Current Reviews, 2012, 8, 39-49 39 Modulation of in Acute Ischemic as Treatment Challenges

Joaquin Jordan1, Laura Moreno-Parrado1,2, David Anton-Martinez1,3, Kurt A. Jellinger4 and Maria F. Galindo*,5

1Grupo de Neurofarmacología, Dpto Ciencias Médicas, Fac de Medicina, Universidad de Castilla La Mancha, Avenida Almansa, 14, Albacete 02006, Spain 2Sección de Microbiologia, Complejo Hospitalario Universitario de Albacete, Albacete, Spain 3Sección de Bioquímica, Complejo Hospitalario Universitario de Albacete, Albacete, Spain 4Institute of Clinical Neurobiology, Kenyongasse 18; A-1070 Vienna, Austria 5Unidad de Neuropsicofarmacología Translacional, Complejo Hospitalario Universitario de Albacete, Albacete, Spain

Abstract: Stroke is a major cause of death and disability throughout the world. Its pathophysiology is complex and includes excitotoxity, inflammatory pathways, oxidative damage, ionic imbalances, apoptosis and other death mechanisms, angiogenesis, and . The ultimate result of the complex ischemic cascade is neuronal death with irreversible loss of neuronal functions. New developments in stroke pathophysiology have induced significant advances in acute stroke management. Among the extracellular signals, , microglia and cytokines as major consequences of hypoxia may be targets for future . Among the intracellular signals, calcium-induced and as most important factors of ischemic cell death and for dysfunctions of the blood- barrier are important goals for neuroprotective agents. Third messengers, like p53, peroxisome proliferator-activated receptors and nuclear factor kappa-B (NF-kB) also play important roles in the pathogenesis of ischemic cell death, and may be further important targets of modern neuroprotective agents. The final stage of ischemic cell death via apopotosis and other cell death cascades, mainly influenced by energy deficiency and mitochondrial dysfunction may be influenced by antiapoptotic and other strategies as potential new targets for designing newer and more successful therapeutic modalities of acute ischemic stroke. Keywords: Apoptosis, extracellular signals, inflammation, intracellular signals, ROS, stroke.

INTRODUCTION Here we review the currently available knowledge about the inflammatory and death pathways activated in acute The need for better stroke therapies has inspired research stroke and their implications for neuronal cell death via into the cellular and molecular mechanisms of ischemic apoptosis. brain damage, with major focus on the neuronal response under ischemic conditions. The neuronal response is poorly EXTRACELLULAR SIGNALS characterised in stroke patients and details about the molecular mechanisms of neuronal death are mainly based Inflammation on studies in animal models of ischemic injury (global, focal, hypoxia/). This depends on the nature and duration Although less well understood, inflammation is a of the ischemic insult, the location of the cells relative to the consequence of hypoxia and has been considered as a infarcted area and the neuronal subtype being affected. Acute therapeutic target in acute ischemic stroke [4-6]. The central neurological conditions such as cerebrovascular diseases are nervous system (CNS) has for long been regarded as an associated with irreversible loss of and glial cells. immune privileged organ, with the blood–brain barrier Severe or prolonged injury results in uncontrollable cell (BBB) tightly regulating the influx of immune cells and death within the core of lesions. In addition, ischemic cell mediators from the vascular compartment to the brain death is also characterized by a long delay between the insult parenchyma [7]. There are many evidences that and manifestation of major cell damage. This delay varies inflammation and immune response play an important role in greatly, depending on the nature of the insult and the brain the outcome of ischemic stroke patients, and they have been region being affected. In some cases it is as long as several associated with larger brain damage. Brain inflammation is days or even weeks [1, 2] whereas in others it is a few hours characterized by activation of microglia and astrocytes, or less [3]. expression of key inflammatory mediators, but limited invasion of circulating immune cells. Inflammation induces

rapid expression of key inflammatory mediators -cytokines,

*Address correspondence to this author at the Unidad de chemokines and prostaglandins- which in turn up-regulate Neuropsicofarmacología, Complejo Hospitalario Universitario de Albacete, adhesion molecules, increase permeability of the BBB, c/ Hermanos Falcó 37, 02003 Albacete, Spain; Tel: +34 967 597477; Fax: facilitating invasion of peripheral immune cells, induce +34 967 597173; E-mail: [email protected] release of potentially toxic molecules and compromise brain

1875-631X/12 $58.00+.00 © 2012 Bentham Science Publishers 40 Current Immunology Reviews, 2012, Vol. 8, No. 1 Jordan et al. cells. Because the BBB is disrupted after stroke, the immune cytokines, chemokines, play an important role in recruiting system comes into contact with CNS antigens, in both the cells into areas of active inflammation [13]. brain and periphery [5, 8]. The production of arachidonic acid potentially Microglia are primarily involved in immune surveillance exacerbates the injury process by increasing extracellular [8, 9], but when activated have macrophage-like capabilities levels of aspartate and glutamate by inhibiting sodium- including phagocytosis, inflammatory cytokine production, dependent uptake and by stimulating exocytosis of glutamate and antigen presentation [10]. Normally these in synergy with PKC activation. Activation of the neuroinflammatory changes are transient with microglia cascade also leads to the synthesis of eicosanoids which returning to a resting state as the immune stimulus is regulate neuronal channels and the formation of - resolved. Activated microglia secrete a wide range of factors, superoxide free radicals (O2 ). Therefore, cytokines have the some of which can actively trigger apoptosis in neuronal cell capacity to create downstream modulation of cell signaling cultures [11, 12]. At the same time, microglia are also events and at the extreme cell death. reported to increase neuronal survival through the release of Recently it has been shown that patients with acute stroke trophic and anti-inflammatory factors (Table 1). Cytokines had significantly better outcome with treatment can be classified into four major groups: growth factors, compared with placebo [14], by involving minocycline anti- interleukins, interferons, and tumor factor. Cytokine inflammatory properties, especially its ability to suppress receptors, based on their three-dimensional structures, have activation of microglia, that may contribute to cytoprotection been classified into: receptors of the hematopoetin receptor in the CNS [15, 16]. Minocycline neuroprotection has also family, interferon receptors, transforming growth factors been reported in experimental ischemic stroke [17, 18] and (TGF), tumor necrosis factors (TNF) receptors, receptors for excitotoxic conditions [19]. the immunoglobulin (Ig) superfamily, and chemokine receptors. Inflammation after stroke involves leukocytes infiltration in brain parenchyma, specially neutrophils, that contribute to The concept that cytokine expression may be beneficial cerebral damage after ischemia [20] through reperfusion or and/or deleterious is illustrated by the potential dichotomous secondary injury mechanisms. Therefore, adhesion role of the immune response in stroke. The chemotactic molecules in leukocytes and endothelial cells are key

Table 1. Adhesion Molecules

Model Approach Effects Ref.

Selectins Knockout mice Blockade with a P-selectin MCAO ischemia monoclonal antibody Decrease infarct volumes [90] sCRsLex Improvement in neurological outcome and reduction Focal cerebral ischemia Blockage or deficiency cerebral infarcts volumes Global cerebral ischemia Antibodies Reduction survival periods [91] E-selectin Cerebral ischemia Intranasal administration Induction immune tolerance and reduction injury [92] L-selectin Cerebral ischemia Antibodies against L-selectin Ineffective in stroke [93]

Immunoglobulin Superfamily Less cerebral damage [94] ICAM-1 Cerebral ischemia Anti-ICAM-1 Reduction in neutrophil accumulation, apoptosis and [95] neurological deficits Clinical trials Anti-ICAM-1 Side effects and no improvement in outcome at 90 days. Improvement in neurological deficits and reduction in VCAM-1 Focal cerebral ischemia Leukotriene receptor antagonist death by inhibition the ischemia-induced [96] upregulation of VCAM-1

Treatment with VCAM-1 Cerebral ischemia Ineffective in stroke antibodies

Integrins Protection to injury by reduction of the upregulation of In vitro studies Aprotinin [97] neutrophil CD11b expression

Anti-CD11b or Anti-CD18 Reduction in infarct volumes, apoptosis and decreased [98] MCAO in rats monoclonal antibodies. accumulation of neutrophils [99] anti-integrin [100] Clinical studies Negative results in acute stroke anti-CD11b or anti-CD18 [101] Modulation of Apoptosis in Acute Ischemic Stroke as Treatment Challenges Current Immunology Reviews, 2012, Vol. 8, No. 1 41 molecules that contribute to cerebral damage. In Table 1 we chemokines after cerebral ischemia is thought to be resume their effects and function as well as their relevance in deleterious by increasing leukocyte infiltration [22, 23]. In neuroprotetion after stroke (for review see [6, 21]). this context, levels of a variety of chemokines such as monocyte chemoattractant protein-1 (MCP-1), IL-8 and Most inflammatory reactions are mediated by cytokines, which are up-regulated in the brain in response of a variety macrophage inflammatory protein-1 (MIP-1), have been found to increase in animal models of ischemia, and its of stimulus including ischemia, being IL-1, interleukin-6 inhibition or deficiency has been associated with reduced (IL-6), TNF-, interleukin-10 (IL-10) and TGF-, the most injury in transient [23]. Chemokines could studied cytokines related to inflammation in stroke (Table 2). have an important role in homing stem cells to injured Chemokines play important roles in cellular communication regions and could also be involved in marrow derived and inflammatory cell recruitment. Expression of stromal cell migration into ischemic brain [24]. On the other

Table 2. Inflammatory Mediators

Model Approach Effects Ref.

Cytokines Rat primary cortical neuron IL-1 cultures with excytotoxic amino IL-1 prior to injury Attenuation in neuronal death. [102] acids MCAO cerebral ischemia IL-1 deficient mice Smaller infarcts. [103]

Reduction in the size of the infarcts and the [104] MCAO cerebral ischemia IL-1ra severity of neurologic deficits. [105]

TNF-  Experimental stroke Inhibition of TNF-  Reduction in ischemic brain injury. [106] TNF- ab or TNF- Cerebral ischemia   Beneficial after cerebral ischemia. [107] binding protein MCAO model of cerebral Intracisternal Reduction in infarct size and decreased microglial [108] ischemia administration activation. [109] Animal Models of cerebral Administration and gen IL-10 Benefical effects independently of stroke subtype. [110] ischemia transfer [111] Protection from ischemic stroke and reduction of TGF-  Mouse ischemic stroke Overexpression [112] the inflammatory response. Cultured neuron TGF-  Protection from ischemia-like insults. [113]

Reduced infarct volume when it is administered into the penumbra area of rats, 1 hour after [114] MCAO model of cerebral TGF-  MCAO. [115] ischemia No neuroprotective effect when injected in the [116] core area of the infarct.

Chemokines Animal models of ischemia Inhibition or deficiency Injury reduction. [23]

Transient Focal cerebral Fractalkine (CX3CL1) Reduction in infarction size and lower mortality MCP-1 [117] ischemia deficient mice rate.

Cyclooxygenase

Increased the number of healthy neurons in Transient Global ischemia COX-1 inhibition [118] hippocampus. [119] Cerebral ischemia COX-2 inhibitors Improvement neurological outcome. [120] NMDA exposure COX-2 deficient mice Injury reduction. [121]

Matrix Metalloproteinases

Experimental models of Inhibition of MMPs Reduce infarct size and brain edema. [122] ischemia Cerebral ischemia MMP-9 deficient mice Smaller infarcts. [123]

Delayed expression Cerebral ischemia Neurovascular remodeling and stroke recovery. [124] MMPs 42 Current Immunology Reviews, 2012, Vol. 8, No. 1 Jordan et al. hand, chemokines are also signaling molecules that process of neuronal death is called , which was downregulate microglia activity. On such molecule is first described by Olney et al. in the nineteen-seventies [26]. fractalkine (CX3CL1), which is primarily expressed by The presence of glutamate in the synapse is regulated by neurons and which has been shown previously to inhibit active, ATP-dependent transporters in neurones and glia. For secretion of pro-inflammatory cytokines by activated instance, in CNS ischemia a reduction of glucose causes a microglia [25]. decrease in ATP production, leading to an impairment of glutamate uptake. Moreover, the membrane potential of Excitotoxicity Process presynaptic neurones is lost and efflux of excitatory amino acids occurs, contributing to the excessive activation of post- is the principal excitatory synaptic glutamate receptors [27]. In ischaemic stroke the in the mammalian CNS, which activates different types of involvement of excitotoxicity is well established and plays -forming receptors (ionotropic) and G-protein- an important role as a cause of neuronal death. coupled receptors (metabotropic) to develop their essential The principle of excitotoxicity has been well-established role in the brain. Under physiological conditions, in normal experimentally, both in in vitro systems and in vivo, synaptic functioning, activation of excitatory amino acid following administration of excitatory amino acids into the receptors is transitory, and appears to be related to synaptic nervous system. Much attention has been directed at plasticity; indeed, it ensures the establishment of long-term obtaining evidence for a role for excitotoxicity in the potentiation, a process believed to be responsible for the neurological sequelae of stroke, and there now seems to be acquisition of information. However, if, for any reason, little doubt that such a process is indeed a determining factor receptor activation becomes excessive or prolonged, the in the extent of the lesions observed [28, 29]. Several clinical target neurones become damaged and eventually die. This trials have evaluated the potential of antiglutamate drugs to

Table 3. Treatment Strategies for the Management of Acute Ischemic Stroke

Targets Drugs Effects Ref.

Selfotel No efficacy [125] No efficacy NMDA receptor blockers No efficacy [126] No efficacy Lincostinel Numerous side effects

NBQX Robust neuroprotection [127] AMPA receptor blockers ZK 200775 (YM872)

GABA agonist Clomethiazole Failed to show benefit [128] Sodium channels blocker Lubeluzole No effects in reducing deaths [129]

Sodium channels blocker and prevents glutamate release Fosphenytoin Has not been demonstrated

Sodium and calcium channels blocker Sipatrigine (619C89) Not have any favourable effects [130]

Blocks the intracellular increase of calcium Calcium chelator DP-b99 Shown significant improvements

Tirilazad Appeared to marginally worsen stroke [131] Free -scavenger activity Ebselen Effective in improving outcomes [132] Stabilizes the HIF-1 protein expression Deferoxamine Decreased the size of brain damage [133]

NXY-059 Reducing lesion volume and neurologic deficits [134] Free radical-scavenger Failed to confirm the efficacy [135] Antiapoptotic Citicoline Increase in the odds of recovery [136]

Piogitazone Reduce immune reactions and reveals a Activation of PPAR- Thiazolidinedione powerful anti-inflammatory potencial PPAR-  agonist Rosiglitazone Anti-inflammatory (reduces levels of ROS) [72]

Ciclosporin A MPTP blockers Neuroprotective effects [137] Bongkrekic acid

Suppress S-100 Arundic acid Improves neuronal survival after stroke [138] Modulation of Apoptosis in Acute Ischemic Stroke as Treatment Challenges Current Immunology Reviews, 2012, Vol. 8, No. 1 43 improve outcome following acute ischaemic stroke. In the hours from the onset. NO has an inhibitory role in the Table 3 we resume different clinical trails where the efficacy dynamic regulation of the BBB function [48]. Its of the modulation of these targets was assayed. expression is detrimental and therefore its inhibition has yielded in reduced infarct volumes and reduced INTRACELLULAR SIGNALS neurological deficits [49, 50]. Furthermore, knock-out mice for iNOS gene have smaller infarcts than wild Second Messengers type mice when they are submitted to MCAO [49]. NO may cause DNA damage in cerebral ischemia Calcium trough the formation of peroxynitrite [51]. 2+ - Elevation of Ca is assumed to initiate various - The rate of O2 production is affected by pathological processes that causes neuronal dysfunction. mitochondrial metabolic state Oxygen normally Calcium activates a number of Ca2+-dependent that serves as the ultimate electron acceptor and is reduced influence a wide variety of cellular components, like to water. However, electron leak to oxygen through - cytoskeletal proteins or second- messenger synthases. complexes I and III can generate O2 [52], which However, overactivation at NMDA receptors triggers an increases when the electron carriers harbor excess excessive entry of Ca2+, initiating a series of cytoplasmic and electrons, either from inhibition of oxidative nuclear processes that promote neuronal cell death [30-33]. phosphorylation or from excessive calory For instance, Ca2+-activated proteolytic enzymes, like consumption. On the other hand, NADPH oxidase is a , can degrade essential proteins [34]. Moreover, membrane-bound that catalyses the 2+ - Ca /calmodulin kinase II (CaM-KII) is activated, and a production of O2 from oxygen. When microglia are number of enzymes are phosphorylated, which increases activated, the cytosolic subunits (p47, p67, p40 and their activity [35]. All these mechanisms, together with Rac2) [53] translocate to membranes, where they bind enhanced oxidative stress (see below) can induce cell death. to the membrane-associated subunits (p22 and gp91) and assemble the active oxidase to produce - extracellular O2 . Neural damage in response to cerebral vascular dysfunction is mediated by NADPH Considerable evidence suggests that reactive oxygen oxidase. Ischaemic stroke is reduced in mice lacking species (ROS), which are generated when blood flow returns to ischemic brain areas, are mainly responsible for the functional NADPH oxidase [54], and there is a clear role of NADPH oxidase in intracerebral haemorrhage . Oxidative stress is a major factor for [55]. Recently, it has been reported that overactivated neuronal death in ischemia and others disorders [36, 37]. microglia injure oligodendrocytes through NADPH Excess ROS can modify membrane phospholipids, and result oxidase [56]. in cell and tissue injury [38]. Increased ROS and/or cytokines produced from inflammation promote oxidative There is evidence that some antioxidant treatments can modification of fatty acids within membrane phospholipids. attenuate or delay disease progression in animal models of The oxidation of fatty acids and phospholipids causes neurodegenerative diseases. Compounds with free radical detrimental effects on cell signaling and activates scavenging activity (tirilazad, ebselen), iron chelator phospholipase, which stimulates the release of arachidonic (deferoxamine) or free radical trapping properties (NXY- acid (AA), an -6 polyunsaturated fatty acid, and 059) have been examined in experimental models of stroke biologically active pro-inflammatory mediator [39]. In and evaluated clinically as neuroprotective agents (Table 3). addition, AA can induce apoptosis by increasing the The NXY-059 compound (Cerovive®), has been suggested to production of proapoptotic ceramide [40], Ca2+ uptake into act as free radical scavenger and was effective in reducing mitochondria [41], and excess ROS production via direct lesion volume and neurological deficits. The positive results effects on mitochondria [42, 43]. Moreover, AA in the from the first Stroke-Acute-Ischemic-NXY-Treatment presence of iron, a catalyst of auto-oxidation, increases (SAINT-I) trial, which followed many of the Stroke oxidative damage of cells and the subsequent mitochondrial Academic Industry Roundtable (STAIR) guidelines, impairment [44]. reinvigorated the enthusiasm for neuroprotection [57]. However, the SAINT-II trial, has failed to confirm the In this line, excitotocycty may depend, in part, on the - efficacy of NXY-059 in acute ischemic stroke treatment generation of (NO) and superoxide anion (O2 ). adding a new disappointment in the search of a clinically - NO is an important signaling molecule involved in effective neuroprotection for ischemic stroke. While a low physiological processes such as neuronal eight copper chelator crosses the BBB [58], the difficulty of communication, host defense, and regulation of the many antioxidants to penetrate the BBB is one possible vascular tone [45]. NO is synthesized by nitric oxide reason for their lack of efficacy. It has been reported that synthetase (NOS). The beneficial or detrimental feeding rats with CoQ for 2 months failed to increase brain effects of this molecule will depend on where and CoQ levels [59]. when is expressed [46]. After induction of ischemia, the vasodilator effect of NO produced by eNOS, is Third Messengers beneficial because it induces vasodilatation and limits blood flow reduction [47]. However, when ischemia The activation of some transcription factors has been progresses, NO produced by iNOS contribute to brain detected in neurons and microglia in the penumbra area. injury [46]. iNOS is expressed in the postischemic They intricately regulate a variety of genes that modulate brain, reaching its peak level in infiltrating cells 48 cellular functions. Recent studies have shown that 44 Current Immunology Reviews, 2012, Vol. 8, No. 1 Jordan et al. transcription factors like p53, peroxisome proliferator- PPAR agonist rosiglitazone could be a potential activated receptors (PPARs), nuclear factor (NF)-kappaB novel therapeutic agent for stroke [72]. and the hypoxia-inducible factors are contributing to - NF-kB is one of the most important transcription ischemic brain damage [60-62]. factors playing a pivotal role in mediating responses - The p53 protein, also known as "guardian of the to a variety of signals, including oxidative stress and genome", is activated upon stress signals, such as hypoxia-reoxygenation [60-62]. Indeed it has been hypoxia, depolarization and DNA damage [63, 64]. thought of as the ‘central switch’ or ‘central mediator Although the ability of p53 to trigger cell-cycle arrest of the immune response’ controlling the synthesis of was discovered first, its action in controlling different cytokines and chemokines. NF-kB is apoptosis is the most intensely studied. A p53- activated by more than 150 stimuli and results in the dependent apoptosis program was first noted induction of more than 150 genes which influence following irradiation of mouse thymocytes [65, 66]. cell survival and maintenance of normal functional A substantial role for p53 in neuronal cell death integrity [73]. Genes regulated by NF-kB include induced by oxidative stress, DNA damaging agents, enzymes for COX-2, iNOS and prostaglandin or excitotoxicity in vitro, and in experimental models synthase-2, interleukin-6 and -1, dynorphin, and of stroke in vivo [63]. The regulation of p53 functions intercellular and vascular adhesion molecules. is tightly controlled through several mechanisms Inhibition of NF-kB signaling pathway has been including p53 transcription and translation, protein associated with alteration in activity-dependent stability, post-translational modifications, and synaptic plasticity, suggesting that the NFB subcellular localization [63, 67]. p53 activates the signaling pathways are actively involved in expression of genes engaged in promoting growth modulation of important neuropsychological arrest or cell death in response to multiple forms of functions such as synaptic remodeling and plasticity cellular stress [63, 67]. The pro-apoptotic functions of [74]. p53 are largely promoted through transactivation of - The hypoxia-inducible factors (HIFs) are specific target genes such as Bax, PUMA or Noxa transcription factors the expression and stability of [68]. In addition, p53 may mediate cell death through which are regulated by oxygen tension. The present mechanisms independent of transcriptional activity, understanding of HIF regulation has described the for example through interference with endogenous majority of oxygen-controlled HIF regulation survival mechanisms after translocation of p53 to the occurring on the alpha subunit, whereas the beta mitochondria or by transcriptional repression due to subunit is not sensitive to oxygen levels and is interactions with the transcriptional cofactors constitutively present in the nucleus. Certain disease CBP/p300 [67]. Blocking p53 expression states the stabilisation of the HIF subunit even in the demonstrably and efficiently protects against cerebral presence of oxygen. The induction of HIF-1, and the ischemia in experimental models, and this protection consequent expression of its target genes, activates a includes reduction in rates of apoptosis [69]. p53- pivotal signaling pathway involved in molecular and deficient mice show reduced neuronal death after cellular adaptation to hypoxia [75]. Indeed, HIF-1 ischemia [70] and the antisense knockdown of p53 regulates the expression of a wide range of genes resulted in a significant increase in neuronal survival involved in vasomotor control, angiogenesis, after ischemia [69]. erythropoiesis, iron metabolism, cell cycle control, - PPARs are ligand-activated transcription factors of cell proliferation and death, and energy metabolism the nuclear hormone receptor superfamily with 3 [76]. PPAR isoforms (, / and ). PPARs regulate transcription of target genes by heterodimerizing with Decision Stage the retinoid X receptor and binding to PPAR response elements of regulatory promoter regions of target Mitochondria are considered the main link between genes PPARs are endogenous protective factors in cellular stress signals and the execution of programmed cerebral ischemia. Ligands for two forms of the nerve cell death [77, 78]. In addition to generating ATP, receptor, PPAR and PPAR, are clinically available; mitochondria also play critical roles in regulating cellular ligands for PPAR are currently under development. viability and show selective vulnerability to injury. Rosiglitazone and pioglitazone, both PPAR ligands Mitochondria are both targets and important sources of ROS; of the thiazolidinedione class, enhance insulin- cumulative oxidative stress, disrupted mitochondrial mediated glucose uptake and are widely used for respiration and oxidative mitochondrial damage are treatment of type 2 diabetes [71]. PPAR activation associated with and may promote cell death [79, 80]. was recently shown to mitigate the inflammation Dysfunctions of mitochondria disturb cell function, cause associated with chronic and acute neurological DNA damage, and may initiate cell death. Furthermore, insults. After focal ischemia, PPAR expression is mitochondrial membrane permeabilization (MMP) is a increased in the brain, especially in the peri-infarct critical event during apoptosis, representing the "point of no area. Activation of PPAR with pioglitazone or return" of the lethal process. The MMP determines whether thiazolidinedione might also reduce immune reactions cells will succumb to or survive the injury, and represents a and at the same time reveals a powerful anti- 'point of no return' in mitochondrial cell death [81]. inflammatory potential in ischemic . The Cytochrome c released from mitochondria into the

Modulation of Apoptosis in Acute Ischemic Stroke as Treatment Challenges Current Immunology Reviews, 2012, Vol. 8, No. 1 45

Lack of oxygen

↓Aerobic metabolism ↑Anaerobic metabolism

↓ATP

ATP-reliant ion transport pumps

depolarization

↑[Ca2+] intracellular

Glutamate releasing

stimulates

AMPA receptors NMDA receptors

↑↑[Ca2+] intracellular

Free radicals, reactive oxygen species EXCITOTOXICITY (ROS) , endonucleases, ATPases, and phospholipases

Mitochondria break down

Caspase-dependent apoptosis Necrosis

brain reperfusion

Inflammatory response BBB damage

Edema

Fig. (1). Changes observed during ischemia. 46 Current Immunology Reviews, 2012, Vol. 8, No. 1 Jordan et al. cytoplasm binds to Apaf-1 to initiate the formation of an stress as major factors of both blood-brain barrier , which then binds pro-caspase-9. The dysfunctions and ischemic neuronal cell death. Third oligomerization of caspase-9 on the apoptosome activates messengers, like p53, peroxisome proliferator-activated the protease. The active caspase-9 cleaves 2 “executioner” receptors (PPAR) and NF-kB as important regulators of cell caspases, caspase- 3 and caspase-7, that then go on to cleave death and survival, they all may be important targets for new key substrates within the cell [82]. therapeutic approaches. Modulation of cell death after acute ischemia is finally determined by impaired bioenergetics and Mitochondria consume nearly 85% to 90% of a cell’s mitochondrial dysfunction, giving evidence that provide oxygen to support oxidative phosphorylation by harnessing molecular dysfunction to mitochondria as a central platform oxidized fuel to the synthesis of ATP. Depending on the in the execution of diverse cellular events, including cell availability of intracellular ATP, the cell death pathway may switch from apoptosis to necrosis. While apoptosis is the death, although presenting promising therapeutic targets in stroke and other ischemic brain diseases [79]. The present predominant cell death pathway in the presence of adequate review has summarized the role of adhesive molecules and ATP, overwhelming depletion of ATP results in necrotic cell of inflammatory mediators in various experimental models death [83]. Marked changes in ATP and related energy of cerebral ischemia, their approaches and effects in therapy metabolites develop quickly in response to occlusion of a and neuroprotection, and has critically summarized the pros cerebral artery, as expected from limitations in the delivery of oxygen and glucose [84]. However, these alterations are and cons of recent treatment strategies in the management of acute ischemic stroke, only very few of which showed some often only partially reversed on reperfusion despite improved marginal effects, whereas the majority of these treatment substrate delivery. Ischemia-induced decreases in the modalities failed to show significant and convincing mitochondrial capacity for respiratory activity probably efficacies. Considering the current controversy surrounding contribute to the ongoing impairment of energy metabolism the management of acute ischemic stroke, further studies in during reperfusion and possibly also to the magnitude of changes seen during ischemia (Fig. 1). both experimental models and human stroke victims are necessary in order to elucidate the reasons for the failure of Calcium-induced Cyt c release, as occurs in neurons many clinical trials despite promising neuroprotective during stroke and ischemia, involves rupture of the clinical studies. New and controlled trials should be designed mitochondrial outer membrane and can be blocked by to critically evaluate the major mechanisms underlying inhibitors of the mitochondrial permeability transition pore stroke pathophysiology with emphasis on new targets for (MPTP). MPTP blockers, such as cyclosporin A (CsA) and designing successful treatment modalities. bongkrekic acid, have shown neuroprotective effects in animal models of ischemia. Several inhibitors of Cyt c ACKNOWLEDGEMENTS release have shown promise in models of CNS apoptosis. This work was supported by SAF2008-05143-C03-1 Execution Phase from Ministerio de Ciencia e Innovación; Investigación sobre drogodependencias, Ministerio de Sanidad y Consumo In general, antiapoptotic strategies under evaluation for and 04005-00 and PI2007/55 Consejería de Sanidad from neuroprotection include strategies to prevent caspase- Junta de Comunidades de Castilla-La Mancha (to J.J.) and dependent apoptosis (eg, caspase inhibitors) or strategies to by “CCM Obra Social y Cultural-FISCAM’ and prevent caspase-independent apoptosis [85]. ‘Incorporación de grupos emergentes” FIS CARLOS III Caspase-dependent and caspase-independent mechanisms (EMER07/023) and FIS-FEDER (PI080693) (to M.F.G.). of cell death are implicated in focal cerebral ischemia. Increased expression of Fas and of mediators of the extrinsic CONFLICT OF INTEREST caspase-dependent pathway have been shown following focal ischemia. Increased expression of caspase-1, -3, -8, and Declared none. -9, and of cleaved caspase-8, has been observed in the penumbra [86]. The role of apoptosis in ischemia is further REFERENCES supported by reports that the inhibition of caspase-3 reduces [1] Du C, Hu R, Csernansky CA, Hsu CY, Choi DW. Very delayed infarct size after transient focal ischemia [87, 88]. infarction after mild focal cerebral ischemia: a role for apoptosis? J Cereb Blood Flow Metab 1996; 16: 195-201. CONCLUSIONS AND FUTURE DIRECTIONS [2] Kirino T, Tamura A, Sano K. Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuronal cell death following acute ischemia may exhibit Neuropathol 1984; 64: 139-47. [3] McGee-Russell SM, Brown AW, JBB. A combined light and morphological features of apoptosis, autophagy or necrosis, electron microscope study of early anoxic-ischemic cell change in pointing to the existence of multiple non-apoptotic cell death rat brain. 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Received: June 15, 2010 Revised: October 26, 2010 Accepted: November 4, 2010