either DNA methylation (Endres et al. 2000 ) or histone deacetylation has neuroprotective effects 408 P. Li et al.

Fig. 19.4 Preconditioning-triggered neuroprotective signaling events in the nucleus. Preconditioning stimuli activate several transcriptional factors, including NF-κ B, HIF-1, and CREB in the nucleus, and lead to neuroprotection. (1) Preconditioning-induced NF-κ B activation is mediated through multiple receptors including toll-like receptor 4 (TLR4 ), NMDA receptor (NMDA-R ), EPO receptor (EPO-R ), and TNF-α receptor (TNF- a R ). However, the activation of NF-κ B could lead to both detrimental and beneﬁ cial effects on the ischemic injury. The upregulated expression of A1AR has been shown to be neuroprotective. (2) PI3K and ERK1/2 signaling pathways mediate the preconditioning-induced rise in HIF-1, which upregulates the gene expression of EPO, VEGF, and iNOS and leads to neuroprotection. (3) Preconditioning also activates the PKA, MEK, and nNOS signaling cascades; increases Ca2+ in ﬂ ux through CaMK; and activates VEGF-R2, all of which induce the activation of CREB. CREB regulates the gene expression of neuroprotective factors such as Bcl-2, BDNF, and PGC-1 α. (4) Preconditioning stimuli may also exert its neuroprotection through microRNA regulation. MicroRNA-200 ( miR-200) increases following preconditioning and induces HIF-1 expression. In contrast, MicroRNA-132 ( miR-132) is downregulated in response to preconditioning and leads to subsequent increase of a neuroprotective molecule MeCP2. (5) On the nuclear membrane, PPAR-γ can be activated through the iNOS pathway following preconditioning. The activated PPAR-γ upregulates the expression of GLT-EAAT2, which in turn increases the glutamate uptake and contributes to neuroprotection. In addition, preconditioning decreases the DNA damage events by enhancing the DNA repair pathways, such as BER and NHEJ

in experimental models of stroke (Yildirim et al. 2008 ) . These epigenetic modiﬁ cations seem to contribute to preconditioning-induced genomic reprogramming via facilitating widespread regulation of transcription. Further studies on epigenetic modiﬁ cation may identify essential mechanisms underlying genomic reprogramming by preconditioning. 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 409

19.8.3 Transcription Factors

19.8.3.1 NF-k B

Overview of NF-k B

NF- k B is an inducible transcription factor that plays a pivotal role in neural development, learning and memory, and synaptic plasticity in the normal CNS. The activation of NF-k B was reported to upregulate genes encoding proteins involved in both cell survival and cell death. It also acts as a major mediator of proin fl ammatory responses in glial cells through regulating iNOS, proin fl ammatory cytokines, and cell adhesion molecules. NF- kB is ubiquitously expressed and consists of preformed DNA-binding dimers. There are fi ve different NF-k B subunits in mammals. The most common subunits expressed in neurons are p50 and p65, forming homo- and heterodimers in various combinations. In unstimulated cells, NF-k B remains in the cytoplasm by interacting with inhibitory I- kB proteins. Upon stimulation, I- kB is phosphorylated, polyubiquitinated, and then degraded by the proteasome. After degradation of I-k B, NF- kB is released and translocated into the nucleus to stimulate gene transcription. A large array of stimuli can activate NF-k B in both neurons and glial cells.

The Dual Nature of NF-k B in Brain Preconditioning

NF-k B is activated in neurons and glial cells in brain disorders such as stroke. The activation of NF-k B could be either detrimental or bene fi cial to the ischemic cerebral injury. Mice de fi cient in the p50 subunit of NF-k B showed a signi fi cant reduction in infarct size in both transient and permanent stroke models, indicating a detrimental role of NF-k B in cerebral ischemia (Schneider et al. 1999 ; Nurmi et al. 2004 ) . In addition, NF- kB-induced proinfl ammatory responses mediate many of the deleterious effects of cerebral ischemia (Wang et al. 2007a ) . Furthermore, preconditioning with sevo fl urane was shown to confer neuroprotection via suppressing the NF-k B-mediated in fl ammatory response after focal ischemic brain injury (Wang et al. 2011 ) . However, there are three lines of evidence that the activation of NF-k B is also involved in the development of brain preconditioning, indicating its benefi cial role in brain tolerance. First, ischemic or chemical preconditioning induced a rapid and transient activation of NF-k B by increasing its DNA-binding activity and nuclear translocation in vivo and in vitro (Blondeau et al. 2001 ; Ravati et al. 2001 ; Jiang et al. 2003 ) . Second, hypoxic preconditioning was shown not only to increase the phosphorylation of NF-k B (Bigdeli and Khoshbaten 2008 ) but also to persistently upregulate its expression (Rybnikova et al. 2008 ) . Finally, inhibition of NF-k B abolished preconditioning-induced delayed neuroprotection, suggesting that NF-k B activation is required for the signal transduction that underlies the development of brain tolerance (Blondeau et al. 2001 ; Ravati et al. 2001 ) . 410 P. Li et al.

In contrast to adult brain, NF-k B activation by LPS preconditioning may sensitize neonatal brain to hypoxic injury (Wang et al. 2007b ) . Neonatal hyperbilirubinemia, due to accumulation of unconjugated bilirubin (UCB), is a common pathological condition in newborns. Hypoxic or ischemic preconditioning augments the UCB- induced NF- kB-mediated inﬂ ammatory response in astrocytes (Falcao et al. 2007 ) . In ﬂ ammation contributes considerably to the pathogenesis of perinatal brain injury, and NF-k B activation by preconditioning may therefore lead to an increased risk of brain damage during the perinatal period.

Possible Mediators and Pathways by Which They Regulate Preconditioning-Induced NF-k B Activation

There are several possible mediators of preconditioning-induced NF-k B activation, such as TNF-a , EPO, TLR4, and adenosine A1 receptors. Hypoxic or ischemic preconditioning resulted in an upregulation of TNF-a converting enzyme (TACE), serum TNF-a level, and neuronal TNF receptor 1 (TNFR1) (Pradillo et al. 2005 ; Bigdeli and Khoshbaten 2008 ) , whereas TACE inhibitor BB1101 or TNFR1 antisense blocked preconditioning-induced NF-k B as well as the protective effect (Pradillo et al. 2005) . Signals from EPO and NMDA receptors were also shown to trigger the activation of NF-k B via different pathways. EPO-induced NF-k B activation was mediated by Jak2, which led to phosphorylation of the inhibitor I-k B (Digicaylioglu and Lipton 2001) , whereas the NMDA receptor activated NF- k B- dependent signaling through BDNF and its cognate receptor TrkB (Jiang et al. 2003 ) . In addition, ischemic preconditioning-induced NF-k B activation was prevented in TLR4-de fi cient mice, suggesting that the TLR4 signal is likely to participate in the regulation of NF-k B activation (Pradillo et al. 2009 ) . The adenosine A1 receptor (A1AR) was also identifi ed as a neuroprotective target of NF-k B. The NF- k B p50 subunit-de fi cient mice expressed reduced levels of A1AR and blocked A1AR induction by LPS treatment (Jhaveri et al. 2007 ) . Finally, preconditioning- induced transient NF-k B activation may lead to the direct transcriptional activation of inhibitory protein I-k B and thus inhibit the activation of more NF-k B after secondary injury (Blondeau et al. 2001 ) . These data suggest that NF-k B lies at the intersection of cell death and survival pathways in cerebral preconditioning, and that its role and precise mechanism need to be further evaluated.

19.8.3.2 Hypoxia-Inducible Factor-1 (HIF-1)

The Transcription Factor HIF-1

HIF-1 is a transcription factor that regulates the expression of hundreds of genes in response to reduced oxygen availability. A 120-kDa O 2 -regulated HIF-1a and a 91- to 94-kDa constitutively expressed HIF-1 b subunit constitute the heterodimeric HIF-1. 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 411

Under normoxic conditions, HIF-1 a is subjected to hydroxylation by prolyl-4 hydroxylase domain (PHD) family proteins which generate the binding site for the component of the ubiquitin ligase complex. This process is fully dependent on oxygen. Polyubiquitination targets HIF-1a for proteasomal degradation. In low-oxygen conditions, however, the rate of hydroxylation and degradation declines. The non- hydroxylated proteins then accumulate, dimerize with the HIF-1 b subunit, and regulate the expression of a number of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism (Semenza 2011 ) .

Preconditioning-Induced HIF-1 Activation Is Neuroprotective

HIF-1 activation has been established to be neuroprotective, and HIF-1-mediated responses are involved in preconditioning. It was demonstrated that hypoxic preconditioning induces a rapid increase both in HIF-1a protein (Liu and Alkayed 2005 ) and HIF-1 a and HIF-1 b mRNA (Bergeron et al. 2000; Shao et al. 2005 ) . The increased HIF-1a protein led to an increased HIF-1 nuclear translocation (Bernaudin et al. 2002a ) and DNA-binding activity (Ruscher et al. 2002 ; Prass et al. 2003 ; Shao et al. 2005 ) . The enhanced activity of HIF-1 induced an increased expression of target genes, including EPO and VEGF (Bernaudin et al. 2002a ; Prass et al. 2003 ; Shao et al. 2005 ) . In primary cultured astrocytes, hypoxic preconditioning also could markedly increase the activity of HIF-1 (Ruscher et al. 2002 ) , which not only mediates astrocyte tolerance against oxygen-glucose deprivation (Liu and Alkayed 2005 ) or oxidative injury (Chu et al. 2010 ) but also provides paracrine neuroprotective effect on neurons (Ruscher et al. 2002) . Apart from hypoxic preconditioning, increased HIF-1 activity was also shown to be involved in the neuroprotection induced by hyperbaric oxygen preconditioning (Gu et al. 2008 ; Peng et al. 2008 ) and various pharmacological types of preconditioning (Hua et al. 2003; Mu et al. 2005; Li et al. 2008a, b; Limatola et al. 2010 ) . These studies reveal that HIF-1 mediates both glial and neuronal responses to preconditioning. However, studies with neuron-speci ﬁ c HIF-1a knockout mice showed contradictory results. Although HIF-1 a inactivation in calcium/calmodulin-dependent protein kinase (CaMK) II a -positive neurons signi ﬁ cantly increased the ischemia-induced brain injury in mice, it did not affect the development of ischemic tolerance in response to hypoxic preconditioning (Baranova et al. 2007 ) . Several in vitro studies indicated the possibility that HIF-1 activation in CaMKIIa -negative cells such as glia contributes to the hypoxic preconditioning-induced neuroprotection instead. In sum, further in vivo research to delve into the neuronal versus glial roles of HIF-1 in preconditioning is warranted.

How Does Preconditioning Regulate HIF-1 Activation?

It is established that hypoxic or ischemic preconditioning upregulate HIF-1 a via inhibiting its degradation. Hypoxic preconditioning in neonatal rat brain induced an 412 P. Li et al. upregulation of PHD-2, the predominant PHD isoform responsible for regulating HIF-1 a after hypoxia, following the increase in HIF-1a protein level (Jones et al. 2006 ) . Such hypoxia-induced modulation of PHD expression was almost absent in an HIF-1-defi cient cell line (Hofbauer et al. 2003 ) , indicating that HIF-1 may be regulated by its own degradation mechanism. The enhanced HIF-1 activity after preconditioning was accompanied by the increased expression of well-known target genes, including those for EPO, VEGF, and iNOS (Bernaudin et al. 2002a ; Mu et al. 2003 ; Prass et al. 2003 ; Li et al. 2008a, b ; Chu et al. 2010 ) , all of which play important roles in the establishment of tolerance. A recent study showed that the administration of an anti-HIF-1 compound YC-1 could diminish the accumulation of VEGF as well as the protective effect (Chu et al. 2010 ) . In astrocytes, cytochrome P450 2C11, an arachidonic acid epoxygenase, was identi fi ed as a target gene of HIF-1 because there is a speci fi c and direct interaction between P450 2C11 and HIF-1 a. The increased expression of P450 2C11 was preceded by an increase in HIF-1 a protein, and HIF-1-linked upregulation of P450 2C11 may contribute to the induction of astrocytic tolerance (Liu and Alkayed 2005 ) . Studies showed that some signaling pathways may also contribute to the preconditioning-mediated induction of HIF-1 a. Inhibition of the ERK1/2 pathway could block the thrombin (Hua et al. 2003 ) or iso fl urane (Li et al. 2008a ) preconditioning-induced HIF-1a upregulation. In addition, a PI3K inhibitor signi fi cantly reduced the enhanced expression of HIF- 1 a induced by ginkgolide B and ischemic preconditioning (Wu et al. 2009 ) . These studies demonstrate that PI3K and ERK1/2 signaling pathways mediate the preconditioning-induced rise in HIF-1.

19.8.3.3 cAMP Response Element-Binding Protein (CREB)

Transcription Factor CREB and Its Association with Preconditioning

CREB is a transcription factor which can bind to certain DNA sequences called cAMP response elements. It is constitutively and ubiquitously expressed in neurons. In response to various extracellular stimuli, CREB is activated by phosphorylation at speciﬁ c protein residues such as Ser133 and Ser144. In neurons, CREB targets certain neuroprotective genes, such as Bcl-2 and BDNF. It is established that CREB acts as an important mediator of synaptic plasticity, neuronal growth, and survival in both the developing and mature nervous system (Rami et al. 2008 ) . CREB activation is also implicated to be essential in brain preconditioning, which is supported by the following. The level of CREB phosphorylation was increased after sublethal transient global ischemia (Mabuchi et al. 2001 ; Hara et al. 2003 ) , preconditioning ischemia by middle cerebral artery occlusion (Meller et al. 2005 ) or hypoxic exposure (Gao et al. 2006 ; Churilova et al. 2010 ) . The phosphorylation of CREB was consistently induced in cultured neurons after exposure to glutamate (Mabuchi et al. 2001; Lin et al. 2008) or oxygen-glucose deprivation preconditioning (Meller et al. 2005 ; Lee et al. 2009 ) . Phosphorylated CREB appeared to translocate 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 413 to the nucleus (Gao et al. 2006) and triggers the activation of cAMP response element (CRE)-mediated gene transcription (Mabuchi et al. 2001 ) . The administration of CRE-decoy oligonucleotides decreased CREB-DNA-binding activity, suppressed upregulation of CREB target genes, and abolished the neuroprotective response by preconditioning both in vitro and in vivo (Hara et al. 2003 ; Meller et al. 2005 ; Lin et al. 2008 ) . A dominant negative CREB mutant inhibited both the preconditioning-induced CRE transcriptional activity as well as neuroprotection (Terasaki et al. 2010 ) , suggesting that normal CREB activation is required for the acquisition of brain tolerance.

The Signaling Pathways Involved in the Preconditioning-Induced Activation of CREB

In response to preconditioning, CREB activation is mediated by several possible signaling pathways. Treatment with low doses of NMDA stimulated CREB- dependent gene expression (Soriano et al. 2006 ) , whereas an NMDA receptor antagonist MK-801 inhibited CREB phosphorylation induced by preconditioning (Mabuchi et al. 2001 ) . Recent studies reported that NR2A subtype but not NR2B subtype-containing NMDA receptors contribute to the preconditioning-induced CREB activation and subsequent upregulation of CREB target genes (Chen et al. 2008 ; Terasaki et al. 2010 ) . Activation of NMDA receptors led to Ca2+ in ﬂ ux and subsequent phosphorylation of CREB through CaMK (Mabuchi et al. 2001 ) . Removal of extracellular Ca2+ or inhibition of CaMK reduced preconditioning- induced CREB activation both in cultured neurons and in whole brain (Mabuchi et al. 2001 ; Meller et al. 2005 ) . Apart from CaMK, other kinases such as protein kinase A (PKA) and MEK were also shown to be responsible for ischemic preconditioning-induced CREB activation (Meller et al. 2005 ) . Recently, it was reported that VEGF-A/VEGFR-2 signaling contributes to the CREB activation after ischemic preconditioning both in neurons and endothelial cells (Lee et al. 2009 ) . In addition, nNOS was shown to be involved in the hypoxic preconditioning-induced CREB phosphorylation because CREB was not activated in nNOS knockout mice (Gutsaeva et al. 2008 ) . Some of the well-known neuroprotective targets of CREB include Bcl-2 and BDNF. The preconditioning-induced upregulation of these CREB target genes was correlated with elevation of CREB phosphorylation (Mabuchi et al. 2001 ; Meller et al. 2005 ; Chen et al. 2008 ; Lin et al. 2008 ; Terasaki et al. 2010 ) . Administration of CRE-decoy oligonucleotide signi ﬁ cantly blocked the preconditioning-induced rise in Bcl-2 expression (Mabuchi et al. 2001 ; Meller et al. 2005 ; Lin et al. 2008 ) . In addition, CREB is an important transcription factor regulating the expression of nuclear coactivator PGC-1a , which has an impact on nuclear-mitochondrial communication. It has also been suggested that hypoxic preconditioning-stimulated CREB activation contributes to mitochondrial biogenesis via regulating PGC-1a expression (Gutsaeva et al. 2008 ) . 414 P. Li et al.

19.8.4 MicroRNA

19.8.4.1 The Gene Expression Regulator: MicroRNA (miRNA) and Its Role in Cerebral Ischemia

MiRNAs are a recently discovered family of noncoding short RNA molecules that negatively regulate posttranscriptional gene expression by binding to complemen- tary mRNAs and repressing translation in eukaryotes. It is evident that miRNAs are able to regulate the expression of at least one-third of human genes (Lewis et al. 2005 ) , and play critical roles in the development and function of normal brains (Saugstad 2010 ) . Several brain-speci ﬁ c miRNAs have been discovered in mouse and human differentiating neurons, suggesting that these miRNAs serve to establish and maintain normal neuronal protein expression pro ﬁ les. Many studies have implicated miRNAs in ischemic and traumatic brain injuries, as well as various neurodegenerative disorders (Saugstad 2010 ) . Just as one example, it was shown that the cerebral miRNA expression pattern was altered after focal cerebral ischemia (Jeyaseelan et al. 2008 ; Dharap et al. 2009 ) .

19.8.4.2 Preconditioning-Induced miRNA Regulation May Contribute to Neuroprotection

Recent studies reported that miRNAs mediate endogenous neuroprotection afforded by cerebral ischemic preconditioning (Dharap and Vemuganti 2010 ; Lee et al. 2010 ; Lusardi et al. 2010) . The levels of miRNAs were altered quickly following a transient middle cerebral artery occlusion in a rat model of ischemic preconditioning (Dharap and Vemuganti 2010 ) . Some of these miRNAs remained altered up to 3 days after preconditioning ischemia. The regulation of miRNA by preconditioning can decrease or increase protein levels depending on whether the miRNA is raised or lowered. Using a mouse model of ischemic preconditioning, Lee et al. found that, from a total of 360 miRNAs, two miRNA families (miR-200 and miR-182) were upregulated selectively at 3 h after ischemic preconditioning (Lee et al. 2010 ) . Transfections of these two miRNA family members were shown to protect mouse neuroblast cells against oxygen-glucose deprivation damage by upregulating HIF-1a . PHD2 is one of the targets of the miR- 200 family and plays a critical role in degrading HIF-1 a . Therefore, ischemic preconditioning may induce HIF-1a upregulation through miR-200 family-mediated translational repression of PHD2 (Lee et al. 2010 ) . In addition to increases in some miRNAs, there are also decreases in other miRNA levels with preconditioning. For example, the global transcriptional regulator, methyl CpG-binding protein 2 (MeCP2), has been identifi ed as the most prominent target of the fall in miRNAs in ischemic preconditioned cortex (Lusardi et al. 2010 ) . This was supported by the observation that multiple miRNAs are downregulated by ischemic preconditioning target MeCP2 in a rat model (Saugstad 2010 ) . Among the 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 415 downregulated miRNAs, miRNA-132 has been demonstrated to negatively regulate the expression of MeCP2. An ischemic preconditioning-induced decrease in miR- 132 is consistent with the fi nding that there is a rapid increase in MeCP2 protein in preconditioned mouse cortex, but with no correlating changes in its mRNA expression (Lusardi et al. 2010 ) . Since MeCP2 knockout mice showed increased susceptibility to ischemia, it was suggested that the specifi c miRNA/MeCP2 interaction is involved in the preconditioning-induced neuroprotection (Lusardi et al. 2010 ) .

19.8.5 DNA Damage and Repair

19.8.5.1 Oxidative DNA Damage in Ischemic Brain Injury and Preconditioned Brain

DNA damage is present in pathological conditions such as stroke and traumatic brain injury. Accumulating evidence suggests that cerebral ischemia-induced DNA damage plays a critical role in neuronal cell death. Endogenous oxidative DNA damage, in the form of base damage and strand breaks, can be detected in the ischemic brain during stages preceding the manifestations of cell death and is believed to trigger cell death via various intracellular signaling pathways. The common forms of oxidative DNA damage include base modi ﬁ cations, such as 8-oxodeoxyguanine (8-oxo-dG) and 8-hydroxy-2¢ -deoxyguanosine (8-OHdG), apurinic/ apyrimidinic (AP) site lesions, and single-strand breaks. Several of these DNA lesions occur in the ischemic brain and are therefore proposed as biomarkers of the oxidative stress induced by stroke (Li et al. 2011b ) . Sublethal ischemic preconditioning was shown to markedly attenuate the nuclear accumulation of oxidative DNA damage, including 8-OHdG, 8-oxo-dG, AP sites, and DNA strand breaks, induced by subsequent ischemia that was otherwise lethal (Baek et al. 2000 ; Li et al. 2006b, 2007 ) . Consequently, harmful DNA damage-responsive events, such as NAD depletion and p53 activation, were also reduced in preconditioned brains (Li et al. 2006b ) .

19.8.5.2 DNA Repair Pathways and Their Role in Preconditioning-Induced Neuroprotection

Most of the DNA damage induced by ROS in mammalian cells are repairable and can be reversed via different DNA repair pathways. In the brain, there are three major DNA repair mechanisms: DNA excision repair, including base excision repair (BER) and nucleotide excision repair; direct reversal of DNA damage; and the non- homogenous endpoint jointing (NHEJ) pathway (Li et al. 2011b ) . These DNA repair systems, particularly the base excision repair (BER), are endogenous defense mechanisms to combat oxidative DNA damage. Loss of DNA repair exacerbates neuronal loss after stroke, while upregulation of DNA repair capacity may be a critical mediator of endogenous neuroprotection. 416 P. Li et al.

BER is the primary DNA repair pathway with the ability to ﬁ x base lesions that arise due to oxidative damage. Ischemic preconditioning markedly increased the expression of essential BER enzymes including DNA polymerase- b, AP endonu- clease (Liebelt et al. 2010 ) , X-ray cross-complementing group (XRCC), and 8-oxoguanine DNA glycosylase (OGG1), as well as the BER-mediated DNA repair activity (Baek et al. 2000 ; Li et al. 2006b, 2007 ) . Ischemic preconditioning also induced the neuronal expression of Ku70, a multifunctional DNA repair protein involved in NHEJ-mediated double-strand break repair (Sugawara et al. 2001 ) . These results suggest that activation of DNA repair pathways may contribute to the ischemic preconditioning-induced neuroprotection by enhancing the endogenous repair of oxidative DNA damage.

19.9 Summary

A plethora of experimental evidence suggests that the sublethal insult provided by preconditioning elicits multiple protective signals originating from various subcellular organelles to help the cells tolerate the upcoming lethal insult. In the mito- + chondria, moderately increased ROS, the opening of mtK ATP channels, inhibition of the mitochondrial permeability transition pores, mitochondrial biogenesis, and mitophagy are all implicated in the protection triggered by preconditioning. In addition, upregulation of chaperone proteins, enhanced ERAD, and moderate Ca2+ release in the ER may stimulate multiple pro-survival signaling cascades and contribute to the neuroprotection induced by preconditioning. Degradation of selective misfolded and aggregated proteins by autophagy in the lysosome and by polyubiquitin tagging to the proteasome also are essential components of preconditioning- afforded neuroprotection. Preconditioning modi fi es Golgi apparatus-associated Ca 2+ signaling and activates the peroxisome proliferator-activated receptors on the nuclear membrane. In the nucleus, preconditioning increases DNA repair capacity, alters the expression of certain transcriptional factors, changes the epigenetic modi fi cations, and induces gene remodeling, all of which render the brain resistant to subsequent injury. From the manifold nature of these presumably simultaneous responses, it is clear that the preconditioned cell mounts an exquisitely coordinated series of molecular attacks against cellular injury in which many, if not all, organelles participate. More research will be required to elucidate the fi ne details of the mechanisms underlying preconditioning in the organelles discussed above. For example, how important is organelle turnover in the battle against cell death in terminally differentiated cells such as neurons? What role do mitochondrial fi ssion and fusion play in preconditioned neurons in the protection against ROS, DNA damage, protein misfolding, and ER stress? In what ways do organelles cross com- municate across their various locations within the cell and how is the cytoskeletal framework involved in this process? How do protein aggregations and insoluble inclusions in the brains of those elderly patients with neurodegenerative diseases clog these means of communication between subcellular organelles? Presumably, 19 How Do Subcellular Organelles Participate in Preconditioning-Conferred... 417 these elderly humans are also at a high risk for stroke due to their age and may have compromised preconditioning defenses as a result of their neurodegeneration. Further research will also determine how the organelles that were not discussed above, such as the nucleolus, ribosomes, cytoskeleton, and centriole, participate in the protection afforded by preconditioning. Such research will aid in rational drug design to target speci fi cally those subcellular organelles that play the most vital role in preconditioning. Alternatively, it may be possible to engage a broader network of organelles with cocktail drugs in order to mimic the full complexity and robustness of endogenous adaptation to sublethal ischemic injury in the mammalian brain.

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Srinivasan Narayanan* , Jake T. Neumann* , Kahlilia C. Morris-Blanco , Miguel A. Perez-Pinzon , and Hung Wen Lin

20.1 Introduction

Cerebral ischemia, most notably in the form of stroke, is the leading cause of morbidity and mortality resulting in long-term disability in the USA. Approximately 800,000 strokes occur each year and 87% of all strokes in the world are caused by embolism, thrombosis, or systemic hemorrhage/hypoperfusion, all of which cause cerebral ischemia (Roger et al. 2011 ) . The medical cost for the treatment of stroke in the USA was estimated to be $25 billion in 2007 (Roger et al. 2011 ) . Due to this great burden, a fundamental understanding of cerebral ischemia and the inciting cellular dysfunction is imperative for the development of new therapies to combat this growing epidemic. One main consequence of cerebral ischemia is the loss of cellular bioenergetics, which is the conversion of oxygen and glucose to adenosine triphosphate (ATP) through oxidative phosphorylation by mitochondria. Mitochondria are not the only source of ATP; however, no other cellular organelle produces ATP at a more efﬁ cient rate or quantity than mitochondria (Williamson et al. 1976 ) . The principal and initial detriment during cerebral ischemia is that ATP production becomes a limiting factor to maintain normal cellular processes (Busto and Ginsberg 1985 ) . Neurons in the

*Authors Srinivasan Narayanan and Jake T. Neumann both are equally contributed.

S. Narayanan • J. T. Neumann • K. C. Morris-Blanco • H. W. Lin (*) Cerebral Vascular Disease Research Laboratories, Department of Neurology, Leonard M. Miller School of Medicine , University of Miami , Miami , FL 33136 , USA e-mail: [email protected] M. A. Perez-Pinzon Cerebral Vascular Disease Research Laboratories, Neuroscience Program, Department of Neurology, Leonard M. Miller School of Medicine , University of Miami , Miami , FL 33136 , USA

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 429 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_20, © Springer Science+Business Media New York 2013 430 S. Narayanan et al. brain are dependent on proper mitochondrial function due to the high metabolic energy demand and decreased ability to respire anaerobically. Neurons require high levels of ATP to maintain axonal/dendritic outgrowth, ionic gradients, and produce, release, and reuptake neurotransmitters (Dagani and Erecinska 1987 ; Mattson and Kroemer 2003 ; Chan 2006 ) . During cerebral ischemia, the availability for oxygen and glucose becomes limited to the neuron, leading to diminished ATP production. Oxygen is depleted within 10 s, glucose is depleted 2–4 min, while ATP levels are completely exhausted within 5 min of cerebral ischemia (Richmond 1997 ) . In addition to cellular bioenergetics, mitochondria also play an intricate role in the initiation and propagation of the apoptotic intrinsic pathway (Green et al. 2011 ) . Apoptosis (programmed cell death) is propagated through cytosolic Ca 2+ in ﬂ ux by a variety of mechanisms including increased protease activation, reactive oxygen species (ROS) production, depolarization of the mitochondrial membrane potential, and opening of the mitochondrial permeability transition pore (MPTP) (Baumgartner et al. 2009; Loor et al. 2011) . Maintaining normal mitochondrial function could prove to be of great therapeutic value following many pathologic processes (i.e., cerebral ischemia), and could be accomplished through novel therapies such as ischemic preconditioning (IPC). IPC is the intrinsic neuroprotective response from a brief sublethal event, which increases the tolerance toward a future ischemic event. This chapter focuses on the role of IPC on brain mitochondria relating to neuronal apoptosis, physiology, and signaling pathways involved in IPC-mediated neuroprotection of the mitochondria following ischemia.

20.2 Mitochondria and Synaptic Dysfunction

Electrical information is transmitted from one neuron to another via the synapse. Under normal oxygen levels (normoxia), an action potential from the neuron is perpetuated from the axon to the dendrites; subsequently, neurotransmitters such as glutamate, acetylcholine, or g -aminobutyric acid (GABA) are released from presynaptic terminals to adjacent postsynaptic neurons or targets (non-neuronal), activating respective ion channels or receptors. The basis of neurotransmission involves ion gradients established by various ion pumps and transporters on the cell membrane, which either exchange ions or use ATP as an energy source to extrude ions against their own gradient. In the brain, neurons are surrounded by glial cells, which provide substrates to neurons and aid in the removal of glutamate from the synapse through the use of the glutamate-aspartate transporter. This transporter shuttles one glutamate and two Na+ ions into the glial cell, as one K+ ion is extruded (Storck et al. 1992) . The importance for the regulation of these ion gradients is observed during periods of extreme cellular stress (i.e., cerebral ischemia), whereby a decrease in mitochondrial respiration and increase in the cytoplasmic levels of Ca2+ can be detrimental to neurons (Dagani and Erecinska 1987 ) . Cerebral ischemia triggers severe stress in neurons, as cerebral blood ﬂ ow is interrupted to a focal or global region of the brain decreasing oxygen/glucose levels. 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 431

Oxygen/glucose deprivation (OGD) leads to decreased mitochondrial ATP production and potentially resulting in apoptosis (see mitochondria and apoptosis section) (Kass and Lipton 1982 ; Eguchi et al. 1997 ; Taylor et al. 1999 ) . Before ATP levels are signi fi cantly decreased in ischemia, a small transient depolarization and enhanced synaptic excitability increases the release of neurotransmitters (Fujiwara et al. 1987 ; Leblond and Krnjevic 1989 ) . In addition, decreased ATP production and increased presynaptic depolarizations lead to decreased cytoplasmic ATP caused by the consumption of ATP by various ATPases in the extracellular membrane, mitochondria, and endoplasmic reticulum (ER). Consequently, during severe and prolonged ischemic conditions, the Na+ /K+ ATPase function is attenuated. The Na+ /K+ ATPase normally counteracts cytoplasmic Na+ accumulation from the glutamate transporter, where glutamate and two Na+ ions in the synapse are exchanged for one K+ ion (Szatkowski et al. 1990 ) . The inability for glutamate reuptake results in enhanced glutamate in the synapse, increasing the activity of N -methyl- d -aspartate (NMDA) and a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Benveniste et al. 1984) . NMDA receptors are widely accepted as the excitotoxic response to excessive amounts of glutamate, through the massive infl ux of Ca 2+ (Rothman 1984 ; Silver and Erecinska 1990 ) . The increase in cytoplasmic Ca2+ leads to an amplifi ed amount of Ca2+ sequestered by the mitochondria and eventually causes the induction of apoptosis (Deshpande et al. 1987 ; Martins et al. 1988 ) . As neurons gain access to the necessary oxygen/glucose during and after reperfusion, recovery from ischemia is dependent on the ability of the mitochondria to resume ATP production with the replenishment of oxygen/glucose. ATP production after reperfusion allows ATPases to extrude cations to the cytosol and re-establish proper pH and ion gradients (Nishijima et al. 1989 ; Eleff et al. 1991 ) . As ion gradients are re-establish after ischemia and subsequent reperfusion, the glutamate- aspartate transporter sequesters extracellular glutamate into glia and neurons, decreasing NMDA receptor activation and in fl ux of Ca2+ -ending Ca 2+ -induced NMDA-mediated excitotoxicity (Fig. 20.1 ).

20.3 Ischemic Preconditioning

Ischemic preconditioning (IPC) is a neuroprotective mechanism whereby mild ischemic insults protect against subsequent lethal ischemia. IPC has been extensively studied in the heart and brain but also confers protection in hepatic, renal, skeletal muscle, and intestinal tissue. The induction of IPC activates multiple pathways involved in cellular defense and particularly diminishes mitochondrial dysfunction, an aberration intimately tied to neuronal degeneration following ischemic injury. Activation of IPC has been suggested to attenuate apoptosis (Piot et al. 1997; Tomasevic et al. 1999 ) , improve mitochondrial Ca2+ buffering capacity (Tanaka et al. 2009 ) , and prevent damage mediated by ROS (Ravati et al. 2001 ) . These mitochondrial adaptations are thought to be one of the major mechanisms by which IPC mediates ischemic protection and will be presented in detail. 432 S. Narayanan et al.

Cerebral Ischemia

ATP

Loss of Na+ /K+ ATPase

Presynaptic Ca2+ influx

Glutamate release

NMDA receptor activation

Neuronal cytosolic Ca2+ influx

Mitochondrial Ca2+ overload

MPTP opening

Cyt c release

Apoptosis

Fig. 20.1 Pathogenesis of cerebral ischemia leading to apoptosis. Cerebral ischemia leads to widespread loss of ATP production and thus failure to maintain ion homeostasis between extracellular and intracellular compartments. This leads to uncontrolled Ca2+ in ﬂ ux and neurotransmitter vesicular fusion with the membrane of the presynaptic terminal. If excitatory glutamate is released, NMDA receptors become activated resulting in Ca2+ inﬂ ux at the postsynaptic nerve terminals. Cytosolic Ca2+ activates a host of deleterious molecules, leading to protease activation, DNA degradation, and mitochondrial Ca2+ overload. This mitochondrial Ca2+ leads to MPTP opening, the + irreversible step toward apoptosis. Opening of mK ATP and K Ca channels dissipate mitochondrial membrane potential, which increases the Ca2+ buffering capacity of mitochondria. Together, these mechanisms allow the cell to stave off apoptotic signaling during an ischemic challenge (ATP, adenine triphosphate; Ca2+ , calcium; NMDA, N -methyl- d -aspartate; MPTP, mitochondrial perme- + ability transition pore; mK ATP , mitochondrial ATP-linked potassium channel; KCa , mitochondrial calcium linked potassium channels, Cyt c , cytochrome c ) 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 433

20.3.1 IPC and Apoptosis

Glutamate excitotoxicity and Ca2+ overload generated during ischemic injury facilitate opening of the MPTP (Rothman 1984 ; Garthwaite and Garthwaite 1986) , an irreversible step in the apoptotic cascade that leads to whole-cell metabolic derangement (Zoratti and Szabo 1994 ) . The MPTP complex consists of regulatory protein cyclophilin D, voltage-dependent anion channel (VDAC), and adenine nucleotide translocase (ANT). VDAC may be affected by glutamate excitotoxicity and Ca2+ overload, as Ca2+ binds to VDAC sites and facilitates opening of the MPTP (Gincel et al. 2001 ) . The opening of the MPTP results in uncoupling of oxidative phosphorylation, mitochondrial swelling, and subsequent release of proapoptotic molecules such as Bak and Bax to the cytosol (Vander Heiden et al. 1997 ) . The induction of IPC has been suggested to prevent opening of MPTP (Javadov et al. 2003 ) , thus inhibiting the activity of apoptotic signaling molecules such as caspase 3 and Bcl-xL following lethal ischemic episodes (Miyawaki et al. 2008 ) . IPC-mediated inhibition of MPTP is also regulated by the induction of hexokinases (Zuurbier et al. 2005 ) , enzymes that associate with the outer membrane of mitochondria. Hexokinases prevent binding of proapoptotic molecules to VDAC which decreases MPTP activation (Majewski et al. 2004a, b ) . Neuroprotection can also be achieved through IPC-mediated mitochondria + + protection through the ATP-sensitive K (mK ATP ) channel in mitochondria, which is analogous to the cell surface ATP-linked potassium channels found in cardiac myocytes (Inoue et al. 1991; Garlid et al. 1997) . These channels allow K + entry into the mitochondrial matrix leading to mitochondrial uncoupling and decreased + membrane potential. The induction of IPC activates mK ATP by upregulation of the + enzyme that phosphorylates mK ATP [protein kinase C e (PKC e )] (Raval et al. + 2007 ) . Opening of mK ATP by IPC results in cytoprotection in multiple tissue types such as the gut, heart, liver, brain, and kidney (Garlid et al. 1997; Fryer et al. 2000; Liu et al. 2002 ; Hai et al. 2005 ; Zhang et al. 2011a ) . Numerous studies have + suggested that activation of mK ATP channels have antiapoptotic properties (Grover et al. 1989 ; Kowaltowski et al. 2001 ; Murata et al. 2001 ; Liu et al. 2002 ) , whereby + 2+ opening of mK ATP channels decreases Ca accumulation in mitochondria inhibiting the opening of MPTP following ischemia (Murata et al. 2001 ) . Activation + of mK ATP channels also suppresses apoptotic signaling by hindering translocation of Bax and cytochrome c release (Liu et al. 2002 ) and induces preconditioning through mitochondria swelling and ROS generation (Carroll et al. 2001 ) . + Mitochondrial K ATP -speci ﬁ c activators reduce ischemic injury in the brain by increasing regional cerebral blood ﬂ ow, attenuating ROS, and maintaining the mitochondrial membrane potential (Mayanagi et al. 2007 ) . Together, these results + suggest that mK ATP channels may play a major role in IPC-mediated protection and could be the subject of future therapeutic interventions to ameliorate damage resulting from cerebral ischemia (Fig. 20.2 ) . 434 S. Narayanan et al.

PKC

GSK-3b HK Cyt c

HK MPTP Activation VDAC VDAC

ANT ANT CyD CyD

MPTP

Fig. 20.2 Induction of ischemic preconditioning prevents mitochondrial permeability transition pore opening. The MPTP is composed of four major subunits (HK, VDAC, ANT, CyD). VDAC interacts with GSK-3 b ( green arrow), while phosphorylation of GSK-3 b through PKC e prevents this interaction ( red bar ). HK-VDAC complex is facilitated through IPC (green arrow ) and prevents VDAC activation of proapoptotic mechanisms. The induction of IPC inhibits MPTP activation resulting in dissociation of HK from VDAC resulting in cytochrome c release (right image ). (MPTP, mitochondrial permeability transition pore; VDAC, voltage-dependent anion channel; GSK-3b , glycogen synthase kinase 3 beta; PKCe , protein kinase C e; HK, hexokinase; IPC, ischemic preconditioning; ANT, adenine nucleotide translocase; CyD, cyclophilin D; Cyt c , cytochrome c ) (Color ﬁ gure online)

20.4 IPC and the Endoplasmic Reticulum

During normoxia, the levels of Ca2+ in the extracellular space (1–2 mM) are similar to those inside the lumen of the ER, in contrast to low levels of cytosolic Ca2+ (100 nM). Rises in cytosolic Ca2+ are buffered by voltage-gated uniporters located in the inner mitochondrial membrane and sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) in the ER membrane (Paschen et al. 1996 ; Colegrove et al. 2000 ; Collins et al. 2001 ) . During ischemia, depressed mitochondrial respiration reduces the amount of available ATP; as a consequence, SERCA is unable to pump Ca 2+ into the ER. However, mitochondria continue to buffer this rise in cytosolic Ca2+ by increasing mitochondrial Ca 2+ uptake via voltage-gated uniporters (Reynolds 1999 ; Pivovarova et al. 2004 ) . Following cerebral ischemia, excess Ca2+ continues to ﬂ ood the postsynaptic terminal via glutamate-NMDA receptor activation (Nicholls and 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 435

Ward 2000 ) , where the Ca2+ uniporter continues to uptake Ca2+ into the mitochondria until a Ca2+ threshold is reached, resulting in collapse of the inner membrane and opening of the MPTP (Nicholls 1978 ; Hunter and Haworth 1979 ) . The detrimental consequence of MPTP opening is a result of the massive mitochondrial in fl ux of Ca2+ , which opens the large conductance of the Ca2+ -activated potassium + channel (KCa ) in the inner mitochondrial membrane to infl ux K into the mitochondria + (Siemen et al. 1999 ; Xu et al. 2002 ) . During cerebral ischemia, this KCa -mediated K in fl ux depolarizes the mitochondrial membrane potential (MMP) and decreases Ca2+ sequestration (Holmuhamedov et al. 1998 ) ; however, IPC activation induces hyperpolarization of MMP and increases the Ca 2+ buffering capacity in hippocampal neurons of the mitochondria for future neuroprotection (Tanaka et al. 2009) . Moreover, in isolated rat hearts, the induction of IPC conferred cardioprotection following ischemia through the activation of K Ca (Cao et al. 2005) . In addition, K Ca activation decreased MPTP opening in the mitochondria, improving cardiac function following ischemia (Cao et al. 2005) . These cardiac studies may be extended to the brain suggesting that IPC may ameliorate toxic cytosolic loading of Ca2+ following ischemia. The damaging effects of Ca2+ on the mitochondria occur from the inability of the ER to continue to sequester Ca2+ during periods of reduced ATP; however, the ER is also the location where new protein is synthesized/folded. Under normoxia, high ER Ca2+ levels allow for proper protein synthesis and folding, where the disruption of ER Ca 2+ levels can inhibit cell division and induce apoptosis (Ghosh et al. 1991 ; Short et al. 1993 ; Waldron et al. 1994 ) . Under physiological Ca2+ levels, an ER chaperone glucose-regulated protein (GRP) 78 binds and inactivates stress proteins such as PKR-like ER kinase, inositol-requiring enzyme, and activating transcription factor 6 (Harding et al. 1999; Shen et al. 2002) . During conditions of ER stress (i.e., cerebral ischemia), these kinases and transcription factors become active and initiate a cascade of events to protect the cell from damage, termed the “unfolded protein response” (UPR) (Kozutsumi et al. 1988 ; Dorner et al. 1989 ) . The UPR protects the proteins from aggregation and assists in their folding. IPC induction decreases the sensitivity of the ER to ischemic stress by transiently upregulating GRP78 protein levels (Hayashi et al. 2003 ) . This increased tolerance to ischemia results from the excess levels of GRP78 binding to unfolded proteins and allows for enhanced recovery during reperfusion (Burda et al. 2003 ) .

20.5 Signaling Pathways Leading to Mitochondrial Neuroprotection

20.5.1 Reactive Oxygen Species

During mitochondrial respiration, ROS are normally produced in complex I and III (Boveris and Chance 1973 ; Philipson et al. 1985 ) , and scavenged by superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase. Superoxide and 436 S. Narayanan et al. hydrogen peroxide are the most prevalent ROS generated by the mitochondria (Floyd 1990 ) , producing highly reactive hydroxyl radicals (Li and Jackson 2002 ) . After ischemia, the production of ROS occurs at an increased rate over the cells’ ability to remove ROS leading to enhanced superoxide/hydrogen peroxide production (Piantadosi and Zhang 1996) . ROS are also elevated after IPC induction, (Ravati et al. 2000 ) conferring cytoprotection in cardiac and neuronal tissues through activation of nitric oxide synthase (NOS) (Andoh et al. 2000) , PKC (Korichneva et al. 2002; Kabir et al. 2006 ) , enhanced Ras/Raf/ERK 1/2 (Guyton et al. 1996 ; Milligan et al. 1998 ) , nuclear factor- kB (NF-k B) (Milligan et al. 1998 ; Ravati et al. 2001 ) , and tensin homolog (PTEN). PTEN can then inhibit the PI3K/Akt pathway (Cai and Semenza 2005 ) and modulate Bcl-2 family of proteins (Tang et al. 2005 ) , which regulate apoptosis. ROS can directly modify PKCs through sulfhydryl oxidation (Korichneva et al. 2002 ) , where PKC inhibitors prevent the cytoprotection mediated through free radicals suggesting that ROS targets PKC-mediated IPC-induced protection (Liu et al. 2008 ) . IPC induction in cardiac tissue is dependent on the activation of PKCe (Kabir et al. 2006) , where in neuronal cultures, IPC induction activates PKCe and ERK1/2 (Kim et al. 2007) and enhances NF- kB activation (Kim et al. 2010 ) , resulting in the modulation of mitochondrial apoptotic factors Bcl-2, Bax, and p53 in the brain (Chen et al. 1996; Dixon et al. 1997 ; Nijboer et al. 2008 ) . ERK1/2 activation also enhances levels of manganese superoxide dismutase (MnSOD, a mitochondrial enzyme that scavenges ROS generation by the mitochondrial electron transport chain) in cortical neuronal cultures, decreasing ROS accumulation during reperfusion (Scorziello et al. 2007 ) . The generation of ROS from ischemic stress also induces damage to mitochondrial DNA (Yakes and Van Houten 1997; Ohtaki et al. 2007) . The induction of IPC upregulates DNA repair enzymes via the base-excision repair pathway in neuronal mitochondria to increase the repair capacity for future ischemic events (Englander et al. 2002 ) .

20.5.2 Protein Kinase C

The consequences of cerebral ischemia have been linked to the induction of speci ﬁ c isoforms of PKC (Korichneva et al. 2002 ; Kabir et al. 2006 ) modulating IPC- mediated neuroprotection (Reshef et al. 2000; Raval et al. 2003 ) . PKC e and PKC d belong to a family of serine/threonine kinases that have opposing roles in cellular apoptosis and IPC (Chen et al. 2001 ; Inagaki et al. 2005 ; Duquesnes et al. 2011 ) . The induction of IPC upregulates PKCe (Raval et al. 2003 ; Lange-Asschenfeldt et al. 2004 ) , as the inhibition of PKCd abrogates IPC-mediated neuroprotection, and peptide activators of PKCe mimic IPC in organotypic hippocampal slice cultures (Raval et al. 2003 ; Della-Morte et al. 2011 ) . The neuroprotective role of PKC e + includes phosphorylation of the mK ATP channel to increase mitochondrial matrix K + levels (Costa et al. 2005 ) , increased ERK1/2 phosphorylation and COX-2 induction (Kim et al. 2007 ) , alter the levels of phosphorylation of the mitochondrial respiratory chain proteins (Dave et al. 2008 ) , inhibition of the plasmalemma Na+ / K + -ATPase and voltage-gated Na+ channels (Dave et al. 2009 ) , and modulation of GABA synapses (DeFazio et al. 2009 ) . 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 437

Despite the protective effects of PKCe , PKC d also has putative roles in cell survival. During ischemia, the levels of PKCd increase; however, the induction of IPC upregulates 26S proteasome, an enzyme that degrades PKCd (Asai et al. 2002 ; Durrant et al. 2004 ; Churchill et al. 2010 ) . In addition, IPC induction in myocardial tissue prevents the accumulation of PKCd in mitochondria allowing for mitochondrial accumulation of PKCe (Churchill et al. 2010 ) . Therefore, this evidence suggests that the cytoprotective effects of IPC are dependent on the induction of PKCe over PKCd following ischemic stress.

20.5.3 Nitric Oxide

Nitric oxide (NO) is a gasotransmitter that regulates several physiological processes, such as vasodilation of vascular smooth muscle (Furchgott and Zawadzki 1980 ) , neurotransmission (Bredt et al. 1990 ) , posttranslational modi ﬁ cations via S-nitrosylation of proteins, and cellular apoptosis (Melino et al. 1997 ) . NO is the by-product of the conversion from l -arginine to l -citrulline via NOS originating from the urea cycle (Palacios et al. 1989 ) . There are three isoforms of nitric oxide synthase (NOS): endothelial NOS, inducible NOS, and neuronal NOS (Knowles and Moncada 1994 ) . NOS is activated during ischemia (Morikawa et al. 1992 ) by the Ca2+ /calmodulin complex after entry of cytosolic Ca2+ (Bredt and Snyder 1990 ; Schmidt et al. 1992 ) . Under normoxic conditions, NO can S-nitrosylate caspase-3 to prevent apoptosis (Zhou et al. 2005) and activate guanylate cyclase to stimulate cyclic guanosine monophosphate (cGMP) production and subsequent activation of protein kinase G (PKG). IPC-induced neuroprotection has been suggested to occur through activation of all NOS isoforms (Centeno et al. 1999; Gidday et al. 1999 ; Cho et al. 2005 ; Scorziello et al. 2007 ) . Inhibition of NOS via Nw -nitro-L-arginine or Nw -nitro-L-arginine methyl ester hydrochloride attenuates preconditioning (Gonzalez-Zulueta et al. 2000 ) , enhancing ROS formation to induce NO, activation + of PKG, with subsequent activation of mK ATP channels (Han et al. 2002 ; Costa et al. 2005 ) . In addition, NO also can activate Ras, as a consequence, and stimulate ERK1/2 posttranscriptional expression of MnSOD (Gonzalez-Zulueta et al. 2000 ; Santillo et al. 2001 ) and Trx (mitochondrial redox protein) (Andoh et al. 2002, 2003) . Taken together, MnSOD and Trx serve to attenuate oxidative stress, thereby preventing cell damage. Therefore, the role of NO is diverse and can affect many crucial components of the preconditioning response.

20.5.4 Nitric Oxide and the Mitochondria

NO inactivates the electron transport chain (ETC) in mitochondria by inhibiting electron entry into the ETC, thus generating low levels of ROS by decreasing the ETC activity (Bolanos et al. 1995 ) . This attenuates Ca2+ overload, ROS generation, and MPTP activation in mitochondria (Burwell et al. 2006; Nadtochiy et al. 2007 ) . 438 S. Narayanan et al.

NO competes with oxygen to inhibit cytochrome c activity when oxygen is limited, potentially activating ROS generation in preceding subunits of the ETC (Palacios- Callender et al. 2004 ) . In addition to the direct targets of NO during IPC, NO can S-nitrosylate cytochrome oxidase and dynamin-related protein 1 (drp-1) (Westermann 2009) , which plays a role in mitochondrial ﬁ ssion and fragmentation resulting in cell death. S-nitrosylation of Bcl-2 results in Bcl-2 activation and thus cellular apoptosis (Azad et al. 2006 ) . NO can also result in ETC-mediated complex I inhibition. This limits the amount of ROS formed and thus mediates opening of the MPTP (Shiva et al. 2007 ) , enhancing cell survival.

20.6 Transcriptional Activators Regulate Mitochondrial Protection

Genomic reprogramming induced by IPC plays an integral role in the induction of mitochondrial tolerance against future lethal ischemic insults. For example, IPC upregulates gene transcripts involved in the regulation of ETC complexes and mitochondrial biogenesis (McLeod et al. 2004 ) . The induction of IPC has been suggested to regulate the expression of microRNAs (miRNA) (Dharap and Vemuganti 2010 ) , which are small noncoding RNA that regulate posttranscriptional gene expression. Predictions using bioinformatic analyses strongly suggest that the miRNAs modulated by IPC speciﬁ cally target miRNAs encoding transcriptional regulators (Lusardi et al. 2010 ) . Inhibiting the expression of transcriptional regulators during IPC increases infarct size and abolishes IPC-mediated protection following lethal ischemia (Strohm et al. 2002 ) . A number of transcriptional regulators [i.e., peroxisome proliferator-activated receptors (PPARs)] activated by IPC have been shown to enhance mitochondrial activity and protection.

20.6.1 Peroxisome Proliferator-Activated Receptors

PPARs are transcription factors that belong to the nuclear hormone receptor family. Numerous isoforms have been identi fi ed, including PPARa , PPARb / d , and PPARg (Issemann and Green 1990 ; Dreyer et al. 1992 ) . Various roles have been attributed to the PPAR superfamily, such as regulating insulin sensitivity, glucose homeostasis, oxidation of fatty acids, and maintaining integrity of vasculature (Kim et al. 2002 ; Hafstad et al. 2009 ; Yin et al. 2011 ) . Blockade of PPARg or PPARa inhibits IPC- mediated protection (Lotz et al. 2011 ) , which highlights the importance of these transcription factors in ameliorating injury following ischemia. In non-invasive magnetic resonance imaging studies, PPARa and PPARb knockout mice developed larger infarct following middle cerebral artery occlusion (MCAO) (Pialat et al. 2007 ) than their counterparts. Similarly, neuronal-specifi c PPARg knockout mice developed 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 439 increased brain damage and functional neurological impairment after induction of MCAO (Zhao et al. 2009 ) . Pharmacological activation of PPARg decreased apoptosis- induced cell death in neuroblastoma cell lines following OGD via 14-3-3 e, a protein that aids in binding Bad and thus preventing apoptosis (Wu et al. 2009b ) . In addition, PPARg overexpression and activation in neuroblastomas induced maintenance of mitochondrial membrane potential and decreased production of ROS and inhibition of apoptotic metabolites (cytochrome c, caspases) following OGD (Wu et al. 2009a ) . In microglia cultures, PPARg activation increased activity of antioxidant enzymes such as SOD1, catalase, and glutathione reductase, effectively decreasing H2 O2 production (Zhao et al. 2009 ) . Furthermore, PPARg de fi cient mice displayed decreased levels of UCP1 (Zhao et al. 2009 ) , a protein involved in enhancing mitochondrial oxygen consumption following IPC (Nadtochiy et al. 2006 ) . These results suggest that IPC-induced PPAR-regulation provides mitochondria with a multilateral defense against ROS by modulating apoptosis, bioenergetics, and antioxidant protection.

20.6.2 Peroxisome Proliferator-Activated Receptor g Coactivator-1a

PPAR g coactivator-1a (PGC-1a ) is a transcriptional coactivator that binds to PPAR g and modi ﬁ es the expression of several genes, many of which are intimately tied to metabolism and adaptation from hypoxia (Arany et al. 2005 ; Gutsaeva et al. 2008 ) . PGC-1a knockout mice exhibited mitochondrial dysfunction and increased susceptibility to heart failure supporting the critical regulatory role of PGC-1a in energy and oxidative homeostasis (Arany et al. 2005 ; Lehman et al. 2008 ) . NMDA receptor- mediated Ca2+ inﬂ ux or ROS production following ischemic episodes both led to subsequent PGC-1 a activation (Luo et al. 2009 ) . PGC-1a has been suggested to confer neuroprotection following lethal OGD (Luo et al. 2009 ) . During transient global ischemia, PGC-1a mediated increased mitochondrial biogenesis through the activity of nuclear transcription factor 1 and mitochondrial transcription factor 1 (key regulators of mitochondrial transcription) (Gutsaeva et al. 2008 ) . NOS knockout mice failed to demonstrate the same increases in mitochondrial biogenesis following transient ischemia (Gutsaeva et al. 2008 ) , suggesting PGC-1a -mediated biogenesis is an NO-dependent process. PGC-1a increased mitochondrial UCP1 (Barbera et al. 2001 ) , which enhanced recovery following ischemia-reperfusion (Hoerter et al. 2004 ) . PGC-1 a also increased the expression of UCP2 and UCP3 (St-Pierre et al. 2006) isoforms involved in decreasing ROS formation (Haines et al. 2010; Toime and Brand 2010 ) . Overexpression of PGC-1a increased antioxidant compounds like MnSOD, thioreduxin reductase, thioreduxin 2, and catalase in bovine neuronal cells and aortic endothelial cells (Weinberg et al. 2000 ; Wright et al. 2007 ) . The activation of these antioxidants and cytoprotective genes suggests that PGC-1a may serve a therapeutic role following cerebral ischemia and may be an important contributor to IPC-induced neuroprotection (Fig. 20.3 ). 440 S. Narayanan et al.

IPC

PKCe PI3K Bax/Bad PGC-1a

GSK-3b P

P + + + H H + H H+ H MPTP ATP ATP + ETC Synthase UCP1 mK

+ + ADP+Pi ATP + H H K H+

Fig. 20.3 Induction of ischemic preconditioning modulates mitochondrial function inducing cytoprotection. The induction of IPC activates several molecular mediators that alter mitochondrial function in efforts to prevent MPTP opening. IPC induction activates an uncoupling effect in mito- + chondria through mK ATP opening and activation of UCP-1. The in ﬂ ux of cations results in mild depolarization of the MMP and attenuation of ROS formation. In addition, IPC attenuates activation of proapoptotic factors, such as Bax/Bad and phosphorylated GSK-3 b, suppressing MPTP opening (red bars ), while the induction of IPC can activate molecular factors such as PI3K, PKCe , and PGC-1a (green arrows) (IPC, ischemic preconditioning; MPTP, mitochondrial permeability + transition Pore; mK ATP , mitochondrial ATP-linked potassium channel; UCP, uncoupler protein; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; Bax, B cell lymphoma- 2-associated X protein; Bad, B cell lymphoma-2-associated death promoter; GSK-3b , glycogen synthase kinase 3 beta; PI3K, phosphoinositide 3-kinase; PKC e, protein kinase C e; PGC-1a , peroxisome proliferator-activated receptor g coactivator 1-a ) (Color ﬁ gure online)

20.6.3 Hypoxia-Inducible Factors

Adaptation to hypoxia can be regulated by hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1b and hypoxia-induced HIF-1a . Under normoxia, the presence of oxygen and 2-oxo- glutarate activates prolyl hydroxylases (PHD) and acetyltransferase arrest-defective-1, which hydroxylize and acetylate HIF-1a (Jeong et al. 2002 ; Metzen et al. 2003) . These modiﬁ cations are recognized by the von Hippel–Lindau protein (pVHL), which target HIF-1a for ubiquitinated protein degradation (Huang et al. 1998; Maxwell et al. 1999 ) . If oxygen levels decrease due to hypoxic conditions, 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 441 non-hydroxylated HIF-1a translocates to the nucleus and forms a complex with HIF-1 b to bind to hypoxia-responsive elements in the DNA to upregulate a wide array of genes. These upregulated genes include vascular endothelial growth factor (VEGF) (Kelly et al. 2003 ) , inducible nitric oxide synthase (iNOS) (Zhang et al. 2011b) , erythropoietin (EPO) (Blanchard et al. 1992 ) , and enzymes involved in cellular metabolism (i.e., pyruvate dehydrogenase kinase 1) (Papandreou et al. 2006 ) . To date, limited research has been performed on cerebral ischemia models; however, the induction of IPC increased the activation of HIF-1a in various IPC-induced tissue models (Bergeron et al. 2000 ; Vazquez-Valls et al. 2011 ) . Active HIF-1a in smooth muscle is responsible for the increase in COXIV-2 gene (which encodes COXIV-2) and the mitochondria protease LON gene expression (protease that is required for the degradation of COXIV-1) (Fukuda et al. 2007 ) . In heterozygous- null HIF-1 a mice, the presence of HIF1-a was required for cardiac IPC-induced ROS production, PTEN oxidation, Akt (protein kinase B) activation, and protection against ischemia-reperfusion injury (Cai and Semenza 2005 ; Cai et al. 2008 ) . Akt activation prevented MPTP opening, thereby decreasing reperfusion injury in cardiomyocytes (Hausenloy et al. 2005 ) . In addition, HIF-1a activation from in vivo IPC-induced renal cells increased Bcl-2 protein expression and prevented mitochondria release of apoptotic proteins (e.g., cytochrome c) (Yang et al. 2009 ) . Thus, HIF-1 a plays a critical role in adapting the cell to stressful conditions and may prove to be an important target of activation following IPC as more information regarding its regulation and mechanism of action is elucidated (Fig. 20.4 ).

20.6.4 Sirtuin 1

Sirtuins (Sirts) are NAD+ -dependent histone deacetylases that regulate gene transcription involved in metabolism, life span, and DNA repair (Yang et al. 2007; Boily et al. 2008 ; Ho et al. 2009 ; Mao et al. 2011 ) . Sirtuins, specifi cally mammalian Sirt1, are activated by IPC and are required for IPC-mediated protection against ischemic injury (Della-Morte et al. 2009 ; Nadtochiy et al. 2011 ) . Sirt1 interferes with apoptotic pathways through inhibition of pro-apoptotic transcription factors such as p53 (Vahtola et al. 2010 ) , involved in Bax activation and FOXO1 (Chen et al. 2009 ) . Sirt1 has also been suggested to enhance HIF-2a activity during hypoxic conditions through direct deacetylation (Dioum et al. 2009 ) . Additionally, Sirt1 aids in cellular coping mechanisms against oxidative stress by increasing HIF-2a -mediated MnSOD (Dioum et al. 2009 ) and enhancing PGC-1a activity (Gerhart-Hines et al. 2007 ; Chen et al. 2009) . Sirt1, along with PGC-1a , localizes to the mitochondria (Aquilano et al. 2010) and associates with nucleoids (structures located in the mitochondrial matrix containing mitochondrial DNA). Interaction of Sirt1 and PGC-1 a with mitochondrion- specifi c transcription factor A was also suggested (Aquilano et al. 2010 ) , indicating that Sirt1 and PGC-1a may associate with mitochondrial transcription. Sirt1 and PGC-1a were previously defi ned as nuclear proteins that regulate transcription of various genes; however, this new evidence suggests Sirt1 and PGC-1 a may also regulate mitochondrial function through more direct mechanisms. 442 S. Narayanan et al.

HIF-1α HIF-1β Hypoxia

O2 HIF-1

ROS PHD PHD

OH OH

Mitochondria Proteasome Nucleus

Degradation Hypoxic gene Attenuation of Apoptotic signals transcription Mitochondrial adaptation to hypoxia

Fig. 20.4 Induction of ischemic preconditioning protects mitochondria via HIF-1. IPC and hypoxia increase ROS production, which is naturally produced primarily from complex I and III of the ETC in mitochondria. ROS inhibits PHD, allowing for accumulation of HIF-1 (HIF-1 is composed of two subunits, HIF-1a and HIF-1b , which bind to form the active HIF1 protein). HIF-1 mediates mitochondrial protection by decreasing proapoptotic stimuli and adapting mitochondrial respiration to hypoxic conditions. HIF-1 is also a transcription factor and translocates to the nucleus under IPC or hypoxic conditions to upregulate nuclear transcription of several genes involved in adaptation to hypoxia. In the presence of oxygen, PHDs are not suppressed, enhancing hydroxylation and subsequent degradation of HIF-1 through VHL, which targets HIF-1 for ubiquitination and thus promoting proteasomal processing and degradation (IPC, ischemic preconditioning; ROS, radical oxygen species; ETC, electron transport chain; PHD, prolyl hydroxylase; HIF, hypoxia-inducible factor; VHL, von Hippel–Lindau)

20.6.5 AMP-Activated Protein Kinase Pathway

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme activated during bioenergetic stress that suppresses ATP depletion while promoting ATP production. AMPK modulates nuclear transcription factors in order to inhibit protein synthesis (Ng et al. 2011 ) while stimulating glycolysis and fatty acid oxidation (Russell et al. 2004; Janovska et al. 2008) . AMPK activity is also protective against lethal ischemic stress. Pharmacological activation of AMPK reduced myocardial infarct following ischemia-reperfusion injury (Kim et al. 2011 ; Paiva et al. 2011 ) and improved neuronal viability after hypoxic deprivation (Zhang et al. 2010 ) . 20 Ischem ic Preconditioning-Mediated Signaling Pathways Leading to Tolerance… 443

AMPK is activated by IPC (Nishino et al. 2004 ) and is required for IPC-mediated protection (Peralta et al. 2001 ) . The speci ﬁ c targets of AMPK during IPC have not been elucidated, but AMPK activity has been associated with mitochondrial protection. During IPC, AMPK inhibits ATP breakdown while enhancing energy metabolism (Peralta et al. 2001) . Following ischemia-reperfusion injury, AMPK activation inhibited MPTP opening (Paiva et al. 2011 ) and apoptotic death (Kim et al. 2011 ) . AMPK may function as a master regulator of other transcriptional activators involved in IPC-mediated mitochondrial protection. For example, AMPK activates PPAR a (Bronner et al. 2004 ; Meng et al. 2011 ) , regulates mitochondrial biogenesis through direct phosphorylation of PGC-1a (Jager et al. 2007 ; Canto et al. 2009 ) , is required for HIF-1 transcriptional activity during hypoxic conditions (Jung et al. 2008 ) and stimulates Sirt1 activity by enhancing NAD+ availability (Fulco et al. 2008 ; Canto et al. 2009 ) .

20.6.6 Nuclear Factor Erythroid-2 Related Factor

In addition to cellular adaptation to hypoxia, attenuating ROS following ischemia is another important target. Nuclear factor erythroid-2 related factor (Nrf2) is a transcription factor involved in protecting the cell from the damaging effects of oxidative stress and is involved in IPC (Liverman et al. 2004 ; Bell et al. 2011 ) . In mice, 50% reduction in cerebral blood ﬂ ow enhanced Nrf2 activity, upregulating oxidative stress response genes such as oxidative stress-induced (OSI) protein, heat shock protein (HSP)-84, and transthyretin (Liverman et al. 2004 ) . Accumulation of ROS in ischemic tissues prevented Nrf2 degradation through phosphorylation (Leonard et al. 2006) , thus allowing Nrf2 to induce transcription of antioxidant enzymes (Favreau and Pickett 1991 ; Li and Jaiswal 1992 ; Prestera et al. 1995 ; Thimmulappa et al. 2002 ) . NO has been suggested to induce Nrf2 translocation from cytosol to nucleus through S-nitrosylation of cysteine residues in cultured rat pheochromocy- toma cells (Um et al. 2011 ) . In addition, the use of S -nitroso- N -acetylpenicillamine (NO donor)-activated PKC-dependent phosphorylation was also suggested as another pathway for Nrf2 nuclear translocation (Um et al. 2011 ) . The interactions of Nrf2 and acetyl-l -carnitine (ALCAR) have been recently described. ALCAR is involved in transporting fatty acids to mitochondria, attenuating neuronal damage following cerebral hypoxia through Nrf2-mediated pathways in adult rats (Hota et al. 2011) . The addition of ALCAR also increased mitochondrial biogenesis via pERK-dependent upregulation of PGC-1 a and also enhanced Nrf1, which stimulated Nrf2 activity (Hota et al. 2011 ) . In mixed neuronal/astrocyte cultures, H 2 O2 administered at levels akin to ischemia-reperfusion stimulated Nrf2 target genes sul ﬁ redoxin (Srxn1 ) and heme oxygenase 1 (Hmox1 ) (Calabrese et al. 2005 ; Hota et al. 2011 ) . Knocking out Nrf2 abrogated this gene induction following exposure to H2 O2 (Hota et al. 2011 ) . This suggests IPC, which similarly stimulates transient levels of oxidative stress, may induce neuroprotection through Nrf2- dependent expression of antioxidant enzymes, protecting cells from the damaging 444 S. Narayanan et al. effects of ROS. In particular, Nrf2, in conjunction with HIF-1, may result in an effective therapy against cerebral ischemia, forming both an immediate and sustained cellular response to thrive under ischemic conditions.

20.7 Conclusion

Cerebral ischemia induces numerous processes that attempt to protect the neuron from innate cellular damage. OGD leads to mitochondria dysfunction and inadequate ATP levels as various ATPases attempt to remove/pump ions and maintain ion gradients. The in ﬂ ux of Ca2+ , via NMDA receptors, persists as glutamate increases in the synapse, further depolarizing the plasma membrane to activate VGCC. This, in turn, deposits additional Ca2+ in the cytosol, which is sequestered by the mitochondria and incites an array of detrimental processes ultimately leading to cell death. There is a complex interaction between many pathways and signaling cascades that regulate mitochondrial adaptation and hence cellular adaptation to hypoxia. The exploitation of these mechanisms through IPC could provide a therapy that increases brain viability following cerebral ischemia, while decreasing the long-term neurological impairment typically associated with stroke and other cerebral vascular-related diseases. The burden of cerebral ischemia is alarming, and ascertaining knowledge regarding the precise mechanisms governing cytoprotection following ischemia could prove to be invaluable in developing future therapies.

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Ann M. Stowe and Jeffrey M. Gidday

21.1 Overview of the Neurovascular Unit

Stroke is a major cause of death and long-term adult disability in the United States (Lloyd-Jones et al. 2010 ) , and as a result, there is an emphasis on understanding the anatomical and functional neuronal plasticity that contributes to recovery of function following ischemic injury (Braun et al. 2007 ; Dancause and Nudo 2011) . This focus also holds true in preclinical models of stroke, wherein reductions in infarct volume and improvements in functional outcome are the gold standard for a “successful” intervention. But while preservation of neuronal viability is fundamental to minimizing injury, particularly as it manifests as functional de ﬁ cits, neurons are not the only CNS cell type that contribute to both the progression of injury and the success of repair following stroke (Faraci 2003 ) . It is now recognized that every neuron is organized into a larger neurovascular unit (NVU; see reviews del Zoppo 2009a, b, 2010 ; Takahashi and Macdonald 2004 ) . The NVU is the anatomical basis for the functional interactions between neurons and blood vessels that underlie not only the regulation of local and regional cerebral blood ﬂ ow (CBF) but many other responses dependent on neurovascular communication, both under normal conditions and in response to injury.

A. M. Stowe Department of Neurology & Neurotherapeutics , University of Texas Southwestern Medical Center , Dallas , TX 75390 , USA J. M. Gidday (*) Department of Neurosurgery , Washington University School of Medicine , St. Louis , MO 63110 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 457 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_21, © Springer Science+Business Media New York 2013 458 A.M. Stowe and J.M. Gidday

Microvessels (small arterioles, venules, and capillaries) also protect neurons from blood-borne pathogens by cellular specialization to form the blood–brain barrier (BBB), which includes tightly coupled endothelial cells surrounded, predominantly, by astrocyte end-feet adherent to the abluminal side of the microvessels (see reviews Faraci 2011 ; Jin et al. 2010 ) . Thus, the functional BBB can be considered to extend the traditional NVU to be comprised of not only neurons and endothelial cells but astrocytes, pericytes, resident immune cells, and the interconnecting basal lamina.

21.2 NVU-Speci ﬁ c Injury in Ischemic Stroke

Many of the established hallmarks of poststroke ischemic injury quanti fi ed in animal studies actually occur within the NVU, including in fl ammation, vasogenic edema, BBB disruption, and perfusion dysregulation. Progression to some of these phenotypes begins within hours of the ischemic event, most notably with the upregulation of proin fl ammatory cytokines (e.g., interleukin-1b [IL1b ], tumor necrosis factor- a [TNF- a]), chemokines, and endothelially expressed cellular adhesion molecules (e.g., intracellular adhesion molecule-1 [ICAM-1], P-selectin) (An and Xue 2009 ; Andjelkovic et al. 2003 ; Bowen et al. 2006; Ding et al. 2005 ) . During the acute stages of stroke, proin fl ammatory mediators exacerbate microvascular injury in a feedforward mechanism, as many of the released cytokines, chemokines, and proteases upregulate each other or work in a synergistic manner toward tissue destruction (Lee et al. 2009 ; Malik et al. 2008 ; Stowe et al. 2009 ) . Over the early hours to days following stroke onset, these proinfl ammatory mediators promote the recruitment of blood-borne leukocytes, which roll along venules, become adherent, and eventually diapedese into the ischemic parenchyma. Leukocytes are key players in postischemic infl ammation and secondary NVU injury following stroke (Danton and Dietrich 2003 ; Man et al. 2007 ; Huang et al. 2006 ) . Even recruited leukocytes that only roll along or adhere to venules, but do not undergo diapedesis, contribute to the loss of BBB integrity by either oxidative injury, secondary to reactive oxygen species (ROS) production, or proteolytic injury from the release of destructive enzymes (Dallegri and Ottonello 1997 ; Shapiro 2002 ) . These leukocyte-derived proteases, such as neutrophil elastase and matrix metalloproteinases (MMPs), degrade the basal lamina and extracellular matrix, further increase leukocyte recruitment, and promote additional leukocyte diapedesis into surrounding tissue (Gidday et al. 2005 ; Henriksen and Sallenave 2008 ; Stowe et al. 2009 ; ) . Ischemic injury also affects endothelial and astrocytic expression and distribution of structural proteins. In rat brain cerebral endothelial cultures, oxygen- glucose deprivation (OGD) redistributes junctional proteins (e.g., zonula occludin-1 [ZO-1]) from a membrane localization to a more diffuse pattern, which anatomically weakens the endothelium (An and Xue 2009 ) . Transient stroke in mice also decreases total expression of occludin, another integral membrane protein located within the BBB tight junctions (Hua et al. 2008; Wacker et al. 2012 ) . 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 459

Integrins on endothelial cells and astrocytic end-feet, which anchor both cell types to the intervening basal lamina, creating a physical barrier against paracellular ef fl ux, are also downregulated following stroke injury (del Zoppo 2009a ; Ding et al. 2006a) . The loss and/or displacement of these proteins, coupled with the presence of leukocyte-derived degradative enzymes, culminates in the breakdown of the BBB and increased hemispheric edema after stroke, documented in both transient (Hua et al. 2008 ; Ding et al. 2006a ; Bigdeli et al. 2007 ; Dong et al. 2009 ; Stowe et al. 2011 ) and permanent (Hoshi et al. 2011 ; Ikeda et al. 1999 ; Masada et al. 2001 ; Zhang et al. 2006 ; ) preclinical stroke models, as well as in cell culture studies (Lenzser et al. 2005 ) . The physiological role of the NVU in regulating CBF is also compromised following stroke. The cerebral vasculature favors a state of partial vasoconstriction (i.e., myogenic tone) under normal physiologic conditions (Fagan et al. 2004 ) . Loss of tone occurs immediately following ischemia and, in combination with early ROS production, results in vasodilation of the arterioles and, to a lesser extent, venules. For portions of the ischemic infarct that become reperfused, areas of high, unregu- lated CBF are created that contribute to the progression of vasogenic edema. The subsequent mechanical compression of edematous parenchyma upon microvascular perfusion, and the further dysregulation of autoregulatory mechanisms, including loss of nitric oxide (NO) production (Fagan et al. 2004 ) , results in a substantial decline in CBF within the ischemic hemisphere during the fi rst hours following stroke. In preclinical studies, one hour after permanent middle cerebral artery occlusion (pMCAo), regional CBF (rCBF) and oxygen consumption are signi fi cantly reduced relative to the contralateral, healthy cortex (Chi et al. 2010 ) . Reductions in rCBF continue through 2 weeks following stroke onset and occur not only within the area of ischemia but in peri-infarct regions of the affected hemisphere as well (Dawson et al. 1999 ; Furuya et al. 2005 ) . Longer-term mechanisms of NVU recovery include the process of angiogenesis, or the formation of new blood vessels from the preexisting microvascular bed. Angiogenesis occurs over the days and weeks following stroke onset, particularly in the peri-infarct region, in an effort to reintroduce blood to the hypoperfused tissue (Gustavsson et al. 2007 ; Wei et al. 2001 ; Zhang et al. 2000 ) , a bene fi cial effect of NVU-based plasticity also evident following clinical stroke (Krupinski et al. 1994 ) . Infl ammatory mediators, BBB disruption, vasogenic edema, and hypoperfusion additionally contribute to apoptosis and necrosis within the NVU, although cell- speci fi c quanti fi cation of cellular death within the BBB is dif fi cult to achieve in vivo. Cell culture studies show, however, that ischemic injury promotes the apoptosis of some portion of endothelial cells (An and Xue 2009; Andjelkovic et al. 2003 ; Zhang et al. 2007a ) , astrocytes (Chu et al. 2010 ; Du et al. 2011 ; Liu and Alkayed 2005 ) , and oligodendrocyte precursor cells (Deng et al. 2003 ) , in addition to the loss of overall functionality within the NVU. Results from studies in a number of in vivo and in vitro preclinical stroke models make it increasingly apparent that preconditioning (PC) prior to ischemia can ameliorate virtually all of the aforementioned NVU-speci fi c stroke pathologies. Those fi ndings will be reviewed below in more detail. 460 A.M. Stowe and J.M. Gidday

21.3 Documented NVU-Based Tolerance Phenotypes in Response to Preconditioning

This book focuses on the protective effects of PC to reduce ischemic and other injuries within the CNS. But it should be noted that ischemic tolerance is likely characterized by enhancing the viability and functionality of all the cells within the CNS, not only neurons; such a concept is consistent with the robust magnitude of protection typically afforded by PC. In addition, PC does not merely delay stroke-induced ischemic injury but provides a sustained protection that remains even weeks following stroke onset (Furuya et al. 2005 ; Gustavsson et al. 2007 ) . The remainder of this chapter will discuss both the protective effects of PC on cells of the NVU and how the cells of the NVU collectively mediate PC-induced protection, independent of the nature of the preconditioning stimulus. It is this “back-and-forth” communication between neurons and microvessels that underlies the physiological function of the NVU. Specifi cally, the physical coupling of astrocytes to both endothelial cells and neurons suggests the capability of bidirectional communication between neurons and microvessels (del Zoppo 2009b ) . That such bidirectional communication also in fl uences outcome is re fl ected by studies of ischemic tolerance that demonstrate enhanced survival of neurons in parallel to bene fi cial effects of PC on endothelium (Table 21.1 ) and/or astrocytes (Table 21.2 ).

21.3.1 Acute Vascular In ﬂ ammation

As mentioned previously, early sequelae of ischemic injury include upregulation of a host of proin fl ammatory mediators, anatomical disruption of the BBB, and recruitment of leukocytes from blood into brain parenchyma. Considerable evidence has accumulated indicating that preconditioning leads to epigenetic responses that counter several postischemic vascular infl ammatory sequelae. As examples, brief ischemic PC, 3 days prior to transient middle cerebral artery occlusion (tMCAo), attenuates the message-level expression of a number of proin fl ammatory molecules by as much as 90% at 24 h of reperfusion (Bowen et al. 2006 ) . The ischemia- induced upregulation of endothelial ICAM-1 (An and Xue 2009 ; Andjelkovic et al. 2003) and vascular cell adhesion molecule-1 (VCAM-1) is inhibited by PC in endothelial cell cultures (An and Xue 2009 ) , a phenotype also documented in vivo in a sevo fl urane PC model (Yu et al. 2011 ) . Reductions in the poststroke elaboration of infl ammatory mediators correlate with decreases in the total number of OX42/Cd11b + macrophages, neutrophils, and activated microglia that diapedese into the ipsilateral cortex (Bowen et al. 2006 ) . Exercise PC virtually eliminates the number of diapedesed leukocytes in the ipsilateral hemisphere when measured 2 days after reperfusion, in conjunction with reducing the number of ICAM-1-positive microvessels (Ding et al. 2005 ) , while preconditioning with dietary fenofi brate, an activator of nuclear PPAR- a receptors, 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 461 (continued) delivery and consumption delivery 2 , ICAM-1 expression , ICAM-1 expression a prior to stroke. PC reduced prior to stroke. a induced vasodilation in ex vivo MCA vivo ex in induced vasodilation edema, BBB permeability pharmacologic absence of histamine; PC enhances via histamine VEGF expression through 2 week poststroke expression permeability, MMP-9 activity MMP-9 activity permeability, ipsilesional CBF ZO-1, minimized ICAM-1 and VCAM-1 expression factors TNF- IV, poststroke edema; reduced IV and poststroke prior to stroke; expression integrin poststroke preserved PC decreased cell mortality, ICAM-1 expression ICAM-1 expression PC decreased cell mortality, cit, in genetic or fi neurologic de PC did not decrease IV, PC reduced IV in WT, but not KO, mice not KO, but PC reduced IV in WT, 3, BMEC WT nNOS KO, WT nNOS KO, OGD OGD Mouse bEND. 3 × 10-min tMCAo tMCAo Rat increased P450 2 C11; no change in PC decreased IV; LPS pMCAo Rat (SHR) increased local CBF 24 h post-pMCAo PC decreased IV, 5-min tMCAo pMCAo Mouse, eNOS and Hyperoxia tMCAo Rat Intermittent and continuous hyperoxia reduced IV, 2003 LPS tMCAO Rat PC reversed ischemia-induced impairment of Ach- LPS pMCAo Rat (SHR) and eNOS CBF, enhanced peri-infarct PC reduced IV, 10-min tMCAo tMCAo Rat (SHR) PC decreased IV, in fl ammatory mRNA, transcription OGD OGD Rat BMEC PC preserved cell viability, maintained membrane-bound exercise Treadmill tMCAo Rat on EC and astrocytes expression integrin PC upregulated Electroacupuncture tMCAo Rat PC decreased IV, neurologic de fi cit, edema, BBB Treadmill exercise tMCAo Rat PC upregulated TNF- Hypoxia tMCAo Mouse, HDC KO, Iso fl urane pMCAo Rat PC increased ipsi rCBF, O 2002 1999 2003 2007 2007 2005 2006 2009 2009 2005 2006b Vascular-based protection 2011 2010 Atochin et al. Bigdeli et al. et al. Bowen Furuya et al. Fan et al. Fan Dawson et al. Dawson Table Table 21.1 Reference Alkayed et al. An and Xue et al. Andjelkovic PC method et al. Takeda Injury model Animal/cell lines Chi et al. Results Ding et al. Ding et al. Dong et al. 462 A.M. Stowe and J.M. Gidday prior to stroke; PC decreased mortality and cell death prior to stroke; after H/I via eNOS CEC and neurons; PC blocked by PI-3 K inhibition CEC and neurons; PC blocked induced angiogenesis; NOS inhibition was without induced angiogenesis; NOS inhibition was on CBF response effect and increased CBF, PC decreased IV, poststroke, and after No PC in iNOS KOs vasoreactivity. peroxynitrite is pharmacologically decomposed viability by VEGF-induced CREB phosphorylation BBB disruption, edema decreased poststroke stroke; VEGF, ROS, NO, NFkB binding, in fl ammatory fl NO, NFkB binding, in ROS, VEGF, not HO-1, increase after PC mediators, but tolerance despite nNOS or iNOS inhibition by arterial spin labeling/MRI BBB disruption; increased postischemic ZO-1 and occludin expression PC increased ROS via NADPH oxidase prior to stroke; oxidase prior to stroke; via NADPH PC increased ROS nox2 KO, WT nox2 KO, Newborn rat Newborn not nNOS or iNOS, PC increased pAkt and eNOS, but Newborn rat Newborn pro-angiogenic genes, upregulated CBF, PC preserved rat Newborn PC decreased BBB disruption, loss of MAP2+ cells Newborn rat Newborn PC decreased brain injury via eNOS; PC-induced Newborn rat Newborn in CECs PC increased HSP72 expression ischemia ischemia ischemia ischemia ischemia tMCAo Mouse cit, edema, mortality, fi neurologic de PC decreased IV, hypoxia Rat PC decreased BBB disruption; inhibited increases in 2 agonist) Hypoxia Hypoxia/ bCCAo bCCAo Gerbil PC upregulated eNOS, Akt-Ser473 phosphorylation in Hyperthermia Hypoxia/ CoCl Diazoxide bCCAo Rat/rat CEC PC depolarized CEC mitochondrial membrane prior to Hypoxia Hypoxia/ 2007 2004 15-min tMCAo tMCAo Mouse PC increased ipsi rCBF during occlusion, as measured Hyperthermia Hypoxia/ LPS tMCAo Mouse, iNOS and Pam3CSK4 (TLR OGD OGD Rat CEC OGD PC protects against OGD-induced loss of CEC 2007 LPS Hypoxia/ 2008 2005 1999 (continued) 2006 1999 2007

2008 2009 2010 Table 21.1 Table Hoyte et al. Hoyte Hashiguchi et al. Sakatani et al. Lee et al. Lenzser et al. Lin et al. Kunz et al. Kunz Gustavsson et al. Gustavsson Reference Gidday et al. PC method Hua et al. Injury model et al. Ikeda Animal/cell lines Results Kalpana et al. 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 463 (continued) diapedesis in ipsilateral cortex, as well as monocyte as well monocyte diapedesis in ipsilateral cortex, in blood activation bCCAo in CEC eNOS expression contraction and acetylcholine-induced relaxation of isolated carotid arteries adherence, diapedesis, BBB disruption and eNOS activity increased post-SAH NO levels before and after SAH; tolerance lost in eNOS-null treatment prior to hypoxic PC mice or with L-NAME cit, via fi neurologic de PC decreased IV, stroke, SphK2 BBB disruption, HSP70 expression in CEC BBB disruption, HSP70 expression disruption and reduced impairments diapedesis, ICAM-1, ROS, in endothelial-dependent relaxation reduced endothelial desquamation and edema; by NOS inhibition tolerance blocked PC decreased IV, activated microglia, and neutrophil activated PC decreased IV, PC decreased vasospasm, neurologic de fi cit; PC fi neurologic de PC decreased vasospasm, and WT) ouse rolling, cit, leukocyte fi neurologic de PC decreased IV, Perinatal rat PC abrogated the reduction in potassium-mediated Mouse/rat CEC BBB PC decreased CEC death and reduced in vivo Rat postischemic rCBF, Remote or local PC improved ere asphyxia loading ischemia MCAo/pMCAo M MCAo/pMCAo ypoxia t ypoxia LPS tMCAo Mouse bCCAo bCCAo Gerbil 6 h after brain regions PC increased rCBF in several Hypoxia SAH Mouse (eNOS KO 3-min tMCAo/LPS tMCAo Rat LPS PC requires NO, leads to high PC reduced IV; tMCAo pMCAo Rat PC decreased IV, neurologic de fi cit, peri-infarct edema, Brief asphyxia Sev Hypoxia tMCAo Mouse post SphK2 CEC protein prior to stroke; PC upregulated 2004 Brief ischemia Forebrain Dipyridyl pMCAo/iron Repetitive h 2006 2011 Feno fi brate tMCAo Rat PC decreased IV, microglia activation, neutrophil 2000 2001 2010 2009 2005 2008 2011 2009 Stowe et al. Stowe Ouk et al. Rosenzweig et al. et al. Vellimana Strackx et al. et al. Vlasov Reference Masada et al. Methy et al. Nakamura et al. PC method Puisieux et al. Injury model Animal/cell lines Results et al. Wacker 464 A.M. Stowe and J.M. Gidday

NADPH cerebral CEC inducible NOS, oxygen- glucose brain microvascular vascular endothelial vascular iNOS OGD VEGF BMEC knockout, knockout, subarachnoid hemorrhage, KO SAH nitric oxide synthase, channel agonist NS1619 and blocked by channel agonist NS1619 and blocked Ca NOS channel antagonist PX Ca expression and enzymatic activity and enzymatic activity expression and decreased AIF increased pAkt and p-survivin, translocation from mitochondria to nucleus BK junction and adherens junction protein expression in junction and adherens protein expression a sphingosine kinase-2-dependent manner and hypoperfusion hyper- and of astrocytes activation apoptosis, degeneration, mimicked effects Ethanol PC protective monocytes. by BK delayed fashion MMP9, and reversed loss of occludin MMP9, and reversed matrix metalloproteinase, bilateral common carotid artery occlusion, regional cerebral blood ﬂ ow, ow, ﬂ cerebral blood regional MMP rCBF bCCAo blood–brain barrier, blood–brain barrier, apoptosis-inducing factor apoptosis-inducing factor vascular cell adhesion molecule, vascular wild-type, AIF BBB WT VCAM infarct volume, volume, infarct IV sphingosine kinase 2, reactive oxygen species, reactive preconditioning, SphK2 ROS nuclear factor kappa-light-chain-enhancer of activated B cells, kappa-light-chain-enhancer of activated nuclear factor PC spontaneously hypertensive rat, spontaneously hypertensive intracellular adhesion molecule, permanent middle cerebral artery occlusion, NFkB SHR ICAM pMCAo toll-like receptor, receptor, toll-like TLR endothelial NOS, zonula occludin-1, eNOS

10-min tMCAo pMCAo Rat (SHR) CBF in a PC decreased IV and increased peri-infarct knockout, knockout, lipopolysaccharide, Hypoxia OGD Human CEC PC decreased OGD-induced loss of CEC viability, histidine decarboxylase, bCCAo pMCAo Rat PC decreased edema, BBB disruption, and both MMP9 KO ZO-1 2006 Brief bCCAo Ethanol/NS1619 bCCAo bCCAo Rat Mouse PC reduced Postconditioning leukocyte reduced rolling/adherence, the neuronal extent of postischemic Hypoxia tMCAO Mice PC reduced BBB permeability to IgG, altered tight LPS ﬂ urane Sevo tMCAo Rat PC protected BBB, reduced adhesion molecules, MMP2, (continued) HDC 2006 2007a 2008 2010 2002

2011 transient middle cerebral artery occlusion, neuronal NOS, Zhang et al. Zhao and Nowak Yu et al. Yu Table 21.1 Table Reference Kato et al. et al. Wang et al. Wang PC method Injury model Animal/cell lines Results tMCAo endothelial cells, Zhang et al. deprivation, deprivation, growth factor, factor, growth endothelial cells, nNOS nicotinamide adenine dinucleotide phosphate-oxidase, 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 465 (continued) PC decreased ,

a astrocytes upregulate more HIF-2, which is required for EPO upregulate astrocytes production to protect neurons AQP4 on astrocyte end-feet on astrocyte AQP4 increased GFAP/astrogliosis cell death, improved barrier indices, and blocked loss TJ barrier indices, and blocked cell death, improved proteins via astrocytes via HO-1; increased Akt signaling HIF-1 of activated PC decreased neuronal death and levels microglia together, exhibited synergistic protection synergistic exhibited together, microglia of tolerance; minimized activated tolerant hippocampi exhibited at 2 days poststroke, stroke; increased Jun immunoreactivity neurons and astrocytes neurons and astrocytes decreased cytokines, IV, neurologic de ﬁ cit via GLT-1 cit via GLT-1 ﬁ neurologic de IV, decreased cytokines, PC upregulates VEGF, EPO more in astrocytes than neurons; EPO more in astrocytes VEGF, PC upregulates PC decreased IV and BBB breakdown; in vitro PC decreased IV and BBB breakdown; astrocytes astrocytes astrocyte astrocyte cocultures Rat astrocytes Rat astrocytes fragmentation DNA cleavage, PC decreased cell death, PARP

3 O 2 Mouse astrocytes PC increased cell viability and VEGF secretion; stabilized 2 As O 2 OGD Mouse astrocytes PC by hyperthermia or ginkgolides alone decreased cell death; ginkgolides Brief tMCAo/OGD pMCAo/OGD Mouse/CEC and Hypoxia OGD WT, KO mouse bCCAo bCCAo Rat PC alone moderately increased activated microglia; poststroke, 3NP pMCAo Rat PC decreased edema and postischemic AQP4 immunoreactivity;

bCCAo bCCAo Gerbil PC increased hippocampal Jun-related immunoreactivity prior to 10 min tMCAo × 3 tMCAo Rat in HSP70 upregulated PC 2, 3, 5 days prior decreases IV; bCCAo bCCAo Gerbil PC increased GFAP-positive hippocampal astrocytes in regions EPO NO, staurosporine, Ceftriaxone DFO, EDHB tMCAo H Rat PC Poststroke, prior to stroke; expression PC increased GLT-1 Thrombin tMCAo Mouse not BBB disruption; increases PC decreased IV and edema, but Hypothermia/ 2011 2006 2011 1996 1994 1995b Glial-based protection 2005 1995a 2007 2010 2009 2011 Diaz et al. Chen et al. Hoshi et al. Kato et al. Hirt et al. Table Table 21.2 Reference et al. Chavez PC method Injury model Du et al. Animal/cell lines Results Gesuete et al. Kato et al. Kato et al. Chu et al. Chu et al. 466 A.M. Stowe and J.M. Gidday

Cx43 ethyl- bilateral EDHB erythropoietin, bCCAo permanent middle EPO pMCAo desferrioxamine, DFO cerebral endothelial cells, heat shock protein, CEC uptake, ef fl ux ATP; in vivo, PC increased Cx43 in vivo, ux ATP; fl ef uptake, 2+ HSP vascular endothelial growth factor, factor, endothelial growth vascular glucose transporter-1, glucose transporter-1, required Ca decreased astrocyte death via EETs decreased astrocyte microglia poststroke of GLT-1 via upregulation immunoreactivity proliferation in striatum and neocortex, contributing to contributing proliferation in striatum and neocortex, tolerance not GLUT3 but and decreased IV in WT only via adenosine VEGF a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, In vitro, PC increased survival, Cx43; Cx43 hemichannel In vitro, PC increased survival, PC increased HIF-1, P450 2 C11 prior to OGD; post-OGD, glutamate uptake PC decreased cell death, increased astrocytic PC enhanced 2-DG uptake, and GLUT1 mRNA on astrocytes, on astrocytes, and GLUT1 mRNA PC enhanced 2-DG uptake, GLUT1 AMPA heme oxygenase-1, Cx43/ glial fi brillary acidic protein, fi glial HO-1 mouse, WT Cx30 KO, astrocytes astrocytes astrocyte cocultures astrocyte astrocyte coculture infarct volume, volume, infarct GFAP IV Rat astrocytes/ hypoxia-inducible factor, hypoxia-inducible factor, 3-nitropropionic acid 3-nitropropionic acid glucose transporter-3, glucose transporter-3, HIF / 3NP 2 aquaporin, O 2 GLUT3 AQP preconditioning, knockout, PC tMCAo KO staurosporine, poly (ADP-ribose) polymerase, STS PARP 2-deoxyglucose, wild-type, WT 2-DG glutamate transporter-1, glutamate transporter-1, pMCAo Rat and reduced accumulation of CD11b-positive PC reduced IV, a nitric oxide, GLT-1 NO Hypoxia TNF- OGD/rosiglitazone OGD OGD bCCAo Cultured rat Rat neuron/ bCCAo Rat PC decreased hippocampal cell death; increased GLT-1 epoxyeicosatrienoic acid, Tamoxifen/hypoxia Tamoxifen, H bCCAo bCCAo Gerbil PC increased extent of postischemic microglia/macrophage fl urane Sevo Repetitive hypoxia Anoxia tMCAo Rat neuron/ Rat Reduced astrocyte and microglial activation EET (continued)

2008 2001 2011 2008

transient middle cerebral artery occlusion, oxygen-glucose deprivation, oxygen-glucose deprivation,

2005 1997 2007 2007b Table 21.2 Table common carotid artery occlusion, 43, connexin Nawashiro et al. Nawashiro Romera et al. et al. Yu OGD tMCAo 3,4-dihydroxybenzoate, Liu and Alkayed et al. Yu Zhang et al. Reference Lin et al. PC method Injury model Animal/cell lines Results Liu et al. cerebral artery occlusion, 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 467 reduces endothelial ICAM-1 expression and neutrophil diapedesis in the ischemic area (Ouk et al. 2009 ) . Attenuation of poststroke leukocyte rolling and adherence in cortical microvessels by ethanol PC is mediated by large conductance, calcium- activated potassium (BK Ca) channels, present on all cells of the NVU, although specifi c activation of these channels on nonneuronal NVU cells may serve to minimize ROS and infl ammatory cytokine production (Wang et al. 2010 ) . Our previous work also documents a signifi cant decline in leukocyte rolling and adherence to postcapillary venules, as well as diapedesis into the parenchyma, 24 h after tMCAo when this ischemic event is preceded by a repetitive hypoxic PC stimulus, in conjunction with complete attenuation of BBB disruption (Stowe et al. 2011 ) . While acute phase infl ammation is considered detrimental during the initial hours and days following stroke onset, milder infl ammatory responses may contribute to various PC stimuli-induced changes in gene expression that establish the protective phenotype (Bowen et al. 2006) (see also Chap. 18 by Vartanian). In fact, several successful PC stimuli that protect the NVU either upregulate, or are themselves, proinfl ammatory mediators of poststroke injury, whereas the magnitude of their elaboration poststroke is often reduced in ischemia-tolerant brains. One of the most well-studied PC stimuli is lipopolysaccharide (LPS), a potent inducer of CNS leukocyte diapedesis in high doses (Puntambekar et al. 2011 ) . The ability of LPS to drive the gene expression necessary for ischemic tolerance is thought to derive from its ability to stimulate ROS production by NADPH oxidase, in neurons and endothelial cell—but not astrocytes (Kunz et al. 2007) , to activate toll-like receptors (Marsh et al. 2009; Stevens et al. 2008 ) , to increase endothelial nitric oxide synthase (eNOS) expression (Furuya et al. 2005; Lin et al. 2010; Puisieux et al. 2000 ) , to increase Akt levels (Lin et al. 2010 ) , and to activate microglia (Rosenzweig et al. 2004 ) . LPS PC leads to reductions in the number of activated microglia and neutrophils in the ipsilateral hemisphere, as well as monocyte and neutrophil activation in peripheral blood, following transient focal stroke (Rosenzweig et al. 2004 ) . TNF-a mRNA and protein are upregulated by exercise PC in all cortical and subcortical regions over the course of 2–3 weeks of intervention, but exercise PC ultimately minimizes the upregulation of TNF- a or ICAM-1 expression poststroke (Ding et al. 2005 ) . Even histamine, fundamentally known to all allergy suf- ferers, acts to trigger the protective effects of hypoxic PC, as mice null for the histamine-synthesizing enzyme histidine decarboxylase fail to establish tolerance to tMCAo (Fan et al. 2011 ) .

21.3.2 BBB Disruption

As mentioned above, while proin ﬂ ammatory mediators and leukocytes exacerbate poststroke BBB disruption, stroke-induced injury also includes an anatomical disas- sembling of BBB structure that can be blocked by prior PC (Bigdeli et al. 2007 ; Ding et al. 2006b; Dong et al. 2009; Hoshi et al. 2011; Hua et al. 2008; Ikeda et al. 1999 ; Kalpana et al. 2008 ; Lenzser et al. 2005 ; Masada et al. 2001 ; Stowe et al. 468 A.M. Stowe and J.M. Gidday

2011; Vlasov et al. 2005 ; Yu et al. 2011; Zhang et al. 2006 ) . This barrier-protective phenotype is achieved in a number of rodent stroke models, despite the variety of PC stimuli that promote it, including hypoxia (Stowe et al. 2011 ; Wacker et al. 2009 ) , intermittent normobaric hyperoxia (Bigdeli et al. 2007 ) , the mitochondrial ATP-sensitive K+ channel activator diazoxide (Lenzser et al. 2005 ) , exercise (Ding et al. 2006a ) , electroacupuncture (Dong et al. 2009 ) , toll-like receptor ligands (Hua et al. 2008 ) , hyperthermia (Ikeda et al. 1999 ) , the iron chelator dipyridyl (Methy et al. 2008 ) , sevo fl urane (Yu et al. 2011 ) , and brief ischemic PC (Gesuete et al. 2011; Masada et al. 2001 ; Vlasov et al. 2005 ; Zhang et al. 2006 ) . The molecular basis whereby these different PC stimuli ultimately lead to this vasculoprotective effect still requires further elucidation, but some clues have emerged. OGD PC in cultured brain microvessel endothelial cells (BMEC) preserves membrane-bound ZO-1 expression at the tight junctions and minimizes F-actin stress fi ber formation to maintain the integrity of the endothelial cell (An and Xue 2009) . Preservation of pre- and postischemic tight junction protein expression and/or localization has also been noted in vivo in response to various PC stimuli (Hua et al. 2008 ; Wacker et al. 2012 ; Yu et al. 2011) . PC minimizes the poststroke upregulation of MMP-9, which contributes to basal lamina degradation in nonpre- conditioned animals (Dong et al. 2009 ; Yu et al. 2011 ; Zhang et al. 2006 ) . Modulation of aquaporin channels (Hoshi et al. 2011 ) , increases in sphingosine- 1-phosphate (Wacker et al. 2012 ) , alterations in endothelial HSP70 expression levels (Masada et al. 2001 ) , and reductions in NFk B and ROS (Kalpana et al. 2008) are also implicated in PC-induced protection. The involvement of NO in regulating vascular permeability and the extent to which NO is implicated in PC-induced ischemic protection in both neonates and adults (Atochin et al. 2003 ; Furuya et al. 2005; Gidday et al. 1999; Hashiguchi et al. 2004 ; Kunz et al. 2007 ; Lin et al. 2010 ; Puisieux et al. 2000 ; Vlasov et al. 2005 ; Vellimana et al. 2011 ) suggest this mediator participates in the improvements in BBB integrity that characterize the tolerant brain, but studies have yet to directly test this hypothesis. Ultimately, areas of BBB disruption strongly correlate to areas of neuronal injury during stroke recovery (Ikeda et al. 1999) , suggesting that the ability of preconditioning to preserve BBB integrity could have benefi cial effects on neuronal survival. The specifi c contributions of PC-treated astrocytes to BBB preservation are individually addressed below.

21.3.3 Postischemic Vasoreactivity, Autoregulation, and Cerebral Blood Flow

Following focal stroke, an overall reduction in ipsilateral rCBF occurs; a prolonged period hypoperfusion also follows global ischemia. Although the mediators of these responses are multifactorial, a downregulation of NO production in the hours following ischemia contributes importantly to this phenotype (for reviews, see (Faraci 2003 ; Laude et al. 2002) ). Augmentation of NO levels can preserve tissue at risk of ischemic 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 469 injury, as NO mediates cerebrovascular dilation as a counter to myogenic tone, inhibits leukocyte adherence and diapedesis as well as platelet aggregation, and upregulates pro-survival transcription factors. In the CNS, NO is made by three isoforms of nitric oxide synthase (NOS), inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS). Evidence from several studies implicates eNOS as the primary mediator for PC-induced upregulation of NO production. In particular, LPS PC enhances eNOS expression prior to (Furuya et al. 2005 ; Puisieux et al. 2000 ) and after stroke (Furuya et al. 2005) , concomitant with an attenuation in the extent of peri-infarct blood fl ow reduction over the initial days poststroke (Furuya et al. 2005 ) . Ischemic PC upregulates hippocampal eNOS predominantly in endothelial cells (Lin et al. 2010 ) , although also transiently in hippocampal neurons (Hashiguchi et al. 2004) . Furthermore, hypoxic PC-induced tolerance to ischemia in newborn rat pups involves eNOS-derived NO (Gidday et al. 1999) , as does tolerance to subarachnoid hemorrhage (SAH)-induced vasospasm in adult mice (Vellimana et al. 2011 ) . In confi rmation, the genetic or pharmacologic inhibition of eNOS blocks PC-induced protection (Atochin et al. 2003 ; Gidday et al. 1999 ; Hashiguchi et al. 2004 ; Lin et al. 2010) , which, at least in the neonatal rat brain, is unaffected by either iNOS or nNOS inhibition (Gidday et al. 1999 ; Lin et al. 2010 ) . Perhaps the mechanistic involvement of eNOS depends to some extent on the PC stimulus, age, or type of ischemic insult, or it could be that the production of NO from other NOS isoforms is important under some conditions, since not all studies support a proprietary role for eNOS-derived NO in PC-induced tolerance. In fact, several investigations provide evidence supporting the involvement of iNOS. For example, LPS PC, which induces eNOS in the aforementioned studies (Furuya et al. 2005 ; Puisieux et al. 2000 ) , salvages CBF in peri-infarct regions during ischemia in wild-type mice but not in iNOS nulls (Kunz et al. 2007 ) . Moreover, in rats, inhibition of iNOS during iso fl urane PC reverses the improvement in rCBF measured in the ischemic hemisphere following pMCAo (Chi et al. 2010 ) . Neither iNOS knockout mice nor wild types treated with the putative iNOS inhibitor aminoguanidine exhibit the PC-induced preservation of postischemic functional hyperemia observed in untreated wild types (Kunz et al. 2007 ) . Finally, in neonatal rats, inhibition of NOS was without effect on the ability of hypoxic PC to improve peri-ischemic CBF (Gustavsson et al. 2007 ) , suggesting an NO-independent mechanism for establishing tolerance, not requiring any NOS isoform. The production of ROS in ischemia, an important contributor to the dysregulation of NVU functioning, is lessened in the preconditioned brain (for review, see Busija et al. 2008 ) . Similar to the induction of proin fl ammatory cytokines described above, hypoxic PC initially requires the formation of free hydroxyl radicals in the brain to serve as a proximal triggers for induction of the protective phenotype (Boy et al. 2011 ) . Mild oxidative stress, mediated through increased NADPH oxidase activity, is also required for successful ethanol PC to upregulate signaling mechanisms for establishing tolerance (Becker 2002 ) . In effect, the levels of ROS production in response to the PC stimulus are substantially lower than those occurring in response to the primary ischemic event. ROS scavengers can also play a role in establishing the tolerant phenotype, in that focal ischemic PC increases superoxide 470 A.M. Stowe and J.M. Gidday dismutase (SOD) in conjunction with a reduced infarct volume following pMCAo (Wacker et al. 2012 ) . Dietary feno fi brate PC for 2 weeks prior to stroke reduces poststroke levels of ROS, which also serves to increase the bioavailability of NO to maintain vasoreactivity in the cerebral endothelium (Ouk et al. 2009 ) . As with proinfl ammatory mediators, some ROS production may be necessary for establishing recovery mechanisms. In fact, NO and free radicals may work in concert during preconditioning to increase vasodilation and trigger protective signaling mechanisms that promote improved CBF both during and after subsequent severe ischemia (Kunz et al. 2007 ; Laude et al. 2002 ) . The upregulation of NO, downregulation of ROS, and preservation of the BBB may contribute to improvements in intra- or postischemic CBF in preconditioned animals. With respect to intra- or end-ischemic CBF, hypoxic PC attenuates the extent of ipsilateral CBF reduction during the ischemic insult both in the hypoxia/ ischemia newborn rat pup model (Gustavsson et al. 2007 ) and in adult mice with tMCAo (Fan et al. 2011) , the latter of which is mediated by the PC-induced production of vascular endothelial growth factor (VEGF). Brief ischemic PC improves intra-ischemic rCBF during tMCAo in mice, as measured with perfusion-weighted MRI, which correlates to a smaller infarct volume at 24 h (Hoyte et al. 2006 ) . However, improvements in intra-ischemic fl ow are not a universal fi nding: In rat tMCAo, intra-ischemic rCBF, as determined by iodoantipyrine autoradiography, is not affected by prior brief ischemic PC (Alkayed et al. 2002 ) . The effects of LPS PC on levels of ischemic CBF are also controversial: In one study, no effect was noted 15 min after MCAo (Dawson et al. 1999 ) , but in another, intra-ischemic CBF was higher in preconditioned mice, a phenotype not observed in iNOS nulls (Kunz et al. 2007 ) . In addition, iso fl urane PC increases both oxygen delivery and oxygen consumption within the ischemic hemisphere, suggesting a higher amount of viable cells utilizing oxygen due to higher rCBF and increased oxygen supply (Chi et al. 2010 ) . Hypoxic PC vasodilates main cerebral arteries by 8% and leptomeningeal anastomoses by 27%, without alteration in vessel wall anatomy (Woitzik et al. 2006 ) , although how this increase in perfusion prior to stroke onset contributes to the preservation of intra- and/or postischemic CBF in animals with PC remains unclear. With respect to PC’s effects on postischemic CBF, in rat models of pMCAo, isofl urane PC signifi cantly increases rCBF in the focal ischemic area at one (Chi et al. 2010) , three (Zhao and Nowak 2006) , and 24 h (Dawson et al. 1999 ) after ischemic onset. Peri-infarct rCBF is enhanced in several hippocampal sub fi elds, as well as in several other regions of the gerbil brain, 6 h after global ischemia when animals are preconditioned (Nakamura et al. 2006 ) . Finally, the magnitude of both postischemic reactive hyperemia and postischemic hypoperfusion following global forebrain ischemia in rats is abrogated by an ischemic postconditioning protocol (Wang et al. 2008) . Despite possible variations in induction pathways for the improvements in CBF documented to occur early following PC-induced ischemic tolerance, at least one study demonstrates that enhancements in peri-infarct CBF do not appear to be transient but can persist through 2 weeks following pMCAo (Furuya et al. 2005 ) . 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 471

Improvements in postischemic vascular reactivity also characterize the ischemia-tolerant brain. In mice, the impaired postischemic functional responses to whisker stimulation, as well as pial arteriolar dilation to hypercapnia and topical acetylcholine, were prevented by LPS PC (Kunz et al. 2007) . Impaired endothelium-dependent dilations to acetylcholine were also abrogated in middle cerebral arteries isolated from rats preconditioned with LPS (Takeda et al. 2007) or fenoﬁ brate (Ouk et al. 2009 ) . A similar retention of acetylcholine-induced dilation of isolated arteries was reported for the carotid of postnatal day 4 rats subjected to brief intrauterine asphyxia PC on embryonic day 17, prior to severe perinatal asphyxia on the day of birth (Strackx et al. 2010 ) . Finally, the prolonged vasoconstriction of large arteries following SAH (i.e., vasospasm) is attenuated by hypoxic PC secondary to eNOS upregulation (Vellimana et al. 2011) , serving as yet another reﬂ ection of reduced macro- and microvascular dysfunction in the tolerant brain.

21.3.4 Angiogenesis and Vascular Remodeling

There are few investigations into the long-term effects of PC on angiogenesis, as most preclinical studies focus on the acute and subacute stages of stroke injury and repair. What we have learned to date is that, over the course of weeks, exercise PC upregulates the expression of several integrin subtypes in microvessels and adjacent astrocytes and preserves these expression patterns following stroke onset (Ding et al. 2006a) . In a separate series of endothelial cell culture experiments by the same group, it was demonstrated that the gradual administration of TNF-a to these cultures, which mimics the slow increase in TNF-a during exercise PC, alters integrin expression in a manner similar to that occurring in vivo after exercise, suggesting a proangiogenic role for TNF-a in cortical angiogenesis prior to and after stroke injury (Ding et al. 2006a ) . In another study, neonatal rats with prior hypoxic PC immediately upregulate angiogenesis-related genes, and signifi cant increases in cortical capillary volume are evident even 24 h after the PC stimulus (Gustavsson et al. 2007 ) . To promote the translation of preclinical PC stimuli to ef fi cacious clinical treatments, more experiments investigating the in fl uence of any PC stimulus on long-term recovery mechanisms within the NVU, such as angiogenesis and other neurovascular plasticity responses, are desperately needed.

21.3.5 Direct Endothelial Cytoprotection

In addition to positively affecting postischemic in ﬂ ammation, BBB breakdown, tissue perfusion and vascular reactivity, and angiogenesis, PC may exert direct cytoprotective actions on cerebral endothelial cells themselves, which may contribute directly or indirectly to the aforementioned vasculoprotective phenotypes. For example, several kinds of PC stimuli reduce endothelial desquamation in 472 A.M. Stowe and J.M. Gidday response to forebrain ischemia in rats (Vlasov et al. 2005 ) . The expression of the cytoprotective HSP72 in endothelial cells of the neonatal rat brain is implicated in hypoxic-ischemic tolerance in this model (Sakatani et al. 2007) . Mechanistically, studies in cultured cerebral endothelial cells indicate that phosphorylation of the cyclic AMP response element-binding protein (CREB) transcription factor secondary to VEGF-A/VEGFR-2 signaling induced by OGD PC protects bEnd.3 cells from OGD-induced cell death (Lee et al. 2009) , and that phosphorylation of the inhibitor-of-apoptosis (IAP) gene family member survivin, secondary to Akt activation in response to hypoxic PC, protects human BMECs from OGD-induced apoptosis (Zhang et al. 2007a ) .

21.3.6 Preconditioning and Ischemic Tolerance in Glial Cells of the NVU

To paraphrase the beginning of this chapter, neither neurons nor vascular cells participate alone in the function of the NVU, nor in its injury, protection, and recovery following ischemia. Astrocytes play a critical role in these processes, as functional contributors to the maintenance of the intact BBB, as the cellular source of paracrine-targeted molecular signals, and as metabolic way stations for neurotransmitters and neuromodulators, to name just a few. Recent evidence is emerging that astrocytes also respond to preconditioning stimuli and contribute in a positive way to the overall ischemia-tolerant phenotype (also recently reviewed by Trendelenburg and Dirnagl 2005 ; Vangeison and Rempe 2009 ) . As examples, reactive astrocytes increase after ischemia in the ischemia-tolerant hippocampus of gerbils (Kato et al. 1994 ) and also exhibit increased Jun-related immunoreactivity (Kato et al. 1995a ) . Integrins on astrocytic end-feet are upregulated by exercise PC, strengthening the adherence of end-feet processes to the basal lamina to bolster BBB integrity (Ding et al. 2006a ) . In addition, in vitro PC of astrocytes, but not endothelial cells, preserves barrier integrity and ZO-1 membrane localization following OGD (Gesuete et al. 2011 ) . Another membrane protein, connexin 43 (Cx43), which forms gap junctions in astrocytes, also forms hemichannels on the astrocytic membrane that are required for hypoxic PC-induced protection both in astrocyte cell culture and in an in vivo mouse model of transient focal stroke (Lin et al. 2008 ) . PC upregulates Cx43 hemichannels as well as their recycling, improving the ability of astrocytes to increase extracellular Ca2+ in fl ux and ATP effl ux (leading to elevated extracellular adenosine) following ischemic injury (Lin et al. 2008 ) . The anatomical location of astrocytes within the NVU also allows astrocytes to mediate water distribution across the BBB through expression of a water channel, aquaporin 4 (AQP4), on their end-feet. Thrombin PC reduces vasogenic edema following tMCAo but still allows for BBB disruption (as quantifi ed by IgG extravasation) early during reperfusion, coincident with enhanced AQP4 protein expression on astrocytic end-feet ipsilateral to the stroke (Hirt et al. 2009 ) . 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 473

Preconditioning with a succinate dehydrogenase inhibitor, 3-nitropropionic acid (3NP), increases astrocytic GFAP immunoreactivity in the ipsilateral hemisphere, concomitant with a reduction in cerebral edema; both occur in conjunction with a reduction in AQP4 expression on peri-infarct reactive astrocytes (Hoshi et al. 2011) . The authors suggest these seemingly contradictory fi ndings to those in the aforementioned thrombin PC study may be due to the ROS-generating mechanism of 3NP PC and may show that enhanced reactive astrocytosis in 3NP PC specifi cally reduces the need for AQP4 expression. Understanding the infl uence of AQP4 expression on edema formation is critical to discriminate the origin of two very distinct poststroke pathologies, vasogenic edema and BBB disruption, as both are not necessarily coincident with respect to either timing or location in areas of ischemic injury (Lo et al. 1994 ) . Also, given the heterogeneity of astrocytic responses to hypoxic PC in hippocampal astrocytes compared to cortical astrocytes (Liu and Alkayed 2005 ) , further studies investigating other responses of astrocytes to particular PC stimuli, and how they actually contribute to the subsequent NVU protective phenotype (e.g., decreased edema vs. decreased BBB disruption), are warranted. Several signaling pathways initiated in astrocytes appear to contribute to NVU protection following ischemic injury. Preconditioning by a number of stimuli leads to the stabilization of HIF-1 a expression in astrocytes, and the upregulation of VEGF (Chu et al. 2010; Gesuete et al. 2011 ) , as well as other growth factors (Gesuete et al. 2011) , possibly in an effort to provide neuroprotection to adjacent neurons (Chu et al. 2010; Kato et al. 1994) . Astrocyte-based HIF-1 transcription positively infl uences the survival of neurons in coculture (Vangeison et al. 2008 ) , while HIF-2 transcription upregulates erythropoietin (EPO) to also protect neurons in a paracrine manner (Chavez et al. 2006) . HIF-1-regulated genes include anti- apoptotic, glycolytic, and angiogenic proteins, all capable of contributing to the ischemia-tolerant phenotype. In addition, studies in cultured astrocytes indicate that brief ischemic PC leads to the HIF-1-mediated induction of P450 2C11 epoxygenase in astrocytes, which in an autocrine fashion contributes to astrocyte tolerance (Liu and Alkayed 2005 ) . Hypoxic PC increases metabolism (glucose uptake) in cocultures of rat hippocampal neurons and astrocytes, consistent with an increase in the message level of the glucose transporter GLUT1 in both astrocytes and neurons (Yu et al. 2008) . While only neurons respond to PC with elevations in HSP70 expression prior to tolerance, the fi nding that both astrocytes and neurons exhibit elevated HSP70 during ischemic tolerance suggests that astrocytic expression of this chaperone protein is causal to the overall protection (Chen et al. 1996) . Finally, PC with EPO induces protection in astrocyte cultures by upregulating heme oxygenase-1 (HO-1) mRNA and protein, as well as upregulating levels of the pro-survival kinase Akt (Diaz et al. 2005 ) . The location of astrocytes within the NVU not only allows for secretion of growth factors to promote survival but also positions them for protecting against poststroke excitotoxicity. Preconditioned astrocytes reduce glutamate excitotoxicity through the PPARg receptor-mediated upregulation of plasma membrane-bound glutamate transporter-1 (GLT-1) transporters (Romera et al. 2007 ) . The astrocytic isoform of 474 A.M. Stowe and J.M. Gidday

GLT-1 is predominantly responsible for maintaining low extracellular glutamate levels following ischemia. Preconditioning with ceftriaxone, a b -lactam antibiotic that activates GLT-1, reduces infarct volume and promotes functional recovery for weeks following stroke onset (Chu et al. 2007 ) . Con fi rming the importance of this mechanism, PC-induced protection in this model and in another using brief ischemia for preconditioning (Zhang et al. 2007b ) is inhibited by blocking GLT-1 activation in preconditioned animals. Histology following global ischemia shows that the preservation of CA1 neurons in PC-treated brains is associated with GLT-1- expressing astrocytes that are tightly wrapped around the pyramidal neurons, a morphological characteristic not found in control animals (Yu et al. 2008 ) . Collectively, these fi ndings suggest a PC-induced anatomical outgrowth of astrocytic processes to envelop neurons after ischemia in order to minimize injury secondary to the increased capacity for astrocytes to take up extracellular glutamate. While not specifi cally a part of the NVU as currently defi ned, several studies have investigated the role of other glial cell populations in PC-induced protection. With respect to microglia, brief ischemic PC activates microglia both prior to and immediately following stroke onset (Kato et al. 1995b ; Liu et al. 2001 ) ; the former may be indicative, perhaps, of a mild, PC-induced tissue “injury” (Liu et al. 2001 ) . The number of activated microglia appearing in the infarcted hemisphere of the postischemic brain are generally found to be reduced in preconditioned animals (Nawashiro et al. 1997 ; Ouk et al. 2009 ; Kato et al. 1994, 1995b; Rosenzweig et al. 2004; Yu et al. 2011) , consistent with the notion that reductions in microglial activation following the ischemic insult are another de fi ning feature of the ischemia-tolerant brain. Nevertheless, the extent to which microglia contribute to the overall protection that characterizes preconditioning-induced ischemic tolerance, and the underlying mechanisms for such an effect, requires further elucidation. Currently, only a handful of studies have investigated the effect of PC on oligodendrocytes, or oligodendrocyte precursor cells (OPCs). In one culture model of primary rat OPCs, OGD PC actually exacerbated cell death following exposure of the cell line to a lethal OGD stimulus (Deng et al. 2003 ) . It remains unclear, at present, whether the “sublethal” OGD PC employed in this study was really optimally titrated, given that any number of “stressful” PC stimuli can, when applied at a magnitude and/or duration greater than that required to induce protective epigenetic responses, actually make cells more vulnerable to subsequent injury. In a separate study in a human oligodendrocyte cell line (MO3.13), HIF-1 a gene transcription is upregulated following chemical PC, which increases the levels of survival-promoting factors including EPO, VEGF, and cellular apoptosis-inhibitory factors 1–3, ultimately affording protection against TNF-a -induced injury (Yao et al. 2008 ) . Interestingly, endothelial cell secretion of as yet unidenti fi ed factors promotes survival in neighboring OPCs in what has been termed the “oligovascular niche” (Arai and Lo 2009 ) . As an example of this, endothelial cells in culture enhance OPC proliferation, without affecting differentiation, and provide protection from OGD- induced injury via the pro-survival SRC-Akt signaling pathways in OPCs (Arai and Lo 2009) —additional evidence of the infl uence of the NVU on cellular survival after stroke-induced ischemic injury (Table 21.3 ). 21 Preconditioning the Neurovascular Unit: Tolerance in the Brain’s Nonneuronal Cells 475 glutamate receptor, GluR in fl ux into cells via AMPA/ fl in 2+ -induced upregulation of HIF-1 and -induced upregulation a vascular endothelial growth factor factor endothelial growth vascular VEGF new protein synthesis (EPO, VEGF), improved survival survival protein synthesis (EPO, VEGF), improved new kainate receptor; decreased GluR2 erythropoietin, EPO a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA hypoxia-inducible factor, hypoxia-inducible factor, HIF Human MO3.13 PC enhanced TNF- a oligodendrocyte precursor, precursor, oligodendrocyte TNF- OPC 2 tumor necrosis factor, tumor necrosis factor, TNF OGD OGD or excitotoxicity Rat OPC PC increased cell death, Ca DFO/CoCl 2003 Oligodendrocyte precursor cell-based protection 2008 oxygen-glucose deprivation, oxygen-glucose deprivation, desferrioxamine, OGD DFO Reference Deng et al. PC method Injury model Animal/cell lines Results Table Table 21.3 Yao et al. Yao 476 A.M. Stowe and J.M. Gidday

21.4 Summary and Conclusions

It is widely accepted that injury from stroke does not affect just one type of cell or involve one pathogenetic mechanism; in turn, preconditioning should also be recognized as a stimulus that induces epigenetic changes in all resident brain cells (not to mention circulating and inﬁ ltrating immune cells) that, acting together, contribute to the ischemia-tolerant state. Thus, the variety of proinﬂ ammatory, oxidative, excitotoxic, apoptotic, and perfusion-based mechanisms that act in concert on all the cells of the neurovascular unit to cause ischemic damage are, individually and collectively, uniquely affected by prior preconditioning. Understanding the molecular- genetic basis of these adaptive changes in vascular and glial cell populations, in addition to those operative in neurons, is required to fully appreciate, and leverage therapeutically, the full extent of endogenous cerebroprotection.

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Cherine H. Kim , Han Chen , and John H. Zhang

22.1 Introduction

The delicate and complex architecture of the brain presents signi fi cant challenges for neurosurgery. Beneath the skull and meninges, the brain is highly susceptible to mechanical injury. Incision, retraction, and electrocauterization—all necessary neurosurgical maneuvers—can cause collateral damage to healthy tissue at the periphery of the operative site. Brain edema and hemorrhage are serious complications that commonly develop following neurosurgical procedures and may lead to further injury from hypoperfusion or cell death (Bruder and Ravussin 1999 ) . Currently, surgical brain injury (SBI) is not specifi cally treated but is rather left to heal on its own. The complications of SBI may also hinder therapeutic approach. The current medicolegal climate has led to defensive medical practice by physicians in high-risk specialties. In a study published in the Journal of the American Medical Association, nearly 75% of polled neurosurgeons admitted to avoiding certain procedures or high-risk patients (Studdert et al. 2005 ) . Diminishing perioperative risks could expand the possibility of more aggressive surgical interventions. In addition, with the rising costs of medical care, any measures to simplify perioperative care would be invaluable. Even neurosurgical patients without life-threatening complications must be monitored closely in the critical care unit. Thus, the benefi ts of limiting complications of SBI extend beyond improving morbidity and mortality.

C. H. Kim (*) School of Medicine , Loma Linda University , Loma Linda , CA , USA e-mail: [email protected] H. Chen Department of Neurological Surgery , University of New Mexico , Albuquerque , NM , USA e-mail: [email protected] J.H. Zhang Loma Linda University, Loma Linda, CA, USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 485 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_22, © Springer Science+Business Media New York 2013 486 C.H. Kim et al.

While preconditioning strategies have demonstrated promising neuroprotective effects for stroke and traumatic brain injury, they are of limited clinical use because these injuries are not anticipated. In contrast, the predictable nature of SBI offers the opportunity to establish preemptive measures. To date, developing less invasive surgical methods has been the predominant strategy for limiting SBI, with relatively little research focused on understanding its biochemical pathophysiology. Thus, therapeutic strategies for SBI have yet to be adequately explored. In this chapter, we will describe SBI’s animal model, review its pathophysiology, and discuss potential preconditioning therapies for SBI.

22.2 Surgical Brain Injury Model

Despite its widespread impact and predictability, SBI is clinically addressed by rather nonspeci fi c postoperative care. In order to facilitate the study of speci fi c treatments for SBI, the in vivo model for SBI was recently developed by John H. Zhang and colleagues (Matchett et al. 2006 ; Jadhav et al. 2007a ; Jadhav and Zhang 2008 ) . This rodent model was designed to mimic surgically induced brain injury. In brief, the rodent brain is exposed in a small window in the cranium through which tissue resection is performed. The margins of resection are delineated by distance from the bregma, as shown in Fig. 22.1 . This partial right front lobectomy simulates the injuries sustained by neural tissue from standard neurosurgical maneuvers. The model provides for a consistently measureable edema by brain water content in perilesional tissue and neurological defi cits following SBI. The model mimics injuries characteristic of general neurosurgical procedures, producing reproducible brain tissue loss and injury that is representative of routine neurosurgeries in clinical practice and provides a platform from which to study the molecular mechanisms involved in SBI pathophysiology as well as potential therapeutic agents.

Fig. 22.1 Partial right frontal lobectomy. Two incisions ( red lines) are made leading away from the bregma (white ×) along the sagittal and coronal planes 2 mm lateral and 1 mm proximal to the sagittal and coronal sutures, respectively 22 Preconditioning for Surgical Brain Injury 487

22.3 Pathophysiology of Surgical Brain Injury

Surgical brain injury is caused by both primary and secondary injury mechanisms. Primary injury, in ﬂ icted directly by mechanical forces at the time of injury, is largely inescapable and irreversible. Secondary injury entails a cascade of cellular and metabolic disruption triggered by the primary insult. Typical biochemical disturbances of secondary injury involve the generation of toxic and inﬂ ammatory molecules, such as reactive oxygen species, prostaglandins, and cytokines. The principal complications that result from such mechanisms after SBI are brain edema, cell death, and hemorrhage.

22.3.1 Brain Edema

Localized brain edema has been shown to be a major postoperative complication of SBI. Many rodent studies indicate that the brain water content of tissue surrounding the resection site is increased by approximately 3% during the fi rst three postoperative days and gradually resolves by a week after surgery (Matchett et al. 2006 ; Yamaguchi et al. 2007) . This sequence was further supported with measured apparent diffusion coef fi cients by magnetic resonance imaging (Matchett et al. 2006 ) . Brain edema is believed to develop from both vasogenic and cytotoxic mechanisms. In vasogenic edema formation, loss of BBB integrity causes extravasation of proteins from the vascular compartment and subsequent fl uid accumulation in brain tissue. Cytotoxic edema is formed by fl uid accumulation in cells from improper ion balance. Brain edema is followed by increased intracranial pressure, which may lead to local ischemia, herniation, and cell death. SBI leads to abnormal extravasation of proteins from the vasculature, suggesting loss of BBB integrity (Matchett et al. 2006 ; Jadhav et al. 2007a, b; Jadhav and Zhang 2008 ; Lee et al. 2008a ; Bravo et al. 2008 ; Di et al. 2008 ; Hao et al. 2009 ) . While a clear mechanism for SBI-induced BBB disruption has yet to emerge, growth factors and in fl ammatory pathways have been implicated in previous studies. SBI is characterized by increased expression of vascular endothelial growth factor (VEGF) and decreased expression of zona occludens-1 (ZO-1), a tight junction protein; inhibition of ERK1/2 phosphorylation suppresses VEGF expression and salvages ZO-1 expression (Jadhav et al. 2007b ) . Another growth factor, erythropoietin, exacerbates brain edema in SBI (Matchett et al. 2006 ) . Other studies have shown increased extracellular matrix degradation by matrix metalloproteinases in SBI, compromising the basement membrane of endothelial cells (Yamaguchi et al. 2007 ; Jadhav et al. 2008 ) . A key enzyme in the production of prostaglandins and other in fl ammatory mediators, cyclooxygenase-2 (COX-2) appears to play a role in the induction of brain edema in SBI as well (Jadhav et al. 2009, 2010) . Lipid peroxidation by reactive oxygen species (ROS) has been implicated in BBB disruption (Lo et al. 2007 ; Lee et al. 2008b, 2009) . Infl ammatory mediators are notorious participants in BBB regulation, yet they alone do not dictate the development of edema following SBI. Hyong et al. 488 C.H. Kim et al. demonstrated the capacity of rosiglitazone to decrease in fl ammatory markers, such as tumor necrosis factor-a , interleukin- b , and myeloperoxidase, following SBI; however, brain edema and BBB disruption persisted (Hyong et al. 2008 ) .

22.3.2 Cell Death

Apoptosis and other forms of cell death have also been implicated in SBI. Several studies have reported apoptotic changes at the perilesion site in SBI (Matchett et al. 2006; Bravo et al. 2008 ; Sulejczak et al. 2008 ) . Matchett et al. demonstrated apoptotic neuronal death by positive triple immunostaining for terminal deoxynu- cleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), cleaved caspase 3, and NeuN (Matchett et al. 2006 ) . Neuronal apoptosis was later shown to be accompanied by astrogliosis in the perilesional area (Sulejczak et al. 2008 ) .

22.3.3 Perioperative Hemorrhage

Hemorrhage is a major obstacle in neurosurgery and likely contributor to SBI. Postoperative hemorrhage in SBI may trigger injury by disrupting the BBB by similar mechanisms as observed in the intracerebral hemorrhage and subarachnoid hemorrhage stroke models. Intraoperative bleeding can obscure the surgical ﬁ eld and cause local ischemic insult, as well as systemic cardiovascular and respiratory insta- bility. Extensive losses may require blood transfusions, prolonging the surgery. Procedure duration, extended by efforts to stem bleeding, is itself a risk. Achieving hemostasis is unquestionably critical in all types of surgeries but poses unique challenges in neurosurgery. Many surgical methods for controlling bleeding, such as clamping or suturing, are unacceptable in neurosurgery because of the delicate nature of brain tissue. Since Harvey Cushing ﬁ rst introduced its surgical use in 1928, electrocauterization has been the primary neurosurgical method of reducing intraoperative bleeding (Voorhees et al. 2005 ) . While a groundbreaking addition to neurosurgery, electrocauterization itself can cause the destruction of healthy brain tissue either directly or by thermal injury.

22.4 Potential Preconditioning Therapies for Surgical Brain Injury

Currently, clinical management of surgical brain injury is limited to nonspeciﬁ c postoperative care. Many promising therapeutic agents and strategies to mitigate complications of SBI have been observed in animal models (summarized in Table 22.1 ); however, nearly all of these studies utilized pre- or posttreatments. 22 Preconditioning for Surgical Brain Injury 489 BWC ↑↑ BWC, BWC, (continued) ↓ NS p-ERK1/2, ↑↑ ↓ ↓ lipid peroxida- ↑ p-ERK1/2, neurological score BWC, ↑ neurological score NS, NS ↓ BWC ↑ ↓ ↓ ↓ VEGF, VEGF, BWC ↑ ↓ ↓ LPO ↓ neuroscore BWC brain water content (BWC) brain water BWC, VEGF, VEGF, BWC, BWC, BWC, LPO ZO-1, BWC ↑ ↑ ↑ ↑ ↑ ↑ ↑ NS, ZO-1, ↑↑ ↓ (NS) tion (LPO) ↑ ↓↓ ↓ ↑ Tx: No effect Tx: No effect Tx (150 mg/kg dose): SBI: SBI ﬁ ndings: Tx: -histidine 1,000 mg/kg l i.p, thioperamide 5 mg/kg i.p) Before surgery 1 dose, 60 min Before surgery before surgery Starting 1 day prior to surgery Starting 1 day prior to surgery (5 mg/kg i.p) One dose apocynin (Tx): Harmful, Treatment SBI: 30 min before surgery KO: Starting 6 days prior to surgery Starting 6 days prior to surgery Tx: No effect 1 h before surgery Tx (5, 15 mg/kg dose): One dose ( surgery Immediately following Tx: Three daily doses, starting 2 days 45 min before surgery 45 min before surgery Treatment: or apocynin pretreatment or apocynin posttreatment Rat (male) Mouse (male) Erythropoietin pretreatment oxidase KO NADPH daily doses (5,000 U/kg i.p) Four SBI: Rat (male) PP1 pretreatment Rat (male) One dose (1.5 mg/kg i.p) Rat (male) Pretreatment Simvastin SBI: Melatonin pretreatment daily doses (i.p) Seven One dose (5, 15, or 15 mg/kg i.p) SBI: SBI: Mouse (male) L-histidine and thioperamide Rat (male) pretreatment MMP inhibitor-1

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2008 Experimental animal studies of therapeutic agents for surgical brain injury brain injury Experimental animal studies of therapeutic agents for surgical 2008a 2008b 2007 J Neurosci Methods J Neurosci Lett Neurosci J Neurosurg Suppl Acta Neurochir Suppl Acta Neurochir Brain Res Brain Neurosurg Lo et al. ( ( Lo et al. Bravo et al. ( ( et al. Bravo Name, Year, Journal ( Matchett et al. Animal Model Treatment SBI ﬁ ndings, treatment outcome Table Table 22.1 Jadhav et al. ( ( et al. Jadhav Lee et al. ( ( Lee et al. Lee et al. ( Yamaguchi et al. ( ( et al. Yamaguchi 490 C.H. Kim et al.

a , ) a a

HIF-1 , b GSH, NS, ↓ TNF- a aqua- ↑ ↑ ↑ ↑ IL-1 (HIF-1 ↓ hypoxia- a , cyclooxge- myeolperoxi- TNF- ↑ B ↑ ↑ ↑ a k MDA, MDA, BWC, COX-2, COX-2, ↓ ↓ ↓ NS, NS, NS, NF- ↓ ↓ ↓ TNF- ↓ NS, ↓ , ↑

a B b k BWC, BWC, BWC, malondialdehyde (MDA), MPO, ↑ ↑ ↑ ↑ BWC, IL-1 glutathione (GSH), AQ-4 NF- TNF- ↓ ↓ nase-2 (COX-2) nase-2 (COX-2) inducible factor-1 dase activity (MPO), dase activity ↑ ↓ porin-4 (AQ-4) ↓ ↑ ↓ no effect showed Tx: Tx: SBI: SBI: Other concentrations of drug 2.5 ATA) 2.5 ATA) SBI: 2 and 4 h after surgery and 4 h after surgery 300 mg/kg i.p.) 300 mg/kg i.p) Immediately following surgery surgery Immediately following Tx (150 mg/kg): Five daily doses (1 h O Five Starting 5 days prior to surgery Starting 5 days prior to surgery 30 min before, 30 min after, 30 min before, after, One dose (75, 150, or surgery Immediately following Tx (150 mg/kg): One dose (75, 150, or preconditioning minoguanidine posttreatment Aminoguanidine posttreatment Rat (male) Rosiglitazone pretreatment Three doses (1 or 6 mg/kg i.p.) Rat (male) SBI: A Rat (male)

Mouse (male) Hyperbaric oxygen Stroke ) )

) ) 2008

2009 2009 (continued) 2008 Brain Res Brain Lett Neurosci Res Brain Di et al. ( ( Di et al. ( et al. Jadhav Name, Year, Journal ( Hyong et al. Animal Model Treatment SBI ﬁ ndings, treatment outcome Table 22.1 Table Hao et al. ( ( Hao et al. 22 Preconditioning for Surgical Brain Injury 491

SBI presents a unique opportunity to test neuroprotection by preconditioning therapies that may prove clinically relevant. In the following text, we propose the use of hyperbaric oxygen, inhalatory anesthetic, and N-methyl-D-aspartate as preconditioning agents for SBI.

22.4.1 Hyperbaric Oxygen

To date, hyperbaric oxygen (HBO) is the only preconditioning therapy that has been successful in the surgical brain injury model. Jadhav et al. investigated brain edema and neurological outcome with HBO preconditioning (HBO-PC) treatment in mice subjected to SBI (Jadhav et al. 2009, 2010) . The mice were treated with 100% for 1 h at 2.5 ATA for fi ve consecutive days prior to surgery. Examined at 24 and 72 h after surgery, the HBO-treated mice subjected to SBI showed both decreased brain water content and improved neurological status, which was assessed by 21-point sensorimotor scoring and wire-hang and beam-balance tests. The authors suggested that the benefi ts of HBO-PC for SBI are mediated by COX- 2, as its inhibition reversed the effects, and SBI-induced COX-2 overexpression was attenuated by HBO-PC. HBO preconditioning has demonstrated neuroprotective effects in several models of stroke and brain injury; however, its mechanism is not fully understood. Most studies have shown that the key benefi t of HBO-PC is apoptosis inhibition. Ostrowski et al. suggested that HBO-PC reduced the activation of the p38 apoptotic pathway and upregulated the expression of neurotrophins, such as brain-derived neurotrophic factor (Ostrowski et al. 2008 ) . Hypoxia-inducible factor-1a (HIF-1) has also been implicated as a mediator of HBO-PC. A transcription factor expressed in response to hypoxia, HIF-1 governs genes responsible for adaptive responses (Carmeliet et al. 1998 ) ; however, the actions of HIF-1 are complex, as excessive accumulation of HIF-1 results in the activation of proapoptotic and proin fl ammatory pathways (Li et al. 2005; Halterman et al. 1999) . Jadhav et al. observed increased HIF-1a expression following SBI that was reduced by 5 days of HBO-PC at 2.5 ATA (Jadhav et al. 2009 ) . In contrast, Peng et al. demonstrated that the same HBO-PC treatment upregulated HIF-1 a expression in a mouse hypoxia model (Peng et al. 2008 ) . In short, HIF-1 a needs more experimental study to clarify its role in HBO-PC. HBO-PC has also been shown to activate endogenous antioxidants. HBO appears to present an oxidative challenge to which the brain upregulates the activity of endogenous antioxidants, such as superoxide dismutase (SOD), in global ischemia models (Nie et al. 2006; Wada et al. 2001 ) . The induction of antioxidant systems prior to injury would bolster the ability to limit oxidative damage and cell membrane disruption by ROS following brain insult, preserving BBB integrity (Fig. 22.2 ). Historically, the clinical use of HBO-PC has been controversial because of oxygen toxicity issues. HBO generated oxidative stress on vulnerable tissues, such as the lung, is a valid concern; however, the hazards of HBO may be somewhat overstated. 492 C.H. Kim et al.

Surgical Brain Injury

HBO-PC and Inhalatory Anesthetic-PC Direct Trauma ↑Inflammatory Mediators (COX-2,MPO)

↑Apoptosis

↑ROS

↑Lipid ↓Tight Peroxidation Junction Proteins

↓BBB Integrity

↑Neuronal ↑Intracranial ↑Brain Edema Death Pressure

Fig. 22.2 Proposed scheme of surgical brain injury pathophysiology and potential preconditioning interventions. HBO hyperbaric oxygen, PC preconditioning, COX-2 cyclooxygenase-2, MPO myeloperoxidase, ROS reactive oxygen species

Research studies and clinical trials have quelled fears of toxicity and established that HBO treatment pressure at 1.5 ATA is well tolerated without permanent pulmonary complications (Holbach et al. 1972, 1977; Rockswold et al. 1992, 2007) . The variability in HBO-PC dosing regimen presents another challenge; studies have differed in exposure pressure, timing, number, and duration of sessions. This complicates the translation of HBO-PC into the clinic. Thus, the challenges of introducing HBO-PC as an acceptable therapy for SBI are to establish efﬁ cacy and safety of treatment.

22.4.2 Inhalatory Anesthetics

Preconditioning with inhalatory anesthetics has shown some promising neuroprotective results in experimental stroke models—studies have shown reduction of infarct volumes and improvement of neurological de ﬁ cit scores in cerebral ischemia (Zhao and Zuo 2004 ; Zheng and Zuo 2004 ; Payne et al. 2005 ; Liu et al. 2006 ; Kapinya et al. 2002 ) . Some studies have suggested the induction of nitric oxide synthase as a potential mechanism for inhalatory anesthetic preconditioning. Kapinya et al. demonstrated the 22 Preconditioning for Surgical Brain Injury 493 increase of inducible NO synthase (iNOS) from inhalatory anesthetic preconditioning in an ischemic model; the reduction of infarct was eliminated with the administration of an iNOS inhibitor (Kapinya et al. 2002 ) . Another study by Zhao and Zuo showed analogous results in a neonatal hypoxia-ischemia model (Zhao and Zuo 2004 ) . KATP channels in the brain, especially those of mitochondrial origin, appear to play a major role in reducing neuronal death from brain injury. Preconditioning with inhalatory anesthetics is thought to be neuroprotective through KATP channel opening and activation; the inhibition of K ATP channels has been shown to abolish the beneﬁ cial effects of inhalatory anesthetic preconditioning in both in vitro and in vivo studies (Kaneko et al. 2005 ; Kehl et al. 2004 ; Xiong et al. 2003 ) . Adenosine A1 receptor activation has been suggested to be a trigger for KATP channel opening in inhalatory anesthetic preconditioning (Liu et al. 2006 ) ; however, downstream effects of KATP channel opening that mediate neuroprotection are not well understood. Studies investigating the action of inhalatory anesthetic preconditioning of cardiac tissue have demonstrated improved mitochondrial energy generation and increased ROS production that was dependent on mitochondrial KATP channel opening (O’Rourke 2004 ; Tanaka et al. 2003 ) . Thus,

KATP channel-mediated neuroprotection seen in inhalatory anesthetic preconditioning may also be mediated by ROS production, a pathway shared by other therapies, such as HBO-PC or ischemic preconditioning. More research is needed to establish downstream effects of KATP channel activation in brain. Most preconditioning studies have utilized isofl urane because of its availability and limited systemic side effects. The few studies that have studied the neuroprotective effects of halothane preconditioning have shown confl icting results in rodent cerebral ischemia models. Some have suggested that halothane may offer a therapeutic window of neuroprotection (Sarraf-Yazdi et al. 1999 ; Baughman et al. 1988 ; Warner et al. 1993, 1995) ; however, the argument for its clinical relevance is a moot point, as halothane is no longer manufactured in the United States because of its potential hepatotoxicity (Kitano et al. 2007 ) . Sevofl urane has recently been utilized for preconditioning studies and has shown encouraging results in hypoxia-ischemia models (Payne et al. 2005 ; Kehl et al. 2004) . Xenon has also emerged as promising preconditioning agent for neuroprotection (Bantel et al. 2009 ; Limatola et al. 2010 ) . The potential induction of ROS by inhalatory anesthetic preconditioning is of particular interest for the treatment of SBI. HBO-PC has demonstrated ROS ability to activate endogenous antioxidant systems. Inhalatory anesthetics offer a potentially less hazardous and more convenient way to precondition by oxidative challenge (Fig. 22.2 ). Further studies are needed to examine the effects of inhalatory anesthetic preconditioning on cerebral edema and BBB integrity.

22.4.3 N -Methyl-D -Aspartate

Glutamate is a major excitatory neurotransmitter of the central nervous system (CNS) that is involved in the pathophysiology of brain injuries. Glutamate concentrations have been reported to rise signi ﬁ cantly following various types of 494 C.H. Kim et al.

CNS injury, such as ischemia or trauma (Beneveniste et al. 1984 ; Katayama et al. 1990; Liu et al. 1991 ) . Excessive activation of ionotropic N-methyl-D-aspartate (NMDA) receptors by glutamate in the postsynaptic cell causes an in fl ux of sodium and calcium that overwhelms the cell’s ability to maintain the ion hemostasis. The accumulation of intracellular sodium may result in cellular edema, while intracellular calcium can cause the inappropriate activation of regulatory cascades that mediate cell death (Choi 1994, 1995; Choi et al. 1987 ) . In addition, glutamate-induced over-activation of the CNS augments the metabolic needs of neural tissue. In vitro studies have shown NMDA preconditioning (NMDA-PC) to protect cultured neurons from subsequently administration of a neurotoxic concentration of glutamate (Chuang et al. 1992 ) . Calcium involvement in NMDA-PC was confi rmed by Raval et al. in ( 2003 ) . Their in vitro study demonstrated that calcium chelation abolished the neuroprotective effects of NMDA-PC. NMDA preconditioning has been shown to reduce neuronal death from not only excitotoxic damage but from oxidative insults as well (Smith et al. 2008 ) . Most in vivo NMDA-PC studies have focused on investigating its anticonvulsant effects; however, one has examined its effi cacy in traumatic brain injury. In a 2010 study, Costa et al. observed that NMDA preconditioning improved neurological function in the mouse traumatic brain injury model (Costa et al. 2010 ) . Though its clinical applicability has yet to be established, NMDA-PC has shown promising results in both in vitro and in vivo models of ischemia and CNS injury. The importance of glutamate-induced excitotoxicity in the pathophysiological sequelae of SBI is yet unknown; however, glutamate’s role in other similar insults, like traumatic brain or spinal cord injury, suggests that limiting glutamate signaling may prove benefi cial in SBI as well. NMDA-PC could theoretically limit apoptosis and brain edema of SBI by a mechanism similar to that has been observed in ischemia and traumatic brain injury (Fig. 22.3 ). The ability of NMDA-PC to protect against neuronal death could prove clinically useful in limiting the complications of SBI.

22.5 Future Directions

The anticipatable timing of surgical brain injury provides a unique opportunity for preemptive intervention, but clinical medicine has yet to utilize preconditioning methods to protect the brain from SBI. Before these therapies can be tested by clinical trial, further in vivo experimental studies are needed to evaluate preconditioning agents and to provide a better mechanistic understanding of SBI pathophysiology. The pathophysiological understanding of SBI is still sparse compared that of other stroke or brain injury models. To date, SBI studies have implicated certain potential pathways, such as the p38 apoptotic and the COX-2-mediated in ﬂ ammatory pathways. Further studies are needed to expand on upstream and downstream mediators of these signaling pathways in the pathogenesis of SBI. 22 Preconditioning for Surgical Brain Injury 495

Fig. 22.3 Proposed mechanism of NMDA preconditioning on surgical brain injury. NMDA preconditioning (NMDA-PC ) attenuates glutamate over-activation, reducing cell edema and death. NMDA-R NMDA receptor, SBI surgical brain injury

HBO preconditioning has been shown to attenuate brain edema formation and neurological deterioration in surgical brain injury (Jadhav et al. 2009, 2010 ) . While HBO-PC has demonstrated promising results in animal studies, it remains to be seen whether it is feasible therapy in the clinical setting. A hyperbaric facility may not be easily accessible to neurosurgical patients, limiting the applicability of HBO-PC for SBI. In such a case, it would be important to examine other preconditioning therapies activating similar endogenous protective mechanisms.

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Esther Shohami and Michal Horowitz

23.1 Introduction

Traumatic brain injury (TBI) represents the leading cause of death and disability in young people (Myburgh et al. 2008 ) . The acute mechanical primary injury is not the sole factor determining the functional outcome (Leker and Shohami 2002 ; Bramlett and Dietrich 2007 ) ; rather, the forces applied to skull and brain at the time of impact lead to the activation of a complex cascade of molecular and biochemical events that evolve over time and produce secondary brain damage. Whereas some of these processes are transient and short acting, others may be active even for months after the insult. Moreover, similar mechanical impacts do not necessarily result in similar impairment of function, and the variability between individuals in the consequence of TBI may represent differences in the ability to cope with the secondary damage. During the past decade, our understanding of the cellular and molecular changes that occur after TBI has signiﬁ cantly increased, and the notion that endogenous neuroprotective cascades are also set in motion after TBI has been explored. One may speculate that the balance between the harmful and protective processes determines the ﬁ nal outcome of TBI, and that the vulnerability of the injured brain to a given insult depends upon its ability to recruit its own endogenous neuroprotective mechanisms. The activation of neuroprotective pathways can result either from a direct response to harmful signals, such as increased intracellular calcium levels, which is a direct consequence of the insult, or from a pre-injury exposure to a precondition

E. Shohami, Ph.D. (*) Department of Pharmacology, Institute of Drug Research , Hebrew University of Jerusalem , Jerusalem 91120 , Israel e-mail: [email protected] M. Horowitz Laboratory of Environmental Physiology , Hebrew University of Jerusalem , Jerusalem 91120 , Israel

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 499 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_23, © Springer Science+Business Media New York 2013 500 E. Shohami and M. Horowitz that provides enhanced intrinsic defensive ability. Cascades that are involved in the regulation of cell fate, repair, plasticity, memory, and motor skills, activated by the protein kinases such as mitogen-activated protein kinase (MAPK), Akt, and GSK have a special impact on the balance between injury and repair that determines the ﬁ nal outcome of the cell, tissue, and organism after TBI (Neary 2005 ) . Numerous studies have demonstrated the profound effect of preconditioning by brief ischemic or thermal exposures on the outcome of brain ischemic injury. The beneﬁ cial consequences of such procedures have been demonstrated in different in vitro (Liu et al. 2000) and in vivo models (e.g., Glantz et al. 2005; Dirnagl et al. 2003; Blanco et al. 2006 ) . The present chapter focuses on two representative neuroprotective mechanisms: (1) the endogenous “on-demand”-induced synthesis of endocannabinoids (eCBs) and their activities as a potential system for neuroprotection after TBI and (2) long-term heat acclimation as a global preconditioning paradigm that induces “cross-tolerance” against a variety of stressors, including TBI, to convey improved functional outcome. Whether the shared pathways between these seemingly different pathways play a role is discussed.

23.2 Endocannabinoids: An On-Demand “Self-Protecting” Family

Following the discovery of the chemical nature of D 9 -tetrahydrocannabinol (THC), the active plant-derived cannabinoid (Mechoulam and Gaoni 1965 ) , and its receptors CB1 and CB2 (Devane et al. 1988; Matsuda et al. 1990; for a recent review see Onaivi 2009 ) , the endogenous ligands for these receptors were identi fi ed in the mammalian brain as signaling lipids (Devane et al. 1992 ; Mechoulam et al. 1995 ) . For over two decades, our knowledge of the structure and functions of the endocannabinoids (eCBs) system has signi fi cantly expanded. The eCB system consists of ligands, such as anandamide (arachidonoylethanol- amide, AEA) and 2-arachidonoylglycerol (2-AG), receptors (CB1, CB2, possibly also TRPV1 and GPR55), transporters, and enzymes, which are responsible for the synthesis (N -acyl-ethanolamine phospholipids-PLD, NAPE-PLD; diacylglycerol lipase, DGL) and degradation of these mediators (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL) (Piomelli 2003 ; Mackie 2006 ) . The role of the eCB in brain function has long been the focus of investigations that shed light on their synthesis and metabolism, the nature of their activity (as neurotransmitters or regulators), and the signaling pathways in their target cells. The eCB has a multiplicity of actions, mainly in the brain, under both physiological and pathological conditions. Unlike “classical” neurotransmitters, the eCBs are not stored in presynaptic vesicles but rather are produced “on demand,” with increased intracellular Ca++ as the major trigger for synthesis (Di Marzo et al. 1999 ) . The eCBs are effi ciently removed from their sites of action by cellular uptake via a speci fi c transporter and rapid catalytic inactivation or bioconversion to arachidonic 23 Intrinsic Neuroprotection in Traumatic Brain Injury 501 acid (see Hansen et al. 2002 ) . The primary ligands produced in the brain are AEA (Devane et al. 1992) and 2-AG (Mechoulam et al. 1995; Sugiura et al. 1995 ) which activate both the CB1 and CB2 receptors. CB1, the most abundant G-protein- coupled receptor (GPCR) in the brain, is mainly located at nerve terminals, thus activating signaling pathways that produce presynaptic inhibition (Piomelli 2003 ) . The CB2 receptors are expressed predominantly in nonneuronal cells and are upregulated mainly under neuroinfl ammatory conditions (Pacher and Hasko 2008 ) . The brain tissue concentration of 2-AG is approximately 200-fold higher than that of AEA (Bisogno et al. 1999 ) , and the rank order for the distribution of both eCBs in different areas is similar: highest in brainstem, striatum, and hippocampus and lower in cortex, diencephalon, and cerebellum. Whereas 2-AG is a full agonist with low af fi nity at both CB1 and CB2 receptors, AEA is a partial CB1 agonist and relatively weak activator of CB2 (see Hwang et al. 2010 ) . Calcium-stimulated formation of NAPEs, which is the precursor pool of AEA and other related metabolites, is a key event in the biosynthetic pathway that accounts for the neuronal availability of eCBs. Accordingly, pathophysiological events, such as TBI or stroke that lead to high intracellular calcium, induce a marked increase in the levels of these mediators (Hansen et al. 2001 ) . Therefore, not surprisingly, a large body of evidence demonstrates a marked increase in eCB levels in response to such pathogenic events as kainic-acid-induced seizures; 6-hydroxydopamine, NMDA, or glutamate toxicity; shock-induced stress; ischemia; and trauma. The question of whether the activation of the eCB system in response to pathological insult (a) plays a role in the compensatory repair mechanism of the brain mediated via CB receptors signaling (for review, Bahr et al. 2006 ; Hwang et al. 2010 ) or (b) is part of the harmful cascades induced by the insult was extensively investigated in the past decade. To address this question, following brain insults, investigators either (a) administered synthetic 2-AG, AEA, or agonists/antagonists of the CB1/CB2 receptors or (b) manipulated the levels or signaling of the eCB by inhibition of the degrading enzymes/transporters or deletion of the relevant enzymes or receptors.

23.2.1 TBI Increases eCB Levels

Following traumatic and ischemic/reperfusion brain insults, the catabolism of membrane phospholipids increases, leading to the accumulation of degraded products, including NAPE, the precursor pool for eCB, while the level of other phospholipids is decreasing (Hansen et al. 2002 ) . The fi rst reports on the accumulation of eCB in the brain of experimental models of TBI were published a decade ago. Panikashvili et al. ( 2001 ) found that 2-AG levels are signi fi cantly elevated in the ipsilateral hemisphere of mice 1 h after closed head injury (CHI), peaking to tenfold increase at 4 h and remaining high (sixfold) for at least 24 h post injury. In contrast to TBI insult, no increases in AEA and 2-AG levels were detected up to 24 h in a model of acute neuronal damage induced by ouabain (Van der Stelt et al. 2001 ) , yet exogenous AEA in a dose-dependent manner 502 E. Shohami and M. Horowitz reduced in vivo cellular swelling during early phase, post induction of excitotoxicity in the same model. Hansen et al. (2001 ) , using rat neonate models of necrosis, apoptosis, and concussive head trauma, demonstrated the accumulation of AEA precursor and other N -acylethanolamine phospholipids 4 and 24 h after the insult. Using a model of forebrain ischemia, Melis et al. ( 2006 ) reported that 2-AG but not AEA is produced in the ventrotegmental area (VTA). The signi fi cantly increased 2-AG levels were not due to the increase in diacylglycerol lipase (DAG) activity but rather due to the increased availability of DAG precursors for 2-AG, resulting from the ischemia-induced upregulation of PI-PLC. Using in vitro and in vivo models of ischemia and several pharmacological interventions, Melis et al. (2006 ) proposed that the eCB system might serve as a neuroprotective mechanism by its “on-demand” nature of release in response to ischemia and its ability to suppress excessive glutamate release. In contrast, Muthian et al. ( 2004 ) found an increase in AEA, but not in 2-AG, 30–120 min after transient middle cerebral artery occlusion in rats. Treatment with CB1 antagonists before injury led to improved outcome, suggesting a destructive role for AEA (Muthian et al. 2004 ) .

23.2.2 Enhancement of Endogenous eCB Is Neuroprotective

To explore the role of anandamide signaling in vivo, several investigators have targeted its degrading enzyme to augment and extend its brain activities. To this end, mice lacking the enzyme FAAH2/2 and administered exogenous anandamide, exhibited robust CB1-dependent behavioral responses, such as hypomotility, analgesia, catalepsy, and hypothermia (Cravatt et al. 2001 ) . Supporting this notion, Coomber et al. ( 2008) recently reported that inhibition of eCB metabolism attenuates enhanced hippocampal neuronal activity induced by kainic acid. Hwang et al. ( 2010) provide data showing that selective FAAH inhibitors have therapeutic potential against neuropathological states, including TBI and stroke as well as neurodegenerative diseases, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. Enhancement of the eCB activity may also be achieved by inhibiting their transporter. Thus, the inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor palmitylsulfonyl ﬂ uoride (AM374) were shown to enhance (MAPK) activation in cultured hippocampal slices. After an excitotoxic insult to the slices, these combined inhibitors protected against cytoskeletal damage and synaptic decline, similar to the effect produced by the CB1 agonist (Karanian et al. 2005 ) . On the other hand, a reduction of the eCB activity may be achieved by inhibiting their signaling either by speci ﬁ c antagonists or by knockout of the receptor. Thus, CB1 −/− mice did not respond to treatment with 2-AG after CHI, and their functional recovery was less pronounced than that of their wild-type controls (Panikashvili et al. 2005 ) . The accumulating data demonstrate that neuronal injury activates eCB signaling as an intrinsic neuroprotective response via activating signaling pathways downstream from CB receptors and promoting neuronal maintenance and function. 23 Intrinsic Neuroprotection in Traumatic Brain Injury 503

23.2.3 CB1 Receptor Agonists Are Neuroprotective

The earlier studies on the potential neuroprotective effects of cannabinoids performed in vitro showed that cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures and protect rat hippocampal neurons from excitotoxicity (Shen et al. 1996 ; Shen and Thayer 1998 ) . Anandamide protects cerebral rat cortical neurons from in vitro ischemia (Sinor et al. 2000 ) . The protection of neurons against secondary excitotoxicity was suggested to be due to the closing of calcium channels. Gilbert et al. exposed rat hippocampal neurons in culture to an excitotoxic pattern of synaptic activity and showed a signi fi cant reduction in cell death when cultures were exposed to either THC or to the synthetic agonist WIN 55212-2 (Gilbert et al. 2007) . In excitotoxically lesioned organotypic hippocampal slice cultures, the CB1 receptor antagonist, AM251, blocked the neuroprotection mediated by WIN (Koch et al. 2011 ) . Reports from the in vitro studies encouraged researchers to explore the neuroprotective effects of cannabinoids in vivo. The synthetic cannabinoid agonist WIN- 55212 reduced hippocampal neuronal death and infarct volume after transient global cerebral ischemia in the rat (Nagayama et al. 1999 ) . Leker et al. (2003 ) reported that the agonist HU-210 improved functional recovery and reduced infarct volume after permanent middle cerebral artery occlusion. The latter agonist is coupled to ERK activation and stimulates the PI3K downstream target protein kinase B (PKB, also known as Akt) (Galve-Roperh et al. 2002 ) . Along the same line, the acute administration of THC increased the Ser473 phosphorylation of the kinase Akt in mouse hippocampus, striatum, and cerebellum. This effect was blocked by the selective CB1 antagonist rimonabant (Ozaita et al. 2007 ) . Activation of the PI3K/Akt pathway could modulate the expression and activity of genes involved in cell survival, highlighting the CB1-induced neuroprotection that is afforded by endogenous and synthetic CB1 agonists. These fi ndings are important for the understanding of the control of the cell death/survival decision via the eCB system. Following our fi nding (Panikashvili et al. 2001 ) that a tenfold increase of 2-AG 4 h after CHI persists for at least for 24 h (sixfold higher than in controls), we decided to treat mice 1 h post injury with synthetic 2-AG. The treatment attenuated blood–brain barrier permeability and edema formation, along with reduced infarct volume and neuronal cell death at the CA3 hippocampal region. Moreover, the neurobehavioral status of the mice at 24 h displayed greater recovery and persisted for a long duration (up to 3 months). The CB1 antagonist SR141716A partly inhibited 2-AG protection, albeit at a relatively high dose, suggesting that these effects are not mediated solely via the CB1 receptor (Panikashvili et al. 2001, 2005 ) . In mice treated with 2-AG, the levels of the proinfl ammatory cytokines tumor necrosis factor-alpha (TNF-a ), interleukin- 1 b IL-1b , and IL-6 were not elevated to the same extent as in controls (Panikashvili et al. 2006 ) . The robust activation of NF-kappaB (NK-k B), the major infl ammatory transcription factor, was completely abolished by 2-AG (Panikashvili et al. 2005 ) . Several authors have highlighted the importance of 2-AG on the regulation 504 E. Shohami and M. Horowitz of microglia during disease (e.g., Benito et al. 2005; Cabral and Marciano-Cabral 2005; Carrier et al. 2004; Ehrhart et al. 2005; Franklin and Stella 2003 ) . In view of these reports, 2-AG could be considered as a potent modulator of CNS diseases, for which infl ammation and autoimmunity are the causes of CNS damage (Centonze et al. 2007 ) . Modulating cerebral blood fl ow is another possible mechanism by which the eCB could exert a protective effect. The ability of eCB to alter cerebral vascular resistance was recorded in a model system of human brain endothelial cells (HBEC) in which 2-AG was shown to inhibit the ET-1-induced Ca 2+ infl ux into HBEC. In view of these observations, we have demonstrated that HBEC express CB1, CB2, and TRPV1 receptors, and that 2-AG functions as a vasorelaxant that may counteract the powerful vasoconstrictor ET-1 (Golech et al. 2004; Chen et al. 2000 ) . Together, these studies provide an endogenous pathway for regulating endothelial- dependent vascular reactivity, which may be important in pathological conditions, such as TBI.

23.2.4 Nonneuronal CB2 Receptors also Provide Neuroprotection

In the brain, CB2 receptors are expressed predominantly in nonneuronal cells and are upregulated mainly under neuroinfl ammatory conditions. Whereas in health, the normal expression of CB2 is hardly detected in the brain, the receptors are upregulated in activated microglia, leading to increased cell proliferation along with a reduction of the release of proinfl ammatory agents like TNF-a and nitric oxide (NO) (for review: Stella 2010 ) . Leukocyte activation and extravasation into the brain parenchyma, including rolling, adhesion to the endothelium, and transmigration, are among the early responses to traumatic or ischemic brain injury. The activation of CB2 receptors by synthetic specifi c agonists (such as O-3853, O-1966) signifi cantly attenuated these processes and afforded neuroprotection in models of ischemic stroke (Pacher and Hasko 2008; Zhang et al. 2007 ) . The nature of the CB receptors, which activate the agonist-mediated response in glial cells, is still not fully explained, and CB-like receptors are implicated in the regulation of their response. Several reports described the presence of CB-like receptors in cultured astrocytes; however, their role in vivo is yet to be determined (Stella 2010 ) . N -Arachidonoyl- l -serine (AraS) is a brain component that is structurally related to the endocannabinoid family which improves functional outcome, reduces cerebral edema, and decreases lesion volume after CHI. AraS leads to ERK and Akt phosphorylation and the induction of their downstream antiapoptotic pathways after CHI. These protective effects are reversed by speci fi c CB2 antagonists and are probably related to indirect signaling via CB2R, as the agonist does not directly bind to the known CB receptors (Cohen-Yeshurun et al. 2011 ) . AraS can thus be considered as another, novel, eCB with neuroprotective pro fi le. 23 Intrinsic Neuroprotection in Traumatic Brain Injury 505

23.2.5 Summary: Neuroprotection via Supply of eCB

The expression and function of the eCB and their respective receptors in the brain, on neurons, astrocytes, microglia, and the cerebrovasculature point to their role in multiple functions. The formation and accumulation of eCB in response to injury, along with their multipotent properties as antioxidants, vasodilators, anti-infl ammatory agents, inhibitors of excitotoxicity, as well as their role in neurogenesis, suggest that the “on-demand” formation of eCB may represent an intrinsic neuroprotective and neuroregenerative response. Interestingly, these lipid mediators, via their speci fi c receptors and coupling mechanisms, activate intracellular signaling cascades that are associated with promoting cell survival. On some of these cascades, we will focus in the next section of this chapter, in which the speci fi c mode of long-term preconditioning that affords “intrinsic neuroprotection” will be described.

23.3 Heat Acclimation Predisposition of Neuroprotection via Phenotype Consolidation

23.3.1 Preconditioning: Inherent Protective Mechanisms Induced by Preceding Sublethal (Multiple) Stressors

Cells have many mechanisms for adapting to and surviving stress. Some of these coping mechanisms are constitutive; others are inducible and therefore require time for their expression. In 1986 , Murry et al. discovered that intermittent brief ischemic episodes protect the heart from a subsequent sustained ischemic insult and suggested that in cardiac patients, the multiple angina episodes that often precede myocardial infarction in man may delay cell death after coronary occlusion and thereby allow for greater salvage of myocardium through reperfusion therapy. The preceding sublethal intermittent ischemic episodes provided a cardiac preconditioning effect. Less than 10 years later, brain preconditioning was widely recognized as a powerful cytoprotective mechanism, thereby providing an innovative approach for the discovery of novel cerebroprotective strategies (Obrenovitch 2008 ) . This preconditioning effect, during which protection is conveyed via repetitive short episodes of sublethal stress (Sommerschild Hilchen and Kirkeboen 2002 ; Xi et al. 2001 ; Szalay et al. 2007 ; Perez Pinzon et al. 1999 ) , is considered part of a rapid adaptive mechanism induced by many physiological and pharmacological stressors (e.g., hypoxia, ischemia, heat stress, various anesthetic drugs) and is shared by many cells and organs. Recently, a new concept of post-conditioning has also developed, whereby brief repetitive cycles of ischemia with intermittent reperfusion that follows immediately after prolonged ischemia elicits tissue protection in different organs like heart, liver, kidneys, and the brain (Kaur et al. 2010 ) . The broad range of organs that already demonstrate a post-conditioning effect may imply that the family of mechanisms assigned to post-conditioning is another conserved stress protective response. 506 E. Shohami and M. Horowitz

The recognition that preconditioning is achieved via diverse stressors and provides protection against novel stressors introduced the term cross-tolerance , a term encom- passing wide forms of preconditioning (and perhaps post-conditioning) manipulations (Kirino 2002 ; Horowitz 2007 ; du Toit et al. 2008 ; Hausenloy and Yellon 2007 ) . The vast number of studies on preconditioning focused on short/limited time bound- aries (classical PC), in which short-term cross-tolerance (2–3 days) is conferred following a brief recurrent sublethal stress application. This short-term protective process comprises a fi rst window of protection that lasts for approximately 1 h and involves the activation of immediate salvage signaling pathways that are later replaced by transcriptional activation and an enhanced expression of cytoprotective components, thereby affording protection for several days (second window of protection) (Bolli 2007 ; Hausenloy and Yellon 2007 ) . Universal pathways are shared by all types of preconditioning (e.g., adenosine for the fi rst window and HSP72 or NO for the second window of protection). Gene chip analyses revealed, however, that additionally, substantial subsets of the differentially expressed genes are unique either to each preconditioning stimulus or tissue/organ, thus suggesting that different preconditioning manipulations may determine speci fi c neuroprotective phenotypes (Stenzel-Poore et al. 2007 ) . In contrast to the classical preconditioning effects, we have demonstrated that long-term acclimation to adverse thermal environment (heat acclimation) or exercise training confers long-lasting protection (for approximately 2–3 weeks), which is memorized via epigenetic mechanisms against a diversity of novel stressors, comprising heat stress, ischemia, TBI, hyperoxia, ionized irradiation, and noise (Paz et al. 2004 ) in both the heart and the brain (Horowitz 2007 ) . Heat acclimation cross- tolerance (HACT) is the outcome of long-term changes that are developed while shaping the acclimatory homeostasis (Horowitz 2007 ) . HACT differs from classical preconditioning, gaining its maximal capacity only when heat acclimatory homeostasis has been achieved (4 weeks). HACT at that phase has a memory and after loss of acclimation within exposure of 2 days regains the acclimating conditions (Tetievsky et al. 2008 ; Tetievsky and Horowitz 2010 ) . During the initial acclimating phase when the classical preconditioning achieves its maximal effect, subjection of the short-term heat-acclimated phenotype to a novel stressor aggravates injury. HACT occurs in a wide range of taxonomic groups, including invertebrate worms (Treinin et al. 2003 ) , insects (Bayley et al. 2001 ) , ectothermic vertebrates (Todgham et al. 2005) , and rodents (Horowitz 2007 ; Bromberg and Horowitz 2004 ; Shein et al. 2007a ) , implying that this feature is evolutionarily conserved.

23.3.2 Brief Description of the Process of Heat Acclimation- Physiological-Molecular Linkage

23.3.2.1 Introduction

Heat acclimation is achieved via persistent exposure to environmental heat. In terms of the physiological regulatory effectors, the heat-acclimated phenotype displays 23 Intrinsic Neuroprotection in Traumatic Brain Injury 507 reduced metabolic and heart rates, lower temperature thresholds for activating heat dissipation effectors, and increased cardiovascular reserves and capacity of the evaporative cooling system. Collectively, the acclimatory pro fi le is a transition from an early transient, “inef fi cient” state—the hallmark of short-term heat acclimation (STHA)—to a state in which cellular machinery and integrative processes are highly ef fi cient, when acclimatory homeostasis has been reached (Horowitz 1998, 2002, 2010 ) . The acclimation process leads to hardening of the cellular responsiveness to heat stress and delays thermal injuries (Selye 1955 ; Horowitz 2002 ) . To better understand the HACT phenomenon, understanding the concepts and the cellular/molecular mechanisms of thermal hardening assists us in unraveling heat-acclimation-mediated neuroprotection.

23.3.3 Conceptual Model of Heat-Acclimation-Mediated Cytoprotection

During heat acclimation, an initial marked elevation in hypothalamic (and core) temperature is followed by steady state stabilization at a lower level (Horowitz and Meiri 1985 , 1993 ) . In parallel, the onset phase of acclimation is marked by a transient transcriptional upregulation of genes implicating enhanced membrane depolarization. In turn, enhanced neuronal excitability and perturbation in cellular maintenance prevails (Schwimmer et al. 2006 ) . This physiological setup is the switch that leads to the evolution of the heat-acclimated phenotype. In the hypothalamus and the heart, which have been extensively studied, constitutive downregulated expression of genes related to various metabolic activities, including mitochondrial energy metabolism and food intake, and marked upregulation of a large cluster of genes linked with the immune response are noteworthy (Schwimmer et al. 2006 ) . This transcriptome map, which agrees with the metabolic features of the acclimated status and suggests that neuromodulation affects thermoregulatory thresholds, involves the reprogramming of gene expression. In this dynamic setup, cytoprotective genes play a major role. The transient excitable STHA switches on the buildup of large reserves of cytoprotective molecules including heat shock protein species (HSPs) (Maloyan et al. 1999 ; Openheim et al. 1996 ) or the hypoxia-inducible factor (HIF-1a ) (Maloyan et al. 2005 ; Shein et al. 2005 ) and the activation of genes associated with antiapoptosis and antioxidation, all display faster rate of transcription upon further stress (Horowitz et al. 2004; Horowitz 2007 ) . Both features coincide with improved heat endurance of the acclimated phenotype. Collectively, heat acclimation causes a two-tier response. Although the increase in reserves suggests that the cell is now endowed with “on-call” cytoprotective molecules without the need for de novo synthesis, the abrupt component, namely, faster transcription, also improves the renewal rate of stress-protein reserves (Horowitz 2010 ) . Given that the HACT depends on on-call shared pathways responding to both heat (the adaptagent) and novel stressor, understanding the cytoprotective features of the heat-acclimated phenotype provides us with a tool to examine in detail heat acclimation 508 E. Shohami and M. Horowitz

Fig. 23.1 Physiological and functional outcome measures after TBI in normothermic (NT) and heat-acclimated (HA) mice. (a ) Cerebral water content (edema) (b ) Improved neurobehavioral function (neurological severity score, NSS) (c ) Improved cognitive function expressed as time of exploration of a novel object. (d ) Body (colonic) temperature 4 and 24 h after TBI (Adapted from Shein et al. 2005, 2007b, c )

as a “global preconditioning paradigm” that induces protective predisposition against TBI via a “cross-tolerance” mechanism.

23.3.4 Physiological Evidence of Neuroprotection

The effects of heat acclimation on the outcome of TBI have been examined in rodents (rats, mice) subjected to CHI. Following CHI neuroprotection in the heat- acclimated phenotype vs. normothermic controls was evidenced by a greater recovery of motor and cognitive functions, as well as reduced levels of neuropathological parameters such as brain edema, blood–brain barrier disruption, infarct size, and neuronal cell loss (Fig. 23.1 ). Collectively, the heat-acclimated animals demonstrated signifi cantly less damage following injury as compared with normothermic controls (Shohami et al. 1994a ; Shein et al. 2005; Umschwief et al. 2010 ) . Interestingly, CHI also induces temporal hypothermia and an ability to maintain hypothermic state for a longer period of time as compared with non-acclimated 23 Intrinsic Neuroprotection in Traumatic Brain Injury 509 mice (Shein et al. 2008 ) . Taken together, our earlier fi ndings, which are predominantly a descriptive confi rmation of neuroprotection, led us to explore the potential physiological/cellular links to the mechanisms of neuroprotection.

23.3.5 Do Enriched Cytoprotective Pathways Play an Important Role in Shaping the Neuroprotective Features of the Heat-Acclimated Phenotype

We focus in this section on four central functional categories that were studied extensively by our team: (1) antioxidation, (2) HIF-1-erythropoietin receptor signaling (both stress and metabolic effectors), (3) antiapoptosis, and (4) anti-in ﬂ ammatory capacities and the expression of bene ﬁ cial neurotropic factors.

23.3.5.1 Antioxidation

Reactive oxygen species (ROS) have been extensively studied and proposed as key mediators in the pathogenesis of ischemia and trauma (e.g., Chan 2001 ) . The activation of multiple, evolutionary-conserved, cellular ROS defense strategies and the oxidative/reductive balance ultimately determine the fate of a cell. Although ROS production was shown in numerous TBI models to exacerbate adverse outcomes (Shohami et al. 1997 ) , ROS-neutralizing compounds were protective when given soon after trauma (e.g., Beit-Yannai et al. 1996 ) . The low molecular weight antioxidants (LMWA) are one of the defense systems that developed during evolution to prevent ROS from killing cells. We used a method that measures the overall, rather than the individual, antioxidant activity, which is based on the determination of the total reducing power of the biological tissue or ﬂ uid (Kohen and Nyska 2002 ) . The results showed that the chemical nature of LMWA is not altered by heat acclimation. Interestingly, the concentrations of the basal LMWA were lower in heat-acclimated rats (Beit-Yannai et al. 1997 ) ; however, while in the non-acclimated animals LMWA levels decreased, probably due to consumption, during the post-TBI period, high levels of antioxidants were sustained in the heat-acclimated group for up to 1 week following trauma. Long-term heat acclimation (LTHA) likely diminishes the need for LMWA accumulation in a mechanism that still remains to be resolved.

23.3.5.2 Hypoxia-Inducible Factor and Its Downstream Signaling

The HIF-1a , a transcriptional activator and the master regulator of oxygen homeostasis, is essential for the development of the heat-acclimated phenotype in hearts of both the nematode Caenorhabditis elegans (Treinin et al. 2003 ; Maloyan et al. 2005 ) and mice (Bromberg and Horowitz 2004 ) suggesting an 510 E. Shohami and M. Horowitz evolutionary conserved feature. Cai et al. (2003 ) demonstrated that erythropoietin (Epo), a major HIF-1-targeted pathway, induces cardioprotection against ischemia- reperfusion injury in mice. Preceding this fi nding, Bernaudin et al. reported that Epo and its receptor (EpoR) are expressed in a variety of tissues, including the nervous system (Bernaudin et al. 1999 ; Bernaudin et al. 2000 ) thus implicating the protective role of the HIF-1a –Epo cascade in neuroprotection. Indeed, Epo was shown to confer neuroprotection in the CHI model (Yatsiv et al. 2005 ) as well as in other modes of brain insults in experimental animals and humans (e.g., Siren et al. 2001 ; Ehrenreich et al. 2002 ) . Heat-acclimated rats and mice demonstrated constitutive elevation of HIF-1a in cell nuclei; in turn, when following ischemic or traumatic insults, EpoR transcriptional activation coincided with enhanced cardioprotection and neuroprotection, respectively (Maloyan et al. 2005; Shein et al. 2005 ) . Recently, our team succeeded to ablate HACT-mediated neuroprotection in the CHI mouse model via the inhibition of HIF-1a dimerization (Umschweif et al. 2011 ) . In a matched normothermic group, the same inhibitor did not affect either functional recovery or lesion volume which was similar to those in the saline-treated normothermic group. In additional series of experiments, Shein et al. ( 2008 ) demonstrated that treatment with anti-Epo antibody induced no effect in heat-acclimated mice, whereas in non-acclimated mice, increased edema formation was evident (Shein et al. 2008 ) . Collectively, these studies support our suggestion that the neuroprotective role of the HIF-1a –Epo cascade in the acclimated phenotype represents an intrinsic neuroprotective mechanism that depends both on the establishment of larger cytoprotective protein reserves during the acclimation process and on an intensifi ed acute response. The described HIF-1a acclimation protective profi le is similar to that described for HSPs after heat acclimation in the heart (Maloyan et al. 1999 ; Horowitz et al. 2004 ) and brain (Openheim et al. 1996 ) .

23.3.5.3 Antiapoptosis

The main cell-death event observed immediately after insult is necrosis, whereas the second phase of cellular death is mainly associated with apoptosis (Yakovlev and Faden 2001 ) . TBI-induced apoptosis has a critical role in brain-cell death, with neurons being the most vulnerable target (Zhang et al. 2005 ) . Phosphorylation (activation) of the kinase Akt leads to further downstream effects that may oppose apoptosis and promote cell survival. We reported that heat acclimation led to a post-TBI increase in the levels of phosphorylated Akt, as compared with control, normothermic TBI mice (Shein et al. 2007b ) . Moreover, the inhibition of Akt phosphorylation abolished the heat-acclimation-induced functional benefi ts. Hence, we suggested that Akt phosphorylation is an essential step in the induction of heat- acclimated neuroprotection after TBI. Given our fi ndings that heat acclimation provides a cellular antiapoptotic environment (Horowitz et al. 2004 ; Assayag et al. 2010 ) and thus contributes to one important aspect of thermotolerance, the bridge between these data and our 23 Intrinsic Neuroprotection in Traumatic Brain Injury 511 investigation on apoptosis pro fi le in the heat-acclimated closed head injury phenotype was short. Indeed, our results show decreased caspase-3 activity and a reduced number of terminal dUTP-biotin nick end labeling (TUNEL)-positive cells in the HA mice, a marker of apoptosis, indicating that post-TBI apoptosis was attenuated in this phenotype (Umschwief et al. 2010) . We further focused on the mitochondrial Bcl-2-originated (intrinsic) apoptotic pathway. When lethal signals predominate, Bcl-2 family members interact with the mitochondria to induce apoptosis via increasing outer mitochondrial membrane permeabilization, leading to the release of cytochrome c (cyt c ), which causes conformational changes in Apaf-1, and to activation of the caspase-9–caspase-3 (the death executioner) cascade. The Bcl-2 proteins are upstream to “the point of no return” in the intrinsic pathway of cellular apoptosis (Kutuk and Basaga 2006 ) . Both the Bcl-xL (death suppressor) and BAD (Bcl-2-associated death promoter) proteins are associated with mitochondrial outer membrane permeability. Hence, the transcripts and encoded proteins of these genes profi les post-CHI provide us with cues to the antiapoptosis effects detected in the heat-acclimated phenotype. At the end of the heat acclimation period and preceding TBI, both Bcl-xL and the Bcl-xL/BAD ratio were signi fi cantly higher than in the matched controls. Following trauma, this elevated ratio probably contributes to the bene fi cial response, partially because of a rapid posttraumatic decline of BAD transcription. Similar results were obtained for ischemic/reperfusion and heat-stressed insults in the hearts, thus emphasizing that HACT neuroprotection is only one component in a setup of a whole body preconditioning effect. Given our concept of a two-tier stress response in the Bcl-xL–BAD interaction, Bcl-xL reserves increase with acclimation, whereas BAD decline is the component that holds the acute response. Notably, elevated HSP70 reserves and an enhanced anti-in fl ammatory environment both contribute to the antiapoptotic environment seen in the HA-CHI phenotype.

23.3.6 The Anti-in ﬂ ammatory Capacity and the Expression of Bene ﬁ cial Neurotropic Factors

The presence of infl ammatory cells around the traumatic region may increase cellular damage via the release of toxic enzymes that increase cell membrane damage and by the release of chemoattractants to leukocytes. In many experimental TBI models and clinical studies, a rapid, robust increase of in fl ammatory mediators (Shohami et al. 1994b ; Hutchinson et al. 2007 ) and induction of chemokines and adhesion molecules are reported. All these factors, in turn, activate immune and glial cells. The local infl ammatory response is now believed to play an important role in mediating and exacerbating secondary tissue damage after TBI. The concept of a “double-edged sword” evolved with regard to the concomitant bene fi cial and adverse effects of proinfl ammatory mediators that depend on the kinetics of their expression and posttraumatic regulation in the injured brain (Shohami et al. 1999 ; Morganti-Kossmann et al. 2002 ; Lenzlinger et al. 2001 ) . Accordingly, investigating 512 E. Shohami and M. Horowitz the levels of pro- and anti-infl ammatory cytokines in heat-acclimated and normothermic mice revealed that the former do indeed display changes in pro- and anti- in fl ammatory capacity as part of the cross-tolerance response. Heat acclimation establishes higher pre-injury levels of the anti-in fl ammatory cytokines interleukin-10 (IL-10) and interleukin-4 (IL-4) and reduces the post-injury expression of the proinfl ammatory cytokine TNFa mRNA (Shein et al. 2007c ) . The brain-derived neurotrophic factor (BDNF), discovered in the early 1950s as a member of the neurotrophin family, regulates the survival, proliferation, and maintenance of function in various neuronal cell populations (Bibel and Barde 2000 ; Binder and Scharfman 2004 ; Sharma 2006 ) . Several studies have demonstrated the bene fi cial effects of BDNF administration in spinal cord injury and TBI models (Blesch and Tuszynski 2007; Faden et al. 2004; Mahmood et al. 2002 ; Nakajima et al. 2007; Vavrek et al. 2006 ) . Glial cells express BDNF (Conner et al. 1997 ) , and the bene fi cial roles of glial cells—microglia in particular—may be associated with their ability to supply trophic factors (Kempermann and Neumann 2003 ) like BDNF. Interestingly, heat-acclimated mice express higher levels of BDNF at the end of the 30 days acclimation period before injury is induced. During the post-injury period, heat acclimation induces an increase in the amount of BDNF-positive ramifi ed microglia, but a global increase of this factor was not noted. Our fi ndings suggest that the BDNF-positive rami fi ed subpopulation of microglia may indeed play a part in HA-induced neuroprotection

23.4 Concluding Remarks: Do HACT and eCBs Share Similar Neuroprotective Pathways?

In this review, we shed some light on the nature of two intrinsic neuroprotective mechanisms: on-demand synthesis of eCB and the development of the HACT phenotype (Fig. 23.2 ). We showed that HACT brings about constitutive changes and enriches some key cytoprotective pathways, such that upon the extra stress infl icted by TBI, the intrinsic capacity of the brain to cope with the consequences of the trauma is greater than in normothermic TBI animals. Thus, higher ef fi ciency of antioxidants, augmented Akt phosphorylation, and antiapoptotic and anti- in fl ammatory capacities are presented in heat-acclimated TBI mice, leading to decreased pathological (e.g., edema, BBB disruption, lesion volume, cell death) and functional impairments (neurological and cognitive, see Fig. 23.1). Notably, the HACT developed gradually during the process of heat acclimation, which renders protection to novel stressors. In contrast, the eCB is rapidly activated upon insult (within hours) to provide neuroprotection. It is interesting to note that the eCB system described in the fi rst part of this chapter displays similar properties, for example, signaling via Akt phosphorylation, and shares the activities as antioxidants, antiapoptotic, and anti-in fl ammatory agents with those elicited by LTHA, further corroborating the nature of their intrinsic neuroprotective mechanism(s). 23 Intrinsic Neuroprotection in Traumatic Brain Injury 513

Brain injury Ca+2 CB -receptor eCB Signaling pathway NArPE HA Constitutive pathways

HA acute response pathways Secondary damage eCB eCB

P-ERK

HIF 1 Bcl-xL P-Akt Bad

Cytochrome c Acute protective EpoR Caspase 3 response Constitutive Cell elevation of : survival cytoprotective proteins and anti-inflammatory cytokine

Heat Acclimation, 1mo 34ºC eCB activation, Hours Fig. 23.2 Endogenous neuroprotective mechanisms. Right panel depicts the “on-demand” TBI- induced release of eCB and their CB receptors mediated activation of cell survival signaling. Left panel displays heat-acclimation-mediated cross-tolerance evolved during the long-term heat acclimation process. Survival signaling comprises constitutive and TBI-mediated acute responses. Secondary damage is attenuated by both mechanisms; however, while eCB pathways are activated promptly upon exposure to TBI, heat-acclimation-mediated cross-tolerance is a global preconditioning paradigm which evolves before the insult. Additionally, acute response mechanisms are activated in the heat-acclimated mice following the insult

References

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David C. Henshall and Eva M. Jimenez-Mateos

24.1 Epilepsy

24.1.1 Overview

Epilepsy is a common, chronic neurologic disorder characterised by recurrent, spontaneous seizures (Chang and Lowenstein 2003 ) . Seizures are caused by abnormally synchronised discharges of populations of neurons in the brain and are the result of transient imbalances between excitation and inhibition. Epilepsy affects about 1% of the population, with worldwide prevalence estimated at 50 million. At least 40 clinically distinct syndromes are recognised, with temporal lobe epilepsy (TLE) as the most common form in adults. Epilepsy is controlled using anti-epileptic drugs which include phenytoin, valproic acid and carbam- azepine, among others. Treatment is ineffective, however, in about a third of patients for whom the main alternative is surgical resection of the epileptic focus (e.g. hippocampectomy). Common causes of acquired epilepsies include traumatic brain injury, birth and neurodevelopmental abnormalities and infection, whereas a genetic component is presumed to account for most primary (idio- pathic) epilepsies (Chang and Lowenstein 2003 ) .

Chapter as part of a book entitled Innate Neuroprotection for Stroke, part of the Stroke Book Series, Springer. D. C. Henshall (*) • E. M. Jimenez-Mateos Department of Physiology & Medical Physics , Royal College of Surgeons in Ireland , 123 St. Stephen’s Green , Dublin 2 , Ireland e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 521 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_24, © Springer Science+Business Media New York 2013 522 D.C. Henshall and E.M. Jimenez-Mateos

24.1.2 Prolonged Seizures and Brain Damage

Epileptic seizures rarely last more than a few minutes. Prolonged seizures – status epilepticus (SE) – can develop if the normal mechanisms for seizure termination fail or where there is non-compliance with epilepsy medication. Prevalence rates suggest SE affects about 20 per 200,000 population per year (DeLorenzo 2006 ) . Treatment of SE is with anticonvulsant drugs such as benzodiazepine and barbiturates, although a number of anti-epileptic drugs are also used. SE can be highly damaging to the brain. There is overwhelming evidence from animal studies that SE causes neuronal death (Wasterlain et al. 1993 ; Meldrum 1997 ) . Prolonged seizures are also profoundly harmful to the human brain (Fujikawa et al. 2000 ) . The hippocampal CA1, CA3 and hilar subﬁ elds are particularly vulnerable, whereas the CA2 pyramidal neurons and the granule neurons of the dentate gyrus are somewhat resistant. Damage caused by SE in an otherwise normal brain can itself lead to the development of a chronic epileptic state. Excitotoxicity, the main mechanism underlying seizure-induced neuronal death, occurs due to prolonged over-activation of glutamate-gated channels such as N-methyl- d -aspartate (NMDA) receptors (Meldrum 2002 ; Fujikawa 2006 ) . Programmed cell death pathways are also important, with evidence that seizures activate apoptosis-associated signalling pathways culminating in caspase-dependent and caspase-independent neuronal death (Henshall and Simon 2005 ; Engel and Henshall 2009 ) . In this chapter, we describe the origins of animal models of epileptic tolerance, the different preconditioning stimuli and the methods for triggering SE. We discuss current understanding of the molecular mechanisms, reﬂ ect on the similarities and differences with ischemic tolerance and examine potential future directions for research on epileptic tolerance.

24.2 Epileptic Tolerance

24.2.1 Discovery

Epileptic tolerance is most often modelled by exposing rats or mice to single or repeated brief, non-harmful seizures one or more days before an episode of SE. The notion that brief seizures could protect against prolonged seizures is somewhat of an anathema in epilepsy. This is because “seizures beget seizures” (Gower’s hypothesis), and repeated sub-convulsive stimulation of the brain (kindling) can have a potent pro-epileptic effect. Early life seizures have also been shown to lead to permanently enhanced brain excitability (Chen et al. 1999 ) . Nevertheless, evidence that prior exposure to seizures could, under certain cir- cumstances, protect against SE emerged shortly after the ﬁ rst studies on ischemic tolerance. Conference proceedings appeared ﬁ rst (Kelly and McIntyre 24 Preconditioning for Epilepsy 523

1990, 1991; Najm et al. 1992) along with in vitro reports (Chuang et al. 1992 ; Marini and Paul 1992 ) . The ﬁ rst full publication on epileptic tolerance in vivo was by Kelly and McIntyre in 1994, who described the effect of SE in animals which had previously been kindled (Kelly and McIntyre 1994 ) . In their study, rats underwent hippocampal kindling before SE induced by systemic kainic acid (KA). They found kindled animals had dramatic reductions in damage to the piriform cortex, substantia nigra and hippocampus (Kelly and McIntyre 1994 ) . Of note, the interval between stimulation (preconditioning) and SE (challenge) was up to 28 days. This was quite different to the window in ischemic tolerance which usually lasts only a week. A year later, however, Sasahira and colleagues demonstrated epileptic tolerance in rats using similar intervals between preconditioning and challenge to those effective in ischemic tolerance (Sasahira et al.

1995) . They used bicuculline, an antagonist of g -amino butyric acid (GABA)A receptors, to induce seizures which were then terminated by diazepam, followed by a second bicuculline challenge 1, 3, 5 or 7 days later. In animals with 3- or 5-day intervals between preconditioning and challenge, hippocampal injury was strongly reduced. Notably, the animals in the 1- and 3- but not the 5-day interval groups underwent less severe seizures at the time of the second challenge of SE. Nevertheless, epileptic tolerance was af ﬁ rmed; seizures could indeed protect against seizure-induced neuronal death.

24.3 Models of Epileptic Tolerance

24.3.1 Different Models, Protein Synthesis Dependence and Time Window

Table 24.1 presents a selection of the various murine models of epileptic tolerance. Two of the main features of tolerance are the requirement for new protein synthesis and the time period between preconditioning and challenge (Dirnagl et al. 2003 ; Gidday 2006) . Epileptic tolerance has been shown to require de novo protein synthesis on the basis that cycloheximide reduces protection in epileptic tolerance (Emerson et al. 1999 ; Rejdak et al. 2001 ; Jimenez-Mateos et al. 2008 ) . An interval is also required between seizure preconditioning and SE which is most commonly between 24 and 72 h (see Table 24.1 ). Longer intervals are effective in several models, particularly kindling, whereas shorter gaps such as 8 h have been shown to be insuf ﬁ cient time to generate tolerance (Boeck et al. 2004 ) .

24.3.2 Kindling

Following Kelly and McIntyre’s original studies, other groups have demonstrated that kindling can be used to protect against SE using related paradigms (Andre et al. 2000a ; 524 D.C. Henshall and E.M. Jimenez-Mateos ) ) ) ) ) 1994 ) ) b ) ) NMDA NMDA ) ) 2001 ) ) ) ) 2010 1991 1999 2007 1995 1997 2007 2001 2004 2000a, 1998 2003 2005 Borges et al. ( ( et al. Borges Ogita et al. ( ( Ogita et al. lipopolysaccharide, LPS Kondratyev et al. ( S kainic acid, KA pro fi led by microarray antagonist features References Mechanisms/modulators or other features quinolinic acid electroconvulsive shocks, electroconvulsive QA ECS day, day, d Effect of PC on SE Effect severity? Yes (adjusted for) Reduced sensitivity to PILO Andre et al. ( a intra-hippocampal, Interval Interval between PC and SE i.h. pilocarpine (given systemically), pilocarpine (given PILO Rat 1 d (adjusted for) Yes Used “double” PC paradigm and preconditioning, and PILO intracerebroventricular, PC PILO Rat Not speci fi ed KA (i.a.) Mouse 1 d No Pro fi led by microarray Hatazaki et al. ( KA (systemic) Rat KA (systemic) Rat 1 d QA (i.c.v.) 1 d Mouse Not reported 1 or 2 d ERK and p38 MAPK Yes, decreased decreased Yes, ( Najm et al. Jiang ( et Boeck et al. al. ( KA (systemic) Rat 1, 3 or 7 d No ( Emerson et al. i.c.v. -aspartate, Murine models of epileptic tolerance Murine models of epileptic tolerance d This study explains only that the challenge was given 24 h after kindling had been acquired 24 h after kindling had been acquired given only that the challenge was This study explains a intra-amygdala, -methyl- Table Table 24.1 PC Kindling KA (systemic) Rat Challenge (SE) Species Up to 28 d (adjusted for) Yes First proof of tolerance ( and McIntyre Kelly KA i.h. Systemic KA (systemic) KA (i.c.v.) Rat Bicuculline 1 or 7 d Bicuculline decreased Yes, LPS Rat NPY overexpression 3 or 5 d PILO decreased Yes, ( El Bahh et al. unchanged at 5 day SE severity ( Sasahira et al. Rat 3 d No Dmowska et al. ( ECS KA (systemic) Rat 3 or 7 d No i.a. Downregulation of Bcl-X Rapid kindling KA (systemic) Rat 3 weeks Not reported to KA reported Reduced sensitivity ( Penner et al. NMDA (systemic) NMDA KA (systemic) Mouse 1 d Cross-tolerance Hypoxia Not reported Bicuculline by A1 receptor blocked Tolerance Rat 14 d Yes Sieklucka et al. ( N Key: Key: 24 Preconditioning for Epilepsy 525

Penner et al. 2001 ) . In particular, strong protection of limbic structures including the hippocampus was observed when the amygdala was the site of kindling and when SE was induced by the cholinergic mimetic pilocarpine (Andre et al. 2000a ) .

24.3.3 Electroconvulsive Seizures

Electroconvulsive seizures (ECS) are used for the treatment of severe depression in humans and are an attractive preconditioning tool because they are known to up-regulate survival pathways and change neuronal morphology and function (Follesa et al. 1994 ; Brecht et al. 1999 ; Ploski et al. 2006) . ECS delivered before SE is an effective means for producing epileptic tolerance. Kondratyev et al. reported minimal electroshocks delivered via orbital (corneal) electrodes signiﬁ cantly reduced limbic damage produced by systemic KA-induced SE in rats (Kondratyev et al. 2001 ) . However, changes to the intensity and/or route of electroshock administration can produce opposite consequences. Andre and co-workers found that brain damage was actually enhanced by electroshocks delivered via ear clips before SE (Andre et al. 2000a, b) . This emphasises the often thin line between modelling epileptic tolerance and provoking an increase in excitability which works counter to tolerance.

24.3.4 Systemic KA

Systemic injection of KA is a common method for seizure preconditioning. In rats, a low dose of 5 mg/kg KA is suf ﬁ cient (Blondeau et al. 2000 ; Blondeau et al. 2001 ) or higher doses if terminated promptly with an anticonvulsant (Najm et al. 1998 ; Jiang et al. 2005; Borges et al. 2007) . A double episode of seizure preconditioning with systemic KA has been reported to be even more effective (Borges et al. 2007 ) , and other glutamate agonists also appear to work for preconditioning, such as NMDA (Ogita et al. 2003 ) (Table 24.1 ). Higher KA doses are needed in mice, such as 15 or 20 mg/kg. For example, seizure preconditioning in mice with systemic KA 1 day before SE induced by intra-amygdala KA reduced hippocampal damage by ~50–60% (Hatazaki et al. 2007 ; Jimenez-Mateos et al. 2008 ; Tanaka et al. 2010 ) . An example of both seizure damage after SE in mice and the protection afforded in epileptic tolerance is provided in Fig. 24.1 .

24.3.5 Centrally Administered Excitotoxins

Direct injection of low doses of excitotoxins into the brain can also be used for seizure preconditioning. For example, intra-hippocampal KA in rats can protect the contralateral hippocampus against damage caused by intracerebroventricular (i.c.v.) 526 D.C. Henshall and E.M. Jimenez-Mateos

Fig. 24.1 Seizure preconditioning protects the hippocampus against status epilepticus . Representative photomicrographs showing hippocampal damage labelled using Fluoro-Jade B staining (seen as black dots ) in SJL mice 24 h after status epilepticus . The image in the top panel is from an animal which underwent status epilepticus induced by intra-amygdala KA without seizure preconditioning. The lower panel is an image of the hippocampus from a mouse which received seizure preconditioning (20 mg/kg KA, i.p.) 24 h before status epilepticus. Scale bar, 800 m m (Adapted from Tanaka et al. 2010 . Copyright © 2010 Elsevier Inc.) 24 Preconditioning for Epilepsy 527

KA (El Bahh et al. 2001 ; Lere et al. 2002 ) . SE induced by i.c.v. quinolinic acid, coupled with low-dose systemic NMDA as preconditioning, has also been used to model tolerance (Boeck et al. 2004 ; de Araujo Herculano et al. 2011 ) .

24.3.6 Cross-Tolerance

Seizures are not the only means to protect against SE in epileptic tolerance. A non-epileptic stimulus can take the place of a seizure and protect against SE – “cross-tolerance”. Sieklucka et al. showed that bicuculline-induced seizures were suppressed in rats that had been exposed to brief global ischemia 2 weeks prior (Sieklucka et al. 1991) . Others have also shown that preconditioning with ischemia reduces brain injury caused by SE (Emerson et al. 1999) . Lipopolysaccharide (LPS) injection, a potent activator of ischemic tolerance (Tasaki et al. 1997 ; Rosenzweig et al. 2004 ) , is also effective in place of seizure preconditioning in epileptic tolerance (Dmowska et al. 2010 ) . Likewise, epileptic seizures can protect against an episode of ischemia (Tow ﬁ ghi et al. 1999 ).

24.3.7 How Far Does Protection Extend?

The hippocampus has been the region of most interest in epileptic tolerance, but only few studies have examined whether neuroprotection extends along its full ros- tro-caudal extent (Kelly and McIntyre 1994 ; Kondratyev et al. 2001 ; Tanaka et al. 2010 ) . This is important because there may be differences in inter-region connectivity and vulnerability to seizure-induced neuronal death between rostral (septal) and ventral (temporal) hippocampus. When kindling was the preconditioning stimulus, the dorsal hippocampus was most protected (Kelly and McIntyre 1994 ) . In contrast, the ventral hippocampus was more protected against SE-induced damage when systemic KA was used for preconditioning (Tanaka et al. 2010 ) . This suggests different methods of seizure preconditioning may exert greater or lesser effects on one or other pole of the hippocampus. Seizure preconditioning can also protect non-hippocampal structures, including the substantia nigra and piriform cortex (Kelly and McIntyre 1994 ; Andre et al. 2000a ) . However, most groups have either not com- mented on extra-hippocampal regions (Sasahira et al. 1995; Najm et al. 1998 ; El Bahh et al. 2001 ; Ogita et al. 2003 ; Hatazaki et al. 2007 ; Dmowska et al. 2010 ) or reported minimal protection outside of the hippocampus (Borges et al. 2007 ) .

24.3.8 Transferability of Tolerance Models

In certain models, individual strains of rats and mice display remarkable differences in vulnerability to seizure-induced neuronal death (Schauwecker 2002 ; 528 D.C. Henshall and E.M. Jimenez-Mateos

Xu et al. 2004 ) . A reasonable question is whether the same model of epileptic tolerance can be reproduced in another species or strain. We addressed this by comparing C57BL/6 with SJL mice using the same epileptic tolerance model, evoking seizure preconditioning with systemic KA and SE with intra-amygdala KA (Tanaka et al. 2010 ) . Epileptic tolerance was readily modelled in SJL mice. The same interval between preconditioning and challenge could be used (24 h), although a higher dose of KA was needed for preconditioning to obtain optimal protection (Tanaka et al. 2010) . As with C57BL/6 mice, SE severity was not altered in preconditioned SJL mice. Whether other epileptic tolerance paradigms are transferable between strains or species is not known, but these ﬁ ndings again support the broad conservation and experimental robustness of the paradigm.

24.4 Epileptic Tolerance and Epileptogenesis

Epileptogenesis is the process initiated by an injury to the brain which later culminates in the emergence of spontaneous recurrent seizures (Pitkanen and Lukasiuk 2011 ) . The term is also used to describe the increase in seizure susceptibility or lowering of seizure thresholds produced by kindling. Neuronal death is a common feature of epilepsy-precipitating injuries, so it would be reasonable to expect epileptic tolerance to mitigate or prevent emergence of epilepsy. Does epileptic tolerance affect epileptogenesis? Here, we have to exclude models where preconditioning has a strong effect on seizure severity during SE because reducing SE duration in animal models is itself anti-epileptogenic (Pitkanen et al. 2005) . The ﬁ rst study to look at the effect of seizure preconditioning on epilepsy development after SE found no anti-epileptogenic effect (Andre et al. 2000a ) . It is possible that this was because the model did not protect the hippocampal hilar region. Indeed, the hilus contains some of the most vulnerable neurons in the hippocampus, loss of which has been suggested to be the minimal substrate for epileptogenesis (Sloviter 1987, 2008; Bumanglag and Sloviter 2008) . LPS-induced epileptic tolerance was also reported not to alter the course of epileptogenesis (Dmowska et al. 2010 ) . Anti-epileptogenic effects of epileptic tolerance have been found, however, and in association with preservation of hilar neurons. Analysis of spontaneous seizure rates after SE induced by intra- amygdala KA showed that mice previously given seizure preconditioning with systemic KA experienced ~70% fewer epileptic seizures compared to non-preconditioned SE animals (Jimenez-Mateos et al. 2008 ) . A subsequent study which covered a longer period after SE found that rates of spontaneous seizures eventually converge between these groups (Jimenez-Mateos et al. 2010 ) . This was in association with gradual expansion of the hippocampal lesion towards the injury- group level suggesting either more neuroprotection must be achieved for permanent anti-epileptogenesis or a way to sustain protection must be applied (Jimenez-Mateos et al. 2010 ) . 24 Preconditioning for Epilepsy 529

24.5 Mediators of Epileptic Tolerance

24.5.1 Neuroprotection Secondary to a Reduction in the Severity of SE

It is quite common for the duration and/or severity of SE to be reduced in seizure- preconditioned animals (Sieklucka et al. 1991 ; Kelly and McIntyre 1994 ; Sasahira et al. 1995 ; Najm et al. 1998 ; El Bahh et al. 2001 ; Lere et al. 2002 ) . For example, Kelly and McIntyre reported only 63% of kindled rats went into SE following systemic KA, compared to 97% of controls (Kelly and McIntyre 1994 ) . Reduced SE intensity was also reported by others when kindling was used (Andre et al. 2000a ) . This most likely arises because the preconditioning stimulus boosts inhibition in the brain, for example, by increasing GABA levels (Sieklucka et al. 1991, 1992 ) . Another mechanism could be reduction in KA receptor binding sites (Savage et al. 1984 ; Thompson et al. 1988 ) . But if the goal is to explore endogenous neuroprotection mechanisms, rather than identify anticonvulsant mechanisms, this becomes a confounder – less seizures, less damage. Several preconditioning stimuli, including ECS (Andre et al. 2000a ; Kondratyev et al. 2001 ) , LPS (Dmowska et al. 2010 ) and single-dose systemic KA in mice (Hatazaki et al. 2007 ; Tanaka et al. 2010 ) , are not associated with signi ﬁ cant changes to the intensity of SE in tolerance. Presumably, the preconditioning stimulus in these models does not cause lasting activation of inhibition or downregulation of pro-excitatory genes in the period before SE.

24.5.2 Adenosine

Adenosine was one of the earliest mediators proposed for ischemic tolerance (Heurteaux et al. 1995 ; Plamondon et al. 1999 ) . Adenosine is thought to protect via presynaptic A1 receptors, which exert an anti-excitatory effect in the brain by reducing glutamate release (Boison 2008b ) . Both activation of central adenosine A1 receptors and blocking adenosine degradation have powerful anticonvulsant and anti-epileptogenic effects (Li et al. 2007 ; Boison 2008a ) . A1 receptor activation leads in turn to K ATP channel activation which may lead to induction of heat shock protein (HSP) 70 (Blondeau et al. 2000 ) . Administration of an adenosine A1 receptor antagonist was shown to block tolerance induced by systemic NMDA in a mouse model (Ogita et al. 2003 ) .

24.5.3 Transcription Factors: NfkB and Others

Activation of transcription factors has been associated with epileptic tolerance, and these may function as central regulatory points in fl uencing expression of possibly 530 D.C. Henshall and E.M. Jimenez-Mateos dozens of downstream target genes. Nuclear factor k B (Nfk B) was among the fi rst to be associated with epileptic tolerance. Seizure preconditioning resulted in a sustained increase in Nfk B activity in the hippocampus, whereas inhibition of Nf kB blocked tolerance (Blondeau et al. 2001 ) . The specifi c target genes of Nf k B have not been identi fi ed. Bcl-2 is an attractive possibility and has been linked previously with ischemic tolerance (Shimazaki et al. 1994 ; Shimizu et al. 2001 ) . Another is brain-derived neurotrophic factor (BDNF), which was implicated from in vitro studies as a target of Nf k B in epileptic tolerance (Marini et al. 1998 ) . However, NfkB is not activated in some tolerance models (Ogita et al. 2003 ) , and other transcription factors may also be involved. NMDA activates cyclic AMP response element-binding protein (CREB) and the nuclear activator protein 1 (AP1) transcription factor at doses which generate tolerance against systemic KA (Ogita and Yoneda 1994; Yoneda and Ogita 1994; Ogita et al. 2003 ) . Other transcription factors include c-Jun, which are induced following ECS (Brecht et al. 1999) . Extracellular signal regulating kinase (ERK) and the p38 mitogen-activated protein kinase have also been proposed as mediators of epileptic tolerance (Jiang et al. 2005 ) . Transcriptional silencers may be just as important. As discussed below and in other chapters, the transcriptional response to tolerance is dominated by downregulation of genes, so identi fi cation of silencing mechanisms is critical. Recent work in ischemia has identi fi ed the polycomb-group proteins (PcG) in tolerance (Stapels et al. 2010 ) . Studies on this family have not been undertaken in epileptic tolerance.

24.5.4 NPY

Neuropeptide Y (NPY) has been a focus of several studies in epileptic tolerance. NPY is contained within interneurons in the hippocampus and has powerful anti- epileptic effects (Vezzani et al. 2002 ) . Indeed, recent viral vector studies showed intra-hippocampal overexpression of NPY-reduced epileptogenesis (Noe et al. 2008) . Increased expression of NPY has been reported after seizure preconditioning in rats by intra-hippocampal KA (El Bahh et al. 1997, 2001 ) . Borges et al. also detected an increase in NPY after preconditioning in rats with systemic KA (Borges et al. 2007) . Studies have yet to prove, however, if NPY induction is required for tolerance.

24.5.5 Growth Factors

Growth factors can exert potent neuroprotective effects via neurotrophin receptors against seizure-induced brain injury and epilepsy development (Tandon et al. 1999 ; Lahteinen et al. 2004; Barton and Shannon 2005) . Induction of growth factors has been reported in several seizure-preconditioning paradigms. This includes ECS 24 Preconditioning for Epilepsy 531

(Follesa et al. 1994 ; Ploski et al. 2006 ) and following systemic KA (Borges et al. 2007) . BDNF is also protective in an in vitro model with features of epileptic tolerance (Marini et al. 1998 ) .

24.5.6 HSPs

Heat shock proteins are another class of neuroprotective molecule implicated in epileptic tolerance. There is remarkable spatio-temporal matching of HSP up-regulation after ischemic preconditioning with the acquisition of tolerance (Chen et al. 1996 ) , and HSP overexpression is known to protect against seizure-induced neuronal death in vivo (Yenari et al. 1998 ; Akbar et al. 2003 ; Tsuchiya et al. 2003 ) . Seizure preconditioning induces HSPs over a time frame compatible with the acquisition of a tolerant state after systemic KA (Blondeau et al. 2000 ; Borges et al. 2007 ) . Functional studies are needed, however, to determine if there is a causal role for HSPs in epileptic tolerance.

24.5.7 Bcl-2 Family Proteins

There is strong evidence that modulation of Bcl-2 family proteins contributes to ischemic tolerance. Indeed, antisense targeting of Bcl-2 or upstream CREB blocks ischemic tolerance (Shimazaki et al. 1994 ; Shimizu et al. 2001; Meller et al. 2005 ) . Downregulation of the pro-apoptotic member Bim has also been implicated (Meller et al. 2006 ) . Expression of certain Bcl-2 family members is altered by seizure preconditioning. Kondratyev and co-workers showed ECS prevented up-regulation of pro-apoptotic

Bcl-XS after SE (Kondratyev et al. 2001 ) . ECS also downregulates pro-apoptotic Bim (Shinoda et al. 2004 ) and up-regulates anti-apoptotic Bcl-w (Murphy et al. 2007 ) . These changes may be functionally important because loss of Bim reduces whereas loss of Bcl-w increases vulnerability to SE-induced neuronal death in vivo (Murphy et al. 2007, 2010 ) . Expression of several other Bcl-2 family proteins is not changed by seizure preconditioning, including Bid, Bad and Bcl-X L (Shinoda et al. 2004 ; Murphy et al. 2007 ) .

24.5.8 Clinical Correlates of Epileptic Tolerance

There is evidence that transient ischemic attacks can protect the human brain from stroke and are the clinical correlate of ischemic tolerance (Moncayo et al. 2000 ) . Is there a clinical correlate of epileptic tolerance? Many patients with TLE continue to experience seizures, so these could function as seizure preconditioning. Indeed, acute cell death is rarely present in resected hippocampus from TLE patients 532 D.C. Henshall and E.M. Jimenez-Mateos

(Henshall et al. 2000, 2004 ; Mathern et al. 2002 ) . One potential clinical correlate of epileptic tolerance is regulation of the Bim gene. Expression of Bim is signi ﬁ cantly lower than normal in the hippocampus from TLE patients, and this can be experimentally reproduced in rats using ECS to evoke brief non-harmful seizures (Shinoda et al. 2004) . We may speculate that a similar mechanism may run down Bim levels in patient brain, helping to evade cell death despite ongoing epileptic seizures.

24.6 Transcriptome of Epileptic Tolerance

24.6.1 Expression Pro ﬁ ling in Tolerance

By the early 2000s, hypothesis-driven gene-centric approaches had built up a substantial but nevertheless incomplete picture of how tolerance protected the brain. Experimental approaches capable of capturing a comprehensive view of the biological mechanisms underpinning tolerance were needed. Stenzel-Poore and co-workers used microarrays to characterise the transcriptome of ischemic tolerance. From this, we learned that ischemic preconditioning re-programmed the way the brain responded to ischemia. In particular, transcriptional downregulation was an unex- pectedly important feature, with energy-hungry processes such as metabolism and channel/transport activity most suppressed (Stenzel-Poore et al. 2003, 2007 ) .

24.6.2 Microarray Features of Epileptic Tolerance

Transcriptome analysis offered a similar opportunity to understand epileptic tolerance. Studies published in 2007 profi led the transcriptome in various hippocampal subfi elds following seizure preconditioning. Borges et al. found up-regulation was prominent in a rat model, with the affected processes including signalling, tissue structure, neurotransmission and metabolism (Borges et al. 2007 ) . In a similar model in mice, the affected genes included some involved in apoptosis, chromatin remodelling and the cell and ubiquitin cycles (Hatazaki et al. 2007 ) . These studies helped de fi ne the transcriptional changes triggered by preconditioning, but they missed a crucial aspect of the pathophysiology of epileptic tolerance: the response of the preconditioned brain to the harmful seizure challenge. Only that analysis fully captures the protected phenotype. We undertook this experiment next and contrasted the normal transcriptional response to SE to the pro fi le in animals previously preconditioned and then subject to SE (Jimenez-Mateos et al. 2008 ) . The results showed there were almost 50% more genes regulated in tolerance compared to the normal response to SE alone (Jimenez-Mateos et al. 2008 ) . Moreover, 42% of the genes regulated in tolerance were unique and not regulated in the SE-only group (Jimenez-Mateos et al. 2008 ) . Thus, seizure preconditioning had substantially changed how the brain responded to SE. 24 Preconditioning for Epilepsy 533

Fig. 24.2 Insights into epileptic tolerance from gene expression pro ﬁ ling . (a ) Graphs show numbers of genes differentially up- and downregulated in the CA3 sub ﬁ eld of the hippocampus in epileptic tolerance. Mice were subject to status epilepticus alone (injury) or given seizure preconditioning 24 h before status epilepticus (tolerance) and mRNA analysed by microarray 24 h later. Note that the majority of differentially regulated genes in tolerance are downregulated (Data from Jimenez- Mateos et al. 2008 . Copyright © 2008 Elsevier Inc.) ( b ) Comparison of gene overlap between challenge (focal cerebral ischemia or status epilepticus ) and tolerance (preconditioning plus challenge). Whereas in ischemic tolerance just a few genes were common between injury and tolerance (Data from Stenzel-Poore et al. 2007 ) , in the setting of epileptic injury, there was substantial overlap. Nevertheless, substantial numbers of genes regulated in epileptic tolerance were unique and not regulated by status epilepticus alone

Among the differentially regulated genes in epileptic tolerance, 73% were downregulated compared to control (Jimenez-Mateos et al. 2008) . Thus, a key feature of ischemic tolerance – gene suppression – was also present in epileptic tolerance. Figur e 24.2a presents numbers of genes differentially up- and downregulated between injury and tolerance for key processes. Note that the majority of differentially regulated genes in tolerance were downregulated. Gene numbers alone, however, are not sufﬁ cient to identify over-represented biological processes because they do not take into account the numbers of changed transcripts relative to the overall abundance of genes in a given category. Adjustment for this using Z-score bioinformatics revealed calcium signalling, transporter activity and neurotransmitter genes were the biological processes most prominently suppressed in epileptic tolerance (Jimenez-Mateos et al. 2008 ) . The single most over-represented category of molecular process among the differentially suppressed genes in the transporter category was extracellular glutamate-gated ion channel activity. The synapse was the cell compartment most over-represented with downregulated genes differentially expressed in tolerance (Jimenez-Mateos et al. 2008 ) . Also notable was the under-representation of suppression of metabolism in epileptic tolerance. Thus, the neuroprotection in epileptic tolerance was associated with an anti-excitotoxicity phenotype. 534 D.C. Henshall and E.M. Jimenez-Mateos

24.6.3 Microarray Features of Epileptic Versus Ischemic Tolerance

When you compare gene overlap between injury and tolerance in epilepsy with ischemia, it is clear that there is less “re-programming” to injury in the former (Fig. 24.2b ). That is, the difference between injury and tolerance is much greater in ischemia than SE, implying stronger re-programming in ischemia. Are the same gene categories affected in ischemic and epileptic tolerance? Downregulation of transport genes (which include channels) is found in both models of tolerance, so this could be a required element. In contrast, the suppression of metabolism and defence genes seen in ischemic tolerance is less prominent in epileptic tolerance. This supports tailoring of the genomic response according to the nature of the preconditioning or challenge (Stenzel-Poore et al. 2007 ) . Loss of energy-expensive functions may best protect against ischemia, whereas activation of processes to maintain metabolism and defence may suit protection against SE. Up-regulation of certain genes may also refl ect homeostatic mechanisms to replenish proteins degraded during the challenge. The transcriptional profi le of epileptic tolerance contained a large number of genes of unknown function (Jimenez-Mateos et al. 2008 ) . Once fully characterised, these genes may continue to be a source of insight into the tolerance mechanism. Another question being pursued is whether the gene expression response in tolerance has a central “coordinating” mechanism. Chromatin remodelling, epigenetic modi fi cations such as DNA methylation and the molecular effectors of these processes such as repressor element-1 silencing transcription factor (REST or NRSF) and polycomb-group proteins (PcG) are attractive candidates for future study (Spencer et al. 2006 ; Ooi and Wood 2007 ; Mehler 2008 ; Zukin 2010 ) . If these can be pharmacologically targeted, then we have a better chance of recapitulating the full range of molecular changes which underlie neuroprotection in tolerance.

24.7 Final Perspectives and Future Directions

Epileptic tolerance was discovered around the same time as its better-understood “cousin” ischemic tolerance. Many ideas have been drawn from ischemia and explored in epilepsy. What has emerged is that epileptic tolerance is just as highly conserved, can be just as protective and may involve some of the same basic biology. Just as in ischemic tolerance, epileptic tolerance features re-programming to injury in which turn-off is more prominent than turn-on, but some of the gene targets are different implying tailoring to the nature of the preconditioning or challenge. The longer-term consequences of tolerance are, by contrast, better researched in epilepsy than ischemia, with studies having addressed the impact of the protection on the development of co-morbidities, namely, epilepsy. 24 Preconditioning for Epilepsy 535

What are the challenges for the next few years? We must move from transcript to protein. After preconditioning, which proteins are made that contribute to the phenotype of tolerance? What is the proteome after SE in animals previously preconditioned? Technology is fast enabling us to answer these questions. Indeed, the proteome of ischemic tolerance was recently reported (Stapels et al. 2010 ) . The availability of new protein labelling techniques such as click chemistry (Dieterich et al. 2007) alongside the latest generation of quantitative proteomics will allow the next stage of discovery in epileptic tolerance to be realised.

Acknowledgments The authors thank Martha B. Johnson and Roger P. Simon for support with comparative bioinformatics between ischemic and epileptic tolerance and Suzanne Miller-Delaney for careful editing. The authors wish to acknowledge the support of Science Foundation Ireland (08/IN1/B1875).

References

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Steven Roth and John C. Dreixler

25.1 Background

A promising mechanism for neuroprotection in retinal tissue is to activate an endogenous protective capacity, which robustly attenuates ischemic injury, by utilizing ischemic preconditioning (IPC) (Kamphuis et al. 2007a ; Roth et al. 1998 ; Roth 2004; Sakamoto et al. 2006) . This exciting fi nding has important clinical implications. Retinal ischemia is a component of a number of disease states that result in blindness, with no effective preventive or treatment strategy at present to prevent the ischemic damage. Central retinal artery occlusion (CRAO) due to thrombus or embolism develops into retinal ischemia, often with visual acuity £ 20/400, at an incidence of 1–15/10,000 yearly (Chen and Lee 2008 ; Chen et al. 2011 ; Rudkin et al. 2009 ) . Severe visual loss increases health-care costs, mortality, and depression and decreases productivity, independence, and quality of life. CRAO is also often the fi rst indication of signi fi cant atherosclerosis, whose prevalence increased 35% from 1993 to 2007 ( www.hcupnet.ahrq.gov ). Current therapy, which attempts to increase retinal blood fl ow, has not improved outcome. Inner retinal ischemia is also, signifi cantly, a fi nal common pathway in major, chronic vision-threatening diseases including retinal vein occlusion and diabetic retinopathy (DR) (Barber et al. 2011) . With the increasingly aging population, DR is expected to increase fourfold by 2050 ( www.cdc.gov ), a major health-care burden affecting >10 million new US persons yearly. Therapeutic delay impedes adequate treatment of CRAO. In chronic disease such as DR, diffi culties with administration, dosage, and adverse effects limit treatments (Tang and Kern 2011 ) . Despite decades of research, there has not been much solid progress in the treatment of both acute and chronic retinal

S. Roth, M.D. (*) • J. C. Dreixler, Ph.D. Department of Anesthesia and Critical Care , The University of Chicago , 5841 South Maryland, MC4028 , Chicago , IL 60637 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 541 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_25, © Springer Science+Business Media New York 2013 542 S. Roth and J.C. Dreixler ischemic disease, leaving millions with severely disabling visual loss and little hope of visual recovery. Although the time course of acute ischemic insult, e.g., CRAO, and chronic ischemic insult, e.g., DR, is quite different, they share pathophysiology (e.g., capillary closure, macular edema), as well as, most importantly, a fi nal common path to visual loss (Zheng et al. 2007 ) . This fi nal common ischemic pathway includes infl ammation, glial activation, oxidative stress, and neurodegeneration (Adamis and Berman 2008 ; Kern 2007 ; Madsen-Bouterse et al. 2010 ; Nishijima et al. 2007 ) . There have been a number of obstacles that have limited development of neuroprotective agents for retinal ischemia. A drug would have to be given within a reasonable time window of the ischemic event. Second, all attempts to date are characterized by incomplete protection and the risk of nonspeci fi c effects, including toxicity. An alternate approach is to use endogenously available means of neuroprotection. Ischemic preconditioning (IPC) has the promise of uncovering such a neuroprotective strategy because it relies upon utilizing the retina’s endogenous capacity for neuroprotection. While IPC involves a transient ischemic stimulus prior to the prolonged, damaging ischemia, recently, “post-conditioning” (Post-C) has been shown to ameliorate ischemic damage; in post-conditioning, the ischemic stimulus has been applied as late as 24 h after retinal ischemia (Dreixler et al. 2011c ; Fernandez et al. 2009a ) . Clinical application of IPC or Post-C could take the form of a transient ischemic stimulus or their pharmacological mimicking. Both strategies have been promising in animal experiments involving acute ischemia (Biermann et al. 2010 ; Roth et al. 2006 ; Zhu et al. 2007 ) , and recently, a form of ischemic conditioning has been applied in rat models of glaucoma and diabetic retinopathy (Belforte et al. 2011; Fernandez et al. 2011) . In both instances, there was evidence of improvement in retinal function.

25.2 Models for Studying Endogenous Neuroprotection in the Retina

Harnessing endogenous neuroprotection has the potential advantages of speci fi city, avoidance of side effects, and perhaps even enhanced protection by using the endogenous properties of the tissue. Ischemic preconditioning requires a brief period of ischemia, which does not produce any damage. Studying mechanisms of IPC protection in the retina has advantages such as the retina’s accessibility, reproducibility of the model, and capability to examine function, structure, blood fl ow, and biochemical and molecular mechanisms of neuroprotection. A brief, non-damaging period of ischemia is applied in the rodent retina. In the literature, there are several different models. In one, IPC is produced by occlusion of the central retinal artery for 5 min, and in the other, intraocular pressure is increased transiently above systolic arterial pressure (Roth et al. 1998 ; Zhang et al. 2002; Zhu et al. 2002 ) . Twenty- four or seventy-two hours later IPC is followed by a prolonged (45–60 min), damaging occlusion of the retinal circulation. The advantage of this model is that 25 Ischemic Pre- and Post-conditioning in the Retina 543 the transient ischemic stimulus is easily applied and produces no retinal injury. Another model involves exposure to systemic hypoxia (Zhu et al. 2002 ) . The advantage of this approach is that the eye is not manipulated at all, and it is possible to produce long-lasting neuroprotection. Moreover, when adult mice were repeatedly preconditioned with systemic hypoxia over a 2-week period, there was a signi fi cant preservation of retinal function and structure even when retinal ischemia was induced as late as 30 days after the last hypoxic preconditioning stimulus (Zhu et al. 2007) . Although not the same as IPC, an interesting related fi nding is that brief exposure to bright light protects the retina against subsequent phototoxicity from prolonged bright light (Liu et al. 1998 ) . It has been demonstrated that the protection by IPC is time-related. A 24- or 72-h separation between IPC and ischemia is associated with functional and histological protection, whereas a separation of 1 week is not. Conversely, a 1-h period between IPC and ischemia results in the retina being almost totally destroyed (Roth et al. 1998) . In the mouse model of hypoxic preconditioning, protection lasts only 24 h (Zhu et al. 2002) . It is apparent that IPC in the retina differs from that described in the myocardium, where two different phases of IPC are present, an early and a late phase (Martin and Walter 1996 ) . In the myocardium, the early protection by IPC is observed within minutes after preconditioning and dissipates within 2–3 h, while the late phase is more slowly developing and lasts for several days. Similarly, both acute and delayed preconditioning has been found in the brain (Nandagopal et al. 2001 ) . The lack of early preconditioning in the retina suggests that the mechanisms responsible for protection from ischemic damage, while sharing some common features, are organ-dependent. Based upon the time window for IPC in the retina, the mechanism appears to involve the expression of protein(s) that is protective. This hypothesis was con fi rmed by the fi nding that inhibition of protein synthesis results in blockade of the IPC protective effect (Roth et al. 1998 ) . Alternatively, downregulation of pro-apoptotic gene expression has been demonstrated (Zhang et al. 2002 ) . However, the neuroprotection likely includes a complex series of changes in gene expression (Kamphuis et al. 2007a, b ) .

25.3 Endogenous Signaling Pathways Involved in IPC

To date, multiple signaling pathways have been identi ﬁ ed in retinal IPC. Early experiments in a rat model of IPC and ischemia using pharmacological antagonists of adenosine receptors demonstrated a requirement for adenosine receptors A1 and A2a, stimulation of which appears to constitute an early event in the initiation of neuroprotection by IPC in the retina (Ghiardi et al. 1999; Li and Roth 1999) . Also involved in the initiation phase of IPC is opening of mitochondrial KATP channels (mKATP), as demonstrated by pharmacological antagonism of pharmacologically mediated channel opening (Roth et al. 2006 ) . The roles of two PKC isoforms, -d and -e , in the retinal neuroprotective pathways initiated by IPC have been demonstrated 544 S. Roth and J.C. Dreixler

(Dreixler et al. 2008 ) . The PKCd inhibitor, rottlerin, and specifi c siRNA targeted against the two PKC isoforms attenuated retinal neuroprotection by IPC. Both proteins are operative in in vivo signaling downstream from the opening of mKATP channels. Both PKC subtypes exhibited protective mechanisms that block the apoptotic damage caused by retinal ischemia. Moreover, the activation and translocation of two other PKC isoforms, - a and - g , play an important role in retinal IPC (Ding et al. 2009 ) . Two studies have demonstrated the novel involvement of opioid receptors in IPC, opening up a new signaling pathway which has still not been extensively investigated. The administration of morphine sulfate prior to retinal ischemia in rats attenuated ischemic injury, an effect that was blocked by naloxone (Husain et al. 2009, 2011 ) . Peng et al. showed more speci fi cally that pharmacological antagonism of the delta opioid receptor attenuated the neuroprotective effect of hypoxic preconditioning in the retina (Peng et al. 2009 ) . Cell survival mechanisms play a key role in IPC. p38, a MAPK, regulates critical processes in cell death/survival, including transcription, protein degradation and localization, mRNA stability, apoptosis, cytoskeletal dynamics or cell migration, and in fl ammation, with most of these described in vitro (Coulthard et al. 2009 ) . Challenging the notion that p38, upon activation by cellular stress, primarily mediates cell death (Bossy-Wetzel et al. 2004 ; Roth et al. 2003 ) , we demonstrated the essential role of p38a in IPC (Dreixler et al. 2009a ) . Transient retinal p38 activation was also neuroprotective (Dreixler et al. 2009a) , and subsequently it was also shown in heart (Khan et al. 2010 ) . Thus, p38 is an example of how to fi nely tune cellular signaling to facilitate cell survival. Some insight into the mechanisms of activation, control, and downstream signaling from p38 that control cell survival has been demonstrated by the role of the mitogen-activated protein kinase phosphatase-1, MKP-1, in retinal IPC (Dreixler et al. 2011b ) . Akt, a serine/threonine kinase, is a central signaling molecule regulating cell growth, proliferation, migration, protein synthesis, transcription, glucose metabolism, angiogenesis, and survival (Manning and Cantley 2007 ) . We showed for the fi rst time that specifi c Akt isoforms are essential for ischemic tolerance. Interfering RNA targeting Akt2 or Akt3 blocked IPC (Dreixler et al. 2009b ) . Facilitating cell survival and neuronal repair, Akt overexpression enhanced motor neuron regeneration (Namikawa et al. 2000 ) , dopaminergic neuron trophism (Ries et al. 2009 ) , and attenuated apoptosis (Ries et al. 2006 ) . Akt attenuated p38’s pro-apoptotic effect in vitro (Widenmaier et al. 2009 ) . But the mechanisms of Akt-related neuroprotection are largely unstudied. There are other signaling molecules involved in IPC including nitric oxide synthase (NOS) and heat shock proteins. In a mouse model of retinal IPC (Zhu et al. 2002 ) , the use of constitutive nitric oxide synthase (NOS) pharmacological inhibitors and knockout mice strains found that both endothelial NOS (eNOS) and neuronal NOS (nNOS), but not inducible NOS (iNOS), are involved in ischemic tolerance (Zhu et al. 2006 ) . Additionally, in a hypoxic preconditioning model in the mouse retina (Zhu et al. 2002 ) , the protective role of the HIF-1a signaling pathway in hypoxic preconditioning via increased hemeoxygenase-1 25 Ischemic Pre- and Post-conditioning in the Retina 545

(HO-1) protein expression was demonstrated (Zhu et al. 2007 ) . Related to these ﬁ ndings, the time course of production of heat shock protein 27 (HSP27) after IPC closely modeled the time window for IPC neuroprotection in a rat IPC and ischemia model, and injection of cobalt chloride induced HSP27 and mimicked IPC via production of HIF-1 (Li et al. 2003 ; Whitlock et al. 2005a, b ) .

25.4 The Mechanisms of Endogenous Neuroprotection in the Retina: Putting It Together

Insights learned from the study of the mechanisms of retinal ischemic preconditioning are also applicable to related conditions such as glaucoma and are applicable as a model for ischemia in other areas of the central nervous system. From these studies, the goal is to further understand the mechanisms of ischemic tolerance and, second, to decipher the endogenous mechanisms responsible for ischemic tolerance, and then to attempt to activate them to provide neuroprotection. The mechanism of neuroprotection could involve either an increase in protective proteins or a decrease in the expression of pro-apoptotic genes (Chen and Simon 1997 ) . We have shown attenuation of apoptosis after prolonged ischemia in the preconditioned retina, with decreased TUNEL cells in the inner retina and decreased expression of caspases and PARP accompanying the functional and histological neuroprotection of IPC (Zhang et al. 2002 ) . The protective proteins in retinal IPC have not been described yet, but among those that might be involved are heat shock proteins (HSPs), growth and transcription factors, nitric oxide, and glutamate receptors (Chen and Simon 1997 ; Nandagopal et al. 2001 ) . Interestingly, preconditioning the retina with light, but not hyperoxia, was protective against damage upon prolonged exposure to light. In this model, expression of growth factors and MAP kinases increased with the light exposure (Liu et al. 1998) . The expression of the 32-kD small heat shock protein heme oxygenase 32 (HO-1) is increased in the retina in response to repetitive hypoxic preconditioning (Zhu et al. 2007) and light exposure (Kutty et al. 1995) , as well as in the brain following ischemia, trauma, and subarachnoid hemorrhage (Beschorner et al. 2000) . Although increased expression of HO-1 correlates with the time course of IPC protection, the results are suggestive but do not necessarily prove that HO-1 is protective in these models; further experiments are necessary to demonstrate causality. Similarly, expression of another small HSP, HSP27, correlated with the time window of IPC protection, while HSP70 and HSP90 expression did not (Li et al. 2003 ) . We have described in detail the signal transduction pathways that initiate IPC (Li et al. 2000 ) . Our experiments have shown that activation of both adenosine A1 and A2a receptors is required for IPC’s protective effects (Ghiardi et al. 1999; Li and Roth 1999 ) . This ﬁ nding was surprising considering other results showing opposing effects of adenosine receptor subtypes after prolonged retinal ischemia 546 S. Roth and J.C. Dreixler

(Li et al. 1999 ) and suggests the possibility that redundant pathways are present that can be stimulated to produce IPC. Nevertheless, it is clear that adenosine is one of the early initiators of preconditioning. In addition, downstream from adenosine receptors, the following steps occur: activation of protein kinase C (PKC), opening of mKATP channels, and the stimulation of de novo protein synthesis (Ettaiche et al. 2001 ; Li et al. 2000 ) . However, the order in which these events occur, the subtypes of PKC involved, and the proteins produced are currently active areas of research. Blockade of nitric oxide synthase (NOS) and of the production of hydroxyl radicals did not affect IPC protection, suggesting that NO and OH− radicals are not involved (Li et al. 2000 ) . In this model of ischemic preconditioning and ischemia, we have examined the effect of IPC on blood fl ow in the retina following ischemia. IPC was followed 24 h later by ischemia for 30, 60, 75, or 90 min. Rats were anesthetized and mechanically ventilated to maintain constant arterial pH, PCO2, and PO2 . Using radioactive micro- spheres, we measured the blood fl ow at 60 or 150 min after ischemia. It was determined that IPC prevented post-ischemic hypoperfusion when ischemia was 60 min or less in duration. This result shows that preservation of tissue perfusion is another mechanism whereby IPC is protective (Lin and Roth 1999 ) . In summary, IPC requires adenosine, de novo protein synthesis, and mediators including protein kinases B and C, nitric oxide synthase, MAPK p38, HSP27, HIF- 1 a, and mKATP channel opening. Other signaling pathways originating from stimulation of delta opioid receptors are involved. There appears to be a redundancy in signaling pathways which enables IPC to be induced by a number of diverse stimuli. Additionally, IPC operates by preserving post-ischemic blood fl ow. In the future, insights gained from this research may provide the opportunity to design more effective neuroprotective strategies.

25.5 Post-conditioning

A more recent, related advance is that post-conditioning (Post-C, transient ischemia after the damaging ischemia) immediately after ischemia-reperfusion (Dreixler et al. 2010 ; Fernandez et al. 2009a ) , or more interestingly as late as 24 h after ischemia (Dreixler et al. 2011c ) , facilitated robust functional recovery after ischemia. Since Post-C, conceptually related to IPC, changes the post-ischemic state to favor retinal survival, its direct applicability to acute or chronic ischemia is readily apparent. Early results suggest mechanistic similarities with IPC. For example, glutamate clearance, via attenuated glutamate uptake and glutamate synthase activity, is involved (Fernandez et al. 2009b) . Moreover, both protein kinase B/Akt and MAPK p38a are essential for neuroprotection in Post-C immediately after ischemia-reperfusion (Dreixler et al. 2011a ) . From an understanding of the mechanisms of Post-C, it may be possible to design speciﬁ c neuroprotective strategies that will be applicable long after prolonged ischemia has occurred. 25 Ischemic Pre- and Post-conditioning in the Retina 547

25.6 Summary

Studies over the past decade have demonstrated in animal models the robust neuroprotective effects of pre- and post-ischemic conditioning in the retina. Multiple signaling pathways are involved, and novel mechanisms including the role of opioid receptors have been elucidated. Moreover, ischemic conditioning attenuated the loss of retinal function in diabetic retinopathy and in glaucoma, suggesting its potential relevance to chronic retinal disease as well as acute ischemia. While ischemic preconditioning is dif ﬁ cult to apply clinically, post-ischemic conditioning is directly relevant. Moreover, as mechanisms are further elucidated, it may soon be feasible to pharmacologically mimic IPC or post-conditioning, which should yield novel treatments for ischemic retinal disorders.

Acknowledgments Dr. Roth’s research has been supported by National Institutes of Health (Bethesda, Maryland) grant EY10343, the University of Chicago Institute for Translational Medicine, the Glaucoma Research Foundation (San Francisco, California), the Foundation for Anesthesia Education and Research (Rochester, Minnesota), and the American Heart Association (Dallas, Texas). There are no proprietary interests.

References

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component of the beta-cell pro-survival effects of glucose-dependent insulinotropic polypep- tide. J Biol Chem 284:30372–30382 Zhang C, Rosenbaum DM, Shaikh AR, Li Q, Rosenbaum PS, Pelham DJ, Roth S (2002) Ischemic preconditioning attenuates apoptosis following retinal ischemia in rats. Invest Ophthalmol Vis Sci 43:3059–3066 Zheng L, Gong B, Hatala DA, Kern TS (2007) Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes. Invest Ophthalmol Vis Sci 48:361–367 Zhu Y, Ohlemiller KK, McMahan BK, Gidday JM (2002) Mouse models of retinal ischemic tolerance. Invest Ophthalmol Vis Sci 43:1903–1911 Zhu Y, Ohlemiller KK, McMahan BK, Park TS, Gidday JM (2006) Constitutive nitric oxide synthase activity is required to trigger ischemic tolerance in mouse retina. Exp Eye Res 82: 153–163 Zhu Y, Zhang Y, Ojwang BA, Brantley MA Jr, Gidday JM (2007) Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1. Invest Ophthalmol Vis Sci 48:1735–1743 Part VI Clinical Applications Chapter 26 Clinical Cerebral Preconditioning and Postconditioning

Cameron Dezfulian

26.1 An Introduction to Preconditioning

Preconditioning is exposure of an organ to a sublethal physiologic stress (e.g., brief transient ischemic accident or TIA) which then induces adaptations designed to protect that organ against a subsequent similar but more severe, normally lethal stress (e.g., prolonged stroke). Thus, a brief organ stress results in cellular adaptations that place that organ in a protected state, able to withstand more severe subsequent injury than was possible prior to the preconditioning stimulus. From an evolutionary perspective, this would be a highly desirable adaptation designed to permit tolerance to future similar insults which may be anticipated given the harbinger of the initial insult. Ischemic preconditioning (IPC) was initially noted in 1986 where four 5-min cycles of coronary occlusion substantially (75%) reduced the area of infarction resulting from a more prolonged, 40-min coronary occlusion and reperfusion which immediately followed (Murry et al. 1986) . Of note, IPC was not protective against severe (3-h) coronary occlusion. The ﬁ rst report of cerebral IPC followed several years later (Kitagawa et al. 1990 ) . Research in subsequent years has revealed two distinct windows of cerebral protection by IPC ( Durukan and Tatlisumak 2010 ) : an early, acute phase where protection is present within minutes of IPC but fades after a few hours, and a remote (in many studies more effective) window of protection occurring generally >24 h after IPC and fading within a week. A comprehensive discussion of the mechanisms whereby IPC operates against cerebral ischemia is beyond the scope of this chapter, but this topic has been recently reviewed (Dirnagl et al. 2009; Durukan and Tatlisumak 2010) . IPC appears to trigger production of oxygen free radicals and NO, adenosine, and changes in calcium

“What does not kill me makes me stronger.” Friedrich Nietzsche, Twilight of the Idols , 1888. C. Dezfulian (*) Department of Critical Care Medicine , University of Pittsburgh , Pittsburgh , PA , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 553 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_26, © Springer Science+Business Media New York 2013 554 C. Dezfulian

Fig. 26.1 Methods of preconditioning and postconditioning . Classical ischemic preconditioning utilizes brief, sublethal focal or global ischemia to produce protection against subsequent more prolonged, lethal ischemia. Modi ﬁ cations of this means of inducing ischemic tolerance substitute other sublethal stressors in the place of the ischemia to produce similar degrees of protection. These include the use of pharmacological agents (pharmacological preconditioning), other physiological stressors such as heat stress or endotoxin exposure, or ischemia delivered to a remote part of the body. In clinical practice remote ischemic preconditioning and postconditioning generally utilizes limb ischemia through inﬂ ation of a blood pressure cuff to pressures higher than the systolic blood pressure to produce limb ischemia. Ischemic preconditioning can be applied minutes (acute) or days (remote) before the lethal ischemia in effect producing two windows of protection. The application of direct or remote ischemia, or pharmacological agents, early in reperfusion has been referred to as postconditioning and produces similar protection against lethal ischemia

fl ux which in turn activate secondary intracellular signaling pathways. These pathways include the activation of important protein kinases such as PI3K, MAPK, ERK, Akt, and PKC as well as changes in mitochondria through K-ATP channel opening and resistant to mitochondrial permeability transition. The acute phase of IPC is believed to be the result of posttranslational modi fi cations (e.g., phosphoryla- tions by activated kinases), whereas delayed IPC appears to trigger new protein synthesis of genes involved in angiogenesis, energy metabolism, vasomotor control, in fl ammation, and cell survival (e.g., growth factors). Cerebral IPC has been investigated in a variety of animal models which have de fi ned effective means whereby IPC can be delivered. Furthermore, numerous investigations have extended the means by which the brain may be preconditioned (protected) beyond classical IPC, as described above (Fig. 26.1 ). Additional stresses closely related to IPC include: 26 Clinical Cerebral Preconditioning and Postconditioning 555

¥ Remote ischemic preconditioning (RIPC) Ð Brief ischemia and reperfusion delivered to another organ or a limb resulting in a preconditioned state remotely, such as in the brain, conferring ischemic tolerance. This is extremely important to the clinical application of preconditioning and will be reviewed separately below. ¥ Pharmacological preconditioning (PPC) Ð Various drugs have been reported to produce a preconditioned state within the heart and brain. These drugs may trigger adaptive signals similar to those induced by IPC. Much of the clinical application of PPC has centered around the use of volatile anesthetics (e.g., iso fl urane, des fl urane), and these will be reviewed separately below. ¥ Cross-tolerance Ð Other forms of physiologic stress have been noted to induce some of the same protective pathways as IPC and confer similar subsequent ischemic tolerance. The stressors include insults unrelated to ischemia such as spreading depression, heat stress, hyperbaric oxygen, and LPS as well as stressors that recapitulate parts of ischemia such as hypoxia or mitochondrial respirator chain inhibition. Clinical trials of cross-tolerance in the brain have not been conducted or proposed, and this topic will not be reviewed in this chapter. ¥ Ischemic post-conditioning (IPost) Ð Ischemic post-conditioning is the application of short bursts of ischemia during reperfusion which may provide neuroprotection through similar mechanisms as IPC. This fi eld of research is rapidly expanding in similar directions as IPC and offers the clinical advantage of application after the event is known to have occurred. IPost will be addressed separately at the conclusion of this chapter.

26.2 Translating Experimental IPC Results to Clinical Trials

The delivery of classical IPC to prevent cerebral ischemic injury has varied in numerous models of focal and global ischemia (Fig. 26.2 ). IPC itself can be delivered as a brief focal or global insult. Global ischemia is most commonly induced through a single episode of brief (2Ð5-min) two-vessel (common carotid arteries) occlusion, although multiple shorter (1-min) global ischemic exposures have been employed to produce IPC. Longer exposures appear injurious. In most studies, global IPC is provided 48 h before lethal ischemia although immediate and 24-h protection has been demonstrated. Global 2-vessel IPC is effective in rodent models where the circle of Willis is not intact or exhibits poor collateral fl ow (gerbils, mice). Effective global IPC in rats, who like humans have a patent circle of Willis, generally requires either a 4-vessel occlusion or the addition of hypotension (mean arterial pressure <50 mmHg) to more prolonged (5Ð10-min) 2-vessel occlusion to prevent signifi cant collateral blood fl ow. Global IPC has been demonstrated to protect against subsequent ischemic brain injury resulting from longer periods of similar global brain ischemia, cardiac arrest (systemic global ischemia), and stroke (focal ischemia). Providing analogous global IPC in humans is impractical outside the setting of aortic clamping during cardiopulmonary bypass and in the elderly risks plaque embolization from carotid atherosclerosis. 556 C. Dezfulian

Fig. 26.2 Varieties of experimental ischemic preconditioning. Preconditioning is provided to an animal (typically a rodent) through a brief, sublethal focal or global ischemia. The classically used example of focal ischemia is middle cerebral artery occlusion rendering ischemia over much of the ipsilateral temporal and parietal cortex. Focal preconditioning is typically delivered as a series of 1Ð3 brief (5Ð10 min) artery occlusions with similarly timed period of reperfusion in between. This contrasts with global ischemic preconditioning where the sublethal ischemia is nearly always delivered as a single, shorter (2Ð5 min) occlusion of either both common carotids (mice, gerbils), all 4 cerebral vessels (rats) or both common carotids accompanied by hypotension (rats, occasionally mice). This is designed to deprive the entire brain from blood ﬂ ow. Subsequent lethal ischemia is similarly instituted using longer ischemic times. In focal models, the lethal ischemia ranges from 60 to 120 min and on occasion permanent ischemia without reperfusion. In global models, the ischemia time is on the order to 5Ð10 min. Protection against lethal ischemia is present at the two distinct time periods shown: acutely within minutes of the preconditioning, extinguishing within a few hours, and remotely beginning at 24 h after preconditioning and extending often to a week later

Focal ischemia has been used to trigger IPC protection against subsequent prolonged focal ischemia. Focal IPC has primarily been studied in rodent models of transient middle cerebral artery (MCA) occlusion of 5Ð10-min duration to protect against subsequent longer (60Ð120-min or permanent) MCA occlusion. In these studies, focal IPC is usually performed in 2 or 3 cycles of brief ischemia followed immediately or 24Ð48 h later by prolonged or permanent stroke in the same vascular distribution. The protection noted within these studies has some evidence of clinical relevance on the basis of studies demonstrating neuroprotection against stroke after a TIA discussed below. It is important to note that nearly all preclinical studies performed on rodents have been done on young adult male animals. The male predominance results from evidence that female rats have smaller infarcts than males after similar middle cerebral artery occlusion (Alkayed et al. 1998 ) , and that estrogen therapy provides neuroprotection against focal ischemia in male and ovariectomized female animals (Gibson et al. 2006 ) . In heart, IPC shows no added protection in younger females, but protection returns with aged females (Turcato et al. 2006; Ostadal et al. 2009 ) . 26 Clinical Cerebral Preconditioning and Postconditioning 557

In other forms of cardiac PPC and IPC, the stimulus needed to precondition females appears to exceed that for males and protection lasts for shorter durations (Pitcher et al. 2005 ) . Gender comparisons using PPC in focal cerebral ischemia have yielded con ﬂ icting results (Kitano et al. 2007 ; Limatola et al. 2010 ) . Global IPC protected 4-month-old rats against global ischemia, but protection was signi ﬁ cantly diminished at 24 months (He et al. 2005 ) . Thus, clinical trials of IPC need to pay close attention to recruitment of females and elderly in their cohorts with consideration for subgroup analyses.

26.3 Practical Considerations for IPC Clinical Trial Design

The study of cerebral IPC serves two important purposes related to clinical translation. The fi rst is the identi fi cation of endogenous pathways of protection against cerebral ischemia which theoretically could be triggered using appropriate therapeutic agents. In this setting, IPC continues to serve as a means by which potential therapeutic targets are discovered in hopes of creating effective ischemic neuroprotective drugs. The second is its direct application, and that of derivatives such as RIPC, PPC, and IPost, as therapies against cerebral ischemia. The requirement of IPC to precede cerebral ischemia mandates foreknowledge of an ischemic event. Thus, IPC can only be effective against planned cerebral ischemia such as that occurs during surgical carotid endarterectomy, embolization or clipping of aneurysms, and cardiopulmonary bypass for open heart surgery. The more recent discovery of ischemic protection from ischemic post - conditioning (IPost) provides an alternative whereby similar methods may confer neuroprotection after the occurrence of ischemia. IPost will be discussed separately below. The application of IPC can be technically challenging. In clinical trials of IPC in patients on cardiopulmonary bypass, IPC often is achieved by aortic clamping (Hausenloy and Yellon 2009 ) . This technique is feasible when the chest is open for surgery and the patient is on bypass but outside of these unique conditions is impractical. Catheter-based methods of therapy such as deployment of stents, sclerosing agents, or thrombolytics permit for prior IPC to be delivered using the catheter (via balloon in fl ation) to achieve transient vessel occlusion resulting in ischemia. This method of delivering IPC could be employed prior to embolization of an aneurysm or intra-arterial delivery of thrombolytics during cerebral infarction. The most common anti-cerebral ischemic therapy given at present is intravenous thrombolysis, and in this setting, delivering IPC by manipulating the intracerebral arteries is risky and impractical. Even in the case of carotid endarterectomy where there is ready access to the carotid artery, delivery of IPC is still problematic due to concerns over plaque embolization which the procedure is meant to prevent. Even when technical considerations can be met, the use of IPC in humans requires a high standard of attention to patient safety concerns which was not necessary in the antecedent preclinical trials. Ischemia and reperfusion clearly has the potential to result in irreversible cerebral injury when a certain threshold is exceeded, 558 C. Dezfulian which makes the use of IPC in the sensitive cerebral circulation dangerous and requires clinicians to demonstrate their “protective” IPC is not in fact injurious. The use of MRI has demonstrated that some “benign” TIAs actually result in infarction (Oppenheim et al. 2006 ) . Indeed, experimental “sublethal” ischemia, when examined using more sensitive injury detection methodologies or longer durations of follow-up, clearly shows the hallmarks of injury (Sommer 2008 ) . Animal studies demonstrate that some combinations of brief ischemia can yield more severe, rather than less severe, subsequent ischemic injury (Tomida et al. 1987 ) . Careful phase I trials of IPC are thus required to establish safety of IPC regimens before widespread clinical testing. These trials have not yet been performed whereas phase I results are available for the use of RIPC in cerebral ischemia (Koch et al. 2011 ) where the risks are substantially less. As IPC is translated into the clinical arena, it will be important to assess for cerebral injury using more than one sensitive injury marker and to follow up patient outcomes beyond hospital discharge. Examples would include patient follow-up at 3 or 6 months for neurocognitive testing as well as cerebral MR imaging in addition to these tests and serum biomarkers (e.g., NSE, S100B) obtained at the time of recovery from the index ischemia. Clearly, this will add to the costs required for clinical trials, which is problematic at a time of research spending limitations. Since humans are much more heterogeneous than laboratory animals, a number of issues require special attention in translating preclinical IPC to the bedside. Speci fi c patient populations may be more sensitive to injury during IPC or may benefi t less from its protections and thus will need to receive different protocols (or be excluded) from interventions aimed at the bulk of the population. These subpopulations include the elderly (He et al. 2005 ; Della Morte et al. 2008 ) and females (Song et al. 2003 ; Kitano et al. 2007 ) where both animal and clinical data have called into questions the effectiveness of IPC. Patients with antecedent neurovascular or neurodegenerative disease have been excluded from all trials of IPC to date but are at the highest risk for injury after procedures such as coronary artery bypass grafting and may require modi fi ed treatment protocols. Attention must be paid to the medications patients are consuming which may interfere with or mimic IPC. For example, a recent interventional cardiology trial of RIPC excluded 93/336 patients screened due to their use of glibenclamide or nic- orandil which antagonizes and mimics IPC protection, respectively (Hoole et al. 2009 ) . Ironically, >95% of patients enrolled in this trial, which demonstrated cardioprotection by RIPC, were on statins, which also have been considered pharmacological preconditioning (PPC) agents against cerebral ischemic injury (Domoki et al. 2009 ; Die et al. 2010 ) . Where to draw the line on inclusion and exclusion of patients based on their medication pro fi les becomes increasingly problematic as ever more drugs are implicated as PPC agents and makes the need for empiric clinical data greater. Many of the technical and safety issues discussed above have been avoided through the use of remote IPC (RIPC) or PPC. RIPC will be discussed more extensively below, but brie fl y it avoids many of the issues discussed above by delivering the IPC to a limb extremity (thigh or arm) where the risk of irreversible injury is 26 Clinical Cerebral Preconditioning and Postconditioning 559 minimal. This method thus minimizes the risk for signi fi cant adverse events while providing a readily accessible site for IPC delivery. For these reasons RIPC, if proven clinically effective, will likely be dominant over classical IPC in the clinical arena and trials utilizing RIPC are being conducted at a much faster pace than trials of classical IPC. PPC is another alternative to IPC which permits ready drug delivery with improved safety margins. The most classically used PPC agents are the halothane anesthetics which also permits administration as part of the anesthetic regimen for the procedure being performed. However, all pharmacological agents come with their own adverse effects and drug interactions and require individual evaluation. It is also possible that these agents may not be as effective as IPC against ischemic injury. Clinical trials of pharmacological preconditioning will be discussed separately below. It is important to note that direct clinical comparisons between IPC, PPC, and RIPC are lacking.

26.4 TIA as a Model of Classical IPC in Stroke

Transient ischemic accidents (TIAs) are brief, focal neurological defi cits believed to be caused by arterial thrombosis which resolve without permanent neurological sequelae through endogenous thrombolysis. TIA is associated with a 10.5% risk of stroke in the subsequent 90 days based on one large cohort study and 15Ð20% based on a meta-analysis. In most IPC studies, these are defi ned as episodes lasting <60 min since longer episodes have been associated with CT evidence of infarction in 80% of cases where subsequent follow is performed (Bogousslavsky and Regli 1985 ) . One study which permitted longer duration of TIA (median 120 min) also failed to show protection against subsequent stroke (Johnston 2004 ) . Thus, TIA is by de fi nition a brief, noninjurious period of ischemia which is followed by a stroke and is a human model of IPC with subsequent lethal cerebral ischemia. Several studies (Weih et al. 1999 ; Moncayo et al. 2000 ; Arboix et al. 2004 ; Johnston 2004 ; Wegener et al. 2004 ; Schaller 2005 ; Della Morte et al. 2008 ) have sought to test the hypothesis that TIA protects the brain against subsequent injury from stroke through IPC (Table 26.1 ). Since the diagnosis of TIA is a clinical one made on the basis of history and physical exam and in many of these studies performed after a stroke has occurred, the risk of recall bias is signi fi cant. Nonetheless, studies designed to test this hypothesis as a proof of principle that IPC exists in humans are fairly consistent in supporting the IPC’s existence with one notable exception (Johnston 2004 ) . These studies mirror similar comparisons done in patients suffering myocardial infarction, which show that preceding angina reduces myocardial injury (Ottani et al. 1995 ; Andreotti et al. 1996 ) . Some noteworthy consistencies emerge from this comparison. TIAs appear to protect most when they are recent (within a week), multiple (2Ð3 but not more), and of brief duration (<20 min). This replicates experimental IPC fi ndings. Furthermore, TIA is not effective in protecting against lacunar strokes which are smaller and tend to differ in etiology. TIA also does not appear to protect in the 560 C. Dezfulian 72 h ³ 4 weeks from 6 h to 2 years better outcomes than TIA ³ after stroke cardioembolic strokes cardioembolic strokes best result 1 week of stroke (120 min median) TIA to stroke interval ranged interval TIA to stroke TIA <4 weeks before stroke had TIA <4 weeks before stroke No change if TIA < or TIAs protective in large artery, artery, in large TIAs protective 1Ð3 TIAs lasting 0Ð20 min within TIA duration 20Ð270 min 2) ³ thrombolysis in myocardial infarction grade grade thrombolysis in myocardial infarction TIMI Glasgow Coma Scale, Glasgow stroke presentation and better mRS stroke cant fi GCS. mRS and GCS were not signi analysis in multivariate antecedent TIA despite similar perfusion ow) fl recanalization (TIMI cits. Faster fi de of lesions and better admission discharge NIHSS and mRS in patients with prior TIA based on prior TIA of discharge independent scale) observed in patients independent scale) observed with 1Ð3 antecedent TIAs lasting 10Ð20 min t fi (reduced bene within 1 week of stroke in 1Ð4 weeks) mRS, and NIHSS at mean 13-month ow), fl NIHSS unavailable follow-up follow-up; on 41% patients without TIA not lacunar strokes or >7 days before stroke alters the or >7 days before stroke occurrence of disability (mRS Antecedent TIA was associated with less severe associated with less severe Antecedent TIA was Reduced infarcts by MRI in patients with Reduced infarcts No change in NIHSS or mRS at the time Favorable outcomes (no or mild disability; Favorable recanalization (TIMI improved TIA pre-stroke mRS in nonlacunar but Prior TIA improved Failed to con fi rm that TIA <1 day, 1Ð7 days, rm that TIA <1 day, fi to con Failed GCS modi fi ed Rankin scale, fi modi mRS without TIA in same vascular without TIA in same vascular territory as stroke in any vascular territory vascular in any in patients >65 years old ipsilateral TIA (<60 min) thrombosis treated with urokinase or without prior TIA on TIA characteristics ) Nonlacunar stroke with prior TIA NIH Stroke Scale, NIH Stroke ) Anterior circulation stroke with ) Nonlacunar stroke with TIA 2008 ) Nonlacunar and lacunar stroke with ) Case:control (1:3) of patients with/ 2000 2004 NIHSS ) TIA with subsequent stroke based ) due to cerebral artery Stroke 2004 1999 Clinical studies of stroke outcomes in patients with and without antecedent TIA outcomes in patients with and without antecedent TIA Clinical studies of stroke 2004 2005 Moncayo et al. ( ( Moncayo et al. Schaller ( Abbreviations: Wegener et al. ( ( et al. Wegener ( Arboix et al. Table Table 26.1 Study reference ( et al. Weih Patient/TIA characteristics Major outcomes Subgroup analysis Johnston ( Della Morte et al. ( ( Della Morte et al. 26 Clinical Cerebral Preconditioning and Postconditioning 561 elderly brain (Della Morte et al. 2008) consistent with human results in angina-MI (Abete et al. 1997) and animal data in brain IPC (He et al. 2005 ) . TIA also appears to provide a direct neuroprotection (smaller infarct with similar perfusion defect) and a faster thrombolysis (Wegener et al. 2004 ; Schaller 2005) similar to what has been noted in the heart (Andreotti et al. 1996 ) . Finally, it is noteworthy that these effects were seen in these studies despite controlling for a number of medications linked to PPC such as aspirin, statins, and nitroglycerin. Taken as a whole, these studies support the hypothesis that IPC is an effective means of providing ischemic protection in brain. They suggest IPC should be performed within a week of the anticipated insult and that several (2Ð3), brief (10-min) cerebral arterial occlusions will be most effective in patients aged <65.

26.5 Clinical Trials of Remote and Pharmacologic Preconditioning

Clinical trials of PPC in brain are hard to characterize since so many agents have been characterized as being preconditioners. Included in this list are a number of medications routinely used in the primary prevention of stroke, including antihy- pertensives, lipid-lowering agents (statins), and aspirin. The fact that IPC has been noted in patients on these drugs (in the form of TIA before stroke, see above) makes it unlikely that these agents are mimicking the same pathways of ischemic tolerance. Although many studies have demonstrated activation of similar protective pathways as IPC (K-ATP channels, NO, and PKC activation), there is no evidence of superiority and little evidence of an additive bene fi t when PPC and IPC are combined (Guo et al. 1994 ; Carr et al. 1997; Mosca and Cingolani 2000; Unal et al. 2003 ; Redel et al. 2009 ) . Furthermore, inhibitors have variable effects on IPC vs. PPC protection bringing into question the notion that the mechanisms are indeed the same. To this extent, a comprehensive discussion of PPC in the brain is beyond the scope of this chapter but has been reviewed recently (Dirnagl et al. 2009) . PPC has been most extensively evaluated in a clinical setting in the context of inhaled volatile anesthetics such as sevo fl urane, iso fl urane, en fl urane, des fl urane, and, rarely, halothane. A meta-analysis of trials of inhaled anesthetics in cardiopulmonary bypass (Yu and Beattie 2006 ) demonstrated reductions in myocardial injury (troponin extrusion) with use of des fl urane and sevo fl urane and a trend to mortality bene fi t. In most of these studies, these agents were used throughout the case as anesthesia (compared to IV anesthesia generally with propofol) and in concert with narcotics (also implicated in PPC). Thus, it is not clear whether this represents preconditioning since the agent is administered before, during, and after the procedural ischemia in most cases. An interesting and more convincing trial of PPC showed that infusion of nitroglycerine 24Ð28 h prior to an exercise stress test, long after the medication would be metabolized and its effects gone, resulted in improved exercise stress test performance in patients with coronary artery disease (Jneid 562 C. Dezfulian et al. 2005 ) . Good clinical trials of PPC in cerebral ischemia are lacking but offer the theoretical advantage of easier delivery than IPC with similar ef fi cacy and different, perhaps more predictable, adverse effects. RIPC in clinical practice generally refers to the use of a blood pressure cuff to produce limb ischemia by occluding blood fl ow to an arm or leg for 5Ð10 min in 2Ð3 cycles prior to a planned ischemic procedure. Cuff pressures are generally the greater of 200 or 20 mmHg above the patient’s systolic blood pressure. In a recent phase I trial in preparation for use of RIPC before treatment of subarachnoid hemorrhage (Koch et al. 2011 ) , these in fl ation pressures created some pain, but this is tolerated. The main adverse effects related to occlusion of IVs and interruption of monitoring (oxygen saturation and blood pressure) when the upper extremities were used, so the authors switched to the lower extremities without problems. As with IPC, RIPC has been most extensively tested in recent randomized controlled trials aimed at producing cardioprotection, and most of this work has been nicely summarized in recent reviews (Walsh et al. 2007 ; Hausenloy and Yellon 2009 ) . RIPC shows promise in reducing myocardial injury (troponin extrusion) in adults and children undergoing cardiac surgery and elective angioplasty. The mechanism of action is believed to be liberation of humoral mediators (e.g., adenosine or bradykinin) or activation of the autonomic nervous system. Based on the ease of delivery and safety of RIPC, it is clearly the preferred method of IPC delivery assuming similar ef fi cacy. A number of recent randomized clinical trials involving neurologically relevant endpoints highlight the enthusiasm for RIPC. Walsh and colleagues randomized adults undergoing carotid endarterectomy to RIPC delivered by sequential compression of each thigh for 10 min with cuff pressures suf fi cient to obliterate the foot pulses by Doppler (Walsh et al. 2010 ) . This pretreatment resulted in signifi cant reductions in deterioration of patients’ saccadic latency, used as a surrogate for mild brain injury. In a randomized trial of 40 patients with cervical spondylotic myelopathy requiring decompression, Hu and colleagues demonstrated that three 5-min upper right limb ischemia-reperfusions to 200 mmHg right after anesthesia induction resulted in reductions in neuron-specifi c enolase and S100B release and a more rapid clinical recovery (Hu et al. 2010 ) . Finally, Jensen and colleagues performed RIPC with 4 cycles of 5 min on each hind limb ischemia-reperfusion just prior to 1-h deep hypothermic circulatory arrest in Yorkshire pigs, resulting in reductions in brain lactate and more rapid EEG recovery and behavioral scores (Jensen et al. 2011 ) . RIPC in this model was not only neuroprotective but also cardioprotective. These three trials clearly demonstrate the promise of RIPC which, coupled to its noninvasive nature and safety, makes this the most feasible form of delivering IPC to the brain.

26.6 Ischemic Post-conditioning (IPost)

IPost is the application of brief bursts of ischemia and reperfusion after the lethal ischemic injury during the reperfusion phase. Similar to IPC, this technique was initially shown to be protective in heart (Na et al. 1996 ) and subsequently brain 26 Clinical Cerebral Preconditioning and Postconditioning 563

(Zhao et al. 2006 ) . Most animal studies of IPost have demonstrated the requirement for IPost initiated within the fi rst minute after reperfusion which is believed to be mechanistically due to some benefi ts derived from gradual reperfusion (less oxidant injury) and some from activation of similar reperfusion injury salvage kinase (RISK) pathways (Hausenloy and Yellon 2007 ; Hausenloy and Yellon 2009 ) as in IPC such as activation of Akt, ERK 1/2, and MAPK. Numerous animal and clinical trials of IPost have been conducted primarily in the last decade and have been the subject of recent reviews (Ovize et al. 2010 ; Sandu and Schaller 2010 ; Zhao 2010 ) . Overall, IPost appears to provide the same degree of cardioprotection and neuroprotection as IPC, though the requirements for execution immediately at reperfusion are stricter. There are, however, reports of IPost-mediated neuroprotection against focal ischemia delayed as late as 6 h after reperfusion (Ren et al. 2008 ) and against global injury up to 2 d later (Burda et al. 2006; Danielisova et al. 2006 ) . At present, clinical trials of IPost for neuroprotection have not been published, but the in vivo and in vitro studies are promising (Zhao 2010 ) . IPost shares a number of analogies to IPC beyond the mechanistic similarities. Both pharmacological and remote IPost have been described, akin to IPC. The former is very diffi cult to distinguish from simple pharmacologic drug therapy for neuroprotection and is a topic too broad this chapter. Remote IPost is intriguing in that similar to RIPC, this modality can be delivered distal to the site of desired protection (i.e., brain) providing a similar degree of protection and can be delivered after an ischemic event is detected. This permits safe and practical application of a therapy in the prehospital setting in patients suffering acute brain ischemia. This hypothesis was tested in a recent randomized trial of prehospital remote IPost delivered by upper arm blood pressure cuff infl ation to 200 mmHg for 4 cycles of 5-min ischemia and 5-min reperfusion in patients suffering suspected acute myocardial infarction. The results demonstrated signifi cant improvements in vessel patency upon presentation for percutaneous intervention as well as higher degrees of salvage of myocardium after reperfusion. The improved vessel patency by remote IPost is reminiscent of results noted in clinical trials of RIPC in heart and brain described above. Remote IPost thus holds signi fi cant promise for clinical application in the prehospital setting for patients being transported to the hospital with strokes or in cardiac arrest.

26.7 Summary

Cerebral IPC permits the use of endogenous mechanisms of ischemic tolerance after brief ischemic stress to protect against anticipated acute ischemic brain injury in elective high-risk surgery. Stroke outcomes data after antecedent TIA supports experimental evidence that IPC is clinically effective. IPost offers similar protective mechanisms with the ability to intervene on the broader range of acute ischemic brain insults which cannot be anticipated, such as stroke, cardiac arrest, intracranial hemorrhage, and brain trauma. Existing evidence shows similar degrees 564 C. Dezfulian of neuroprotection provided by all these modalities (IPC, PPC, IPost, RIPC, and remote IPost), and experimental evidence provides guidance on the methods whereby pre- and post-conditioning may best be delivered and the patient populations in which modiﬁ cations may be needed. Remote IPC and IPost provide the greatest clinical promise for novel, effective therapies since they can be readily applied in most patients with minimal risk using basic equipment, namely, a sphyg- momanometer to a limb. Not surprisingly, clinical trials of RIPC and remote IPC have been advancing most rapidly in the past 5 years with encouraging results. The future will likely see a number of clinical trials of remote IPost to treat the most common ischemic brain injuries as well as continued mechanistic work in the ﬁ eld of IPC to elucidate additional pharmacological targets intended to mimic the brain’s endogenous protective machinery.

References

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George Kwok Chu Wong , Matthew Tak Vai Chan , and Wai Sang Poon

27.1 Background

In the neuroscience ﬁ eld, preconditioning refers to endogenous mechanisms for brain protection against brain injury and cerebral ischemia. As de ﬁ ned by Dirnagl et al. ( 2009) , preconditioning is a clinical procedure by which a noxious stimulus close to but below the threshold of damage is applied to the tissue. Shortly after preconditioning (known as early, rapid, or classical preconditioning) or after a delay of 1Ð3 days, during which protein-synthesis-dependent protection (delayed preconditioning) (Chen et al. 1996 ) is induced, tolerance or resistance to the same, similar, or even different noxious stimuli given beyond the threshold of damage develops locally or distally. Preconditioning thereby protects against subsequent injury. Most stimuli leave an unprotected in-between period and never last more than a few days. Preconditioning can also be viewed as a prophylactic neuroprotection strategy. Rapid preconditioning is appealing because the technique can be applied therapeutically in the setting of surgery involving a high risk of ischemic complications, such as cardiovascular or cerebrovascular surgery. Preconditioning

G. K. C. Wong , M.D. (*) Division of Neurosurgery , Prince of Wales Hospital, The Chinese University of Hong Kong , 4/F Clinical Science Building , Hong Kong , Hong Kong Department of Surgery , Prince of Wales Hospital , 4/F Clinical Science Building, 30Ð32 Ngan Shing Street , Shatin, New Territories , Hong Kong e-mail: [email protected] M. T. V. Chan , FANZCA Department of Anesthesia and Intensive Care , Prince of Wales Hospital, The Chinese University of Hong Kong , Hong Kong , Hong Kong W. S. Poon , FRCS Division of Neurosurgery , Prince of Wales Hospital, The Chinese University of Hong Kong , 4/F Clinical Science Building , Hong Kong , Hong Kong

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 567 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_27, © Springer Science+Business Media New York 2013 568 G.K.C. Wong et al. is distinguished from postconditioning, which consists of intermittent interruption of blood fl ow to an organ to lessen the injury associated with focal ischemia and the resulting rapid reperfusion. Interest in preconditioning for cerebral ischemia was revived upon publication of the Stenzel-Poore et al. report in The Lancet in 2003 (Stenzel-Poore et al. 2003 ) . In their experiment, mice were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning) or 60 min (injurious ischemia) or to preconditioning followed 72 h later by injurious ischemia (eight mice per condition). RNA was extracted from cortical regions of the ischemic and nonischemic hemispheres. Three pools per condition were generated, and RNA was hybridized to oligonucleotide microarrays for comparison of ischemic and nonischemic hemispheres. Real-time PCR and Northern blotting were used to validate results. The microarray revealed changes in gene expression with little overlap between the different experimental conditions. Injurious ischemia induced upregulation of gene expression; 49 (86%) of the 57 regulated genes showed increased expression in the ischemic hemisphere. By contrast, preconditioning followed by injurious ischemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation. These adaptations were thought to be similar to the neuroprotective strategies in hibernating and hypoxia- tolerant states. Subsequent evidence from genomic studies (Stenzel-Poore et al. 2007 ) suggested that diverse stimuli that trigger preconditioning achieve neuroprotection through a common process that depends on fundamental reprogramming of the response to injury. Such reprogramming of the genomic response leads to the induction of novel neuroprotective pathways not ordinarily found in the setting of ischemia. The nature of the preconditioning stimuli determines the phenotype of neuroprotection, a phenotype that parallels protective adaptations also found in certain physiologic conditions where the preconditioning stimuli exist at levels that can induce injury. Human preconditioning after transient ischemic attack and seizure has been well described. Ischemic preconditioning was discovered in the heart in 1986 (Murry et al. 1986 ) and in the brain in 1990 (Kitagawa et al. 1990 ) . In a retrospective case-control study involving 148 stroke patients, a favorable outcome (Glasgow Outcome Scale score of 5) was signifi cantly associated with prior transient ischemic attack (Wang et al. 2003) . This is clear evidence that ischemic preconditioning improves ischemic tolerance in the human brain. Epilepsy tolerance was fi rst demonstrated by Kelly and Mclntyre, who showed that brain damage can be reduced when status epilepticus is preceded by a preconditioning seizure (Jimenez- Mateos and Henshall 2009; Kelly and Mclntyne 1994 ) . In their study, rats were subjected to hippocampal kindling, i.e., repeated electrical stimulations until generalized convulsions are elicited, and status epilepticus was then induced by systemic kainic acid. The kindled rats showed a dramatic reduction in damage to the piriform cortex, substantia nigra reticulate, and hippocampus; proposed 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage... 569 mediators/mechanisms included transcription factor Nfk B, altered ion-channel expression, upregulation of growth factors and other protective genes, and suppression of anti-apoptotic Bcl-2 family proteins. In this chapter, we explore the current status of research in preconditioning strategies in surgery.

27.2 Coronary Bypass

In coronary artery bypass graft surgery, adverse neurological events have devastating consequences with fi ve- to tenfold increases in mortality, two- to fourfold increases in length of hospital stay, and three to six times the need for use of intermediate- to long-term care facilities (Roach et al. 1996 ) . The most severe adverse cerebral outcomes after cardiac surgery, classi fi ed as type I outcomes, include stroke, transient ischemic attacks, coma, and stupor. Of these, the most important is frank stroke. Type II outcomes include neurocognitive abnormalities such as memory de fi cit, agitation, confusion, and diminished emotional health (Smith 2006 ) . These outcomes may be classi fi ed as subtle and subclinical, but they are signi fi cant and can be persistent. The Multicenter Study of Perioperative Ischemia Research Group and the Ischemia Research and Education Foundation Investigators reported from their study of 2,108 patients that adverse cerebral outcomes occurred in 129 (6.1%) patients and that half of these adverse outcomes were strokes. Type II outcomes were probably underestimated due to lack of detailed neurocognitive assessments. Proposed etiologies include marked hemodynamic fl uctuations; cerebral embolism of atherosclerotic plaque, air, fat, and platelet aggregates; cerebral hyperthermia after the discontinuation of cardiopulmonary bypass; as well as in fl ammatory and neurohumoral derangement after surgery. The use of intermittent ischemic arrest for myocardial protection has been advocated as equivalent or superior to cold cardioplegia. Intermittent ischemia, however, requires frequent manipulation of the aorta with repetitive clamping and partial occlusion. The repeated aortic trauma may result in debris from the aortic wall being shed into the systemic circulation. Thus, its application is suggested only in patients with no aortic and cerebrovascular disease. In a randomized clinical trial comparing a single-clamp technique with intermittent ischemic arrest for myocardial preservation, the incidence of perioperative cerebral microemboli and that of postoperative neuropsychological disturbance as measured by the Luria-Nebraska Neuropsychological Battery were comparable between the two techniques (Musumeci et al. 1998 ) . Biochemical analysis suggested that intermittent ischemic arrest provides more effective myocardial preservation. Further support of the hypothesis of preischemic preconditioning is derived from a report of better preservation of ATP content in the myocardium during subsequent periods of ischemia during intermittent ischemic arrest for coronary surgery (Yellon et al. 1993 ) . 570 G.K.C. Wong et al.

The relation between ischemic preconditioning and free radicals in clinical patients has also been studied (Zhong et al. 2001a ) . Experimental studies have suggested that free radicals play a role in ischemia-reperfusion injury, including myocardial stunning and arrhythmias and pulmonary perfusion injury. In a randomized clinical study of 43 patients, the protocol for ischemic preconditioning was two cycles of 2-min ischemia followed by 3-min reperfusion. Free radicals were measured using electron spin resonance spectroscopy. They found that the free radical generation in coronary sinus blood in the ischemic preconditioning group was 9.7 and 16.6% after the ischemic preconditioning protocol and 10 min after declamping and 6.8 and 13.3% in the controls. The free radicals in arterial samples were 21, 14, 10, and 9% at 10 min and 1, 2, and 24 h, respectively, after reperfusion. The cardiac index and right ventricular ejection fraction were improved by ischemic preconditioning. Thus, although ischemic preconditioning had no effect on free radical generation after surgery, it protected against postoperative stunning, suggesting that free radicals might act as one of the triggers for ischemic preconditioning (Zhong et al. 2001b ) . Myocardial revascularization on the beating heart has gained popularity because it avoids the damage induced by cardiopulmonary bypass, which involves hypothermia, aortic cannulation, and cross-clamping. According to the review by Laurikka et al. ( 2002) , surgical procedures on the beating heart require less blood to be transfused and afford shorter hospital stays, lower rates of organ dysfunction, fewer hemorrhagic complications, and lower mortality rates in high-risk patients. Surgical procedures performed on the beating heart reduce the cost of surgery. However, such surgery may be associated with a less than optimal operative fi eld, limited access, and thus an incomplete procedure. In addition, the ischemic period during grafting may cause contractile dysfunction and vascular endothelial injury and risk myocardial necrosis. Ischemic preconditioning is proposed as the solution because it has been shown experimentally to delay myocardial damage, preserve high-energy phosphates, suppress arrhythmias, and improve postischemic functional recovery. In a randomized clinical study of 32 patients with left anterior descending coronary artery or two-vessel disease who were to undergo off-pump coronary artery bypass grafting, ischemic preconditioning (two cycles of regional 2-min ischemic preconditioning in the left anterior descending coronary artery) resulted in complete postoperative recovery of stroke volume. Ischemic preconditioning also decreased the immediate myocardial enzyme release and prevented the postoperative increase in heart rate, supporting the hypothesis that ischemic preconditioning increases myocardial tolerance toward subsequent ischemic insult. Although local ischemic preconditioning is an effective cardioprotective strategy, mechanical methods for achieving such preconditioning are not accepted by most cardiac surgeons for fear of cardioemboli. Thus, extensive research has been aimed at identifying pharmacological agents to mimic ischemic preconditioning in the hope of achieving the same bene fi t (Wang et al. 2003 ) . There is evidence that mitochondrial adenosine triphosphate-sensitive potassium channels play a major role in ischemic preconditioning. Diazoxide, a selective channel opener, has been shown to confer cardioprotection in animal models of myocardial infarction and in vitro 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage... 571 cultured human cardiomyocytes subjected to ischemia-reperfusion stimulus. Diazoxide was also tested in a 40-patient randomized clinical trial (Wang et al. 2003 ) . All were stable angina patients scheduled for isolated elective coronary artery bypass graft surgery. Diazoxide (1.5 mg/kg) administered prior to the start of cardiopulmonary bypass conferred additional myocardial protection beyond that provided by cardioplegia alone, as manifested by better hemodynamic recovery and a tendency toward decreased release of creatine kinase postoperatively. Hyperbaric oxygen conditioning through reactive oxygen species generation has also been proposed as an alternative to mechanical ischemic preconditioning. In a randomized study of 81 patients given hyperbaric oxygen therapy prior to fi rst-time elective on-pump cardiopulmonary bypass coronary artery bypass grafting, the preconditioning led to signifi cantly decreased pulmonary vascular resistance, increased stroke volume, improved myocardial left ventricular stroke work, decreased postoperative myocardial injury, reduction in the length of stay in the intensive care unit, reduction in blood loss and transfusion, reduction in low cardiac output syndrome and inotrope use, decreased incidence of atrial fi brillation, fewer pulmonary complications, and fewer wound infections (Yogaratnam et al. 2010 ) . However, this method of treatment is usually not available in cardiac surgical units, making a further multicenter randomized control study dif fi cult to conduct. Remote ischemic preconditioning is probably the most attractive and promising treatment. Remote ischemic preconditioning is a phenomenon whereby brief ischemia of one organ or tissue confers protection of another organ or tissue against sustained ischemia-reperfusion insult. Remote ischemic preconditioning by brief lower limb ischemia and reperfusion was reported clinically for the fi rst time in 37 pediatric patients undergoing corrective surgery for congestive heart defects (Cheung et al. 2006 ) . The preconditioning procedure was shown to reduce troponin release 24 h postoperatively. A single-blinded randomized controlled study of 57 adult patients undergoing elective coronary artery bypass graft surgery was reported in 2007 (Hausenloy et al. 2007 ) . Remote ischemic preconditioning consisted of three 5-min cycles of right upper limb ischemia, achieved with an automated cuff in fl ator placed on the upper arm and in fl ated to 200 mmHg, with an intervening 5 min of reperfusion during which the cuff was de fl ated. The investigators found that remote ischemic preconditioning signifi cantly reduced overall serum troponin-T release at 6, 12, 24, and 48 h after surgery. Similar reductions in troponin-T release over the 72-h perioperative period were reported in 2009 from a randomized clinical trial that involved 45 patients (Venugopal et al. 2009 ) . The mechanisms are not yet clarifi ed but may be related to humoral mediators or recruitment of a neuronal pathway.

27.3 Subarachnoid Hemorrhage

Although aneurysmal subarachnoid hemorrhage accounts for only 3Ð5% of strokes, its profound consequences and unique window of intervention have justifi ed its classifi cation as a separate entity (Wong et al. 2008 ) . Early aneurysm occlusion, 572 G.K.C. Wong et al. expert endovascular neurosurgery and microsurgery, and neurointensive care are now the standards of care. Despite these practices, aneurysmal subarachnoid hemorrhage accounts for 11% of patient mortalities and 27% of long-term disabilities (Langham et al. 2009 ) . Even in patients with a “favorable neurological outcome,” our local study suggested 27Ð44% cognitive dysfunction, not to mention the higher proportion of disabled patients who are left with such dysfunction (Wong et al. 2009 ) . The impact of cognitive dysfunction is of paramount importance, as more than half of the impaired patients are between 40 and 60 years of age and are at a point in their working lives at which a signi fi cant career change would be diffi cult. Delayed cerebral ischemia remains an important cause of morbidity and disability after aneurysmal subarachnoid hemorrhage. Although current therapies have improved prognosis in patients with delayed cerebral ischemia, outcomes remain poor. Interventions such as transluminal balloon angioplasty and endothelin antagonists effectively target angiographic vasospasm but at times fail to improve outcomes. Thus, the relation between angiographic vasospasm and neurologic outcome may be associative in addition to causative, and neurologic injury may not be entirely explained by ischemia. However, it should be emphasized that angiographic vasospasm plays an important role in the pathogenesis of delayed cerebral ischemia as patients with signifi cant angiographic vasospasm frequently develop ischemia and infarction in this distribution and their outcome is poor. Oral nimodipine provides modest protection against cerebral infarction and the ensuing outcome, and research efforts have been directed at fi nding adjunctive preventive measures. Activation of innate neurovascular protective mechanisms by preconditioning could be a novel therapeutic approach against subarachnoid hemorrhage-induced vasospasm and delayed cerebral ischemia. In a murine model (Vellimana et al. 2011 ) , preconditioning nearly completely prevented vasospasm and neurological de fi cits. Preconditioning also prevented subarachnoid hemorrhage-induced reduction in nitric oxide availability and increased endothelial nitric oxide (eNOS) synthase activity in mice with and without subarachnoid hemorrhage. These effects were lost in wild-type mice treated with nitric oxide inhibitor and in eNOS-null mice. These data supported notion that eNOS-derived nitric oxide, a known mediator of vascular preconditioning, counters the pathogenesis of cerebral vasospasm. Making a limb transiently ischemic, i.e., remote ischemic limb preconditioning, has been shown to induce ischemic tolerance in a distant organ. Experimental animals undergoing brief limb ischemia have smaller infarcts than animals not exposed to this intervention before stroke. Subarachnoid hemorrhage with risk of delayed ischemia is an ideal candidate for this treatment because of the unique time window (fi rst 72 h) that exists after the initial hemorrhage. In a phase Ib study of the safety and feasibility of remote ischemic limb preconditioning after subarachnoid hemorrhage, 33 patients underwent limb preconditioning every 24Ð48 h for 14 days (Koch et al. 2011 ) . Limb preconditioning consisted of three 5-min in fl ations of a blood pressure cuff to 200 mmHg around a limb followed by 5-min reperfusion of the patient’s leg. Ischemia times were then escalated to 7.5 and 10 min. No session was 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage... 573 prematurely terminated, and no objective signs of neurovascular injury were observed. Similar principles have been tested in abdominal aortic aneurysm repair (Ali et al. 2007 ) and coronary artery bypass graft surgery. This study paves the way for ef fi cacy study of remote limb preconditioning and its ability to reduce delayed cerebral ischemia and hence improve neurological outcomes.

27.4 Carotid Revascularization

Ischemic stroke accounts for 85% of all strokes and is one of the leading causes of disability and death worldwide. According to a recent review by Young et al. ( 2011 ) , stroke accounts for 1 in every 18 deaths in the USA and leaves nearly 30% of those af fl icted permanently disabled. In the USA alone, nearly 800,000 individuals each year suffer a stroke, with one-quarter of those patients suffering a recurrent stroke. Despite advances in stroke prevention, imaging, treatment, and rehabilitation, the costs of care continue to rise. In 2008, the cost of stroke care in the USA was $65 billion/year. The estimated cost of care for patients with stroke in 2010 was $73.7 billion, with overall costs expected to exceed $2 trillion by 2050. One of the key etiologies is carotid atherosclerosis; severe carotid stenosis is the most important risk factor for recurrent stroke. Results of large-scale randomized controlled clinical trials and meta-analyses of pooled patient data have made carotid endarterectomy the standard of care for patients with symptomatic severe carotid stenosis. Carotid angioplasty with stenting has emerged as an alternative treatment, driven in part by patient, physician, and hospital preferences for “less invasive” procedures. Despite antiplatelet prophylaxis, periprocedural thromboembolic complication remains the major cause of morbidity and mortality associated with carotid endarterectomy or carotid angioplasty and stenting. Research efforts have been directed toward reducing these complications and making these procedures more cost-effective. Remote ischemic preconditioning is a physiologic therapy whereby brief ischemia-reperfusion episodes attenuate damage by subsequent prolonged ischemic insult. The myocardial protective effects have been previously discussed, and similar protective effects are expected for carotid endarterectomy, which includes a period of temporary carotid occlusion. A 70-patient pilot randomized clinical trial was recently reported (Walsh et al. 2010) . Direct ischemic preconditioning of the brain is technically possible during carotid endarterectomy, simply by repetitive clamping and unclamping of the internal carotid artery. However, the risk of plaque disruption and embolization resulting in stroke can be devastating. Remote ischemic preconditioning is thus an attractive option for carotid revascularization. In the pilot study, remote ischemic preconditioning was performed immediately after the induction of anesthesia. An infl atable cuff was placed around one thigh. The posterior tibial or dorsalis pedis artery was insonated with a handheld Doppler probe. The cuff was then infl ated until the Doppler signal was completely obliter- ated. Each leg was then subjected to 10 min of ischemia followed by reperfusion. 574 G.K.C. Wong et al.

At least 10 min elapsed before clamping of the carotid artery for carotid endarterectomy. Deterioration in saccadic latency (a surrogate marker of brain injury) was half that of patients who did not undergo remote ischemic limb preconditioning. The exact mechanisms are unknown but are thought to be related to humoral and neural pathways as well as systemic anti-in fl ammatory and anti-apoptotic responses. The pilot results are encouraging, and large-scale trials are suggested to determine the possible clinical bene fi t. For carotid angioplasty and stenting, direct ischemic preconditioning is feasible with deployment of a proximal balloon occlusion system. In a series of 43 patients in whom a proximal balloon occlusion system was used for carotid angioplasty and stenting, initial occlusion was associated with symptoms of cerebral hypoperfusion in 10 patients, but the symptoms reversed within 10 min of balloon defl ation (Faries et al. 2008 ) . There was no recurrence of neurologic symptoms with subsequent balloon reinfl ations lasting 6Ð22 min. Thus, direct ischemic preconditioning can be a useful tactic to allow carotid angioplasty and stenting in patients “not tolerating” temporary balloon occlusion.

27.5 Intracranial Aneurysm Surgery

Intracranial aneurysm rupture accounts for 85% of all spontaneous subarachnoid hemorrhages, and treatment of the intracranial aneurysm to prevent rebleeding and facilitate subsequent treatment of other complications such as delayed cerebral ischemia and hydrocephalus is the key to successful patient management. Aneurysm occlusion can be achieved by applying a microsurgical clip to aneurysm neck (exo- vascular route) or X-ray-guided intravascular coil deployment in the aneurysm body (endovascular route) (Wong et al. 2011 ) . Intraprocedural rupture is associated with morbidity and mortality and should be avoided. Temporary parent artery clip occlusion is a well-established technique to defl ate the aneurysm and avoid re-hemorrhage during clip application for a dissecting aneurysm. However, temporary artery clip occlusion poses a risk of cerebral ischemia and infarction resulting in new neurological defi cits, especially with a duration of more than 10 min. The risk is signifi cant even under intravenous brain-protection anesthetics (propofol, etomidate, and phenobarbital individually or in combination) (Lavine et al. 1997 ) . Other strategies include those that are physiologically based (hyperoxygenation, hemodilution, and hypervolemia) and pharmacologically based (nimodipine, erythropoietin, and others) (Frietsch and Kirsch 2004 ) . Direct ischemic preconditioning is another promising strategy. Our research group previously evaluated the effects of ischemic preconditioning, produced by 2-min proximal artery clip occlusion followed by 30-min reperfusion, on brain tissue gases and acidity during microsurgery in patients with aneurysm dissection (Chan et al. 2005a) . All 12 patients underwent surgery within 72 h after the onset of hemorrhage. All patients received nimodipine infusion at 1Ð2 mg/h together with target controlled infusions of propofol and remifentanil. Neuromuscular blockade 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage... 575 was achieved with rocuronium infusion. Normocapnia and normothermia were maintained with an inspired oxygen concentration set at 30%. After craniotomy, a calibrated multiparameter catheter was inserted into the ipsilateral middle frontal gyrus. Patients were randomized to routine care (n = 6) or ischemic preconditioning ( n = 6). Immediately prior to temporary parent artery clip occlusion, a bolus of 2Ð3 mg/kg thiopentone was given to produce electroencephalographic burst suppression. Arterial pressure was maintained within 20% of baseline pressure with phenylephrine infusion. The duration of temporary parent artery clip occlusion in treating the dissections by aneurysm neck clipping ranged from 5 to 15 min. Brain tissue oxygen and acidity decreased and brain carbon dioxide tension increased in all patients, but the rates of change in brain tissue oxygen and acidity were signi fi cantly lower in patients with ischemic preconditioning. Brain tissue oxygen in all patients receiving routine care fell below 10 mmHg before release of the temporary artery clip, whereas only one-third of patients with ischemic preconditioning showed ischemic change during brain tissue oxygen measurement. We hypothesized that the neuroprotective effect is derived from N-methyl-D-aspartate activation during ischemic preconditioning. We advocate this simple and effective technique for brain protection when prolonged proximal artery occlusion is required during complex aneurysm surgery. Another interesting agent is magnesium. Magnesium is a vasodilator. In animal models, magnesium blocks voltage-dependent calcium channels and reverses experimental vasospasm. The later bene fi t is clinically evident in patients with preeclamp- sia. In such patients, magnesium infusion increases cerebral blood fl ow and reduces the risk of seizure. We previously studied the interaction between magnesium infusion and temporary parent artery occlusion during microsurgery in 18 patients (Chan et al. 2005b ) . The study method was similar to that of the above-mentioned study. All patients underwent surgery within 72 h after the onset of hemorrhage. The patients were randomly assigned to receive either magnesium (n = 9) or saline infusion ( n = 9). For patients receiving magnesium, 20 mmol magnesium sulfate was administered over 10 min, and this was followed by continuous infusion at 4 mmol/h. The plasma magnesium concentration in patients receiving active treatment increased to 2.1 ± 0.5 mmol/L, whereas that in patients receiving placebo was 0.8 ± 0.3 mmol/L. Magnesium infusion increased baseline brain tissue oxygenation from 16 ± 14 to 24 ± 8 mmHg. With temporary parent artery clip occlusion, brain tissue oxygenation and acidity decreased promptly in all patients. Postocclusion brain tissue oxygenation was signi fi cantly greater for patients receiving magnesium infusion. The duration of temporary parent artery clipping ranged from 5 to 14 min. We hypothesized that the improved brain oxygenation resulted from dilation of small leptomeningeal arteries and improved collateral blood fl ow. Thus, magnesium infusion may be a useful adjunct when prolonged temporary parent artery occlusion is anticipated. The other method of aneurysm occlusion is coil embolization. Sometimes, temporary balloon occlusion of the parent vessel at the aneurysm neck is necessary for coil deployment in wide-necked aneurysms. Although no study has focused on patients undergoing balloon-assisted coil embolization of an aneurysm, similar direct ischemic preconditioning and magnesium infusion should work in these patients also. 576 G.K.C. Wong et al.

27.6 Conclusions

The results of clinical research into preconditioning for coronary artery bypass graft surgery, repair of subarachnoid hemorrhage, carotid revascularization, and intracranial aneurysm surgery are encouraging and should be further explored. Both direct and remote ischemic preconditioning are promising strategies for clinical application.

Financial Disclosure Nil.

References

Ali ZA, Callaghan CJ, Lim E et al (2007) Remote ischemic preconditioning reduces myocardial and renal injury after elective abdominal aortic aneurysm repair: a randomized controlled trial. Circulation 116:I98ÐI105 Chan MT, Boet R, Ng SC et al (2005a) Effect of ischemic preconditioning on brain tissue gases and pH during temporary cerebral artery occlusion. Acta Neurochir Suppl 95:93Ð96 Chan MT, Boet R, Ng SC et al (2005b) Magnesium sulfate for brain protection during temporary cerebral artery occlusion. Acta Neurochir Suppl 95:107Ð111 Chen J, Graham SH, Zhu RL et al (1996) Stress proteins and tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab 16:566Ð577 Cheung MM, Kharbanda RK, Konstantinov IE et al (2006) Randomized controlled trial of the effects of remote ischemic preconditioning in children undergoing cardiac surgery: ﬁ rst clinical application in humans. J Am Coll Cardiol 47:2277Ð2282 Dirnagl U, Becker K, Meisel A (2009) Preconditioning and tolerance against cerebral ischemia: from experimental strategies to clinical use. Lancet Neurol 8:398Ð412 Faries PL, DeRubertis B, Trocciola S et al (2008) Ischemic preconditioning during the use of the PercuSurge occlusion balloon for carotid angioplasty and stenting. Vascular 16:1Ð9 Frietsch T, Kirsch JR (2004) Strategies of neuroprotection for intracranial aneurysms. Best Pract Res Clin Anaesthesiol 18:595Ð630 Hausenloy DJ, Mwamure PK, Venugopal V et al (2007) Effect of remote ischemic preconditioning on myocardial injury in patients undergoing coronary artery bypass graft surgery: a randomised controlled trial. Lancet 370:575Ð579 Jimenez-Mateos EM, Henshall DC (2009) Seizure preconditioning and epileptic tolerance: models and mechanisms. Int J Physiol Pathophysiol Pharmacol 1:180Ð191 Kelly ME, Mclntyne DC (1994) Hippocampal kindling protects several structures from the neuronal damage resulting from kainic acid-induced status epilepticus. Brain Res 634:245Ð256 Kitagawa K, Matsumoto M, Tagaya M et al (1990) “Ischemic tolerance” phenomenon found in the brain. Brain Res 528:21Ð24 Koch S, Katsnelson M, Dong C et al (2011) Remote ischemic limb preconditioning after subarachnoid hemorrhage: a phase Ib study of safety and feasibility. Stroke 42:1387Ð1391 Langham J, Reeves BC, Lindsay KW et al (2009) Variation in outcome after subarachnoid hemorrhage: a study of neurosurgical units in UK and Ireland. Stroke 40:111Ð118 Laurikka J, Wu ZK, Lisalo P et al (2002) Regional ischemic preconditioning enhances myocardial performance in off-pump coronary artery bypass graft. Chest 121:1183Ð1189 Lavine SD, Masri LS, Levy ML et al (1997) Temporary occlusion of the middle cerebral artery in intracranial aneurysm surgery: time limitation and advantage of brain protection. J Neurosurg 87:817Ð824 27 Preconditioning Strategy: Coronary Bypass, Subarachnoid Hemorrhage... 577

Murry CE, Jennings RB, Reimer KA (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124Ð1136 Musumeci F, Feccia M, MacCarthy PA et al (1998) Prospective randomized trial of single clamp technique versus intermittent ischemic arrest: myocardial and neurological outcome. Eur Cardiothorac Surg 13:702Ð709 Roach GW, Kanchuger M, Mangano CM et al (1996) Adverse cerebral outcomes after coronary bypass surgery. N Engl J Med 335:1857Ð1863 Smith PK (2006) Predicting and preventing adverse neurological outcomes with cardiac surgery. J Card Surg 21(Suppl 1):S15ÐS19 Stenzel-Poore MP, Stevens SL, Xiong Z et al (2003) Effect of ischaemic conditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states. Lancet 362:1028Ð1037 Stenzel-Poore MP, Stevens SL, King JS (2007) Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis. Stroke 38:680Ð685 Vellimana AK, Milner E, Azad TD et al (2011) Endothelial nitric oxide synthase mediates endogenous protection against subarachnoid hemorrhage-induced cerebral vasospasm. Stroke 42(3): 776Ð782 Venugopal V, Bognolo G, Yellon DM (2009) Remote ischaemic preconditioning reduces myocardial injury in patients undergoing cardiac surgery with cold blood cardioplegia: a randomised controlled trial. Heart 95:1567Ð1571 Walsh SR, Nouraei SA, Tang TY et al (2010) Remote ischemic preconditioning for cerebral and cardiac protection during carotid endarterectomy: results from a pilot randomized clinical trial. Vasc Endovascular Surg 44:434Ð439 Wang X, Wei M, Kuukasjarvi P et al (2003) Novel pharmacological preconditioning with diazoxide attenuates myocardial stunning in coronary artery bypass graft. Eur J Cardiothorac Surg 24: 967Ð973 Wong GK, Ng R, Poon WS (2008) Aneurysmal subarachnoid haemorrhage: a clinical review. Surg Pract 12(2):51Ð55 Wong GK, Wong R, Mok VC et al (2009) Clinical study on cognitive dysfunction after spontaneous subarachnoid haemorrhage: patient pro ﬁ les and relationship to cholinergic dysfunction. Acta Neurochir (Wien) 151:1601Ð1607 Wong GK, Kwan M, Ng R et al (2011) Flow-diverters for treatment of intracranial aneurysms: current status and ongoing clinical trials. J Clin Neurosci 18(6):737Ð740 Yellon DM, Alkhulai ﬁ AM, Pugsley WB (1993) Preconditioning the human myocardium. Lancet 34:276Ð277 Yogaratnam JZ, Laden G, Guvendik L et al (2010) Hyperbaric oxygen preconditioning improves myocardial function, reduces length of intensive care stay, and limits complications post coronary artery bypass graft surgery. Cardiovasc Revasc Med 11:8Ð19 Young KC, Jain A, Jain M et al (2011) Evidence-based treatment of carotid artery stenosis. Neurosurg Focus 30:E2 Zhong KW, Tarkka MR, Eloranta J et al (2001a) Effect of ischaemic preconditioning, cardiopulmonary bypass and myocardial ischaemic/reperfusion on free radical generation in CABG patients. Cardiovasc Surg 9:362Ð368 Zhong KW, Tarkka MR, Eloranta J et al (2001b) Effect of ischemic preconditioning on myocardial protection in coronary artery bypass graft patients: can the free radicals act as a trigger for ischemic preconditioning? Chest 119:1061Ð1068 Chapter 28 HBO Preconditioning for TBI and Stroke Patients

Qiang Wang and Lize Xiong

28.1 Introduction

As one of the top killers of humans, cerebral ischemia claims hundreds of thousands of lives every year throughout the world. Major breakthroughs in the pathophysiology of ischemia/reperfusion (I/R) injury have prompted the “brain attack” campaign—a worldwide campaign to enlighten patients about the early symptoms of stroke. However, prevention is more important than treatment. Preconditioning, a potent endogenous protective mechanism, activates multiple endogenous signaling pathways that can increase cellular tolerance to several potentially noxious stimuli and results in protection against ischemia and has attracted great interest in the last two decades. Unfortunately, the idea of exposure to brief periods of ischemia before an anticipated cerebral ischemic event does not appear to be clinically relevant. Although some investigators have applied the ischemic preconditioning technique in cardiac surgery, we could not expect it be directly applied to the brain. In the 1990s, Kitagawa et al. (1990 ) found that neuronal cells in the gerbil hippocampus developed resistance to ischemic damage after a brief period of reversible ischemia. This report was the fi rst cited to demonstrate the ischemic tolerance in brain by ischemic preconditioning. Recently, a number of substances and other treatments have proved effective for inducing ischemic tolerance in the brain. The term “preconditioning” has been expanded to include pretreatment with other physical stimuli such as hyperbaric oxygen (HBO) and acupuncture. Numerous animal experiments and clinical trials indicate the former may be benefi cial for stroke. However, the results differ signi fi cantly and even seem to con fl ict, especially in clinical trials. Thus, the

Q. Wang • L. Xiong (*) Department of Anesthesiology , Xijing Hospital, Fourth Military Medical University , Xi’an 710032 , China e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 579 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_28, © Springer Science+Business Media New York 2013 580 Q. Wang and L. Xiong clinical application of these methods is as yet unknown because of their unclear ef ﬁ cacy, toxicity, and adverse side effects; therefore, further clinical trials are critically needed to resolve these issues. Therefore, in this chapter, we aim to review some recent HBO research in stroke and traumatic brain injury (TBI), trying to provide a framework that can be used to generate testable hypotheses and treatment strategies that are linked to the ischemic tolerance in brain.

28.2 Application of Hyperbaric Oxygen in Patients with Stroke

HBO therapy is defi ned as a treatment in which a patient intermittently breathes 100% oxygen while inside a chamber that is pressurized to a pressure greater than one atmospheric absolute (ATA). HBO therapy was fi rst documented in 1662; since that time, reports of bene fi cial effects from increased pressure have increased accordingly. This therapy was used to treat many conditions, including burns, gas gangrene, soft tissue infection, anemia, crush injury, and ischemic stroke. Numerous basic studies, clinical trials, and animal experiments have suggested that HBO has potential benefi cial effects on ischemic stroke and I/R injury resulting from traumatic brain injury (TBI). However, because of the lack of solid clinical evidence about the therapy’s effectiveness, safety, and relative mechanisms, the application of HBO has only been approved for medical conditions that for which the effectiveness and low risk of HBO therapy has been confi rmed by extensive clinical trials.

28.2.1 HBO Therapy for Ischemic Stroke

In 1969, a 40-year-old Caucasian male naval of fi cer was diagnosed with an arterial block of the right middle cerebral artery via cerebral angiograms after clear onset of clinical symptoms. Although he regained partial use of his left lower extremity after 1 month of conventional therapy, the upper extremity and facial paresis did not show signi fi cant improvement (Hart and Thompson 1971 ) . As we know, cerebrovascular accidents from thrombotic or embolic phenomena create inadequate perfusion to a portion of the brain. The resultant decrease in perfusion causes a neuronal de fi cit, which appears to be due primarily to the hypoxia. Since HBO has the ability to deliver oxygen to ischemic tissue, its function does not directly depend on the ability to perfuse the injured area. Investigational studies indicate HBO to be an encouraging new therapy for improving function of central neural system function (Lambertsen et al. 1935 ; Smith and Lawson 1962 ; Heyman et al. 1966 ) . Based on this theory, this desperate patient was treated with HBO therapy (2.5 ATA, 2 h daily). His upper motor ability improved gradually. Four months later, he returned to work at full duty. Does this sound like a miracle? 28 HBO Preconditioning for TBI and Stroke Patients 581

Today, HBO therapy is increasingly used in a number of areas of medical practice. In ischemic events such as stroke, HBO therapy has been to shown to promote oxygen delivery, inhibit leukocyte activation, decrease cerebral edema, lipid peroxidation, and decrease the volume of brain infarction, improve outcomes, and maintain the blood–brain barrier (BBB) (Al-Waili et al. 2005 ; Sukoff and Ragatz 1982 ; Thom 1993; Sunami et al. 2000 ) . The goal of HBO therapies is considered essentially to salvage the penumbra to minimize cognitive and physical sequelae associated with acquired brain injury in order to enhance the patient’s quality of daily life. Typically, clinical treatments involve pressurization to between 1.5 and 3.0 ATA for periods between 60 and 120 min once or twice per day for varying durations. Numerous basic and animal models have shown bene ﬁcial effects of HBO in cerebral I/R injury.

HBO therapy entails the inhalation of high concentrations of O2 (94–100%) at pressures higher than sea level. Its effects result from the combined action of hyperoxia and hyperbaria, when pressurized pure O2 becomes dissolved in all body fl uids, especially in the plasma and cerebrospinal fl uid (Leach et al. 1998 ) . As local anoxia and energy failure are the initiating cellular stage of ischemia, the inhalation of oxygen at increased atmospheric pressures might produce a marked elevation in arterial blood oxygen partial pressure and content. The delivery of hyperoxygenated blood to ischemic areas to aid in their recovery is vital to the concept of HBO therapy. However, clinical studies remain limited and somewhat controversial. This section reviews the most relevant literature in two speci fi c areas: ischemic stroke and trauma-related I/R injury. Therapeutic treatment for acute ischemic stroke can be approached in two basic ways: fi rst, to restore or improve blood fl ow in an occluded vascular territory and, second, to direct therapy at cellular and metabolic targets (Fisher 1995 ) . Interruption of a cerebral artery reduces, even abolishes, the delivery of oxygen to the brain. Therefore, cerebral histopathologic damage depends on the degree and duration of hypoxia resulting from vascular impairment. The key is to salvage ischemic penumbra, fi rst demonstrated by Furlan et al. ( 1996 ) , which is a rim of mild to moderately ischemic tissue that exists between normally perfused brain and the evolving infarct. In other words, the ischemic penumbra has potential to be salvaged to some extent based on the presence of collateral vessels and perfusion pressures. The border of the penumbra may be a predictor for I/R injury recovery. There are many recent clinical trials on HBO therapy, and it seems that it may help salvaging the penumbra in ischemic tissues. But previous studies on this subject showed con fl icting results. In order to clarify the safety, effectiveness, and feasibility of HBO therapy, we focus on several studies that meet the criteria for the highest level of evidence as prospective, randomized, controlled, double-blinded clinical trials or trials with a large sample sizes in this chapter. We hope that our discussion of these trials may be useful to clinicians who are unsure of whether their patients will bene fi t from this therapy. Neubauer and his team ( 1980) enrolled 122 patients in the 1980s; 86 men and 36 women were included who suffered an acute or complete stroke. The average age of patients was 66 years with a range from 44 to 88 years. All patients were given a complete neurological examination and computed tomography (CT) scans, electro- 582 Q. Wang and L. Xiong encephalograms (EEGs), skull x-rays, and cerebral spinal fl uid examinations when indicated. Outcome evaluation included ambulation problems, dysphasia, and days in hospital. The observation period was 10 years. Patients were treated with 1 h of HBO exposure every 12 h. Some patients clearly improved while in the chamber and regressed when out of the chamber, and their exposure was prolonged up to 2 h, or they were treated more frequently during each 24-h period. Results showed that most patients who received HBO therapy had improved motor function and spent fewer days in the hospital (standard treatment 287 days vs. HBO treatment 177 days), and 62% patients in the HBO group had better daily life qualities, such as behavior, communication ability, and willingness to participate in social life. In addition, HBO therapies did not show signi fi cant adverse side effects. Thus, the therapy is extremely safe. Unfortunately, because it lacks a control group, analyses based on their data cannot indicate that all patients benefi t from HBO therapy. However, its results do suggest that patients with complete stroke bene fi t from HBO. The fi rst prospective randomized controlled trial was reported in 1991 by Anderson et al. ( 1991) . Their trial enrolled 39 patients with onset of neurological de fi cits due to ischemic cerebral infarction who were randomized to two groups: HBO (n = 20) and sham (n = 19). Outcome measures included a validated graded neurological examination and quantifi cation of infarct size measured by head CT scan. Though all 39 patients received contemporary standards of nursing care, it should be noted that patients in the HBO group seemed to have more severe insults and, accordingly, more medical care demands. Their results showed a trend toward better outcomes in the sham group. That is, HBO therapy might be harmful to patients. This condition may have resulted in the study observations due to the unstable health of the HBO patients. However, these patients were not followed for long. Thus, it is still unclear whether HBO bene fi ts or risks patients. In August 1995, Nighoghossian and investigators (1995 ) published another double-blind pilot study, in which 21 men and 13 women were included over 3-year enrollment period. Patients’ onsets strongly suggested that they suffered from middle cerebral artery occlusion. Patients were randomized into HBO (1.5 ATA 40 min daily) and sham (air) groups. Mortality and changes in three healthcare scales, the Orgogozo scale, the Rankin disability scale, and the Trouillas scale, 6 months and 1 year later were used as outcome measures. At inclusion, there were no signifi cant differences between groups in age, time from stroke onset to randomization, or Orgogozo scale scores. In the fi rst week, four patients from sham group and three from the HBO group were excluded because of neurological deterioration or myocardial infarction. The mean Orgogozo scale score for the HBO group was signifi cantly better at 1 year. However, there was no signifi cant improvement observed in the other two scales. Based on their results, they hypothesized that HBO therapy might be safe and improve outcomes. However, they also noted that a larger clinical trial might be needed in order to con fi rm their hypothesis. The deeper we research, the more questions we have. Another persistently controversial study was reported by Rusyniak et al. ( 2003 ) . in 2003. Thirty-three patients were randomized to treatment for 60 min in a monoplace hyperbaric chamber pressurized with 100% oxygen to 2.5 ATA in the HBO group or 1.14 28 HBO Preconditioning for TBI and Stroke Patients 583

ATA in the sham group. Their results suggested that HBO might be harmful to ischemic stroke patients. Drs. Zhang et al. (2003 ) (Badr et al. 2001 ) and Jain ( 2003) questioned the pressure chosen in this study. In animal experiments, 3 ATA HBO reduced infarct volumes and improved neurological outcomes within 6 h, but increased infarct volumes and aggravated neurological de fi cits when applied at 12 or 24 h after the onset of reperfusion. In Rusyniak’s research (Rusyniak et al. 2003 ) , only 15% patients were treated within 6 h, which may have resulted in bias. Previous studies in stroke patients indicated that 100% oxygen at a pressure of 1.5 ATA is better tolerated by brain injured by acute ischemia than higher pressures. This pressure had a favorable effect on glucose and energy metabolism of the brain, as well as on the clinical course. It seems fi tting those patients who received oxygen at 1.14 ATA did better than those who received it at 2.5 ATA, since the former is closer to the ideal pressure of 1.5 ATA. Dr. Jain (2003 ) also suggested that establishing another group treated with 1.5 ATA may be better. Dr. Rusyniak responded that the most suitable pressure for the treatment of ischemic stroke is unknown, and their decision to use a sham treatment at 1.14 ATA was based on the desire to blind the study as much as possible. Because of the differing opinions on this issue, signifi cant progress is still needed. Rockswold and colleagues (2010 ) reported on a prospective, randomized clinical trial comparing the effect of hyperbaric to normobaric hyperoxia in 2010. They examined 74 patients with severe traumatic brain injury (TBI). Five of them were excluded when they experienced brain death. The remaining 69 patients were randomized into hyperbaric (n = 26, 1.5 ATA), normobaric (n = 21), and control groups (n = 22). The numbers in each group were not equal as a result of dif fi culties obtaining accurate jugular venous blood gases. Brain tissue oxygen partial pressure (PO2 ), intracranial pressure (ICP), microdialysate lactate, glucose, pyruvate, and glycerol were continuously monitored as outcome measures. The global metabolic measures were cerebral blood fl ow (CBF), cerebral metabolic rate of oxygen (CMRO2 ), etc. The article concluded that HBO had a larger effect than normobaric hyperoxia on oxidative cerebral metabolism related to its ability to produce a brain tissue PO2 > 200 mmHg. In addition, there were no signs of cerebral oxygen toxicity after HBO treatment.

28.2.2 Future of HBO Therapy for Stroke

Widespread use of HBO treatment has emerged for many diseases including stroke since the 1960s. HBO therapy has demonstrated value as adjunct treatment in patients with ischemic cerebral stroke, including those who have undergone surgery on the extracranial portion of the major cerebral vessels (Elinskii et al. 1984 ) . Two pilot case studies have been reported which indicated HBO therapy to be effective in treating patients with hemorrhagic stroke and chronic brain injury (Lim et al. 2001 ; Hardy et al. 2007 ) . Furthermore, abundant animal studies and greater clinical experience over the last two decades have produced indications for which HBO therapy might appear benefi cial. However, there are no solid convincing conclusions reported, which 584 Q. Wang and L. Xiong leads to persistent controversy. Subsequently, enthusiasm for using HBO in stroke has waned, especially after Rusyniak (Rusyniak et al. 2003 ) reported his research. Though ischemic stroke may provide a good rationale for the use of HBO, there is no convincing evidence that HBO therapy improved outcomes after stroke or brain injury. Considering that HBO is expensive, not universally available, not risk- less, and lacks solid supporting evidence, the therapy Evaluation Committee for the Insurance Corporation of British Columbia has suggested that the medical literature does not support the use of HBO for traumatic brain injury and stroke (Alternative Therapy Evaluation Committee for the Insurance Corporation of British Columbia 2003) . The “guideline for the early management of adults with ischemic stroke,” published in 2007, notes that data do not support the routine use of HBO in the treatment of patients with acute ischemic stroke at present. These two authoritative documents may slow the pace of future use of HBO as a treatment for stroke. The major criticism of HBO might be related to the lack of pre- and postischemic stroke period controlled clinical trials, the unknown mechanisms, and potential adverse side effect. According to clinical practices, acute ischemic stroke treatment trials may have narrow time windows in which potential benefi cial effects can result. Effectiveness of HBO therapy may largely depend on how soon the patient was delivered to hospital and received treatment after the onset of symptoms. Severity of insult and greater demands for medical care can also result in deviations. Furthermore, effi cacy, feasibility, and possible tissue toxicity are equally important. The clinical application of HBO for ischemic stroke remains to be fully understood due to the confl icting results reported in current clinical trials. Further prospective, randomized, controlled, double-blinded, and multicenter trials are needed to determine whether HBO therapy in stroke patients might be still a competitive treatment for ischemic stroke or I/R injuries resulted from TBI. The answer may give clinicians another option in treatment on these diseases.

28.2.3 HBO Preconditioning in Patients with Stroke

Protection is de fi nitely superior to treatment. Although ischemic preconditioning is con fi rmed to be effective in animal models, it is not always feasible in clinical practice. Researchers introduced HBO preconditioning into the campaign against cerebral I/R injury nearly two decades ago. In 1993, Sterling et al. (1993 ) . provided one of the earliest experimental evidence that HBO pretreatment (also known as HBO preconditioning) prior to reperfusion was able to induce myocardial protection in rabbits. Following this, Wada et al. (1996 ) . in 1996 demonstrated the phenomenon of ischemic tolerance induced by repeated HBO preconditioning in the gerbil hippocampus. Thereafter, the protective effect of HBO has also been observed in other experimental models and organs, such as transient middle cerebral artery occlusion in rats, spinal cord ischemia in rabbits, and liver I/R injury in rats (Kim et al. 2001 ; Xiong et al. 2000 ; Dong et al. 2002 ; Nie et al. 2006 ; Chen et al. 1998 ; Yu et al. 2005) . In the past decade, ischemic tolerance induced by HBO preconditioning is 28 HBO Preconditioning for TBI and Stroke Patients 585 getting increasing attention, along with its therapeutic effects when applied after the ischemic injury. However, most previous studies on the protective effects of HBO preconditioning are based on data from animals. So far did only four clinical studies examine the effect of HBO preconditioning on human ischemic injuries (Sharifi et al. 2004 ; Yogaratnam et al. 2010 ; Alex et al. 2005 ; Li et al. 2011 ) . The fi rst clinical program to address the effect of HBO preconditioning was the Hyperbaric Oxygen Therapy in Percutaneous Coronary Intervention (HOT-PI). In this study (Shari fi et al. 2004 ) , patients diagnosed with unstable angina or acute myocardial infarction were randomized into control or HBO groups. Patients in the HBO group received adjunctive HBO treatment 2 h before or immediately after percutaneous coronary intervention (PCI) followed by another treatment less than 18 h after the fi rst HBO treatment. The HBO treatment consisted of 100% oxygen at 2 ATA for 90 min. The authors demonstrated that patients who received adjunctive HBO therapy had a lower clinical restenosis rate. In addition, the incidence of adverse cardiac events such as myocardial infarction, coronary artery bypass graft (CABG), and death was signi fi cantly higher in the control group compared to the HBO group in the coming 8 months of PCI. In another study (Yogaratnam et al. 2010 ) , 81 patients who had fi rst-time elective on-pump CABG were divided into two groups: 40 in the control group and 41 in the HBO group. Prior to CABG, the HBO group received twice 30-min HBO preconditioning, separated by a 5-min interval. The HBO treatment consisted of 100% oxygen at 2.4 ATA. The results showed that the HBO group had a decreased pulmonary vascular resistance (PVR) before cardiopulmonary bypass (CPB) and increased stroke volume (SV) and left ventricular stroke work (LVSW) when CPB had been withdrawn. The HBO group also attenuated the increase in cardiac troponin T (cTnT), spared inotrope administration, and reduced incidence of cardiac output syndrome and atrial fi brillation, which indicated that HBO preconditioning alleviated myocardial injuries and improved postoperative myocardial function. There have been only two clinical studies on HBO preconditioning-induced cerebral protection. Alex et al. ( 2005) . observed the effects of HBO preconditioning on neuropsychometric dysfunction and systemic in fl ammatory responses after CPB. In this prospective trial, 33 patients (HBO group) were preconditioned with HBO (2.4 ATA) before on-pump CABG. Thirty-one patients received air (1.5 ATA) as control. Patients in both groups had 3 sessions in the hyperbaric chamber at 24, 12, and 4 h before CPB. In each session, two 30-min HBO preconditioning or air treatments were given with a 5-min interval. Infl ammatory markers and neuropsychometric assessment were measured as predictors of neuropsychometric dysfunction. The results showed that the control group had markedly postoperative increases in in fl ammatory markers such as soluble E-selectin, CD18, and heat shock protein 70 (Hsp 70), which was not observed in the HBO group. Neuropsychometric dysfunction was also signifi cantly higher in the control than in the HBO group. This indicated that that HBO preconditioning could reduce neuropsychometric dysfunction and the in fl ammatory response after CPB. Recently, Li and colleagues (2011 ) have demonstrated that HBO preconditioning resulted in both cerebral and cardiac protective effects in patients undergoing 586 Q. Wang and L. Xiong on-pump CABG surgery. However, no such protective effects were observed in off- pump CABG patients. Their prospective, randomized, single-blinded study enrolled 49 patients. Patients were randomized to either control (n = 25) or HBO groups ( n = 24). Both groups were further divided into on-pump and off-pump subgroups (15 vs . 10 in control group and 14 vs. 10 in HBO group, respectively). The HBO group received preconditioning once per day (35 min for twice, with a 5-min interval, 2 ATA) for 5 days before surgery. Compared to those patients in the control on-pump subgroup, the HBO on-pump subgroup had lower levels in S100b protein, neuron- speci fi c enolase (NSE), and serum cTnI, associated with reduced postoperative intensive care unit length of stay and fewer dosages of inotropic drugs. Nevertheless, no similar differences were noted between the two off-pump subgroups. Another Chinese study reported that HBO preconditioning could improve hypoxic tolerance and fatigue at high altitude in young volunteers (Cui et al. 2008 ) . To observe the infl uence of HBO preconditioning on antioxidase activity, blood lactate clearance, and exercise tolerance, Cui et al. measured the levels of superoxide dismutase (SOD), nitric oxide (NO), nitric oxide synthase (NOS), lactate, blood urea nitrogen (BUN), and malondialdehyde (MDA) in blood from young volunteers, and the EMG- bicycle-ergometer was taken as a tool to determine fatigue. They concluded that HBO preconditioning could enhance antioxidase activity, increase blood lactate clearance, and reduce fatigue, and that these effects lasted for 8 days.

28.2.4 Clinical Prospects for HBO Preconditioning

Although present trials seem to be encouraging, the future of HBO preconditioning medicine remains unclear. We note that in the studies mentioned above, the primary endpoints were most often biomarkers instead of clinical outcomes. Do these biomarkers predict patient outcomes? As we know, every coin has two sides. Thus, HBO, just as pharmacological preconditioning methods (Yogaratnam et al. 2008 ) , may also have potential risks or adverse effects if not properly used. For safe clinical use, we require an optimal HBO-preconditioning protocol, including exact preconditioning dosing and timing, which are poorly understood (Table 28.1 ). Training and equipment are also obstacles for HBO promotion. HBO therapy requires speci fi c devices and specially trained physicians, which limits HBO therapy to some large medical centers. When patients are at high risk for an ischemic attack and HBO is not available, are there any alternatives able to mimic the protection of HBO? In an experimental study, Zhang et al. ( 2004) found that long periods of inhaling high concentration oxygen before cerebral ischemia could also induce ischemic tolerance in rats. Other studies on the mechanisms of HBO preconditioning suggested that numerous proteins and molecules are involved in the process. On the other hand, these studies provided novel targets for the developments of new drugs and new therapies, which may ultimately replace the need for HBO. Under the principles of evidence-based medicine, the greatest evidence is provided in the form of prospective randomized controlled trials (RCTs). The purpose 28 HBO Preconditioning for TBI and Stroke Patients 587 No Side effects Yes Yes No No ), etc. 2 , ICP, glucose, Yes No 2 at 24 h and 90 days improvements at 24 h and 90 days improvements Serum cardiac troponin I (cTnI) Serum catalase Neuron-speci fi c enolase (NSE) cerebral blood fl ow (CBF), cerebral metabolic rate of oxygen (CMRO Serum S100 beta On-pump by 5-min interval, 5 days by 5-min interval, Sham: 1.14 ATA Sham: 1.14 ATA outcome scale effects, adverse Mortality, 1 h/12 h; More frequent within 24 h Mortality Days in hospital 1 h/every 8 h 1 h/every of infarct Volume 40 min/day Rankin) Trouillas, Healthcare scale (Orgogozo, Con 25) sham 16) NBO 21; con 22) sham 20) sham 17) Design Treatment Outcome measures Effectiveness Clinical trials on hyperbaric oxygen preconditioning CABG 49 patients (HBO 24; Stroke Stroke 33 patients (HBO 17; Rockswold (2010) Rockswold RCDB 17 min 1.5 ATA, Brain tissue PO TBI 69 patients (HBO 26; Table Table 28.1 Author (year) Disease/surgery Neubauer (1980) Stroke Clinical review Anderson (1991) Stroke 1.5 ATA RCDB 122 patients 39 patients (HBO 19; 2 h/12 h 1.5 ATA Motor function improvement Quality of daily life Graded neurological examination Yes No No No Nighoghossian (1995) Stroke RCDB 34 patients (HBO 17; 1.5 ATA Mortality Yes No Rusyniak (2003) RCDB 1 h single 2.5 ATA, The percentage of participants with Li (2011) PRCSB 2, separated × 35 min 2 ATA, 588 Q. Wang and L. Xiong of RCTs is not only to prove laboratory fi ndings, but also to address clinical issues in real populations and situations. We expect further well-designed multicenter RCTs to promote HBO preconditioning from bench to bedside.

References

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Qiang Wang and Lize Xiong

29.1 Introduction

Stroke is the second most common cause of death and results in a large number of people with disability worldwide. Strokes are either ischemic or hemorrhagic, but more than 80 % of stroke cases are caused by cerebral ischemia (Donnan et al. 2008) . Although many neuroprotective agents have been proved to reduce infarction volume and improve neurological recovery in basic research with animal models of stroke, few of them show effects in clinical trials (Lu et al. 2002 ; Lees et al. 2006) . Moreover, most of the currently used pharmacotherapies are focused on treatment after ischemia. Therefore, it is a huge and urgent medical need to develop novel and rational strategies aimed at preventing stroke as well as reducing impairments caused by ischemia/reperfusion (I/R) injury. The phenomenon of preconditioning-induced ischemic tolerance provides a new idea for the prevention of I/R injury. The preconditioning effect is that a brief exposure to sublethal or noninjurious stimuli can increase resistance to the subsequent, prolonged, and lethal damage (Murry et al. 1986 ) . There are various kinds of preconditioning measures, such as ischemia (regional or remote), hypoxia, endotoxin, cytokines, and anesthetics. Accumulating preclinical evidences have demonstrated that these preconditioning methods, especially ischemia preconditioning, could induce neuroprotection and myocardial protection against I/R injury. But from the clinical point of view, all the mentioned preconditioning ways have limitations and adverse effects to be applied in patients, especially the patients with severe illness. Electroacupuncture (EA) is a novel therapy based on traditional acupuncture combined with modern electrotherapy. Due to the bene ﬁ cial effects of acupuncture to different brain and heart diseases, EA has been used in treatment as an improvement

Q. Wang ¥ L. Xiong (*) Department of Anesthesiology , Xijing Hospital, Fourth Military Medical University , Xi’an 710032 , China e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 591 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_29, © Springer Science+Business Media New York 2013 592 Q. Wang and L. Xiong on traditional acupuncture. Evidence shows that EA not only reduces the myocardial injury but also signiﬁ cantly promotes recovery of neurological function and, thus, improves their quality of life (Chou et al. 2009 ) . In recent years, numerous studies have shown that EA also induced preconditioning effect, inducing ischemic tolerance as well (Xiong et al. 2003 ; Wang et al. 2005) . Since EA is economical, easily performed, and has few negative side effects, it is clinically applicable for prevention and not just treatment of ischemic cardiac/ cerebral disease. In this review, we discuss evidence of EA preconditioning-induced neuroprotection both from animal experiments and clinical trials, parameters of EA preconditioning, and some mechanisms involved in this effect. Because the heart shares lots of characters with the brain, especially in I/R injury, and researchers have done lots of work of EA preconditioning on the heart, the myocardial protective effect of EA preconditioning is included.

29.2 “Preventative Acupuncture” and EA Preconditioning

As an indispensable part of traditional Chinese medicine, acupuncture has played an important role in the prevention and treatment of diseases throughout history. “Treating before sick” is the plain idea of preventive medicine in traditional Chinese medicine. “Preventative acupuncture” is an approach using acupuncture to “treat before sick,” namely, applying acupuncture in healthy or mildly sick patients, to stimulate the meridians of the body and enhance the body’s resistance to disease, in order to prevent disease or to reduce the extent of damage following disease (Wang and Liang 2008) . Integrated with electrotherapy, EA is conducted by insert- ing acupuncture needles into acupoints and then changing electric stimulation parameters, including the stimulation frequency, current intensity, pulse width, and pulse interval (Napadow et al. 2005 ) . In 2003, Xiong and colleagues (2003 ) fi rst reported that repeated EA stimulation at the Baihui acupoint (GV20) before cerebral ischemia in rats could signifi cantly reduce infarct volume caused by transient middle cerebral artery occlusion (MCAO) (38.3 ± 25.4 mm in the EA group vs. 220.5 ± 66.0 mm in the control group and 168.6 ± 57.6 mm in the anesthetized group) and improve later neurological outcomes. The results shown that EA stimulation before ischemia could produce an effect similar to ischemic preconditioning and induce ischemic tolerance. Later, this group showed that preconditioning with a single EA session could induce tolerance to focal cerebral ischemia in rats. At the same time, Jiang K et al. ( 2003 ) found that EA preconditioning on Hegu (LI4), a well-known acupoint commonly used in Oriental medicine for the treatment of neuronal injury resulting from hypoxia-ischemia, could induce neuroprotection in neonatal hypoxic-ischemic rat brains. There are many similarities between EA preconditioning and ischemic preconditioning, such as the common species and organs and the regularity of stimulating time and pattern. Furthermore, both ischemic preconditioning and EA 29 Electroacupuncture Preconditioning for Stroke Patients 593 preconditioning can produce the acute and delayed neuroprotection. Wang et al. (2005 ) found that a single EA stimulation at Baihui acupoint for 30 min could induce biphasic tolerance against focal cerebral ischemia: the acute phase occurred 2 h after EA preconditioning while the delayed ischemic tolerance was observed 24 h after the stimulus. EA preconditioning is not neuroprotective only in ischemic cerebral injury. Recently, researchers found that EA preconditioning could reduce pathological injury in hippocampal neurons, decrease the expression of activated caspase-3, reduce the number of apoptotic neurons in the CA1 area, and improve learning and memory in rats exposed to high-sustained positive hypergravity (+Gz) (Feng et al. 2010) . The results showed that EA preconditioning could ameliorate +Gz-induced impairment of learning and memory by inhibiting neuronal apoptosis. In addition, EA preconditioning was also observed in the heart. Tsou M et al. (2004 ) applied EA to bilateral Neiguan (PC6) before or during myocardial I/R induced by ligating and reperfusing the left anterior descending coronary artery. The results showed that there were signifi cant reductions in cardiac enzymes, the duration of arrhythmia and mortality rate in rats that were either preconditioned or treated with EA on PC6, compared with those that did not undergo EA.

29.3 Parameters of EA Preconditioning

The electric stimulation parameters (frequency, pulse width, current intensity, and duration) and their quantifi cation are important parts of the research on the principles and application of EA. Studies have shown that electric stimulation of different parameters may have different effects on some bodily functions. Nested study design was adopted to test the in fl uence of different parameters and their combination on EA preconditioning-induced cerebral ischemic tolerance in rats. This study at last confi rmed the optimum electrical stimulation parameters of EA preconditioning: sparse-density wave of 2/15 Hz, current intensity of 1 mA, 30 min/ day for 5 consecutive days (Yang et al. 2004 ) . The results showed that varying the frequency and waveform of the stimulus could produce different protective effects, but there were no signifi cant differences in infarct volume between rats who received stimulation from 1 to 3 mA current intensity, indicating that frequency and waveform were probably more important parameters than current. Sparse- density wave had the most obvious neuroprotective effect, followed by intermittent wave, and the continuous wave’s neuroprotective effect was relatively poor. The probable reason for this is that continuous wave tended to induce tolerance of the electric stimulus, whereas sparse-density wave could stimulate the release of different types of neurochemicals by transforming between low-, medium-, and high-frequency stimuli. Therefore, the sparse-density wave EA stimulation could generate neuroprotective effect at different targets via the activation of different signaling pathways. 594 Q. Wang and L. Xiong

29.4 Acupoint Speci ﬁ city of EA Preconditioning

Based on meridian theory, an acupoint is relatively specifi c to certain functions or certain organs, and different effects occur when different acupoints are stimulated. The neuronal speci fi city of acupoints has been tested by functional magnetic resonance imaging (fMRI), providing neurobiological evidence for the existence of acupoint speci fi city (Wu et al. 2002 ; Na et al. 2009 ) . Lu et al. (2002 ) found that EA preconditioning of the Baihui acupoint could induce more robust neuroprotection against cerebral I/R injury than stimulation 1 cm lateral to the Baihui acupoint or non-meridian points at the distal limbs. The Baihui acupoint was chosen because the theory of meridians in traditional Chinese medicine indicates that the Du meridian is closely related to the brain and spinal cord, and Baihui is one of the acupoints of the Du meridian. At the same time, in modern medical terms, the Baihui acupoint (GV20) is in the projection area of the motor and sensory cortex, as well as in the projection area of the anterior cerebral artery. Therefore, Baihui is probably an important acupoint in preventing and treating cerebral diseases. Similarly, EA preconditioning at the Weizhong acupoint (BL40) was more benefi cial for spinal cord I/R injury in rabbits than preconditioning at the Tsusanli acupoint (ST36) (Lei et al. 2003 ) . Hegu (LI4) was chosen for the treatment of neuronal injury resulting from hypoxia-ischemia (Jiang et al. 2003 ) , and Neiguan (PC6) was preferred in EA preconditioning-induced cardioprotection for its effect on heart disease (Tsou et al. 2004 ) .

29.5 Mechanisms of EA Preconditioning

EA is pleiotropic and EA preconditioning could have multiple complicated in ﬂ uences on the physiology of brain and on the pathophysiology of cerebral ischemia. EA preconditioning enhanced the activity of mitochondrial respiratory enzymes and reduced the production of reactive oxygen species (ROS), consequently improving the function of the respiratory chain and antioxidant capacity in the ischemic penumbra (Zhong et al. 2009 ; Siu et al. 2004) . In addition, it increased the levels of anti-apoptotic genes like Bcl-2 while decreasing the levels of pro- apoptotic genes such as c-Jun and c-Fos, inhibiting subsequent apoptotic cascades (Jiang et al. 2004) . Jiang et al. ( 2003) also found that the antagonizing effect of EA preconditioning on cerebral hypoxia/ischemic injury may be related to its activation of KATP , inhibiting the neuronal apoptosis induced by the immediate genes c-Fos and c-Jun at early injury stages. Recent investigations have shown that the endocannabinoid system may be a new mechanism of EA preconditioning-induced neuroprotection. EA preconditioning increased the release of 2-arachidonylglycerol (2-AG) and N -arach- idonoylethanolamine-anandamide (AEA) and upregulated the expression of cannabinoid receptor 1 (CB1) in brain. Selective CB1 antagonist AM251 blocked the neuroprotective effects of EA preconditioning, and CB1 short interfering RNA (siRNA) attenuated EA preconditioning-induced cerebral ischemic tolerance. 29 Electroacupuncture Preconditioning for Stroke Patients 595

Meanwhile, preconditioning with 2-AG and AEA also reduced infarct size and improved neurological outcomes (Wang et al. 2009 ) . Activation of the CB1 receptor triggers signal transduction events that can infl uence ischemic compensatory responses. Cellular responses that elicit neuroprotection may involve CB1 receptors and their link to a variety of signaling elements, including the Gi/Go family of G-proteins, mitogen-activated protein kinase (MAPK), kinase (MEK1/2), and its substrate, extracellular signal-regulated kinase (ERK1/2). Further studies have demonstrated that the neuroprotection due to EA preconditioning could be abolished by U0126 (a specifi c inhibitor of the MEK1/2) or TAT-e V1Ð2 (an e PKC-selective peptide inhibitor). The blockade of CB1R by a CB1 receptor antagonist AM251 reversed the activation of ERK1/2 and e PKC from EA preconditioning. These fi ndings suggested that the ERK1/2 and e PKC pathway might be involved in EA pretreatment-induced cerebral ischemic tolerance via the cannabinoid CB1 receptor (Du et al. 2010 ; Wang et al. 2011 ) . Moreover, BBB integration and stress reactions are involved in the neuroprotection after EA preconditioning. The expression and activity of matrix metalloproteinase-9 (MMP-9), one of the matrix metalloproteinases (MMPs), decreased after EA preconditioning; subsequently, brain edema and BBB damage were signifi cantly alleviated (Dong et al. 2009 ) . Level of heat shock protein 70 (HSP70) in the cortex was elevated in the EA preconditioning group. Enkephalin and opioid receptors (Xiong et al. 2007 ) , adenosine, and adenosine receptor (A1) (Wang et al. 2005 ) have also been implicated in EA preconditioning-induced cerebral ischemic tolerance. Previous studies indicate that the N -methyl-d -aspartate receptors (NMDARs) are responsible for glutamate-induced excitotoxicity in the post-ischemic brain (Briz et al. 2010) . EA preconditioning suppressed the expression of NR1, a subunit of the NMDAR, which may contribute to its effect in reducing apoptosis and protecting cerebral neurons (Meng et al. 2008 ) . Further study suggested that reduced NR1 expression could be reverted by speci fi c inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway, but inhibition of the ERK pathway did not show the same effects (Sun et al. 2005 ) . Therefore, EA preconditioning attenuates glutamate excitotoxicity by modulating the PI3K pathway. The mechanism of EA preconditioning in the heart is not entirely identical to brain. Studies have shown that EA preconditioning can signifi cantly attenuate the incidence of arrhythmia and enhance myocardial cAMP and Gs alpha protein after I/R injury (Gao et al. 2006a, 2007 ) . This attenuating effect was signi fi cantly inhibited by intraperitoneal pretreatment with propranolol, a speci fi c beta-adrenoceptor antagonist (Gao et al. 2006b ) . These results indicated that EA preconditioning is antiarrhythmic after myocardial I/R and is mediated by the post-receptor signaling pathway of the beta-adrenergic receptor. Later, the same group found that adenylate cyclase, protein kinase A, and L-type Ca 2+ channel, the beta-adrenoceptor signaling 2+ 2+ components modulating intracellular Ca ([Ca ]i ), were involved in mediating the neuroprotection of EA preconditioning in rat hearts isolated and subjected to simulated global ischemia and reperfusion (Gao et al. 2008 ) . Preconditioning with EA can effectively resist myocardial I/R-induced arrhythmia and intracellular calcium oscillation in the rat (Qin et al. 2008 ) . 596 Q. Wang and L. Xiong

29.6 Clinical Application of EA Preconditioning

Although EA preconditioning has been shown to reduce cerebral ischemic injury in numerous preclinical studies, it is unclear whether EA preconditioning could also be applied in clinical practice. The latest clinical trials provided some evidence for the effectiveness of EA preconditioning in patients (Table 29.1 ). To study neuroprotection after EA preconditioning, Lu et al. ( 2010 ) . enrolled 32 patients requiring selective craniocerebral tumor resection and randomly assigned them to EA or control groups. Patients in the EA group received EA stimulation at Fengfu (Du16) and Fengchi acupoint (GB20) for 30 min, 2 h before surgery. Patients in the control group received no preconditioning. The results showed that the serum levels of S100beta and NSE in the EA group were signi fi cantly lower than in the control group at the end of the surgery and 24 h post-surgery. This indicated that EA preconditioning may have potential protective effects on surgical brain damage. In the same year, Yang and colleagues (2010 ) also designed a randomized controlled trial to investigate cardioprotection of EA preconditioning in patients undergoing heart valve replacement surgery. EA or sham stimuli was applied at bilateral Neiguan (PC6), Lieque (LU7), and Yunmen (LU2) for 30 min each day for 5 consecutive days before surgery. The level of serum cardiac troponin I was signifi cantly decreased in the EA group at 6, 12, and 24 h after aortic cross-clamp removal. Meanwhile, EA preconditioning also reduced inotrope use at 12, 24, and 48 h after intensive care unit arrival and shortened intensive care unit stay time. These results demonstrated that EA preconditioning may alleviate cardiac I/R injury in adult patients undergoing heart valve replacements. These two clinical trials have indicated that EA preconditioning may have bene fi cial effects on patients undergoing surgery. But, this evidence is limited since the number of enrolled patients is small and both of these trials were conducted in a single center. Thus, multiple center randomized controlled trials are needed to provide further evidence on EA preconditioning.

29.7 Future Prospects of EA Preconditioning

Available data indicates that EA preconditioning can reduce ischemic cerebral injury and improve neurological outcomes. Like other preconditioning methods, EA preconditioning can induce biphasic (acute and delayed) tolerance against cerebral ischemia. However, unlike other preconditioning methods, neuroprotection induced by EA preconditioning is parameter-dependent and acupoint-speciﬁ c. Multi-level, multi-pathway, and multi-target mechanisms have been identi ﬁ ed in neuroprotection after EA preconditioning. Compared with other preconditioning methods, EA preconditioning is cheap, safe, easily-applied, and more likely to be accepted by patients and has strong clinical applicability. 29 Electroacupuncture Preconditioning for Stroke Patients 597 Yes Yes No and NSE Serum level of S100beta Serum level Serum cardiac troponin I Yes No mA, 2/15 Hz 30 min .8Ð1.9 mA, 5/30 Hz 30 min Acupuncture points Fengchi Lieque Neiguan Yunmen Postoperative condition 60 patients (EA 30; Con 30) Acupuncture points The inotrope score Clinical trials on electroacupuncture preconditioning Craniocerebral tumor resection 32 patients (EA 16; Con 16) replacement) (2010) (valve Yang RC Fengfu 0 Table Table 29.1 Author (year) Disease/surgery Lu (2010) Design RC Treatment Outcome measures 1Ð4 Effectiveness Side effects 598 Q. Wang and L. Xiong

Further studies must be focused on (1) the duration and ef fi cacy of EA preconditioning in inducing ischemic tolerance; (2) the infl uence of gender, age, and pathological status such as diabetes on the EA preconditioning-mediated neuroprotection; and (3) translation from bench to bedside. Thus, much work remains. However, if we successfully fi nd satisfying answers to the above three key problems, we may turn the wishes of patients suffering from stroke to live a better life into a reality through EA preconditioning.

References

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Michael Katsnelson and Sebastian Koch

30.1 Introduction

Stroke is one of the leading causes of mortality and the leading cause of disability in the United States and is a prevalent and devastating pathological entity worldwide. Alongside primary prevention, acute pharmacological or mechanical intercessions, maximization of collateral blood ﬂ ow, and neuroprotection, the innovative idea of inducing an arti ﬁ cial stress to help the body weather the ongoing or impending pathophysiological one (ischemic conditioning) has been discovered and extensively studied in the laboratory in many species and target organs (Gulker et al. 1977; Murry et al. 1986 ; Reimer et al. 1986 ; Cochrane et al. 1999 ; Kume et al. 1996 ; Lee et al. 2008; Iliodromitis et al. 2007 ; Yin et al. 2009 ; Dave et al. 2001, 2005 ; Giovanardi et al. 2009 ; Chen et al. 2008 ; Hausenloy 2009 ; Mio 2010 ; Zhang et al. 2011 ; Fisher et al. 1994 ) . The translation from the lab bench to the bedside has been a rather slow and tenuous process. In this chapter, we will discuss the proof of concept and clinical promise of ischemic conditioning, highlight the challenges of translating this exciting concept to clinical practice, and review the latest clinical efforts of pre- and postconditioning the heart (often, current cardiac trials tend to outline future cerebral experimental directions) and the brain.

M. Katsnelson (*) Department of Neurology , University of Miami , 1150 NW 14th Street, PAC, Suite#609 , Miami , FL 33136 , USA e-mail: [email protected] S. Koch Associate Professor of Clinical Neurology , University of Miami Miller School of Medicine , 1120 NW 14th Street, Room 1365 , Miami , FL 33136 , USA e-mail: [email protected]

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 601 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4_30, © Springer Science+Business Media New York 2013 602 M. Katsnelson and S. Koch

30.2 Clinical Rationale

One of the most established tenets of modern science is the theory of evolution and its central premise of natural selection and the survival of the fi ttest. These postulates rest on the fact that since the beginning of life on this planet, all living organisms have been subjected to different levels and degrees of environmental change. These have ranged from minuscule alterations in the intracellular environments to apocalyptic cosmic events. The outcome, nevertheless, usually remained the same. Those life-forms that could endure, adapt fast enough, and even thrive in spite of and often due to the new environmental stress would survive; other organisms, that could not, would perish. All living things today, by defi nition, are the survivors – they have the molecular tools to weather a range of environments and changing conditions. This basic adaptive strategy allows most organisms which are subjected to a mild form of environmental stress to be more tolerant of a more lethal subsequent insult. This can be seen with different levels of stress in many different species. The delib- erate induction of such tolerance is known as preconditioning as the inducing stimulus precedes the noxious or lethal insult and was fi rst discovered a quarter century ago (Gulker et al. 1977 ; Murry et al. 1986) . Further studies have revealed protective effects when the conditioning is initiated during (perconditioning) (Schmidt et al. 2007 ) or even after (postconditioning) (Na et al. 1996 ; Andreka et al. 2007 ) clinical injury. Many different stresses can lead to such protection. These include mild forms of ischemia (ischemic conditioning), hypoxia/hyperoxia, drugs (pharmaceutical conditioning), and hypo- or hyperthermia (Raval et al. 2008 ; Dirnagl et al. 2009 ) . Ischemic conditioning has emerged as one of the most powerful protective techniques discovered so far and has made the furthest forays into clinical practice – thus, it will be the focus of our discussion. Ischemic preconditioning was fi rst demonstrated in the myocardium, where transient occlusions of a coronary artery in the canine model followed by a permanent coronary occlusion resulted in a decrease in the size of the myocardial infarct size (Murry et al. 1986) . In the myocardium, ischemic preconditioning can achieve a remarkable degree of protection, with reductions in infarct size of 50–70% (Downey and Cohen 2009 ) . Protecting another important organ, the brain, has also been achieved in preclinical models. Moreover, it was discovered that cross- tolerance can be established between different organs. Short sublethal ischemia and reperfusion in one organ can induce ischemic tolerance at different locations of the body: some in the adjoining tissue and some that are distinct and distant to the sight of conditioning. It was fi rst described in cardiac muscle in 1993 where four 5-min cycles of occlusion and restoration of fl ow of a circumfl ex coronary artery reduced the subsequent size of MI in a different coronary territory (Przyklenk et al. 1993 ) . This phenomenon is known as remote ischemic preconditioning and provides an easily accessible and noninvasive way of inducing mild ischemia (Hausenloy and Yellon 2008 ) . 30 Clinical Trials of Ischemic Conditioning 603

30.2.1 Human Conditioning: Proof of Concept

Several small clinical observational trials suggest that a preconditioning response can be elicited from the human brain. In 12 patients with aneurysmal subarachnoid hemorrhage, transient 2-min cerebral artery occlusions intraoperatively, prior to vessel occlusion for aneurysm clipping, appeared to induce metabolic evidence of ischemic tolerance (Chan et al. 2005 ; Chen et al. 2005) . After the artery occlusion, the preconditioned tissue showed a slower decline in tissue pH and oxygenation, leading the authors to speculate that a relatively simple intervention may offer protection during complex neurovascular surgery. Similarly, 10 patients undergoing carotid stenting who developed neurological symptoms after initial balloon occlusion of the carotid artery were able to tolerate subsequent carotid occlusions without recurrence of symptoms (Faries et al. 2008 ) . The authors attributed this to a preconditioning response. Very similar observations have also been noted during coronary angioplasty and stenting where repetitive coronary artery balloon occlusion resulted in progressively diminishing anginal pain (Inoue et al. 1996 ) . Ten years ago, a study of human volunteers showed that remote noninvasive ischemic preconditioning (four 5-min inﬂ ation of a blood pressure cuff to 200 mmHg with 5-min cycles of reperfusion in between) demonstrated a safe and practical induction of preconditioning in humans in vivo (Kharbanda et al. 2001, 2002 ) . Postconditioning with noninvasive remote conditioning in humans has likewise been shown (Loukogeorgakis et al. 2007 ) . Recently, almost a dozen clinical studies utilizing this method to protect both the heart and the brain have been published with many more clinical trials on the way (Table 30.1 ). With such a strong and promising number of basic science and animal studies as well as proof of concept studies in humans, why has the widespread clinical application of conditioning been so slow to materialize?

30.3 Challenges of Translational Research

Although the preclinical experimental models of preconditioning provide a convincing body of evidence, there remain a signi fi cant number of obstacles to translating this successfully to the standard of care at the bedside with many logistical hurdles yet to overcome and questions to answer. Most of the preclinical data was conducted on animals that are relatively young and disease-free, whereas most of the clinical uses would apply to an older population of humans walking around with many poorly controlled medical conditions. With little to no prior pathology, an “exogenous” preconditioning treatment in the healthy nonhuman subjects may produce impressive and easily produced results. Would the degree of protection or even the existence of a response at all be different if the proverbial laboratory rat was a diabetic hypertensive who was a couch potato with a lifelong addiction to cigar smoking and Krispy Kremes? This question is important: the presence of disease may in itself have an unexpected bene fi t of serving 604 M. Katsnelson and S. Koch 22% in myocardial ↓ 23% renal impairment ↓ = 0.11) postoperative inotropic support postoperative p ↓ group infarction, acute coronary syndromes signi fi cantly reduced vs. 53% changes, or renal lung protection 43% troponin at 72 h troponins Perioperative 35% troponin at 48 h 27% troponin at 8 h 27% in troponin, and Reduced troponin, reduction in stroke cant) troponin at 72 h fi 26% (not signi CK-MB and troponin at 6, 24, 48 h Troponin, No adverse events noted in the treatment events No adverse ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ were NSE 24, 72, and 110 h after surgery ischemia occlusion ycles of 10-min iliac artery

subarachnoid hemorrhage subarachnoid hemorrhage www.controlled-trials.com www.controlled-trials.com SAH , ) ) Coronary bypass of 5-min arm ischemia 3 cycles ) ) CABG surgery of 5-min arm ischemia 3 cycles ) Coronary bypass fl urane Sevo ) ) Coronary bypass of 5-min arm ischemia 3 cycles in troponin release, EKG No difference ) ) Pediatric cardiac surgery ischemia of 5-min leg 2 cycles ) ) +/− valvoplasty CABG surgery of 5-min arm ischemia 3 cycles ) ) Carotid endarterectomy ischemia of 10-min leg 2 cycles deteriorations, (32% saccadic latency Fewer ) ) Coronary angioplasty of 5-min arm ischemia 3 cycles 2007 ) ) pump CABG surgery Off of 5-min arm ischemia 4 cycles ) ) after SAH Vasospasm of 5-, 7.5-, or 10-min leg 3 cycles 2010 2009 2010 ) Abdominal aneurysm repair 2 ) c CABG surgery of 5-min arm ischemia 3 cycles ) ) Cervical decompression surgery of 5-min arm ischemia 3 cycles S-100B at 6 h and 1 day; 2006 2010 2010 2009 2010 2011 Completed and ongoing clinical trials with remote ischemic limb preconditioning Completed and ongoing clinical trials with remote ischemic limb preconditioning 2007 2010 2010 www.clinialtrials.gov www.clinialtrials.gov : neuron-speci fi c enolase, Source Hausenloy et al. ( ( et al. Hausenloy ( Ali et al. ( Hoole et al. ( et al. Wagner ( Hong et al. NSE Frassdorf et al. ( ( Frassdorf et al. ( Rahman et al. ( Ali et al. Cerebral ( et al. Walsh ( et al. Koch Trial Trial Cardiac ( Cheung et al. Clinical setting Intervention Outcome Table Table 30.1 Hu et al. ( ( Hu et al. Thielman et al. ( ( Thielman et al. 30 Clinical Trials of Ischemic Conditioning 605 as a physiological stressor and potentially eliciting a preconditioning response. In other words, a typical “vasculopath,” an individual with vessels diseased throughout his body (brain, carotid heart, peripheral vessels), may have already maximized his benefi ts from ischemic preconditioning naturally – further stress may produce negligible or no results. Studies have provided some interesting data for this hypothesis. A chronic occlusion of both carotid arteries in rats led to subsequent smaller strokes than in controls who did not undergo such treatment (Kim et al. 2008 ) . A long-term adaptive response may have been induced in the myocardial muscle of swine who underwent chronic coronary artery occlusion (McFalls et al. 2006 ) . The mechanism may involve induced chronic mitochondrial resistance to ischemia, with results present even after 10 weeks. A human experiment found subjects exposed to 4 weeks of living at a high altitude (atmospheric hypoxia) had a sustained amount of increased antioxidant enzyme activity compared to the amounts measured in those living at sea level (Sinha et al. 2009 ) . It appears that preconditioning responses already occur in vivo due to a variety of stressors and may have non-transient effects. If a chronic state of “endogenous” preconditioning in many patients is already in place, will extra efforts by clinicians lead to further (or any) bene fi ts? In animals, the data is confl icting: in one study of rats with prior exposure to hypoxia, adding another preconditioning stimulus (an acute occlusion of a coronary artery) did not lead to any additional protection in reducing a subsequent myocardial infarction size compared to controls with just the chronic exposure and without the acute stimulus (Neckar et al. 2002 ) . Similar results were achieved in another study where several months of coronary artery stenosis mitigated further effects of preconditioning (Watanabe et al. 2006 ) . More promising results were obtained in the subarachnoid hemorrhage rat model (Ostrowski et al. 2005 ) . Hyperbaric oxygen, which was administered after a subarachnoid hemorrhage (an “endogenous” preconditioning event), still resulted in an additional induction of protection with improved neurological function and neuronal histology, a reduction in oxidative stress, and an improved cerebral blood fl ow (Ostrowski et al. 2006 ) . It may be that many protective cascades exist, and by stressing the organism in a different way, we recruit a distinct (but complimentary) adaptive system for the added benefi t. It is also possible that by stressing the organism during or after a signifi cant clinical event (perconditioning and postconditioning, respectively), a whole different set of protective networks (the details of which have been poorly understood to date) may be activated. These possibilities clearly require further study. In humans, the answer appears more promising. Clinical data on this topic may suggest that even in the diseased state, some degree of preconditioning is possible. One study showed that subjects who became symptomatic during balloon occlusions of the carotid artery during carotid stenting, were able to tolerate subsequent occlusions without the development of symptoms (Faries et al. 2008 ) . A critical reader of such a fi nding may wonder if this proves further preconditioning or simply represents a recruitment of cerebral collaterals, another important but distinct compensatory mechanism. Aortic cross clamping and subsequent muscular injury, in another study, proved less deleterious in patients with a history of atherosclerotic iliofemoral stenosis than in patients without it (Badhwar et al. 2004 ) . These fi ndings suggest that preconditioning in states of chronic ischemia may be possible. 606 M. Katsnelson and S. Koch

Another logistical concern is that in the lab animal, stroke is induced at a speci fi c time and in a controlled environment – preconditioning at a particular point before- hand is a matter of routine. In clinical practice, quite the opposite holds true. A vast majority of strokes in humans happen without warning, thus making the use of preconditioning as a viable preventative intervention logistically dif fi cult, at least prior to most cases of stroke or MI. Some clinical settings are known for a higher risk of a cardiac or cerebral ischemia, making them a natural setting for clinical trials (Fisher et al. 1994; Dirnagl et al. 2009) . Ideally, the risk of stroke has to be relatively high and predictable so that preconditioning can be initiated prior to the possibility of such an event. Examples include procedures such as cardiac bypass surgery, carotid stenting, or endarterectomy, which all have a well-documented elevated risk for these events. Another window of opportunity exists before the vasospasm (which can lead to delayed cerebral ischemia and brain injury) that occurs in a signi fi cant number of aneurysmal subarachnoid hemorrhages. The proper setting for a planned clinical trial of preconditioning is important. Carotid artery stenting, for example, carries a small but defi nitive risk of stroke during and after the procedure – 3% in asymptomatic and 6% in symptomatic disease ( 1991 ; 1995 ) . With such a low incidence of the condition of interest, a properly powered trial would require a large number of patients and span over many years. Coronary artery bypass grafting, another setting of a possible large-size clinical trial for preconditioning, also has several potential limitations (Djaiani 2006 ) . It has been associated with both stroke (about 2%) and cognitive decline (15–36% at 6 weeks) (Newman et al. 2001 ; Djaiani et al. 2007 ) . However, detecting, diagnosing, and properly documenting cognitive changes require rigorous time-consuming and expensive neuropsychological testing – a real obstacle to demonstrating even preliminary results. Cardiac surgery may not be the sole cause of neurocognitive injury, and progression of the widespread vascular disease as the culprit of cognitive may play a bigger role than previously thought (Selnes et al. 2005 ; Potter et al. 2004 ) . “Native” preconditioning from existing vasculocclusive disease, again, may be a confounding variable. Another one of the potential hurdles for preconditioning are the medications that are used to combat the disease. Most patients are taking numerous pharmacologically active substances, both by prescriptions from their physicians and in the form of “natural” vitamins and supplements. The role of these substances in the body’s preconditioning response is largely unknown – they may already accomplish a lion’s share of the work or conversely counteract any protective effect – and needs to be better studied and understood. For example, in the past 15 years, statins have become the mainstay of both acute and chronic therapy for different manifestations of atherosclerotic disease. In one study, the effects of pre- and postischemic conditioning on the infarct size were reduced in rats that were taking the lovastatin chronically or acutely (Kocsis et al. 2008 ) . Chronic pretreatment with the drug decreased the postischemic conditioning response but showed little effect on preconditioning. Conversely, acute treatment reduced the effectiveness of pre-ischemic conditioning, but had less consequence on post-conditioning. Given the expanding ranks and sophistication of our medical arsenal, interactions between medications and ischemic conditioning will need to be taken into account. 30 Clinical Trials of Ischemic Conditioning 607

Secondary cascades of injury after an ischemic event are still not completely known and may vary from organ to organ: the brain is likely much less forgiving than the heart or skeletal muscle, and once these processes are under way, pre- and postconditioning may do little to ameliorate or reverse ongoing damage and disruption. Another important consideration is that not all forms of preconditioning will be available to be easily and commonly translated to the clinical setting and the amount, timing, and degree of optimal ischemic conditioning are yet to be determined. In the laboratory, multiple methods of preconditioning confer a protective effect. These include direct ischemic preconditioning through transient arterial occlusions of cerebral vessels; occluding blood ﬂ ow to different tissues, that is, an arm or leg in remote limb preconditioning; inducing hypoxia or hyperoxia with the use of hyperbaric chamber; or pharmacological preconditioning. Direct conditioning of ischemic organs through vascular occlusion or clamping is usually impractical and not safe in a medically critical or unstable patient. Hyperbaric chambers are also not readily available or always logistically viable. Remote preconditioning through the induction of limb ischemia and pharmacological preconditioning are the most practical considerations for clinical use. Side effects such as local tissue damage or distal venous occlusion in remote ischemic preconditioning are distinct possibilities, and their frequency needs to be monitored and controlled. A pharmaceutical agent with proven preconditioning properties would simplify the method of delivery and make it possible to precondition patients in any setting and for prolonged periods of time, if need be. Per- and postconditioning in cerebral ischemia has less animal data and is not well studied in human subjects. The amount of bene ﬁ t from preconditioning will likely vary based on individuals’ genetic variability, the level of their response to preconditioning, the severity of pathological stress and cellular damage that was incurred, and the timing and type of preconditioning. More clinical data in humans is needed before clinical trials can be relied on to give accurate and reproducible results. Industry-sponsored trials may be a challenge since no blockbuster drug is readily produced from a tourniquet or a blood pressure cuff (Faries et al. 2008 ; Inoue et al. 1996 ) .

30.4 Current Clinical Trials

Given the potential limitations outlined above, however, most ischemic conditioning studies completed to date are on the heart (Table 30.1 ). As with most other novel pharmacological and interventional revascularization therapies of the past 30 years, the testing of the myocardium precedes that of the cerebrum. The cardiac studies provide important directions and outline potential pitfalls, although the similarities between an essential human muscle and the most complex known biological structure are dwarfed by the differences in their unique cellular environments and biochemical networks. In this section, we will examine several landmark ischemic conditioning trials of both organs. 608 M. Katsnelson and S. Koch

A postconditioning trial was conducted in 30 patients with STEMI undergoing percutaneous coronary interventions (PCI) in 2005 (Staat et al. 2005 ) . Half of the participants were postconditioned with six 30-s cycles of in fl ating the angioplasty balloon after the stenting of an affected coronary vessel. Creatine kinase was reduced by 36% in the next 72 h. Postconditioning has been shown to also have longer term protective effects, with higher left ventricular ejection fraction and reduction in MI size at 1 year and 6 months, respectively (Thibault et al. 2008 ) . Preconditioning trials of a similar population of patients likewise showed promising results (Rentoukas et al. 2010 ; Botker et al. 2010 ) . In one trial, 251 patients with STEMI and primary PCI were randomized to a treatment (four 5-min arm cuff infl ations and defl ations applied even before the patient arrived to the ER) and a control group (no such treatment) with the treatment group showing more cardiac tissue saved by SPECT scan (Botker et al. 2010 ) . In 2007, a fi rst successful remote ischemic preconditioning trial in adults undergoing planned CABG surgery was published (Hausenloy et al. 2007 ) . Fifty-seven patients were randomized to either receive three 5-min in fl ations of a blood pressure cuff to 200 mmHg, effectively cutting off circulation or having sham in fl ation with no pressure administered before the start of surgery. Reduction in the serum levels of troponin T of 43% was achieved in the 72-h area under the curve. Half a dozen studies since then have mostly shown similar encouraging results, and more comprehensive trials are currently under way. In a relatively large study of 242 subjects, limb preconditioning prior to elective coronary angioplasty and stenting reduced periprocedural troponin release as well as clinical cardiac and cerebral ischemic events at 6 months (Hoole et al. 2009 ) . During this study, however, no clinical cerebral events were reported showing again the relatively low event rate following this type of intervention. A recent prospective study of 162 subjects undergoing ischemic preconditioning before on-pump coronary surgery has been completed (Rahman et al. 2010 ) . Three cycles of 5-min ischemia and reperfusion of the arm failed to produce statistically signifi cant results in troponin release, hemodynamic, renal, or lung protection between the treatment and control group after surgery. This study underscores the non-heterogeneity of study protocols; patient selection and modest understanding of neuroprotective properties of other medical interventions still pose signi fi cant challenges that need to be overcome. Pharmacological preconditioning in the heart has shown variable results, with cyclosporine showing the greatest promise (Mewton et al. 2010 ; Piot et al. 2008 ) . Hyperbaric preconditioning prior to cardiac surgery has shown potential effect in small preliminary studies (Jeysen et al. 2011 ) . Resveratrol may present the next worthy target to explore (Della-Morte et al. 2009 ; Saleh et al. 2010 ) . To date, only a few human clinical trials of ischemic conditioning in the nervous system have been completed. A randomized clinical trial of patients undergoing carotid endarterectomy has been undertaken involving 70 subjects (Walsh et al. 2010 ) . Patients were preconditioned using two 10-min cycles of the lower extremities after the induction of anesthesia but before surgery. Fewer saccadic latency deteriorations, serving as a surrogate marker of ischemia, were recorded in the treatment group, but the results were not statistically signi fi cant (32% vs. 53% p = 0.11). 30 Clinical Trials of Ischemic Conditioning 609

Another randomized controlled trial assessed remote ischemic preconditioning as a protective mechanism before spinal cord surgery (Hu et al. 2010 ) . Forty adult cervical myelopathy patients were randomized to three 5-min cycles of upper limb ischemia or a control group. The rates of serum S-100B measured at 6 h and 1 day after surgery and neuron-specifi c enolase 24, 72, and 110 h after surgery were signifi cantly reduced in the treatment arm. A statistically signifi cant improvement in recovery at 7 days and 1 and 3 months after the procedure was also noted. Patients with subarachnoid hemorrhage are at high risk for secondary ischemic injury and stroke in the two or so weeks following the initial symptom onset, largely through cerebral vasospasm. The risk is well defi ned and occurs over a relatively short period and with a high enough incidence to make it a viable outcome to study. Our group has conducted a phase IB study of 33 patients after an SAH who underwent remote ischemic limb preconditioning (Koch et al. 2011 ) . A gradated protocol of 5-, 7.5-, and 10-min occlusions of the upper or lower extremity in three cycles with reperfusion in between was undertaken. No clinical side effects were noted, and these procedures were well tolerated in this critically ill group. A phase II trial is currently under way.

30.5 Conclusion

Ischemic conditioning remains an enticing therapeutic target in protection from ischemia. A robust body of preclinical data and a compelling study of the potential underlying mechanisms argue for further clinical trials. Such trials are already under way in cardiac medicine and are beginning to be undertaken for the protection of nervous tissue ischemia. Additional laboratory investigation of the impact of native disease, concomitant medication use, and the effect of the aging brain on the preconditioning response may be desirable and of signifi cant value in further defi ning potential limitations. If, as it now appears, remote limb preconditioning continues to evolve as the most likely candidate for clinically de fi nitive trials of preconditioning in cardiac disease, this method should deserve equal consideration for clinical trials of cerebral protection.

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mechanism: ﬁ rst demonstration of remote ischemic perconditioning. Am J Physiol Heart Circ Physiol 292:H1883–H1890 Selnes OA, Grega MA, Borowicz LM Jr, Barry S, Zeger S, Baumgartner WA, McKhann GM (2005) Cognitive outcomes three years after coronary artery bypass surgery: a comparison of on-pump coronary artery bypass graft surgery and nonsurgical controls. Ann Thorac Surg 79:1201–1209 Sinha S, Singh SN, Saha M, Kain TC, Tyagi AK, Ray US (2009) Antioxidant and oxidative stress responses of sojourners at high altitude in different climatic temperatures. Int J Biometeorol 54:85–92 Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L’Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre- Fouet X, Ovize M (2005) Postconditioning the human heart. Circulation 112:2143–2148 Thibault H, Piot C, Staat P, Bontemps L, Sportouch C, Rioufol G, Cung TT, Bonnefoy E, Angoulvant D, Aupetit JF, Finet G, Andre-Fouet X, Macia JC, Raczka F, Rossi R, Itti R, Kirkorian G, Derumeaux G, Ovize M (2008) Long-term beneﬁ t of postconditioning. Circulation 117:1037–1044 Thielmann M, Kottenberg E, Boengler K, Raffelsieper C, Neuhaeuser M, Peters J, Jakob H, Heusch G (2010) Remote ischemic preconditioning reduces myocardial injury after coronary artery bypass surgery with crystalloid cardioplegic arrest. Basic Res Cardiol 105:657–664 Wagner R, Piler P, Bedanova H, Adamek P, Grodecka L, Freiberger T (2010) Myocardial injury is decreased by late remote ischaemic preconditioning and aggravated by tramadol in patients undergoing cardiac surgery: a randomised controlled trial. Interact Cardiovasc Thorac Surg 11:758–762 Walsh SR, Nouraei SA, Tang TY, Sadat U, Carpenter RH, Gaunt ME (2010) Remote ischemic preconditioning for cerebral and cardiac protection during carotid endarterectomy: results from a pilot randomized clinical trial. Vasc Endovascular Surg 44:434–439 Watanabe K, Yaoita H, Ogawa K, Oikawa M, Maehara K, Maruyama Y (2006) Attenuated cardioprotection by ischemic preconditioning in coronary stenosed heart and its restoration by carve- dilol. Cardiovasc Res 71:537–547 Yin Z, Gao H, Wang H, Li L, Di C, Luan R, Tao L (2009) Ischaemic post-conditioning protects both adult and aged Sprague–Dawley rat heart from ischaemia-reperfusion injury through the phosphatidylinositol 3-kinase-akt and glycogen synthase kinase-3beta pathways. Clin Exp Pharmacol Physiol 36:756–763 Zhang W, Miao Y, Zhou S, Jiang J, Luo Q, Qiu Y (2011) Neuroprotective effects of ischemic postconditioning on global brain ischemia in rats through upregulation of hippocampal glutamine synthetase. J Clin Neurosci 18:685–689 Epilogue

This brings to a close our collection of perspectives on innate tolerance in the CNS. We solicited these chapters from their respective authors in mid-to-late 2011; thus, by the time the reader holds this book in their hands, the fi eld of pre- and postconditioning in the CNS will surely have advanced in distinct and signifi cant ways that are not represented in this compendium. Nevertheless, we cannot help but feel a certain satisfaction that many of the main leaders in the fi eld Ð the best minds in the business, so to speak Ð agreed to provide detailed summaries of the many different facets of this jewel of a concept. And that, as a result, this book provides a uniquely comprehensive resource for those interested in the study and application of endogenous protection, both newcomers and experienced alike. Evident from the many fi ndings reported herein, our knowledge about innate cytoprotection in the CNS has grown rapidly since this fi eld emerged onto our collective conscience in the 1990s. Capturing the current status of the fi eld after two decades of investigation was the underlying purpose of this book. In brief, in addition to what we continue to learn from anoxia-tolerant animals, species that hibernate, and studies of endogenous protection in heart and other tissues (Part I), we have developed a number of reliable animal and culture models to study this phenomenon (Part III), and our mechanistic understanding about the inducers, the signaling pathways, the genomics, and the effectors of the ischemia-tolerant phenotype (Part IV) Ð and the many stimuli that induce such a phenotype (Part II) Ð is expanding rapidly. Some scientists are now assessing the ability of triggering innate tolerance to protect against epilepsy, trauma, subarachnoid and intracerebral hemorrhage, spinal cord injury, and retinal disease (Part V). Not surprisingly, excitement about translating what we have learned at the bench to the clinic is running high, with patient subgroups and treatment protocols de fi ned (Part VI) and having just returned from our second biennial “Translational Preconditioning Workshop” meeting. And yet, paradoxically, one might also say that, even at the preclinical level, we are just scratching the surface of this fi eld and that we have so much more to learn in order to maximize the potential bene fi ts of innate tolerance for combating human disease. Perhaps these ongoing advancements will be represented in a second edition of this book…

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 615 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4, © Springer Science+Business Media New York 2013 616 Epilogue

In closing, as editors, we want to express our sincere gratitude to all of the chapter authors for their wonderful summaries. Without their scholarship and their willingness to take with utmost seriousness our requests of each of them for the highest quality of contribution, the value of this book would surely be diminished. So, many thanks to them, and thanks as well to all of our readers for your interest in this stimulating topic.

Ð Jeffrey M. Gidday, Miguel A. Perez-Pinzon, and John H. Zhang. Author Index

A Addink, A.D.F. , 29 Aarts, M. , 343, 344, 354 Adeleine, P. , 582 Aasum, E. , 438 Adelson, P.D. , 532 Abas, F. , 322 Aderem, A. , 353 Abcouwer, S.F. , 541 Adjodah, D. , 545 Abdel-Karim, I. , 585 Adler, D. , 585 Abdullah, M.S. , 581 Adler, M.W. , 504 Abe, H. , 8, 235 Adolph, E.F. , 5 Abe, J. , 56, 57 Adornetto, A. , 436, 437 Abe, K. , 8, 10, 544 Agarwal, N. , 186, 191, 200, 545 Abe, M. , 319 Agnello, D. , 219 Abe, O. , 222, 275, 459–461, 467–470 Agrapart, M. , 192 Abe, T. , 462, 467–471 Aguilar-Bryan, L. , 62 Abel, E.D. , 439 Aguirre-Chen, C. , 297 Abete, P. , 106, 558–561 Ahlemeyer, B. , 370, 409, 431, 436 Abo-Ramadan, U. , 278 Ahlstrom, H. , 373 Ábrahám, H. , 272 Ahmad, F. , 439 Abramovitch, R. , 491 Ahmed, S.H. , 4, 221 Abu, A.M. , 293 Ahn, J.Y. , 294 Aburatani, H. , 366, 368–370, 372, 373 Ahn, M.Y. , 440 Acikgoz, O. , 219 Ahn, Y.H. , 438 Acker, H. , 440 Ahn, Y.I. , 443 Acker, T. , 192 Ahroon, W.A. , 5 Ackery, A. , 301 Ahuja, T. , 342, 347, 351 Acquaviva, A.M. , 441 Ai, J. , 301 Adair Kirk, T.L. , 467 Aibiki, M. , 118, 119, 121, 123 Adamczyk, P. , 375 Aiello, L.P. , 370 Adamczyk, S. , 171, 322, 331, 391 Ainslie, P.N. , 112 Adamek, P. , 78, 604 Aiyagari, V. , 310 Adamis, A.P. , 542 Aizenman, E. , 5, 368 Adamo, D.S. , 113 Akagawa, K. , 431, 435 Adamopoulos, S. , 83 Akao, M. , 60, 433 Adams, J. , 353 Akawi, O. , 441 Adams, M.R. , 25 Akbar, M.T. , 367, 531 Adams, R.E. , 310 Akca, O. , 170, 171, 492, 493 Adams, R.J. , 135, 457 Akimov, S. , 172 Adamson, C. , 501, 502 Akita, J. , 542

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 617 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4, © Springer Science+Business Media New York 2013 618 Author Index

Akiyama, T.E. , 406 An, S.S. , 184, 201 Akthar, A.M. , 604 Ander, B.P. , 375 Alam, J. , 443 Andersen, N.H. , 11, 53, 82, 608 Albani, D. , 231, 234, 465, 468, 472, 473 Anderson, B.J. , 113 Albert, M.R. , 3 Anderson, D.C. , 582, 587 Albrecht, M.A. , 434 Anderson, D.L. , 492 Albrecht, R.F. , 493 Anderson, L.L. , 5 Albright, C.D. , 23 Anderson, M.F. , 189, 197 Aldur, M. , 300 Anderson, R.E. , 143 Alestalo, K. , 11 Anderson, T.R. , 42, 63 Alex, J. , 585 Andersson, L.C. , 188, 198, 201 Alexander, M.S. , 441, 542, 546 Andjelkovic, A.V. , 313, 458–461 Alexandrov, A.V. , 56, 57, 319, 323 Andoh, T. , 436, 437 Alexandrovich, A.G. , 340, 344, 370, 502–504, Andrade, A.L. , 350 507–512 Andre, V. , 523–525, 527–529 Al ﬁ eri, A. , 299 Andre-Fouet, X. , 52, 53, 63, 79, 80, 318, 608 Ali, A.A. , 604 Andreka, G. , 53, 602 Ali, K.M. , 299 Andreka, P. , 53, 602 Ali, N. , 78, 604 Andreotti, F. , 559, 561 Ali, Z.A. , 573, 604 Andres, A.M. , 396 Aliprandi, M. , 530 Andrew, R.D. , 42 Alkan, T. , 184, 185, 194, 264, 322 Andrews, M.T. , 37 Alkayed, N.J. , 231, 235, 275, 411, 412, 459, Andrews, S.B. , 434 461, 466, 470, 473, 556 Andrukhiv, A. , 62 Alkhulai ﬁ , A.M. , 6, 77, 243, 569 Andrukhiv, K. , 64, 74 Allain, R. , 175 Aneesh, B. , 583 Allard, M. , 487, 489 Ang, E.T. , 106, 108 Allen, B.S. , 71 Angora, H. , 214 Allen, C.L. , 139 Angoulvant, D. , 63, 79, 80, 608 Allen, M.W. , 581 Anju, B. , 184, 186, 462, 467, 468 Almog, S. , 500, 501 Annella, T. , 437 Al-Mubarak, B. , 443 Annunziato, L. , 186, 191, 192, 231, 235, 331, Alonso, O.F. , 10, 235, 272, 312, 323, 505 436, 437 Alonso, R.J. , 582–584 Anrather, J. , 462, 467–471 Alsbo, C.W. , 346 Anthony, D.C. , 11 Alshaher, M. , 561 Antos, C.L. , 64 Alt, F.W. , 441 Anttila, V. , 11 Altay, T. , 5, 44, 184–186, 194, 196, 198, 202, Anvari, M. , 299 203, 293, 459, 463, 467, 468 Anzivino, M. , 10 Althaus, J. , 265 Ao, Z. , 544 Altieri, M. , 6, 531 Aoki, M. , 399 Alvanou, A. , 184–186, 194 Aoyama, K. , 112 Al-Waili, N.S. , 581 Appelboom, G. , 167 Amamoto, T. , 144 Applebaum, A. , 57 Amberger, N. , 6 Aquilano, K. , 441 Ambrosino, G. , 501 Ara, J. , 184, 185 Ambrosio, G. , 61 Arab, S. , 14, 68, 70 Amerson, B.S. , 72 Aragno, M. , 406 Ames, A. III. , 279 Aragones, J. , 190, 194 Aminova, L.R. , 190, 192, 220, 297 Arai, K. , 110, 474 Ammar, A.D. , 175 Araki, T. , 7, 8, 352, 465, 472–474, 531, 532 Ammini, C.V. , 370 Arany, Z. , 439 An, G. , 8 Arboix, A. , 559, 560 An, P. , 458–461, 468 Ardell, J.L. , 55, 56 Author Index 619

Ardizzone, T.D. , 310 Avkiran, M. , 75 Argaud, L. , 64, 75 Avramovich, A. , 235, 500 Argent, C.C. , 405 Avvedimento, E.V. , 437 Arheden, H. , 81 Awan, S. , 172 Arimura, A. , 272 Ayer, R.E. , 294, 487, 489 Ariyurek, Y. , 382 Ayigari, V. , 137 Armendariz-Borunda, J. , 441 Ayoub, I. , 190, 297 Armin, S.S. , 297 Ayub, A. , 63 Armstrong, S.C. , 57 Azad, N. , 438 Aronowski, J. , 279, 439 Azad, T.D. , 188, 201, 292, 293, 297, 300, Arora, S. , 232, 236, 353 463, 468, 469, 471, 572 Arras, M. , 73 Aztiria, E. , 299 Arrowsmith, J.E. , 175 Azuma, Y. , 45 Arslan, E. , 561 Azzam, N. , 9, 504 Arthur, P.G. , 184, 194, 195, 245 Arundine, M. , 354 Arvin, B. , 9 B Asaga, T. , 184, 185, 188, 191, 197 Baba, H. , 195, 512 Asahi, M. , 110 Baba, M. , 352 Asai, A. , 437 Babb, T. , 524, 525, 527, 529 Asakai, R. , 64 Babcock, D.J. , 44 Asanuma, H. , 60, 75 Babsky, A.M. , 60 Asavaritkrai, P. , 188, 190 Babu, G.G. , 82 Asayama, J. , 58 Babusikova, E. , 184, 185, 200 Ashford, C.A. , 23 Bach, S. , 369 Ashley, E. , 52, 77, 78, 604, 608 Bachmann, S. , 192 Ashley, M.D. , 231, 235, 295, 319, 322, 325–327, Bachoo, R. , 439 330, 342, 348–350, 352–354 Badaut, J. , 245, 465, 472 Ashraf, M. , 63 Bader, M. , 72 Ashra ﬁ an, H. , 53, 602 Badhwar, A. , 605 Ashwood, T. , 591 Badin, R.A. , 367 Ashworth, A. , 57 Bading, H. , 342, 343 Asimakis, G.K. , 59 Badr, A.E. , 583 Assaraf, M.I. , 465, 473 Bae, E.K. , 380 Assayag, M. , 510 Bae, H.S. , 594 Astrup, J. , 23, 24, 280 Bae, M.H. , 440 Ates, E. , 245 Bae, M.K. , 440 Atkinson, D.E. , 20 Baek, S.H. , 415, 416 Atochin, D.N. , 231, 235, 461, 468, 469 Baes, M. , 190, 194 Atsina, K. , 442, 443 Baethmann, A. , 279 Attanasio, G. , 5 Bahjat, F.R. , 274 Attigah, N. , 273 Bahk, J.H. , 78, 604 Attwell, D. , 344, 453 Bahr, B.A. , 501, 502 Au, M. , 441 Bai, X.G. , 596 Auchampach, J.A. , 60, 62 Bai, Y. , 504 Auer, R.N. , 273, 279 Baier, H. , 25 Auh, S. , 45 Bailey, D.M. , 187, 202 Aupetit, J.F. , 52, 53, 79, 80, 318, 608 Baines, C.P. , 56, 57, 60, 61, 63, 75, 390, Auroy, Y. , 166 393, 394 Auvergne, R. , 527, 529, 530 Bajgar, R. , 391 Auwerx, J. , 443 Bakajsova, D. , 42 Auyeung, W.W. , 443 Baker, A.J. , 301 Avci, E. , 296 Baker, C.J. , 271, 273 Avezaat, C.J. , 294 Baker, J.E. , 250 620 Author Index

Baker, T.J. , 9 Barros, R.C. , 46 Bakondi, B. , 11 Barry, S. , 606 Balafonova, Z. , 63 Bar-Tana, J. , 443 Balasingam, V. , 9 Bartel, D.P. , 380 Balazs, R. , 21 Bartels, M. , 10, 466, 474 Baldelli, S. , 441 Bartkowski, H. , 270 Balducci, C. , 530 Barton, M.E. , 530 Balduini, W. , 401 Bartrons, R. , 443 Balentine, J.D. , 270, 319 Bas, O. , 156, 157 Balfour, W.M. , 5 Basaga, H. , 511 Balshaw, D. , 83 Basarsky, T.A. , 344 Banasik, A. , 6 Bass, R. , 511 Banci, L. , 436 Bassel-Duby, R. , 439 Banerjee, A. , 57, 601 Bastide, M. , 4, 9, 463, 467–471, 474 Banerjee, S. , 63 Bateman, M.C. , 352 Banke, N.H. , 439 Bates, T. , 529 Banning, A.P. , 82 Batteur, S. , 10 Bantel, C. , 171, 172, 493 Baudry, M. , 523–525, 527, 529 Bao, J. , 185, 201 Bauer, B. , 11, 52, 65, 602 Bao, W.M. , 459, 464, 468 Bauer, C. , 185, 191, 201 Bao, Y.C. , 441, 608 Baughman,V.L. , 493 Bao, Z.Q. , 56 Baumgartner, H.K. , 430 Baohan, A. , 544 Baumgartner, W.A. , 606 Bar, P.R. , 368, 501 Baur, J.A. , 441 Baram, T.Z. , 522 Baxter, G.F. , 52, 54, 57, 63, 64, 68–75, 83, 563 Barancik, M. , 57, 438 Baxter, P.S. , 354, 443 Barancik, T. , 56 Baydoun, A.R. , 63 Baranova, K. , 184, 185, 187, 192, 193 Bayley, M. , 506 Baranova, O. , 184, 191, 192, 411, 465, 473 Bayraktutan, U. , 139 Barba, I. , 75 Baysal, K. , 373 Barbe, M.F. , 4 Bazzoni, G. , 231, 234, 465, 468, 472, 473 Barber, A.J. , 541 Beal, M.F. , 247 Barber, P.A. , 235, 250, 273, 275, 340, 462, 470 Beale, J. , 581 Barber, R.D. , 192 Beard, G.L. , 192 Barbera, M.J. , 439 Beart, P.M. , 184, 186, 199, 201, 411, 412, 459, Barbour, B. , 453 465, 473 Bard, L. , 340 Beas-Zarate, C. , 441 Barde, Y.A. , 512 Beattie, W. , 561 Bardutzky, J. , 10 Beauchamp, N.J. , 310 Bare, O. , 441 Beauchamp, P. , 468, 470 Bari, F. , 195, 294, 459, 462, 467–469 Bechmann, I. , 192, 412 Bari, M. , 504 Becker, A. , 431, 436 Barker, P.B. , 310 Becker, D.P. , 494 Barkmeier, A.J. , 504 Becker, K.J. , 9, 293, 363, 407, 469, 553, 561, Barlow, C. , 429 567, 602, 606 Barnard, M.R. , 52, 77, 78, 313, 604, 608 Becker, L.B. , 61, 83, 390 Barnes, B.M. , 37, 38, 44, 45 Becker, R.C. , 68 Barnes, R.W. , 175 Bedanova, H. , 78, 604 Baron, J.C. , 581 Bederson, J.B. , 270 Barone, F.C., 4, 5, 8, 9, 118, 151, 152, 165, 231, Beeson PB , 3 234, 270, 271, 274, 278, 349, 368, 544 Begley, R. , 436 Barr, H.W. , 175 Behl, N. , 353 Barreto-Chang, O.L. , 319 Behrends, M. , 56, 57 Barrientos, A. , 436 Beis, I. , 57 Author Index 621

Beit-Yannai, E. , 507, 509, 510 Bernard, S.A. , 120 Belayev, L. , 235, 272, 273 Bernardi, G. , 226, 227 Belda-Iniesta, C. , 328 Bernášková, K. , 274 Beley, A. , 185, 187, 194, 200, 463, 468 Bernassola, F. , 437 Belforte, N. , 542 Bernaudin, M. , 8, 113, 184, 185, 189–192, Belhomme, D. , 175 197–199, 202, 203, 250, 260, 264, Belkin, D.A. , 19, 25 265, 297, 323, 366, 369–374, 407, Bell, K.F. , 443 411, 412, 510, 542, 546 Bell, R.M. , 52, 56, 83 Berne, R.M. , 8 Bellahcene, M. , 436 Bero, C. , 219, 220 Bellail, A. , 184, 185, 191, 197–199, 202, 510 Berrendero, F. , 501 Bellomo, R. , 120 Berridge, M.J. , 434 Bellver-Estelles, C. , 523–525, 527–529, Berrocal, M. , 405 531–534 Berthezene, Y. , 438 Belmaker, R.H. , 142 Berti, R. , 353 Belosjorow, S. , 56, 57 Bertoni-Freddari, C. , 395 Belousova, O.B. , 296 Bertrand, N. , 185, 187, 194, 200, 463, 468 Beltowski, J. , 174 Beschorner, R. , 545 Belyaev, N.D. , 534 Besshoh, S. , 354 Bembry, J. , 504 Bestmann, L. , 73, 175 Benarroch E , 4 Betz, A.L. , 9, 109, 214, 218, 280, 310 Bendek, G. , 229 Beuf, O. , 438 Beneveniste, H. , 494 Bevilacqua, L. , 441 Beni, S.M. , 502, 503 Bezstarosti, K. , 605 Benito, C. , 504 Bhardwaj, A. , 296 Bennett, C.N. , 143 Bhargava, P., 505 Bennett, I.L., Jr. , 3 Bhasin, R.R. , 310 Bennett, M.V. , 346, 433 Bhaskar, P. , 443 Bennett, S. , 585 Bhat, R.V. , 328 Benoit, J.N. , 62 Bhateja, P. , 232, 236 Benolken, R.M. , 143 Bhatia, R. , 280 Ben-Shabat, S. , 500, 501, 503 Bhatt, A.J. , 184, 185, 187, 197, 199, 265 Benveniste, H. , 431 Bhopale, V.M. , 299 Bequet, F. , 110 Bian, J.H. , 435 Berchner-Pfannschmidt, U. , 440 Bian, J.S. , 175 Berchtold, N.C. , 106 Biasi, C. , 29 Berdeaux, A. , 73 Bibel, M. , 512 Berenshtein, E. , 61 Bickler, P.E. , 27, 41, 42, 44, 45, 167, 169, Berg, G. , 75 184, 187–189, 193, 194, 202, Berg, M. , 340 245, 248, 401 Berg, R.M.G. , 187, 202 Biedermann, A.J. , 42 Berg-Dixon, S. , 441 Biegon, A. , 340, 344 Berge, R.K. , 438 Bienengraeber, M. , 61 Bergen, A.A.B. , 541, 543 Bienias, J.L. , 141, 144 Berger, J.P. , 406 Biermann, J. , 175, 542 Bergeron, M. , 8, 44, 113, 184, 186, 191, 194, Biernaskie, J. , 352 260, 264, 265, 297, 411, 441 Bigdeli, M.R. , 298, 409, 410, 457, 461, 467, 468 Bergk, A. , 6, 138, 184, 559, 560 Bigini, P. , 219 Bergman, T.A. , 583 Bignami, A. , 23 Bergmann, A.G. , 234 Billiar, T.R. , 303 Berk, B.C. , 56, 57 Binder, D.K. , 512 Berkson, H. , 25 Bindokas, V. , 542–544 Berman, A.J. , 542 Birbaumer, N. , 457 Berman, N.E. , 443 Birch, D.G. , 140 622 Author Index

Birch, E.E. , 140 Bonanni, L. , 433 Birnbaum, M.J. , 442 Bond, A. , 7, 345, 351 Birnbaum, Y. , 245 Bonhoeffer, T. , 353 Bishop, S.P. , 584 Bonilla, L.G. , 435 Bishop, T. , 190, 194 Bonnefoy, E. , 52, 53, 63, 79, 80, 318, 608 Bisogno, T. , 500–502 Bonnekoh, P. , 270 Biswal, S. , 443 Bonner, C. , 523–525, 527–529, 532–534 Bittigau, P. , 501, 502 Bonner, H.P. , 531 Bjerve, K.S. , 144 Bonner, T.I. , 500 Black, K.L. , 151 Bono, F. , 491 Blackburn, M.R. , 46 Bonser, R.S. , 78, 604, 608 Blackstone, E. , 46 Bontemps, L. , 79, 80, 608 Blair, N.S. , 63, 75 Boocock, D.J. , 251 Blair, S.N. , 105 Bootman, M.D. , 434 Blake, J. , 37 Bopassa, J.C. , 75 Blanchard, K.L. , 441 Bopp, M. , 185 Blanck, T.J. , 166 Bordelon, Y.M. , 138 Blanco, C. , 463, 471 Bordet, R. , 4, 9, 235, 322, 331, 406, 463, Blanco, M. , 500 467–471, 474 Bland, R.J. , 530, 544 Bordone, M.P. , 542, 546 Blazquez, C. , 443 Borecky, J. , 435 Blesch, A. , 512 Borger, M.A. , 606 Blevins, T.L. , 369 Borges, K. , 524, 525, 527, 530–532 Block, F. , 524, 527, 529 Born, J.G. , 348 Blomeyer, C. , 438 Boronow, J. , 142 Blondeau, N. , 10, 134, 137, 138, 141, 143, Borowicz, L,M., Jr. , 606 147–152, 154, 155, 157, 158, 245, Borowski, A. , 604 348, 409, 410, 525, 529–531, 546 Borsotto, M. , 546 Bloom, F.E. , 270 Bortolotto, Z. , 524, 527, 529 Bluhmki, E. , 136 Boscarino, C. , 14, 68 Blumenthal, J.A. , 606 Bosch-Marce, M. , 441 Bocharova, L.S. , 352 Boscia, F. , 186, 191, 192, 331 Bochner, B.S. , 10 Bossy-Wetzel, E. , 544 Boeck, C.R. , 494, 523, 524, 527 Botker, H.E. , 11, 52, 53, 68, 82, 83, 608 Boengler, K. , 73, 74, 78, 394, 604 Bottcher, M. , 11, 53, 82, 608 Boer, J.M. , 382 Bottger, C. , 503 Boerema, A.S. , 38 Böttiger, B.W. , 279 Boet, R. , 293, 574, 575, 603 Bottini, A.G. , 582, 587 Bo ﬁ lis, E. , 57 Bouchama, A. , 124 Bogdahn, U. , 469 Bouckova, R. , 9 Bognolo, G. , 571 Boudina, S. , 58, 439 Bognolo, J. , 78 Bouhidel, O. , 73 Bogousslavsky, J. , 6, 531, 559, 560 Bouillaud, F. , 439 Bogoyevitch, M.A. , 57 Boule, J.L. , 367, 375, 467 Bohm, F. , 81 Boulouard, M. , 184, 199, 203, 323 Bohr, V.A. , 441 Bouma, H.R. , 38 Boily, G. , 441 Bourbonnais, D. , 583 Boison, D. , 11, 348, 529 Boutelle, M.G. , 280 Boker, D.K. , 301 Bouvier, F. , 81 Bolanos, J.P. , 437 Bouwman, R.A. , 390 Bolay, H. , 272, 273 Boveris, A. , 435 Boling, W. , 10 Bowen, K.K. , 185, 189, 231, 234, 299, Bolli, R. , 6, 56, 57, 63, 83, 436, 506 364–366, 368, 370, 371, 373, Bolte, C.S. , 65 414, 458, 460, 461, 467 Author Index 623

Boy, S. , 469 Bronner, M. , 443 Boyd, J. , 352 Brookes, P.S. , 437–439, 441 Boyle, D.W. , 190, 195 Brooks, N. , 373 Boyle, J.R. , 604 Brott, T.G. , 313 Boyle, N. , 4 Browder, I.W. , 367, 458, 459, 462, 467, 468 Boysen, G. , 272 Brown, E. , 4 Bracey, M.H. , 502 Brown, J.H. , 57 Bradley, J. , 542 Brown, Q.B. , 502 Brahmachari, H.D. , 5 Brown, T.M. , 135, 457 Brambrink, A.M. , 213, 220, 251 Brownstein, M.J. , 500 Bramham, C.R. , 108 Bruce, A.J. , 10, 523 Bramlett, H.M. , 245, 499 Bruce, I.C. , 322, 325, 464, 470 Brana, C. , 527, 529, 530 Brück, W. , 218, 273 Branco, L.G. , 46 Brucklacher, R.M. , 265 Brand, M.D. , 439 Bruckner, B.A. , 370 Brandt, U. , 62 Bruder, N.J. , 170, 171, 485 Branston, M. , 24 Bruer, U. , 184, 245 Branston, N. , 24 Bruggen, N. , 459 Brantley, M.A., Jr. , 185, 186, 191, 194, 200, Bruhl, M.L. , 56 542, 543, 545 Bruhn, T. , 340 Brantner, C.A. , 434 Brum, P.C. , 437 Brar, B.K. , 57 Brunder, D.G. , 280 Bratton, A. , 544 Brunecker, P. , 6 Braun, C. , 457 Brunet, J.F. , 465, 472 Braun, J. , 273 Brunette, E. , 342, 347, 351 Bravo, T.P. , 487–489 Brunskill, E.W. , 63, 75 Brealey, D. , 296 Brusselmans, K. , 491 Brecht, S. , 525, 530 Bryan, J. , 62 Bredt, D.S. , 437 Bryan, M. , 529 Breese, N.M. , 504 Bryan, R.N. , 431 Breier, G. , 192 Bryant, A.J. , 171 Brener, S.J. , 76 Brzozowski, T. , 245 Brennan, T.A. , 485 Bu, X. , 184–188, 193, 544 Brenner, M. , 9, 10 Buchan, A.M. , 235, 251, 270, 273, 275, 282, Brethorst, S. , 344 340, 349, 462, 470 Breuer, A. , 500, 501, 503 Buchan, K.G. , 76 Brew, K. , 110 Buchholz, J.N. , 41, 44 Brew, S. , 296 Buchthal, B. , 343 Bridges, R.J. , 24 Buck, L.T. , 27, 28, 194, 195, 245, 371 Brierley, J.B. , 149, 229, 230, 273 Buckberg, G.D. , 71 Brilstra, E.H. , 295 Buckley, N.J. , 534 Brind, J. , 142 Buechert, A. , 73 Brines, M. , 218, 219, 250, 510 Buerk, D.G. , 299 Brinkhous, A.D. , 279, 493 Buettner, G.R. , 56, 60 Brint, S. , 270–272 Bugyi, H. , 71 Brisman, M.H. , 271, 273 Bulckaen, H. , 235 Brismar, H. , 184 Bullard, R.W. , 40 Brison, R.J. , 297 Bult Ito, A. , 341 Briz, V. , 595 Bumanglag, A.V. , 528 Brizendine, E.J. , 582–584 Bunk, E.C. , 353 Broderick, J.P. , 313, 375 Bunn, H.F. , 440, 441 Brombacher, F. , 273 Buonocore, G. , 401 Bromberg, Z. , 506, 509 Burchill, S.A. , 544 Brom ﬁ eld, E. , 148 Burda, J. , 245, 323, 326, 327, 435, 563 624 Author Index

Burda, R. , 323, 326, 435 Camins, A. , 441 Burge, C.B. , 412 Campbell, B. , 541 Burgess, J.R. , 148 Campbell, C.A. , 278 Burgin, W.S. , 319, 323 Campbell, M. , 369 Burguete, M.C. , 370 Campbell, S.J. , 11 Burke, R.E. , 544 Campbell, W.B. , 25 Burke, T.J. , 601 Campochiaro, P.A. , 441 Burley, D.S. , 73, 74 Campos, K.M. , 171 Burne, M.J. , 443 Canas, P.T. , 170 Burr, G.O. , 144 Cannon, J.R. , 215 Burton, J.R. , 74 Cansev, M. , 141 Burwell, L.S. , 437, 438 Cantley, L.C. , 544 Bushnell, C. , 375 Canto, C. , 443 Busija, D.W. , 195, 371, 391, 433, 459, 462, Cantrell, A.R. , 42 467–469 Canuelo, A. , 190 Busto, R. , 38, 124, 235, 272, 273, 280, 297, Canzoniero, L.M. , 44, 436, 437 323, 347, 375, 429, 601 Cao, C.M. , 435 Butler, D. , 501, 502 Cao, C.X. , 111, 367 Butler, G.J. , 581 Cao, G. , 184, 187, 196, 216, 299, 367 Butler, K.L. , 73 Cao, J. , 328 Butter ﬁ eld, D.A. , 443 Cao, L. , 166, 168, 169, 172 Buttino, N. , 4 Cao, X. , 57, 436 Buyse, J. , 190, 194 Cao, Z. , 391 Byers, D.M. , 353 Cappello, S. , 74, 76 Byrav, D.S. , 3 Carafoli, E. , 22 Byus, C.V. , 467 Cardenas, A. , 234 Cardenas, S. , 190 Cardoso, S. , 195 C Cardwell, E.M. , 56 Cabeza, N. , 559, 560 Carey, H.V. , 37, 41 Cabral, G.A. , 504 Carhuapoma, J.R. , 310 Cacciatore, F. , 561 Carloni, S. , 401 Caffrey, J.L. , 72, 73 Carlson, S. , 227, 271 Cagnotto, A. , 219 Carlson, Z.A. , 47 Cahill, J. , 291, 299 Carmeliet, P. , 184, 185, 190, 194, 197, 199, Cai, X. , 443 201, 491 Cai, Z. , 436, 441, 510 Carmona, J.J. , 441 Caidahl, K. , 81 Carnethon, M. , 457 Cairns, C.B. , 601 Caroli, A. , 492 Cairns, R.A. , 441 Carpenter, K.L. , 511 Cakici, I. , 78 Carpenter, M.K. , 10 Calabrese, V. , 443 Carpenter, R.H. , 604, 608 Calabresi, P. , 226, 227 Carr, C.S. , 561 Calabro, A. , 319 Carr, D. , 473 Calderone, A. , 346, 356 Carraway, M.S. , 185, 193, 195, 395, 413, 439 Cale, A.R. , 585, 586, 608 Carreira, R.S. , 396 Calisaneller, T. , 299 Carrey, Z. , 581 Callaghan, C.J. , 573, 604 Carrick, E. , 353 Calvani, M. , 443 Carrier, E.J. , 504 Calvert, J.W. , 46, 299, 300, 438, 491 Carroll, J. , 606 Calzada, N. , 80 Carroll, R. , 443 Camacho, S.A. , 62 Carson, M.J. , 467 Camara, A.K. , 60, 61 Carswell, H.V. , 273 Cameron, J.A. , 9, 353, 368, 403, 531 Carvalho, C. , 195 Author Index 625

Casabona, G. , 330 Chang, H.-M. , 185, 201 Casado, E. , 328 Chang, K.-C. , 184, 185, 193, 194 Casagrande, S. , 510 Chang, L.K. , 531 Cassano, S. , 437 Chang, W. , 373 Cassard-Doulcier, A.M. , 439 Chang, Y.C. , 232, 237, 412, 413, 458, 462, 472 Cassulini, R.R. , 303 Chang, Y.S. , 411 Castellani, R.J. , 38, 40 Channon, K. , 82 Castillo, J. , 234, 500 Chao, M.L. , 38 Castillo, M.R. , 341 Chao, P.L. , 190, 201 Castillo, P.E. , 346 Chaparro Huerta, V. , 441 Castren, E. , 247, 530 Chappell, J.E. , 143 Cattano, D. , 172, 493, 557 Chatterjee, P.K. , 3 Catterall, W.A. , 42 Chaudhry, K. , 106, 110, 112, 118 Cavallaro, G. , 436 Chaurasia, O.P. , 443 Cave, A.C. , 60 Chavarria, I. , 504 Cavone, L. , 232, 235 Chavez, I.J. , 81, 318 Cebeira, M. , 501 Chavez, J.C. , 137, 184, 190–192, 265, Cebrian, M.E. , 390 465, 473 Cejas, P. , 328 Chavko, M. , 327 Cen, Y. , 443 Che, W. , 56, 57 Centeno, J.M. , 437 Che, X.M. , 370 Centonze, D. , 226, 227, 504 Chen, B. , 214, 222, 443 Centrella, M. , 373 Chen, B.S. , 340 Cepek, L. , 219, 510 Chen, B.Y. , 594 Ceranowicz, P. , 313 Chen, C. , 173 Cernak, I. , 512 Chen, C.-F. , 184, 185, 193, 194, 201 Cerra, C.E. , 583 Chen, C.H. , 299, 436 Cerski, C.T. , 601 Chen, C.J. , 441, 594 Cevik, C. , 78 Chen, C.S. , 541, 542 Cha, J.Y. , 438 Chen, C.Y. , 603 Chader, G.J. , 545 Chen, D. , 9, 368, 531 Chaer, R.A. , 603, 605, 607 Chen, G. , 299, 326 Chakravarthula, P. , 377 Chen, H.M. , 4, 124, 272, 319, 322, 332, 395, Chalabreysse, L. , 64 584, 601 Chalk, J.B. , 278 Chen, J.J. , 4, 6, 8, 114, 117, 120, 122, 123, Chan, A. , 184, 194, 195 184, 187, 196, 216, 231, 235, 248, Chan, B.P.L. , 368 275, 277, 344, 367, 399, 433, 436, Chan, C.Y. , 26, 27 439, 460, 464–466, 468, 473, 474, Chan, D.C. , 430 503, 531, 545, 567 Chan, M.T. , 293, 574, 575, 603 Chen, K. , 522 Chan, P.H. , 8, 184, 194, 195, 280, 297, 327, Chen, L.J. , 392, 399, 436 328, 435, 458, 466, 473, 474, 509 Chen, M. , 342, 343, 413, 435, 532, 585, 591, Chance, B. , 435 594, 596 Chance, G.W. , 143 Chen, M.F. , 584 Chandel, N.S. , 430, 433, 443 Chen, M.L. , 81 Chandler, K.E. , 534 Chen, P.J. , 584 Chandra, M. , 561 Chen, P.S. , 412, 413 Chandra, S. , 278 Chen, P.X. , 436 Chandran, S. , 443 Chen, Q. , 60, 342, 343 Chang, A.Y. , 185, 193, 201 Chen, R-L. , 78, 79, 282, 441 Chang, B.S. , 521 Chen, S. , 167, 168, 172–174, 353, 392, 403, Chang, C.Z. , 173, 293, 298, 299, 487, 491–493, 584–586, 592, 593, 595, 491, 495 596 Chang, G.W. , 440 Chen, S.D. , 6 626 Author Index

Chen, S.T. , 270, 319 Chock, P.B. , 437 Chen, S.Y. , 594 Chodosh, L.A. , 192 Chen, W. , 56, 61, 173, 174, 294, 299, 390 Choe, W. , 443 Chen, W.-H. , 184, 185, 194 Choi, A.M. , 443 Chen, X. , 293, 318, 323–325, 435 Choi, D.W. , 8, 24, 44, 271, 342, 347, Chen, X.C. , 459, 464, 468 349–351, 494 Chen, X.J. , 342, 343 Choi, H. , 584 Chen, Y. , 42, 353, 504, 510, 595 Choi, I. , 45 Chen, Y.C. , 439 Choi, J.H. , 120 Chen, Y.E. , 438, 439 Choi, J.S. , 247, 248 Chen, Y.-S. , 185, 191, 200, 544 Choi, K.Y. , 438 Chen, Y.T. , 439 Chokshi, S.K. , 221, 277 Chen, Y.Y. , 458 Chong, K.Y. , 389 Chen, Z. , 184, 185, 197, 199, 461, 467, Chopp, M. , 4, 10, 121, 123, 124, 152, 272, 470, 601 277, 459, 512 Chen, Z.F. , 10 Chopra, K. , 70 Chen, Z.-Y. , 188, 190 Choquet, D. , 340 Chenevert, T.L. , 310 Chou, P. , 592 Cheney, J.A. , 392 Choudhuri, R. , 443 Cheng, D. , 319, 327 Chow, C. , 442 Cheng, H.C. , 120, 544 Chow, M.M. , 299 Cheng, J. , 168, 176, 557, 558 Chowdhry, S. , 443 Cheng, O. , 173, 247 Christian, S.L. , 40–42, 44, 45, 341 Cheng, Q. , 214, 222 Christiansen, E.H. , 11, 53, 82, 608 Cherednichenko, G. , 62 Christiansen, K. , 144 Cherepanov, V. , 70 Christie, L.A. , 106 Chesselet, M-F. , 230, 273 Christopher, Q. , 184, 186, 191, 198 Cheung, M.M.H. , 14, 53, 68, 70, 77, 78, 262, Christopher, T.A. , 57 571, 602, 604 Christou, I. , 323 Cheung, W.M. , 439 Chu, C.J. , 107, 113 Chevion, M. , 61 Chu, H. , 592 Chi, J.W. , 185, 193, 201 Chu, K.A. , 185, 191, 380, 401, 414, 465, Chi, O.Z. , 118, 169, 221, 277, 459, 461, 474, 594 469, 470 Chu, M. , 171, 460, 464, 466, 468, 474 Chi, S. , 437 Chu, P.W.Y. , 184, 186, 199, 201, 411, 412, Chi, X. , 391 459, 465, 473 Chianelli, M.S. , 542, 546 Chu, X. , 5, 189, 364, 366, 370–372, 531, 532 Chiari, P. , 64 Chua, R. , 436 Chiariello, L. , 5 Chuang, D.M. , 494, 523 Chiariello, M. , 61 Chuang, K.H. , 594 Chiarugi, A. , 232, 235 Chujo, K. , 184, 185, 188, 191, 197 Chien, C.T. , 441 Chun, Y.S. , 584 Chien, K.R. , 57 Chun, Z.X. , 303 Chigasaki, H. , 584 Churchill, E.N. , 437 Chileuitt, L. , 279 Churilova, A.V. , 184, 185, 187, 193, 199, 412 Chin, S.L. , 439, 594 Cidlowski, J.A. , 247 Chinn, H.I. , 5 Ciechanover, A. , 402 Chiu, C.C. , 142 Ciesielski, T.M. , 367, 375, 467 Chiu, J.H. , 166, 173, 295, 491, 584, 593, 594 Cieszkowski, J. , 313 Chiu, T.-H. , 184, 185, 193, 194, 201 Cimarosti, H. , 264 Chiueh, C.C. , 436, 437 Cingolani, H.E. , 561 Cho, J.H. , 294 Cinti, S. , 439 Cho, S. , 367, 437 Ciriolo, M.R. , 441 Cho, T.H. , 438 Ciullo, I. , 437 Author Index 627

Cizkova, D. , 435 Collins, T.J. , 434 Ckakraverty, D. , 524, 525, 527, 529 Collip, J.B. , 4 Clagett, G.P. , 175 Collis, M.G. , 27 Clandinin, M.T. , 143 Collman, R.G. , 458 Clark, A.W. , 110 Colohan, A. , 487–489 Clark, J.B. , 231, 235, 437, 461, 468, 469 Colohan, A.R. , 293, 295–297, 299, 487–489, Clark, J.C. , 458–461, 467, 468, 471, 472 491, 605 Clark, J.E. , 436 Colombrita, C. , 443 Clark, J.F. , 310 Colon, G.P. , 214, 218, 310 Clark, R.K. , 9 Comas, T. , 342, 348 Clark, R.S. , 510, 532 Compton, D.R. , 500, 501 Clarke, D.D. , 20 Conder, M.L. , 433 Clarke, K. , 438 Cong-Yina, D. , 174, 274 Clarke, M. , 294 Connell, B.J. , 399, 400, 608 Clarke, S. , 63, 443 Conner, J.M. , 512 Clarke, S.C. , 82, 604, 608 Connolly, E.S. , 273 Clarke, S.J. , 61, 393 Conroy, R.M. , 528 Clayton, C.E. , 348, 349, 353, 531 Constantinides, C. , 280 Clemens, J.A. , 436 Constantino, L.C. , 494 Clement, H. , 303 Contestabile, A. , 47 Clement, J.P. , 62 Conti, V.R. , 59 Clemmensen, P. , 80, 318 Cook, D.A. , 301 Clergue, F. , 166 Cook, H.W. , 353 Clerk, A. , 57 Coomber, B. , 502 Clevenger, R. , 438 Cooper, A.L. , 157 Clifford, S.C. , 440 Cooper, D.J. , 499 Cliffton, K. , 25 Cooper, D.Y. , 580 Clifton, G.L. , 42 Cooper, N.R. , 113 Cloft, H.J. , 302 Cooreman, M. , 47 Coburn, M. , 47 Copin, J.C. , 458 Cochrane, J. , 601 Corasaniti, M.T. , 437 Cockman, M.E. , 440 Corbett, D. , 118, 124, 244, 352 Codaccioni, J.L. , 170, 171 Corcoran, M.E. , 524, 525, 530 Cognet, L. , 340 Cordell, W.H. , 582–584 Cognolato, D. , 319 Cordis, G.A. , 56, 58, 61 Cohen, H.R. , 194 Corleone, D. , 491 Cohen, J. , 221 Cornog, J.L. , 23 Cohen, M.V. , 55–57, 60, 62, 63, 72–76, 326, Corradetti, R. , 117 436, 437, 602 Correia, S.C. , 195 Cohen, O. , 506 Correll, M.P. , 328 Cohen, R.E. , 402 Correnti, E.E. , 144 Cohen-Cory, S. , 108 Corvera, J.S. , 53, 71, 75, 79, 317, 326 Cohen-Gadol, A.A. , 488 Cosar, M. , 156, 157 Cohen-Yeshurun, A. , 504 Coserea, I. , 273, 343 Colbourne, F. , 273 Costa, A.D. , 62, 64, 74, 436, 437 Cole, D.J. , 279, 280 Costa, C.L. , 64, 74 Cole, T.J. , 603 Costa, T. , 494 Colegrove, S.L. , 434 Costi, A. , 63 Coleman, T. , 250 Cotman, C.W. , 24, 106 Coles, J.G. , 14, 68, 70 Cottin, Y. , 52, 53, 80, 318, 608 Collard, J.G. , 215 Cottrell, JE. , 170, 171 Colledge, M. , 353 Coughlin, S.R. , 214, 215 Collen, D. , 491 Coulborne, F. , 118 Collino, M. , 406 Coulet, F. , 192 628 Author Index

Coulson, J.M. , 534 D Coulthard, L.R. , 544 da Dalt, L. , 297 Couplan, E. , 439 da Silva, R. , 175 Cousins, S.L. , 340 Dabringhaus, A. , 469 Couture-Lepetit, E. , 75 Dacey, L.J. , 175 Coven, D.L. , 442 Dach, N. , 166, 173, 295, 491 Cowen, M. , 585, 586, 608 Dagani, F. , 430 Cox, P.N. , 77, 78, 604 Dahl, M.B. , 29 Coy, P.E. , 443 Dahl, N.A. , 5 Coyle, P. , 270 Dahlgren, N. , 229 Craigon, M.H. , 192 Dahmani, S. , 170 Crane, J. , 467 Dai, H.B. , 184, 185, 197, 199, 461, 467, 470 Cravatt, B.F. , 502 Dai, S. , 457 Crawford, D.C. , 344, 350 Dai, W. , 68–70 Crawford, S. , 441 Dai, Z.G. , 170 Crea, F. , 5 D’Alecy, L.G. , 38 Crestanello, J.A. , 60, 61 Dale-Nagle, E.A. , 185, 201 Crichiutti, G. , 297 Dalkara, T. , 234, 247, 272, 273 Critz, S.D. , 57, 60, 62, 74, 436, 437 Dallegri, F. , 458 Croal, B.L. , 76 Dallner, G. , 141, 143, 144 Croisille, P. , 63, 608 D’Alonzo, A.J. , 6, 81, 433 Crompton, M. , 63 Damiano, S. , 436, 437 Crook, J.E. , 557, 558, 561 D’Amico, M. , 69 Crooks, P. , 244 Dana, A. , 54 Crosson, C.E. , 186, 191, 200, 544, 545 Danbolt, N.C. , 168 Crow, R.J. , 279 Dancause, N. , 457 Crowder, C.M. , 5, 189, 196, 197, 201 D’Andrea, D. , 61 Crowell, R.M. , 274 Dang, C.V. , 441 Crozatier, B. , 436 Danial, N.N. , 63 Crozier, R.A. , 353 Danielisová, V. , 323, 326, 435, 563 Csermely, P. , 505 D’Annunzio, V. , 75 Csiszar, A. , 47 Danton, G.H. , 458 Csont, T. , 606 Danysz, W. , 24, 351 Cuda, G. , 437 Darbenzio, R.B. , 6, 81, 433 Cuello, F. , 75 Darian-Smith, C. , 352 Cui, G.Y. , 173, 297 Darling, C.E. , 65 Cui, H.F. , 595 Darrow, R.M. , 545 Cui, J.H. , 248, 367, 586 Das, A. , 72 Cui, L. , 72–75, 80, 185, 191, 196, 198, Das, B.K. , 3 326, 443 Das, D.K. , 6, 56–58, 61 Cui, Y.J. , 172, 173, 436 Das, K. , 530 Cui, Z. , 439 Das, M. , 61, 63, 443 Cukerman, E. , 14, 68, 70 Dasgupta, N. , 5, 196, 197, 201 Culmsee, C. , 370 Dash, P.K. , 299 Cummins, R. , 227, 271 Dashkin, O. , 42, 436 Cung, T.T. , 52, 53, 63, 79, 80, 318, 608 Dashnyam, S. , 436 Cuomo, O. , 331 Dausmann, K.H. , 37 Currie, R.W. , 4, 8, 64, 165, 231, 234, 274, 278, Daut, J. , 62 349, 399 Davalos, A. , 234, 591 Curry, A. , 106, 110 Dave, J.R. , 353 Cuthbertson, B.H. , 76 Dave, K.R. , 27, 42, 244–246, 248, 249, 251, Cutillas, B. , 443 347, 349, 375, 391, 392, 433, 436, Cuzzocrea, S. , 406 441, 494, 601, 602, 608 Cybulska, R. , 524, 527–529 Davenpeck, K.L. , 10 Author Index 629

Davicioni, E. , 442 Deguchi, K. , 10 David, G. , 40 Dehdashti, A.R. , 294 Davidsen, L. , 23 Dehn, J. , 41 Davidson, B.L. , 9 Dehpour, A.R. , 68, 69, 71 Davidson, B.R. , 293 Deike-Glindemann, J. , 47 Davidson, S.M. , 52, 75, 83, 393, 442, 443 del Prete, A. , 186, 191, 192 Davies, B. , 221 Del Pulgar, T.P. , 503 Davies, K. , 459 del Zoppo, G.J. , 9, 109, 151, 247, 457, 459, 460 Davila-Roman, V.G. , 175 Delacey, M. , 57 Davis, A.E. , 11 Delaney, K. , 352 Davis, A.M. , 72, 74 Deleeuw, G. , 429 Davis, D.P. , 368 Deli, M.A. , 231, 234, 465, 468, 472, 473 Davis, D.S. , 467 Della-Morte, D. , 251, 347, 436, 441, 558–561, Davis, R.L. , 270 602, 608 Davis, S.M. , 59, 591 Dell’aversano, C. , 436, 437 Davis, W. , 106, 109, 110 DeLorenzo, R.J. , 522 Dawn, B. , 57, 63 Delplanque, B. , 157, 158 Dawood, F. , 14, 68 Delucinge Vivier, C. , 343 Dawson, D.A. , 3, 4, 9, 10, 261, 275, 459, Delyani, J.A. , 60 461, 470 DeMarco, M.P. , 171 Dawson, T.M. , 8, 9, 219, 250, 437, 543, 545 Demarest, K. , 502 Dawson, V.L. , 8, 9, 437, 543, 545 Dembinski, A. , 313 Day, A.L. , 272 Dembinski, M. , 313 de Araujo Herculano, B. , 527 Dembowski, C. , 219, 510 de Beaumont, L. , 583 Demchenko, I.T. , 185, 193, 195, 231, 235, de Belleroche, J.S. , 367 439, 461, 468, 469 de Blier, I.G. , 175 Demchuk, A.M. , 319, 323 de Bock, K. , 190, 194 Demirkan, F. , 561 de Cabo, R. , 47, 441 Demougeot, C. , 185, 187, 194, 200 de Castro, J. , 328 Dempsey, R.J. , 231, 234, 370 de Freitas, G.R. , 6, 531, 559, 560 den Dunnen, J.T. , 382 de Hert, S.G. , 175 Dendorfer, A. , 70, 71 de Menezes, R.X. , 382 Deneve, J. , 73 de Petrocellis, L. , 500 Deng, H.W. , 68–70 de Simone, C. , 61 Deng, J. , 171, 190, 195 de Simone, G. , 457 Deng, W. , 459, 474, 475 de Simoni, M.G. , 231, 234, 465, 468, 472, 473 Deng, X. , 174 de Tribolet, N. , 294 Deng, Z. , 438 de Vera, A. , 532 Denko, N.C. , 441 de Vreede-Swagemakers, J.J. , 250 Densem, C.G. , 82, 604, 608 de Vries, H.E. , 247, 368 Deplanque, D. , 4, 9, 235, 463, 467–469 de Zavalia, N. , 542 Dereski, M.O. , 4 Deal, D.D. , 171 Deriu, G.P. , 319 Dean ﬁ eld, J.E. , 11, 603 Derksen, J.W. , 368 Deaton, D. , 6 Derlon, J.M. , 581 Debruyne, D.N. , 150, 152, 154 Deroose, C. , 190, 194 Deckwerth, T.L. , 47 DeRose, G. , 605 DeCoster, M.A. , 187, 201 DeRubertis, B. , 574, 603, 605, 607 Deem, S. , 296 Derumeaux, G. , 63, 79, 80, 608 Defazio, R.A. , 42, 246, 347, 433, 436, 441, Derwall, M. , 47 602, 608 Deshpande, J.K. , 431 De ﬁ llo, A. , 492 DesRoches, C.M. , 485 Deftereos, S. , 53, 82, 608 Desvergne, B. , 438 DeGirolami, U. , 274 Devane, W.A. , 500, 501 630 Author Index

Devaskar, S.U. , 370 Ding, S.-J. , 173, 186, 198, 411, 412 DeVries, A.C. , 229 Ding, Y. , 106–110, 113, 120, 458–461, 467, Dewerchin, M. , 190, 194, 491 468, 471, 472 Dhakshinamoorthy, S. , 443 Ding, Y.F. , 69 Dhananjay, S. , 184, 186, 462, 467, 468 Ding, Y.H. , 106, 109–111, 458–461, 467, 468, Dharap, A. , 380, 414, 438 471, 472 Dhodda, V.K. , 185, 189, 364–366, 368, 370, Ding, Z. , 461, 467 371, 373 Ding Y-C , 120 Di, C. , 601 Dingledine, R. , 524, 525, 527, 530–532 Di, F. , 487, 490 Dinh, Y.R. , 299 Di Filippo, C. , 69 Diolez, P. , 439 Di Lisa, F. , 52, 72, 563 Dioum, E.M. , 441 Di Marzo, V. , 500, 502 Dippel, D.W. , 294 Di Napoli, M. , 353 Diringer, M.N. , 310 Di Paola, R. , 406 Dirkx, R. , 190, 194 Di Raimondo, D. , 222 Dirnagl, U. , 5, 6, 134, 136, 138, 139, 141, 165, Di Renzo, G. , 186, 191, 192, 331 167, 169, 184–186, 191, 195, 198, Di Salvo, C. , 52, 73, 77, 78, 604, 608 201, 248, 261, 273, 280, 293, 363, di Sciacca, R. , 222 391, 407, 472, 492, 493, 500, 523, Diacono, J. , 561 553, 561, 567, 602, 606 Diana, M. , 319 Ditzel, D.A. , 343 Diao, C. , 120 Divac, I. , 24, 25 Diaz, Z. , 465, 473 Divoux, D. , 184, 185, 189, 191, 199, 366, 369, Dichgans, J. , 8, 184, 187, 199, 200 371, 373, 374, 510 Dick, O. , 343 Dixon, E.P. , 436 Dicker, P , 531 Dixon, J.E. , 56 Dickerson, F. , 142 Dixon, K.C. , 23 Dickson, E.W. , 65, 68 Djaiani, G.N. , 606 Die, J. , 558 Dmowska, M. , 524, 527–529 Diehl, J.A. , 194 Dobrota, D. , 184, 185, 200 Diehl, K. , 220 Dodd, P.R. , 347 Diemer, H. , 494 Doddrell, D.M. , 278 Diemer, N.H. , 340, 346, 351, 431 Dodoni, G. , 394 Diener, H.C. , 591 Doeller, J.E. , 46 Dieterich, D.C. , 535 Dohi, K. , 436 Dietrich, W.D. , 10, 38, 118, 124, 270, 272, Dolatshad, H. , 531 273, 280, 312, 323, 458, 499, 505 Doliba, N.M. , 60 Diez-Juan, A. , 190, 194 Doll, C. , 26 Digicaylioglu, M. , 218, 410 Dominiak, P. , 70, 71 Dijk, F. , 541, 543 Domoki, F. , 195, 294, 391, 469, 558 Dijkstra, C.D. , 368 Domorakova, I. , 323, 326 Dikic, I. , 353 Domowicz, M.S. , 114, 118, 119 Dillman, J.F. , 8, 340, 342, 437 Donald, A. , 603 Dillman, W.H. , 531 Donato, M. , 75 Dillmann, W. , 8 Dong, C. , 294, 296, 302, 353, 558, 562, 572, Dimitriu, C. , 542 604, 609 Dinakarpandian, D. , 110 Dong, H.L. , 167, 169, 171–174, 459, 461, 467, Dinarello, C.A. , 9 468, 562, 584, 585, 595, 604, 609 Ding, A.-S. , 184, 186, 194, 195, 466, 473, 474 Dong, X.W. , 170 Ding, D.W. , 185, 186 Dong, Y. , 443 Ding, J. , 544 Donnan, G.A. , 59, 134, 136 Ding, L. , 353 Donohoe, P.H. , 245 Ding, N. , 544 Dor, Y. , 491 Ding, Q. , 171 Dore, S. , 311 Author Index 631

Dorn, G.W. , 63, 75, 436 Dung, S.-W. , 184, 185, 194 Dorner, A.J. , 435 Dungey, A.A. , 605 Doron, H. , 508, 512 Dunleavy, M. , 531 Dos, S.P. , 58 Dunwiddie, T.V. , 46 Dost, T. , 74, 436 Duong, H.K. , 312 Dough, R.H. , 580 Dupuis, B. , 4, 9 Douglas, R.M. , 392 Duquesnes, N. , 436 Doutheil, J. , 399, 434 Duranski, M. , 438 Downey, G.P. , 70, 461, 467 During, M.J. , 530, 544 Downey, J.M. , 6, 52, 55–57, 60–64, 70, 72–76, Durrant, D. , 437 83, 326, 436, 437, 584, 602 Durukan, A. , 234, 278, 553 Doyle, K.P. , 274 Duszczyk, M. , 184, 185, 347, 351 Drabek, C.M. , 25 Dutka, D.P. , 77, 82, 604, 608 Dragatsis, I. , 184, 191 Dutton, G.R. , 140 Draghiciu, O. , 38 Duverger, D. , 271 Dreier, J.P. , 280, 295 Dworetzky, B. , 148 Dreixler, J.C. , 542–544, 546 Dysarz, F.A. , 500 Drejer, J. , 431, 494 Dzavik, V. , 53, 602 Drenger, B. , 61 Dzeja, P.P. , 60, 61, 435 Dresselaers, T. , 190, 194 Drew, G.M. , 68–71 Drew, K.L. , 38, 40–47, 249, 341, 436 E Drexler, H. , 74 Earm, Y.E. , 437 Dreyer, C. , 438 Ebel, D. , 604 Driva, M. , 53, 82, 608 Ebrahimi, F. , 68, 69, 71 Droge, W. , 61 Eckardt, K.U. , 192 Drori, S. , 395, 439 Edgar, D.M. , 37 Drummond, G.R. , 280 Edlund, C. , 141, 143, 144 Drummond, J.C. , 279, 368 Edmands, S.D. , 169 Du, A. , 175 Edwards, R.J. , 63 Du, E. , 544 Eells, J.T. , 58, 433 Du, F. , 121, 123, 184, 186, 191, 198, 459, 465 Eerbeek, O. , 433 Du, J. , 173, 185, 186, 188, 491, 595 Egemen, N. , 300 Du, X. , 302 Eguchi, Y. , 431 du Mesnil de Rochemont, R. , 319 Ehlers, M.D. , 353 du Toit, E.F. , 506 Ehrenreich, H. , 218, 219, 222, 510 Duan, C.L. , 185, 186, 194 Ehrhart, J. , 504 Duan, L. , 391, 392 Eid, T. , 218, 219 Duchatelle, I. , 184, 185, 191, 197–199, 202, 510 Eidelman, L.A. , 235, 500 Duchen, M.R. , 63, 64, 75 Einhaupl, K.M. , 6 Ducruet, A.F. , 167 Eklind, S. , 262 Dudkin, K.N. , 184, 185 Eklof, B. , 23 Duehmke, R. , 82, 604, 608 Ekstrom von Lubitz, D.K. , 351 Duffy, T.E. , 229 Ekuni, R. , 157 Dugan, L.L. , 8, 349, 350 El Bahh, B. , 524, 527, 529, 530 Duldner, J.E. , 313 El Shafei, H. , 76 Duman, N. , 219 El Shafei, M. , 57 Duman, R.S. , 237, 525, 531 Elbelghiti, R. , 63, 608 Dumas, T.C. , 323 Eldaif, S. , 73 Dumont, A.S. , 299 Elder, M.G. , 435 Dumont, R.J. , 299 Eleff, S.M. , 431 Duncan, R. , 354 Eliasziw, M. , 175 Duncan, R.C. , 272 Eli-Berchoer, L. , 506, 507, 509, 510 Duncker, D.J. , 65, 68, 69, 605 Elinskii, M.P. , 583 632 Author Index

Ellefsen, S. , 29, 30 Fahnestock, M. , 528 Ellinger, H. , 63 Fajardo, L.F. , 111 Elliot, P.J. , 353 Falcao, A.S. , 410 Elliott, P.J. , 353, 443 Fan, D.F. , 173 Ellis, S.G. , 76 Fan, G.C. , 53, 71 Ellisman, M.H. , 544 Fan, L. , 558 Ellison, J.A. , 4, 8, 165, 231, 234, 274, 278, Fan, M. , 184, 186, 194, 195, 466, 473, 474 349, 399 Fan, Q. , 81 El-Mitwalli, A. , 319, 323 Fan, X.-L. , 184, 186 Eloranta, J. , 577 Fan, Y. , 112 Elrod, J.W. , 46 Fan, Y.H. , 459, 461, 467, 468, 595 Else, P.L. , 141 Fan, Y.-Y. , 184, 185, 197, 199, 461, 467, 470 Elsner, R. , 25 Fando, J.L. , 435 Elvert, G. , 192 Fandrey, J. , 440 Emerson, M.R. , 523, 524, 527 Fang, H. , 184, 194, 195, 342, 347, 351, 382, Emmel, G.L. , 580 466, 473, 474 Emsley, R. , 142 Fang, L. , 185–187, 193 End, E. , 581 Fang, Q. , 299 Endres, M. , 106, 136, 273, 407 Faraci, F.M. , 4, 221, 457, 458, 468 Engel, T. , 522, 531 Faraco, G , 232, 235 Engelman, R.M. , 6, 58, 61 Fares, W. , 585 Englander, E.W. , 436 Faries, P.L. , 574, 603, 605, 607 Englmeier, U. , 192 Farooqui, A.A. , 143, 144 Engstrom, T. , 80, 318 Farr, C.D. , 231, 235, 380, 414, 415, 438 Ennis, S.R. , 234, 310, 313, 459, 463, 467, 468 Fathali, N. , 298 Enomoto, Y. , 319 Fattoretti, P. , 395 Epema, A.H. , 38 Faulkner, C.L. , 231, 235, 380, 438 Eramo, N. , 61 Favreau, L.V. , 443 Erdem, E. , 272, 273 Fazal, M.T. , 76 Erecinska, M. , 20, 21, 430, 431 Feccia, M. , 569 Ergul, A. , 459 Federoff, H.J. , 265, 297, 473, 491 Ergun, R. , 297 Fedorko, L. , 606 Erhardt, J.A. , 5, 368 Fei, L. , 174, 274 Ericsson, A. , 373 Feierman, J.R. , 23 Erinjeri, J.P. , 459 Feige, J.N. , 443 Esposito, A. , 61 Feindt, P. , 604 Esposito, E. , 186, 191, 192, 331 Fekete, S. , 184, 185 Estey, C. , 441 Fekete, V. , 606 Etinger, A. , 500, 501 Felberg, R.A. , 323 Ettaiche, M. , 546 Feldman, A.B. , 8, 340, 342, 437 Evans, D.A. , 141, 144 Felger, R.S. , 25 Evans, M. , 441 Feliciello, A. , 437 Evans, S. , 352 Feng, J.Q. , 73, 328, 436 Evenson, K.R. , 105–106 Feng, R. , 332 Feng, S.F. , 173, 593 Feng, X.Y. , 342, 343 F Feng, Y. , 184, 185, 187, 197, 199, 265 Fabian, R.H. , 280 Feng, Z.C. , 26, 368 Fabricius, M. , 280 Fenstermacher, J.D. , 271 Faden, A.I. , 510, 512 Fenton, R.A. , 68 Faeh, D. , 184, 187–189, 193, 194, 202 Fenton, W.S. , 142 Fagan, S.C. , 459 Ferdek, P. , 430 Fagernes, C.E. , 30 Ferdinandy, P. , 52, 72, 74, 606 Fahlman, C.S. , 44, 167, 169, 184, 187–189, Fergusen, S. , 291 193, 194, 202, 248, 401 Ferguson, G.G. , 175 Author Index 633

Ferguson, T.B. , 457 Flowers-Ziegler, J. , 370 Ferikova, M. , 323, 326 Floyd, R.A. , 436 Fernandes, J.S. , 157 Fluegel, D. , 141, 148 Fernandez, D.C. , 542, 546 Fogarty, E.F. , 297 Fernandez, L. , 70 Foley, C. , 56, 57 Fernandez-Lopez, D. , 376, 410 Follesa, P. , 525, 532 Fernandez-Ruiz, J.J. , 501 Folsom, A.R. , 105 Ferraguti, F. , 530 Fonarow, G.C. , 136 Ferrand-Drake, M. , 435 Fong, W.H. , 439 Ferrandon, A. , 523–525, 527–529 Fonnum, F. , 25 Ferrara, N. , 561 Fonseca, R. , 353 Ferreira, J.C. , 437 Font, G. , 53, 602 Ferrera, R. , 75 Fontaine, R.H. , 184, 185, 197, 199, 201 Ferriero, D.M. , 8, 44, 112, 184–186, 191, Fontana, L. , 441 193–195, 297, 441 Fontos, G. , 53, 602 Ferrini, D. , 559 Forbes, R.A. , 62, 392 Fesahat, F. , 299 Forbes, T.L. , 605 Feuerstein, G.Z. , 4, 8, 9, 57, 134, 137, 231, Ford, E. , 457 234, 270, 271, 274, 278, 327 Ford, S.E. , 492, 582, 587 Feurstein, G.Z. , 118 Forsten, H. , 441 Feuvray, D. , 561 Forstermann, U. , 437 Fezza, F. , 501, 504 Fossati, G. , 232, 235 Fidalgo, A.R. , 184, 185, 202 Fossati, S. , 232, 235 Fiebach, J.B. , 6, 319 Fossett, D. , 296 Fieles, W.E. , 328 Fouad, K. , 512 Figueredo, V. , 62 Fowler, J.H. , 443 Figueredo-Cardenas, G. , 273 Fox-Talbot, K. , 441, 510 Filatov, I. , 296 Fraisl, P. , 190, 194 Finazzi-Agro, A. , 437 Franceschi, L. , 319 Findlay, J.M. , 301 Francis, J.M. , 82 Finet, G. , 52, 53, 63, 79, 80, 318, 608 Francis, K.R. , 185, 191, 196, 198 Finfer, S.R. , 499 Franck, G. , 22 Fink, D.J. , 23 Frangou, S. , 142 Fink, S.L. , 370, 531 Frank, M. , 184, 185 Finkelstein, A. , 141, 144 Franke, T.F. , 327, 328 Finkelstein, M. , 581 Franklin, A. , 504 Fischer, G. , 73 Frascarolo, P. , 175 Fishbein, K.W. , 64 Fraser, C. , 541 Fisher, D.A. , 351, 352 Fraser, P.A. , 299 Fisher, J.K. , 63 Frassdorf, J. , 172, 604 Fisher, M. , 45, 59, 134, 234, 272, 581, 601, Frassetto, S.S. , 248 606 Frasure-Smith, N. , 142 Fishman, M.C. , 234 Fratelli, M. , 510 Fisk, L. , 184, 185, 200 Fredholm, B.B. , 27, 529 Fiskum, G. , 247 Freeman, D.A. , 38 Fitzgibbons, J.C. , 184, 185, 189 Freeman, S. , 506 Fix, A.S. , 340 Frei, U.A. , 192 Flack, J.E., 3rd. , 6 Freiberger, J.J. , 172, 173 Flaherty, M.P. , 63 Freiberger, T. , 78, 604 Flamme, I. , 192 Freie, A.B. , 5, 44, 184–186, 188, 192, 194, Flannery, R.J. , 299, 433 196, 198, 202, 203, 293, 458, 459, Flegal, K. , 135 463, 467, 468, 470 Flores-Soto, M.E. , 441 Frelinger, A.L. , 313 Floris, S. , 368 Frenneaux, M.P. , 53, 78, 602, 604, 608 Flowers, K. , 585 Frere, J.M. , 22 634 Author Index

Freret, T. , 184, 185, 189, 199, 203, 323, 366, G 369, 371, 373, 374 Gabel, S. , 61 Frerichs, K.U. , 38, 40, 327, 371 Gadamski, R. , 184, 185, 347, 351 Freyer, D. , 219, 411 Gadian, D.G. , 367 Friberg, H. , 247 Gafken, P. , 353 Friedman, N. , 506, 507, 510 Gage, A.T. , 184, 187, 188 Friel, D.D. , 434 Gagnon, P.M. , 185, 201 Frietsch, T. , 296, 574 Gagnon, R.C. , 9 Frigatti, P. , 319 Gahide, G. , 63, 608 Frndova, H. , 77, 78, 604 Gai, N. , 503 Froozandeh, M. , 457, 461, 467, 468 Gainer, H. , 23 Frotnczak-Baniewicz, M. , 488 Gaisa, N. , 47 Fry, P.A. , 340, 344 Gaitanaki, C. , 57 Fryer, R.M. , 56, 58, 433 Gala, R. , 231, 235, 319, 322, 325–327, 330 Frys, K. , 437 Gale, K. , 524, 525, 527, 529, 531, 532 Fu, H.B. , 367 Gall, C.M. , 512 Fu, Q. , 64 Gallagher, K.P. , 6, 55 Fu, W.X. , 595 Gallego, J. , 184, 185, 197, 199, 201 Fu, Y.C. , 6, 441 Gallez, B. , 190, 194 Fuchs, E.C. , 343 Gallily, R. , 511 Fuji, H. , 53, 64 Gallo, E.F. , 462, 467–471 Fujibayashi, Y. , 184, 185, 194 Gallucci, F. , 558–561 Fujihara, H. , 195 Galluzzi, L. , 430 Fujikawa, D.G. , 522 Galofre, M. , 595 Fujisawa, S. , 299, 433 Galson, D.L. , 441 Fujita, M. , 60, 75, 303 Galvani, M. , 559 Fujita, T. , 279–281 Galve-Roperh, I. , 503 Fujito, T. , 603, 607 Gambhir, S. , 370 Fujiwara, H. , 59, 61 Gamoh, S. , 144 Fujiwara, N.H. , 302, 431, 462, 472 Gan, Y. , 399, 460, 464, 466, 468, 474 Fukaya, M. , 344 Ganote, C.E. , 57 Fukuda, R. , 441 Gant, V.A. , 443 Fukuda, S. , 173 Ganzella, M. , 523, 524, 527 Fukui, S. , 10, 118 Ganzhorn, J.U. , 37 Fukumura, D. , 491 Gao, C.-Y. , 185, 186, 197, 199, 411 Fukunaga, K. , 187, 230, 462, 468, 469 Gao, F. , 57 Fukunaga, R. , 6, 236, 579 Gao, G. , 186, 193, 297, 412, 413 Fukunaga, Y. , 342, 343 Gao, H. , 171, 274, 460, 464, 466, 468, 474, Fulco, M. , 443 601 Fuller, S.J. , 57 Gao, J.H. , 595 Fuller-Bicer, G.A. , 73 Gao, L. , 231, 235, 586 Funakoshi, T. , 172, 173 Gao, Q. , 322, 325, 435, 464, 470 Furchgott, R.F. , 437 Gao, R. , 442 Furie, K. , 135, 457 Gao, W. , 391 Furlan, M. , 581 Gao, X.M. , 293, 311, 318, 319, 321, 323–325, Furlan, R. , 504 327, 328, 330, 331, 375, 494, 523, Furuta, T. , 271 563 Furuya, K. , 3, 4, 9, 10, 222, 275, 366, Gao, Y. , 184, 187, 196, 201, 216, 299, 367, 398, 368–370, 372, 373, 459–461, 412, 413, 460, 464, 466, 468, 474 467–470 Gaoni, Y. , 500 Furuya, N. , 402 Gao-Yu, C. , 174, 274 Fusco, F. , 273 Garcia, C. , 175 Fütterer, C. , 167, 169, 170, 280, 298, Garcia, J.A. , 441 492, 493 Garcia, L. , 248 Author Index 635

Garcia, S. , 81, 318 Gerloff, C. , 457 Garcia-Dorado, D. , 52, 72, 75 Germano, S.M. , 270 Garcia-Eroles, L. , 559, 560 Geromanos, S. , 189, 381, 530, 535 Garcia-Roves, P.M. , 439 Gerstenblith, G. , 506, 507, 509, 510 Garcia-Yebenes, I. , 376 Gertz, K. , 273, 408 Gardiner, E. , 585 Gerwins, P. , 373 Gardner, T.W. , 541 Geschwind, D.H. , 10 Garg, N. , 270 Gess, B. , 412 Garlick, P.B. , 60 Gessa, G.L. , 502 Garlid, K.D. , 6, 58, 62, 64, 74, 81, 433, 436, 437 Gesuete, R. , 231, 234, 465, 468, 472, 473 Garner, C.C. , 352 Gething, M.J. , 435 Garnier, P. , 185, 187, 194, 200, 248 Geyer, A.B. , 399 Garthwaite, G. , 433 Gezercan, Y. , 297 Garthwaite, J. , 433 Ghadban, C. , 503 Gasche, Y.G. , 458 Ghai, H.S. , 27, 371 Gáspár, T. , 195, 294, 390–392, 433, 469, 558 Ghaleh, B. , 73 Gass, A. , 319 Ghasemi, M. , 68, 69, 71 Gass, P. , 279 Ghezzi, P. , 9 Gassmann, M. , 185, 191, 201 Ghiardi, G.J. , 543, 545 Gateau-Roesch, O. , 64, 75 Gho, B.C. , 65, 68, 69, 245 Gatting, M. , 213, 220 Ghosh, T.K. , 435 Gattullo, D. , 74, 76 Giala, M. , 184–186, 194 Gaughan, C.L. , 431 Giang, D.K. , 502 Gaunt, M.E. , 77, 604, 608 Giannopoulos, G. , 53, 82, 608 Gautier, S. , 463, 467, 470, 471, 474 Giannotta, S.L. , 294 Gaver, V. , 606 Giardina, C. , 294, 299 Gavilanes, A.W. , 463, 471 Gibson, C.L. , 556 Gawron, A. , 524, 527–529 Gibson, D. , 500, 501 Ge, P.F. , 399, 400, 403 Gibson, D.G. , 234 Ge, Y.G. , 80, 81 Gibson, G. , 76 Geiger, P.C. , 439 Gibson, P.H. , 76 Geiser, F. , 37 Gibson, S. , 56 Geisler, S. , 396 Gidday, J.M. , 5, 8, 9, 44, 138, 139, 165, Gele, P. , 235 184–189, 191–194, 196, 198, Gelly, C. , 439 200–203, 213, 220, 248, 259, 260, Gelpi, E. , 70, 443 262, 264, 266, 271, 292, 293, 297, Gelpi, R.J. , 75 300, 302, 309, 312, 313, 411, 437, Genade, S. , 57 441, 458, 459, 462, 463, 467–471, Gendron, T. , 154 523, 541–545 Geng, D.F. , 159 Gietz, A. , 273 Geng, J.X. , 466, 474 Giffard, R.G. , 8, 349, 350, 367 Geng, X. , 6 Gigante, P.R. , 167 Genis, A. , 506 Gilbert, G.L. , 503 Genka, C. , 60 Gilbert, S.F. , 4 Gensert, J. , 190 Giles, T. , 352 Geocadin, R.G. , 184–186 Gill, D.L. , 435 Georgieff, M. , 280 Gillespie, C. , 457 Gerace, E. , 322, 326, 330 Gillespie-Brown, J. , 57 Gerard, L. , 608 Gillum, R.F. , 106 Gerard, R.D. , 441 Gin, T. , 293, 603 Gerasimenko, J.V. , 430 Gincel, D. , 433 Gerasimenko, O.V. , 430 Ginis, I. , 9, 184, 187, 500, 511 Gerhart-Hines, Z. , 441, 443 Ginsberg, M.D. , 38, 124, 136, 235, 270, 272, Gerich, F.J. , 392 273, 280, 297, 323, 429, 601 636 Author Index

Gioffre, P.A. , 5 Gonzales, E.R. , 184, 185, 201, 271, 467 Giordano, F.J. , 442 Gonzalez, G. , 62, 75 Giovanardi, R.O. , 601 Gonzalez, J.M. , 280 Giralt, M. , 439 Gonzalez, P. , 25 Girgis, J. , 512 Gonzalez-Baron, M. , 328 Giricz, Z. , 606 Gonzalez-Barroso, M.D. , 439 Gissel, C. , 434 Gonzalez-Zulueta, M. , 8, 340, 342, 437 Giulian, D. , 9 Goodman, M.D. , 73 Giunta, F. , 172, 493 Goodman, Y. , 391 Givel, F. , 438 Gopalan, B. , 10, 189, 202, 366, 368, Gladwin, M.T. , 438 373–377, 467 Glantz, L. , 235, 500 Gopher, A. , 500, 501 Glassman, M. , 56, 57 Gorbunova, V. , 441 Glazier, S.S. , 232, 235 Goren, B. , 184, 185, 194, 322 Gleiter, C.H. , 218 Gorgias, N. , 184–186, 194 Glimcher, L.H. , 397 Gorji, A. , 341 Globus, M.Y-T. , 38, 272, 323 Gorog, D.A. , 436 Glos, J. , 37 Gorospe, M. , 436 Gluschenko, T.S. , 184, 185, 187, 192–196, Gorus, F. , 190, 194 199, 409 Gosling, P. , 78, 604, 608 Gnaiger, E. , 190, 194 Goto, M. , 55, 56, 60, 61 Go, A. , 457 Goto, S. , 273 Gobbi, M. , 530 Goto, Y. , 270 Godman, C.A. , 294, 299 Gotoh, J. , 3, 4, 9, 10, 275, 459, 461, 470 Godukhin, O.V. , 184, 186, 193, 195 Gottlieb, M. , 323, 326, 327, 563 Goebel, U. , 175, 542 Gottlieb, R.A. , 63, 75 Goel, G. , 112 Gottlieb, S.F. , 584 Goemans, C. , 510 Gottlob, K. , 443 Goertz, A. , 280 Gottschalk, B. , 6, 559–561 Gokmen, N. , 219 Gotz, T. , 544 Goldberg, A.L. , 402 Gou, X. , 595 Goldberg, M.E. , 4 Gould, E. , 113 Goldberg, M.P. , 8, 349–352 Gower, D.J. , 4 Golden, J.A. , 184, 185 Goyagi, T. , 231, 235, 275, 461, 470 Goldstein, B. , 47 Gozal, Y. , 61 Goldstein, I.M. , 5, 541–543 Grabb, M.C. , 8, 245, 246, 342, 347, 349, 351 Goldstein, L.J. , 299 Graber, S. , 352, 353 Golech, S.A. , 504 Grabiec, U. , 503 Golshani, K.J. , 274 Graf, R. , 279–281 Goma, F.M. , 64 Grafe, M.R. , 274 Gomez, L. , 64 Graham, C.E. , 544 Gomez-Pinilla, F. , 113 Graham, D.I. , 228, 232, 235, 270, 273 Gomi, T. , 601 Graham, S.H. , 4, 6, 8, 231, 235, 275, 277, 340, Gomi, Y. , 461, 471 344, 367, 369, 399, 436, 465, 473, Gomis, P. , 299 503, 530, 531, 567 Gonatas, N.K. , 23 Grahn, D.A. , 37 Goncalves, L.M. , 442, 443 Grande, L.A. , 107, 113 Gonenc, S. , 219 Graumann, J. , 535 Gong, B. , 542 Graupner, G. , 166, 173, 295, 491 Gong, K.-R. , 185, 186, 197, 199 Gray, J.J. , 184, 187, 202 Gong, Y. , 294, 310 Gray, L.J. , 556 Gonoi, T. , 62 Greasley, P.J. , 504 Gonye, G.E. , 377 Greeley, G.H. , 436 Gonzales, C. , 230, 273 Green, D.R. , 78, 430, 604, 608 Author Index 637

Green, R. , 606 Guida, N. , 331 Green, S. , 438 Guillet, B.A. , 170, 171 Green, T.K. , 43 Gulbins, E. , 435 Greenberg, D.A. , 10, 503, 523, 524, 527, 529 Gulker, H. , 601, 602 Greenberg, J.H. , 276, 277, 280 Gunaydin, B. , 78 Green fi eld, J.C., Jr. , 5, 51 Guo, A,C. , 561 Greenlund, K. , 457 Guo, M. , 106, 110, 118, 184, 194, 195, 466, Greenmayre, J.T. , 24, 25 473, 474 Greensmith, L. , 370 Guo, Q. , 171, 322, 331, 373 Greer, D.M. , 124 Guo, R.X. , 436 Greer, J.J. , 438 Guo, S.X. , 170 Grega, M.A. , 606 Guo, W. , 186, 198 Grego, F. , 319 Guo, X. , 184, 185, 195, 199 Gregory, A.D. , 107, 113 Guo, Y.L. , 57, 63, 373, 436 Gregory, C.A. , 11 Gupta, H. , 5, 541–543 Greisman, S.E. , 3 Gupta, K. , 443 Gressens, P. , 170, 184, 185, 197, 199, 201 Gurd, J.W. , 354 Grider, M. , 113 Gurvitz, V. , 235, 500 Grieb, P. , 488, 523 Gustavsson, M. , 189, 197, 264, 265, 459, 460, Grif fi n, G. , 500, 501 462, 469–471 Grif fi n, S.C. , 585, 586, 608 Gustaw, K. , 201 Grif fi ths, E.J. , 61–63, 443 Gute, D.C. , 5 Grif fi ths, T. , 340 Gutilla, M. , 46 Grigg, J. , 541 Gutsaeva, D.R. , 185, 193, 195, 395, 413, 439 Grigoriadis, N. , 509, 510 Gutzwiller, F. , 184, 187–189, 193, 194, 202 Grimm, C. , 185, 191, 192, 201, 250 Guvendik, L. , 571, 585, 586 Grisar, T. , 22 Guyton, K.Z. , 436 Grisar-Charlier, J. , 22 Guyton, R.A. , 53, 71–75, 79, 317, 326 Grisham, M.B. , 436 Guzman, M. , 443, 503 Groc, L. , 340 Guzy, R.D. , 430 Grocott, H. , 175, 606 Gwak, M.S. , 172, 221 Grodecka, L. , 78, 604 Gwinn, R. , 7, 235 Groen, R.J. , 295 Gyi, L. , 4 Groenendaal, F. , 198, 436 Gysembergh, A. , 57, 68 Grooms, S.Y. , 356 Grosfeld, A. , 190, 194 Gross, E.R. , 298 H Gross, G.J. , 55, 56, 58, 60–62, 65, 68–70, 83, Ha, J. , 443 298, 433, 502 Ha, T. , 367, 458, 459, 462, 467, 468 Gross, R.W. , 439 Ha, Y. , 184, 201 Grossman, R.G. , 351 Ha, Z.D. , 586 Groszer, M. , 185, 191, 201 Haase, N. , 457 Grotta, J.C. , 279, 319, 323, 439, 591 Habib, M.M. , 293 Grover, G.J. , 6, 62, 81, 433, 561 Hacke, W. , 130 Grundy, E. , 52, 77, 78, 604, 608 Hackett, D.R. , 559, 561 Gu, G.J. , 173, 411 Hackett, S.F. , 441 Gu, J.L. , 57, 172, 440, 493 Hadam, J. , 524, 525, 527, 529 Gu, Q. , 171 Haddley, K. , 534 Gu, Y. , 294 Hadingham, S.J. , 278 Gu, Z. , 110 Hafner, G. , 3 Guagliano, E. , 443 Hafner, M. , 354 Guan, Y.S. , 173, 294 Hafstad, A.D. , 438 Gudino-Cabrera, G. , 441 Hagberg, H. , 189, 197, 222, 260, 261, 410, Gueugniaud, P.Y. , 64 459, 460, 462, 469–471 638 Author Index

Haghir, H. , 341 Hansen, A.J. , 23, 24 Hagioka, S. , 42 Hansen, B.M. , 27 Hagve, M. , 438 Hansen, H.H. , 501, 502 Hahn, H. , 436 Hansen, H.S. , 501, 502 Hahn, J. , 486–489 Hansen, S.H. , 501, 502 Hai, S. , 433 Hansen, T.M. , 11, 53, 82, 608 Haile, M. , 166 Hansen-Schwartz, J. , 295 Hailpern, S. , 457 Hansson, G.K. , 9 Haines, B.A. , 439 Hansson, O. , 367 Hajizadeh, S. , 457, 461, 467, 468 Hanus, L. , 500, 501, 503 Hajrasouliha, A.R. , 68, 69, 71 Hao, W. , 487, 490 Hakim, A.M. , 7, 273–275, 277, 349 Hao, Y. , 231, 235 Hale, S.L. , 245, 353 Haque, R.M. , 167 Halestrap, A.P. , 61–63, 75, 393, 394, 443 Hara, K. , 8 Halkos, M.E. , 53, 71, 72, 75, 79, 317, 326 Hara, S. , 192 Hall, A.C. , 169 Hara, T. , 401, 412, 413 Halle, E. , 273 Harada, H. , 232, 237 Hallenbeck, J.M. , 3–5, 9, 10, 38, 40, 45, 109, Harada, K. , 10 111, 118, 134, 138, 139, 141, 165, Harada, N. , 299 169, 184, 187, 275, 327, 367, 371, Harch, P.G. , 297 459, 461, 466, 470, 474, 500, 511, Hardemark, H.G. , 591 523, 527 Harder, D.R. , 231, 235, 275, 461, 470 Halley, M. , 124 Harding, H.P. , 435 Halliwell, B. , 220 Hardingham, G.E. , 342, 343, 443 Halow, J. , 62 Hardingham, N.R. , 343 Halpain, S. , 352, 353 Hardy, P. , 583 Halsey, J.H. , 274 Harken, A.H. , 601 Halterman, M.W. , 265, 297, 491 Harker, C.T. , 38 Hamada, H. , 10 Harkin, D.W. , 245 Hamada, J. , 187, 412, 413, 462, 468, 469 Harms, L. , 6 Hamakubo, T. , 366, 368–370, 372, 373 Harold, F.M. , 20 Hamann, G.F. , 109 Harper, M.E. , 441 Hambleton, M.A. , 63, 75 Harries, M.D. , 188, 201, 292, 293, 297, 300, Hamblin, M. , 438 463, 468, 469, 471 Hamernik, R.P. , 5 Harrington, C.A. , 5, 10, 41, 189, 313, 343, Hamill, C.E. , 220 364, 366, 368, 370–374, 467, 532 Hamilton, R.W. , 581 Harris, A.L. , 192 Hammerling, U. , 436 Harris, A.Z. , 340, 343 Hammond, B.J. , 21 Harris, J. , 52, 77, 78, 604, 608 Hamrick, S.E.G. , 185, 193, 195 Harris, K.A. , 605 Han, B.H. , 188, 201, 463, 468, 469, 471 Harris, R.A. , 190, 194 Han, C. , 173 Harris, V.A. , 8 Han, D.H. , 439 Harris, W.S. , 141 Han, H.C. , 71, 602 Harrop, A. , 544 Han, J. , 56, 57, 437, 544 Hart, G.B. , 580 Han, R.Q. , 294 Harten, S.K. , 190, 194 Han, S. , 184–188, 193, 544 Hartings, J.A. , 280 Han, X. , 184, 187, 195, 439, 466, 472 Hartl, F.U. , 4, 353 Hanada, M. , 328 Hartley, D.M. , 8, 349, 350 Hand, S. , 26 Hartnett, K.A. , 5, 368 Handa, N. , 6, 236, 579 Hartung, H.P. , 10 Handschin, C. , 439, 443 Hartwig, J.H. , 353 Hanley, D.F. , 184–186, 310 Harukuni, I. , 556 Hanley, P.J. , 62 Hasan, D. , 294 Author Index 639

Hasbani, M.J. , 351, 352 Heesom, K.J. , 61 Hashemi, P. , 280 Hegde, A.N. , 353 Hashiguchi, A. , 187, 462, 468, 469 Heidarianpour, A. , 457, 461, 467, 468 Hashimoto, M. , 37, 38, 45, 144, 270–273, Heidbreder, M. , 70, 71 279, 581 Heidenreich, R. , 192 Hashimoto, N. , 274, 277 Heijnen, C.J. , 198, 436 Hasko, G. , 501, 504 Heim, C. , 524, 527, 529 Hasselblatt, M. , 219, 510 Heim, T. , 143 Hassels-Berger, J. , 24 Heine, M. , 340 Hassen, G.W. , 245 Heinen, Y. , 73 Hassoun, S.M. , 322, 331 Heisler, N. , 25 Hata, R. , 6, 236, 579 Heiss, W. , 24 Hatala, D.A. , 542 Heiss, W-D. , 279–281 Hatase, O. , 346 Hekmati-Moghadam, S.H. , 299 Hatazaki, S. , 523–525, 527–529, 531–534 Heldmaier, G. , 37 Hatch, T.F. , 140 Helftenbein, G. , 438 Hattori, K. , 229 Heller, H.C. , 37, 352 Hattori, R. , 58 Hellwig-Burgel, T. , 440 Hatzinikolas, G. , 442 Helm, G.A. , 302 Haug, F.M. , 392 Helms, M.J. , 606 Hausenloy, D.J. , 11, 52, 53, 56, 59, 63, 64, Helqvist, S. , 80, 318 67, 71–75, 77, 78, 82, 83, 388, 393, Helton, R. , 265 396, 441, 506, 557, 562, 563, 571, Hemmert, J.W. , 544 601, 602, 604, 608 Hemmings, B.A. , 328 Hawel, L. , 467 Henchcliffe, C. , 544 Hawkinson, J.E. , 343 Hendershot, L. , 435 Haworth, R.A. , 435 Heneka, M.T. , 406 Hay, N. , 443 Henn, V. , 218 Hayakawa, T. , 24 Hennelly, M.M. , 544 Hayashi, T. , 10, 328, 399, 400, 435 Hennemann, B. , 469 Haydon, P.G. , 344 Henning, O. , 68 Hayer, S. , 343 Henrich-Noack, P. , 214, 273 Hayer-Hartl, M. , 4 Henriksen, P.A. , 458 Hayes, J.D. , 443 Henry, D.E. , 433 Hayes, K. , 106, 113 Henry, M.M. , 58, 433 Hayward, M.D. , 52, 73, 77, 78, 237, 604, 608 Henry, T.D. , 81, 318 He, D.Y. , 594 Henshall, D.C. , 9, 313, 353, 367, 368, 375, 463, He, G.H. , 271 467, 474, 522–529, 531–534, 568 He, H. , 439 Heo, J.H. , 605 He, P. , 184, 185, 197, 199, 461, 467, 470 Hepp, S. , 392 He, Q. , 328 Herbert, J.M. , 491 He, R.R. , 69 Herculano, B. , 494 He, W. , 184, 186, 191, 198 Herdegen, T. , 525, 530 He, X.H. , 441 Herlenius, E. , 184 He, Y.Y. , 4, 8, 173, 221, 270, 271, 311, 401 Hermes, M. , 604 He, Z. , 272, 279, 557, 558, 561 Hermes-Lima, M. , 38 Headrick, J.P. , 60, 72 Hernandez, F. , 175 Heads, R.J. , 56, 57 Hernandez, L. , 62 Heald, P.J. , 23 Hernando, V. , 75 Heales, S.J. , 437 Herndon, R.M. , 24 Heard, S.O. , 68 Heron, A. , 248 Hecht, N. , 197, 470 Herrmann, O. , 273 Heck, P.M. , 82, 558, 604, 608 Herson, P.S. , 280 Heerdt, P. , 166 Hertz, R. , 443 640 Author Index

Herwig, A. , 38 Ho, P.M. , 457 Herzog, N. , 71 Hobbs, T.R. , 274 Hespel, P. , 190, 194 Hochachka, P.W. , 22, 25, 26, 28, 41, 371 Hess, D.C. , 459 Hockings, P.D. , 278 Hetz, C. , 63 Hoerter, J. , 439 Hetzel, F.W. , 4 Hof, P.R. , 27 Heurteaux, C. , 8, 10, 117, 138, 141, 143, Hofbauer, K.H. , 412 147–152, 154, 155, 246, 348, 525, Hoff, J.T. , 151, 173, 214–220, 294, 299, 529–531, 546 309–314 Heusch, G. , 52, 56, 57, 62, 63, 72–74, 78, Hoffman, D.R. , 140 83, 604 Hoffman, E.P. , 443 Hewitt, K. , 348 Hoffman, M.S. , 185, 201 Hewitt, M. , 342, 347, 351 Hoffman, R.A. , 37 Heyman, A. , 580 Hoffman W.E. , 493 Heynen, S. , 191, 192 Hoffmann, O. , 367 Hibbeln, J.R. , 142, 144 Hofmann, F. , 343 Hickey, E.J. , 263, 367 Hofmann, K. , 430 Hickey, R.W. , 184, 187, 196, 401 Hofmann, P.A. , 390 Hicklin, D.J. , 184, 185, 197, 199, 201 Hogan, E.L. , 270, 319 Hicks, CA. , 7, 345, 351 Hogan, M.J. , 274, 277, 348 Hidalgo, J. , 188, 198, 201 Hogg, R.T. , 441 Hideg, K. , 544 Hogins, J. , 344, 350 Hiebert, G.W. , 512 Hogue, C.W., Jr. , 175 Higashi, H. , 431 Hohnadel, E.J. , 459 Hightower, L.E. , 294, 299 Holbach, K.H. , 492 Higuchi, T. , 42 Holbrook, N.J. , 436 Higuti, T. , 62, 433 Hollen, L. , 41 Hil ﬁ ker-Kleiner, D. , 73, 74 Hollis, V.S. , 438 Hill, M.D. , 340 Holloszy, J.O. , 439 Hill, M.L. , 5, 51, 601 Holman, R.T. , 140, 144 Hillard, C.J. , 502, 504 Holmes, E.W. , 5, 51 Hillis, G.S. , 76 Holmes, G.L. , 529, 530 Hillje, A.L. , 353 Holmstrom, K.M. , 396 Hillman, G.R. , 280 Holmstrup, M. , 506 Hills, C.P. , 351 Holmuhamedov, E.L. , 6, 60, 435 Hine, C. , 441 Holmvang, L. , 80, 318 Hines, J.J. , 5, 51 Holtby, H.M. , 77, 78, 604 Hirai, K. , 63 Holtsberg, F.W. , 10 Hiraizumi, Y. , 436 Holtzclaw, B.J. , 124 Hirakawa, M. , 42, 581 Holtzman, D.M. , 9, 185, 188, 437, 462, 468, 469 Hiramatsu, K. , 270 Homen, D.S. , 367, 375, 467 Hirata, A. , 60, 75 Hong, D.M. , 78, 604 Hirata, N. , 388 Hong, H. , 382 Hirata, T. , 172, 173 Hong, J.M. , 601 Hirohashi, K. , 433 Hong, S. , 531 Hirota, K. , 441 Hong, S-C. , 270 Hirota, Y. , 60 Hong, S.K. , 71, 602 Hirsch, N. , 436 Hong, S.M. , 112 Hirt, L. , 465, 472 Hong, S.S. , 184, 186, 193, 200, 201 Hiwatari, M. , 461, 471 Hong-Li, X. , 487, 490 Ho, C.J. , 232, 237, 441 Honma, M. , 544 Ho, D.Y. , 370, 531 Honmou, O. , 10 Ho, K.L. , 4 Hoole, S.P. , 82, 558, 604, 608 Ho, L.-T. , 184, 185, 193, 194, 201 Hooper, J. , 77 Author Index 641

Hopkins, S.J. , 111, 511 Hu, C.-J. , 192, 395 Hori, M. , 53, 60, 64, 75 Hu, C.P. , 70 Horiguchi, T. , 371 Hu, F. , 184, 187, 195, 466, 472 Horikawa, Y. , 76 Hu, G. , 106 Horiuchi, Y. , 512 Hu, H. , 216, 217, 294, 299 Horky, L.L. , 134, 136 Hu, L.F. , 175 Horn, J.W. , 340 Hu, Q. , 279, 299 Hornik, C.P. , 327, 328 Hu, R. , 173, 174, 297 Horowitz, M. , 312, 506–512 Hu, S.L. , 171, 173, 174, 297, 562, 604, 609 Horrobin, D.F. , 142–144 Hu, W. , 172, 173 Horrocks, L.A. , 143, 144 Hu, W.-W. , 184, 185, 197, 199 Horsburgh, K. , 273, 443 Hu, W.N. , 586 Horstrup, J.H. , 192 Hu, W.W. , 461, 467, 470 Hoschtitzky, J.A. , 52, 603 Hu, X. , 184, 185, 191, 196, 198, 415 Hoshi, A. , 459, 465, 467, 468, 473 Hu, Z. , 436 Hoshi, K. , 603, 607 Hua, F. , 174, 274, 367, 458, 459, 462, 467, 468 Hoshida, S. , 53, 64 Hua, Y. , 173, 214–220, 234, 294, 296, 299, Hoshikawa, T. , 167 310–315, 401, 411, 412, 459, 463, Hoshino, T. , 462, 472 467, 468 Hossain, M. , 171, 172 Huan, L. , 594 Hossmann, K-A. , 118, 270, 279 Huang, C. , 396 Hota, K.B. , 443 Huang, C.-C. , 185, 201, 232, 237, 263, 264, Hota, S.K. , 443 458, 462, 467–469, 472 Hothersall, J.S. , 436 Huang, C.F. , 271 Hou, H. , 504 Huang, C.H. , 593, 594 Hou, L. , 167, 169, 172–174, 298, 493, 584, 592 Huang, C.K. , 56 Hou, W.G. , 173 Huang, F.P. , 294, 310 Hou, Y.Z. , 185, 186, 193 Huang, H.-S. , 185, 191, 200, 544 Houkin, K. , 10 Huang, J. , 231, 235, 278–280, 458 Housman, C. , 527 Huang, L.E. , 349, 440 Hov, D.A. , 30 Huang, N.C. , 280 Hovda, D. , 494 Huang, P.L. , 185–187, 193, 231, 234, 235, Howard, A.B. , 11 278, 461, 468, 469 Howard, M.R. , 534 Huang, Q. , 56, 57 Howard, V. , 457 Huang, R. , 79 Howell, K. , 443 Huang, S.Y. , 57, 142 Howells, D.W. , 134, 136 Huang, W.J. , 123 Howlett, A.C. , 500 Huang, W.Q. , 440 Howng, S.L. , 293, 298, 299 Huang, X. , 184, 186 Hoyer, K. , 439 Huang, Y. , 74, 114, 115, 117, 123, 167, 169, Hoyos, B. , 436 171, 221 Hoyt, R.F., Jr. , 438 Huang, Z.G. , 63, 118, 234 Hoyte, L.C. , 235, 273, 275, 340, 462, 470 Hübel, K. , 279–281 Hrehorovska, M. , 435 Hughes, D. , 534 Hruza, Z. , 4 Huh, J-T. , 271, 273 Hsieh, C.C. , 584 Hui, K.K. , 592 Hsieh, J. , 319, 328, 330, 331 Hui, L. , 487, 490 Hsu, A.K. , 56, 58, 68–70, 433 Huisamen, B. , 57 Hsu, C.Y. , 4, 8, 221, 270, 271, 319 Hulbert, A.J. , 141 Hsu, F.-C. , 184, 185, 193, 194 Hunninghake, G.W. , 56, 60 Hsu, F.P. , 487, 489 Hunt, M.A. , 296 Hsu, S. , 151 Hunter, B. , 10, 189, 366, 368, 373, 374, 467 Htun, P. , 57 Hunter, C. , 169, 221, 277, 459, 461, 469, 470 Hu, B.R. , 346, 353, 595 Hunter, D.R. , 435 642 Author Index

Hunter, T. , 190, 353 325–327, 330 Huo, X. , 185, 186, 197, 199 Inoue, T. , 603, 607 Hurn, P.D. , 167, 168, 176, 229, 231, 235, 275, Inserte, J. , 75 280, 431, 461, 470, 493 Insko, M.A. , 47 Hurtado, O. , 234, 376, 407, 466, 473 Inubushi, T. , 68 Hurwitz, S. , 148 Inukai, T. , 512 Husain, S. , 544 Iqbal, A. , 78, 604 Huster, G. , 313 Irvin, S.M. , 503 Hutchinson, P.J. , 511 Irwin, M.G. , 81 Hwang, B.Y. , 167 Isaev, N.K. , 167, 169, 170, 184, 186, 198, Hwang, D. , 141 219, 280, 298, 492, 493 Hwang, J.W. , 166, 168, 501, 502 Ishida, A. , 530, 531 Hwang, P.M. , 437 Ishida, H. , 60 Hwang, S.L. , 293, 298, 299 Ishida, K. , 173 Hylland, P. , 27, 29, 30 Ishida, M. , 59 Hyong, A. , 487–490 Ishida, T. , 5 Ishii, K. , 541 Ishii, Y. , 271 I Ishikawa, A. , 8, 235 Iacomino, G. , 437 Ishikawa, Y. , 172, 173 Iadecola, C. , 9, 135, 136, 139, 437, 462, Israel, M. , 390 467–471 Israeli, D. , 431 Iceman, K.E. , 42, 436 Issemann, I. , 438 Ide, K. , 111 Itabashi, H.H. , 522 Ide, T. , 348 Ito, M. , 584 Ieong, E. , 171, 172 Itoh, K. , 501 Iglesias, R. , 439 Itoyama, Y. , 8, 464, 465, 472–474 Iijima, T. , 431, 435 Itti, R. , 79, 80, 608 Iimura, O. , 56 Iurato, L. , 184 Ikeda, F. , 353 Ivanova, N.E. , 583 Ikeda, T. , 263, 399, 459, 462, 467, 468 Iwabuchi, S. , 349 Ikeda, Y. , 443 Iwai, M. , 184, 187, 196, 299 Ikegaya, Y. , 352 Iwai, T. , 58 Ikeno, F. , 436 Iwama, T. , 319 Ikenoue, T. , 459, 462, 467, 468 Iwana, G.K. , 506 Ikonomidou, C. , 501, 502 Iwanaga, Y. , 184, 185, 188, 191, 197 Iliodromitis, E.K. , 57, 83, 436, 601 Iwao, Y. , 431, 435 Im, Y.B. , 373 Iwase, H. , 63, 430 Imai, I. , 461, 471 Iwata, N. , 230 Impey, S. , 9, 368, 531 Iwatsubo, T. , 352 Imura, N. , 192 Izumiyama, M. , 464 Inada, H. , 300 Izumo, S. , 56 Inagaki, K. , 436 Izuta, H. , 399 Inagaki, N. , 62, 117 Inamasu, J. , 118 Inamura, K. , 431 J Inazawa, J. , 62 Jaattela, M. , 367 Ingram, D.D. , 106 Jabehdar-Maralani, P. , 68, 69, 71 Ingwall, J.S. , 439 Jaburek, M. , 62, 74 Inners-McBride, K. , 59 Jacewicz, M. , 270–272, 275, 279–281 Inohara, H. , 63, 75 Jackson, D.C. , 25, 26, 28, 29, 44 Inoue, D. , 58 Jackson, R.M. , 436 Inoue, I. , 62, 391, 433 Jacobus, W.E. , 431 Inoue, K. , 231, 235, 295, 319, 322, Jaderstad, J. , 184 Author Index 643

Jadhav, V. , 173, 174, 294, 299, 486–491, 495 Jeoung, N.H. , 190, 194 Jager, S. , 439, 443 Jeppesen, L.L. , 4 Jaggi, A.S. , 245, 298, 505 Jessick, V.J. , 342, 348–350, 352–354, 403 Jagiella, W.M. , 582, 587 Jesunathadas, R. , 173, 487, 490, 491, 495 Jahn, H.M. , 353 Jeyabalan, A.P. , 438 Jahng, G.H. , 594 Jeyabalan, G. , 175 Jahr, C.E. , 345 Jeyaseelan, K. , 414 Jain, A. , 573 Jeysen, Z.Y. , 608 Jain, K.K. , 583 Jhaveri, K.A. , 410 Jain, M. , 573 Ji, C. , 373 Jain, R.K. , 491 Ji, S.P. , 524, 525, 530 Jaiswal, A.K. , 443 Ji, X. , 120 Jakob, H. , 78, 604 Jia, H.M. , 80 Jakob, R. , 64, 74 Jia, J. , 439 Jallo, J.I. , 504 Jia, S.H. , 461, 467 Jamil, K. , 47 Jiang, B.H. , 190 Janaky, T. , 606 Jiang, D. , 184, 185, 195, 199 Janero, D.R. , 501, 502 Jiang, G. , 190 Jang, J.H. , 443, 603 Jiang, H. , 169, 506, 509 Janier, M. , 438 Jiang, J. , 184–186, 188, 331, 601 Janmey, P.A. , 353 Jiang, K.W. , 592, 594 Jannier, V. , 170 Jiang, N. , 277 Jannsen, B.J. , 280 Jiang, Q. , 459 Janoff, A. , 4 Jiang, R. , 53, 72–75, 319, 322, 332 Janovska, A. , 442 Jiang, T. , 80 Jantze, H. , 188, 201 Jiang, W. , 80, 524, 525, 530 Januzzi, J.L., J.r. , 76 Jiang, X. , 185, 193, 195, 247, 344, 350, 409, Jardine, K. , 441 410, 412 Jaroshevich, O. , 184, 185, 187, 193 Jiang, Y. , 217, 312 Jarpe, M.B. , 56 Jiao, X. , 46 Jarretou, G. , 151, 152, 155 Jickling, G. , 375 Jarvis, C.R. , 42 Jikuya, T. , 319 Jauch, E. , 375 Jimenez-Mateos, E.M. , 523–529, 531–534, 568 Javadov, S.A. , 63, 443 Jin, B.-K. , 230, 273 Jaworska-Adamu, J. , 524, 527–529 Jin, K.L. , 8, 10, 369, 436, 503, 530, 531 Jay, T.M. , 25 Jin, R. , 458 Jeffrey, R.R. , 76 Jin, X.H. , 586 Jeftinija, K. , 344 Jin, Y. , 353 Jeftinija, S. , 344 Jin, Z.G. , 595 Jegger, D. , 73 Jin, Z.Q. , 74 Jelkmann, W. , 440 Jinka, T.J. , 46 Jenkins, D.P. , 77 Jinka, T.R. , 41, 47 Jenkins, K. , 144 Jneid, H. , 561 Jenkins, L.W. , 510 Jo, N. , 542 Jennings, N.M. , 546 Joh, H.-D. , 231, 235, 275, 461, 470 Jennings, R.B. , 5, 6, 19, 51, 58–61, 64, 165, 243, Johansen, F.F. , 340, 346 259, 505, 553, 568, 591, 601, 602 Johansson, D. , 26, 29, 30 Jensen, F.E. , 459, 474, 475 Johnnides, M.J. , 184, 187, 196, 299 Jensen, H.A. , 11, 562 Johnson, C.A. , 340 Jensen, J.S. , 80, 318 Johnson, G.L. , 56 Jeon, D. , 380 Johnson, M.B. , 523, 525, 528, 532–534 Jeon, H. , 301 Johnson, M.R. , 500 Jeon, Y.T. , 78, 166, 168, 604 Johnson, R.S. , 184, 191 Jeong, J.W. , 440 Johnson, S.B. , 140 644 Author Index

Johnston, K.M. , 583 Kaheinen, P. , 441 Johnston, M.V. , 459, 460, 462, 469–471 Kaidoglou, K. , 184–186, 194 Johnston, S.C. , 559, 560 Kaimaktchiev, V. , 367 Joly, S. , 191, 192 Kain, T.C. , 605 Jonas, E.A. , 433 Kaiser, R.A. , 63, 75 Jonas, S. , 137, 601, 606 Kaku, Y. , 319 Jonassen, A.K. , 57 Kalaycioglu, S. , 78 Jones, A.W. , 464, 467 Kalb, R.G. , 8, 340, 342, 437 Jones, D.L. , 544 Kalb ﬂ eisch, J.H. , 367, 458, 459, 462, 467, 468 Jones, M. , 106 Kalehua, A.N. , 9 Jones, N.M. , 184, 186, 191, 194, 199, 201, Kalil, A.N. , 601 264, 265, 412, 459, 465, 473 Kallenberg, K. , 6, 138, 559, 560 Jones, R.H. , 606 Kallmes, D.F. , 302 Jones, S. , 601, 606 Kalmar, B. , 370 Jones, T.A. , 107, 113 Kalogeris, T.J. , 464, 467 Jones, T.E. , 439 Kalpana, S. , 184, 186, 462, 467, 468 Jones, W.K. , 53, 63, 71 Kaltoft, A.K. , 11, 53, 82, 608 Joo, H. , 437 Kaluza, J. , 459 Jordan, B. , 190, 194 Kamada, H. , 280 Jordan, J.E. , 71 Kamada, T. , 8, 53, 64 Jorgensen, E. , 80, 318 Kamelgard, J. , 61 Jorgensen, H.S. , 4, 118 Kametsu, Y. , 273, 277 Joseph, B. , 171, 172 Kaminski, N.E. , 500, 501 Joshi, R. , 3, 294, 299 Kamiya, T. , 10 Jougla, E. , 166 Kamme, F. , 353 Jourdain, A. , 487–489 Kammersgaard, L.P. , 118, 120 Jovanovic, A. , 60, 435 Kamphuis, W. , 541, 543 Jovanovic, S. , 60, 435 Kan, Y.W. , 299 Jovanovic, V. , 6, 559–561 Kanamaru, K. , 300 Jover, T. , 346, 356 Kanchuger, M. , 569 Joye, E. , 438 Kaneko, T. , 169, 303, 391, 493 Juettler, E. , 10 Kanemitsu, H. , 272 Juhasz, E.D. , 53, 602 Kang, B. , 373 Juhaszova, M. , 64, 394 Kang, H. , 166, 168 Julian, T. , 21 Kang, I. , 443 Julier, K. , 175 Kang, J.X. , 148, 184, 187, 195, 466, 472 Jump, D.B. , 140 Kang, K.M. , 380 Jung, H.H. , 167, 168, 170, 172, 326, 331 Kang, N. , 184, 187, 195, 466, 472 Jung, K.-H. , 185, 191, 380, 465, 474 Kang, Y.J. , 299 Jung, S.N. , 443 Kang, Z. , 173, 174, 491 Jung, W.S. , 594 Kano, T. , 232, 237, 462, 472 Jungehulsing, G.J. , 6 Kanwar, M. , 542 Junier, M.P. , 370 Kanzik, I. , 78 Jurgensen, J.S. , 192 Kao, L.W. , 582–584 Jurgenson, U. , 141, 144 Kao, R.L. , 367, 458, 459, 462, 467, 468 Justicia, C. , 110, 370 Kaoukis, A. , 53, 82, 608 Juvonen, T. , 11 Kapadia, R. , 373 Kapinya, K.J. , 167, 169, 170, 184, 185, 191, 198, 201, 219, 220, 280, 298, 492, 493 K Kaplan, B. , 270, 272 Kaasik, K. , 46 Kaplan, S.S. , 271 Kabir, A.M. , 436 Kappel, A. , 192 Kabir, A.N. , 57 Kappelle, L.J. , 368 Kader, A. , 271, 273 Karabacakoglu, A. , 296 Author Index 645

Karadenizli, Y. , 78 Keedy, A. , 291 Karanian, D.A. , 501, 502 Keenan, S. , 510 Karavolias, G.K. , 57, 83 Keep, R.F. , 173, 214–220, 234, 280, 294, 296, Kareva, T. , 544 299, 309–315, 411, 412, 458–461, Karibe, H. , 118 463, 467, 468 Karikó, K. , 276 Kehl, F. , 170, 171, 438, 492, 493 Karliner, J.S. , 74 Keith, B. , 192 Karmazyn, M. , 64 Keith, R.A. , 328 Karsch, M. , 184, 186, 198, 219 Kelbaek, H. , 80, 318 Karski, J. , 606 Keller, H. , 438 Karwowski, J. , 603, 605, 607 Kelley, J. , 367, 458, 459, 462, 467, 468 Kasaoka, S. , 303 Kelley, M. , 74 Kass, I.S. , 170, 171, 431 Kelly, B.D. , 441 Kassell, N.F. , 270 Kelly, D.A. , 26 Katakam, P. , 390–392 Kelly, D.P. , 439 Katakam, P.V. , 195, 433, 469 Kelly, M.E. , 10, 522–524, 527, 529, 568 Katayama, Y. , 462, 463, 470, 472, 494 Kelly, P.A.T. , 273 Katchanov, J. , 273 Kelly, P.J. , 279, 280 Kathiresan, S. , 76 Kelsey, N.A. , 159 Kato, H. , 7, 8, 243, 244, 347, 352, 399, 461, Kelty, J.D. , 121, 123 464, 465, 471–474 Kemp, M. , 77 Kato, K. , 8 Kempermann, G. , 512 Katoh, H. , 584 Kempski, O. , 213, 220 Katsnelson, M. , 294, 296, 302, 558, 562, 572, Kennedy, C. , 38 604, 609 Kennedy, J. , 251 Katsumata, T. , 463, 470 Kenneth, N.S. , 192 Katsura, K. , 391, 463, 470 Kensler, T.W. , 443 Kattenhorn, M. , 603 Kent, K.C. , 603, 605, 607 Kaufman, R.J. , 435 Kent, T.A. , 280 Kaul, D. , 367 Keogh, B. , 52, 73, 77, 78, 604, 608 Kaur, H. , 505 Keogh, C. , 185, 191, 196, 198 Kaveeshwar, U. , 5 Kerendi, F. , 53, 71, 72, 75, 79, 317, 326 Kavelaars, A. , 198, 436 Kern, T.S. , 541, 542 Kawagoe, J. , 8 Kerr, R.S. , 294 Kawaguchi, K. , 436 Kerstein, J.R. , 493 Kawaguchi, M. , 167, 279 Kertz, B. , 142 Kawahara, K. , 349 Kesaev, S.A. , 583 Kawahara, N. , 222, 260, 275, 276, 348, 366, Keshert, E. , 491 368–370, 372, 373, 437, 459–461, Ketterman, A.J. , 57 467–470 Kettner, N.W. , 592 Kawai, C. , 59 Kety, S.S. , 21, 23 Kawai, K. , 342, 348 Keum, S. , 271 Kawai, N. , 118, 280, 342, 372, 530, 531 Kevin, L.G. , 61, 388, 390 Kawakatsu, H. , 56, 57 Key , L, 373 Kawamata, T. , 279 Keyl, P.M. , 310 Kawano, H. , 280 Khalid, A.M. , 438 Kawano, T. , 280 Khalil, D. , 113 Kawaraguchi, Y. , 167 Khaliulin, I. , 61 Kayama, T. , 112, 531 Khan, M. , 184, 201, 544 Kazim, S. , 69 Khan, S.N. , 82, 544, 604, 608 Ke, Y. , 184, 186, 191, 198, 459, 465 Kharatishvili, I. , 528 Kearn, C.S. , 504 Kharbanda, R.K. , 11, 52, 53, 68, 70, 77, 78, Keating, J. , 280 82, 571, 602–604, 608 Keberle, H. , 219 Khaspekov, L. , 245 646 Author Index

Khazaei, M.R. , 353 Kimura, K. , 6, 236, 579 Kheireddin, A.S. , 296 Kimura, M. , 310 Kholodilov, N. , 544 Kimura, Y. , 174 Khoshbaten, A. , 409, 410, 457, 461, 467, 468 Kin, H. , 53, 72–75, 326 Khot, U.N. , 250 Kindy, M.S. , 10 Khoury, J.C. , 375 King, G.L. , 370 Kida, M. , 59 King, J.S. , 506, 532–534, 568 Kienzle, M.F. , 458 Kingsman, S.M. , 192 Kieran, N.E. , 443 Kinni, H. , 106, 113, 123 Kiessling, M. , 270, 271, 346, 347 Kino, T. , 370 Kietzmann, T. , 297 Kinoshita, M. , 68 Kikuchi, H. , 270, 274, 277 Kinsolving, C.R. , 23 Kilian, S.A. , 56, 57 Kipar, A. , 534 Kim, A.S. , 442, 443 Kipnis, E. , 171 Kim, C. , 272, 278 Kirchner, J.L. , 606 Kim, C.H. , 584 Kiriazis, G. , 184–186, 194 Kim, C.S. , 166, 168 Kirino, T. , 123, 134, 139, 222, 229, 275, 342, Kim, D.H. , 443 348, 366, 368–370, 372, 373, 437, Kim, E. , 245, 247, 436, 437 459–461, 467–470 Kim, E.H. , 465, 474, 605 Kirk, M.A. , 582–584 Kim, E.J. , 436, 443, 602 Kirkeboen, K.A. , 505 Kim, G.T. , 584 Kirkorian, G. , 63, 79, 80, 608 Kim, H. , 108 Kirsch, J.R. , 167, 296, 493, 574 Kim, H.F. , 142 Kirschner, D.L. , 43 Kim, H.I. , 438 Kis, B. , 294, 371, 391, 392, 433, 459, 462, Kim, H.J. , 184, 201, 299, 352, 503 467, 468, 558 Kim, H.S. , 443 Kishi, K. , 62, 433 Kim, J. , 60, 75, 185, 191, 390, 443 Kiss, L. , 46 Kim, J.A. , 352 Kissela, B. , 457 Kim, J.H. , 78, 604 Kitagawa, K. , 6, 8, 138, 236, 243, 244, 259, Kim, J.M. , 465, 474 351, 412, 413, 553, 568, 579 Kim, J.W. , 438, 441 Kitajima, H. , 319 Kim, J.Y. , 415, 416 Kitakaze, M. , 52, 60, 75, 83, 175 Kim, K.N. , 184, 201 Kitano, H. , 167, 168, 176, 280, 493, 557, 558 Kim, K.S. , 438 Kitaoka, T. , 310 Kim, K.W. , 440 Kittner, S. , 457 Kim, M.S. , 185, 191, 380, 465, 474, 584 Kiviluoma, K. , 11 Kim, N. , 437 Kiyama, H. , 544 Kim, S. , 214, 218, 310 Kjoller, C. , 346 Kim, S.-J. , 185, 191, 465, 474, 544 Klahn, S.L. , 41 Kim, S.H. , 64, 440, 441, 605 Klatzo, I. , 274, 276 Kim, S.O. , 57 Klaus, J. , 231, 235, 275, 461, 470 Kim, S.S. , 443 Klaus, J.A. , 231, 235, 278–280 Kim, S.U. , 185, 191 Kleim, J.A. , 113 Kim, S.Y. , 438 Klein, N. , 603 Kim, W.K. , 504 Klein, R.M. , 443 Kim, Y. , 124 Kleinrok, Z. , 201 Kim, Y.I. , 71, 562, 602 Klenk, E. , 144 Kim, Y.J. , 231, 235, 601 Klesse, L.J. , 8, 340, 342, 437 Kimbro, J.R. , 279 Klinger, M. , 440 Kimes, A.S. , 556 Kloc, M. , 64 Kimoto, S. , 601 Kloner, R.A. , 11, 52, 57, 65, 83, 106, 245, 602 Kimura, A. , 118, 119 Kluger, M.J. , 38 Kimura, H. , 174 Klumpp, S. , 370, 431, 436 Author Index 647

Klune, J.R. , 303 Kondo, S. , 501 Knab, R. , 6 Kondo, Y. , 192 Knable, M. , 142 Kondoh, T. , 262 Knerlich, F. , 218 Kondratyev, A. , 524, 525, 527, 529, 531 Knigge, T. , 506 Konishi, N. , 167 Knight, R.A. , 57, 437 Kono, G. , 272 Knoblach, S.M. , 512 Konstantinov, I.E. , 14, 53, 68, 70, 77, 78, 293, Knoblich, J.A. , 353 571, 602, 604 Knochel, J.P. , 124 Konstas, A.A. , 120 Knowles, R.G. , 437 Kooyman, G.L. , 25 Knuckey, N.W. , 184, 194, 195, 201 Korcsmaros, T.B.C. , 505 Knudsen, D.J. , 271 Korematsu, K. , 273 Knuuttila, J. , 530 Koren, L. , 141, 144 Ko, S.Y. , 465, 474 Korf, H.W. , 503 Kobara, M. , 58 Korichneva, I. , 436 Kobayashi, C. , 192 Korolchuk, V.I. , 402 Kobayashi, H. , 437 Korsmeyer, S.J. , 63 Kobayashi, K , 230 Korthuis, R.J. , 5, 464, 467 Kobayashi, S. , 8, 512 Korzhevskii, D.E. , 463, 468, 472 Kocaogullar, Y. , 296 Koshimizu, M. , 58 Koch, C.J. , 491 Kossmann, T. , 511 Koch, J.M. , 585 Kossyvakis, C. , 53, 82, 608 Koch, M. , 503 Kosten, T.A. , 502 Koch, S.E. , 73, 294, 296, 302, 558, 562, 572, Koster, S. , 492 604, 609 Kottenberg, E. , 78, 604 Kochanek, P.M. , 510 Koudstaal, P.J. , 294 Kochkina, E.G. , 184, 185, 200 Kovac, S. , 341 Kocsis, G.F. , 606 Kovacs, I.A. , 505 Kocsis, J.D. , 10 Kovacs-Simon, A. , 606 Kodai, S. , 433 Kowall, N.W. , 473 Kodama, T. , 366, 368–370, 372, 373 Kowaltowski, A.J. , 58, 433 Kodesh, E. , 507, 510 Kowluru, R.A. , 542 Koegel, I. , 531 Kozuka, M. , 230 Koehler, R.C. , 231, 235, 278–280, 431 Kozutsumi, Y. , 435 Koerner, I.P. , 213, 220 Kraemer, M. , 469 Ko ﬂ er, J. , 229 Kraemer, P.J. , 10 Kogure, K. , 7, 8, 244, 272, 347, 352, 399, 465, Kraig, R.P. , 114, 118, 119 472–474 Krajewski, S. , 248 Kohama, S.G. , 274 Kramer, A.F. , 106 Kohen, R. , 235, 500, 503, 509 Kramer, B. , 601, 602 Köhler, H. , 506 Kranias, E.G. , 53, 71 Kohr, G. , 343 Kraus, D.W. , 46 Koizumi, H. , 464 Krause, G.S. , 243, 250 Koizumi, J. , 271 Kraydieh, S. , 10, 270, 272, 312, 505 Kok, J.W. , 38 Kremastinos, D.T. , 57, 436, 601 Kokubo, Y. , 8 Krenz, M. , 60, 62 Kolar, F. , 605 Kreutz, S. , 503 Kollmar, R. , 10, 118 Krey, G. , 438 Kolocassides, K.G. , 83 Krieg, T. , 62, 73, 75, 326 Kolvekar, S.K. , 52, 73, 77, 78, 603, 604, 608 Krieglstein, J. , 247, 370, 431, 436 Komatsu, M. , 401 Kriegstein, A.R. , 494 Kominami, E. , 401 Krige, D. , 192 Kondapalli, J. , 430 Kristensen, S.D. , 11, 53, 82, 608 Kondo, N. , 9 Kristensen, T.A. , 30 648 Author Index

Kristenson, H.J. , 41, 42, 45 Kyto, V. , 441 Kristensson, K. , 141, 143, 144 Kyuwa, S. , 271 Kristiansen, S.B. , 52, 53, 68, 602, 603 Krnjevic, K. , 46, 431 Kroemer, G. , 430 L Kroenke, C.D. , 274 Labrande, C.N. , 170 Kroese, F.G. , 38 Lacerda, L. , 73 Krueger, C. , 367 Lachman, L.B. , 9 Krug, M. , 188, 201 Lackland, D. , 457 Krumnikl, J.J. , 279 Laclau, M.N. , 58 Krumschnabel, G. , 29 Laden, G. , 571, 585, 586 Krupinski, J. , 459 Laderoute, K.R. , 442 Krusell, L.R. , 11, 53, 82, 608 Lafuste, P. , 190, 194 Krýsl, D. , 274 Lagouge, M. , 443 Kubo, T. , 63, 75 Lagreze, W.A. , 175, 542 Kubota, Y. , 541 Lahey, S.J. , 175 Kucinski, T. , 6, 319 Lahteinen, S. , 530 Kudo, I. , 192 Lai, C.C. , 389 Kudo, Y. , 372 Lai, J.C. , 392 Kuhn, R. , 347 Lai, M.K. , 441 Kuhn, T.B. , 40, 46 Lai, P.H. , 594 Kuipers, S.D. Lai, T.W. , 343, 344 Kukreja, R.C. , 72, 505 Lai, Y. , 401 Kulinskii, V.I. , 348 Laigle, C. , 151, 152, 155 Kullar, P. , 77 Lam, A. , 296 Kumar, P. , 459 LaManna, J.C. , 25, 26, 38, 44, 45, 190, 191 Kumar, S. , 57, 367, 459 Lambertsen, C.J. , 580 Kumar, V. , 367 Lambrechts, D. , 190, 194 Kumar, Y. , 293 Lamers, J.M. , 605 Kume, M. , 601 Lamming, D.W. , 441 Kumral, A. , 219 Lamy, C. , 558 Kunimoto, M. , 192 Lan, J.Q. , 8–11, 189, 231, 235, 344, 348, 368, Kunis, D.M. , 531 380, 381, 438, 530–532, 535 Kuno, A. , 74 Land, J.M. , 437 Kuntscher, M.V. , 245 Landreth, G.E. , 406 Kunz, A. , 261, 462, 467–471 Landucci, E. , 322, 326, 330, 345, 347 Kuppusamy, P. , 544 Lang, F. , 435 Kuramoto, K. , 319 Lange, C. , 191, 192 Kuraoka, M. , 271 Lange, M. , 406, 438 Kuroiwa, T. , 270 Lange, P.S. , 190, 297 Kuroki, M. , 300 Lange-Asschenfeldt, C. , 245, 246, 248, 347, Kurtz, C.C. , 41 349, 436, 601 Kusnierz-Cabala, B. , 313 Langeberg, L.K. , 353 Kutty, G. , 545 Langham, J. , 572 Kutty, R.K. , 545 Langley, B. , 190, 297 Kutuk, O. , 511 Langlois, J.B. , 438 Kuukasjarvi, P. , 568, 570, 571 Langridge, J. , 189, 381, 530, 535 Kuwabara, K. , 8, 351 Lankila, P. , 188, 198, 201 Kuwagata, M. , 541 Lanneau, D. , 402 Kuzuya, T. , 53, 64 Lanz, S. , 192 Kwan, A.L. , 293, 298, 299 Lao, N. , 174, 491, 584 Kwan, M. , 574 Laprais, M. , 463, 467, 470, 471, 474 Kwong, K.K. , 592 Laragione, T. , 219 Kyrzopoulos, S. , 83 Larsen, H.K. , 30 Author Index 649

Larsen, T.S. , 438 Lecour, S. , 52, 73, 83 Larson, E.B. , 106 Lee, A.W. , 541, 542 Laser, M. , 545 Lee, B. , 397 Laskey, W.K. , 80 Lee, B.I. , 605 Lasley, R.D. , 61 Lee, B.Y. , 581 Lassen, J.F. , 11, 53, 82, 608 Lee, C. , 443 Lassen, N.A. , 23, 24 Lee, C.C. , 46 Lassonde, M. , 583 Lee, C.D. , 105 Latchman, D.S. , 53, 57, 64, 367 Lee, C.M. , 328 Lathrop, D.A. , 83 Lee, E.M. , 412 Laties, A.M. , 543, 545 Lee, G. , 74 Latini, S. , 117 Lee, H.C. , 190, 201 Lattermann, R. , 280 Lee, H.M. , 436 Lau, A. , 340 Lee, H.S. , 299 Lau, D. , 343 Lee, H-T. , 232, 237, 262, 412, 413, 458, 462, Laude, K. , 468, 470 472, 544 Laudenbach, V. , 184, 185, 197, 199, 201, 265 Lee, J. , 167, 168, 170, 172 Laudizi, L. , 297 Lee, J.A. , 396 Launay, J.-M. , 175 Lee, J.C. , 57 Laurer, H.L. , 511 Lee, J.D. , 56, 57 Laurikka, J. , 570 Lee, J.E. , 112, 367 Lauritzen, I. , 8, 117, 138, 141, 143, 148–150, Lee, J.H. , 299 155, 348, 529 Lee, J.J. , 326, 331, 535 Lauritzen, M. , 280 Lee, J.S. , 601 Lauterborn, J.C. , 512 Lee, J.W. , 294 Lavail, M.M. , 194 Lee, K.H. , 542, 543 Lavine, S.D. , 294, 574 Lee, K.J. , 440 Lawrence, D. , 78 Lee, K.M , 231, 235 Lawrence, M.S. , 370 Lee, K.R. , 214, 218, 310 Laws, E.R. , 488 Lee, K.S. , 10, 270 Lawson, C.S. , 6, 64 Lee, L.Y. , 190, 201 Lawson, D.D. , 580 Lee, M. , 45, 184, 201 Laxenaire, M.C. , 166 Lee, N.T. , 438 Lazarevich, E.V. , 184, 185 Lee, P.J. , 443 Lazarewicz, J.W. , 184, 185, 194, 347, 351 Lee, R.M. , 437 Lazariotto, M. , 406, 438 Lee, S. , 487–490 Lazdunski, M. , 8, 10, 117, 138, 141, 143, Lee, S.-T. , 185, 191 147–152, 154, 155, 348, 525, Lee, S.H. , 231, 235 529–531, 546 Lee, S.K. , 185, 191, 380, 465, 474 Lazou, A. , 57, 112, 601 Lee, S.R. , 110, 192 Le, Y.Z. , 191, 192 Lee, S.T. , 380, 414, 465, 474 Le Gal La Salle, G. , 527, 529, 530 Lee, S.Y. , 436 Leach, R.M. , 581 Lee, T. , 541 Leaf, A. , 148 Lee, W.D. , 544 Leaf, J. , 71 Lee, W.H. , 190, 195 Leanza, G. , 299 Lee, Y. , 396 Leasure, J.L. , 106, 113 Lee, Y.-J. , 45, 185, 191, 200, 544 Leavitt, B.J. , 175 Lee-Lewandrowski, E. , 76 Leber, K. , 279 Lees, K.R. , 136, 591 Leblond, J. , 431 Lefer, D.J. , 438 Lebuffe, G. , 63, 235, 322, 331 Legos, J.J. , 5, 368 Lechler, E. , 38 Lehman, J.J. , 439 Lecomte, J.M. , 583 Lehmann, K.A. , 221 Leconte, C. , 184, 199, 203, 323 Lehmann, S. , 367 650 Author Index

Lehnardt, S. , 367 Lewen, A. , 8, 328 Lehning, E.J. , 431 Lewis, B.P. , 412 Lehot, J.J. , 64 Lewis, M. , 142 Lehotsky, J. , 399, 404, 405 Ley, K.F. , 299 Lei, B. , 170, 171 Leys, D. , 4, 9 Lei, D. , 185, 201 Leza, J.C. , 234 Lei, Y. , 594 Lezoualc’h, F. , 436 Leibowitz, R.T. , 10 Lhermitte, M. , 4, 9, 235 Leiphart, J.L. , 532 L’Huillier, I. , 52, 53, 80, 318, 608 Leist, M. , 251 Li, B. , 5, 79, 349, 541–543, 545, 546, 586 Leite, J.P. , 532 Li, C.Q. , 61, 80, 367, 401, 436, 458, 459, 462, Leker, R.R. , 370, 499, 503, 504 467, 468 Lekic, T. , 298, 487, 489 Li, D. , 175 LeMay, D.R. , 38 Li, F. , 173, 174, 272, 297, 332 Lemieux, H. , 190, 194 Li, G.C. , 6, 458, 558 Lenhard, S.C. , 278 Li, H. , 194, 397 Lennmyr, F. , 373 Li, J.L. , 14, 53, 68, 70, 71, 77, 78, 106, Lenz, F.A. , 504 108–110, 120, 167, 173, 174, Lenzlinger, P.M. , 511 184–188, 193, 197, 199, 299, 397, Lenzser, G. , 392, 459, 462, 467, 468 406, 441–443, 458–461, 467, 468, Leonard, M.O. , 443 471, 472, 544, 602, 604 Leonard, S.S. , 438 Li, L. , 74, 167, 169, 172, 187, 190, 221, 298, Leone, T.C. , 439 326, 331, 353, 595, 601 Leone-Kabler, S.L. , 28 Li, M. , 159, 189, 343, 381, 530, 535 Leong, S. , 143 Li, M.-M. , 184, 186 Lepola, P. , 11 Li, M.H. , 603 Leprivier, G. , 442 Li, N. , 415, 416 Lere, C. , 527, 529, 530 Li, P. , 171, 415, 439, 460, 464, 466, 468, 474 Lerin, C. , 441 Li, Q. , 63, 174, 299, 542–545 Lerner-Marmarosh, N. , 56, 57 Li, Q.F. , 169, 411, 412 Leroux, P. , 184, 185, 197, 199, 201 Li, Q.J. , 466, 474 Lesage, F. , 155 Li, R.C. , 57, 63 Lesperance, F. , 142 Li, S. , 53, 71, 332 Lesperance, P. , 142 Li, S.Q. , 466, 474 Lesser, J.R. , 81, 318 Li, S.R. , 294 Lessov, N.S. , 5, 41, 189, 274, 313, 343, 364, Li, T. , 529 366, 367, 370–372, 374, 375, 463, Li, W. , 174, 184, 349, 415, 416 467, 474, 527, 532 Li, W.B. , 466, 474 Lester, R.A. , 345 Li, W.M. , 80 Leung, A.W. , 75 Li, X. , 111, 171, 402, 595 Leung, M.C. , 594 Li, Y. , 53, 71, 123, 147, 171, 277, 443, 491, Leung, P.Y. , 10, 189, 202, 366, 368, 545, 585 373–377, 467 Li, Y.-X. , 186, 198 Levant, G. , 608 Li, Y.J. , 68–70 Levi, E. , 436 Li, Y.P. , 173, 411 Levijoki, J. , 441 Li, Y.X. , 411, 412 Levin, S.G. , 184, 186, 193, 195 Li, Y.Z. , 562, 604, 609 Levine, S.R. , 123 Li, Z. , 173, 594 Levy, D.E. , 229 Lian, Q. , 173, 491 Levy, M.L. , 294, 574 Liang, F.X. , 592 Levy, R.B. , 57 Liang, H.L. , 395 Lewandowski, E.D. , 439 Liang, H.W. , 322, 325, 464, 470 Lewandrowski, K. , 76 Liang, J. , 185, 186, 188 Lewczuk, P. , 219, 510 Liang, W. , 171, 460, 464, 466, 468, 474 Author Index 651

Liao, J. , 319, 328, 330, 331 Lindsey, K. , 545 Liao, J.-F. , 184, 185, 193, 194 Ling, F. , 120 Liao, J.R. , 594 Lingle, D.M. , 61 Liao, M. , 353 Link, A.J. , 535 Liao, S. , 53, 65, 71 Linseman, D.A. , 159 Libal, N.L. , 168, 280 Liou, G.I. , 544 Liberman, M.C. , 5 Liou, J.Y. , 439 Licata, G. , 222 Lipp, P. , 434 Lichtenwalner, R.J. , 369 Lipsky, R.H. , 530, 531 Liebelt, B. , 106, 112, 123, 399, 416 Lipton, P. , 21, 24, 25, 42, 43, 431 Lieberman, A.P. , 9 Lipton, S.A. , 410, 544 Liem, D.A. , 69 Liron, T. , 436 Lienhart, A. , 166 Lisabeth, L. , 457 Ligumsky, M. , 500, 501 Lisalo, P. , 570 Li-Jun, Z. , 174, 274 Lissandron, V. , 404, 405 Likosky, D.S. , 175 Lit, L. , 302 Lilly, G. , 184, 186, 462, 467, 468 Little, S.P. , 436 Lim, A.L. , 441 Liu, B. , 353 Lim, E. , 573, 604 Liu, C.L. , 114, 117, 123, 346, 353, 400, 403, Lim, J. , 583 543, 545 Lim, K.H. , 63, 443 Liu, D. , 375, 443, 494 Lim, K.L. , 403 Liu, F. , 344 Lim, K.Y. , 414 Liu, G.S. , 55, 57, 62, 63, 72, 443 Lim, S.C. , 184, 194, 195 Liu, H. , 368 Lim, S.W. , 166, 168 Liu, J. , 9, 10, 78, 111, 112, 184, 187, 297, Lim, S.Y. , 75, 388, 393 367, 437, 466, 474, 500, 531, 583, Lim, T.W. , 78, 172, 604 585, 592 Lim, W.K. , 583 Liu, J.R. , 6 Lim, Y.J. , 78, 213, 221, 604 Liu, J.S. , 8 Limatola, V. , 172, 411, 493, 557 Liu, K. , 173, 531 Lin, A.M.Y. , 184, 185, 190, 193, 194, 201 Liu, L. , 194, 343, 344, 354 Lin, B. , 243 Liu, M. , 53, 71, 411, 412, 459, 466, 473 Lin, C.H. , 412, 413 Liu, P. , 78 Lin, C.L. , 293, 298, 299 Liu, P.K. , 248 Lin, C.-S. , 230, 273 Liu, P.P. , 14, 68, 70 Lin, G. , 79 Liu, S.H. , 80, 81, 595 Lin, H.W. , 347, 436, 602 Liu, T. , 9 Lin, H.-Y. , 263, 264, 462, 467–469 Liu, W. , 173, 216, 217, 294, 311 Lin, J. , 192, 439, 465, 473, 546 Liu, X. , 118, 169, 277, 319, 322, 332, 391, Lin, J.G. , 592 459, 461, 469, 470, 595, 601 Lin, J.H.C. , 184, 187, 194, 195, 261, 397, Liu, X.H. , 8 466, 472 Liu, X.L. , 80 Lin, L.C. , 441 Liu, X.M. , 433 Lin, M.T. , 123 Liu, Y. , 7, 8, 55, 56, 62, 74, 80, 81, 167, 168, Lin, S.J. , 251 170, 173, 174, 185, 186, 188, 244, Lin, T.N. , 8, 439 248, 274, 343, 344, 352, 354, 390, Lin, W.Y. , 261, 262 392, 435, 436, 491–493, 586, 592, Lin, X.M. , 80 593, 595 Lin, Y. , 346 Liu, Z. , 173, 297 Lincoln, T.M. , 74, 436, 437 Liu, Z.-H. , 184, 186 Lindell, S.L. , 41 Liu, Z.L. , 154, 156 Linden, M.D. , 313 Liverman, C.S. , 443 Lindsay, K.W. , 572 Lizasoain, I. , 234, 376, 466, 473, 500 Lindsberg, P.J. , 409 Ljunggren, B. , 244 652 Author Index

Lliff, B. , 46 Lowry, T. , 523, 524, 527, 529 Lloyd, P.G. , 110 Lu, A. , 10, 189, 192, 302, 310, 366, 466, 474 Lloyd-Jones, D. , 135, 457 Lu, B. , 595 Lo, E.H. , 109, 110, 136, 137, 139, 226, 247, Lu, C. , 443 473, 474 Lu, D. , 512 Lo, R.T. , 295 Lu, G.-W. , 185–187, 193–195, 197, 199, Lo, S,C. , 594 260, 348 Lo, W. , 487, 489 Lu, H. , 510 Lobner, D. , 347, 349, 351 Lu, L. , 343 Lochner, A. , 57, 506 Lu, M. , 175 Lodge, D. , 7, 345, 351 Lu, N. , 544 Lodge, N.J. , 6, 81, 433 Lu, Q. , 544 Loeffert, J.E. , 8, 369, 530, 531 Lu, R. , 70 Loeschcke, H.H. , 580 Lu, S. , 171, 274, 409, 460, 464, 466, 468, 474 Loewenson, R.B. , 582, 587 Lu, T.J. , 342, 343, 413 Lof ﬂ er, M. , 62 Lu, W.Y. , 193, 343, 349, 351, 354 Lofman, C.O. , 29 Lu, Y. , 593, 595 Lofye, M.T. , 72 Lu, Z.H. , 190, 493, 591, 592, 594–596 Logan, J.G. , 22 Luan, R. , 601 Lohse, L.A. , 38 Luan, X.D. , 106, 110, 118, 120, 458, 460, Lolait, S.J. , 500 461, 467 Lonborg, J. , 80, 318 Lucas, D.R. , 339 London, R.E. , 60 Lucchesi, B.R. , 6 Long, G.G. , 340 Lucchinetti, E. , 73 Longa, E.Z. , 227 Luckl, J. , 280 Longatti, P. , 299 Lucus, P. , 581 Loor, G. , 430 Luders, H.O. , 524, 525, 527, 529 Loor, Y. , 124 Ludman, A.J. , 77, 78, 83 LoPachin, R.M. , 431 Ludolph, A.C. , 348 Lopez, B.L. , 57 Ludwig, L.M. , 493 Lorbar, M. , 68 Ludwig, P.S. , 279, 493 Lorenz, J. , 53, 71 Luerding, R. , 469 Lorenzo, P. , 234, 466, 473 Lui, W.Y. , 584 Losano, G. , 72, 74, 76 Lukasiuk, K. , 528 Lottermann, A. , 523, 524, 527 Lukic, V. , 10 Lotz, C. , 406, 438 Lund, T. , 438 Lou, N. , 184, 187, 195, 466, 472 Lundberg, A.M. , 531 Louden, C.S. , 57 Lundby, C. , 187, 202 Loufouat, J. , 64, 75 Lundin JI, Checkoway H , 4 Louhelainen, M. , 441 Lundy, E.F. , 38 Loukogeorgakis, S.P. , 11, 562, 603 Luo, B.Y. , 322, 325, 464, 470 Lounis, B. , 340 Luo, H. , 173, 174, 399 Loupatatzis, C. , 435 Luo, M. , 80 Louzy, M. , 175 Luo, Q. , 322, 331, 601 Lovatt, D. , 184, 187, 195, 466, 472 Luo, T.F. , 399 Lovell, M.B. , 605 Luo, W. , 79 Lovett-Barr, M.R. , 185, 201 Luo, Y. , 120, 171, 172, 263, 415, 416, 439 Low, E. , 583 Luo, Z.J. , 231, 235, 604, 609 Lowenstein, D.H. , 10, 521 Lupu, F. , 190, 194 Lower, C. , 47 Lurton, D. , 524, 530 Lowicka, E. , 174 Lusardi, T.A. , 231, 235, 342, 348, 350, Löwl, D. , 167, 169, 170, 184, 185, 191, 198, 352–354, 380, 414, 415, 438 201, 219, 220, 280, 298, 492, 493 Lust, R. , 74 Lowry, O. , 24 Lutcavage, M.E. , 25 Author Index 653

Lutz, B. , 502 Magistretti, P.J. , 27 Lutz, P.L. , 19, 25–28, 30, 44, 248, 347 Mahadik, S.P. , 144 Lv, F.L. , 367 Maher, E.R. , 440 Lv, S. , 173 Mahfouz, M. , 146 Lyden, P.D. , 214, 222, 591 Mahmood, A. , 512 Lysko, P.G. , 9 Mai, K.H. , 443 Lythgoe, M.F. , 367 Mai, M. , 4 Lytle, B.W. , 76 Mai, W. , 443 Lyuboslavsky, P. , 220 Maidatsi, P. , 184–186, 194 Maier, C.M. , 118 Mailer, K. , 64 M Maiti, S. , 352, 353 Ma, D. , 171, 172, 184, 185, 202, 493 Majda, B.T. , 201 Ma, G.Q. , 392, 586 Majewski, N. , 443 Ma, J. , 184, 194, 195, 270–272, 367, 458, 459, Majid, A. , 271 462, 466–468, 473, 474 Majmundar, A.J. , 190, 192, 194 Ma, J.-X. , 186, 191, 200, 545 Makanji, S.S. , 501 Ma, J.F. , 6 Makela, T. , 11 Ma, L. , 595 Makita, K. , 169, 493 Ma, M.-C. , 185, 191, 200, 544 Makris, N. , 592 Ma, Q. , 279 Makriyannis, A. , 501, 502 Ma, R. , 167, 169 Maldonado, R. , 503 Ma, X.L. , 57, 80 Maleeff, B. , 57 Ma, Y.L. , 38, 44, 45 Malhotra, A.D. , 184–186 Ma, Z.-M. , 184, 186, 195 Malhotra, S. , 250 Maboudou, P. , 4, 9, 235 Malik, M. , 458 Mabuchi, T. , 109, 351, 412, 413 Malkoff, M. , 323 MacAllister, R.J. , 11, 52, 77, 78, 603, 604, 608 Mallard, C. , 189, 197, 222, 459, 460, 462, MacArthur, P.H. , 438 469–471 MacCarthy, P.A. , 569 Mallolas, J. , 466, 473 MacDonald, E. , 291 Malmqvist, K.G. , 431 MacDonald, J.F. , 343, 349, 351, 354 Maloyan, A. , 507, 509, 510 Macdonald, R.L. , 291, 295, 301, 457 Maly, J. , 556 Maceren, R.G. , 9, 185, 188, 437, 462, 468, 469 Man, S. , 458 MacFarlane, P.M. , 185, 201 Manabe, T. , 344 MacGillivray, T.E. , 76 Manalo, D.J. , 510 Machiyama, Y. , 21 Mancardi, D. , 72, 74, 76 Macia, C. , 63, 608 Mandelbaum, A. , 500, 501 Macia, J.C. , 79, 80, 608 Mandemakers, W. , 395 Mackay, K. , 57 Mandriota, S. , 192 MacKenzie, E.T. , 184, 185, 189, 191, Mangano, C.M. , 569 197–199, 202, 271, 510 Mangino, M.J. , 41 Mackie, K. , 504 Mani-Babu, S. , 64 Macleod, M.R. , 59, 134, 136 Manieri, M. , 439 Madan, A. , 185, 193, 195 Manintveld, O.C. , 605 Madden, L. , 585 Mann, G.E. , 63, 299 Maddock, H.L. , 63, 75 Mannaioni, G. , 220 Maderdrut, J.L. , 272 Manning, B.D. , 544 Madsen-Bouterse, S.A. , 542 Mano, M. , 442 Maeda, H. , 64 Mans ﬁ eld, C.M. , 171 Maeda, N. , 439 Mansur, K. , 544 Maehara, K. , 605 Manta, S. , 157 Magalhaes H , 37 Mantz, J. , 170 Magarinos, A.M. , 352 Mao, M. , 187, 190 654 Author Index

Mao, X. , 5 Martin, S.L. , 37 Mao, X.O. , 10 Martin R.D. , 487–489 Mao, X.R. , 189, 196, 201 Martin Villalba, A. , 409 Mao, Z.Y. , 154, 156, 441 Martinez, J.M. , 142 Maraire, N. , 271, 273 Martinez-Lara, E. , 190 Marais, E. , 57 Martinez-Romero, R. , 190 Marangell, L.B. , 142 Martino G, Pluchino S , 10 Marangos, P. , 270 Martins, E. , 431 Marban, E. , 60, 62 Martins, J.G. , 142 Marban, E . , 433 Martins, W.C. , 527 Marber, M.S. , 53, 56, 57, 63, 64, 436 Martone, M.E. , 346 Marcel, Y.L. , 144 Maruki, Y. , 431 Marcet, M.M. , 542, 544, 546 Marunouchi, T. , 9 Marchal, G. , 581 Maruoka, N. , 184, 185, 194 Marchi, A.G. , 297 Maruyama, Y. , 605 Marchuk, D.A. , 271 Marx, W.F. , 302 Marciano-Cabral, F. , 504 Marxsen, J.H. , 440 Marcon, M. , 606 Masada, T. , 109, 112, 215, 234, 313, 459, 463, Marcos, D. , 411 467, 468 Marcos, H.J.A. , 542 Mascagni, P. , 232, 235 Marcoux, F.W. , 274 Mascareno, E. , 57 Marek, P. , 271 Mascaro, J.G. , 78, 604, 608 Marelli, A. , 457 Mash, D. , 27 Mareš, J. , 274 Mashiko, T. , 299, 346, 433 Marescaux, C. , 523–525, 527–530 Masino, S.A. , 46 Maricq, H. , 270, 319 Masmejean, F.M. , 170 Marie, C. , 185, 187, 194, 200, 463, 468 Mason, R. , 502 Marini, A.M. , 8, 523, 530, 531 Masri, L.S. , 294, 574 Marinkovic, I. , 278 Massa, S.M. , 4 Mark, D.B. , 606 Mastour, N. , 465, 472 Markarian, G. , 524, 525, 527, 529 Masuzawa, T. , 372 Markgraf, C.G. , 270 Mata, M. , 23 Markus, R. , 541 Matchett, G.A. , 314, 486–489 Marone, L.K. , 175 Mateo, P. , 439 Marret, S. , 184, 185, 197, 199, 201 Mathern, G.W. , 532 Marsala, J. , 327 Mathews, E. , 544 Marsh, B.J. , 10, 189, 366–368, 373–375, 467 Matiasova, M. , 323, 326 Marshall, C.J. , 57 Matějovská, I. , 274 Marshall, J.C. , 461, 467 Matoba, S. , 58 Marsicano, G. , 502 Matrone, C. , 186, 191, 192 Martel, M.A. , 354 Matsuda, L.A. , 500 Marti, H.H. , 184, 185, 191, 197–199, 202, Matsuda, N. , 396 218, 510 Matsui, T. , 439, 441 Martignetti, V. , 437 Matsuki, N. , 352 Martin, A. , 4, 9, 147 Matsumori, Y. , 112, 531 Martin, B.J. , 65, 68 Matsumoto, K. , 473 Martin, B.R. , 500–502, 504 Matsumoto, M. , 6, 8, 138, 236, 348, 553, Martin, D. , 3, 9, 367, 527 568, 579 Martin, E. , 279 Matsumoto, T. , 58 Martin, H. , 543 Matsunaga, M. , 436 Martin, J.L. , 27, 56, 57 Matsushima, K. , 7, 274, 275, 277 Martin, L.J. , 229, 247 Matsushima, S. , 524–529, 532 Martin, M.J. , 442 Matsuwaki, T. , 271 Martin, R.D. , 298, 314, 487, 489 Mattern, R. , 545 Author Index 655

Mattson, M.P. , 10, 391, 401, 430, 443 McIntosh, T.K. , 511 Matus, B. , 173, 487, 490, 491, 495 McIntyre, D.C. , 10, 522–524, 527–529 Matus, S. , 397 McKay, L.I. , 247 Matz, P.G. , 299 McKhann, G.M. , 606 Mauger, D. , 527 McKleroy, W. , 184, 187, 202 Maulik, N. , 6, 56–58 McLaughlin, B. , 5, 138, 368 Maulucci-Gedde, M. , 494 McLean, J. , 352 Maurer, M. , 167, 169, 170, 280, 298, 492, 493 McLeod, C.J. , 395, 438 Maxwell, K.M. , 5, 541–543, 546 Mcllwain, H. , 23 Maxwell, P.H. , 190, 192, 194, 440, 491 Mclntyne, D.C. , 568 May, J.D. , 582–584 McMahan, B.K. , 185, 188, 542–544 May, M.J. , 436 McMahon, S. , 9 Mayanagi, K. , 391, 392, 433 McMullen, D.C. , 45 Mayer, M.L. , 24 McMurtrey, R.J. , 167, 169 Mayhan, W.G. , 175 McNamara, J.O. , 529 Maynard, K.I. , 279 McQuillen, P.S. , 185, 193, 195 Maysami, S. , 11 Meade, C.A. , 273 Mayser, H. , 185, 191, 201 Meadows, J.J. , 435 Mazarakis, N.D. , 192 Mean, R. , 280 Maze, M. , 171, 172, 184, 185, 202, 493 Mechirova, E. , 323, 326 Mazzone, M. , 190, 194 Mechoulam, R. , 500–504 Mc Kendrick, M.W. , 220 Medellin, G. , 59 McAdoo, D.J. , 494 Meden, P. , 272 McAdoo, J. , 172, 173 Medhi, B. , 3 McAllister, A.C. , 271 Medja, F. , 184, 185, 197, 199, 201 McAllister, A.M. , 493 Meduru, S. , 544 McAllister, J.P. , 458, 460, 461, 467 Meemann, T. , 604 McAuliffe, J.J. , 171, 172, 263 Meesmann, W. , 601, 602 McBurney, M.W. , 441, 443 Megow, D. , 184, 186, 198, 219, 273 McCalden, T. , 279 Megyesi, J.F. , 301 McCarron, R.M. , 504 Mehler, M.F. , 534 McCarter, S.D. , 605 Mehta, S.L. , 439 McCarthy, T.L. , 373 Meigs, J. , 457 McClanahan, T. , 65, 68 Meijer, A.J. , 433 McCloskey, D.P. , 113 Meijer, L. , 369 McColl, B.W. , 273 Meiri, U. , 507 McCollum, P.T. , 585 Meisel, A. , 184–186, 191, 195, 198, 201, 273, McCrone, P. , 142 293, 363, 367, 391, 407, 553, 561, McCrossan, Z.A. , 391 567, 602, 606 McCulloch, J. , 228, 270, 273 Meisel, C. , 273 McCullough, J.R. , 433 Melck, D. , 500 McCullough, L.D. , 187 Meldrum, B.S. , 340, 522 McDermott, M.F. , 544 Mele, E. , 437 McDermott, M.M. , 457 Melino, G. , 437 McDonald, E. , 4 Melis, M. , 502 McDonald, M.C. , 406 Meller, R. , 5, 9, 41, 189, 231, 235, 260, 295, McDonald, T. , 62, 435 319, 322, 325–327, 330, 342, 350, McDonnell, P.C. , 9 352–354, 364, 366, 368, 370–372, McEwen, B.S. , 352 400, 403, 404, 412, 413, 523–525, McFalls, E.O. , 605 527–529, 531–534 McFarlane, C. , 493 Mellergard, P. , 120 McFarlin, B.K. , 111 Mello, M.M. , 485 McInerney, J. , 442 Meloni, B.P. , 184, 194, 195, 201 McIntosh, C.H. , 544 Meltzer, J. , 175 656 Author Index

Melvin, L.S. , 500 Miletich, D. , 493 Memezawa, H. , 272 Milite, D. , 319 Menasche, P. , 175 Millen, J.E. , 25 Mendonca, B.P. , 494 Miller, A.J. , 9, 340, 343 Meng, F. , 170, 171 Miller, B.A. , 184, 185, 201 Meng, P.Y. , 595 Miller, C.C. , 297, 491 Meng, R. , 443 Miller, E.J. , 442, 443 Meng, X. , 393 Miller, W.H. Jr , 465, 473 Menn, B. , 369 Millhorn, D.E. , 193 Mennerick, S. , 344, 350 Milligan, S.A. , 436 Mennini, T. , 219 Millili, J. , 61 Mentzer, R.M., Jr. , 52, 61, 83 Milne, J.C. , 443 Mentzer R, Jr. , 83 Milner, E. , 188, 201, 292, 293, 297, 300, 463, Menzies, F.M. , 402 468, 469, 471, 572 Merasto, S. , 441 Milsom, W.K. , 46 Meratan, A.A. , 298 Milton, S.L. , 28, 44 Mercado, M.L. , 137 Milyakova, E.A. , 184, 185 Mercantonio, S. , 279 Min, Y. , 595 Merino, V.F. , 72 Minakina, L.N. , 348 Merkus, D. , 605 Minami, M. , 9–11, 313, 367, 368, 375, 412, Merritt, C.R. , 23 413, 463, 467, 474, 527, 531 Mertens, E. , 175 Minamino, T. , 60, 75 Mervaala, E. , 441 Minamiyama, Y. , 433 Meschia, J.F. , 557, 558, 561 Minassi, A. , 502 Messer, Z. , 192 Minners, J.O. , 438 Messi, G. , 297 Minnerup, J. , 137 Messing, R.O. , 10, 42 Mint, J.J. , 78, 604 Mestril, R. , 8 Mio, Y. , 601 Methy, D. , 463, 468 Miranda, L.F. , 184, 191, 411 Metzen, E. , 440 Miresmaeili, S.M. , 299 Mewton, N. , 63, 608 Mishima, T. , 431, 435 Meyer, F.B. , 293 Mishima, Y. , 232, 237 Meyer, R. , 296 Mitchell, G.S. , 185, 201 Meyermann, R. , 545 Mitchell, H.M. , 114, 118, 119 Mezt, T.E. , 65, 68 Mitchell, M.B. , 393 Miao, B. , 342 Mitsui, T. , 319 Miao, Y. , 322, 331, 601 Mitsunami, K. , 68 Michael, A. , 57 Mittelbronn, M. , 545 Michael-Titus, A.T. , 148 Miura, I. , 59 Michalkiewicz, M. , 530 Miura, T. , 56, 443 Michalkiewicz, T. , 530 Miwa, A. , 544 Michel, M.C. , 56, 57 Miwa, H. , 344 Michelangeli, F. , 404 Miyagawa, Y. , 232, 237 Michelson, A.D. , 313 Miyake, S. , 45 Michenfelder, J.D. , 22 Miyamoto, E. , 230 Mickel, M. , 62 Miyamoto, O. , 346 Mielke, J.G. , 353 Miyaoka, H. , 436 Mielke, K. , 525, 530 Miyashita, K. , 8, 235 Mies, G. , 276 Miyawaki, T. , 299, 433 Miki, T. , 56, 62, 443 Miyazaki, B. , 271 Mikoshiba, K. , 6, 236, 579 Miyazawa, T. , 118, 299, 491, 584 Mikuni, N. , 524, 525, 527, 529 Mizukami, Y. , 172, 173 Milani, H. , 157 Mizunoya, W. , 370 Miles, L. , 263 Mocanu, M.M. , 52, 56, 57, 73, 74, 83, 441–443 Author Index 657

Mocchetti, I. , 525, 530–532 Morioka, M. , 187, 230, 462, 468, 469 Mochly-Rosen, D. , 27, 57, 331, 349, 436, Morioka, T. , 9 437, 494 Morisaki, K. , 118 Mockridge, J.W. , 56, 57, 63 Morita, K. , 42 Modo, M. , 299 Moriwaki, G. , 184–186 Moesgaard, B. , 501 Morley, P. , 154, 342 Moffat, C. , 441 Moro, M.A. , 234, 376, 466, 473 Moffett, J.R. , 353 Moroni, F. , 232, 235, 322, 326, 330, 345, 347 Mohammad, G. , 542 Morooka, S. , 603, 607 Mohrhauer, H. , 144 Morrice, N. , 353 Mok, V.C. , 572 Morris, M.C. , 141, 144 Mokrushin, A.A. , 184, 185 Morrison, J. , 46 Molkentin, J.D. , 63, 75 Morrison, M. , 46 Moller, K. , 187, 202 Mortelmans, L. , 190, 194 Molnar, C. , 42 Mortensen, U.M. , 52, 603 Molnar, E. , 606 Mosca, S.M. , 561 Molyneux, A.J. , 294 Moskowitz, M.A. , 11, 63, 136, 139, 231, 234, Monaghan, D.T. , 24 235, 247, 278, 437, 461, 468, 469 Monassier, J.P. , 63, 608 Mossiat, C. , 463, 468 Moncada, S. , 437, 438 Mostafa, K. , 463, 467, 470, 471, 474 Moncayo, J. , 6, 531, 559, 560 Mostafa, M. , 544 Mondola, P. , 437 Mostoslavsky, R. , 441 Moneta, D. , 530 Motohashi, O. , 214 Monette, R. , 342, 348 Motsch, J. , 279 Monick, M.M. , 56, 60 Moubarik, C. , 170, 171 Monsein, L.H. , 431 Moucharra ﬁ e, S. , 438 Montgomery, D.L. , 583 Mounayer, C. , 296 Monyer, H. , 8, 273, 349, 350 Mouri, G. , 523, 525, 528, 531–534 Moolman, J.A. , 57 Movsesyan, V.A. , 512 Moon, S.K. , 594 Mu, D. , 185, 187, 190, 193, 195, 411, 412 Moons, L. , 190, 194, 491 Mu, J. , 442 Moore, E.M. , 120 Mudalgiri, N.R. , 73 Moore, J. , 68–70 Mukasa, A. , 366, 368–370, 372, 373 Moore, J.T. , 47 Mukherjee, S. , 144 Moore, J.W. , 192 Mukuda, T. , 370 Moore, L. , 81, 318 Mule, F. , 530 Moore, S.A. , 140 Mullen, M. , 603 Morais, V.A. , 395 Mullenheim, J. , 604 Morales, C. , 75 Muller, B. , 6 Morales, J.R. , 466, 473 Mullins, P.G. , 278 Morawetz, R.B. , 274 Mumford, P.L. , 348 Moreira, P.I. , 195 Mundy, C.R. , 192 Moret, J. , 296 Munk, K. , 11, 53, 82, 608 Morgan, J.P. , 56 Munns, S.E. , 184, 194, 195 Morganti-Kossmann, M.C. , 511 Munoz, J.R. , 11 Mori, A. , 14, 68 Munro, S. , 404 Mori, M. , 5, 41, 189, 313, 343, 364, 366, Muntean, D. , 64 370–372, 532 Muntoni, A.L. , 502 Mori, M.A. , 157 Munyao, N. , 349 Mori, T. , 504 Murad, F. , 437 Morikawa, E. , 272, 437 Muramatsu, H. , 276 Morikawa, S. , 68 Murata, M. , 60, 433 Morimoto, N. , 399 Murata, T. , 184, 185, 194 Morimoto, T. , 124, 601 Murata, Y. , 462, 472 658 Author Index

Murdaugh HV, J.r. , 25 Nakagawa, T. , 63, 75 Murphy, A.N. , 247 Nakagomi, T. , 272, 342 Murphy, B.M. , 353, 531 Nakahara, T. , 303, 541 Murphy, E. , 56, 60–62 Nakajima, H. , 512 Murphy, N. , 524, 525, 527, 529, 532 Nakajima, T. , 8, 235 Murphy, S. , 140 Nakakimura, K. , 348 Murphy, S.F. , 175 Nakamachi, T. , 436 Murphy, S.J. , 167, 168, 176, 280, 493 Nakamura, H. , 280, 463, 470 Murphy, S.P. , 556 Nakamura, K. , 319, 372, 401 Murphy, T.H. , 352 Nakamura, M. , 167, 348 Murray, H.N. , 6, 81, 433 Nakamura, S. , 462, 472 Murry, C.E. , 5, 6, 19, 51, 58, 59, 61, 64, Nakamura, T. , 220, 310, 312, 314, 372 165, 243, 259, 505, 553, 568, Nakamura, Y. , 443 591, 601, 602 Nakane, M. , 437 Muselmann, C. , 184, 185, 191, 198, 201, Nakane, S. , 501 219, 220 Nakano, A. , 57, 62, 63 Mussolino, M.E. , 106 Nakano, S. , 244 Musters, R.J. , 390 Nakao, Y. , 3, 4, 9, 10, 275, 459, 461, 470 Musumeci, F. , 569 Nakata, N. , 8, 244, 347 Muthian, S. , 502 Nakatani, Y. , 192 Muzzi, D.A. , 293 Nakaya, H. , 60, 62 Mwamure, P.K. , 52, 77, 78, 571, 604, 608 Nakaya, Y. , 280, 299 Myburgh, C. , 142 Nakayama, H. , 4 Myburgh, J.A. , 499 Nakayama, M. , 436 Mychaskiw, G. , 583 Nakayama, Y. , 310 Myhr, K.M. , 144 Nakazawa, A. , 367 Mykytenko, J. , 72–75 Nakazawa, H. , 60 Nakazawa, T. , 271 Nalin, C. , 367 N Nalivaeva, N.N. , 184, 185, 200 Na, B.J. , 594 Namba, K. , 42 Na, H.S. , 71, 562, 602 Namba, N. , 62 Nadaud, S. , 192 Namikawa, K. , 544 Nadler, J.V. , 529 Nandagopal, K. , 9, 543, 545 Nadtochiy, S.M. , 437, 439, 441 Nao, B. , 65, 68 Nagahiro, S. , 230, 273 Napadow, V. , 592 Nagai, M. , 10 Narasimhan, P. , 297 Nagai, Y. , 174 Narayan, P. , 61 Nagao, S. , 118 Nardi, A.E. , 142 Nagarkatti, D.S. , 57 Narendra, D. , 396 Nagasawa, H. , 272 Naritomi, H. , 8, 235, 272 Nagase, H. , 62, 110, 391, 433 Narkilahti, S. , 528 Nagata, I. , 270, 274, 277 Nassief, A. , 4, 221 Nagayama, T. , 8, 369, 503, 530, 531 Nasu, I. , 167 Nagerl, U.V. , 353 Navet, R. , 190, 194 Nagoya, J. , 121, 123 Nawashiro, H. , 3, 9, 10, 118, 299, 367, 466, Nahm, S.H. , 71, 602 474, 491, 527, 584 Nairn, A.C. , 353 Naylor, M. , 231, 234, 299, 370, 458, 460, Naito, S. , 24 461, 467 Najm, I.M. , 523–525, 527, 529 Neafsey, E.J. , 399 Nakae, I. , 68 Neary, J.T. , 500 Nakagami, M. , 601 Neckar, J. , 605 Nakagawa, C. , 58 Nedelec, A.S. , 184, 185, 189, 191, 199, 411, 412 Nakagawa, M. , 58 Neder, L. , 532 Author Index 659

Nedergaard, M. , 184, 187, 195, 280, 466, 472 Ning, B. , 382 Neeman, M. , 491 Ning, K. , 353 Nehlig, A. , 523–525, 527–529 Nishijima, K. , 542 Nelles, M. , 71 Nishijima, M.K. , 431 Nelson, A.H. , 271 Nishimura, M.C. , 270 Nelson, D.A. , 120 Nishino, A. , 214 Nelson, R.J. , 38 Nishino, K. , 278 Nelson, S.R. , 523, 524, 527 Nishino, Y. , 443 Némethová, M. , 323, 326, 435, 563 Nishio, S. , 10, 114, 115, 119 Nemets, B. , 142 Nishiura, M. , 8, 235 Nemoianu, A. , 310 Nishiyama, N. , 352, 524, 525, 527, 529, 530 Nemoz, C. , 438 Nishiyama, Y. , 463, 470 Nestler, E.J. , 237 Nishizawa, S. , 295 Netuka, I. , 556 Nishizuka, Y. , 246 Neubauer, R.A. , 581 Nissim, I. , 439 Neubauer, S. , 82 Nissinen, J. , 528, 530 Neuhaeuser, M. , 78, 604 Nithipatikom, K. , 504 Neumann, H. , 512 Nito, F. , 280 Neumann-Haefelin, T. , 319 Nitsch, R. , 367 Newell, J.B. , 76 Niu, G. , 318, 323–325, 563 Newhouse, J.P. , 339 Niwa, Y. , 299 Newman, M.A. , 293 Noble, E.G. , 106 Newman, M.F. , 175, 606 Noble, R.L. , 4 Newton, S.S. , 525, 531 Node, K. , 392 Ng, R. , 571, 574 Noe, F. , 530 Ng, S.C. , 293, 574, 575, 603 Noell, W.K. , 5 Ng, T.L. , 442 Nogawa, S. , 247 Ng, Y.S. , 542 Noguchi, C.T. , 188, 190 Nguemeni, C. , 150, 152, 154, 157, 158 Nogueira, V. , 443 Nguyen, H.V. , 434 Noh, K.M. , 299, 346, 433 Nguyen, K. , 370 Nolte, C. , 319 Ni, Z.H. , 80 Nolte, K.W. , 47 Nicastri, A. , 3 Noma, A. , 62 Nichol, A.D. , 120 Nombela, F. , 466, 473 Nicholls, D.G. , 24, 434–435 Nomura, N. , 299, 491 Nichols, C.T. , 40 Noppens, R. , 213, 220 Nicolay, K. , 368 Norberg, K. , 23 Nie, H. , 174, 491, 584 Nordvik, I. , 144 Nielsen, M. , 346 Noriega, L. , 443 Nielsen, S.S. , 11, 53, 82, 608 Normington, K. , 435 Nielsen, T.T. , 11, 53, 68, 82, 608 Norris, S. , 431 Nielsen-Kudsk, J.E. , 68 Nortje, J. , 511 Nienstedt, J. , 70, 71 Noshita, N. , 8, 328, 416 Nieoullon, A.L. , 170 Nouraei, S.A. , 562, 573, 604, 608 Nighoghossian, N. , 438, 582 Nouvelot, A. , 510 Nightingale, P. , 78, 604, 608 Novakova, O. , 605 Niibori, K. , 60 Novalija, E. , 60, 61, 388, 390 Niinobe, M. , 6, 236, 579 Novikov, M. , 508, 511 Niiro, M. , 7, 235 Nowak, G. , 42 Niitsu, Y. , 270 Nowak, T.S., Jr. , 8, 244, 270–273, 275–282, Nijboer, C.H. , 436 348, 464, 470, 558 Nilsa, R.-D.V. , 193 Nudo, R.J. , 457 Nilsson, G.E. , 19, 26–30, 248, 347 Nunneley, S.A. , 120 Nilsson, L. , 23 Nurmi, A. , 409 660 Author Index

Nuyts, J. , 190, 194 Okten, A.I. , 297 Nyland, H. , 144 Okuda, H. , 524, 525, 527, 529, 530 Nylandsted, J. , 367 Okuno, S. , 435 Nyska, A. , 509 Oldenburg, O. , 62 Old ﬁ eld, E.H. , 297 Oliff, H.S. , 271 O Oliveira, C.R. , 195 Obata, T. , 544 Oliveira, R.M. , 157 Obenaus, A. , 486–489 Oliver, F.J. , 190 Obenovitch, T.P. , 139 Olney, J.W. , 339, 340 Oberley, L.W. , 56, 60 Olsen, T.S. , 4 Obregon, D. , 504 Olson, E.N. , 64 Obrenovitch, T.P. , 25, 259–261, 293, 309, 312, Olsson, R.A. , 55, 72 313, 505 Olsson, T. , 367 O’Collins, V.E. , 134, 136 Olsson, Y. , 274 O’Connell, M.T. , 511 Omae, T. , 272 O’Connor, L. , 403 Omata, N. , 184, 185, 194 Oddo, S. , 403 Omatsu, T. , 271 Odendaal, L. , 606 Ommaya, A. , 274 Odenheimer, D.J. , 270 Onaivi, E.S. , 500 O’Donoghue, M.F. , 502 O’Neill, J.J. , 21 O’Duffy, A.E. , 138 O’Neill, M.J. , 7, 345, 351 Oe, H. , 53, 64 Onesti, S.T. , 271, 273 Oertel, M.F. , 301 Ong, S.B. , 59, 64, 396 O’Farrelly, C. , 443 Onley, D. , 531 Ofengeim, D. , 299, 433 Ono, J. , 184, 185, 188, 191, 197 Ogasawara, M.A. , 193 Oo, T.F. , 544 Ogawa, K. , 605 Ooi, L. , 534 Ogilvy, C.S. , 279 Ooneda, G. , 271 Ogita, K. , 524, 525, 527, 529, 530 Oosthuizen, P. , 142 Ogoh, S. , 112 Openheim, A. , 507, 510 Ogunbayo, O.A. , 404 Opie, L.H. , 73, 506 Oguro, K. , 372 Oppenheim, C. , 558 Oh, J.S. , 184, 201 Ordonez, A.N. , 231, 235, 295, 319, 322, Oh, Y.-S. , 279 325–327, 330, 342, 348–350, Ohara, Y. , 504 352–354, 403, 531 Ohlemiller, K.K. , 185, 188, 201, 542–544 Ordureau, A. , 353 Ohlstein, E.H. , 57 Organisciak, D.T. , 545 Ohnuki, K. , 370 O’Rourke, B. , 60, 62, 169, 433, 435, 493 Ohta, B. , 58 O’Rourke, D.M , 232, 235 Ohta, S. , 56, 57 Orr, A.L. , 38 Ohtaki, H. , 436 Orsini, F. , 231, 234, 465, 468, 472, 473 Ohtsu, H. , 184, 185, 197, 199, 461, 467, 470 Orso, E. , 469 Ohtsuki, T. , 3, 8, 9, 351, 367, 527 Orti, M. , 437 Ohura, M. , 59 Ortmann, A.J. , 185, 201 Oida, Y. , 399 Osbakken, M.D. , 60 Oikawa, M. , 605 Osborne, P.G. , 37, 38, 45 Ojwang, B.A. , 185, 186, 191, 194, 200, 542, O’shea, F. , 296 543, 545 Oshima, T. , 344 Okada, M. , 144, 167 Oshima, Y. , 441 Okada, T. , 280 Oshita, S. , 280 Okado, H. , 544 Osinska, H. , 63, 75 Okamoto, F. , 71 Ostadal, B. , 556 Okauchi, M. , 118 Ost’adal, B. , 605 Author Index 661

Oster-Granite, M.L. , 24 Pamenter, M.E. , 27, 28, 194, 195 Ostrowski, R.P. , 166, 173, 174, 293–295–299, Pan, H.B. , 594 301, 487, 490, 491, 495, 605 Pan, Y. , 473 Osuga, S. , 273, 277 Panagiotidou, A.T. , 603 O’Sullivan, M. , 82, 604, 608 Panagopoulou, V. , 53, 82, 608 Otal, M.P. , 370 Panahian, N. , 234 Otani, H. , 58 Pancani, T. , 345, 347 Otellin, V. , 184, 185 Pancioli, A. , 375 Otori, T. , 276, 277, 463, 470 Pandey, P. , 367 Otsu, K. , 63, 75 Pandya, J.D. , 393 Ottani, F. , 559 Panes, J. , 70 Otto, M. , 3 Pang, L. , 370 Ottonello, L. , 458 Pangalos, M.N. , 137 Ouk, T. , 406, 463, 467, 470, 471, 474 Panikashvili, D. , 501–503 Ouml, R.E. , 26, 29 Panning, B. , 194 Ouyang,Y.B. , 403 Panter, S.S. , 531 Ovadia, H. , 503 Papadakis, M. , 235, 275, 282, 462, 470 Overgaard, K. , 272 Papadia, S. , 343, 413 Ovize, M. , 11, 52, 53, 63–65, 72, 75, 79, 80, Papandreou, I. , 441 83, 318, 563, 602, 608 Papapetrou, P. , 399, 416 Oxman, T. , 245 Papousek, F. , 605 Ozaita, A. , 503 Pappius, H.M. , 276 Ozcan, C. , 61 Parada, L.F. , 8, 340, 342, 437 Özdemir, Y.G. , 272, 273 Paraskevaidis, I.A. , 83 Ozen, O.A. , 156, 157 Parent, J.M. , 10, 369 Parham, S. , 192 Paris, J. , 348 P Park, D.K. , 465, 474 Pablo, J. , 27 Park, E.M. , 437 Pacary, E. , 184, 185, 189, 366, 369, 371, 373, Park, H. , 443 374, 407 Park, H.-K. , 185, 191, 401 Pacher, P. , 501, 504 Park, H.P. , 166, 168 Packard, A.E. , 10, 366, 368, 373, 374, 467 Park, J.M. , 594 Packard, A.E.B. , 189 Park, J.S. , 352 Pac-Soo, C. , 184, 185, 202 Park, J.W. , 584 Paden, C.M. , 340, 344 Park, K.H. , 45, 380 Padmanaban, D. , 431 Park, L. , 462, 467–471 Pagel, I. , 395 Park, M.K. , 299 Pagel, P.S. , 493 Park, P.S. , 271 Pagliaro, P. , 72, 74, 76 Park, S.U. , 594 Pagliei, B. , 441 Park, S.W. , 373 Pain, T. , 62, 392 Park, T.S. , 8, 9, 184, 185, 187–189, 193, 196, Paine, M. , 541 198, 201, 248, 437, 458, 462, 463, Paiva, M.A. , 442, 443 467–469, 544 Palacios, M. , 437 Parker, J.D. , 345 Palacios-Callender, M. , 438 Parker, J.E. , 61 Palladini, G. , 23 Parkinson, F.E. , 27 Pallas, M. , 441 Parks, J.S. , 294 Palmateer, J.M. , 168, 280 Parpura, V. , 344 Palmer, C. , 219, 220 Parrado-Fernandez, C. , 47 Palmer, G.C. , 340 Parratt, J.R. , 55 Palmer, R.M. , 437 Pasceri, V. , 559, 561 Palmeri, M. , 442 Pasch, T. , 73 Palmon, A. , 507, 510 Paschen, W. , 397, 400, 434 662 Author Index

Pasdois, P. , 61 Peng, P.-H. , 185, 191, 200, 544 Passonneau, J. , 24 Peng, W. , 184, 195 Patani, R. , 443 Peng, Y. , 173, 593, 595 Patel, A.M. , 5, 196, 197, 201 Peng, Z.Y, , 173, 411, 491, 595 Patel, H.H. , 68–70, 76, 167, 368 Penick, G.D. , 38 Patel, M.K. , 531 Penix, L. , 522 Patel, P.M. , 279, 280, 368 Penkowa, M. , 188, 198, 201 Patel, S.M. , 184, 185, 202 Penna, C. , 72, 74, 76 Paterno, R. , 437 Pennant, W.A. , 184, 201 Patricelli, M.P. , 502 Penner, M.R. , 524, 525 Patyar, S. , 3 Penrod, C. , 524, 525, 527, 529 Patzer, A. , 406 Pepe, S. , 64 Paucard, A. , 531 Pequignot, F. , 166 Paucek, P. , 6, 58, 62, 81, 433 Peralta, C. , 70, 243, 443 Paul, S.M. , 8, 494, 523 Perdrizet, G. , 294, 299 Pavanello, L. , 297 Pereira, J.G. , 494 Pavlikova, M. , 405 Pereira, M.P. , 466, 473 Pawlak, V. , 343 Perella, M.A. , 117 Payne, C.S. , 326 Perez, E. , 441 Payne, R.S. , 170, 171, 492, 493 Perez, R.S. , 184, 185, 201, 458, 467 Paz, Z. , 506 Perez Asensio, F.J. , 370 Pazdernik, T.L. , 523, 524, 527 Pérez-Pinzón, M.A. , 10, 19, 26, 27, 42, 234, Pearlstein, R.D. , 279, 493 244, 245, 248, 251, 260, 272, 278, Pearson, A.N. , 353 294, 296, 302, 312, 323, 347–349, Pecina, S. , 296 375, 433, 436, 437, 441, 494, 505, Peck, L. , 144, 148 601, 602, 604, 608, 609 Pedal, I. , 545 Perfater, J.L. , 185, 188, 192, 198, 458, 468, Pedata, F. , 117 470 Pedersen, B.K. , 187, 202 Perin, A. , 299 Pedersen, H. , 272 Perkins, G.A. , 544 Pedersen, P.M. , 4 Perkins, R. , 382 Pedersen, W.R. , 81, 318 Perlman, M.E. , 60 Pedraza, N. , 439 Pernow, J. , 81 Pedrazzini, T. , 73 Perra, S. , 502 Pedrono, E. , 278 Perriard, J.C. , 73 Peet, M. , 142 Perry, A. , 11 Peggie, M. , 353 Perry, G. , 38, 40, 195 Pei, D.S. , 342 Peschon, J. , 10 Pei, Z. , 443 Peters, J. , 78, 604 Peirano, P. , 140 Peters, M. , 603 Pek, M. , 27 Petersen, G. , 501 Pek-Scott, M. , 27 Petersen, O.H. , 430 Pelech, S.L. , 57 Petersen, S.E. , 82 Pelham, D.J. , 542, 543, 545 Petersen, S.O. , 506 Pell, T.J. , 68–71 Petit, E. , 8, 184, 185, 189, 191, 197–199, 202, Pellacani, A. , 117 366, 369–374, 510 Pellegrini-Giampietro, D.E. , 322, 326, 330, Petrault, O. , 157 345, 347 Petrillo, R.L. , 581 Pelligrino, D.A. , 9, 185, 188, 437, 462, 468, 469 Petrosino, S. , 502 Pelto-Huikko, M. , 184, 185, 194, 195 Pettet, G.K. , 172 Pena-Tapia, P.G. , 197, 470 Pettit, D.L. , 340, 343 Peng, C.F. , 70 Petzold, A. , 11 Peng, L. , 169, 172, 298 Peugh, J. , 485 Peng, M. , 543, 545 Pevet, P. , 352 Author Index 663

Peynet, J. , 175 Pleasure, D. , 184, 185 Philipp, S. , 72, 74 Plesneva, S.A. , 184, 185, 200 Philipson, K.A. , 435 Plesnila, N. , 229 Phillips-Bute, B. , 606 Ploeg, H. , 69 Pialat, J.B. , 438 Ploski, J.E. , 525, 531 Piantadosi, C.A. , 172, 173, 185, 193, 195, Plum, F. , 229, 230, 273 436, 439 Plumlee, C. , 112 Piazza, T.M. , 41 Pluta, R.M. , 295 Pichiule, P. , 184, 191, 192, 465, 473 Pociej, J. , 438 Pickart, C.M. , 402 Polderman, K.H. , 118, 119 Pickering, J.G. , 441 Pollesello, P. , 506 Pickett, C.B. , 443 Pollock, D.M. , 459 Pickett, W. , 297 Pollock, J.S. , 437 Piersiak, T. , 524, 527–529 Polsky, K. , 230, 273 Pignataro, G. , 11, 186, 191, 192, 231, 235, Polyakova, E.A. , 463, 468, 472 319, 322, 325–327, 330, 331, 340, Pons, S. , 73 380, 438 Pool, A.H. , 530 Pike, M.M. , 274 Poon, W.S. , 293, 571, 603 Piler, P. , 78, 604 Popov, V.I. , 352 Pilipenko, I. , 296 Popp, S. , 170, 171 Pillolla, G. , 502 Porcaro, W.A. , 68 Pinaud, R. , 524, 525 Porcu, M. , 232, 235 Ping, A. , 303 Porter, A.G. , 443 Ping, P. , 56, 57, 63, 393, 394 Porto, I. , 82 Pingping, X. , 322, 331 Poser, W. , 218 Pinsky, D.J. , 273 Post, H. , 56, 57 Pinto, A. , 222 Post, R.L. , 23 Piomelli, D. , 500, 501 Poston, J.N. , 542, 546 Piot, C.A. , 52, 53, 63, 79, 80, 318, 431, 608 Potter, D.E. , 544 Piotin, M. , 296 Potter, G.G. , 606 Piper, C. , 189, 343, 381, 530, 535 Potter R.F. , 605 Pipis, J. , 606 Poulsen, D.J. , 348 Pique, J.M. , 70 Powell-Coffman, J.A. , 506, 509 Piriou, V. , 64 Powers, C. , 459 Pirona, L. , 530 Pozzan, T. , 430 Piroth, Z. , 53, 602 Pradillo, J.M. , 234, 376, 410 Pisano, P.S. , 170, 171 Prado, R. , 42, 270, 280, 347, 375, 601 Piser, T.M. , 503 Prakash, A. , 3 Pistis, M. , 502 Prakash, N. , 4 Pitcher, J.M. , 557 Prange, H. , 219, 222 Pitha, P.M. , 9 Prass, K. , 167, 184–186, 191, 198, 201, 219, Pitkanen, A. , 528, 530 220, 250, 273, 411, 412 Pitkonen, M. , 278 Prats, N. , 70, 443 Pittelli, M. , 232, 235 Pratt, P.F. , 56 Pitts, L.H. , 270 Pratt, S.M. , 439 Pivina, S. , 184, 185, 199 Preckel, B. , 172, 604 Pivorun, E.B. , 38 Prehn, J. , 531 Pivovarova, N.B. , 434 Prehn, J.H. , 523–525, 527–529, 531–534 Pizzo, P. , 404, 405 Preissl, H. , 457 Plamondon, H. , 10, 138, 147, 154, 156, 157, Prendergast, B.J. , 38 348, 525, 529, 531 Prentice, H.M. , 27, 28 Planas, A.M. , 110, 370 Prestera, T. , 443 Plassman, B.L. , 606 Price, C.D. , 251 Plate, K. , 192 Price, M. , 465, 472 664 Author Index

Prigent-Tessier, A. , 185, 187, 194, 200, 463, 468 Querido, J.S. , 112 Priller, J. , 184, 186, 198, 219 Quinn, J.P. , 534 Prince, D.A. , 27 Quinn, R.D. , 175 Pringle, A.K. , 246 Quintero, M. , 438 Prinz, S. , 273 Proctor, J.B. , 73, 75, 326 Prosperetti, C. , 504 R Providencia, L.A. , 442, 443 Raaschou, H.O. , 4 Prywes, R. , 435 Rabadi, M.H. , 113 Przyklenk, K. , 11, 52, 57, 65, 68, 245, 313, 602 Rabb, H. , 443 Ptasznik, A. , 458 Rabin, S.J. , 530, 531 Pu, Q. , 4, 9, 463, 467–469 Racay, P. , 404, 405 Pu, Y. , 442 Racine, R.J. , 528 Pudupakkam, S. , 243 Raczka, F. , 63, 79, 80, 608 Pugh, C.W. , 190, 192, 194, 440 Rademacher, D.J. , 502 Pugliese, A.M. , 117 Radin, N.S. , 151 Pugsley, W.B. , 6, 77, 243, 561, 569 Raffelsieper, C. , 78, 604 Puighermanal, E. , 503 Ra ﬁ kov, A.M. , 583 Puigserver, P. , 441, 443 Rafols, J.A. , 458–461, 467, 468, 471, 472 Puisieux, F. , 4, 9, 235, 463, 467–469 Ragatz, R.E. , 581 Pulsinelli, W.A. , 149, 229, 230, 270–273, 275, Raghavachari, N. , 438 279–281, 349 Rahman, I.A. , 78, 441, 604, 608 Puntambekar, S.S. , 467 Raimondo, S. , 74, 76 Purcell, J.E. , 278 Raisakis, K. , 53, 82, 608 Purcell, N.H. , 63, 75 Raisky, O. , 75 Purshottam, T. , 5 Raivio, J. , 441 Puryear, L.J. , 142 Raizada, M.K. , 370 Pypaert, M. , 442 Rajapakse, N.C. , 371, 391 Pyrgakis, V. , 53, 82, 608 Rajdev, S. , 8 Rajesh, K.G. , 64 Rakhit, R.D. , 57, 63 Q Ralph, G.S. , 192 Qi, D. , 442, 443 Ramchand, C.N. , 142 Qi, J. , 466, 474 Rami, A. , 412 Qi, Y. , 442 Ramos, J.A. , 501 Qi, Z. , 185–187, 193 Ran, R. , 192 Qian, G. , 187, 201 Rancan, M. , 511 Qian, H. , 184, 186, 193, 200, 201 Ransohoff, R.M. , 458 Qian, L. , 437 Raphael, J. , 61 Qian, Z. , 412 Rashid, A. , 78, 604 Qian, Z.M. , 459, 465 Rasley, B.T. , 41, 42, 45 Qian Zhong, M. , 184, 186, 191, 198 Rasmussen, P. , 187, 202 Qin, L.P. , 595 Rasoulian, B. , 298 Qin, Z. , 173, 311, 312, 314 Rastaldo, R. , 72, 74, 76 Qiu, J.H. , 437 Rasulian, B. , 457, 461, 467, 468 Qiu, P. , 166, 168, 169 Ratan, R.R. , 190, 192, 220, 297 Qiu, Y. , 57, 63, 184, 185, 195, 199, 322, Ratcliffe, P.J. , 190, 192, 194, 440, 491 331, 601 Rathvon, M. , 46 Qiu, Z. , 56 Rauca, C. , 188, 201 Qu, Y. , 187, 190 Rauter, M. , 192 Quan Lan, J. , 529 Ravagna, A. , 443 Quaranta, N. , 5 Raval, A.P. , 27, 42, 245, 246, 251, 347, Quarum, M.L. , 345 349, 391, 392, 436, 441, 494, Quast, M.J. , 280 601, 602, 608 Author Index 665

Raval, R.R. , 192 Repine, J.E. , 9 Raval, A.P. , 433 Reshef, A. , 348, 436 Ravati, A. , 409, 431, 436 Revel, D. , 63, 608 Ravizza, T. , 530 Reves, J.G. , 175, 606 Ravussin, P. , 485 Reyes, R. , 111, 118 Ray, U.S. , 605 Reymann, K.G. , 214, 273 Raychaudhuri, S. , 4 Reynolds, I.J. , 434 Rayner, W. , 192 Rezkalla, S.H. , 83 Razdan, R.K. , 504 Rhee, J. , 439 Readnower, R.D. , 393 Rhoden, E.L. , 601 Redaelli, E. , 303 Rhodes, P.G. , 184, 185, 187, 197, 199, 265 Redel, A. , 438, 561 Rhodes, S.S. , 60 Redington, A.N. , 11, 14, 52, 53, 68, 70, 71, Ricciardi, M.J. , 80 77, 78, 82, 293, 602–604, 608 Rice, M.E. , 38, 40, 46 Redman, E. , 441 Richard, V.J. , 58, 59, 61, 468, 470 Rees, P.J. , 581 Richardson, R.J. , 215 Reese, S.A. , 28 Richelme, C. , 138, 154, 348, 525, 529, 531 Reeves, B.C. , 572 Richichi, C. , 530 Reeves, J.G. , 72–75 Richmond, T.S. , 430 Regal, P.J. , 25 Richter, D. , 21 Regli, F. , 559 Rickman, C. , 354 Regli, L. , 294 Ridenour, T.R. , 271 Rehling, M. , 11, 53, 82, 608 Riegler, E.M. , 353 Rehni, A.K. , 232, 236, 245, 298, 322, 353, Riek-Burchardt, M. , 214 400, 403 Riepe, M.W. , 348 Reichensperger, J. , 410 Ries, V. , 544 Reid, D.G. , 278 Riess, M.L. , 60, 61 Reid, K.H. , 5, 19, 184, 194 Rigamonti, A. , 301 Reilly, M. , 8, 191, 198, 199, 366, 369–373 Rigor, B.M. , 5, 19, 184, 194 Reimer, K.A. , 5, 6, 19, 51, 58–61, 64, 165, Rimpilainen, E. , 11 243, 259, 505, 553, 568, 591, Ringwood, L.A. , 438 601, 602 Rinkel, G.J. , 297 Reindhardt, C.P. , 68 Rioufol, G. , 52, 53, 63, 79, 80, 318, 608 Reiner, A. , 273 Ritchie, I.M. , 273 Reinhardt, C.P. , 68 Ritossa, F. , 388 Reiser, G. , 214, 215, 273, 310 Rivas, L.A. , 218 Reith, J. , 4 Rivera, P.M. , 38, 44, 45 Rejdak, K. , 523 Rivera-Cervantes, M.C. , 441 Rejdak, R. , 523 Rivo, J. , 61 Relton, J.K. , 9, 157 Rizwi, F. , 78, 604 Reme, C.E. , 185, 191, 201 Rizzi, M. , 530 Rempe, D.A. , 472, 473 Roach, G.W. , 569 Ren, C. , 175, 293, 311, 318, 319, 321, Robbins, J. , 63, 75 323–325, 327, 375 Roberge, M.C. , 156, 157 Ren, H.M. , 459, 464, 468 Robert, D. , 64, 75 Ren, P. , 173, 411, 491 Roberts, R.L. , 219, 220 Ren, X. , 53, 71 Robertson, F. , 296 Ren, Y. , 270–273 Robertson, H.A. , 524, 525 Ren,C. , 563 Robertson, M.D. , 442 Renshaw, G.M. , 347 Robey, R.B. , 443 Rentoukas, I. , 53, 82, 608 Robin, E. , 4, 9, 25, 171, 322, 331, 391 Renzi, F.P. , 68 Robinson, G.S. , 542 Renzo, G.D. , 231, 235 Robinson, J. , 4 Renzo, G.F. , 436, 437 Robinson, P.F. , 37 666 Author Index

Robinson, R.G. , 270 Roth, D.M. , 76 Rocha, S. , 192 Roth, M.B. , 46 Rocha Araujo, D.M. , 142 Roth, S.U. , 5, 347, 541–546 Roche, H.M. , 146 Rother, J. , 6, 319 Roche, K.W. , 340 Rothman, S. , 340, 431, 433 Rockswold, G.L. , 492, 582, 583, 587 Rothwell, N.J. , 111, 157, 511 Rockswold, S.B. , 492, 583 Rotilio, G. , 441 Rodgers, J.T. , 441 Rouelle, D. , 170 Rodnight, R. , 399 Rougier, A. , 524, 527, 529, 530 Rodriguez, E.R. , 510 Rousou, J.A. , 6 Rodriguez Sinovas, A. , 394 Roussel, S. , 184, 199, 203, 323, 510 Roelke, C.T. , 502 Rovainen, C.M. , 459 Roeser, N.F. , 439 Rovere, C. , 157, 158 Roeskes, C. , 73 Rowan, A. , 441 Roewer, N. , 170, 171, 438, 492, 493 Rowe, J. , 488, 490 Roger, V.L. , 429 Rubaj, A. , 201 Roh, J.-K. , 185, 191, 380, 465, 474 Rubens, J. , 63 Roh, K.J. , 438 Rubino A , 6 Rojanasakul, Y. , 438 Rubio, R. , 8 Rojas, H. , 487, 489 Rudich, A. , 57 Roloff, A.M. , 352 Rudin, D.O. , 140 Romanic, A.M. , 110 Rudkin, A.K. , 541 Romanovskii, D.Y. , 184, 185 Rudolphi, K.A. , 27 Romera, A. , 466, 473 Rueda, D. , 503 Romera, C. , 118, 234, 407, 410, 466, 473 Ruetzler, C.A. , 3, 9, 118, 276, 367, 466, 474, Romey, G. , 141, 143, 148–150, 155 527 Ron, D. , 397, 435 Ruf, T. , 37 Rooyakkers, A. , 343, 344 Ruiz Ramos, R. , 390 Rosamond, W. , 135 Runden Pran, E. , 392 Rose, E. , 55 Ruscher, K. , 184–186, 191, 198, 201, 219, Rosello-Catafau, J. , 70, 443 220, 250, 411 Rosen, A. , 353 Russell, D.A. , 9 Rosen, I. , 229 Russell, R.R. , 442 Rosen, P. , 29 Rustenbeck, H.H. , 219, 510 Rosenbaum, D.M. , 542–546 Rusyniak, D.E. , 582–584 Rosenbaum, P.S. , 5, 541–543, 545, 546 Rutter-Locher, Z. , 442, 443 Rosenberg, G.A. , 137 Rybak, S.L. , 435 Rosenberg, P.A. , 459, 474, 475 Ryberg, E. , 504 Rosenberger, C. , 192 Rybkin, I.I. , 439 Rosenblatt, S. , 437 Rybnikova, E.A. , 112, 184, 185, 187, Rosenmund, C. , 340 192–196, 199, 409, 412 Rosenstein, R.E. , 542, 546 Ryden, L. , 81 Rosenthal, M. , 24–27, 348 Ryu, S. , 231, 235 Rosenzweig, A. , 439, 441 Rzhetskaya, M. , 544 Rosenzweig, H.L. , 5, 41, 189, 313, 364, 366, 367, 370–372, 375, 463, 467, 474, 527, 532 S Rosner, G. , 279–281 Saad, S. , 601 Ross, A.P. , 40–42, 44, 45, 341 Sabina, R.L. , 5, 51 Ross, M.E. , 437 Saboureau, M. , 352 Ross J., Jr. , 57 Sabourin, G. , 441 Rossi, D.J. , 344, 350 Sabourin, L.A. , 349 Rossi, F. , 69 Sabri, M. , 301 Rossi, R. , 79, 80, 608 Sacco, R.L. , 601, 606 Rossi, S. , 69, 504 Sack, M.N. , 438 Author Index 667

Sadasivan, S. , 401 Sancak, T. , 300 Sadat, U. , 562, 604, 608 Sanchez, I. , 608 Sadeghian-Nodoushan, F. , 299 Sanchez-Prieto, J. , 24, 25 Sadeghipour, H. , 68, 69, 71 Sande, P.H. , 542 Sadoshima, J. , 56 Sander, J.W. , 141, 148 Sage, W.M. , 485 Sanders, R.D. , 171, 172 Sah, V.P. , 57 Sandu, N. , 563 Saha, M. , 605 Sandvik, G.K. , 30 Sahibzada, N. , 524, 525, 527, 529, 531 Sang, H. , 166, 168, 169 Sahin, O. , 156, 157 Sankar, R. , 370, 522 Sai Ram, M. , 184, 186, 462, 467, 468 Sanlioglu, S. , 56, 60 Saia, A. , 319 Sano, K. , 272 Sailor, K.A. , 185, 189, 231, 234, 364–366, Sans, M. , 70 368, 370, 371, 373 Santillo, M. , 436, 437 Saini, M. , 124 Santos, M.S. , 195 Saito, A. , 399, 400, 435 Santos, R.X. , 195 Saito, M. , 541 Sapolsky, R.M. , 11, 260, 318–321, 323, 327, Saito, N. , 348, 437 370, 531, 563 Saito, R. , 279–281 Sarandol, E. , 184, 185, 194, 322 Saito, T. , 62, 392 Sarraf-Yazdi, S. , 493 Sakabe, T. , 174, 348, 584 Sartorelli, V. , 443 Sakai, N. , 270 Sarvary, L. , 71 Sakai, Y. , 603, 607 Sarvotham, S.S. , 71 Sakakibara, Y. , 319 Sasaguri, S. , 64 Sakamoto, J. , 56, 443 Sasahira, M. , 523, 524, 527, 529 Sakamoto, K. , 442, 443, 541 Sasaki, H. , 60, 75 Sakamoto, S. , 299 Sasaki, T. , 413 Sakamoto, T. , 141 Sasaoka, N. , 167, 263 Sakatani, K. , 462, 472 Sasayama, S. , 61 Salama, A.I. , 4 Sasik, R. , 368 Saleh, A. , 367 Sastre, A.A. , 220 Saleh, M.C. , 399, 400, 608 Sato, F. , 319 Saleh, N. , 81 Sato, H. , 71 Saleh, T.M. , 608 Sato, J. , 37, 38 Salem, N., Jr. , 144 Sato, M. , 57 Saliba, S. , 441 Sato, R. , 512 Salinas, M. , 323, 326, 435 Sato, S. , 396 Sallenave, J.M. , 458 Sato, T. , 60, 62, 392 Salles, D. , 64 Satoh, K. , 436 Sallustio, V. , 5 Satriotomo, I. , 185, 201 Salmon, P. , 190, 194 Sattler, R. , 147, 343, 349, 351, 354 Salom, J.B. , 370, 437 Saucier, D.M. , 524, 525, 530 Salord, F. , 582 Sauerbruch, S. , 469 Saltzman, H.A. , 580 Saugstad, J.A. , 189, 231, 235, 380, 381, 414, Salvador, M. , 601 438, 530, 531, 535 Samardzija, M. , 185, 191, 192, 201 Saul, I. , 42, 297, 347, 375, 436, 601 Samavati, L. , 56, 60 Saulnier, R. , 184, 199, 203, 323 Sambandam, N. , 439 Saunamaki, K. , 80, 318 Sambrook, J. , 435 Saurin, A.T. , 56, 57 Samoilov, M.O. , 184, 185, 187, 192–196, 199 Sauve, A.A. , 441, 443 Sampat, A. , 546 Savage, D.D. , 529 Samson, F.E. , 523, 524, 527 Savaki, H. , 23 Samuelsson, S.J. , 542 Saver, J.L. , 136 Samuni, A. , 509 Savitz, S.I. , 45 Sancak, B. , 78 Savoie, B. , 546 668 Author Index

Sawada, M. , 9, 229 Schmidt, M.R. , 11, 52, 53, 68, 82, 602, Sawada, T. , 8, 235 603, 608 Sawe, N. , 41, 330 Schmidt, O.I. , 510 Sawitzki, B. , 219 Schmidt-Kastner, R. , 297 Saxena, P. , 293 Schmitt, C. , 457 Saydam, T.C. , 370 Schmitz, B. , 279 Sayen, M.R. , 63, 75 Schneider, A. , 273, 409 Sazonov, I.A. , 296 Schneider, D. , 219, 222 Scalia, R. 46 Schneider, J.P. , 76 Scapagnini, G. , 443 Schneider, K. , 70, 71 Scarabelli, T.M. , 57 Schneider, M. , 190, 194 Scarpulla, R.C. , 395 Schneider, U.C. , 197, 470 Scartabelli, T. , 322, 326, 330, 345, 347 Schnorrenberger, N.K. , 47 Schaal, D.W. , 319 Schock, S.C. , 349 Schäbitz, W.R. , 10, 137 Schoemaker, R.G. , 65, 68–70 Schaefer, S. , 62 Schoen, F.J. , 439 Schaeffer, T. , 278 Schoenborn, R. , 524, 527–529 Schaller, B. , 559–561, 563 Schoffeniels, E. , 22 Schallert, T. , 215, 220, 296, 310 Scho ﬁ eld, P.M. , 82, 604, 608 Schallner, N. , 175 Scholze, C.K. , 192 Schaper, W. , 56, 57, 438 Scholzke, M.N. , 10, 544 Scharbrodt, W. , 301 Schoonderwoerd, K. , 605 Scharff, A. , 184–186, 191, 198, 201, 219, 220, Schormann, T. , 469 411, 412 Schousboe, A. , 431, 494 Scharfman, H.E. , 512 Schreiber, S.S. , 523 Schatz, A.R. , 500, 501 Schreiber, V. , 301 Schau, M. , 440 Schreier, L. , 75 Schaub, M.C. , 73 Schricker, T. , 280 Schauwecker, P.E. , 527 Schroder, F.K. , 492 Schechtman, D. , 436 Schubert, D. , 113 Schechtman, K.B. , 175 Schubert, P. , 27 Scheffer, R.E. , 144 Schubert, W. , 542 Scheinberg, P. , 38 Schuierer, G. , 469 Schell, R.M. , 279 Schuit, F. , 190, 194 Schellinger, P.D. , 6, 219, 222, 319 Schulte, P.M. , 506 Schepers, J. , 368 Schulte, S. , 430 Scheuer, T. , 42 Schultz, D. , 24 Schiavon, E. , 303 Schultz, J.E. , 53, 55, 71 Schindler, C.K. , 531, 532 Schultz, J.J. , 65 Schinzel, A.C. , 63 Schulz, J.B. , 8, 184, 187, 199, 200 Schipper, H.M. , 194, 465, 473 Schulz, R. , 52, 56, 57, 72–74, 83 Schlachetzki, F. , 469 Schumacker, P.T. , 61, 63, 430, 433 Schlack, W. , 172, 604 Schuman, E.M. , 535 Schlanger, S. , 141, 148 Schumann-Bard, P. , 184, 185, 189, 191, 199, Schleimer, R.P. , 10 203, 323, 366, 369, 371, 373, 374 Schley, J.A. , 56 Schurr, A. , 5, 19, 170, 171, 184, 194, 492, 493 Schlief, M.L. , 351, 352 Schutz, H. , 244 Schluesener, H.J. , 545 Schwab, J.M. , 545 Schlumpf, M. , 270 Schwab, S. , 10, 118, 120, 130, 273 Schluter, A. , 439 Schwaber, J.S. , 377 Schmid-Elsaesser, R. , 279 Schwaninger, M. , 10, 273, 343 Schmidt, C.F. , 580 Schwartz, W.J. , 23 Schmidt, H.H. , 437 Schwarzer, C. , 530 Schmidt, J. , 438 Schweizer, U. , 9 Author Index 669

Schwer, C.I. , 175 Shamsutdinova, A.A. , 184, 186, 193 Schwimmer, H. , 507 Shanley, J. , 328 Scognamiglio, A. , 61 Shannon, H.E. , 530 Sconzo, G. , 398 Shannon, R. , 52, 83 Scorziello, A. , 186, 191, 192, 231, 235, 436, Shao, G. , 185, 186, 197, 199, 411 437 Shao, Z. , 61 Scott, B.A. , 5, 196, 197, 201 Shao, Z.H. , 61, 63 Scott, C.W. , 328 Shapira, Y. , 57 Scott, J.R. , 605 Shapiro, H.M. , 279 Sebastian, A.H. , 393 Shapiro, J.I. , 601 Secher, N.H. , 111 Shapiro, L.M. , 82, 604, 608 Seeburg, P.H. , 343 Shapiro, S.D. , 458 Seeliger, M. , 185, 191, 201 Shari fi , M. , 585 Seetharaman, S. , 58, 391, 433 Sharkey, J. , 273 Segal, M. , 435 Sharma, A. , 436 Segev, L. , 327, 328 Sharma, H.S. , 512 Sehara, Y. , 10 Sharp, F.R. , 4, 8, 10, 44, 184, 186, 189–192, Seifalian, A.M. , 293 194, 198, 199, 260, 261, 264, 272, Seifert, E.L. , 441 278, 297, 299, 310, 366, 369–373, Seino, S. , 62 375, 441, 466, 474 Seki, T. , 532 Sharpe, L.G. , 339 Selim, M. , 219, 220 Sharples, L. , 82, 558, 604, 608 Sell, S.L. , 280 Shattock, M.J. , 436 Selnes, O.A. , 606 Shaw, R. , 524, 525, 527, 530–532 Seluanov, A. , 441 Shaw, T.E. , 5, 41, 189, 364, 366, 370–372, 532 Selvanayagam, J.B. , 82 Sheel, A.W. , 112 Selye, H. , 507 Sheen, J.M. , 594 Semenov, D.G. , 184, 185, 194 Sheerin, A.H. , 524, 525, 530 Semenza, G.L. , 8, 44, 184, 186, 190–192, Shehatha, J.S. , 293 194, 220, 297, 411, 436, 441, Shein, N.A. , 312, 503, 506–512 507, 509, 510 Shekhar, S. , 278 Semkova, I. , 247 Sheldon, R.A. , 112, 264 Seo, H.G. , 299 Shelton, J.M. , 439 Seong, J.K. , 438 Shen, A.C. , 60 Sepulveda, M.R. , 405 Shen, H.Y. , 348 Sera fi n, A. , 443 Shen, J. , 322, 325, 435, 464, 470 Sercombe, R. , 299 Shen, M. , 503 Serena, J. , 466, 473 Shen, W.W. , 142 Serlachius, M. , 188, 198, 201 Shen, Y. , 184, 185, 197, 199, 461, 467, 470, 544 Seru, R. , 437 Sheng, H. , 172, 173, 493 Servoss, S.J. , 76 Sheng, R. , 401 Sessler, D.I. , 120 Sheng, W.W. , 441 Seta, K.A. , 193 Shenoy, S.K. , 544 Seth, A. , 184–186 Shi, H. , 460, 464, 466, 468, 474 Settergren, M. , 81 Shi, J. , 595 Severson, D.L. , 438 Shi, L. , 382 Seybold, V.S. , 503 Shi, M.T. , 185, 186 Sgubin, D. , 299 Shi, T. , 593 Sha’a fi , R.I. , 57 Shi, X.Z. , 299 Shah, A.R. , 9, 184, 185, 188, 189, 201, 437, Shibasaki, F. , 172, 173 458, 462, 467–469 Shieh, J.-Y. , 185, 201 Shah, S. , 142 Shigeno, T. , 270 Shaikh, A.R. , 542–546 Shih, L.C. , 9 Shamloo, M. , 342, 431 Shim, Y.H. , 388 670 Author Index

Shima, D.T. , 542 Siddiqui, M.A. , 57, 58 Shima, K. , 10, 299, 491, 584 Sidor, A. , 62, 435 Shimabukuro, T. , 601 Siebler, M. , 6, 319 Shimada, I.S. , 11 Sieklucka, M. , 524, 527, 529 Shimamoto, K. , 443 Sieklucka-Dziuba, M. , 201, 523 Shimazaki, K. , 372, 530, 531 Siemen, D. , 435 Shimizu, K. , 371, 459, 465, 467, 468, 473 Siepmann, K. , 47 Shimizu, M. , 14, 53, 68, 70, 77, 78, 602, 604 Siesjö, B.K. , 20, 21, 23, 229, 244, 272, Shimizu, S. , 8, 63, 75, 369, 431, 530, 531 273, 431 Shimizu, T. , 366, 368–370, 372, 373 Sievers, R.E. , 431 Shimoda, L. , 441 Sieweke, M. , 192 Shimoda, N. , 461, 471 Sigaut, S. , 170 Shimohata, T. , 319, 331 Signore, A.P. , 184, 187, 196, 216, 299, 367, Shimoji, K. , 195, 431 395 Shin, D.S. , 27, 28, 370 Sihra, T.S. , 24 Shin, H.S. , 9 Sikora, J. , 605 Shin, M.L. , 9 Silasi-Mansat, R. , 190, 194 Shinitzky, M. , 141, 148 Silav, G. , 300 Shinmei, S.S. , 522 Silbergleit, R. , 173, 311, 312, 314 Shinmura, K. , 247 Silberman, D.M. , 542 Shinoda, A. , 501 Siles, E. , 190 Shinoda, S. , 531, 532 Silva, D.F. , 303 Shinozaki, Y. , 60, 75 Silva, R.F. , 410 Shintani, Y. , 392 Silvaggi, J.M. , 439 Shioda, S. , 436 Silver, I.A. , 20, 21, 431 Shiomi, H. , 46 Simeonidou, C. , 501, 503, 509, 510 Shiraishi, K. , 340 Simerabet, M. , 171, 322, 331 Shirihai, O.S. , 396 Simkhovich, B.Z. , 57 Shitara, H. , 185, 193, 195, 439 Simler, S. , 525, 530 Shiva, S. , 438 Simmons, D.D. , 185, 201 Shlafer, M. , 38 Simon, D.K. , 439 Shliar, J. , 506, 509 Simon, M.C. , 190, 192, 194 Shoemaker, W.J. , 270 Simon, R.P. , 4–11, 122, 134, 138, 139, 141, Shohami, E. , 235, 312, 340, 344, 370, 165, 169, 189, 202, 231, 235, 274, 499–504, 506–512 275, 277, 300, 313, 319, 322, Shoja, A. , 438 325–327, 330, 340, 344, 348, 349, Shokat, K.M. , 194 364, 366–372, 374–377, 380, 381, Short, A.D. , 435 436, 438, 463, 465, 467, 473, 474, Short, J.G. , 302 500, 503, 506, 522–525, 527–535, Shoshan-Barmatz, V. , 433 545 Shostak, G.A. , 327, 328 Simon, S.H. , 21 Shpargel, K.B. , 259 Simpkins, J.W. , 272 Shreeve, W.W. , 21 Simpson, K. , 297 Shri, R. , 245 Sinclair, D.A. , 441 Shroff, G.R. , 81, 318 Sing, C.F. , 270 Shu, Y. , 184, 185, 202 Singer, J.T. , 235 Shuaib, A. , 591 Singer, O.C. , 319 Shui, Q.X. , 592, 594 Singh, A.K. , 373 Shuke, N. , 37, 38 Singh, D. , 70 Shyr, M.H. , 584 Singh, I. , 373 Shyue, S.K. , 439 Singh, M. , 245 Sick, T.J. , 24, 26, 27, 248, 272, 348, 349, 436, Singh, N. , 232, 236, 245, 298, 322, 505 437, 494, 601 Singh, S.B. , 443 Siddiq, A. , 190, 192, 220, 297 Singh, S.N. , 605 Author Index 671

Singh, T.G. , 232, 236, 353, 400, 403 Sohn, T.K. , 440 Singhal, A.B. , 583 Soivio, A. , 45 Sinha, S. , 605 Sokoloff, L. , 20, 23, 38 Sinn, D.I. , 465, 474 Solaroglu, I. , 486, 487 Sinnamon, W.B. , 38 Sole, S. , 110 Sinor, A.D. , 503 Soleau, S. , 270 Siow, R.C. , 299 Solenkova, N.V. , 74 Sirabella, R. , 186, 191, 192, 331, 436, 437 Sollott, S.J. , 64 Siren, A.L. , 327, 510 Solodushko, V. , 74 Sirén, A.L. , 218, 219 Solomon, R.A. , 271, 273 Sisalli, M.J. , 186, 191, 192 Soltesz, I. , 522 Sitnik, N. , 184, 185, 187, 196 Solway, K. , 10 Sitoh, Y.Y. , 583 Somero, G.N. , 22, 25, 28 Siu, F.K. , 594 Somers, S. , 73 Sivarajah, A. , 406 Sommer, C. , 10, 202, 277, 346, 347, 558 Sivaraman, V. , 73 Sommerschild Hilchen, T. , 505 Sivaswamy, S. , 399 Somogyvári-Vigh, A. , 272 Skeen, J.E. , 443 Soncul, H. , 78 Skou, J.C. , 22 Song, C.X. , 394 Sleph, P.G. , 433 Song, E.-C. , 185, 191, 465, 474 Slivka, A. , 270 Song, E.J. , 440 Sloviter, R.S. , 10, 528 Song, J. , 80 Sluiter, W. , 605 Song, S. , 173, 299, 311, 312, 314 Sluse, F. , 190, 194 Song, X.Y. , 185, 186, 194, 442, 558 Sluzewski, M. , 295 Song, Y.J. , 6 Smallhorn, J.F. , 77, 78, 604 Song, Z. , 322, 331 Smerup, M. , 53, 602 Songur, A. , 156, 157 Smialowska, M. , 184, 185 Sonmez, A. , 219 Smith, A.J. , 494 Sontag, K.H. , 524, 527, 529 Smith, C.B. , 23 Sopa, C. , 524, 525, 527, 529 Smith, C.L. , 434 Sopko, J. , 585 Smith, E.E. , 136 Sorensen, H.R. , 23 Smith, G. , 580 Sorensen, H.T. , 11, 53, 82, 608 Smith, H. , 353 Sorensen, K. , 52, 53, 602, 603 Smith, M.A. , 6, 38, 40, 44–46, 81, 195, 433 Sorensen, P.H. , 442 Smith, M.-L. , 229, 272, 273, 367 Sorensen, P.M. , 23 Smith, P.K. , 569 Sorensson, P. , 81 Smith, R.A. , 494 Soriano, F.X. , 343, 354, 413 Smith, T.K. , 63, 353 Sorimachi, T. , 348 Smrcka, M. , 279 Soubrier, F. , 192 Smul,T.M. , 438 Soucy, J.P. , 583 Snabaitis, A.K. , 75 Souil, E. , 463, 467–469 Sneade, M. , 294 Souktani, R. , 73 Snider, B.J. , 44 Soukup, V.M. , 280 Snipes, J.A. , 294, 392 Souter, M. , 296 Snyder, E.M. , 353 Southard, J.H. , 41 Snyder, S.H. , 24, 437 Soutto, M. , 186, 201, 474, 475 So, N.K. , 531, 532 Souza Pinto, N.C. , 441 Sobrado, M. , 376 Sowter, H.M. , 192 Sobrino, T. , 500 Spandou, E. , 509, 510 Soderberg, M. , 141, 143, 144 Spatz, M. , 9, 184, 187, 500 Soderling, J.A. , 353 Speckmann, E.J. , 341 Sohmer, H. , 506 Spector, A.A. , 140 Sohn, I.S. , 78, 604 Spector, M.D. , 274 672 Author Index

Spees, J.L. , 11 Stenzel-Poore, M.P. , 5, 10, 41, 189, 202, 259, Spelle, L. , 296 260, 274, 300, 313, 343, 348, 364, Spencer, D.D. , 488 366–368, 370–377, 407, 463, 467, Spencer, E.M. , 534 474, 506, 527, 532–534, 568 Spera, P.A. , 4, 8, 165, 231, 234, 274, 349 Stephan, K. , 601, 602 Sperk, G. , 530 Stephanou, A. , 57 Sperling, O. , 348, 436 Stephen, T. , 296 Spicer, Z. , 193 Stephenson, D.T. , 436 Spiegelman, B.M. , 439, 443 Stephenson, F.A. , 340 Spirer, Z. , 141, 144 Sterling, D.L. , 584 Sportouch, C. , 63, 79, 80, 608 Stern, D.M. , 273 Sprague, S. , 106, 110 Stern, M.D. , 506, 507, 509, 510 Spranger, M. , 130 Sternau, R. , 124 Springett, R.J. , 438 Stetler, R.A. , 114, 117, 123, 216, 367, 398, Spyrou, G. , 184, 185, 194 399, 436 Srinivasula, S.M. , 367 Stevens, C.F. , 27 Sriram, S. , 186, 201, 474, 475 Stevens, L. , 144, 148 Srisuma, S. , 443 Stevens, S.L. , 5, 10, 41, 189, 202, 300, 313, Srivastava, S. , 299 343, 364, 366–368, 370–377, 407, St Pierre, J. , 395, 439 467, 506, 532–534, 568 Staab, P.K. , 297 Stevenson, L.A. , 500, 501 Staat, P. , 52, 53, 63, 79, 80, 318, 608 Stewart, J.P. , 534 Stacpoole, P.W. , 370 Stiefel, M. , 219, 510 Stafstrom, C.E. , 530 Stier, G. , 487, 489 Stagliano, N.E. , 234, 260, 278 Stimmelmayr, R., 38, 40 Stahel, P.F. , 510, 511 Stockman, B.A. , 175 Stahl, Z. , 142 Stoffel, W. , 430 Staikopoulos, V. , 442 Stokes, C. , 142 Stamatovic, S.M. , 313, 458–461 Stokoe, J. , 70 Stamova, B. , 375 Stolze, I. , 440 St-Andre, E. , 142 Stone, J. , 4 Stanimirovic, D. , 463, 468 Stone, T.W. , 494 Stanley, A.W. , 55, 72, 584 Storck, T. , 430 Stanton, P.K. , 184, 187, 188 Storey, K.B. , 26, 249 Stapels, M. , 189, 343, 381, 530, 535 Storlien, L.H. , 141 Staudt, M. , 457 Storm-Mathisen, J. , 25 Stecyk, J.A. , 29 Storts, R.W. , 340 Steeds, R.P. , 78, 604, 608 Stowe, A.M. , 5, 44, 184–186, 188, 194, Steenbergen, C. , 56, 60–62, 392, 429 196, 198, 202, 203, 293, 459, Steffens, D.C. , 606 463, 467, 468 Stehlik, C. , 438 Stowe, D.F. , 60, 61 Stein, A.B. , 63 Stowell, C. , 189, 343, 381, 530, 535 Stein, M. , 301 Stoykow, C. , 542 Steinberg, G.K. , 11, 41, 293, 311, 318–321, St-Pierre, J. , 443 325, 327, 328, 330, 331, 375, 531, Strackx, E. , 463, 471 563 Stranahan, A.M. , 113 Steinbusch, H. , 463, 471 Strasser, A. , 403, 531 Steiner, A.A. , 46 Strasser, R.H. , 71 Steiner, T. , 118 Straughan, J.L. , 220 Stella, A.M.G. , 443 Straumann, U. , 218 Stella, N. , 504 Strbian, D. , 234, 278 Stelmasiak, Z. , 523 Streit, W.J. , 9 Stengel, P. , 440 Striggow, F. , 214, 273 Stenslokken, K.O. , 29, 30 Strijbos, P.J. , 157 Author Index 673

Strijdom, J.G. , 57 Sutton, R. , 430 Strniskova, M. , 438 Suzuki, H. , 294, 300 Strobel, G. , 220 Suzuki, K. , 8 Stroev, S.A. , 184, 185, 194, 195 Suzuki, M. , 62, 214 Strohm, C. , 56, 57, 438 Suzuki, R. , 64 Strong, A.J. , 24, 280 Suzuki, S. , 273 Strong, R. , 279, 299, 439 Suzumura, A. , 9 Studdert, D.M. , 485 Svaren, J. , 373 Studer, F.E. , 11 Swafford, A. , 584 Stummer, W. , 106 Swain, J.L. , 5, 51 Stumpner, J. , 438, 561 Swain, R.A. , 110 Su, K.P. , 142 Swan, J.H. , 340 Su, X.W. , 250, 392, 399 Swanson, R.A. , 4, 248, 531 Su, Z. , 382 Swoap, S.J. , 46 Suehiro, E. , 118, 119 Swyer, P.R. , 143 Suehiro, S. , 433 Symon, L. , 24 Su ﬁ anova, G.Z. , 348 Symon, N. , 24 Suga, K. , 431, 435 Szabo, C. , 46, 47 Sugawara, T. , 8, 248, 294, 328, 416 Szabo, I. , 433 Sugden, P.H. , 57 Szabo, K. , 319 Sugimoto, C. , 327, 328 Szalay, M.S. , 505 Sugino, T. , 245 Szantho, G. , 53, 602 Sugioka, K. , 144 Szatkowski, M. , 453 Sugiura, T. , 501 Szekely, L. , 53, 602 Sugiura, Y. , 459, 465, 467, 468, 473 Szelid, Z. , 53, 602 Sugiyama, H. , 370 Szerdahelyi, P. , 61 Sugiyama, K. , 232, 237 Szweda, L.I. , 437 Suh, K. , 192 Sui, J. , 175 Sukagawa, A. , 501 T Sukoff, M.H. , 581 Tada, M. , 53, 64 Sulejczak, D. , 488 Tagaya, M. , 6, 138, 236, 553, 568, 579 Suliman, H.B. , 172, 173, 185, 193, 195, 439 Tahti, H. , 45 Sullivan, P.G. , 393 Tai, K.K. , 391 Sun, G. , 299, 319, 331, 439 Taie, S. , 184, 185, 188, 191, 197 Sun, G.H. , 531 Takabatake, Y. , 603, 607 Sun, G.J. , 595 Takada, Y. , 601 Sun, H.Y. , 300, 326 Takagi, H. , 370 Sun, J.L. , 6, 184, 185, 195, 199 Takagi, K. , 124 Sun, L. , 604, 609 Takahama, H. , 60, 75 Sun, N. , 504, 595 Takahashi, A. , 280, 464 Sun, W. , 184, 195 Takahashi, M. , 457 Sun, X. , 120, 491 Takahashi, R. , 192 Sun, X.C. , 245, 399, 466, 474 Takahashi, T. , 319, 328, 330, 331 Sun, Y.Z. , 594 Takamatsu, S. , 184, 185, 194 Sunami, K. , 581 Takano, H. , 63 Sundaram, M. , 353 Takano, K. , 234 Sundstrom, L.E. , 524, 530 Takano, T. , 184, 187, 195, 466, 472 Sung, J.H. , 331 Takaoka, A. , 68 Sunol, C. , 595 Takaoka, S. , 167 Surh, Y.J. , 443, 603 Takasawa, K. , 351 Sutherland, B.A. , 282 Takashima, S. , 60, 75 Sutherland, G. , 118 Takata, K. , 42, 167 Sutton, E.T. , 391 Takayanagi, K. , 603, 607 674 Author Index

Takeda, K. , 461, 471 Tariq, A. , 301 Takeda, M. , 544 Tarkka, M.R. , 577 Takeda, T. , 436 Tasaki, K. , 3, 9, 118, 367, 466, 474, 527 Takeda, Y. , 42, 279–281, 581 Tasca, C.I. , 494, 527 Takeishi, Y. , 56, 57 Taskapilioglu, O. , 322 Takemura, S. , 433 Tatarkova, Z. , 405 Takeo, S. , 58 Tatlisumak, T. , 234, 278, 553 Takeuchi, O. , 63 Tatsumi, T. , 58 Takeuchi, Y. , 187, 462, 468, 469 Tattersall, G.J. , 46 Taki, W. , 524–529, 531, 532 Taudorf, S. , 187, 202 Talaei, F. , 38 Tauskela, J.S. , 134, 137, 138, 154, 342, 347, Talalay, P. , 443 348, 351 Talantova, M.V. , 544 Tavakoli, S. , 68, 69, 71 Talmor, D. , 57 Tavernier, B. , 171, 322, 331 Tamagawa, M. , 62 Tay, A.S. , 175 Tamaki, N. , 262 Taylor, C.J. , 296 Tamargo, R.J. , 458 Taylor, C.P. , 431 Tamatani, M. , 399 Taylor, C.T. , 443 Tamura, A. , 118, 228, 229, 270, 272, 273, 342 Taylor, T.M. , 184 Tamura, T. , 494 Teasdale, G.M. , 228, 270, 273 Tamura, Y. , 46 Tecce, M.F. , 437 Tan, H.M. , 159 Tee, L.Y. , 44 Tan, W. , 63 Tejada-Berges, T. , 9 Tan, Y.H. , 368 Tekin, D. , 505 Tanabe, J. , 270–272, 275 Ten Broecke, P.W. , 175 Tanahashi, N. , 437 Teoh, R. , 368 Tanaka, A. , 310, 396 Tepikin, A.V. , 430 Tanaka, H. , 346, 356 Terasaki, Y. , 413 Tanaka, K. , 280, 340, 388, 431, 435, 437, 493, Terent, A. , 373 525–529, 531 Terkelsen, C.J. , 11, 53, 82, 608 Tanaka, M. , 61 Ternon, B. , 465, 472 Tanaka, S. , 433 Terzic, A. , 6, 60, 61, 435 Tandon, P. , 530 Tetievsky, A. , 506 Tang, B.L. , 368 Tetzlaff, W. , 512 Tang, J. , 279, 298, 301, 487–491, 541 Thakor, N.V. , 184–186, 195, 199 Tang, R. , 272 Thal, S.C. , 229 Tang, T.Y., 77, 562, 573, 604, 608 Thal, S.E. , 229 Tang, X.L. , 57, 63 Thambo, J.B. , 58 Tang, X.N. , 118, 409 Thamotharan, S. , 370 Tang, X.Q. , 436 Thangnipon, W. , 494 Tang, Y. , 8, 184, 185, 189, 191, 198, 199, 260, Thau Zuchman, O. , 370 264, 302, 366, 369–374, 407 Thayer, S.A. , 352, 503 Tang, Z.L. , 68–70 Thelen, M. , 353 Taniguchi, N. , 8 Themner, K. , 431 Tanno, M. , 436 Theus, M.H. , 185, 191, 196, 198 Tanonaka, K. , 58 Thibault, H. , 63, 79, 80, 608 Tantishaiyakul, V. , 438 Thielmann, M. , 78, 604 Tao, H. , 174 Thiemermann, C. , 3 Tao, J. , 53, 71 Thiersch, M. , 191, 192 Tao, L. , 46, 601 Thimmulappa, R.K. , 443 Tao, T. , 487, 490 Thivierge, J.P. , 342, 347, 351 Tapuria, N. , 293 Thivolet-Bejui, F. , 64 Tarabin, V. , 273 T’Hoen, P.A. , 382 Tariosse, L. , 58 Thom, L.H. , 299 Author Index 675

Thom, S.R. , 296, 299, 581 Tompkins, A.J. , 437, 439 Thomas, C.G. , 340, 343, 493 Tomsick, T. , 313 Thomas, H. , 57 Tong, H. , 56, 390 Thomas, L. , 4 Tong, L. , 167, 169 Thompson, C.B. , 433, 443 Tong, S. , 184–186, 195, 199 Thompson, J.L. , 529 Tong, W. , 173, 382, 487, 488, 490, Thompson, J.W. , 27, 28 491, 495 Thompson, R.C. , 9 Toombs, C.F. , 47 Thompson, R.E. , 580 Topol, E.J. , 76 Thompson, S.J. , 342, 348–350, 352–354, 404 Tornvall, P. , 81 Thorne, C. , 430 Toronen, P. , 530 Thornhill, J. , 118, 124 Torraco, A. , 436 Thornton, J. , 55, 72 Torrealba, J.R. , 41 Thornton, J.D. , 584 Torres, A. , 184, 187, 195, 466, 472 Thuesen, L. , 11, 53, 82, 608 Torres-Mendoza, B.M. , 441 Thuillez, C. , 468, 470 Torrey, D.J. , 353, 531 Thygesen, H.H. , 382 Tortella, F.C. , 353 Tian, H. , 310 Tosaki, A. , 61 Tian, X. , 441 Toutain, J. , 184, 199, 203, 323 Tian, Y. , 375 Touzani, O. , 199 Tikkanen, I. , 441 Touze, E. , 558 Timmler, M. , 348 Tovar, K.R. , 340, 344 Timofeev, I. , 511 Tow ﬁ ghi, J. , 184, 195, 264, 527 Timsit, S. , 369 Townend, J.N. , 78, 604, 608 Tinel, N. , 546 Townsend, P. , 78, 604, 608 Tirosh, A. , 57 Toyoda, T. , 10 Tirrell, D.A. , 535 Trachootham, D. , 193 Tischer-Zeitz, T. , 438, 561 Trani, D. , 76 Titova, E. , 166, 173, 295, 301, 487, Trapp, S. , 171, 172, 493 489, 491 Trautner, S. , 11, 53, 82, 608 Tixier, E. , 184, 199, 203, 323 Traverse, J.H. , 81, 318 Tjulkova, E.I. , 184, 185, 187, 194–196 Traynelis, S.F. , 220 Tjwa, M. , 190, 194 Traystman, R.J. , 168, 229, 231, 235, 275, 431, Tobar, E.T. , 5, 41, 189, 274, 364, 366, 461, 470 370–372, 532 Treggiari, M.M. , 296 Tocco, G. , 523 Treiman, M. , 80, 318 Todd, M.M. , 271, 493 Treinin, M. , 506, 509 Todgham, A.E. , 506 Trembovler, V. , 235, 500, 503, 504, 508–512 Toescu, E.C. , 167 Trendelenburg, G. , 184, 261, 472 Toien, O. , 37, 38, 44–46 Triche, T.J. , 442 Toime, L.J. , 439 Tritto, I. , 61 Toka, O. , 439 Trocciola, S. , 574, 603, 605, 607 Tokuda, M. , 346 Trouillas, P. , 582 Tolon, R.M. , 504 Troy, C.M. , 192 Tolunay, S. , 322 Truettner, J. , 247 Toma, O. , 172 Truong, D.D. , 391 Tomai, F. , 5 Trush, M.A. , 441 Tomasevic, G. , 248, 431 Tsai, B.M. , 557 Tomida, S. , 244, 558 Tsai, C.Y. , 594 Tomino, T. , 280 Tsai, H.D. , 439 Tomita, S. , 184, 185, 188, 191, 197 Tsai, M.-H. , 185, 201 Tomkowicz, B.E. , 458 Tsai, Y.S. , 439 Tomoike, H. , 60, 75 Tsang, A. , 56, 73, 74, 441 Tomonaga, M. , 144, 310 Tsang, V. , 11 676 Author Index

Tsarouchas, K. , 53, 82, 608 Ubogu, E.E. , 458 Tschimmel, K. , 367 Uchida, K. , 512 Tseng, C.-Y. , 185, 201 Udo, H. , 370 Tseng, M.T. , 5, 19, 184, 194 Ueda, H. , 6, 236, 579 Tsenter, J. , 340, 344, 507–510 Ueda, T. , 24 Tsien, J.Z. , 439 Uehara, N. , 232, 237 Tsolaki, M. , 184–186, 194 Ueki, M. , 184, 185, 188, 191, 197 Tsou, M.T. , 593, 594 Ueng, S.W. , 584 Tsuchida, A. , 55 Ugawa, Y. , 459, 465, 467, 468, 473 Tsuchiya, A. , 10 Ukai, R. , 10 Tsuchiya, D. , 531 Uldry, M. , 439 Tsuchiya, T. , 272 Ulevitch, R.J. , 56 Tsujimoto, Y. , 63, 75, 431 Ullmann, C. , 438 Tsujita, T. , 443 Ultsch, G.R. , 25 Tsujita, Y. , 229 Ulus, I.H. , 141 Tsukamoto, O. , 60, 75 Um, H.C. , 443 Tsung, A. , 303 Umemura, K. , 174 Tsunoda, H. , 300 Umezawa, K. , 214 Tsuruta, R. , 303 Umschwief, G. , 508, 510, 511 Tsutsumi, Y.M. , 76 Unal, S. , 561 Tsuzuki, N. , 299, 491 Undar, A. , 250 Tu, H.-C. , 185, 201 Ungerleider, R.M. , 367 Tu, Y.F. , 458, 462, 472 Ungvari, Z. , 47 Tuffy, L.P. , 531 Upadhya, S.C. , 353 Tugoi, I.A. , 184, 185 Upadhyay, U.M. , 458 Tulkova, E. , 184, 185, 187, 193 Urban, P. , 399 Tuma, R.F. , 504 Urenjak, J. , 25 Tuominen, H. , 11 Ursell, P.C. , 431 Tupper, K.Y. , 542, 546 Usansky, H. , 47 Turcato, S. , 556 Ushio, Y. , 187, 230, 273, 462, 468, 469 Turecki, G. , 142 Uske, A. , 294 Turetsky, D.M. , 347, 349, 351 Usov, L.A. , 348 Tureyen, K. , 373 Ustun, M.E. , 296 Turnbull, L. , 556 Uto, A. , 434 Turner, A.J. , 184, 185, 200 Turner, M.S. , 53, 602 Turner, R. , 375 V Turovskaya, M.V. , 193 Vaage, J. , 29, 558 Turovsky, E.A. , 193 Vabulas, R.M. , 4, 353 Turturici, G. , 398 Vadigepalli, R. , 377 Tuszynski, M.H. , 512 Vahtola, E. , 441 Tuttolomondo, A. , 222 Vaidya, A. , 441 Twig, G. , 396 Vajkoczy, P. , 197, 295, 470 Tyagi, A.K. , 605 Valdés, I. , 38, 124 Tymianski, M. , 340, 343, 349, Valencia, I. , 184, 185 351, 354 Valentim, L.M. , 367, 399 Tytell, M. , 4 Valk, T. , 270 Tyul’kova, E.I. , 184, 185, 192, 193 Vallance, P. , 603 Tzeng, Y.W. , 190, 201 Vallet, B. , 171, 322, 331 Vallyathan, V. , 438 Valsecchi, V. , 186, 191, 192 U Valter, K. , 4 Uauy, R. , 140 Van Arsdell, G.S. , 77, 78, 604 Ubl, J.J. , 215 van Bel, F. , 198, 436 Author Index 677

van Beusekom, H.M. , 605 Vartanian, K.B. , 189, 202, 366, 368, van Buiten, A. , 38 374–377 Van Cleemput, J. , 524, 525, 530 Varty, K. , 604 van Dam, A. , 38 Vasquez, J.A. , 6 van Delft, S. , 215 Vass, K. , 558 van den Doel, M.A. , 65, 68, 69 Vataeva, L. , 184, 185 Van den Hove, D. , 463, 471 Vatansever, E. , 184, 185, 194 Van den Thillart, G. , 29 Vaux, E.C. , 440 van der Graaf, Y. , 295 Vavetsi, S. , 53, 82, 608 van der Jagt, M. , 294 Vavrek, R. , 512 van der Kooij, M.A. , 198 Vaynman, S. , 113 van der Meide, P.H. , 368 Vazquez Valls, E. , 441 van der Stelt, M. , 501 Veh, R.W. , 273 van der Veer, E. , 441 Vejlstrup, N. , 80, 318 van der Weerd, L. , 367 Velasco, G. , 503 van der Worp, B.H. , 134, 136 Velazquez, O.C. , 299 van Dijk, E.J. , 294 Veldhuis, W.B. , 368, 501 Van Eyk, J.E. , 62, 435 Veldink, G.A. , 501 Van Geyte, K. , 190, 194 Vellimana, A.K. , 188, 201, 292, 293, 297, 300, van Gijn, J. , 297 463, 468, 469, 471, 572 van Haaften, G.W. , 501 Velly, L.J. , 170, 171, 251 Van Hecke, P. , 190, 194 Vemuganti, R. , 185, 189, 231, 234, 299, van Heijningen, C.L. , 68–70 364–366, 368, 370, 371, 373, 380, van Hinsbergh, V.W. , 215 414, 438, 458, 460, 461, 467 Van Houten, B. , 436 Vendite, D. , 523, 524, 527 van Melle, G. , 6, 531 Venkatachalam, M.A. , 439 Van Meter, K. , 297 Ventureyra, E.C. , 349 Van Meter, M. , 441 Venugopal, V. , 52, 77, 78, 571, 604, 608 van Nieuw Amerongen, G.P. , 215 Verdouw, P.D. , 65, 68, 69 Van Noten, P. , 190, 194 Vergnes, M. , 525, 530 van Ommen G.J. , 382 Verkhratsky, A. , 167 van Rensburg, S.J. , 142 Vermeer, M.A. , 215 van Rooyen, J. , 606 Vernhet, H. , 608 van Soest, S. , 541, 543 Vertesaljai, M. , 53, 602 Van Veldhoven, P. , 190, 194 Veselis, R.A. , 166 Van Waversveld, J. , 29 Vessey, D.A. , 74 Van Winkle, D.M. , 55, 72 Vester, J.W. , 25 Van Wylen, D.G. , 8 Vexler, Z.S. , 112, 367 Vanden Hoek, T. , 61, 390 Vezzani, A. , 530 Vanden Hoek, T.L. , 61, 63, 430 Viader, F. , 581 VandenBerg, P.M. , 113 Vichare, S. , 52, 77, 78, 604, 608 Vandendriessche, T. , 190, 194 Victorov, I.V. , 184, 185, 191, 198, 201, 219, Vander Heide, R.S. , 60 220, 273 Vander Heiden, M.G. , 433 Videen, T.O. , 310 Vandresen-Filho, S. , 527 Vidyadaran, S. , 531 Vangeison, G. , 265, 472, 473 Viera, D. , 137 Vanicky, I. , 327 Vigh, S. , 272 Vankar, G.K. , 142 Vigilanza, P. , 441 Vannucci, R.C. , 184, 195, 264, 265, 527 Vilarim, M.M. , 142 Vannucci, S.J. , 184, 195, 264, 265, 459, 460, Villa, P. , 219 462, 469–471 Villamor, E. , 463, 471 Vara, J.A.F. , 328 Villarroya, F. , 439 Varadarajan, R. , 443 Villringer, A. , 6, 319 Varon, S. , 512 Vinit, S. , 185, 201 678 Author Index

Vinten-Johansen, J. , 52, 53, 70–75, 79, 80, Walker, W. , 302 317, 326 Walkinshaw, G. , 52, 83 Violante, A. , 61 Wallace, M.C. , 175 Viquez, N.M. , 352 Walsh, R.A. , 56, 57 Vivancos, J. , 466, 473, 500 Walsh, S.R. , 77, 562, 573, 604, 608 Viviani, B. , 219 Walski, M. , 488 Vivien, D. , 184, 185, 191, 197–199, 202, 510 Waltenberger, J. , 8, 184, 187, 199, 200 Vlasov, T.D. , 463, 468, 472 Walter, C. , 543 Vles, J.S. , 463, 471 Walter, P. , 194, 397 Vogel, J. , 273 Walton, B. , 603 Vogel, M. , 52, 603 Waltz, A. , 24 Vogt, P.K. , 190 Wan, R. , 443 Volk, H-D. , 273 Wan Sommeren, E. , 175 Vollrath, B. , 301 Wan, Y. , 184, 185, 202 Volpe, J.J. , 459, 474, 475 Wang, B.G. , 174, 294, 299, 322, 331 von Arnim, C.A. , 348 Wang, C.-H. , 57, 167, 168, 170, 172, 184, 185, von Bruehl, M. , 438 187, 193, 194, 201, 274 von der Ohe, C.G. , 352 Wang, C.Z. , 62 von Engelhardt, J. , 343 Wang, D. , 328 von Kummer, R. , 136 Wang, E.Z. , 171, 294, 373 Voorhees, J.R. , 488 Wang, F.-Z. , 120, 184, 186, 194, 195, 466, Vorhees, C. , 263 473, 474, 595 Vorhees, C.V. , 171, 172 Wang, G. , 120, 187, 201 Vos, I.M. , 368 Wang, G.W. , 56 Vos, I.M.P. , 368 Wang, G.Y. , 556 Voskuyl, R.A. , 148 Wang, H. , 120, 171, 215, 297, 409, 442, 460, Vosler, P. , 367 464, 466, 468, 474, 592, 593, 595, Vossen, R.H. , 382 601 Vreugdenhil, E. , 382 Wang, H.N. , 173 Vreugdenhil, M. , 148 Wang, H.Q. , 271 Wang, H.S. , 80 Wang, J. , 170, 171, 311 W Wang, J.-A. , 184 Waataja, J.J. , 352, 503 Wang, J.K. , 326 Wachter, U. , 280 Wang, J.M. , 459 Wacker, B.K. , 184, 185, 188, 192, 198, 458, Wang, J.Q. , 319 463, 468, 470 Wang, J.Y. , 245, 322, 325, 464, 470 Wada, K. , 244, 299, 491, 584 Wang, J.Z. , 367 Wada, T. , 262 Wang, K. , 558 Wada, Y. , 184, 185, 194 Wang, L. , 6, 60, 62, 168, 174, 176, 188, 190, Waddell, T.K. , 461, 467 280, 438, 441, 512, 593 Waeber, C. , 11, 273 Wang, L.-Y. , 192, 232, 237 Waghray, A. , 401 Wang, L.F. , 80, 81 Wagner, K.R. , 310 Wang, M. , 464, 467, 557 Wagner, R. , 78, 604 Wang, M.M. , 294, 296, 314, 315 Waguri, S. , 401 Wang, M.Q. , 294 Wahli, W. , 438 Wang, N.P. , 53, 71–75, 79, 171, 317, 322, 326, Wakeno, M. , 60, 75 331, 373, 544 Wakita, H. , 45 Wang, P.Y. , 55, 310 Waldow, T. , 245 Wang, Q. , 9, 167, 168, 171, 173, 185, 188, Waldron, R.T. , 435 409, 437, 459, 461, 462, 464, Walid, S.M. , 296 467–469, 492, 493, 591–596 Walker, D.M. , 243 Wang, R. , 166, 168, 169, 174 Walker, J.M. , 53, 54, 64, 82, 243 Wang, R.M. , 342, 401 Walker, M.C. , 52, 83, 534 Wang, R.Y. , 106 Author Index 679

Wang, S.T. , 62, 64, 170, 232, 237, 294, 367, Weber, R. , 604 394, 435 Wegener, S. , 6, 559–561 Wang, T. , 56, 57, 80, 184 Wei, C. , 441 Wang, W. , 81, 441, 531, 586 Wei, D. , 293, 325 Wang, X. , 4, 8, 9, 109, 111, 154, 156, 165, Wei, E.Q. , 184, 185, 197, 199, 461, 467, 470 190, 195, 231, 234, 274, 278, 349, Wei, G.Y. , 271, 510 392, 410, 438, 441, 568, 570, 571, Wei, H. , 400 586 Wei, I.H. , 185, 201 Wang, X-J. , 270, 272 Wei, J. , 184, 280, 443 Wang, X.M. , 154, 156 Wei, L. , 184, 185, 191, 196, 198, 271, 459 Wang, Y. , 53, 56, 57, 63, 71, 118, 167, 169, 171, Wei, M. , 53, 71, 568, 570, 571 366, 368–370, 372, 373, 595, 596 Weichselbaum, R. , 367 Wang, Y.H. , 596 Weih, M.K. , 6, 138, 184, 259, 559, 560 Wang, Y.L. , 81, 318 Weihrauch, D. , 388, 493 Wang, Y.M. , 595 Weimar, C. , 219, 222 Wang, Y.P. , 259 Weinberg, J.M. , 439 Wang, Z.P. , 159, 170, 299 Weinbrenner, C. , 57, 71 Wang N-P , 73 Weinstein, P.R. , 8, 112, 227, 271, 279, 299, 531 Wapinsk, I. , 507, 510 Weintraub, N.L. , 53, 71 Wapinski, I. , 506 Weis, J. , 47 Ward, H.B. , 605 Weiss, H.R. , 118, 169, 221, 277, 459, 461, Ward, M.A. , 7, 345, 351 469, 470 Ward, M.W. , 434–435 Weissenborn, K. , 219, 222 Ward, N. , 192 Weisser, J.D. , 392 Ward, P. , 172, 411, 493, 557 Weissmann, G. , 4 Warden, C.H. , 439 Welch, J.L. , 582–584 Warltier, D.C. , 493 Welch, K.M. , 4, 124, 443 Warnecke, C. , 192 Welch, K.M.A. , 272 Warner, D.S. , 172, 173, 271, 279, 493 Welch, W.J. , 4 Warnock, G. , 544 Weller, M. , 8, 184, 187, 199, 200 Warren, D.E. , 28 Wells, D.J. , 531 Wartenberg, K.E. , 219, 222, 296 Wells, K.E. , 531 Warzecha, Z. , 313 Welsh, F.A. , 8, 232, 235, 276, 277 Waschke, K.F. , 167, 169, 170, 280, 298, Welsh-Bohmer, K.A. , 606 492, 493 Wen, C.-Y. , 185, 201 Wasiewski, W.W. , 591 Wen, R. , 543, 545 Wasley, L.C. , 435 Wendel-Vos, G.C. , 105 Wassmann, H. , 492 Weng, Z. , 367 Wasterlain, C.G. , 522 Wenger, R.H. , 190, 218 Watabe, A.M. , 344 Wenzel, A. , 185, 191, 201 Watanabe, K. , 605 Wernecke, K.D. , 6 Watanabe, M. , 56, 344, 391 Werner, C.G. , 345, 347 Watanabe, T. , 63, 75 West, C. , 5, 19, 184, 194 Watchel, M. , 273 West, D. , 76 Waterman, P. , 137 West, G.A. , 274 Watkins, S.C. , 8, 369, 530–532 West, I.C. , 62, 64, 74, 436, 437 Watson, A.J. , 430 Westberg, J.A. , 188, 198, 201 Watson, B.D. , 270, 273, 280 Westbrook, G.L. , 24, 340, 343, 344 Watson, M. , 280 Westerkamp, M. , 47 Waypa, G.B. , 430 Westermann, B. , 438 Webb, C.L. , 9 Westphal, B. , 582, 587 Weber, E. , 271, 345 Wettstein, G. , 402 Weber, J.R. , 367 Whalen, R.E. , 580 Weber, M.L. , 431 Wheeler, T.G. , 143 Weber, N.C. , 172, 604 White, C.W. , 9, 401 680 Author Index

White, D.E. , 544 Wintner, E.A. , 47 White, D.M. , 114, 118, 119 Wise, D.R. , 194 White, J.O. , 435 Wise, G. , 347 White, J.R. , 192 Wisniewski, S.R. , 142 White, P.A. , 52, 53, 602, 603 Wittert, G.A. , 442 White, R.F. , 4, 5, 8, 9, 118, 165, 231, 234, 270, Woitzik, J. , 197, 470 274, 278, 349, 368 Wojcik, C. , 353 Whitlock, N.A. , 186, 191, 200, 545 Wojner, A.W. , 323 Whitman, G.J. , 60, 61 Wolf, T. , 273 Whittaker, P. , 11, 52, 65, 313, 602 Wolfe, C.L. , 431 Whittingham, T.S. , 21, 24 Wolfrum, S. , 70, 71 Wiart, M. , 438 Wolke, L. , 65, 68 Wick, A. , 8, 184, 187, 199, 200 Won, J.S. , 373 Wick, W. , 8, 184, 187, 199, 200 Wong, G.K. , 571, 572, 574 Widenmaier, S.B. , 544 Wong, P.T. , 175 Widmann, C. , 8, 56, 117, 138, 141, 143, Wong, R. , 572 148–150, 154, 155, 348, 409, 410, Wong, T.M. , 435 525, 529, 530 Wong, T.P. , 343, 344 Wiegand, F. , 184 Wong, W.J. , 190, 192, 194 Wieloch, T. , 229, 247, 342, 367, 431 Wong-Riley, M.T. , 395 Wieronska, J.M. , 184, 185 Wood, A. , 137 Wiesener, M.S. , 192, 440 Wood, I.C. , 534 Wieser, W. , 29 Wood, P.L. , 343 Wiggert, B. , 545 Woods, I. , 531 Wightman, K.A. , 340 Woodward, C.L. , 3 Wijeyekoon, B. , 190, 194 Woolsey, T.A. , 459 Wikinski, R.L. , 75 Wootton, L.L. , 405 Wilainam, P. , 11 Wotzlaw, C. , 440 Wilkins, H.M. , 159 Wrang, M.L. , 346, 351 Willam, C. , 190, 194 Wright, D.C. , 439 Willecke, K. , 184, 187, 195, 466, 472 Wright, E. , 9 Willette, R.N. , 9, 271 Wright, M.J. , 56, 57 Williams, A.J. , 353 Wright, T.M. , 442, 443 Williams, B.T. , 601 Wroblewski, J.T. , 24 Williams, D.L. , 367, 458, 459, 462, 467, 468 Wu, A. , 522 Williams, M.T. , 296 Wu, B. , 173 Williams, R. , 603 Wu, C.-L. , 263, 264, 462, 467–469 Williams, V. , 351 Wu, C.W. , 584 Williams-Karnesky, R.L. , 274, 348, 374 Wu, G. , 436 Williamson, E.K. , 433 Wu, H. , 415, 416 Williamson, J.R. , 373, 429 Wu, J.S. , 217, 220, 296, 312, 314, 439 Williamson, J.W. , 111 Wu, K.K. , 439 Wilmshurst, P. , 581 Wu, L.-Y. , 184, 186, 195 Wilson, A.L. , 43 Wu, M. , 167, 169, 298, 493 Wilson, M.A. , 459, 460, 462, 469–471 Wu, M.S. , 441 Wilson, P.V. , 175 Wu, M.T. , 594 Wilz, A. , 11 Wu, P.H. , 439 Wimpee, H. , 62 Wu, R.T. , 190, 201, 328 Windheim, M. , 353 Wu, S.C. , 293, 298, 299 Winfree, C.J. , 273 Wu, W. , 184, 185, 195, 199 Winkler, J. , 469 Wu, W.J. , 63 Winston, S.M. , 237 Wu, W.K. , 159 Winter, B. , 273 Wu, X.M. , 412, 459, 465, 487, 490 Winters, C.A. , 434 Wu, Y. , 402 Author Index 681

Wu, Z. , 441 Xu, N. , 174, 491, 584, 593 Wu, Z.H. , 436 Xu, Q. , 184–188, 195, 436, 466, 472 Wu, Z.K. , 570 Xu, R. , 487, 490 Wurtman, R.J. , 141 Xu, W.G. , 62, 173, 392, 435 Wyatt, A.W. , 63 Xu, X. , 169, 172, 297 Wykoff, C.C. , 440 Xu, X.-J. , 185, 186, 197, 199 Wylie-Rosett, J. , 457 Xu, X.M. , 4, 221 Wyllie, D.J. , 354 Xu, Y. , 439 Wynn, S. , 531 Xu, Z.C. , 184 Xuan, J. , 441 Xuan, W. , 406 X Xuan, Y.T. , 57 Xaus, C. , 443 Xue, D. , 270 Xi, G.M. , 173, 214–220, 234, 271, 294, 296, Xue, F. , 80 299, 309–315, 459, 463, 467, 468 Xue, J.-H. , 270 Xi, L. , 72, 505 Xue, J.J. , 8 Xia, P. , 171, 373 Xue, X.Y. , 303 Xia, Q. , 322, 325, 435, 464, 470 Xue, Y.X. , 458–461, 468 Xia, X.Y. , 399, 459, 462, 467, 468 Xue-Ying, S. , 487, 490 Xia, Y. , 173, 174, 184, 186, 193, 200, 201 Xia, Y.X. , 459, 462, 467, 468 Xia, Y.Z. , 173, 297 Y Xia, Z. , 81, 245, 351, 594 Yabe, T. , 68 Xian, X.H. , 399, 466, 474 Yabuuchi, Y. , 59 Xiang, B. , 274 Yagi, T. , 601 Xiang, J. , 216, 217, 294, 296, 299, 312, 314, 315 Yakes, F.M. , 436 Xiao, J. , 441 Yakovlev, A.G. , 510 Xiao, L. , 70, 186, 198 Yakubchyk, Y. , 349 Xiao, Z.S. , 70 Yam, D. , 141, 148 Xie, J. , 185, 186, 193, 391, 392 Yamada, J. , 319 Xie, Z. , 400 Yamada, K. , 117 Xin, L. , 368 Yamagata, H. , 63, 75 Xin, P. , 53, 71 Yamaguchi, F. , 346 Xing, B. , 319, 322, 332, 601 Yamaguchi, H. , 603, 607 Xiong, J. , 167 Yamaguchi, K. , 346 Xiong, L.Z. , 166–169, 172–174, 184, 186, 298, Yamaguchi, M. , 299, 300, 487, 489 299, 459, 461, 467, 468, 491–493, Yamaguchi, O. , 63, 75 584–586, 591–596, 604, 609 Yamaguchi-Okada, M. , 299 Xiong, Y. , 187, 190 Yamahara, Y. , 58 Xiong, Z.G. , 5, 41, 189, 231, 235, 295, 313, Yamakura, T. , 195 319, 322, 325–327, 330, 340, 343, Yamamoto, A. , 531, 532 349, 351, 354, 364, 366, 370–372, Yamamoto, M. , 443 381, 530–532, 535, 568 Yamamoto, S. , 433 Xiu, J.X. , 56 Yamamoto, T. , 459, 465, 467, 468, 473 Xu, B. , 528 Yamamoto, Y. , 524, 525, 527, 529, 530, 601 Xu, D. , 328 Yamaoka, Y. , 601 Xu, E. , 184, 195 Yamasaki, K. , 61, 433 Xu, F. , 166 Yamasawa, I. , 5, 51, 601 Xu, G.P. , 245, 297 Yamashita, N. , 53, 64 Xu, H. , 121, 123, 189, 192, 302, 375 Yamashita, S. , 173, 216, 217, 299, 303 Xu, J.J. , 4, 173, 221 Yamashita, T. , 10 Xu, K. , 442 Yamawaki, T. , 272 Xu, L. , 73, 80, 403 Yamin, A. , 511 Xu, M. , 63 Yan, C. , 56, 57 682 Author Index

Yan, F.S. , 185, 186, 194 Yap, J. , 73, 78 Yan, G. , 166, 168, 169 Yarimizu, K. , 5 Yan, H.M. , 595 Yarnold, J.A. , 294 Yan, J.H. , 299 Yarov-Yarovoy, V. , 6, 62, 81, 433 Yan, M. , 326 Yasugawa, S. , 230 Yan, Q. , 512 Yasumura, D. , 194 Yan, W. , 173, 401, 402 Yatsiv, I. , 509, 510 Yan, Z.W. , 293, 318, 323–325, 441 Yatsushige, H. , 300 Yanamoto, H. , 270, 274, 277 Yayama, T. , 512 Yanez, N.D. , 296 Ye, R. , 171 Yang, B. , 328 Ye, Z. , 171, 322, 331, 373 Yang, C. , 545, 546 Ye, Z.B. , 441 Yang, C.C. , 441 Ye, Z.G. , 322, 325, 464, 470 Yang, C.F. , 594 Yeh, M.L. , 167 Yang, D. , 544 Yellon, D.M. , 6, 11, 52–54, 56, 57, 59, 63, 64, Yang, E.J. , 147 67–75, 77, 78, 82, 83, 243, Yang, F. , 342, 401 441–443, 506, 557, 562, 563, 569, Yang, G.Y. , 9, 78, 109, 370, 458 571, 602–604, 608 Yang, H. , 441 Yenari, M.A. , 118, 319, 327, 367, 531 Yang, H.S. , 437 Yeo, T.T. , 583 Yang, H.Y. , 390 Yi, J.H. , 373 Yang, J.-J. , 184, 185, 187, 193–195, 466, 472, Yildirim, F. , 408 593–596 Yilmaz, O. , 219 Yang, K. , 214, 222 Yin, C. , 392 Yang, L. , 585, 596 Yin, K.J. , 438 Yang, L.F. , 604, 609 Yin, W. , 184, 187, 196, 264, 299, 395, 583 Yang, L.J. , 373 Yin, X.H. , 342 Yang, M.J. , 326 Yin, Y. , 184–186, 188 Yang, P. , 594 Yin, Z. , 601 Yang, Q. , 171, 595 Ying, W. , 248 Yang, Q.W. , 367 Yip, P.K. , 270 Yang, Q.Z. , 459, 461, 467, 468, 595, Yitzhaki, S. , 396 604, 609 Ylostalo, J. , 11 Yang, S.D. , 272, 296, 584 Yoder, E. , 140 Yang, S.Y. , 322, 325, 464, 470 Yofu, S. , 436 Yang, T. , 189, 202, 231, 235, 343, 366–368, Yogaratnam, J.Z. , 571, 585, 586 374–377, 380, 381, 438, 441, 467, Yokoo, N. , 167 530, 535 Yokota, H. , 346, 356 Yang, W.K. , 184–186, 188, 439, 443, 504 Yokoyama, K. , 169, 391, 493 Yang, X. , 80, 185, 186, 188 Yokoyama, T. , 76 Yang, X.C. , 80, 81 Yoneda, Y. , 524, 525, 527, 529, 530 Yang, X.M. , 55, 56, 60, 62, 72–75, 299, 326, Yonekawa, H. , 185, 193, 195, 439 390, 392, 436 Yonekawa, Y. , 218 Yang, Y. , 391, 530 Yonekura, Y. , 184, 185, 194 Yang, Y.L. , 123 Yong, C. , 443 Yang, Y.R. , 106 Yong, V.W. , 9 Yang, Z.-J. , 231, 235, 278–280 Yonoki, Y. , 541 Yannopoulos, F. , 11, 562 Yoo, M.A. , 440 Yano, S. , 187, 462, 468, 469 Yoon, B.W. , 231, 235 Yan-Ting, G. , 487, 490 Yoon, D.H. , 184, 201 Yao, S.Y. , 186, 201, 474, 475 Yoon, H.J. , 380 Yao, W.X. , 459, 468, 471, 472 Yoon, S. , 80 Yao, Z. , 55, 61 Yoon, Y.W. , 71, 562, 602 Yaoita, H. , 605 Yoshida, N. , 5 Author Index 683

Yoshida, T. , 57 Z Yoshida, Y. , 123, 271, 370 Zacchigna, S. , 190, 194 Yoshikawa, Y. , 271 Zacharowski, K. , 3 Yoshimoto, T. , 214 Zagorski, J. , 10 Yoshimura, M. , 431 Zaha, V. , 442, 443 Yoshimura, S. , 319 Zaid, H. , 433 Yoshinaga, S. , 310 Zai-Hua, X. , 487, 490 Yoshishige, D. , 72, 73 Zak, D.E. , 377 Yoshizumi, M. , 56, 57 Zaleska, M.M. , 137 You, X. , 367 Zaloga, C. , 277 Young, A.B. , 24 Zambrano, N. , 186, 191, 192 Young, A.C. , 500 Zamudio, F.Z. , 303 Young, C.N. , 458, 460, 461, 467 Zanier, E.R. , 231, 234, 465, 468, 472, 473 Young, D.M. , 439 Zapert, K. , 485 Young, J.M. , 168, 176, 557, 558 Zatta, A.J. , 53, 72–75 Young, K.C. , 573 Zaugg, M. , 73 Young, L.H. , 442, 443 Zaun, D.A. , 583 Young, P.R. , 9 Zausinger, S. , 279 Young, S. , 437 Zawadzki, J.V. , 437 Ytrehus, K. , 55, 64 Zaytseva, N.V. , 296 Yu, A.Y. , 8, 44, 184, 186, 191, 194, 297, 441 Zazulia, A.R. , 310 Yu, B. , 171, 172 Zboyan, H.A. , 142 Yu, C. , 561 Zea Longa, E. , 271 Yu, F. , 280, 297 Zechner, C. , 442 Yu, H.M. , 436 Zeger, S. , 606 Yu, H.Y. , 584 Zeng, D. , 78 Yu, J. , 326, 402 Zeng, X.-Z. , 185, 186, 197, 199 Yu, L.M. , 294 Zeng, Y. , 594 Yu, L.N. , 326 Zerbe, R. , 188, 201 Yu, Q. , 171, 460, 464, 466, 468, 474 Zgodzinski, W. , 201 Yu, S. , 184, 194, 195, 466, 473, 474 Zhan, B. , 601 Yu, S.M. , 106 Zhan, L. , 184, 195 Yu, S.P. , 184, 185, 191, 196, 198 Zhan, Q. , 186, 198 Yu, S.Y. , 584 Zhan, R.-Z. , 195 Yu, T.W. , 10 Zhan, X. , 41, 42, 45, 167, 169, 248, 272, Yu, W. , 441 278, 375 Yu, X. , 297, 595 Zhan, Y. , 279 Yu, X.C. , 595 Zhang, A. , 443 Yu, Y. , 595 Zhang, B. , 80 Yu, Z. , 399 Zhang, C. , 56, 57, 194, 439, 542, 543, 545 Yuan, H.B. , 114, 115, 117, 123 Zhang, D. , 433 Yuan, L.B. , 167, 169, 400, 403 Zhang, F. , 114, 117, 123, 367 Yuan, Y. , 171, 193, 322, 331, 373 Zhang, F.J. , 326 Yue, T.L. , 9, 57 Zhang, F.Y. , 459, 464, 468 Yue, Y. , 57, 60, 62 Zhang, G.Y. , 342 Yuen, A.W. , 141, 148 Zhang, H. , 10, 319, 328, 330, 331, 401, Yun, H. , 443 402, 441 Yun, L. , 173 Zhang, H.J. , 583 Yundt, K. , 310 Zhang, H.P. , 167, 169, 400, 403 Yung, W.H. , 184, 186, 191, 198, 459, 465 Zhang, J. , 46, 56, 57, 63, 166, 173, 174, 184, Yun-Jie, W. , 487, 490 186, 193, 200, 201, 231, 235, Yunoki, M. , 10, 114–116, 119–121 278–280, 295, 299, 390, 393, 394, Yvon, A. , 184, 185, 191, 197–199, 436, 438, 439, 486–489, 491 202, 510 Zhang, J.-X. , 184, 185, 197, 199 684 Author Index

Zhang, J.H. , 173, 174, 291, 293–297, 299–301, Zheng, K. , 439 314, 486–491, 495, 583, 605 Zheng, L. , 542 Zhang, J.M. , 53, 71 Zheng, S. , 114, 115, 117, 123, 166–169, Zhang, J.X. , 461, 467, 470 373, 492 Zhang, K. , 184, 186 Zheng, X. , 388 Zhang, L. , 10, 80, 168, 174, 186, 191, 280, Zheng, Y.T. , 56, 57, 167, 169, 298, 493, 586 299, 328, 367, 459, 595 Zheng, Z. , 367 Zhang, L.-Y. , 184, 185, 197, 199 Zhi, J.L. , 436 Zhang, L.K. , 81 Zhitian, Z. , 64 Zhang, L.S. , 401 Zhong, H. , 441 Zhang, L.Y. , 461, 467, 470 Zhong, J. , 190, 195 Zhang, M.M , 69, 319, 322, 332, 466, 474, 504 Zhong, K.W. , 577 Zhang, N. , 184–188, 193 Zhong, L. , 542 Zhang, P. , 121 Zhong, S. , 594 Zhang, Q.G. , 248, 342, 441 Zhou, A. , 189, 381, 530, 535 Zhang, R.J. , 173, 459, 509 Zhou, C. , 279, 299, 300, 491 Zhang, R.L. , 10 Zhou, D. , 187, 201, 231, 235, 299 Zhang, S.J. , 319, 322, 332, 343, 352 Zhou, F. , 38, 40, 249 Zhang, W. , 144, 148, 185–187, 195, 322, 331, Zhou, G. , 56 348, 433, 459, 461, 467, 468, 595, Zhou, H.Z. , 74 601 Zhou, J. , 601 Zhang, X. , 80, 167, 169, 298, 442, 493, 510, Zhou, M.-L. , 188, 201, 292, 293, 297, 300, 524, 525, 530, 583, 586 463, 468, 469, 471 Zhang, X.L. , 441 Zhou, P. , 437, 462, 467–471 Zhang, X.Z. , 586 Zhou, Q. , 187, 201 Zhang, Y.X. , 184–187, 191, 193, 194, 196, Zhou, R.L. , 8 198, 200, 248, 342, 435, 524, 525, Zhou, S. , 322, 331, 601 530, 542, 543, 545, 592, 594 Zhou, W. , 78 Zhang, Z. , 270 Zhou, X. , 78, 184 Zhang, Z.G. , 10, 152, 277, 459 Zhou, Y. , 298, 342, 343, 443 Zhang, Z.X. , 441 Zhou, Z. , 63 Zhang, Z.Y. , 80 Zhu, B.Q. , 74 Zhao, C. , 80 Zhu, C.Z. , 279 Zhao, D. , 319, 322, 332 Zhu, D. , 409, 410 Zhao, H. , 11, 41, 175, 245, 293, 311, 313, Zhu, H. , 328, 601 318–321, 323–325, 327, 328, 330, Zhu, L.-L. , 8, 58, 120, 123, 184, 186, 191, 331, 375, 563 198, 222, 275, 369, 459–461, 465, Zhao, H.G. , 245 467–470, 530, 531 Zhao, H.W. , 40–42, 44, 45, 341 Zhu, M. , 73 Zhao, J. , 299 Zhu, P.J. , 46 Zhao, L. , 270–272, 275–282, 442, 464, 470, Zhu, R.L. , 4, 6, 8, 231, 235, 275, 277, 436, 595 465, 473, 531, 567 Zhao, P. , 167–169, 172, 184, 186, 193, 200, Zhu, W. , 53, 71, 168, 280, 439 201, 221, 262, 263, 443, 492, 493 Zhu, X. , 38, 40, 44, 45, 63, 195, 592, 593, 595 Zhao, Q. , 272 Zhu, Y. , 4, 169, 184–186, 188, 191, 194, 195, Zhao, R.N. , 167, 169 199, 200, 370, 411, 412, 542–545 Zhao, T. , 184, 186, 194, 195, 466, 473, 474 Zhu, Z.H. , 167, 169, 172–174, 298, 491, 493, Zhao, W.S. , 80, 124, 235, 272, 273 584, 586, 591, 592, 594 Zhao, X. , 154, 156, 299, 439 Zhuravin, I.A. , 184, 185, 200 Zhao, Y.L. , 406, 433, 595 Zielasek, J. , 10 Zhao, Z.-Q. , 53, 71–75, 79, 80, 317, 326, 563 Ziembowicz, A. , 184, 185, 347, 351 Zhen, S. , 213, 221 Zilkha, E. , 25 Zheng, H. , 592 Zilles, K. , 341 Zheng, J.Z. , 190 Ziman, B.D. , 64 Author Index 685

Zimmermann, R. , 438 Zou, X. , 595 Zinchenko, V.P. , 193 Zovein, A. , 370 Zini, R. , 73 Zu, L. , 388 Zipfel, G.J. , 188, 201, 463, 468, 469, 471 Zu, Y.L. , 56, 57 Zivin, J.A. , 591 Zucker, I. , 38 Zoer, B. , 463, 471 Zukin, R.S. , 346, 356, 433, 534 Zollinger, A. , 175 Zuo, Z. , 114, 115, 117, 123, 166–172, 213, Zolotukhin, S.P. , 296 221, 298, 326, 331, 373, 492, 493 Zoratti, M. , 433 Zuurbier, C.J. , 433 Zoref-Shani, E. , 348, 436 Zvara, D.A. , 171 Zorov, D.B. , 64, 394 Zwagerman, N. , 106, 110, 112 Zou, F. , 349 Zweier, J.L. , 510 Zou, M. , 343 Zweifach, B.W. , 4 Subject Index

A ATP levels , 28–29

Adenosine A1 receptor (A1 AR) , 45–47, 410 GABA , 30 Adenosine epileptic tolerance , 529 glutamate , 30

Adrenomedullin , 199 K+ATP channels , 29 Alken , 296 neurotransmission , 30 Alpha-linolenic acid (ALA) phosphorylated adenylates , 29 biochemical imbalance , 144–146 telencephalic brain , 29 CA1 neurodegeneration , 154 turtles delayed cross-tolerance , 154 brain , 25–26 intravenous injection , 149–150 diving behavior , 25 K+ channels , 155 metabolic depression , 26–28

K2 P channels , 155 Antiapoptosis , 510–511 natural preconditioner , 153 N -Arachidonoyl-l-serine (AraS) , 504 NFkB , 154 Arctic ground squirrels (AGS) . nutraceuticals , 156–158 See Torpor subchronic treatment , 152 Astrocyte-based HIF-1 transcription , 473 TREK-1 channel , 155 Atmosphere absolute (ATA) , 173, 174 AMP-activated protein kinase Autophagy , 401 (AMPK) , 29–30, 442–443 Angiographic vasospasm , 572 Anoxia resistance B anoxia tolerance (see Anoxia Base excision repair (BER) , 415–416 tolerance) B-cell lymphoma-2 (Bcl-2) , 169, 196, energy consumption 368–369, 511, 531 basal metabolism , 22 Beclin-1 , 402 energy failure , 24–25 Blood-brain barrier (BBB) , 109–110, 198 ion pumps , 22–23 Brain-derived neurotrophic factor sodium pump and electrical and (BDNF) , 108, 247, 512, 530 synaptic activity , 23–24 Brain edema , 487–488 energy production Brain injury ATP , 21–22 cerebral preconditioning , 259 high-energy intermediates , 20–21 ischemic preconditioning , 259 Anoxia tolerance molecular and cellular mechanisms crucian carp ﬁ sh glial response , 261 AMPK , 29–30 vascular response , 261 anaerobic glycolysis , 28 transient ischemic attacks , 259

J.M. Gidday et al. (eds.), Innate Tolerance in the CNS: Translational Neuroprotection 687 by Pre- and Post-Conditioning, Springer Series in Translational Stroke Research, DOI 10.1007/978-1-4419-9695-4, © Springer Science+Business Media New York 2013 688 Subject Index

Brain ischemia postconditioning Cardiopulmonary arrest (CA). See Global distal middle cerebral artery (MCA) cerebral ischemia occlusion Carotid revascularization , 573–574 infarct size , 319, 320 Cell survival, genomics ischemic postconditioning, apoptosis , 368–369 CBF , 319, 321 cell cycle , 369 partial reperfusion , 319 neurogenesis , 369–370 reproducible infarct region , 319 Cellular homeostasis transient occlusion, bCCA , 319 glucose , 370 MCA suture occlusion model ion , 371–372 global ischemia , 322 metabolism , 371 in vitro ischemia , 322 neurotransmitters , 372 Brain preconditioning Central retinal artery occlusion (CRAO) , 541 carotid artery ligation , 236–237 Cerebral blood ﬂ ow (CBF) , 110–111, 470–471 clot busters , 133 Cerebral ischemia electroconvulsive shock (ECS) , 237 mitochondria and synaptic dysfunction , endogenous cellular protective signaling 430–431 pathways , 134 preconditioning focal ischemia , 226–229 and apoptosis , 433, 434 global ischemia , 226–227, 229–230 endoplasmic reticulum , 434–435 ischemic , 225–226 signaling pathwarys, mitochondrial focal preconditioning/focal permanent neuroprotection harmful ischemia , 230–234 mitochondria , 437–438 focal preconditioning/global harmful nitric oxide , 437 ischemia , 235 protein kinase C , 436–437 global preconditioning/focal harmful reactive oxygen species , 435–436 ischemia , 235 tolerance global preconditioning/global harmful adenosine , 8 ischemia , 236 global/focal ischemia , 6, 7 transient focal preconditioning/transient glutamate , 7–8 focal harmful ischemia , 235 neuroprotective effect , 8 ischemic stroke , 138–139 TIAs , 6 neuroprotective agent development failure transcriptional activators, mitochondrial “best-in-class” agents , 136 protection blood ﬂ ow interruption , 135 AMP-activated protein kinase common consequences , 135 pathway , 442–443 drug dose , 137 hypoxia-inducible factors , 440–442 termed ischemic penumbra , 136 nuclear factor erythroid-2 related treatment time , 136 factor , 443–444 work capacity , 135 peroxisome proliferator-activated omega-3 ( see Omega-3) receptor g coactivator-1a , 439, 440 tPA treatment , 133 peroxisome proliferator-activated receptors , 438–439 sirtuin 1 , 441 C Cerebral perfusion CABG surgery . See Coronary artery bypass contralateral perfusion , 277 graft (CABG) surgery hydrogen clearance measurements , 274 Calcitonin gene-related peptide (CGRP) , LPS-preconditioned , 275 68–70 preconditioning-associated CBF changes, cAMP response element-binding protein hypertensive rat , 275, 276 (CREB) , 9, 262, 343, 412–413 CGRP. See Calcitonin gene-related peptide Carassius auratus , 29 (CGRP) Carassius carassius . See Crucian carp Chaperones , 397–398 Cardioprotection. See Ischemic conditioning c-Jun NHP2 terminal kinase (JNK) , 56–57 Subject Index 689

Coronary artery bypass graft (CABG) surgery neuroprotective mechanism , 512, 513 IPC , 77 nonneuronal CB2 receptors , 504 IPost , 79 Endoplasmic reticulum (ER) perioperative myocardial injury , 76 Ca 2+ release , 400–401 preconditioning strategy ER-associated degradation , 400 adverse cerebral outcomes , 569 stress diazoxide , 570–571 chaperones , 397–398 free radicals , 570 glucose-regulated protein 78 , 399–400 hyperbaric oxygen conditioning , 571 heat shock proteins , 398–399 intermittent ischemic arrest , 569 unfolded protein response , 397 myocardial revascularization , 570 Endothelial apoptosis , 299 remote ischemic preconditioning , 571 Endothelial nitric oxide synthase (eNOS) , 188, RIPC , 77, 78 200, 297–298, 469, 572 Crucian carp Epileptic tolerance AMPK , 29–30 centrally administered excitotoxins , anaerobic glycolysis , 28 525, 527 ATP levels , 28–29 cross-tolerance , 527 GABA , 30 electroconvulsive seizures , 525 glutamate , 30 epileptogenesis , 528

K+ATP channels , 29 future aspects , 534–535 neurotransmission , 30 hippocampus , 527 phosphorylated adenylates , 29 kindling , 523, 525 telencephalic brain , 29 mediators of Cyclopentyltheophylline (CPT) , 46 adenosine , 529 Cytosolic glutamate , 24 Bcl-2 family proteins , 531 Cytosolic receptor tyrosine kinases , 56 clinical correlates , 531–532 growth factors , 530–531 heat shock proteins , 531 D neuropeptide Y , 530 Dan , 5 nuclear factor kB , 529–530 Deferoxamine , 219–220 preconditioning stimulus , 529 Delta-opioid receptor , 199–200 murine models of , 523, 524 Des fl urane , 171–172 protein synthesis and time period , 523 Dietary reference intake (DRI) , 144–145 systemic KA , 525, 526 Dietary restriction (DR) , 46 transcriptome of Drum stress , 4 expression pro fi ling , 532 microarray features , 532–534 transferability of , 527–528 E Epileptogenesis , 528 Electroacupuncture (EA) preconditioning EPO. See Erythropoietin (EPO) bene fi cial effects , 591–592 Erk1/2 MAPK , 56 ischemic preconditioning , 592–593 Erythromycin , 220, 251 ischemic tolerance , 592 Erythropoietin (EPO) , 198–199, 218–219, 250 learning and memory impairment , 593 Exercise preconditioning stroke ( see Stroke) amelioration , 106 Electroconvulsive seizures (ECS) , 525 complex exercise , 107 Electroconvulsive shock , 237 forced exercise , 106 Endocannabinoids neuroprotection mechanisms AEA and 2-AG levels , 501–502 anti-and pro-apoptotic signaling brain function , 500–501 pathways , 108 CB1 and CB2 receptors , 501 chronic exercise , 107–108 CB1 receptor agonists , 503–504 clinical application , 113–114 D9 -tetrahydrocannabinol , 500 HIF-1 a , 108, 113 enhancement of , 502 in fl ammatory response , 111–112 690 Subject Index

Exercise preconditioning (Cont.) TLR signaling cascade , 375 metabolic alterations , 112–113 transcriptional pro fi le , 376 neuronal death and survival signaling defense/stress response pathways , 112 heat shock proteins , 364, 365, 367 neurotrophins , 108 in fl ammatory cytokines , 367–368 neurovascular unit integrity (see toll-like receptors (TLRs) , 367–368 Neurovascular unit) effectors of neuroprotection poststroke exercise , 113 epigenetics , 381 simple exercise , 106–107 miRNA , 380–381 stroke-induced brain injury reduction , 105 model of , 378, 379 stroke prevention , 105 priming phase , 378 types of exercise , 106 microarray usuage , 374 Extracellular matrix (ECM) , 109, 458, 487 responses, preconditioning and ischemic tolerance , 364, 366 signaling mediators F cascades , 373 Factor-inhibiting HIF-1 (FIH-1) , 190 transcription factors , 373–374 Field excitatory postsynaptic potentials technological advances , 381–382 (fEPSPs) , 148 transcriptional study , 364, 365 Focal cerebral ischemia , 227–229 Glial-based protection, NVU hypothermia , 114 aquaporin 4 expression , 472–473 physiological variables and anesthesia astrocyte-based HIF-1 transcription , anesthesia confounds , 279–282 473 cerebral perfusion (see Cerebral astrocytes , 472 perfusion) oligodendrocyte precursor cells , tissue damage , 277–278 474, 476 stroke models PC-induced protection , 474 endothelin-induced vasoconstriction , plasma membrane-bound glutamate 273 transporter-1 , 473–474 intraluminal fi lament occlusions , Global cerebral ischemia , 229–230 271–273 clinical applications , 249–250 Macaca mulatta , 274 erythromycin , 251 surgical MCA occlusion , 270–271 erythropoietin , 250 tolerance , 6, 7 ischemic post-conditioning , 245 lethal , 244 mechanisms of G anti-cell death pathways , 247–248 Gamma aminobutyric acid (GABA) , 21, 28, elevated neurotrophic factors , 247 30, 430 suppressed excitotoxicity , 246 Genomics suppressed in fl ammation , 246–247 cell survival triggering pathways , 246 apoptosis , 368–369 natural solutions , 248–249 cell cycle , 369 resveratrol , 251 neurogenesis , 369–370 sublethal , 244 cellular homeostasis in vivo and in vitro models , 244 glucose , 370 Glucocorticoid receptor , 199 ion , 371–372 Glucose-regulated protein 78 (GRP78) , metabolism , 371 399–400 neurotransmitters , 372 Glutamate transporter-1 (GLT-1) , 473–474 converging mechanisms Gold fi sh , 29 blood cell response , 378 Golgi apparatus brain’s reprogrammed genomic Ca 2+ homeostasis, ischemic response , 377 preconditioning , 404–405 shared gene regulation , 375, 376 physiological function , 404 Subject Index 691

H hyperoxygenated blood delivery , 581 Halothane , 169–170 preconditioning ( see Stroke) HBO. See Hyperbaric oxygen (HBO) therapy pressure range , 582–583 Heart. See Ischemic conditioning Hyperthermia Heat acclimation crosstolerance (HACT) clinical implications , 124 phenotype experimental models , 121, 122 acclimatory pro ﬁ le , 506–507 intra-and postischemic hyperthermia , antiapoptosis , 510–511 123–124 anti-in ﬂ ammatory capacity , 511–512 mechanism antioxidation , 509 adenosine receptors , 123 brain-derived neurotrophic factor , 512 HIF-1 a , 123 cytoprotective features of , 507–508 HSPs , 122–123 hypoxia-inducible factor , 509–510 neuroprotection , 121, 122 neuroprotection Hyperthermic preconditioning. See mechanism , 512, 513 Hyperthermia physiological evidence of , 508–509 Hypoperfusion , 111 Heat shock proteins (HSPs) , 122–123, 200, Hypothermia 364, 365, 367 clinical applications endoplasmic reticulum , 398–399 cooling methods , 120 epileptic tolerance , 531 safety , 119 retinal ischemia , 545 timing , 120 Hibernation experimental models

A1 AR agonists , 45–47 cerebral hypoxia/ischemia , 114 arousal blood ﬂ ow , 38, 39 depth and duration , 116 brain injury , 38, 40 rapid vs . delayed tolerance , 115, 116 cerebral ischemia , 38 three-vessel occlusion model , 114 de ﬁ nition , 37 intra-and postischemic hypothermia , dietary restriction , 46 118–119

H2 S exposure , 46 mechanisms hypothermia , 38 adenosine receptors , 117 hypoxia , 38, 46 high-mobility group I (Y) protein , 117

myocardial mitochondrial function , 46 KATP channels , 117 phenotype , 40 neuroprotection , 116, 117 torpor ( see Torpor) TNF- a , 117–118 HIF-1 a . See Hypoxia-inducible factor-1a Hypothermic preconditioning. See (HIF-1a ) Hypothermia High-density lipoprotein (HDL) , 146 Hypoxia. See Hypoxic preconditioning HPC. See Hypoxic preconditioning (HPC) (HPC)

H2 S. See Hydrogen sul ﬁ de (H2 S) Hypoxia-inducible factor , 440–442, HSPs. See Heat shock proteins (HSPs) 509–510 Human brain endothelial cells Hypoxia-inducible factor-1a (HIF-1a ) , (HBEC) , 504 491

Hydrogen sul ﬁ de (H2 S) , 174–175 adults , 191 Hyperbaric oxygen (HBO) therapy , 172–174, Cre-Lox mouse model , 191–192 491–492 exercise preconditioning , 108, 113 bene ﬁ cial effects , 580 FIH-1 , 190 stroke and TBI HIF-2 a isoform , 192 acute ischemic stroke treatment , 581 hyperthermia , 123 clinical trials , 581, 582 mouse retina , 191 complete stroke , 581–582 neonates , 190–191 double-blind pilot study , 582 oxygen-dependent PHDs , 190 future aspects , 583–584 protein , 44 hyperbaric vs . normobaric rat retina , 191 hyperoxia , 583 Hypoxic-ischemic (HI) insult , 167 692 Subject Index

Hypoxic preconditioning (HPC) , 5, 38 I cytoprotection, nonischemic conditions , Immature brain 200–201 anaesthetic , 262–263 effector mechanisms hypoxic preconditioning , 264–266 amyloid-related proteins , 200 ligation and ischemic , 262 angiogenesis , 197 lipopolysaccharide preconditioning , attenuated excitotoxicity , 193 263–264 blood fl ow/tissue perfusion , 197 Immediate early genes (IEG) , 8 enhanced antioxidant capacity , 194 Inhalatory anesthetics , 492–493 EPO , 198–199 Internal carotid artery (ICA) , 272 HSPs , 200 Intracerebral hemorrhage (ICH) humoral mediators , 199 bleeding , 313 in fl ammatory and immunological brain injury mechanisms , 310–311 changes , 196 brain water content, bar graph , 311, 312 metabolic downregulation , 194–195 clinical considerations , 314 opioids , 199–200 hyperbaric oxygen , 311 overt vascular injury reductions , 198 iron-induced brain injury , 312 reduced apoptosis , 196–197 leukocyte in?ltration , 313 VEGF , 199 physical trauma , 312 ERK activity , 264 p44/42 MAPK and kinase inhibition , future aspects , 202–203 312 gene activation and de novo protein post-conditioning , 313–314 synthesis , 264 thrombin , 311 HIF-1 , 265 Intracranial aneurysm surgery induction mechanisms aneurysm occlusion , 574 hypoxia receptor , 186–187 coil embolization , 575 signal transduction pathways , magnesium , 575 187–188 temporary artery clip occlusion , PI3K/AKT signalling , 264 574–575 protection, cerebral ischemia Intraluminal fi lament occlusions CA1 hippocampal pyramidal delayed temperature , 273 cells , 184 endovascular occlusion methods , 272 cell culture , 184 ICA , 272 deferoxamine , 185, 186 overt infarction , 273 hypobaric hypoxia , 185 selective neuronal injury , 273 hypoxia mimetics , 186 IPC. See Ischemic preconditioning (IPC) neonatal brain , 184 IPost. See Ischemic postconditioning (IPost) rapid preconditioning , 186 Ischaemia/reperfusion (I/R) , 40 remote preconditioning , 185 Ischemic conditioning , 52–53, 601 transcriptional activation/genetic clinical application reprogramming CABG surgery ( see Coronary artery delayed stroke tolerance , 189 bypass graft (CABG) surgery) epigenetic transcriptional regulation , coronary revascularization , 82–83 189 patient bene fi t , 83 gene expression , 189 PPCI patients (see Primary percutaneous hippocampal slice , 188 coronary intervention (PPCI)) hypometabolic effects , 189 current clinical trials hypoxia-inducible factor (see CABG surgery , 608 Hypoxia-inducible factor-1a hyperbaric preconditioning , 608 (HIF-1a )) limb preconditioning , 608 non-HIF transcription factors , nervous system , 608 192–193 on-pump coronary surgery , 608 translational potential , 201–202 pharmacological preconditioning , 608 VEGF activity , 265–266 spinal cord surgery , 609 Subject Index 693

STEMI and primary PCI , 608 cardioprotection phases , 53 subarachnoid hemorrhage , 609 cerebral ischemia human conditioning , 603, 604 and apoptosis , 433, 434 IPC ( see Ischemic preconditioning (IPC)) endoplasmic reticulum , 434–435 IPost ( see Ischemic postconditioning clinical trials (IPost)) anti-cerebral ischemic therapy , 557 myocardium , 602 cardiopulmonary bypass , 557 RIC ( see Remote ischemic conditioning experimental “sublethal” ischemia , 558 (RIC)) focal and global ischemia , short sublethal ischemia and 555–557 reperfusion , 602 irreversible cerebral injury , 557–558 translational research pharmacological preconditioning , 558 carotid artery stenting , 606 remote IPC , 558–559 chronic ischemia , 605 serum biomarkers , 558 coronary artery bypass grafting , 606 coronary occlusion , 553 direct ischemic preconditioning , 607 cross-tolerance , 555 endogenous preconditioning delayed phase , 165 treatment , 605 disadvantages , 76 exogenous preconditioning electroacupuncture (EA) preconditioning , treatment , 603, 605 592–593 pharmacologically active focal preconditioning/focal permanent substances , 606 harmful ischemia , 230, 234 Ischemic depolarization (ID) , 42 focal preconditioning/global harmful Ischemic heart disease (IHD). See Ischemic ischemia , 235 conditioning global preconditioning/focal harmful Ischemic postconditioning (IPost) , 11, 555, ischemia , 235 562–563 global preconditioning/global harmful CABG surgery , 79 ischemia , 236 intermittent reperfusion , 71 HBO , 172–174

PPCI patients , 79–82 H2 S , 174–175 signalling pathways ischemic post-conditioning , 555 cell surface receptor activation , 72–73 memory effect , 53 mitochondria , 74–76 mitochondria , 58

transduction pathways , 73–74 mitochondrial KATP channel , 62 stroke mPTP , 63–64 additional proteins , 331 myocardial energy production , 58–59 Akt pathway , 328–330 nitric oxide (NO) , 63 brain ischemia (see Brain ischemia pH , 59–60 postconditioning) pharmacological preconditioning , 555 delayed postconditioning , 322–324 protective mechanism , 175–176

KATP channel , 331 remote ischemic preconditioning , 555 long-term protective effects of , 323, retinal ischemia 325 adenosine receptor , 545–546 MAPK pathways , 330 Akt-related neuroprotection , 544 myeloperoxidase (MPO) activity , 332 blood ﬂ ow , 546 PKC pathways , 330–331 cell survival mechanisms , 544 vs . preconditioning , 326–327 heat shock proteins , 545 preconditioning vs . postconditioning , HIF-1a signaling pathway , 318 544–545 triggers for , 325–326 hypoxia , 543 Ischemic preconditioning (IPC) , 225–226 nitric oxide synthase , 544 acute phase , 165 opioid receptors , 544 CABG surgery , 77 phototoxicity , 543 calcium , 60 PKC isoforms , 543–544 694 Subject Index

Ischemic preconditioning (IPC) (Cont.) M time-related , 543 MAPKs. See Mitogen-activated protein transient ischemic stimulus , 542–543 kinases (MAPKs) ROS , 60–62 Matrix metalloproteinase (MMP) , 110 schematic illustration , 230, 233 Metabotropic glutamate receptors , signalling pathways 346–347 cell membrane surface , 54 Methyl CpG-binding protein 2 (MeCP2) , effectors , 58 414–415 G-protein-coupled receptors , 54 Middle cerebral artery occlusion mediators , 54–58 (MCAO) , 8, 9 metabolic and biochemical Mineralocorticoid receptor , 199

changes , 54 Mitochondrial KATP channel , 62 secondary intracellular , 553–554 Mitochondrial permeability transition pore triggers , 54 (mPTP) , 63–64 surgical strategy , 230–232 de fi nition , 392–393 SWOP , 53, 64–66 inhibition, cardiac preconditioning transient focal preconditioning/transient connexin 43 translocation , 394 focal harmful ischemia , 235 mPTP phosphorylation, PKCe , transient ischemic accidents , 393–394 559–561 ROS production and calcium volatile anesthetics loading , 394 description , 166 Mitochondrial preconditioning des fl urane , 171–172 ATP-sensitive potassium channel halothane , 169–170 BSM-191095 , 392 iso fl urane (see Iso fl urane) Ca2+ -activated K+ channel , 392 sevo fl urane , 170–171 diazoxide , 391 xeon and nitrous oxide , 172 mitochondrial transmembrane Iso fl urane potential , 391–392 animal models , 166 positive feedback loop , 392 preconditioning-induced neuroprotection biogenesis acute and delayed phase , 167–168 hypoxia and ischemia , 395 Bcl-2 , 169 modulation of , 395 glutamate transporter inhibitors , 168 transcriptional regulators , HI insult , 167 394–395

K ATP channels , 168, 169 mitophagy multiple molecules , 168–169 parkin , 396–397 neural tissues , 169 preconditioning-conferred PKC and NOS , 168 protection , 396 permeability transition pore de ﬁ nition , 392–393 J inhibition, cardiac preconditioning , Janus kinase (JAK) , 57–58 393–394 ROS generation of , 388 K HSP70 upregulation , 388–389 Krebs cycle , 21 MAPK/ERK pathway activation , 390–391 PI3K pathway activation , 390 L PKC activation, cardiomyocytes , 390 Lactate dehydrogenase (LDH) , 21 signaling pathways of , 389 Lipopolysaccharide (LPS) , 3–4, Mitogen-activated protein kinases (MAPKs) , 221–222 55–57, 330 Low-density lipoprotein (LDL) , 146 mPTP. See Mitochondrial permeability Lysosome , 401–402 transition pore (mPTP) Subject Index 695

N Non-homogenous endpoint jointing (NHEJ) Neuropeptide Y (NPY) , 530 pathway , 415, 416 Neuroprotection. See also Ischemic Nuclear factor erythroid-2 related factor , preconditioning 443–444 CB1 receptor agonist , 503–504 Nuclear factor-k B (NFk B) , 154 nonneuronal CB2 receptors , 504 dual nature, brain preconditioning , Neurovascular unit (NVU) 409–410 blood-brain barrier formation , 458 mediators of , 410 integrity proin fl ammatory responses , 409 BBB integrity , 109–110 subunits , 409 CBF , 110–111 unstimulated cells , 409 microvasculature and architectural Nucleus framework , 108–109 cAMP response element-binding protein , MMP expression , 110 412–413 primary innate barrier , 108 DNA damage and repair ischemic stroke base excision repair , 415–416 angiogenesis , 459 NHEJ-mediated double-strand break CBF regulation , 459 repair , 416 endothelial and astrocytic oxidative DNA damage , 415 expression , 458 epigenetics , 407–408 proin fl ammatory mediators , 458 genomic reprogramming , 407 structural proteins distribution , 459 hypoxia-inducible factor-1 neuronal viability preservation , 457 astrocytic tolerance, P450 2C11 tolerance phenotypes, preconditioning upregulation , 412 acute vascular in fl ammation , 460, 467 ERK1/2 pathway inhibition , 412 angiogenesis and vascular remodeling , gene expression regulation , 410 471 hydroxylation , 411 BBB disruption , 467–468 hypoxic preconditioning , 411 direct endothelial cytoprotection , neuron-speci fi c HIF-1a knockout 471–472 mice , 411 glial-based protection , 465–466 (see PHD-2 upregulation , 411–412 also Glial-based protection, NVU) PI3K inhibitor , 412 NO and eNOS , 468–469 polyubiquitination , 411 postischemic CBF , 470 microRNA postischemic vascular reactivity , in cerebral ischemia , 414 470–471 neuroprotection , 414–415 ROS production , 469–470 NF-kB vascular-based protection , 460–464 dual nature, brain preconditioning , Nitric oxide synthase (NOS) , 168 409–410 N-methyl- d -aspartate , 493–495 mediators of , 410 N-methyl- d -aspartate glutamate receptors proin fl ammatory responses , 409 AMPA receptors, ischemic tolerance , subunits , 409 340, 341 unstimulated cells , 409 as inducers of tolerance CREB , 342–343 electrical activity, neurons , 343 O postsynaptic dendritic spines loss , 343 OGD. See Oxygen glucose deprivation (OGD) synaptic vs . extrasynaptic , 343–344 Oligodendrocyte precursor cell-based intracellular signaling components , 342 protection , 475 NR1 phosphorylation , 341 Omega-3 overview of , 340 and excitotoxicity spreading depression , 341 EPA and DHA bene fi cial effects , 149 N-methyl-4-isoleucine-cyclosporin epileptic seizures , 147 (NIM811) , 393 fEPSPs’ slope , 148 696 Subject Index

Omega-3 (Cont.) AGS hippocampus , 43–44 hippocampal slices , 148 attenuated excitotoxicity , 43 neuronal protection and cross-tolerance ePKC , 42 phenomenon , 147 euthermic AGS , 42, 44 nutritional de ﬁ ciencies , 149 global cerebral ischemia , 42 pentylenetetrazol-induced seizures , 148 HIF-1 a protein , 44 SCI , 147–148 intracellular calcium , 41 a -linolenic acid Na+ /K + ATPase downregulation , 41 CA1 neurodegeneration , 154 NMDA receptor , 41, 43–44 delayed cross-tolerance , 154 K + channels , 155

K2 P channels , 155 P natural preconditioner , 153 Parkin-mediated mitophagy , 392–397 NFkB , 154 Parkinson’s disease , 391 nutraceuticals , 156–158 Partial right frontal lobectomy , 486 TREK-1 channel , 155 Percutaneous coronary intervention (PCI) , and omega-6, biochemical imbalance 82–83, 585 DRI , 144–145 Perioperative hemorrhage , 488 EPA and DHA , 146, 147 Peroxisome proliferator-activated receptors , erythrocyte sedimentation , 147 438–439 Eskimo population , 147 activation, cerebral ischemic injury , 406 LA and ALA , 144–146 ischemic tolerance , 406–407 LDL and HDL , 146 isoforms of , 405–406 metabolism , 144, 145 Pharmacological preconditioning (PPC) , 555 trans-fatty acids , 146 deferoxamine , 219–220 unsaturated fatty acids intake , 146 erythromycin , 220 preconditioning, chronic erythropoietin , 218–219 administration , 156 lipopolysaccharide , 221–222 PUFAs opioids , 221 balanced intake , 140 TPC ( see Thrombin preconditioning) brain biochemistry , 140–141 Phosphatidyl inositol 3-OH kinase (PI3K) , 56 DHA , 143–144 PI3K-Akt kinase cascades , 56 EPA bene fi cial effects , 141–143 Plamandon , 147 LA and ALA , 144, 145 p38 MAPK , 56–57 metabolism , 144, 145 Polymorphonuclear leukocytes (PMN) , 10 nutrition as risk factor , 139–140 Polyunsaturated fatty acids (PUFAs) rapid preconditioning balanced intake , 140 antiexcitotoxic activity , 151 brain biochemistry , 140–141 brain arteries vasodilation , 151 DHA , 143–144 brain-repair process , 152 EPA bene fi cial effects , 141–143 CA1 neurons , 149 LA and ALA , 144, 145 cellular structural component , 151 metabolism , 144, 145 glutamate excitotoxicity , 149, 150 nutrition as risk factor , 139–140 hyperactivation , 149 PPCI. See Primary percutaneous coronary intravenous injection, ALA , 149–150 intervention (PPCI) multiple temporal and mechanistic Preventative acupuncture , 592 bene fi ts , 152 Primary percutaneous coronary intervention neuroplasticity , 152, 153 (PPCI) , 53 subchronic ALA treatment , 152 IPost , 79–82 in vivo transient model , 149 RIPC , 82 Opioids , 221 Prolyl hydroxylases (PHDs) , 190 Oxygen glucose deprivation (OGD) , 8–9, Protease-activated receptors (PARs) , 215 167, 199 Proteasome acute hippocampal slices , 38 19S cap proteins , 402–403 Subject Index 697

26S proteasome , 402 HIF-1a signaling pathway , 544–545 ubiquitin-proteasome system hypoxia , 543 ischemic tolerance , 403 nitric oxide synthase , 544 neurological disorders , 403 opioid receptors , 544 protein degradation , 403–404 phototoxicity , 543 protein turnover , 402 PKC isoforms , 543–544 substrates , 402 time-related , 543 Protein kinase C (PKC) , 55–56, 168, 436–437 transient ischemic stimulus , 542–543 PUFAs. See Polyunsaturated fatty acids post-conditioning , 546 (PUFAs) RIC. See Remote ischemic conditioning (RIC) RIPC. See Remote ischemic preconditioning (RIPC) R ROS. See Reactive oxygen species (ROS) RDI. See Reference/recommended daily intake (RDI) Reactive oxygen species (ROS) , 435–436 S mitochondrial production, reperfusion , 61 SCI. See Spinal cord injury (SCI) roles , 60 Second window of protection (SWOP) , 53, signalling ROS generation, myocardial 64–66 ischaemia , 61–62 Secretory pathway Ca2+ ATPase1 (SPCA1) , Receptor tyrosine kinases , 56 404–405 Reference/recommended daily intake (RDI) , Seizure , 568–569 144–145 Sevo fl urane , 170–171 Remote ischemic conditioning (RIC) Signal transducers and activators of intramyocardial protection , 65 transcription (STAT) , 57–58 myocardial mechanisms, cardioprotection , Sirtuin 1 , 441 70–71 Soluble amyloid precursor protein (sAPP) , 200 potential humoral factor , 67–69 Spinal cord injury (SCI) , 147–148 potential mechanisms , 67 Stroke. See also Ischemic postconditioning potential neural pathway , 69–70 additional proteins , 331 RIPC ( see Remote ischemic Akt pathway (see Akt pathway) preconditioning (RIPC)) brain ischemia (see Brain ischemia RIPost , 71 postconditioning) RPCT , 71 delayed postconditioning , 322–324 systemic response , 70 electroacupuncture (EA) preconditioning Remote ischemic postconditioning acupoint speci fi city of , 594 (RIPost) , 71 bilateral neiguan , 593 Remote ischemic preconditioning (RIPC) , clinical application of , 596, 597 555. See also Remote ischemic future prospects of , 596, 598 conditioning (RIC) ischemic preconditioning , 592–593 CABG surgery , 77, 78 mechanism , 594–595 heart protection , 52 parameters of , 593 PCI patients , 82–83 HBO preconditioning (see also Hyperbaric PPCI patients , 82 oxygen (HBO) therapy) Remote preconditioning of trauma (RPCT) , 71 antioxidase activity and blood lactate Resveratrol , 251 clearance , 586 Retinal ischemia exercise tolerance , 586 central retinal artery occlusion , 541–542 in fl ammatory markers and ischemic preconditioning neuropsychometric assessment , 585 adenosine receptor , 545–546 ischemic tolerance , 584–585 Akt-related neuroprotection , 544 on-pump CABG surgery , 585–586 blood fl ow , 546 percutaneous coronary intervention , cell survival mechanisms , 544 585

heat shock proteins , 545 K ATP channel , 331 698 Subject Index

Stroke. See also Ischemic postconditioning partial right frontal lobectomy , 486 (Cont.) pathophysiology of long-term protective effects of , 323, 325 brain edema , 487–488 MAPK pathways , 330 cell death , 488 myeloperoxidase (MPO) activity , 332 perioperative hemorrhage , 488 PKC pathways , 330–331 preconditioning therapies , 485–486 vs . preconditioning , 326–327 animal studies , 488–490 preconditioning vs . postconditioning , 318 future aspects , 494–495 triggers for , 325–326 hyperbaric oxygen , 491–492 Stroke Therapy Academic Industry inhalatory anesthetics , 492–493 Roundtable (STAIR) N-methyl-D-aspartate , 493–495 recommendations , 134, 136 SWOP. See Second window of protection ST-segment elevation myocardial infarction (SWOP) (STEMI) , 53. See also Primary Synaptic signaling, ischemic tolerance percutaneous coronary intervention AMPA receptors , 344–346 (PPCI) excitatory transmitters Subarachnoid hemorrhage (SAH) , 200 amino acid glutamate , 339 clinical applications and strategy NMDA receptors (see N-methyl-D- endovascular approach , 295 aspartate glutamate receptors) hypoxic/ischemic preconditioning , inhibitory transmission 294, 295 adenosine , 348–349 ischemic stroke , 295 GABA , 347 limb occlusion , 294 purines , 348–349 coiling , 294 metabotropic glutamate receptors , 346–347 remote conditioning , 295–296 postsynaptic mechanisms of tolerance , conditioning modalities , 293–294 351–352 mechanisms of conditioning presynaptic mechanisms of tolerance , endothelial apoptosis , 299 349–350 endothelial nitric oxide synthase ubiquitin system , 353–354 (eNOS) , 297–298 HO-1 induction , 299 upregulate innate neuroprotective T mechanisms , 298 Takao , 68 ongoing clinical trials , 301–302 Thrombin preconditioning outcome measures of , 300–301 brain edema , 214 preconditioning strategy cerebral ischemia , 214–215 aneurysm , 571–572 endothelial hyperpermeability, Rho kinase cerebral vasospasm , 572 and protein tyrosine kinase , 215 cognitive dysfunction , 572 heat shock protein 27 , 215–216 delayed cerebral ischemia , 572 HIF-1a protein levels , 218 remote limb preconditioning , 572–573 in ﬂ ammatory response, brain injury , 214 traumatic conditioning , 297 PARs , 215 types of conditioning , 292–293 p44/42 mitogen-activated protein , 216, 217 Subcellular organelles primary cultured neurons, OGD , 215, 216 endoplasmic reticulum (see Endoplasmic TNF- a . See Tumor necrosis factor-a (TNF-a ) reticulum) TNF receptor 1 (TNFR1) , 410 golgi apparatus , 404–405 Tolerance mitochondria ( see Mitochondrial anoxia , 5 preconditioning) cerebral ischemic tolerance nuclear membrane , 404–407 adenosine , 8 nucleus ( see Nucleus) global/focal ischemia , 6, 7 proteasome (see Proteasome) glutamate , 7–8 Surgical brain injury (SBI) neuroprotective effect , 8 complications , 485 TIAs , 6 Subject Index 699

cross-tolerance , 10 enhancement of , 502 fever , 3–4 neuroprotective mechanism , 512, 513 in ﬂ ammation , 9–10 nonneuronal CB2 receptors , 504 ischemic postconditioning , 11 HACT phenotype (see Heat acclimation molecular mechanisms , 8–9 crosstolerance (HACT) phenotype) non-cerebral ischemic tolerance, heart , 5–6 HBO therapy (see Hyperbaric oxygen remote , 11 (HBO) therapy) stem cells , 10–11 Tricarboxylic acid cycle (TCA) , 21 trauma , 4 Tumor necrosis factor-a (TNF-a ) , 111, Toll-like receptors (TLRs) , 367–368 117–118 Torpor Turtles blood ﬂ ow , 38, 39 brain , 25–26 cerebral I/R tolerance , 45 diving behavior , 25 euthermy , 40 metabolic depression glycogen/oxidative phosphorylation , 44 adenosine , 27 modeled I/R , 40 channel arrest , 26 OGD tolerance cytosolic Ca +2 , 27 AGS hippocampus , 43–44 electrical activity , 26 attenuated excitotoxicity , 43–44 excitotoxicity , 28 ePKC , 42 GABA levels , 28 euthermic AGS , 42, 44 synaptic activity , 26 global cerebral ischemia , 42 voltage-gated Na+ channels , 27 HIF-1 a protein , 44 Tyrosine kinases , 56 intracellular calcium , 41 Na+ /K+ ATPase downregulation , 41 NMDA receptor , 41, 43–44 U tolerance independent , 41–42 Ubiquitin-proteasome system (UPS) , 400 Transient ischemic attacks (TIAs) , 6 Unfolded protein response (UPR) , 397 ischemic preconditioning , 559–561 preconditioning strategy , 568–569 Transmitter glutamate , 24 V Traumatic brain injury (TBI) Vascular endothelial growth factor (VEGF) , endocannabinoids 197, 199, 487 AEA and 2-AG levels , 501–502 brain function , 500–501 CB1 and CB2 receptors , 501 W CB1 receptor agonists , 503–504 Wagner-Jauregg , 3 D9 -tetrahydrocannabinol , 500 Wenwu , 78