Neuronal and Glial Mechanisms Underlying BBB Dysfunction-Induced Epileptogenesis
Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”
by
Yaron David
Submitted to the Senate of Ben-Gurion University
of the Negev
November 2011
Beer-Sheva
Neuronal and Glial Mechanisms Underlying BBB Dysfunction-Induced Epileptogenesis
Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”
by
Yaron David
Submitted to the Senate of Ben-Gurion University
of the Negev
Approved by the advisor______Approved by the Dean of the Kreitman School of Advanced Graduate Studies______
November 2011
Beer-Sheva
This work was carried out under the supervision of
Professor Alon Friedman
The department of Physiology,
Faculty of Health Sciences
Ben-Gurion University of the Negev.
First and foremost, I would like to thank my mentor, Prof. Alon Friedman, who offered an opportunity to a young student who knew absolutely nothing in the field of neuroscience. Your enthusiasm for the pursuit of knowledge is contagious. I also thank you for being a friend.
I would like to thanks all those people I encountered throughout the years:
Oren Tomkins who brought me to the lab. Uwe Heinemann who kindly opened the gates of the Institute fur Physiologie, in Berlin for me. Sebastian Ivens, for teaching me electrophysiology and being a good friend. Daniela Kaufer and Luisa P. Flores from UC Berkley, without whom I am sure I wouldn‘t have any molecular studies to present. Ilya Fleidervish, with whom every hour is like a semester of teaching. Maya Ketzef, a good friend which never ceases to help me and others. To my many friends in the Beer-Sheva lab: Ofer Prager, Itai Weissberg, Lyn Kamintsky, Jonathan Cohen, Yehudit Gnatek and Nitzan Levi. People in the Charite lab: Ezequiel Lapilover, Karl Schoknecht and Aljoscha Reichert.
And finally, to the many animals that helped my research along the years.
I dedicate this to my dear family, Lea and Hezi my parents and my loving wife, Yifat.
Without whom none of this would have happened.
Contents 1. Abstract ...... 9
2. Introduction ...... 12
2.1. Overview ...... 12
2.2. The Blood Brain Barrier in Health and Disease ...... 12
2.3. Insult Induced Epilepsy...... 14
3. Results ...... 17
3.1. Transcriptome Profiling Reveals TGF-Β Signaling Involvement in Epileptogenesis 17
3.2. Astrocytic Dysfunction in Epileptogenesis: Consequences of Altered Potassium and Glutamate Homeostasis? ...... 43
3.3. Blood Brain Barrier Dysfunction Underlies Stroke Complications ...... 77
3.4. The Axon Initial Segment of Layer 5 Pyramidal Neurons Following Stroke ...... 105
4. Discussion and Conclusions ...... 127
5. Bibliography ...... 131
6. List of Publications...... 140
142...... תקציר .7
List of figures
Figure 2.1: Structure of the Blood-Brain Barrier...... 13 Figure 4.1: Pathogenesis of BBB disruption mediated epileptogenesis...... 127
List of abbreviations
AIS Axon initial segment
BBB Blood-Brain Barrier
BSA Bovine Serum Albumin
CCD Charge-coupled device
CNS Central Nervous System
DOC Deoxycholic Acid
EPSC Excitatory Post Synaptic Current
EPSP Excitatory Post Synaptic Potential
FDR False Discovery Rate
GFAP Glial Fibrillary Acidic Protein
GO Gene Ontology
LED Light Emitting Diode
MCAo Medial Cerebral Artery Occlusion
mRNA Messanger RiboNucleic Acid
qRT-PCR Quantitative Real Time Polymerase Chain Reaction
RBG Rose Bengal
REST Relative Expression Software Tool
SAM Significance analysis of microarrays
SBFI Sodium-Binding Benzofuran Isophthalate
SLE Seizure Like Events
SMAD Mothers Against Decapentaplegic Homolog
TGF-β Transforming Growth Factor beta
TJ Tight Junction
1. Abstract Epilepsy is a common disease of the central nervous system, characterized by the paroxysmal appearance of multiple seizures. Even today, despite our growing understanding of the disease, epilepsy remains an incurable, only partially controlled disease. Epilepsy often develops following insults to the brain, including traumatic brain injuries, ischemia, infections, and tumors. Even today, the exact mechanisms underlying epileptogenesis, i.e., the process by which the healthy brain becomes epileptic, are not known, and there are at present no means to prevent it. Interestingly, in many (if not all) of the epileptogenic insults, vascular pathologies have been described, blood brain barrier (BBB) dysfunction in particular. The BBB is an anatomical and functional barrier that enables the central nervous system to maintain a tightly controlled environment, acting to limit the entry of blood-borne constituents into the brain‘s extracellular space. Recent studies from our laboratory demonstrated that BBB dysfunction is sufficient to induce recurrent paroxysmal epileptiform activity and seizures. Furthermore, it was shown that the penetration into the brain‘s parenchyma of a common blood protein, albumin, is sufficient to induce epileptogenesis, with one experiment suggesting this is mediated through the activation of the Transforming Growth Factor beta (TGF-) signaling pathway.
The main goal of this thesis was to study the mechanisms by which BBB dysfunction induces epileptogenesis.
In my work, I have found that, indeed, direct activation of the TGF-β pathway by the cytokine TGF-β1 results in the appearance of epileptiform activity similar to that observed after brain exposure to albumin. Co-immunoprecipitation revealed binding of albumin to TGF-β receptor II, and Smad2 phosphorylation following cortical application of albumin confirmed downstream activation of this pathway. Transcriptome profiling demonstrated similar expression patterns across large gene groups following both BBB breakdown, and the cortical application of albumin or TGF-β1. Gene changes encompassed genes associated with the TGF-β pathway, astrocytic activation, inflammation, and reduced inhibitory transmission. Importantly, TGF-β pathway blockers suppressed most albumin-induced transcriptional changes and prevented the generation of epileptiform activity.
- 9 - As one of the earliest events observed following either albumin application or other cortical lesions, is the rapid up-regulation the glial fibrillary acidic protein (GFAP), which is found exclusively in astrocytes, in the second part of my work, I investigated the role of astrocytes in our animal model. I have found similar, robust changes in astrocytic gene expression coding for genes associated with potassium and glutamate homeostasis. These changes predict reduced astrocytic clearance capacity for both extracellular glutamate and potassium. Electrophysiological recordings confirmed the reduced clearance of activity-dependent accumulation of both potassium and glutamate 24 h following exposure to albumin. To investigate the consequences of reduced astrocytic uptake of potassium and glutamate on excitatory postsynaptic potentials (EPSPs) I used a computer simulation built within the NEURON environment. Using computer modeling, we predicted that the accumulation of glutamate is associated with frequency-dependent (>100 Hz) decreased facilitation of synaptic potentials, while potassium accumulation leads to frequency-dependent (10–50 Hz) and N- methyl-D-aspartic acid (NMDA)-dependent synaptic facilitation. In vitro electrophysiological recordings during epileptogenesis confirmed frequency-dependent synaptic facilitation leading to seizure-like activity preferentially occurring with stimulation frequencies around 20 Hz. In summary, these data indicate a transcription-mediated astrocytic transformation early during epileptogenesis and suggests that the resulting reduction in the clearance of extracellular potassium underlies frequency-dependent neuronal hyper-excitability and network synchronization.
As BBB dysfunction is commonly observed in the ischemic brain, we explored the role of BBB dysfunction in the pathogenesis of stroke using imaging experiments in the photothrombosis model, exploration of mRNA expression data obtained from rat brains exposed to medial cerebral artery occlusion, and electrophysiological recordings. We observed rapid changes in gene expression following the ischemic insult leading to delayed dysfunction of the BBB surrounding the infarcted brain. Hyperexcitability and seizure like activity appeared only a few days following the ischemic event. We propose that BBB dysfunction may be critically involved in functional recovery after stroke and underlies common clinical complications including hemorrhage, epilepsy, and delayed cognitive and neurological dysfunctions.
- 10 - In the last part of my work, I used an animal model for stroke, to examine changes in the axon initial segment of neurons within the BBB disrupted area. Electrophysiological recordings of action potentials in layer 5 pyramidal neurons from the peri-infarct zone revealed no change in action potential properties: an intact first, presumably axonal upstroke component and a non- significant decrease in firing threshold. In an attempt to characterize the pattern of Na+ channel distribution in the axonal initial segment, we explored the spatial and temporal pattern of the Na+ influx into the axon initial segment elicited by a single action potential using a combination of patch-in-slice recording and high-speed Na+ imaging. In neurons from control animals, Na+ influx was maximal in a region 18 ± 2 µm long, located at a distance of 8 ± 2 µm from the soma and 27 ± 4 µm from the edge of the myelin. Computer simulations indicated that maximal excitability with the minimum number of channels is achieved with the Na+ channel hotspot located at some optimal distance from the soma.
To conclude – in this thesis I revealed early change incurred during the early time points of epileptogenesis. These include the prominent astrocytic changes such as decreased potassium clearance and activation of the TGF-β pathway, which was essential for the development of epilepsy. I could find no functional changes in neuronal intrinsic properties or dysfunction of the axon initial segment following stroke.
I believe and hope that the insights gained by my work could lead to the development of new methods for the identification of patients at risk and facilitate the development of new tools for the prevention and treatment of epilepsy.
Keyword: Blood-Brain Barrier, epilepsy, stroke, glia, astrocytes, TGF-β pathway, RT-PCR, gene array, patch-clamp, potassium glutamate, NMDA, axon initial segment, sodium imaging.
- 11 - 2. Introduction
2.1. Overview The Central Nervous System (CNS) is considered to be one of the most complex systems. It is also one of the greatest wonders of our world how such seemingly inanimate matter as our brain can give rise to thoughts, consciousness, perception, and free-willed actions. These astounding properties and functions are made possible by many cell types, interconnected to form an intricate network able to process and store information.
For many years the prevailing dogma in neuroscience focused on one cell type – the neuron. While it was evident that not only neuron-like cells are present in the CNS, neurons took the center stage and were regarded as the only crucial part for information processing. All the while, other types of cells in the brain – dubbed ―glia‖ by Virchow (1846) – were considered to be the ―glue‖ or scaffold-like structures at best. It is mostly in the last couple of decades that research has begun to shed light on the physiology of glial cells in the CNS.
The effective and coordinated function of neurons and glia in the CNS requires a highly regulated extracellular environment, wherein the concentrations of ions are tightly controlled. Indeed, chemical compounds and ions, whose levels in the blood can fluctuate during exercise or following meals, and which would have little effect on peripheral organs, may be highly toxic to the CNS and must be excluded from the brain extracellular space. Hence, it is essential that the interface between the brain extracellular space and the systemic circulation operates as a dynamic regulator of ion balance, nutrient transport, and barrier to harmful molecules. This is accomplished by the presence of an anatomical and functional barricade, the Blood-Brain Barrier (BBB).
2.2. The Blood Brain Barrier in Health and Disease It was Paul Ehrlich who observed (in the 19th century) that when some water-soluble dyes are injected into the blood stream, they do not stain the brain, the spinal cord, the eyes, or the testes (Ehrlich, 1885). This led some to hypothesize that a barrier, selectively permeable to some compounds while blocking others, exists between the blood stream and these organs. This hypothesis was first met with some criticism, arguing for different affinity of tissue types to these dyes. Finalization of the barrier hypothesis was provided by Edwin E. Goldmann who observed
- 12 - that the hydrophilic dye Trypan blue stained most organs when injected into the periphery, yet spared the CNS and testes (Goldmann, 1909). He conducted another ingenious experiment in which the dye was injected into the subarachnoid space to observe that now, the CNS was completely stained thereby proving the existence of a barrier between the blood compartment and the CNS (Goldmann, 1913).
Thru modern microscopy, we have learned that The Blood-Brain Barrier (BBB) lines most capillaries in the brain (Rubin & Staddon, 1999; Pardridge, 2002; Ballabh et al., 2004; Hawkins & Davis, 2005; Abbott et al., 2006). It is formed during the late embryonic and early neonatal period and gradually becomes impermeable. Whereas proteins are excluded from the early embryonic rodent brain (Saunders et al., 1991), it appears that a significant ionic barrier develops sometime after birth (Butt et al., 1990).
Figure 2.1: Structure of the Blood-Brain Barrier. The BBB is formed by endothelial cells, surrounded by pericytes, a basal lamina, and astrocytic end feet. Tight junctions between endothelial cells limit paracellular access pathways. (Adopted from (Abbott et al., 2006), with permission from the Nature Publishing Group).
The BBB gains its function from a distinct phenotype of endothelial cells on the brain capillary system, and is controlled by interactions with neighboring astrocytes, pericytes, and surrounding neurons, functioning together as a neurovascular unit (Iadecola, 2004). On the circumference of the capillary lies a single layer of endothelial cells, which is morphologically different from those of peripheral capillaries. These endothelia have abundant mitochondria (Oldendorf et al., 1977), lack fenestrations (Fenstermacher et al., 1988), show minimal pinocytic activity (Sedlakova et al., 1999), and are connected thru tight junctions (TJ) (Kniesel & Wolburg, 2000).
- 13 - Surrounding the endothelial cells are pericytes that are attached at irregular intervals. Both endothelial cells and pericytes are enclosed by a basal membrane 30–40 nm thick (Farkas & Luiten, 2001), continuous with the membranes of astrocytic end-feet that surround the cerebral capillaries. The final component of the neurovascular unit is the neighboring neurons, rarely found more than 8–20 μm away from a capillary (Schlageter et al., 1999).
Perturbations in BBB integrity are known to occur under numerous pathological conditions, allowing passage of blood constituents from the brain capillaries into the brain‘s extracellular space. BBB disruption has been documented in patients and animal models with brain tumors or metastases, meningeal or brain infections (Tunkel & Scheld, 1993), epilepsy (Cornford & Oldendorf, 1986), and other conditions such as seizures, cerebrovascular disorders (Klatzo, 1983), autoimmune diseases such as multiple sclerosis (Stone et al., 1995), acute cerebral infarcts (Mark & Davis, 2002). In addition, a leaky BBB was described in patients with neurodegenerative diseases such as Alzheimer‘s (Skoog et al., 1998), as well as in clinical conditions predisposing to neurodegenerative processes, such as head trauma (Barzo et al., 1996; Korn et al., 2005) and psychological stress (Friedman et al., 1996). However, we are only beginning to understand the role of the BBB in the pathogenesis of brain disorders. One of the diseases in which the role of the BBB has been under recent investigation is epilepsy.
2.3. Insult Induced Epilepsy Epilepsy is one of the prevalent disorders of the central nervous system (CNS), reported to occur in 0.5–1.5% of the population. Epilepsy is regarded as a complex of different diseases in which the CNS develops a propensity towards unprovoked seizures. Epilepsy‘s age of onset of has two major incidence peaks, one before the age of 2 and one over the age of 65 years. In the first years of life, most epilepsies result from in-born genetic errors and/or insults in utero, whilst later in life, epilepsy results mostly from acquired cerebral insults.
Many types of brain lesions can transform a healthy brain into an epileptic one, including: stroke, traumatic injury, infection, and brain neoplasia. The period of time from the onset of the lesion until the development of epilepsy is known as the epileptogenesis period.
In the latent period, since some seizures may occur from clinical, prognostic, and pathophysiological viewpoints it is prudent to distinguish these seizures, dubbed acute
- 14 - symptomatic seizures and epilepsy. Acute symptomatic seizures are defined as the seizures occurring within a week following the insult (Sander et al., 1990), in contrast to epileptic seizures – defined as two or more unprovoked seizures occurring after the first week.
The length of the epileptogenic period varies between patients and insults. For example, in stroke patients there is a peak period for developing epilepsy within the 6–12 months following the ischemic incident (Sung & Chu, 1990; Lossius et al., 2005). In cases of epilepsy due to head trauma, 86 percent of patients would have suffered two unprovoked seizures within two years of the traumatic event (Haltiner et al., 1997). Yet, interestingly, the risk for developing epilepsy appears to remain elevated for many years following the traumatic event (Caveness et al., 1979; Haltiner et al., 1997). Studies in survivors of bacterial meningitis show that most patients would have developed epilepsy within the first five years following the event, but, again, life-time risk remains elevated (Annegers et al., 1988).
Such seemingly unrelated events all culminating in the development of epilepsy initiated a search for a common pathogenic element. As already noted above (Section 2.2), one feature common to these brain insults is the breakdown of the blood-brain barrier (BBB). Indeed, a significant and long-lasting BBB breakdown had been observed in both animals (Shapira et al., 1993) and people (Cervos-Navarro & Lafuente, 1991; Tomkins et al., 2001).
To challenge the hypothesis of BBB involvement in the pathogenesis of epilepsy, a model for long-lasting BBB opening via the focal application of bile salts (Greenwood et al., 1991) was established in our lab. It was found that BBB opening results in delayed (~7d following insult) appearance of a hypersynchronous epileptiform activity and that epileptogenesis can be triggered by direct exposure of the brain extracellular space to serum albumin. Both BBB disruption and direct application of albumin on the cortical surface led (within hours) to a robust activation of astrocytes, followed (within few days) by cortical dysfunction and (within weeks) by neuronal degeneration (Seiffert et al., 2004; Tomkins et al., 2007). Since under normal conditions albumin is absent in the brain, the binding partner and its role in activating specific pathways was obscure. A search for albumin binding receptors in other tissues revealed that TGF-β receptors bind albumin in the kidney and are involved in its endocytosis in epithelial cells of the lung
- 15 - (Gekle et al., 2003). This led us to hypothesize that the TGF-β pathway is involved in the process of epileptogenesis.
The aim of my work was to further elucidate the mechanisms involved in epileptogenesis following BBB disruption. The first part of my research involved elucidating the role of TGF- in epileptogenesis using field potential recordings and large scale mRNA expression studies. In the second part of my research I examined the physiological sequelae of albumin on the rat cortex, confronting two hypotheses relevant to the development of hyper-synchronous activity. In the third part of my work, I evaluated the role of BBB disruption in stroke comparing expression changes, electrophysiological recordings, and imaging. In the fourth part, I established the Rose Bengal stroke model in mice to study changes in neurovascular functions in the peri-ischemic, BBB disrupted cortex. I compared alterations in gene expression following BBB dysfunction to those described after stroke; in preliminary experiments using intracellular recordings and fast sodium imaging from neurons in the peri-ischemic region, we challenged the hypothesis that changes in the axon initial segment contribute to alterations in neuronal excitability during epileptogenesis.
- 16 - 3. Results
3.1. Transcriptome Profiling Reveals TGF-Β Signaling Involvement in Epileptogenesis
Abstract Brain injury may result in the development of epilepsy, one of the most common neurological disorders. We previously demonstrated that albumin is critical in the generation of epilepsy after blood–brain barrier (BBB) compromise. Here, we identify TGF-β pathway activation as the underlying mechanism. We demonstrate that direct activation of the TGF-β pathway by TGF-β1 results in epileptiform activity similar to that after exposure to albumin. Coimmunoprecipitation revealed binding of albumin to TGF-β receptor II, and Smad2 phosphorylation confirmed downstream activation of this pathway. Transcriptome profiling demonstrated similar expression patterns after BBB breakdown, albumin, and TGF-β1 exposure, including modulation of genes associated with the TGF-β pathway, early astrocytic activation, inflammation, and reduced inhibitory transmission. Importantly, TGF-β pathway blockers suppressed most albumin-induced transcriptional changes and prevented the generation of epileptiform activity. Our present data identifies the TGF-β pathway as a novel putative epileptogenic signaling cascade and therapeutic target for the prevention of injury-induced epilepsy.
- 17 - Introduction Epilepsy, affecting 0.5–2% of the population worldwide, is one of the most common neurological disorders. Focal neocortical epilepsy often develops after traumatic, ischemic, or infectious brain injury. Although the characteristic electrical activity in the epileptic cortex has been extensively studied, the mechanisms underlying the latent period preceding the occurrence of spontaneous epileptic seizures (epileptogenesis) are poorly understood. After injury, local compromise of blood–brain barrier (BBB) integrity is common (Tomkins et al., 2001; Neuwelt, 2004; Abbott et al., 2006; Oby and Janigro, 2006), as revealed by ultrastructural studies of animal and human epileptic tissue in multiple forms of epilepsy (Kasantikul et al., 1983; Cornford and Oldendorf, 1986; Cornford, 1999; Marchi et al., 2007; van Vliet et al., 2007), raising the possibility that vascular damage, and specifically BBB opening, may serve as a trigger event leading to epilepsy. This hypothesis has been confirmed by animal studies, in which opening of the BBB was sufficient to induce delayed epileptiform activity (Seiffert et al., 2004). Subsequent studies have shown that albumin, the most common serum protein, is sufficient to recapitulate the epileptiform activity induced by BBB disruption.
Furthermore, uptake of serum components such as albumin and IgGs, associated with BBB disruption, has been demonstrated in various cell populations. Albumin is taken up by astrocytes (Ivens et al., 2007; van Vliet et al., 2007), neurons (Marchi et al., 2007; van Vliet et al., 2007), and microglia although to a lesser extent (van Vliet et al., 2007), whereas IgG uptake has been found in neurons (Rigau et al., 2007). In rat lung endothelial cells, albumin endocytosis is mediated by transforming growth factor β receptors (TGF-βRs), leading to phosphorylation of the proximate effector of the canonical TGF-β signaling pathway, Smad2, and translocation of the activated Smad2/Smad4 complex to the nucleus (Siddiqui et al., 2004). TGF-βRs are also implicated in albumin uptake by astrocytes, as blocking TGF-βRs prevents albumin uptake and suppresses albumin-induced epileptiform activity (Ivens et al., 2007). This raises the possibility that albumin activation of the TGF-β signaling pathway serves as the underlying mechanism; however, this hypothesis remains unconfirmed.
Here, we show that activation of the TGF-β signaling pathway is sufficient to induce epileptiform activity. Furthermore, we show that global transcriptional cascades induced by TGF-β1 or albumin exposure before the development of epileptiform activity (during the
- 18 - epileptogenesis window) are nearly identical and can be blocked by application of TGF-βR blockers. Given the pleiotropic effects of the TGF-β signaling pathway, these findings provide a plausible mechanism for epileptogenesis after brain injury and advocate a specific therapeutic target.
- 19 - Materials and Methods In vivo preparation. All experimental procedures were approved by the animal care and use ethical committees at Charité University Medicine, Berlin and Ben-Gurion University of the Negev, Beer-Sheva. The in vivo experiments were performed as described previously (Seiffert et al., 2004; Ivens et al., 2007). In short, adult male Wistar rats (120–250 g) were anesthetized and placed in a stereotactic cage, a 4 mm diameter bone window was drilled over the somatosensory cortex, and the dura was opened. The underlying cortex was then perfused with artificial CSF (aCSF; composition in mm: 129 NaCl, 21 NaHCO3, 1.25 NaH2PO4, 1.8 MgSO4 1.6 CaCl2, 3 KCl, 10 glucose) supplemented with either deoxycholic acid (DOC; 2 mm; Sigma-Aldrich), bovine serum albumin (BSA; 0.1 mm; Merck), corresponding to 25% of serum albumin concentration, or with TGF-β1 (10 ng/ml; Peprotech) for 30 min. Sham-operated animals (perfused with aCSF) served as controls. Only rats with no apparent injury to the cortical surface or bleeding from cortical vessels (as seen under the surgical microscope) at the end of the procedure were used.
To investigate transcriptional changes occurring during the epileptogenesis time window (before the development of epileptiform activity), animals were killed 7/8, 24, or 48 h after treatment. RNA isolated from these animals was used for microarray and quantitative real-time (qRT)-PCR analyses described below. As robust changes in gene expression were observed after the 24 h treatments, a second set of animals including sham-operated controls and animals treated with BSA (0.1 mm) or BSA plus TGF-βR blockers (TGF-βRII antibody, 50 μg/ml; Santa Cruz Biotechnology; SB431542, 100 μm, TGF-βRI kinase activity inhibitor; Tocris Bioscience) were killed 24 h after treatment. RNA isolated from these animals was also used for microarray and qRT-PCR analyses. The last set of animals was treated with 0.1–0.2 mm BSA and killed 24 or 46–50 h after treatment for Smad2-P immunodetection.
In vitro slice preparation. Brain slices for the in vitro experiments were prepared using standard techniques (Pavlovsky et al., 2003; Seiffert et al., 2004; Ivens et al., 2007). Slices were transferred to a recording chamber where they were incubated in aCSF containing BSA (0.1 mm), TGF-β1 (10 ng/ml), or artificial serum (aSerum; composition based on aCSF with the following changes, composition in mm: 0.8 MgSO4, 1.3 CaCl2, 5.7 KCl, 1 l-glutamine, 0.1 BSA). To block the activity of TGF-β1, slices were incubated in aCSF containing SB431542 (10
- 20 - μm) before the addition of TGF-β1 (10 ng/ml). To block TGF-βRs, slices were incubated in aCSF containing SB431542 (10 μm) and TGF-βRII antibody (10 μg/ml) for 30 min followed by incubation in BSA in the presence of TGF-βR blockers. For detection of epileptiform activity, field potentials were recorded 4–12 h after incubation in cortical layer IV using extracellular glass microelectrodes (∼3MΩ) in response to bipolar stimulation at the border of white and gray matter. The time of recording was chosen based on the occurrence of epileptiform activity 4–8 h after albumin exposure in the slice preparation (Ivens et al., 2007).
Albumin and TGF-βRII coimmunoprecipitation. To prepare cortical lysates, brains were isolated from naive adult Wistar rats, dissected in cold saline solution, and lysed in radioimmunoprecipitation assay buffer. BSA (3 μg) was added to lysates to approximately match the amount of precipitating anti-albumin antibodies. Immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore Bioscience Research Reagents) with the following modifications to the standard protocol: the starting amount of protein was increased to 1500 μg, and the incubation time with precipitating antibodies was increased to 90 min. Lysate samples (positive or negative for albumin) were immunoprecipitated with an anti-TGF-βRII antibody (Millipore Bioscience Research Reagents) or an anti-albumin antibody (Biogenesis).
The immunoprecipitated samples were separated with SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was stained with Ponceau S stain to confirm that the IP procedure was successful. It was then destained and blocked with 5% BSA in standard TBS-T buffer overnight at 4°C. TGF-βRII was detected with a rabbit anti-TGF-βRII antibody (Millipore Bioscience Research Reagents) and an alkaline phosphatase-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories). Chemiluminescent detection was done using Lumi-Phos Western Blot Chemiluminescent Substrate (Pierce) and standard x-ray film according to the manufacturer's instructions.
Smad2-P Western blot analysis. Cortical lysate samples from sham-operated controls and animals treated with BSA were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk overnight at 4°C, incubated with a rabbit polyclonal antibody against phospho-Smad2 (Millipore Bioscience Research Reagents) for
- 21 - 48 h at 4°C, and incubated with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories) for 2 h at room temperature.
Microarrays. Total RNA was isolated using the TRIzol reagent (Invitrogen) and prepared using the Affymetrix GeneChip one-cycle target labeling kit (Affymetrix). Biotinylated cRNA was fragmented and hybridized to the GeneChip Rat Genome 230 2.0 Array according to company protocols (Affymetrix Technical Manual). Normalization of the array data was done using GC Robust Multi-Array Average analysis, which takes into account the GC content of the probe sequences (Wu et al., 2004). Functional annotation analysis was performed with the program Database for Annotation, Visualization, and Integrated Discovery (DAVID) 2008 (Dennis et al., 2003) (http://david.abcc.ncifcrf.gov). Unspecific (e.g., cellular process) and redundant terms (e.g., death and cell death) were removed, and the full lists are provided in supplemental Tables 1–8, available at www.jneurosci.org as supplemental material. The GenMAPP 2.0 program (Salomonis et al., 2007, http://www.genmapp.org) was used to visualize genes involved in TGF- β signaling. For the time course analysis, one array was run for each treatment (DOC, BSA, and TGF-β1) for the following time points: 7/8, 24, and 48 h. In addition, a sample from a sham- treated animal (24 h) was run and used to normalize the other arrays. Pairwise Pearson correlation coefficients for the three treatments were determined with Excel (Microsoft). Hierarchical clustering was performed with Gene Cluster and displayed with TreeView software (Eisen et al., 1998).
Arrays were then run for the second set of animals killed 24 h after treatment (sham, n = 2; BSA, n = 3; BSA plus TGF-βR blockers, n = 4). Significance analysis of microarrays (SAM) was performed with a false discovery rate (FDR) threshold of 9.2%. A 1.5-fold change cutoff was also used to filter this list. Genes from this filtered list which demonstrated a log2 ratio difference >0.5 between the albumin and albumin plus blocker treatments were considered part of the attenuated response. The remaining genes were considered part of the unattenuated response. All microarray data are available at the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE12304.
Real-time RT-PCR. mRNA expression levels were determined by quantitative reverse transcriptase-PCR by real-time kinetic analysis with an iQ5 detection system (Bio-Rad). Real-
- 22 - time PCR data were analyzed using the PCR Miner program (Leonoudakis et al., 2008). 18S mRNA levels were used as internal controls for variations in sample preparation. Primer sequences are provided in supplemental Methods, available at www.jneurosci.org as supplemental material.
Statistical analyses. For the electrophysiological data, differences between treated and control slices were determined by the Mann–Whitney U test for two independent samples or the χ2 test using SPSS 13.0. Linear regression analysis for the microarray data was performed with GraphPad Prism. PCR data were analyzed with an unpaired Student's t test (p < 0.05 was considered significant) in Excel (Microsoft) or with the relative expression software tool (REST) (Pfaffl, 2001). REST determines significance of the group ratio results with a randomization test. p < 0.05 was taken as the level of statistical significance.
- 23 - Results
TGF-β signaling is sufficient to induce epileptiform activity To assess the hypothesis that activation of the TGF-β signaling pathway is the mechanism underlying albumin-induced epileptogenesis, we activated this pathway directly by incubating neocortical slices with TGF-β1 (10 ng/ml) in aCSF and performed electrophysiological recordings. These recordings were compared with those of slices treated with aSerum [a solution containing serum levels of electrolytes and 0.1 mm albumin, previously shown to induce epileptogenesis (Ivens et al., 2007)], albumin in aCSF, or aCSF (control). Spontaneous, prolonged, and hypersynchronous interictal-like activity was observed in slices treated with aSerum after 6–10 h (n = 6 of 9 slices, three animals) but never in aCSF-treated slices (Fig. 1 B). When albumin was added to the control aCSF solution, epileptiform activity was recorded only in response to stimulation of the white matter (Fig. 1 C) (n = 8 of 12 slices, six animals). Importantly, epileptiform activity was also recorded after incubation in TGF-β1, which is similar to that seen after treatment with aSerum and albumin in aCSF (Fig. 1 C, n = 5 of 5 slices, four animals; n = 7 of 9 slices, three animals; and n = 8 of 12 slices, six animals, respectively). Recordings were performed 4–12 h after treatment. Although treatment with either TGF-β1 or albumin in aCSF resulted in the appearance of evoked epileptiform activity, only the altered electrolytic solution (i.e., aSerum) resulted in spontaneous activity in the slice preparation. Importantly, when aSerum was applied without albumin, neither spontaneous nor evoked epileptiform activity was recorded. In all three cases (albumin, TGF-β1, and aSerum), the evoked epileptiform activity was all-or-none in nature, paroxysmal, prolonged, and propagating along the cortical slice, similar to that seen after BBB opening with bile salts (Seiffert et al., 2004; Ivens et al., 2007) and typical to that observed in acute models of epilepsy (Gutnick et al., 1982). No epileptiform activity was seen in the control aCSF-treated slices.
To further confirm that the TGF-β1-induced epileptiform activity was dependent on the TGF- βR-mediated pathway, we performed additional trials of the above experiments in the presence of two TGF-βR blockers (SB431542 and anti-TGF-βRII antibody). TGF-βR blockers reduced the number of slices exhibitng epileptiform activity induced by TGF-β1 or albumin by 40% (Fig. 1 C). Moreover, the measured integral of the field potential (albumin, 117.2 ± 35.4 mV*ms; TGF-β1, 84.1 ± 20.1 mV*ms) was significantly lower in slices treated with albumin or TGF-β1
- 24 - in the presence of TGF-βR blockers (albumin and blockers: 23.7 ± 6.9 mV*ms, n = 20 slices, four animals, p = 0.001; TGF-β1 and blockers: 12.5 ± 3.9 mV*ms, n = 8 slices, four animals, p = 0.005) (Fig. 1 D).
Figure 1. TGF-β signaling induces epileptiform activity. A, Photograph of a brain slice displaying electrode positioning. The stimulating electrode was placed at the white–gray matter border. B, Extracellular recordings showing spontaneous interictal-like epileptiform activity after treatment with aSerum containing albumin. Asterisk refers to the region corresponding to the slower time scale shown in the lower trace. C, Evoked responses from slices treated with aCSF, albumin, albumin plus TGF-β receptor blockers, TGF-β1, or TGF-β1 plus TGF-β receptor blockers. TGF-β receptor blockers prevent epileptiform activity induced by albumin or TGF-β1 treatment. D, Comparison of mean event integral (black bars) in the 50–500 ms time range (after stimulation) shows a significant increase in the integral of the delayed epileptiform field potential in the albumin and TGF- β1-treated slices but not in slices treated with TGF-β receptor blockers. The white bars represent the percentage of slices with paroxysmal, epileptiform activity. Error bars indicate SEM. Asterisks indicate p< 0.05.
Albumin binds TGF-βRs and activates the TGF-β pathway To determine whether albumin binds to TGF-β receptors, coimmunoprecipitation using antibodies against albumin or TGF-βRII was performed on cortical lysate samples (obtained
- 25 - from naive rats). An expected band corresponding to TGF-βRII was detected in samples immunoprecipitated with the TGF-βRII antibody. More importantly, this band was also detected in samples preincubated with albumin when immunoprecipitated with the albumin antibody and probed for TGF-βRII (Fig. 2 A). These results reveal a direct interaction between albumin and TGF-βRII. In the canonical TGF-β signaling pathway, Smad2 and/or 3 are phosphorylated after TGF-β receptor activation and form a complex with Smad4, which then translocates into the nucleus and activates transcription (Beattie et al., 2002). To investigate whether albumin activates downstream components of the TGF-β pathway, Smad2 phosphorylation levels in cortical lysates were assessed by Western blot, revealing an increase in Smad2 phosphorylation during the epileptogenic time window in animals exposed to albumin compared with sham- operated controls (Fig. 2 B).
Figure 2. Albumin activates TGF- β pathway. A, Albumin and TGF- βRII immunoprecipitations. Samples treated or untreated with serum albumin were coimmunoprecipitated with antibodies directed against albumin or TGF-βRII. All samples were then probed with an anti-TGF-βRII antibody. The band at 50 kDa is the heavy chain of the precipitating antibody. B, Western blot analysis of Smad2-P 24 and 48 h after albumin treatment. Each band represents a different animal (24 h: controls, n = 2; albumin, n = 3; 48 h: controls, n = 2; albumin, n = 4).
Similar transcriptional profiles follow BBB opening, albumin, and TGF-β1 treatments
- 26 - BBB opening or exposure to albumin in vivo (Ivens et al., 2007), as well as in vitro exposure of neocortical slices to albumin or TGF-β1 (Fig. 1 C), all result in the gradual development of hypersynchronous neuronal epileptiform activity. The delayed appearance of abnormal activity [5–7 h in vitro and >4 d in vivo (Ivens et al., 2007)] suggests a transcription-mediated mechanism. In search of a common pathway and transcriptional activation pattern that underlie epileptogenesis after BBB opening, we performed transcriptome analysis using Affymetrix rat microarrays. RNA was extracted from cortical regions of rats treated with DOC (to induce BBB opening), albumin, or TGF-β1 for various durations (7/8, 24, 48 h). These time points were chosen to evaluate changes in transcription occurring before the appearance of epileptiform activity. Control RNA was extracted from cortical regions excised from sham-operated animals. Hierarchical clustering analysis of these arrays showed that, overall, the three treatments resulted in strikingly similar gene expression profiles, as arrays representing similar time points clustered together regardless of the treatment (Fig. 3 A). These similarities are exemplified in Figure 3 B, which shows a high correlation between the expression profiles for the albumin and TGF-β1 treatments at 24 h (r 2 = 0.75, p < 0.0001).
To identify biological themes common to the three treatments, the gene list was filtered to include genes showing at least a 1.5-fold change in expression and a Pearson correlation coefficient ≥0.95 for pairwise comparisons between all treatments (see Materials and Methods). Hierarchical clustering was performed, and the main clusters were used for gene ontology (GO) analysis with DAVID (Dennis et al., 2003). DAVID calculates the probability that particular GO annotations are overrepresented in a given gene list using a Fisher exact probability test. Molecular function and biological process GO terms with a p value <0.05 containing at least three genes were considered significant. This analysis revealed major gene expression trends that occur in response to all three epileptogenic treatments (Fig. 3 C). Early responses include genes involved in general stress-related cellular, metabolic, and intracellular signaling pathways; early responses persisting to later time points include inflammatory processes as well as genes involved in induction of cell cycle, differentiation, proliferation, and apoptosis; responses at middle to late time points include repression of synaptic transmission and ion transport genes (Fig. 3 C) (for complete GO term annotation results, see supplemental Tables 1–4, available at www.jneurosci.org as supplemental material).
- 27 - Figure 3. Genome-wide transcriptional analysis after epileptogenic treatments. A, Hierarchical clustering of arrays corresponding to 7/8, 24, and 48 h after DOC, albumin, and TGF- β1 treatments. Note how arrays cluster together for each time point across all treatments. Genes showing at least a 1.5- fold change in expression were included. B, Linear regression analysis between TGF-β1 and albumin treatments at 24 h. Only genes with a fold change equal to or >1.5 for the TGF-β1 treatment were included. C, Hierarchical cluster analysis of genes showing correlation (>0.95) between all treatments. Selected clusters were annotated with DAVID to reveal biological themes common to all treatments. Color bar indicates range of log2 ratios.
Gene level expression profiles Selected GO term groups were chosen for further analysis of individual gene expression profiles (Fig. 4). The most dramatic change observed in all treatments across all time points was the early and persistent upregulation of genes associated with immune response activation (Fig. 4 A,B). Inflammatory genes included NF-κB pathway-related genes, cytokines, and chemokines (Il6, Ccl2, Ccl7), transcription factor Stat3, the pattern recognition receptor CD14, and
- 28 - extracellular matrix proteins (Fn1 and Spp1) (Fig. 4 A). Activation of the complement pathway was also prominent (Fig. 4 B) and included C1 subcomponents (C1qa, C1qb, C1qg), the associated protease C1s, Masp1, and C2. A significant neuronal response was prominent in the middle-late time points and included downregulation of genes associated with GABAergic (inhibitory) neurotransmission (including the GABAA receptor subunits, Gabra4, Gabrd, Gabrg1, and Gabrb2, as well as glutamic acid decarboxylase (Gad67) (Fig. 4 C) and modulation of genes associated with glutamatergic (excitatory) neurotransmission (including upregulation of the ionotropic glutamate receptor subunits, GluRδ2 and GluR1 and downregulation of the NMDA receptor subunits, NR2B, NR2A, and NR2C and the metabotropic glutamate receptor, mGluR7) (Fig. 4 D). Furthermore, a variety of voltage-gated ion channels, including calcium, sodium, chloride, and potassium channels, were affected by all three epileptogenic treatments (Fig. 4 E), including a noteworthy downregulation of voltage-gated (Kv7.3 and Kv8.1) and inward-rectifying (Kir3.1) potassium channels. We also found significant modulation of glial- specific genes beginning at the early time point (Fig. 4 F): the cytoskeletal proteins GFAP and vimentin (Vim), and several calcium-binding proteins (S100a6, S100a10, s100a11), were all upregulated while gap junction connexins 30 and 43 (Cx30 and Cx43) and the inward-rectifying potassium channel Kir4.1 were downregulated. Microarray-based gene expression measurements for selected genes were further verified using quantitative real-time PCR. Expression patterns were similar, although the magnitude of the fold changes sometimes differed (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).
- 29 - Figure 4. Gene ontology annotation
analysis. A–F, Log2 ratios for selected genes from GO annotation analysis involved in inflammation (A), complement activation (B), GABAergic transmission (C), glutamatergic transmission (D), voltage-gated ion channels (E), and astrocytes (F). Numbers below data points correspond to the various treatments (7/8, 24, and 48 h).
TGF-β pathway activation underlies epileptogenic transcriptional response Given the high correlation between expression profiles after the three epileptogenic treatments, combined with the biochemical evidence that albumin binds to TGF-β receptors and the physiological evidence that TGF-β1 induces evoked epileptiform activity, we assessed the extent to which each treatment activates transcription of genes known to be associated with the TGF-β signaling pathway using GenMAPP (Salomonis et al., 2007). Forty-three percent of genes analyzed in the TGF-β signaling pathway were modulated by both treatment with albumin and TGF-β1. Genes which showed at least a 1.5-fold change in expression after albumin or TGF-β1 treatment are highlighted in Figure 5. Importantly, 86% of genes modulated by TGF-β1 treatment are also modulated after albumin treatment, indicating a high degree of overlap. To check the specificity of this pathway activation, additional pathways were analyzed. Indeed, there was still a high degree of overlap, but the percentage of genes modulated by both albumin
- 30 - and TGF-β1 treatments was much lower. For example, for the androgen receptor and α6–β4 integrin signaling pathways, only 21.7 and 24% of genes analyzed were modulated by both treatments.
The above evidence indicates that TGF-β signaling is a key mediator of albumin-induced epileptogenesis. To determine if the global transcriptional response seen after albumin treatment is dependent on activation of the TGF-β signaling pathway, we performed an additional set of microarray expression profiles using rats treated with albumin in the absence (n = 3) or presence of TGF-βRI and II blockers (n = 4, TGF-βR1 kinase activity inhibitor SB431542 and anti-TGF- βRII antibody) and killed 24 h after treatment. Although some changes in gene expression resulting from albumin treatment were still present after the blocker treatment, the majority of these changes were absent or attenuated after TGF-β pathway blocker treatment (Fig. 6 A), confirming dependence of the albumin-induced transcriptional response on TGF-β signaling.
Gene ontology analysis was then used to reveal which biological processes were blocked after TGF-β pathway blocker treatment (Fig. 6 A) (for complete GO term annotation results, see supplemental Tables 5–8, available at www.jneurosci.org as supplemental material). Genes in the TGF-β signaling GO term demonstrated a dramatic suppression of the albumin-induced expression changes in the presence of TGF-β signaling blockers (Fig. 6 B). In addition, TGF-β pathway blocker treatment prevented the albumin-induced modulation of genes involved in neuronal processes, immune response, and ion and cellular transport (Fig. 6 B). Several prominent signaling pathways including the NF-κB cascade, Jak-Stat cascade, and MAPKKK cascade were upregulated after albumin treatment but did not show a similar upregulation after albumin treatment in the presence of TGF-β pathway blockers. Quantitative real-time PCR was also performed with these samples to confirm the microarray results (Fig. 6 C). Indeed, TGF-β pathway blocker treatment completely blocked expression changes after albumin exposure for Stat3 and Glt-1 and partially blocked changes for Cx43 and GFAP.
- 31 - Figure 6.Blocking TGF-β signaling prevents albumin- induced gene expression.A, Genomic expression analysis after treatment with albumin or albumin plus TGF-β receptor blockers. SAM was performed with an FDR threshold of 9.2%, and these genes are represented on the heat map. Gene ontology analysis was performed with DAVID for genes showing an attenuated
[(albumin log2ratio) − (albumin + blocker log2ratio) > 0.5] or unattenuated response after treatment with albumin plus TGF-β receptor blockers compared with albumin treatment (see Materials and Methods). B, Fold changes for specific genes from GO analysis. C, qPCR analysis for selected genes after albumin (n = 3) or albumin plus TGF-β receptor blockers (n = 4). Error bars indicate SEM, and asterisks indicate p< 0.05.
- 32 - Figure 5. Albumin alters TGF-β pathway gene expression. TGF-β pathway map generated with GENMAPP software illustrating significant changes (>1.5 or <−1.5-fold change) in gene expression after albumin treatment compared with TGF-β1 treatment. Yellow- labeled genes represent genes up or downregulated after albumin treatment, blue-labeled genes represent genes up or downregulated after TGF-β1 treatment, and green-labeled genes represent genes up or downregulated after both treatments. Gene pathway map created by Nurit Gal and Manny Ramirez, Copyright 2002, Gladstone Institute.
- 33 - Discussion BBB breakdown is a hallmark of vascular injury in the brain and is observed in numerous neurological diseases including traumatic brain injury, stroke, and neurodegenerative diseases (Neuwelt, 2004; Abbott et al., 2006; Oby and Janigro, 2006; Zlokovic, 2008).
Compromise of the BBB is triggered by preceding processes that in turn cause vascular injury. For example, perivascular astrocytes or perivascular microglia could be activated by an initial precipitating event (trauma or ischemia) and cause vascular injury leading to serum albumin extravasation into the brain parenchyma. We have previously shown development of cortical dysfunction—specifically, hypersynchronous neuronal activity after BBB opening or exposure to serum albumin (Seiffert et al., 2004; Ivens et al., 2007). Cortical dysfunction was followed by reduced dendritic branching and neuronal loss several weeks after either treatment (Ivens et al., 2007).
In this study, we now extend these findings to include the appearance of spontaneous, prolonged, and hypersynchronous interictal-like activity after treatment with albumin in a solution containing serum levels of electrolytes (aSerum). We did not observe spontaneous activity when slices were treated with albumin or TGF-β in aCSF, probably attributable to the generally low excitability and lack of spontaneous activity of the deafferented slice preparation (Connors et al., 1982; Gutnick et al., 1982). The changes in electrolyte concentrations in the aSerum solution (e.g., higher K+, lower Mg2+ and Ca2+) are probably sufficient to increase neuronal excitability such that the epileptiform activity, which characterizes the network during activation, appears spontaneously. We have previously shown that spontaneous activity is rarely evoked in slices treated with albumin in aCSF (∼10% of slices) [see also Seiffert et al. (2004)]. Importantly, spontaneous recurrent seizures followed by secondary generalization were also observed in some animals treated with albumin (Ivens et al., 2007). These observations combined with the appearance of epileptiform activity after albumin treatment in vitro likely reflect abnormal network epileptic activity in vivo.
Since serum albumin is sufficient to induce epileptic-like activity, our initial hypothesis was that albumin enters the normally inaccessible CNS environment, binds to TGF-β receptors, and causes a cascade of events culminating in epileptiform activity. Here, we report similar
- 34 - development of epileptiform activity after exposure to TGF-β1, demonstrating the importance of TGF-β pathway activation in this injury model. We further show that albumin binds TGF-βRII and induces the phosphorylation of Smad2. Given the latent period before the appearance of epileptiform activity (i.e., epileptogenesis), we hypothesized that BBB breakdown, albumin, and TGF-β1 share a common mechanism, specifically a transcriptional mechanism involving the TGF-β pathway. Indeed, clustering and gene ontology analysis revealed striking similarities across the three treatments. The most prominent finding from our microarray results is the identification of a key role for TGF-β signaling, with activation of TGF-β-related genes seen in response to TGF-β1, albumin, and BBB breakdown. Blocking TGF-β signaling prevented the majority of the albumin-induced transcriptional responses, allowing us to narrow our gene list and identify the genes that are most relevant to epileptogenesis under these conditions. Furthermore, application of TGF-βR blockers suppressed the development of epileptiform neuronal activity after albumin or TGF-β1 treatment, highlighting this pathway as a novel therapeutic target.
Interestingly, TGF-β1 is generally considered to have a protective function. Previous studies have demonstrated the ability of TGF-β1 to prevent glutamate neurotoxicity in vitro and protect against ischemic injury in vivo (Prehn et al., 1993; Henrich-Noack et al., 1996; Ruocco et al., 1999). However, studies have also suggested TGF-β1 may not always be neuroprotective. Transgenic mice overexpressing TGF-β1 specifically in astrocytes (Wyss-Coray et al., 1995) developed seizures (along with overproduction of extracellular matrix components, severe communicating hydrocephalus, motor incoordination, and early runting). Furthermore, a recent study has demonstrated that blockade of TGF-β–Smad2/3 signaling in peripheral macrophages in a mouse model for Alzheimer's disease results in marked attenuation of cerebrovascular-amyloid deposits (Town et al., 2008). These studies, along with our work, illustrate the complexity and importance of TGF-β signaling in neurological diseases. Additional work is needed to elucidate the mechanisms responsible for the protective and detrimental effects of TGF-β signaling.
Other significant findings from our microarray results include the early upregulation of genes involved in inflammatory processes and the delayed downregulation of genes involved in neuronal processes including synaptic transmission and ion transport. Features of CNS inflammation, such as glial and complement activation, cytokine production, and adhesion
- 35 - protein expression, were all present in our array data. Upregulation of genes involved in activation of the NF-κB pathway and complement cascade reflects a significant innate immune response. In recent years, several studies have shed light on the importance of inflammatory processes in epilepsy. Both NF-κB pathway activation and complement activation have been reported in various epilepsy animal models (Rozovsky et al., 1994; Rong and Baudry, 1996; Lubin et al., 2007; van Vliet et al., 2007), as well as the involvement of other immune response genes including Il6 (Balosso et al., 2008), Ccl2 (Calvo et al., 1996; Manley et al., 2007), Stat3 (Choi et al., 2003), and Fn1 (Hoffman and Johnston, 1998). A study by Rizzi et al. (2003), where status epilepticus (SE) was induced with kainic acid, found cytokines to be causally involved in the SE-induced neuronal damage, as cytokine synthesis preceded hippocampal neuronal injury, and this injury only occurred when cytokines were produced (Rizzi et al., 2003).
Some immune-related genes have also been shown to play a role in neuronal functions. Cytokines such as interleukin-1β (Il1b) and tumor necrosis factor (TNF) have been shown to increase neuronal excitability. Il1b mediates increased neuronal glutamate release (Casamenti et al., 1999), induces phosphorylation of the NMDA NR2B subunit (Viviani et al., 2003; Balosso et al., 2008), and prevents glutamate uptake by astrocytes (Hu et al., 2000). TNF promotes recruitment of AMPA receptors lacking the Glur2 subunit to the neuronal membrane leading to increased calcium influx and also promotes endocytosis of GABAA receptors (Beattie et al., 2002; Stellwagen et al., 2005; Leonoudakis et al., 2008). An upregulation of TNF was observed in our arrays, indicating it may be involved in increasing neuronal excitability during epileptogenesis in addition to its immune-related functions. Another immune-related protein with a possible nonimmune function after injury is C1q, the first component of the classical complement cascade. C1q has recently been shown to play a role in synapse elimination during development and in neurodegeneration (Stevens et al., 2007). Upregulation of several C1q subcomponents in our arrays may be associated with synaptic remodeling and neuronal loss after the generation of the epileptic focus (Ivens et al., 2007).
An imbalance of excitatory and inhibitory transmission resulting in increased network excitability is a feature of most epilepsy models. In our study, several GABAA receptor subunits were downregulated, whereas ionotropic glutamate receptor subunits were upregulated. Genes involved in glutamate transport (Glt-1) as well as potassium and chloride channels were also
- 36 - downregulated. These changes, appearing in the middle-late time points, predict an increase in network excitability. These results are also consistent with a previous microarray study examining gene expression changes during epileptogenesis after electrically induced SE (van Vliet et al., 2007), which found downregulation of GABAA and NMDA receptor subunits.
Our initial findings showing albumin uptake by astrocytes (Ivens et al., 2007) raised the hypothesis that an early glial response precedes the development of abnormal neuronal activity. This hypothesis is reinforced by our microarray data. Glial cells are responsible for the homeostasis of the extracellular environment (i.e., uptake of glutamate and potassium) under normal conditions and for the initiation of inflammatory processes in the CNS when activated after injury. Our results support various studies that have implicated a role for these cells in epileptic activity (Tian et al., 2005; Wetherington et al., 2008). Importantly, the early astrocytic response, before epileptic activity is observed, further strengthens the putative role of these cells in the epileptogenic process itself. The downregulation of the astrocytic glutamate transporter, Glt-1, the inward rectifier potassium channels (Kir 4.1) and connexins Cx26, Cx30, and Cx43, all predict impaired buffering of glutamate and potassium, which in turn will enhance local neuronal excitability. The upregulation of the transcription factor Stat3 predicts astrocytic differentiation (Morita et al., 1995) and transformation (Choi et al., 2003). In addition, upregulation of GFAP and vimentin provide evidence for reactive gliosis found in many epilepsy models, which is characterized by hypertrophy and increased intermediate filament expression (Wetherington et al., 2008). Overall, our microarray data suggest a pronounced immediate glial response followed by a later neuronal response. It remains to be determined whether our treatments induce the release of mediators from astrocytes that in turn activate transcription in neurons as well as in other cell types such as microglia and endothelial cells.
Although many of the molecular changes revealed by our microarray study are shared with different epilepsy models, our study is the first to demonstrate that these changes, after direct vascular injury (i.e., BBB breakdown), are associated with brain exposure to serum albumin and are mediated via the TGF-β signaling pathway. Indeed, using TGF-β pathway blockers, we were able to not only block the transcriptional response after albumin exposure in vivo but also the development of epileptiform activity. Combined, these results present the TGF-β pathway as a novel therapeutic tool for preventing injury-related epileptogenesis.
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- 42 - 3.2. Astrocytic Dysfunction in Epileptogenesis: Consequences of Altered Potassium and Glutamate Homeostasis?
Abstract Focal epilepsy often develops following traumatic, ischemic or infectious brain injury. While the electrical activity of the epileptic brain is well characterized, the mechanisms underlying epileptogenesis are poorly understood. We have recently shown that in the rat neocortex, long- lasting breakdown of the blood-brain barrier (BBB) or direct exposure of the neocortex to serum- derived albumin leads to rapid up-regulation of the astrocytic marker, glial fibrillary acidic protein (GFAP), followed by delayed (within 4-7 days) development of an epileptic focus. We investigated the role of astrocytes in epileptogenesis in the BBB-breakdown and albumin models of epileptogenesis. We found similar, robust changes in astrocytic gene expression in the neocortex within hours following treatment with deoxycholic acid (BBB breakdown) or albumin. These changes predict reduced clearance capacity for both extracellular glutamate and potassium. Electrophysiological recordings in-vitro confirmed the reduced clearance of activity- dependent accumulation of both potassium and glutamate 24 h following exposure to albumin. We used a NEURON model to simulate the consequences of reduced astrocytic uptake of potassium and glutamate on excitatory postsynaptic potentials (EPSPs). The model predicted that the accumulation of glutamate is associated with frequency-dependent (>100 Hz) decreased facilitation of EPSPs, while potassium accumulation leads to frequency-dependant (10-50 Hz) and N-methyl-D-aspartic acid (NMDA)-dependent synaptic facilitation. In-vitro electrophysiological recordings during epileptogenesis confirmed frequency-dependant synaptic facilitation leading to seizure-like activity. Our data indicate a transcription-mediated astrocytic transformation early during epileptogenesis. We suggest that the resulting reduction in the clearance of extracellular potassium underlies frequency-dependent neuronal hyper-excitability and network synchronization.
- 43 - Introduction Epilepsy is one of the most common neurological disorders, affecting 0.5–2% of the population worldwide. While the characteristic electrical activity in the epileptic cortex has been studied extensively, the mechanisms underlying epileptogenesis are poorly understood. Focal neocortical epilepsy often develops following traumatic, ischemic or infectious brain injury. Under these conditions, vascular damage is common and includes local breakdown of the blood- brain barrier (BBB; Abbott et al., 2006; Neuwelt, 2004; Tomkins et al., 2001). The BBB has long been recognized as crucial for maintenance of the brain's micro-environment, but it was only recently documented that disruption of the blood brain barrier plays an important role in the pathogenesis of epilepsy (Seiffert et al., 2004; van Vliet et al., 2007; Marchi et al., 2007). It was found, for example, that in the rat neocortex, long-lasting BBB disruption leads to gradual development (within 4-7 days) of an epileptic focus that persists for weeks (Seiffert et al., 2004; Ivens et al., 2007; Tomkins et al., 2007). Our experiments further indicated that during BBB breakdown, serum-derived albumin diffuses into the brain's extracellular space and is rapidly transported into astrocytes via a specific receptor-mediated mechanism. Albumin uptake by astrocytes was followed by a rapid (within hours) up-regulation of the astrocytic marker glial fibrillary acidic protein (GFAP; Seiffert et al., 2004; Ivens et al., 2007), suggesting that astrocytic dysfunction plays a role in injury-induced epileptogenesis.
Data accumulating from human and animal studies supports the notion that glial cells make an important contribution to the control of neuronal function under both normal and pathological conditions (for reviews see Araque et al., 2001; Seifert et al., 2006; Wetherington et al., 2008). Studies on epileptic tissue show significant alterations in the expression of astrocytic proteins, including increased expression of GFAP (Bordey and Sontheimer, 1998) and reduced expression + of proteins involved in the regulation of extracellular potassium ([K ]o) and glutamate (Hinterkeuser et al., 2000; Schroder et al., 2000). Despite the vast body of data confirming changes in the morphology and function of astrocytes in epileptic tissue, the direct role of these cells in the development of the epileptic network (i.e., in epileptogenesis)remains unclear. In this study, we combined molecular, electrophysiological and computer modeling approaches to investigate the potential role of vascular-injury-induced and albumin-induced early transformation of astrocytes in altered neuronal excitability and epileptogenesis.
- 44 -
Materials and Methods Animals were housed and handled according to the directives of the internationally accredited Animal Care and Use Committees (IACUC) at Charité University Medicine, Berlin, and Ben-Gurion University of the Negev, Beer-Sheva. All experimental procedures were approved by the ethical committees supervising experiments on animals at Charité University Medicine (in-vivo approval no.: G0104/05, in-vitro: T0228/04) and Ben-Gurion University of the Negev (approval no.: BGU-R-71-2006).
In-vivo experiments. The in-vivo experiments were performed as previously described in Seiffert et al. (2004). In brief, adult male Wistar rats (120-250 g) were anesthetized using ketamine and xylazine and placed in a stereotactic frame. A 4-mm diameter bone window was drilled over the somatosensory cortex, the dura was opened and the underlying cortex was perfused with artificial cerebrospinal fluid (ACSF). For the "treated" rats group, the BBB-disrupting agent deoxycholic acid sodium salt (DOC, 2 mM, Sigma-Aldrich, Steinheim, Germany) or bovine serum albumin (0.1 mM, >98% in agarose cell electrophoresis; catalogue no. A7906, Sigma Aldrich, Steinheim, Germany) was added to the ACSF. Albumin concentrations corresponded to 25% of the normal serum concentration [determined to be 0.4 mM for 10 rats, see also Geursen and Grigor (1987); final osmolarity of 303-305 mOsmol/l]. For the sham-operated control group, the cortex was perfused with ACSF. The composition of the ACSF was (in mM): 129 NaCl, 21
NaHCO3, 1.25 NaH2PO4, 1.8 MgSO4, 1.6 CaCl2, 3 KCl, and 10 glucose. Rats were sacrificed at 7–8, 24, or 48 h following treatment, before the onset of epileptiform activity (>4 days, see Seiffert et al., 2004). Microarrays. Total RNA from animals treated with DOC or with albumin was isolated from the somatosensory cortex, directly under the craniotomy area, using the TRIzol® reagent (Invitrogen, Carlsbad, CA), and prepared using the Affymetrix GeneChip one-cycle target labeling kit (Affymetrix, Santa Clara, CA). Biotinylated cRNA was then fragmented and hybridized to the GeneChip Rat Genome 230 2.0 Array according to manufacturer's protocols (Affymetrix Technical Manual). The array data was normalized by using GCRMA (GC Robust Multi-Array Average) or RMA (Robust Multi-Array Average) analysis using the probe annotations distributed by Affymetrix (NetAffx). In addition, we compared these results to those obtained
- 45 - with annotations based on the Unigene build (Dai et al., 2005). For the subset of genes we focused on in this study, we found the expression values obtained from these annotations to be very similar (correlation coefficient over 0.850 for most treatments, p<0.001, data not shown) and therefore the Affymetrix probe set annotations are used throughout the text. One array was run for each treatment (DOC and albumin) and for every contralateral hemisphere for the following time points: 7/8, 24, and 48 h. The data from a sham-treated animal (24 h) was used to normalize the other arrays. To identify genes involved in astrocytic functions, we used GeneCards (http://www.genecards.org), querying for "astrocyte". For comparison of the relative changes in the expression of astrocytic vs. neuronal genes, we used gene sets published by Cahoy and colleagues (2008) of astrocytic and neuronal enriched genes (expressed by S100β+ and S100β-/PDGFRα-/MOG-cells, respectively). Cluster analysis was performed with MATLAB by assessing the expression relationship as the Euclidean distance in N-dimensional space between measurements (N denotes number of gene transcripts). Arrays were then clustered according to distance data, by using the Unweighted Pair Group Method with Arithmetic mean method (UPGMA, Gronau and Moran, 2007). In-vitro astrocytic and neuronal culture preparations. Primary neuronal cortical cultures were prepared from embryonic day 18 rats as reported previously (Kaufer et al., 2004). Briefly, cells were dissociated with a papain solution for 20 min at 37°C. After the removal of the papain solution, the tissue was resuspended in growth medium [MEM with Earle‘s salts containing 2.5% B27 supplement, 0.1% mito serum extender, 5% fetal bovine serum (FBS), 20 mM glucose, and 5 mM L-glutamine] and dissociated by mechanical trituration. The cells were plated, and after 4 h in vitro the cell culture medium was replaced with neurobasal medium supplemented with 2% B27 supplement and 0.5 mM GlutaMAX™. The cells were maintained in
5% CO2 at 37°C. After 7 days in vitro, cytosine arabinofuranoside (AraC) (10 µM) was added to the cultures. After 10 days in vitro, the cells were incubated with 0.4 mM albumin for 24 h at 37°C. For astrocytic cultures, astrocytes were isolated from the cerebral cortices of P0 rat pups. Cells were dissociated with papain and mechanical trituration. The cells were cultured in high- glucose Dulbecco's modified eagle medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and in 5% CO2 (medium was replaced every 3-4 days). After 10 days in vitro, the culture medium was replaced with serum-free high-glucose DMEM (containing 1% penicillin/streptomycin) for 18 h. The cells were then incubated in serum-free medium
- 46 - containing 0.4 mM albumin for 24 h at 37°C.For immunostainingscells were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed in PBS. They were then incubated with 5% normal donkey serum in PBS for one hour at room temperature followed by overnight incubation at 4°C with either mouse anti-NeuN (1:1000; Chemicon, Temecula, CA) or mouse anti-GFAP (1:1000; Cell Signaling Technology, Beverly, MA). The cells were washed in PBS, incubated with donkey anti-mouse Cy3 (1:1000; Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature, and then counterstained with DAPI. Real-time polymerase chain reaction. Total RNA was isolated from the somatosensory cortices of animals treated with DOC or albumin (24 h treatment; n = 3) or from primary cultures (astrocytic and neuronal, n = 3 independent experiments). Expression levels were determined by real-time reverse transcriptase-PCR (RT-PCR) with an iQ5 detection system (Bio-Rad, Hercules, CA) using gene-specific primer pairs. RT-PCR data were analyzed using the PCR Miner program (Zhao and Fernald, 2005), and fold changes in gene expression were represented relative to sham-operated controls (in-vivo samples) or serum-deprived controls (in-vitro samples). Ribosomal 18S RNA (18S rRNA) was used as an internal control for variations in sample preparation. DNase treatment was performed for all samples, followed by first-strand cDNA synthesis (iScript cDNA Synthesis kit, Bio-Rad). PCR reactions were carried out with iQ SYBR Green Supermix (Bio-Rad). Primer specificity was verified by meltcurve analysis. The amplification cycles for 18S, Gja1, GS, SLC1A2, SLC1A3 (GLAST) and Kcnj10 consisted of 40 cyclesof 10 s at 95°C, 30 s at 55°C, and 30 s at 72°C. The amplification cycles for Gjb2 and Gjb6 consisted of 40 cyclesof 10 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Primer sequences (forward, reverse) were as follows: 18S rRNA (GenBank accession number M11188.1, 5‖-CCATCCAATCGGTAGTAGCG-3‖, 5‖ GTAACCCGTTGAACCCCATT-3‖); SLC1A3 (GenBank accession number NM_019225.1; 5‖- GAGGCCATGGAGACTCTGAC- 3‖, 5‖-CGAAGCACATGGAGAAGACA-3‖); GS (GenBank accession number NM_017073.3; 5‖- AGCGACATGTACCTCCATCC-3‖, 5‖ TACAGCTGTGCCTCAGGTTG-3‖); Kcnj10 (GenBank accession number X83585.1; 5‖- GAGACGACGCAGACAGAGAG-3‖, 5‖CCACTGCATGTCAATGAAGG-3‖); Gjb2 (GenBank accession number NM_001004099.1; 5‖-GGTTTGTGATGTGAGCATGG-3‖, 5‖- CTCAGCACACCAAGGATGAA-3‖); Gjb6 (GenBank accession number NM_053388.1; 5‖-
- 47 - GCCAAGATGAGTCACAGCAA- 3‖, 5‖-TCAGAGCTGGATCACAATCG-3‖); Gja1 (GenBank accession number NM_012567.2; 5‖- TCCTTGGTGTCTCTCGCTTT-3‖, 5‖- TTTGGAGATCCGCAGTCTTT-3‖); SLC1A2 (GenBank accession number NM_017215.2; 5‖- GGTCAATGTAGTGGGCGATT-3‖, 5‖-GGACTGCGTCTTGGTCATTT-3‖). In-vitro electrophysiological recordings.For electrophysiological experiments, rats were deeply anesthetized with isoflurane and then decapitated. Brains were quickly removed, and transverse hippocampal-cortical slices (400 µm thick) were prepared using a vibratome (Campden Instruments, Loughborough, UK). Slices were maintained in a humidified, carbogenated (5%
CO2 and 95% O2) gas atmosphere at 36 ± 1°C and perfused with ACSF in a standard interface chamber (Seiffert et al., 2004; Ivens et al., 2007). To mimic the altered ionic environment during BBB disruption, recordings were acquired in a serum-adapted electrolyte solution (sACSF; see Seiffert et al. 2004). sACSF was similar in composition to the ACSF except for different concentrations of MgSO4 (0.8 mM), CaCl2 (1.3 mM), KCl (5.7mM) and glutamine (1 mM). "Treated" slices were incubated with sACSF containing 0.1 mM bovine serum albumin for 2 h before transfer to the perfusion chamber. Electrophysiological recordings were obtained 6-10 h following perfusion with sACSF. Control slices were treated similarly, using sACSF without albumin. For extracellular recordings, glass microelectrodes (~3 MΩ, 154 mM NaCl) were positioned in layer 4 of the neocortex. Slices were stimulated with brief (100 µs) pulses, by using bipolar stimulation electrodes placed at the border between white and gray matter in the same cortical column. Trains of 50 stimuli were applied at 2, 5, 10, 20, 50 and 100 Hz, at 2.5× threshold stimulation intensity. Signals were amplified (SEC-10L; NPI Electronics, Tamm, Germany), filtered at 2 kHz, displayed on an oscilloscope, digitized on-line (CED-1401micro; Cambridge Electronics Design, Cambridge,
UK) and stored for off-line analysis. Extracellular potassium concentrations ([K+]o) were measured with ion-sensitive microelectrodes (ISMEs; Lux and Neher, 1973; Jauch et al., 2002). In vitro intracellular recordingswere obtained from pyramidal neurons (layer 2-3) 23-28 h following the in vivo treatment with albumin or from control rats. Currents were recorded using the whole cell patch configuration, as described previously (Pavlovsky et al., 2003). In brief, glass pipettes were pulled from capillaries using a vertical puller (Narashige, Greenvale, NY) and filled with a solution comprising (in mM): 150 CsCl, 1 MgCl2, 10 HEPES, 4 Na2ATP, 0.1
CaCl2, and 1.1 mM EGTA, pH adjusted to 7.2 with a final osmolarity of 290–310 mOsm. Cells
- 48 - were visualized using infrared differential interference phase contrast video-microscopy. Recordings were performed using AxoPatch 700B (Axon Instruments, Foster City, CA), digitized at 10 kHz and recorded using pClamp 9.2 (Axon Instruments, Foster City, CA). Patch pipette's resistance was 4-5 MΩ. Series resistance was not electronically compensated; however, cells in which series resistance varied by more than 25% were excluded from the analysis. Stimulation protocols were started at least 5 min following impalement to allow intracellular dialysis with the pipette solution. Excitatory post-synaptic currents (EPSCs) were evoked – using a bipolar stimulating electrode positioned <200 µm from the recorded cell – at 75% of the intensity producing maximal EPSCs. N-methyl-D-aspartic acid (NMDA) currents were recorded in the presence of blockers of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid
(AMPA)/kainate (i.e., 30 µM CNQX) and of gamma-aminobutyric acid A (GABAA) receptors (i.e., 10 µM bicuculine methiodide). Cells were voltage clamped to +40 mV to alleviate NMDA receptor blockade and inactivate fast Na+ currents. In some experiments, dihydrokainic acid (DHK, Tocris, Bristol, UK), 100 µM, was added to the extracellular solution to selectively block the astrocyte specific glutamate transporter, SLC1A2 (see Arriza et al., 1994). Computer simulations.A computer model was implemented using the NEURON modeling environment (Hines and Carnevale, 1997) with 20-µstime steps. The model consisted of a multi- compartment isolated cell, simulating a layer 2/3 cortical neuron, using only passive membrane properties. Geometric parameters and spatial relationships of the 74 compartments were modeled after Traub and colleagues (2003). Resting membrane potential was set at -65 mV (determined + + 2 by Na and K conductance); membrane capacitance Cm was 0.9 µF/cm ; and the cytoplasmic resistance was set at 250 Ω/cm2. Simulated excitatory inputs consisted of eight synapses on apical dendrites (located 1368 µm from the soma), contributing currents with AMPA and NMDA kinetics modeled after Saftenku (2005) and Kampa et al. (2004), respectively. AMPA to NMDA maximal current ratios were set at 1 (Myme et al., 2003). Synaptic currents were triggered by a surge of ‗glutamate‘, decaying with first-order kinetics (baseline time constant = 1.2 ms). Down-regulation of uptake mechanisms was simulated by changing the time constant of the decay function, similar to the effect of the application of DL-threo-β-benzyloxyaspartate + (DL-TBOA, Diamond, 2005). To investigate the effects of altered [K ]o, each compartment was + enveloped by a fixed space in which potassium was allowed to accumulate. [K ]o 'diffused' either into the bathing solution or into astrocytes with KIR kinetics. Since KIR channel conductance is
- 49 - + + proportional to [K ]o (Sakmann and Trube, 1984), K influx into ‗astrocytes‘ was determined by + + + the local potassium gradient ([K ]o – [K ]bath) modulated by KIR conductance (log[K ]o;adapted from Ciani et al., 1978).
+ 2 rest + 2 where Ik - momentary K flux (nA/cm ), Ik - resting K flux (nA/cm ), ECS - time constant for potassium diffusion into the extracellular space, astrocytic - time constant for potassium diffusion into astrocytes, F - Faraday constant, C- ratio of astrocytic K+ uptake relative to extracellular diffusion, and V - radius of enveloping extracellular space, set at 20 nm (Egelman and Montague, 1999; Savtchenko et al., 2000). The ionic flux equation describes first-order potassium clearance by both free diffusion and ‗astrocytic‘ uptake (see Kager et al., 2000). Lateral diffusion of K+ ions was not taken into account. To simulate a decrease in astrocytic potassium clearance,
astrocytic was increased to mimic a reduction in astrocytic KIR channels. ―Resting‖ ion + + + + concentrations were set at (in [mM]): [Na ]o, 145; [Na ]i, 12; [K ]o, 3.5; [K ]i, 140. Statistical analysis. Data are expressed as means ± SEM. Differences between treated and control slices were determined by the Mann-Whitney U test for two independent samples. Statistical tests were performed using SPSS 13.0 for Windows. The level of statistical significance was set at p < 0.05, unless otherwise stated.
- 50 - Results
Astrocytic transcriptional changes following BBB opening or exposure to albumin To explore changes in astrocytic gene expression during epileptogenesis, we analyzed gene- array data from DOC- and albumin-treated brains (n = 3 from each treatment) during the first 48 h after treatment and prior to the development of epileptiform activity (Ivens et al., 2007; Seiffert et al., 2004). When compared to sham-operated controls, the two treatments, at each time point, resulted in similar changes in expression of astrocytic-enriched genes with a correlation coefficients between the different treatments (see Methods) of r2 = 0.69, 0.82, 0.85 for 8, 24 and 48 h following treatment, respectively, p<0.0001, Fig. 1a). Unsupervised hierarchical cluster analysis revealed further similarities between changes in transcripts levels in treated cortices (which cluster according to time after treatment) (Fig. 1b) while transcripts changes in the contralateral, untreated, hemispheres are relatively dissimilar and cluster together.
In a recent study, Cahoy and colleagues (2008) created a transcriptome database reflecting cell type-specific, comprehensive mRNA expression levels in astrocytes, neurons and oligodendrocytes. We used these gene lists to classify genes into ―astrocytic‖ or ―neuronal‖ categories. When compared with the ―neuronal‖ category at all examined time points, the ―astrocytic‖ category included a higher average number of genes that underwent a change in expression of more than ±150% (Fig. 1c). Comparison between the results at 8 and 48 h after treatment showed an increase in the average number of genes that reached 150% change in both groups (34 vs. 40 for astrocytes and 21 vs. 28 for neurons, 8 and 48 h after treatment, respectively). We also examined the expression levels of genes reported as over-expressed in reactive astrocytes (Ridet et al., 1997) and found a large overlap with over-expressed genes 8, 24 and 48 h following both treatments (Fig. 1d). These results are consistent with the hypothesis that an early and prominent change in astrocytic gene expression is an important early feature of BBB-breakdown or albumin-induced epileptogenesis (see Discussion).
- 51 - Figure 1. Transcriptional changes in astrocytes following exposure to albumin or BBB disruption. (a) Sham- normalized expression levels of mRNA for genes preferentially expressed in astrocytes at 8, 24, and 48 h following treatment with the BBB disrupting agent DOC (D) or albumin (A). (b) Hierarchical cluster analysis comparing astrocytic gene expression for DOC- treated and albumin- treated cortices at 8, 24, and 48 h following treatment, and the contralateral, non- treated hemisphere (Ctrl Hemi.). (c) Average number of gene transcripts up- or down-regulated by more than 150% grouped by cell-type across all time points. (d) Sham normalized mRNA expression levels of genes coding for known astrocytic activation markers.
- 52 - Altered expression of astrocytic potassium and glutamate regulating genes In their pioneering study, Kuffler and Potter (1964) established that astrocytes are crucial for the control of the brain's extracellular environment. Specifically, these cells limit the + accumulation of [K ]o and glutamate (Oliet et al., 2001; Newman et al., 2004), thus potentially contributing to the regulation of neuronal excitability. We therefore searched our gene array results for changes in the level of expression of several potassium and glutamate homeostasis- related genes. We found that transcripts coding for the predominantly astrocytic (Kcnj10), but + not neuronal (e.g. Kcnj2 or Kcnc1, see Butt and Kalsi, 2006), inward-rectifying K channel (KIR) were down-regulated. In addition, the mRNA coding for the astrocytic glutamate transporters of the solute carrier family 1, subfamily A members SLC1A2 and SLC1A3 (see Su et al., 2003; Chaudhry et al., 1995), but not for SLC1A4, was down-regulated. In contrast, SLC1A1 (preferentially expressed in neurons; see Rothstein et al., 1994) did not show significant changes in expression levels. Glutaminase (Gls, Gls2) and glutamine synthetase (GS), both of which are predominantly expressed in astrocytes (Derouiche and Frotscher, 1991) and are responsible for regulating glutamate levels, were also down-regulated (Fig. 2a). Furthermore, our gene arrays showed that at most time points there was a significant down-regulation of gap junction proteins (Gja1, Gjb2, Gjb6) 24 h following treatment, a finding that indicates reduced spatial buffering capacity (see Wallraff et al, 2006). Real-time RT-PCR confirmed the main observations obtained from the gene arrays, i.e., significant up-regulation of GFAP, and down-regulation of KCNJ10
(KIR 4.1, data not shown and see Fig. 5 in Ivens et al. 2007) as well as KCNJ3, SLC1A2 and SLC1A3, Gja1, Gjb2 and Gjb6 at all time points (connexins 43, 26 and 30, respectively, Fig. 2b).In contrast, glutamine synthetase did not show significant down-regulation (Fig. 2b).
The microarray results hinted at a rapid and robust change in astrocytic gene expression in vivo following BBB breakdown or brain exposure to serum albumin. To further validate the specificity of the astrocytic response to albumin, we exposed cell cultures enriched with either astrocytes or neurons (see Methods) to albumin for 24 h. Significantly, the astrocytic cultures responded with significant down-regulation of the same transcripts found to respond in vivo to albumin (SLC1A3, GS, Gja1, Gjb2 and Gjb6, Fig. 2c). No significant differences in expression levels of the same transcripts were found in the neuronal-enriched culture (except for downregulation of GS and upregulation of Gjb6 mRNA levels, Fig. 2d), supporting the notion that the changes observed in-vivo do indeed reflect an astrocytic response.
- 53 - Figure 2. Alterations in astrocytic potassium and glutamate regulating genes. (a) Sham- normalized mRNA expression levels for genes associated with K+ and glutamate homeostasis at 8, 24, and 48 h following in- vivo treatment with DOC (D) and albumin (A). (b)Sham- normalized mRNA expression levels for selected transcripts (see Results) obtained by real-time RT-PCR 24 h following DOC (grey bars) or albumin (black bars) treatments. (c) Astrocyte enriched cell cultures immunostained for GFAP (red) or NeuN (green). Nuclei visualized with DAPI staining (blue). The graph shows mRNA expression levels in albumin-exposed cultures compared to controls. (d) same as c, for neuron enriched cultures. Abbr.: SLC1a2 – GLT-1,
SLC1a3 – GLAST, Gja1, Gjn2, Gjb6, - connexins 43, 26, 30 respectively, Kcnj3 – KIR3.1
Does epileptogenesis involve reduced glutamate and potassium clearance? To confirm that the transcriptional changes induced by albumin were associated with altered cellular functions, we investigated the clearance of extracellular glutamate and potassium in cortical slices 24 hours following albumin treatment in vivo. To measure synaptic glutamate levels during neuronal activation, we recorded the slowly inactivating (Lester et al., 1990) NMDA currents in cortical neurons by using the whole-cell patch configuration (in the presence of non-NMDA glutamate and GABA receptor blockers, see Methods). Cells were clamped at +40 mV to prevent a potential confounding effect of post-synaptic depolarization due to the + accumulation of synaptic [K ]o. Mean single EPSC rise-time and amplitude were similar in both control and albumin-treated groups [14.5±0.5 vs. 13.2±0.7 ms and 505±100 vs. 492±140 pA, for
- 54 - rise-time (not shown) and amplitude, respectively in treated vs. controls, Fig. 3a, inset], suggesting that no changes in post-synaptic NMDA receptor density or properties at this time point (data not shown). We measured synaptic glutamate elicited by 50 extracellular stimulations at 2, 5, 10, 20, 50 and 100 Hz before and after adding the astrocytic SLC1A2 specific inhibitor, DHK. In neurons from control animals, DHK had no effect on single EPSCs or EPSCs elicited at low stimulation frequencies (<20 Hz). In contrast, stimulation frequencies > 20 Hz resulted in increased NMDA currents (or reduced depression when normalized to the first stimulus, Fig 3b- c, left), suggesting that astrocytic glutamate transporters efficiently reduce synaptic glutamate levels at high frequencies of neuronal activation. The same experiments were then repeated 24 h following cortical application of albumin (i.e. during epileptogenesis). In contrast to the control experiments, DHK had no effect on EPSC amplitude in treated slices (Fig. 3b-c, right), supporting reduced expression of the astrocytic transporter SLC1A2. Repetitive stimulation, however, resulted in a stronger depression of EPSC amplitude in treated slices as compared to controls (see Discussion).
To study K+clearance from the extracellular space, we recorded from control and treated slices (24 h following treatment with albumin) by using ISMEs. We previously reported slower + decay kinetics of [K ]o in response to pressure application in BBB-treated animals (Ivens et al., + 2007). Here we tested for [K ]o accumulation during neuronal activation at different frequencies + of stimulation. In slices from control animals, the increase in [K ]o was limited to 25% of baseline levels (<3.75mM) at all stimulation frequencies with the employed stimulation + intensities and number of stimuli. In contrast, in treated slices [K ]o accumulation was significantly higher at frequencies ≥ 10 Hz, reaching 6.7 mM (Fig. 3d-e).
- 55 - Figure 3: Electrophysiological evidence for reduced glutamate and potassium buffering during epileptogenesis. (a) Single NMDA- mediated EPSCs in control slices and 24 h following albumin treatment in vivo in ACSF (gray) and 10 min following DHK (black). Inset shows mean EPSC amplitude in ACSF. (b) NMDA-mediated EPSCs during train stimulation at 50 Hz in control animals and treated animals. (c) Mean evoked NMDA-mediated EPSC at different stimulation frequencies. + (d) [K ]o levels in control and treated slices during 20 Hz stimulation (e) + Mean [K ]o levels during extracellular stimulation at 2–100 Hz (right). #, p<0.03, *, p<0.001 (n = 6 albumin- treated cells, 5 animals, n = 9 control cells, 7 animals).
Modeling reduced K+ clearance results in frequency-dependent facilitation of excitatory post-synaptic potentials To elucidate the possible contribution of astrocytic dysfunction to neuronal excitability, we developed a NEURON-based model of a post-synaptic neuron and an astrocyte. To evaluate the + role of increased [K ]o accumulation and glutamate accumulation, we focused on examining changes to excitatory synaptic currents in the post-synaptic neuron (see Methods). Excitatory synaptic input was simulated by simultaneous application of glutamate at all 8 distal dendritic processes (Fig. 4a). In light of our experimental data, we simulated the reduction in K+ clearance + by manipulating a [K ]o-regulated potassium removal mechanism (IKIR), while keeping the
- 56 - diffusion component constant. In the absence of neuronal activity, reducing KIR-mediated + potassium clearance had no effect on resting [K ]o and thus had a negligible effect on the rising phase and maximal amplitude of a single excitatory post-synaptic potential (EPSP) (Fig. 4b). Reducing potassium buffering and consequent increased K+ accumulation during repetitive stimulation resulted in enhanced EPSP duration due to slower repolarization (due to a reduced driving force for K+ and a slight increase in NMDA-mediated current, see below and Fig. 4b). + During repetitive activation, the accumulation of [K ]o near the dendritic compartment reached a + maximum of 8.7 (and 16 mM) for reduction to 50% (and 10%) of astrocytic [K ]o buffering + capacity, respectively (Fig. 4c). [K ]o accumulation during repetitive stimulation had a differential effect on AMPA- and NMDA-mediated currents: while the AMPA current showed frequency-dependent depression due to receptor desensitization, the NMDA component was strongly facilitated due to membrane depolarization (Fig. 4b). Reducing astrocytic potassium uptake from the extracellular space to 10% of control values resulted in an increase in total charge transfer mediated by the NMDA component of 44, 344 and 84% at 10, 20 and 100 Hz, respectively, while the AMPA charge transfer decreased by 5, 24, and 15%, respectively (Fig. 4d). Overall, there was a frequency-dependent increase in EPSP amplitude (Fig. 4e) associated with longer decay time (Fig. 4f). Repeated simulations with no NMDA conductance
(GNMDA = 0, with concomitant increased AMPA conductance, to achieve similar depolarization for a single stimulus) resulted in a much smaller facilitation (compare Fig. 4g and h).
- 57 - Figure 4. Application of NEURON-based + model to determine the effects of [K ]o accumulation. (a) Schematic diagram of the modeled layer 2/3 pyramidal neuron containing 74 compartments with 8 synapses (with AMPA and NMDA currents), one at each distal dendrite. (b) Increasing glutamate levels at each of the 8 synapses (top trace representing kinetics of synaptic glutamate level) elicits AMPA-mediated (middle trace) and NMDA-mediated (bottom trace) currents under control conditions (black) and under reduced astrocytic potassium clearance (under a 10-fold decrease of astrocytic K+ clearance, blue trace). (c) Maximal K+concentrations recorded in the vicinity of a distal dendritic compartment during + repetitive stimulation as a function of [K ]o clearance and stimulation frequency. (d) Percent change of total charge transfer by NMDA (black) and AMPA (blue) channels during stimulation at 20 and 100 Hz. (e). Somatic EPSPs under control conditions (black) and under reduced astrocytic potassium clearance (10-fold reduction of control levels, blue trace). (f) 5th EPSP (elicited by stimulation at 4, 20, and 100 Hz) decay time constant at different levels of astrocytic th st potassium clearance rates. (g) Ratio of 5 to 1 EPSP amplitude (P5/P1) at stimulation frequencies of 4–500 Hz at different levels of astrocytic potassium clearance rates. (h) Same as in (g) with GNMDA = 0.
Modeling reduced glutamate clearance results in frequency-dependent depression of excitatory post-synaptic potentials We next used the NEURON model and simulation paradigms described above to test the expected effect of reduced glutamate uptake. We simulated the reduction in glutamate uptake by slowing the transmitter's synaptic decay function. A twofold increase in the glutamate decay time
- 58 - constant resulted in a 48% increase in EPSP amplitude (from 25 to 37 mV) at a single post- synaptic dendrite and a 60% increase in the amplitude of the summated somatic EPSP (Fig. 5a). While for a single stimulation both AMPA and NMDA-components were increased, with repetitive activation, a marked decrease in the AMPA current (due to receptor desensitization, see Otis et al., 1996) and a strong facilitation of the NMDA current (due to post-synaptic depolarization, see Mayer et al., 1984 and Fig. 5b-c) were measured. Somatic EPSP facilitation (ratio of 5th to 1st EPSP amplitude, Fig. 5c) was maximal at 100 Hz with our initial conditions for glutamate clearance. Inhibiting glutamate clearance did not affect EPSP facilitation at low stimulation frequencies (<20 Hz) but reduced it at high stimulation frequencies (>80 Hz). The decreased facilitation was due to a reduced AMPA current through the desensitized receptors, thus keeping the membrane potential below the threshold for NMDA receptor activation. In simulations performed in the absence of NMDA conductance, EPSP facilitation was reduced at most stimulation frequencies, with only a small (<150%) residual facilitation measured at high stimulation frequencies (>100 Hz, Fig. 5d).
Figure 5. Application of NEURON- based model to determine the effects of glutamate accumulation. (a) Somatic EPSP amplitudes for different glutamate time constants. (b) Simultaneous glutamate "application" (kinetics represented in the upper trace) at each of the 8 synapses elicits AMPA-mediated (middle) and NMDA-mediated (bottom) currents under control conditions (black) and a twofold th st increase in glutamate decay time constant (blue). (c) Ratio of 5 to 1 EPSP amplitude (P5/P1) at different stimulation frequencies and varying glutamate decay time constants (values related to control). (d) Same as in (c) with GNMDA = 0.
- 59 - Modeling the concerted effect of reduced potassium and glutamate clearance The simulations showed that while synaptic glutamate levels mainly affected the 1st EPSP in + the train, an activity-dependent increase in [K ]o mainly enhanced EPSP facilitation in a frequency-dependent manner. Since our molecular data indicated a decrease in both potassiumand glutamate buffering mechanisms, we simulated their joint effect on synaptic + transmission. Decreasing the clearance of [K ]o led to maximal EPSP facilitation when stimulating at 20 Hz, while a concurrent twofold reduction in glutamate uptake shifted the optimal frequency for maximal facilitation to 10 Hz (Fig. 6a). Concurrent reductions in + st glutamate and [K ]o clearance led to increases in the duration of the 1 EPSP, which in turn elicited increased and longer NMDA receptor activation per stimulus. The longer EPSPs allowed for a larger charge transfer with longer inter-stimulus intervals (i.e., reduced frequency, Fig. 6b) thus lowering the optimal stimulation frequency. To assess the sensitivity of the synaptic response during repetitive stimulation (at 20 Hz), we used several glutamate decay time + constants and varying levels of [K ]o uptake. We plotted the maximal EPSP amplitude as a + function of [K ]o; Figure 6c demonstrates that increasing synaptic glutamate led to small + increases in the maximal EPSP amplitudes for all levels of [K ]o. However, synaptic facilitation + was decreased with reduced glutamate uptake: thus, synaptic [K ]o accumulation to 10 mM was associated with 40% EPSP facilitation (upon the 5th stimulation) under baseline glutamate clearance, but with only 22% facilitation when glutamate decay time was doubled (Fig. 6d).
Figure 6. Modeling the concerted effect of reduced potassium and glutamate clearance. (a) EPSP facilitation (relative to maximal value) for a 10-fold decrease in + [K ]o clearance (black), a twofold slowing of glutamate decay time constant (red) and for down regulation of both uptake mechanisms (blue) as a function of stimulation frequency. (b) EPSP traces for 10- and 20-Hz trains under a 10-fold decrease in
- 60 - astrocytic K+ clearance (gray and blue traces, respectively) and with both uptake mechanisms down regulated (at 10 Hz, black). Dashed line marks resting potential. (c) Maximal EPSP + amplitude elicited by a train of five stimuli as a function of maximal [K ]o for different glutamate uptake decay time constants (for 1.2, 2.2, 3.2 and 3.5 ms). (d) EPSPs facilitation [ratio of 5th to1st
EPSP amplitude (P5/P1)] for 20 Hz stimulation for different glutamate decay time constants (as in c);
Electrophysiological evidence for frequency-dependent synaptic facilitation during epileptogenesis + Our simulation data predicted maximal EPSP facilitation at 20 Hz when [K ]o clearance is reduced and decreased facilitation (at 50–100 Hz) when the only change induced is glutamate accumulation in the synaptic cleft. We therefore measured field potentials in response to stimulation at various frequencies in brain slices during "epileptogenesis" (exposure to albumin in sACSF) compared to controls (sACSF alone). Comparison of the field potential amplitude and absolute integral during the first five stimuli revealed a significant reduction in both measures only under 100 Hz stimulation [amplitude: 1.13 ± 0.12 vs. 0.46 ± 0.03 mV, 1.44 ± 0.36 vs. 0.47 ± 0.09 mV and area: 2.4 ± 0.2 vs. 0.9 ± 0.02 V*s, 4.4 ± 1.1 vs. 0.5 ± 0.7 V*s, 1st vs. 5th stimulus, control (n = 5) and treated (n = 4), respectively, p<0.05]. Comparing field potential duration (measured at 1/3 maximal amplitude) for the 1st vs. the 5th stimulus among different frequencies did not reveal any changes in control slices. In contrast, in treated slices the field potential was significantly prolonged at 10 and 20 Hz (10 Hz: 7.5 ± 0.4 vs. 9.4 ± 5.5 ms, 6.5 ± 0.7 vs. 13.1 ± 2.6 ms, 20 Hz: 6.0 ± 0.9 vs. 6.8 ± 0.8 ms, 6.6 ± 0.8 vs. 12.9 ± 2.9 ms for 1st vs. 5th stimulus in control and treated, respectively, p<0.05, Fig. 7c). Interestingly, in the "treated" group, stimulation-induced frequency-dependent, long-lasting epileptiform discharges occurred most reliably during 10-Hz stimulation (4 of 4 slices, n = 3 animals), and sometimes at 20 Hz (3 of 4 slices) and 5 Hz (2 of 4 slices), but never at higher frequencies (Fig. 7d). Epileptiform discharges were observed in one control slice without any apparent frequency dependence (5 to 50 Hz, 1 of 5 slices, n = 3 animals, Fig. 7d).
Taken together, our experiments show that exposure to albumin in-vitro induceschanges in neuronal excitability and that evoked network activity facilitates, and often turns into, robust
- 61 - epileptiform discharges upon repetitive stimulation. We found that 10–20 Hz is the most reliable frequency, as was also predicted by our K+ recording data (Fig. 3d) and by our model in the case + of reduction in [K ]o clearance with or without glutamate accumulation (see Fig. 4g and Discussion).
Figure 7. Recording in vitro shows frequency-dependent increased neuronal excitability and hyper-synchronous network activityduring albumin- mediated epileptogenesis. (a, b) Neocortical field potential recordings of brain slices during stimulation trains of 50 pulses at 2, 10 and 100 Hz. Field responses were facilitated in the albumin- treated slices, observed as increased duration of the population spikes (see inset in a, b). (c) Comparison of the average field potential duration (at 1/3 maximal amplitude) for the 5th tothe 1st evoked response reveals maximal facilitation at 10 Hz. (d) Percentage of slices showing prolonged, paroxysmal discharges.
- 62 - Discussion The primary goal of the present study was to study the role of astrocytes in epileptogenesis. With the BBB disruption and albumin-induced models of epileptogenesis, the following findings were obtained early during epileptogenesis: (1) Similar significant changes in astrocytic gene expression occurred following the two treatments. (2) Transcriptional data predicted disturbed homeostasis of extracellular potassium and glutamate. (3) Intracellular recordings confirmed reduced astrocytic glutamate uptake, which did not, however, seem to account for increased + neuronal excitability. (4) Recordings with ISMEs confirmed activity-dependent impaired [K ]o + buffering. (5) A NEURON-based model predicted that reduced [K ]o buffering leads to frequency- and NMDA-R dependent facilitation of EPSPs, with maximal facilitation around 20Hz, while reduced clearance of glutamate results in a modest increase in the amplitude and duration of a single EPSP with decreased facilitation during repetitive stimulation. (6) Finally, extracellular recordings in cortical slices confirmed the frequency-dependent facilitation of synaptic activity predicted by the model and showed epileptiform discharges at preferred stimulation frequencies of 10–20 Hz. Overall, the present study suggests a key role for reduced + astrocytic-mediated clearance of [K ]o in activity-dependent facilitation of synaptic activity during epileptogenesis.
The working hypothesis for the present study is based on our previous experimental results, which demonstrated that BBB breakdown induces epileptogenesis (Seiffert et al., 2004; Ivens et al., 2007; Tomkins et al., 2007) and increases the expression of the astrocytic marker, GFAP, within hours following the epileptogenic treatment. This rapid astrocytic response was observed before the emergence of epileptiform activity, leading to the hypothesis that astrocytes play a role in the epileptogenic process (see also Tian et al., 2005; Ding et al., 2007). Indeed, changes in the structure and function of astrocytes are found in a wide variety of brain insults, including epilepsy, in both animals and man (Schroder et al., 1999; Kivi et al., 2000; Bordey et al., 2001; Herman, 2002; Jauch et al., 2002). However, the role of astrocytic dysfunction in disease progression and neuronal dysfunction is not well understood (for reviews see Heinemann et al., 1999, Seifert et al., 2006 and Schwarcz, 2008). Since BBB breakdown causes albumin extravasation from brain vessels and its specific uptake by astrocytes (Ivens et al., 2007), we put forward the hypothesis that under BBB breakdown astrocytic gene expression is directly
- 63 - modulated by albumin. A role for serum albumin in epileptogenesis is also supported by experiments showing that albumin induced focal epileptiform activity in a dose-dependent manner (Seiffert et al., 2004). Importantly, albumin-induced epileptogenesis is observed only after a window period of several hours (in vitro) or days (in vivo), with no apparent effect on neuronal membrane characteristics, firing properties or amplitude and duration of single EPSCs (Seiffert 2004, Fig. 3 and data not shown). Furthermore, the specific uptake of albumin by astrocytes, together with the rapid changes in the level of astrocyte-specific proteins (Ivens et al., 2007), led us to hypothesize that a concerted astrocytic transcriptional response may underlie epileptogenesis under these conditions. Using microarray technology we confirmed that a large number of astrocytic genes do show significant changes in expression levels as early as 8 h following an epileptogenic event, several days before epileptic activity emerges (Seiffert et al., 2004; Ivens et al., 2007). However, it is plausible that under pathological conditions, different cell populations may express previously unexpressed transcripts, thus confounding our results. Nevertheless, the cell-specific mRNA changes in response to albumin exposure that were evident in our astrocytic- and neuronal-enriched cultures constitute further support for our conclusion that the observed changes do occur preferentially in astrocytes (Fig. 2).The cluster analysis and the strikingly high correlation between expression profiles of both BBB breakdown and albumin treatments at the various time points support the notion that the extravasation of the most abundant serum protein, albumin, through the injured vessels plays a role in the transcriptional modulation of astrocytic genes. An alternative hypothesis – that albumin itself is disruptive to the BBB, leading to the extravasation of some other blood-derived mediator – is less likely, since local application of albumin did not increase BBB permeability to large molecules, as previously measured using systemic injection of Evans-blue (Seiffert et al., 2004).
+ Since astrocytes are known to be key contributors to [K ]o buffering, we searched our microarrays for expression levels of astrocytic K+ channels (Barres et al., 1990). Indeed, Kir 4.1 (but not other K+ channels, see Fig. 2a), previously shown to be expressed in neocortical astrocytes (Hibino et al., 2004; Higashi et al., 2001), was down-regulated, leading to activity- + dependent accumulation of [K ]o in treated cortical slices (Fig. 3 and see Ivens et al., 2007). + Reduced [K ]o clearance has been reported in the injured brain (D'Ambrosio et al., 1999), and a loss of IKIR has been found in reactive astrocytes around freeze lesions (Bordey et al., 2001), after ischemic insults (Koller et al., 2000) and direct injuries (Schroder et al., 1999) , and in epileptic
- 64 - Tsc1 knock-out mice (Jansen et al., 2005) as well as in human subjects with temporal lobe epilepsy (TLE, Bordey and Sontheimer, 1998; Hinterkeuser et al., 2000; Kivi et al., 2000;Jauch et al., 2002). It is noteworthy that KIR 4.1 knock-out mice display seizure activity very early in life consistent with the idea that down regulation of KIR 4.1 channels may contribute to + epileptogenesis (Djukic et al., 2007). The hypothesis that elevated [K ]o could lead to seizure initiation (Fertziger and Ranck, 1970) is not a new one; however, in this study we show that a selective down-regulation of the KIR 4.1 channel occurs prior to the emergence of epileptic activity, thus highlighting the potential role of potassium accumulation in epileptogenesis. To + what extent KIR channels contribute to the spatial buffering of [K ]o is not entirely known and may differ between brain regions. In hippocampal slices, low concentrations of Ba2+ augmented stimulus-induced K+ by 147% (to more than 9mM, Gabriel et al., 1998), while in the neocortex, Ba2+ slowed down the clearance of iontophoretically applied K+ by only 70% (Ivens et al., 2007). Notably, in both preparations, epileptogenesis was associated with a reduced effect of +2 + Ba indicating reduced IKIR. The relatively high [K ]o found during stimulation in the neocortex in our study may reflect impairments in additional buffering mechanisms such as reduced expression of leak K+ channels (not supported by our microarray data but see Pasler et al., 2007), or the down regulation of gap junction proteins (Fig. 2). However, a recent study in connexin knockout mice (which lack gap junctions in the hippocampus) showed an almost conserved capacity for potassium clearance (Wallraff et al., 2006). In addition, due to their rectification + + properties, the contribution of KIR channels to K clearance becomes critical when local [K ]o are high (Chen and Nicholson, 2000; Newman, 1993), thus augmenting their role in buffering + potassium during high frequency stimulation (Fig. 3d). Finally, reduced [K ]o clearance is expected to enhance potassium accumulation due to delayed neuronal repolarization and facilitated synaptic potentials (Fig. 4).
Due to the lack of pharmacological tools that specifically block astrocytic KIR channels, it is difficult to experimentally its role in controlling neuronal excitability. We therefore used computer simulations that predicted that synaptic accumulation of potassiumwill lead to + frequency-dependent facilitation of EPSPs. Importantly, [K ]o accumulation under these conditions did not have an effect on a single EPSC, consistent with the observation of normal field potentials during epileptogenesis (Seiffert et al., 2004). In contrast, EPSCs were strongly facilitated at stimulation frequencies between 10-50 Hz (maximum around 20 Hz) due to
- 65 - membrane depolarization and increased NMDA conductance. Under these conditions, + facilitation (>300%) was associated with synaptic [K ]o levels reaching ~8 mM. This value may seem high considering that during normal neuronal activity (Heinemann et al., 1990) or that recorded in the present study (Fig. 3). Yet these levels were measured using ISMEs at a distance from the narrow synaptic cleft (<200 Å, Egelman and Montague, 1999; Savtchenko et al., 2000). + In fact, the model predicts that during neuronal activity, [K ]o levels in the extracellular space near the soma are three times lower (due to diffusion and astrocytic uptake) than levels in the synaptic cleft; in line with the results of Somjen and colleagues (2008).
In addition to the "potassium hypothesis", our molecular experiments point to reduced expression of astrocytic glutamate transporters in both albumin- and DOC-treated rats. In the normal brain, passive diffusion and transport clears released glutamate into neurons (via members of the solute carrier protein family - SLC1A) and astrocytes (specifically SLC1A2, SLC1A3 and SLC1A4). The relative contribution of each of these mechanisms in the neocortex is unknown. Studies in the hippocampus indicated that astrocytes play a central role in the removal of synaptic glutamate (Bergles and Jahr, 1998) whereas neuronal transport plays a negligible role (Sarantis et al., 1993); leading to the hypothesis that down-regulation of astrocytic glutamate transporters entails glutamate accumulation in the synaptic cleft and increased excitability. A reduction in glutamate uptake mechanisms has previously been reported in various neurodegenerative diseases frequently associated with BBB breakdown (e.g., Zlokovic, 2008; Rothstein et al., 1992). However, evidence for the role of glutamate uptake mechanisms in epilepsy remains inconclusive (Eid et al., 2008). Although Tanaka et al. (1997) demonstrated spontaneous seizures in SLC1A2 knock-out mice, studies of tissue from human TLE patients have failed to provide conclusive evidence for reduced glutamate uptake (Proper et al., 2002; Tessler et al., 1999). Moreover, the consequences of reduced glutamate uptake are still controversial, with considerable heterogeneity between various brain regions and preparations (Hestrin et al., 1990; Sarantis et al., 1993; Turecek and Trussell, 2000; Arnth-Jensen et al., 2002). To determine the functional consequences of the observed transcriptional response on glutamate homeostatic mechanisms, we recorded whole-cell glutamatergic currents before and after application of DHK, a selective inhibitor of the astrocytic glutamate transporter SLC1A2 (Arriza et al., 1994; Rothstein et al., 1994) in slices from control and treated rats. In agreement with previous studies, we found no effect for DHK on single EPSCs or EPSCs evoked by low-
- 66 - frequency stimulation (Hestrin et al., 1990; Diamond and Jahr, 2000; Takayasu et al., 2004). We did, however, find increased glutamatergic currents under DHK with stimulation frequencies higher than 20 Hz in control slices, similar to the effect of TBOA in the hippocampus (Arnth- Jensen et al., 2002). In contrast, in slices from albumin-treated animals, DHK had no effect on EPSCs, supporting the observed down-regulation of SLC1A2. Yet, despite the reduced DHK effect, we did not observe an increase in EPSCs 24 h following the epileptogenic insult. This finding may be explained by activation of compensatory mechanisms (e.g., up-regulation of neuronal glutamate uptake proteins, see Fig. 2a), excessive activation of pre-synaptic mGluR2 (Scanziani et al., 1997; Iserhot et al., 2004) or altered expression of NMDA receptor subunits.
Our computer model predicted that reduced glutamate uptake alone will result in reduced synaptic facilitation due to desensitization of AMPA receptors. These predictions are in agreement with previous studies in brainstem slices showing a frequency-dependent decrease in AMPA currents after incubation with the glutamate uptake blockers, THA and DHK (Turecek and Trussell, 2000), but stand in contrast to recordings in hippocampal neurons showing increased NMDA-mediated EPSPs under similar conditions (Arnth-Jensen et al., 2002). The difference may be because the latter study was conducted with holding voltages of +40 mV, resulting in a higher open probability of NMDA-R operated ion channels. While our molecular and electrophysiological experiments do indicate a reduction in astrocytic glutamate uptake during the early stages of epileptogenesis, both the electrophysiological data and computer simulations suggest that only at high stimulation frequencies (>100 Hz), does sufficient transmitter accumulate to facilitate EPSCs (see below).
Changes in astrocytic membrane potential were not implemented in our model. Extracellular accumulation of potassiumresults in astrocytic depolarization, which inhibits glutamate uptake (or even causes a reversal of the uptake mechanism; see Szatkowski et al., 1990). Simulating a concurrent reduction in both uptake mechanisms, resulted in a reduction of the stimulation frequency at which maximal facilitation occurs (from ~ 20 to 10 Hz). The simulations predicted + that when both [K ]o and glutamate accumulate in the synapse, more NMDA receptors are + activated at any given [K ]o (Fig. 6). In addition, we expect such depolarization to further reduce the cationic-coupled glutamatergic transport, thus enhancing voltage-dependent NMDA currents. The optimal frequency for synaptic facilitation recorded in brain slices exposed to albumin in
- 67 - sACSF was around 20 Hz, which as predicted by the model, was mainly due to reduced + clearance of [K ]o. This stimulation frequency – 20 Hz – also proved to be the minimal + stimulation frequency at which we measured [K ]o accumulation during epileptogenesis. It is striking that repetitive stimulation under these conditions often resulted in seconds-long, seizure- like activity, highlighting the potential role for BBB breakdown and brain exposure to serum albumin in neuronal hypersynchronicity, enhanced excitability and the generation of seizures. A plausible hypothesis would be that the increased, repeated activation of NMDA receptors leads to non-specific synaptic plasticity, thus strengthening excitatory synapses and causing persistent hyperexcitability (Li and Prince, 2002; Shao and Dudek, 2004). This premise also provides a satisfactory explanation for the efficacy of NMDA-R antagonists in improving cortical function in animal studies of brain injury and stroke – conditions in which the BBB is frequently impaired (Hickenbottom and Grotta, 1998; Sonkusare et al., 2005) and seizures are often observed. While further studies are needed to confirm this hypothesis, we propose astrocytic reaction in the injured cortex, and specifically impaired buffering of extracellular K+, as novel targets for the prevention and treatment of injury-related neocortical epilepsies.
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- 76 - 3.3. Blood Brain Barrier Dysfunction Underlies Stroke Complications
Abstract Blood-brain barrier (BBB) dysfunction is typically observed in the peri-infarct region following brain ischemia. Based on imaging experiments in the photothrombosis model, comparison of published gene array data from rat brains exposed to medial cerebral artery occlusion or BBB breakdown, and electrophysiological recordings, we propose that: (1) the ischemic insult is followed by rapid changes in gene expression leading to delayed dysfunction of the BBB within the penumbra; (2) BBB opening is associated with transformation of astrocytes and a local inflammatory response; (3) changes in neuronal functions within the penumbra are delayed and observed as hyperexcitability and seizure like activity. We propose that BBB dysfunction may be critically involved in functional recovery after stroke and underlies common clinical complications including hemorrhage, epilepsy and delayed cognitive and neurological dysfunctions. We thus point to BBB damage and repair as potential targets for the treatment of stroke.
- 77 - Introduction Stroke, a cerebrovascular accident, is characterized by an abrupt disturbance in blood flow to an area of the brain. Stroke is a leading cause of mortality worldwide accounting for 10 to 12% of deaths (Lopez et al., 2006). It is also a major cause for morbidity, with a third of the survivors left disabled or suffering a wide range of sequelae posing a large burden on patients, caregivers and the health system.
Stroke is commonly categorized as either ischemic or hemorrhagic, with the ischemic type being about 5 times more common (Feigin et al., 2003). The ischemic brain is typically characterized by a central core of very low perfusion that undergoes necrotic cell death and a surrounding dysfunctional region known as the ‗stroke penumbra‘ (Ginsberg, 1997). In the penumbra, neuronal dysfunction appears to arise from several entwined processes: energy failure, metabolic disturbances, ionic derangement and the glial and immune response (Dirnagl et al., 1999). It is the fate of this penumbra, which initially consists of viable cells that is pertinent to the patients' recovery. Importantly, the penumbra may either be incorporated into the necrotic region, or recover and escape cell death. Moreover, recovery may not be complete: a primary ischemic stroke may turn hemorrhagic; the peri-infarct zone may turn epileptic or show delayed neurodegeneration. However, to this date, the exact processes underlying these sequelae are not known, there are no specific reproducible markers for predicting the outcome of the penumbra and there is no specific treatment to "salvage" or "re-program" it.
Another frequent hallmark of the penumbra region is increased permeability and dysfunction of the blood-brain barrier (BBB) (Kastrup et al., 1999). The BBB consists of a functional and an anatomical barrier that acts to restrict or facilitate the flow of solutes in and out of the brain. Changes in BBB function have now been documented in most common neurological diseases (Neuwelt, 2004;Abbott et al., 2006; Zlokovic, 2008). In addition, recent studies have demonstrated a direct role of BBB dysfunction in the pathogenesis of cortical dysfunction, epileptogenesis and neurodegeneration (for reviews see (Friedman et al., 2009;Shlosberg et al., 2010) and serum albumin has been suggested to underlie some of the secondary changes by activating transforming growth factor beta (TGF-) signaling.
- 78 - In this paper we explore the hypothesis that BBB dysfunction within the penumbra is a key factor in the development of secondary events and complications following stroke. We explore the consequences of BBB opening, direct cortical exposure to albumin or TGF-1 on large-scale gene expression and compare them with published gene arrays from stroke-treated animals. Finally, we focus on the interactions between the different components of the "neurovascular unit" in the penumbra under dysfunctional BBB. This fundamental concept of the neurovascular unit emphasizes the close relationship between endothelial cells, astrocytes, microglia, neurons and the extracellular matrix. Understanding the interplay of these cells and extracellular components is essential to comprehend the brain‘s response to injury and its functional recovery (del Zoppo, 2009). We further put forward the hypothesis that BBB functions have a critical role in determining interactions within the neurovascular unit and thus highlight it as a potential new target for the treatment of stroke.
- 79 - Methods All experimental procedures were approved by the Beer-Sheva animal ethics committee. If not stated differently, chemicals were purchased from Sigma Aldrich.
Microarray Analysis. In this study, we compare mRNA expression following BBB disruption/cortical albumin application/TGF- application with animals undergoing medial cerebral artery occlusion (MCAo). The experiments were performed as previously described in David et al. (David et al., 2009). In brief, adult male Wistar rats (120-250 g) were deeply anesthetized by intraperitoneal injection of Ketamine (100 mg/ ml, 0.08 ml/100 g) and Xylazine (20 mg/ ml, 0.06 ml/100 g) and placed in a stereotactic frame. A 4-mm diameter bone window was drilled over the somatosensory cortex, the dura was opened and the underlying cortex was perfused with artificial cerebrospinal fluid (ACSF). For the "treated" rats group, the BBB- disrupting agent deoxycholic acid sodium salt (DOC, 2 mM, Sigma-Aldrich, Steinheim, Germany) or bovine serum albumin (0.1 mM, >98 % pure) was added to the ACSF. Albumin concentrations corresponded to 25% of the normal serum concentration [determined to be 0.4 mM for 10 rats, see also Geursen and Grigor (1987); with a final osmolarity of 303- 305 mOsmol/l]. For the TGF- group we used TGF-1 at a concentration of 10 ng/ml in ACSF. For the sham-operated control group, the cortex was perfused with ACSF. The composition of the ACSF was (in mM): 129 NaCl, 21 NaHCO3, 1.25 NaH2PO4, 1.8 MgSO4, 1.6 CaCl2, 3 KCl, and 10 glucose. Rats were sacrificed at 7, 24, or 48 h following treatment, during epileptogenesis (see Seiffert et al., 2004). All microarray data are available at the Gene Expression Omnibus (GEO) website (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE12304.
Stroke microarrays were downloaded from the GEO database – accession number GSE4206, which consist of cortical samples derived from adult Wistar rats 2 hours following MCAo.
Gene Ontology Analysis. Gene Ontology (GO) was used to group genes into functional groups. Significantly up/down regulated genes were selected as genes changing by 1 SD over mean expression level per-treatment. Selected genes were then uploaded to DAVID (Huang et al., 2008;Dennis, Jr. et al., 2003) in order to obtain a list of enhanced GO terms. A GO term was considered to be enhanced when statistical significance was achieved with a Benjamini-corrected p-value of less than 0.05.
- 80 - Induction of Stroke. Stroke was induced by either the MCAo as previously described (Longa et al., 1989) or the Rose bengal (RBG)-induced photothrombosis method. For the RBG model, a craniotomy was performed in male Sprague Dawley rats as described above. The cortex was continuously superfused with ACSF. Body temperature was monitored and maintained at 37 0.5 °C using a heating pad. Focal cerebral ischemia was induced according to an established model of photothrombosis (Watson et al., 1985). Rose bengal was administered intravenously at 7.5 mg/ml saline (0.133 ml/ 100 g body weight) and a circle of approximately 1 mm diameter was then exposed to green laser light (532 nm, Laser 2000 CNI-532) for 15 minutes.
Real-time Fluorescent Imaging. Regional cerebral blood flow (rCBF) and BBB permeability were evaluated as previously published (Prager et al., 2010). Briefly, the non-BBB-permeable dye fluorescein sodium salt (MW: 376.27 Da) was injected into the tail vein (1 mg/ ml saline, 0.08 ml/ 100 g bodyweight) and pial vessels were imaged using a fluorescence stereomicroscope (Zeiss, SteReOLumar V12) and an EMCCD camera (Andor Technology, DL-658 M-TIL) before, during, and after tracer injection (total of 30 s). Images were acquired at a rate of 25 Hz under control condition and 1 to 4 hours following the induction of focal cerebral ischemia (RBG model). Image analysis was performed using MATLAB, for a more detailed discussion of image analysis, see (Prager et al., 2010).
Histolgy and immunohistochemistry.Histology and immunohistochemistry was performed as previously described (Seiffert et al., 2004;Ivens et al., 2007). Briefly, coronal sections (40 m thick) were prepared, mounted and stained with cresyl violet to assess the necrotic region. For evaluation of astrocytic and microglial activation slices were incubated overnight at 4°C using polyclonal rabbit anti-GFAP (1:250, Dako Germany) and polyclonal mouse anti-Iba-1 (1: 250, Wako Pure Chemical Industries, Ltd.) as primary antibodies. Signal detection was achieved by incubation with secondary antibodies (Cy5 goat anti-rabbit IgG (1:100), Alexa Fluor 555 goat anti-mouse IgG (1:100, both Invitrogen Corporation)) for 5 hours at room temperature.
In-vivo recordings. Electroencephalography (EEG) was acquired using an in-vivo recording telemetric system (CA-F40 or F40-EET, Data Science International, United States) as previously reported (Bastlund et al., 2004;Timofeeva and Gordon, 2001). Chronically implanted electrodes were placed on the rats‘ dura 3 mm frontal and 7 mm caudal to bregma. EEG activity was
- 81 - captured for 7 days and later monitored and screened manually for 'Seizure like events' (SLEs) off-line, while the observer was blind to treatment.
In-vitro recordings. Brain slices were prepared by standard techniques (Seiffert et al., 2004). Rats were deeply anaesthetized with isoflurane and decapitated. Brains were quickly removed, and 400 µm-thick coronal slices were prepared from sensory motor cortex using a vibratome (WPI, Vibroslice, Fla, USA). Brain slices were continually perfused with aCSF (constituents see above) in a carbogenated (5 % CO2 and 95% O2), humidified and tempered (34 ± 1°C) atmosphere. For electrophysiological recordings, after at least 90 minutes of incubation, glass microelectrodes (~3 MΩ, 154 mM NaCl) were positioned in layer 4 of the neocortex. Slices were stimulated with brief (100 µs) pulses, by using tungsten bipolar stimulation electrodes placed at the border between white and gray matter in the same cortical column.
- 82 - Results
Vascular Injury in Stroke is followed by Dysfunction of the Blood-Brain Barrier In the RBG stroke model, light-activated RBG (Fig. 1A) induces endothelial damage resulting in the formation of a thrombin clot. In consequence, the regional cerebral blood flow (rCBF) within the "ischemic core" is reduced beneath a critical level eventually causing cellular death (Fig. 1B and Fig. 2A and see below). Surrounding the ischemic core, in the penumbra, rCBF may decrease, remain constant or even increase due to redistributed blood flow and increased metabolic demand (Prager et al., 2010;Armitage et al., 2010). These changes in rCBF, oxygen supply and metabolite clearance induce signaling cascades within endothelial cells (EC) leading to rapid and robust changes in gene expression. Indeed, data from MCAo-induced stroke reveals that within 2 hours after vessel occlusion, the expression of endothelium-related genes is significantly affected and includes those associated with extracellular matrix (ECM) homeostasis (TIMP1, ADAMTS1, SDC1), cell-cell adhesion (SELE, ICAM1, CXCL1, CXCL2, CCL2,CDH1, KITLG, RET, S100A8, S100A9), vascular tone (ADM), stress response (IER3, HSPB1, HSPA1B), immunmodulation (TNFAIP6, PTGS2, IL13RA1, CTSC) and signal transduction (NR4A1, CITED4, TGIF1, ATF3 , FOS, PLCD4, NPY5R) (Fig. 1C). Following a similar time course, vascular leakage becomes prominent in the area surrounding the photothrombotic lesion seen as an accumulation of fluorescein sodium salt in the extravascular compartment (Fig. 1D). Importantly, the increase in BBB permeability was most prominent 2-4 h after the initiation of the injury to the endothelial cells (data not shown), supporting the role of gene expression modifications and subsequent protein synthesis in the underlying process.
- 83 - Figure 1: The blood brain barrier in the stroke’s penumbra and early gene expression changes. A. schematic drawing of the rodent skull, dotted line estimates thrombotic region (left), as induced via light-activated i.v.-injected Rose Bengal (right); B. fluorescent angiography of pial surface vessels before (left) and after (right) photothrombosis; see mal-perfusion within dotted line; C. selection of genes with > 1- fold, 2h after MCAo; d) average intensity maps of fluorescein sodium salt in control and 4 h after photothrombosis reveal increased BBB permeability as the tracer has accumulated in the extravascular compartment.
- 84 - Stroke and BBB Dysfunction are associated with an Inflammatory Response What are the consequences of BBB dysfunction typically observed in the penumbra surrounding the ischemic core? In a recent study we demonstrated that opening of the BBB in- vivo, exposing the cortex to serum-derived albumin or to TGF-, all result in a robust upregulation of astroglial and inflammatory genes (Cacheaux et al., 2009). Importantly, an inflammatory response is also a predominant feature of the peri-infarct region. Histological experiments after RBG treatment show that the ischemic/ necrotic core is characterized by a loss of all cell types (Figure 2A, 3 weeks post stroke). In the area adjacent to the ischemic core, astrocytes and microglia dominate with only few surviving neurons, surrounded by a wider region showing apparently normal neuronal staining (visualized using NeuN staining, see Fig. 2B, 8 days post stroke). In both sub-regions, the astrocytic marker GFAP and the microglial protein IBA1 were elevated compared to the contralateral hemisphere, indicting a prominent inflammatory response (Figure 2 B-D, 8 days post stroke). Similarly, an increased number of astrocytes with no or minimal cell loss can be seen in the BBB disrupted cortex (Seiffert et al., 2004).
- 85 - Figure 2: Inflammatory response following photothrombotic stroke. A. cresyl violet staining revealing necrosis in the ipsilateral hemisphere 3 weeks after ischemia B,C. GFAP, NeuN and DAPI co-staining showing enhanced astrocytic activation in the penumbra (left) 8 days post-stroke; note the clear border between areas with NeuN-positve cells and neuronal loss (B, left); D. Iba-1, DAPI co- staining reveals microglial activation 8 days post stroke.
Microarray analysis of MCAo- BBB-, albumin- and TGF--treated brains reveal striking similarities in the regulation of inflammatory genes: examples of genes up-regulated across treatments are chemokines (i.e. CCL2, CCL3, CCL6, CXCL1, CXCL16, CCL6), cytokines (IL1b, IL33) and adhesion molecules (ICAM1, VCAM) (data not shown). GO terms enhanced across all four treatments include: ‗inflammatory response‘, ‗leukocyte migration‘ and ‗regulation of cell death‘ (Table 1). Thus, microarray analysis predicts a similar inflammatory response following stroke and non-ischemic BBB opening and suggests a role for TGF- signaling in mediating these changes.
- 86 - Table 1. Inflammatory Response to Stroke, BBB-disruption and cortical application of Albumin or TGF-* indicates significant alteration in gene expression with Benjamini- corrected p-value < 0.05.
Go Term Stroke BBB Albumin TGF- disruption
Inflammatory response * * * *
Response to glucocorticoid stimulus * * * *
Response to steroid hormone stimulus * * * *
Immune response * * * *
Response to hormone stimulus * * * *
Leukocyte migration * * * *
Leukocyte chemotaxis * * * *
Regulation of cell death * * * *
Response to cytokine stimulus * * * *
Regulation of programmed cell death * * * *
Regulation of apoptosis * * * *
Cytokine * *
Cytokine-cytokine receptor interaction * * * *
Regulation of acute inflammatory * * * response
Tissue morphogenesis * *
Egf-like domain *
Anti-apoptosis *
- 87 - Delayed Network Dysfunction To measure functional changes within the neuronal network suviving the ischemic event we recorded contineous EEG activity from the penumbra of RBG-treated rats. Recordings revealed increased activity in the high-frequency band (46-90 Hz) from around day 3-4 following treatment (Fig. 3A). The increased energy at high frequenies was due to short, paroxysmal, high amplitude activity lasting 5-30 s which resembled spontaneous seizures (hence termed "seizure- like-events" or SLEs). Interestingly, recordings from rats treated with DOC to open the BBB (where no ischemic lesion was found) documented similar SLEs (Fig. 3A). In vitro recordings in cortical slices from stroke- and DOC-treated animals show typical paroxsysmal, long duration (> 100 ms), all-or-none field potentials in response to brief stimulation of the white matter (Fig. 3B). These typical epileptiform field potentials were similar to those observed in other models of neocortical epilepsy(Gutnick et al., 1982) and confirm that the hyperexcitability observed in-vivo originates in the peri-infarcted or BBB-treated regions.
In search for expression changes related to neuronal hyperexcitability we compared gene arrays from DOC-/albumin-/TGF-1-treated animals with the 2h MCAo microarrays and with previous reports in models of stroke (Table 2). Importantly, BBB opening was sufficient to induce significant changes in gene expression which are likely to influence network excitability. Those genes relate to GABAergic and glutamatergic transmission and ion-channel formation (Table 2). It is worth noting that out of a selection of 44 neuronal-related genes, 27 have changed significantly only at later time points ( 8 h) after stroke while only 6 genes showed acute changes ( 4 h). The number of genes affected by at least one of the three BBB-related models (DOC, albumin, TGF-) was only slighly lower than late after stroke (25 vs. 27, respectively). In the majority of genes a similar tendency for expression changes (i.e. up- or down-regulation) was found in both stroke and isolated lesioning of the BBB (Table 2).
- 88 - Figure 3: Ischemia and BBB-opening alter network properties and neurotransmission- related gene expression A. typical EEG recordings in albumin- and RBG- treated rats; note the synchronic, high- amplitude activity (SLEs); B. typical in- vitro recordings in acute brain slices from DOC- and RBG-treated animals indicating hyperexcitability and hypersynchronicity
- 89 - Table 2. Changes in Neurotransmission-related Gene Expression in Stroke and BBB- opening. “BBB‖ includes DOC-, Albumin- and TGF--treatment; ‖ns‖ = not significant in our analysis of the MCAo microarray; note the downregulation in GABAergic transmission across treatments and the delayed occurrence of gene expression changes. Gene Early Late BBB (4h) (8 h) GABAergic Transmission GABA-A R 3 gabra3 ns GABA-A R 4 gabra4 ns GABA-A R β 1 gabrb1 GABA-A R β 2 gabrb2 ns GABA-A R δ gabrd ns GABA-A -1 gabrg1 ns GABA-A -2 gabrg2 Glutamatergic Transmission GlutR-2 Grid2 ns GluR7b, kainate Grik3 mGluR5 grm5 mGluR7b grm7 ns NMDA-R 1 grin1 ns NMDA-R 2A grin2a ns NMDA-R 2B grin2b ns NMDA-R 2C grin2c ns NMDA-R 3A grin3a ns Ion-Channels K+ channel, Kir3.1 kcnj3 ns K+ Channel (inwardly rectifying, G Protein- kcnj9 activated) K+ Channel I kcnj10 K+ Channel Kv4.3 kcnd3 K+ Channel Kv7.3 kcnq3 ns K+ Channel Kv8.1 kcnv1 K+ Channel Kv 9.1 kcns1 K+ Channel Protein kcnd3 K+ Channel Protein (3145 bp) kcnc1
- 90 - Gene Early Late BBB (4h) (8 h) K+ Channel protein ERG kcnh2 Ca2+ channel (L, v-dependant) cacna1c Ca2+channel 1 (pore-forming) cacna1a Ca2+channel 1 cacna1d Ca2+channel (L, dihydropyridine-sens) cacna2d1 Ca2+channel 2 (L-type) cacnb2 Ca2+channel 3 cacnb3 Ca2+channel, v-dependent, L-type, 1d cacna1d Ca2+channel, v-dependant subunit 2 Cacng3 ns Ca2+ channel, v-dependant, 3.2 cacna1h ns Na+ channel 1 scn1b Na+ channel I scn1a Na+ channel II scn2a1 Na+ channel III scn3a Na+ channel, voltage-gated type II, scn2b ns Na+ channel type IV, scn4b ns Cl-channel-II clic2 Cl- intracellular channel I clic1 Cl- intracellular channel IV clic4 ns
- 91 - Discussion In this study we propose a role for BBB permeability changes in determining the outcome of ischemic brain injury. Our data suggest that BBB dysfunction within the penumbra (1) is a frequent and robust consequence of endothelial injury; (2) is associated with a prominent inflammatory response; (3) is sufficient to induce structural and functional changes within the neuronal network, characterized by hyperexcitability and hypersynchronicity.
Human and animal studies show that BBB dysfunction is a common consequence of ischemic stroke (Stoll et al., 2009; Latour et al., 2004). BBB breakdown in stroke usually has a biphasic nature (Huang et al., 1999; Rosenberg and Yang, 2007) with an initial Primary BBB breakdown which occurs within minutes after injury and results from a direct ischemic damage to endothelial cells (Li et al., 2003); a secondary BBB dysfunction usually develops within hours to days following the primary injury and occurs in brain regions that are not necessarily ischemic (see Fig. 1D and see (Stoll et al., 2009)). An initial RBG plasma half-life of 2 minutes (Klaassen, 1976) makes a delayed photochemical effect altering BBB permeability unlikely. This implies that BBB dysfunction arises from metabolic and inflammatory changes, involving modifications of gene expression within different cellular elements of the neurovascular unit. While the primary BBB injury seems to be unavoidable, secondary dysfunction may be prevented and thus turn out to be a therapeutic target.
Microarray gene analysis 2 h after stroke highlights groups of significantly modulated genes that may be involved in the secondary BBB breakdown (Fig. 1C). Gene groups, which include extracellular matrix homeostasis, cell–cell interactions, cellular signaling, and immune response, were similarly reported in previous reports (Lu et al., 2003; Grond-Ginsbach et al., 2008; Tang et al., 2006; Schmidt-Kastner et al., 2002; Tian et al., 2007; Deli, 2009; Sarabi et al., 2008; Liu et al., 2007). An interesting example is matrix metalloprotease-9 (MMP-9), which is rapidly up- regulated following BBB disruption and stroke and has been associated with BBB breakdown (Heo et al., 1999; Gursoy-Ozdemir et al., 2004). Importantly, minocycline, a tetracycline antibiotic with inhibitory activity on MMP-9, has been shown to reduce BBB breakdown in an animal model (Yrjanheikki et al., 1999; Wang et al., 2002) and is currently being investigated in
- 92 - a clinical trial for stroke (see http://clinicaltrials.gov/show/NCT00630396) with promising preliminary results (Lampl et al., 2007).
The rapid increase in BBB permeability after stroke is followed by a local immune response (for reviews see (Dirnagl et al., 1999; del et al., 2000; Nilupul et al., 2006)), which includes a rapid activation of astrocytes and microglia within the penumbra (Fig. 2 and Nowicka et al. (2008)). Interestingly, a sole BBB opening (without ischemic damage) or brain exposure to serum albumin are sufficient to induce astrocytic activation (Seiffert et al., 2004; Ivens et al., 2007), supporting a key role for BBB dysfunction in the emerging immune response. Indeed, gene expression analysis following BBB disruption revealed an inflammatory response similar to that found following cerebral ischemia, which included upregulation of chemokines, cytokines, and adhesion molecules (Table 1 and see Cacheaux et al. (2009)). In a recent study we further showed that this inflammatory response is mediated by the most abundant serum protein, albumin, which binds to TGF-receptors and activates TGF- signaling (Cacheaux et al., 2009). TGF-1 has also been reported to be highly upregulated in the stroke penumbra (Krupinski et al., 1996); however, experimental data to support (or exclude) the direct influence of TGF- signaling on stroke outcome is sparse. Several groups argue for a protective activity of TGF-1 in cerebral ischemia (Prehn et al., 1993; Henrich-Noack et al., 1996; Ruocco et al., 1999; Ma et al., 2008), while others implicate it with inhibited axonal regeneration and enhanced inflammation after vascular damage (Schachtrup et al., 2010). It was also shown that mice overexpressing TGF-1 develop seizures, hydrocephalus, and motor incoordination (Wyss- Coray et al., 1995). Together, these results indicate TGF-signaling as a potential pathway associated with brain repair mechanisms, and call for additional studies for a better understanding of downstream events and their temporal occurrence in specific cell populations after cerebral ischemia.
An additional mechanism highlighted by our study is associated with cellular adhesion. ICAM-1 is significantly upregulated across our different treatments and is known to be involved in the migration of neutrophils across the endothelium (Greenwood et al., 2002). Endothelium- bound neutrophils release MMP-9, which increases BBB permeability (see above and Gidday et al. (2005)). Administration of cellular adhesion molecule blockers has been demonstrated to decrease the size of the ischemic lesion (Frijns and Kappelle, 2002). However, a clinical trial
- 93 - using Enlimomab (murine ICAM-1 antibody) did not improve patients‘ outcome after stroke and had a detrimental effect, perhaps because of its murine origin (Vuorte et al., 1999). In this context, Selectin-E, a cell-surface protein mediating adhesion of blood neutrophils is also upregulated across treatments and is known to be expressed by activated endothelium upon cytokine stimulation and shown to mediate tissue damage after stroke (Huang et al., 2000). Interestingly, nasal application of Selectin-E in stroke-prone rats prevented secondary thrombosis and hemorrhagic transformation (Takeda et al., 2002; Chen et al., 2003), leading to a clinical phase II trial in patients suffering from stroke or transient ischemic attacks (http://clinicaltrials.gov; Identifier: NCT00012454).
Long-term stroke recovery is crucially dependent on the degree and quality of neural network reorganization. Neuronal circuits from non-ischemic brain regions can take over the tasks of the necrotic cerebrum and their ability for such ―rewiring‖ is associated with a tremendous capacity for plasticity within the penumbra ((Carmichael et al., 2001; Winship and Murphy, 2008) reviewed by Witte et al. (2000); Murphy and Corbett (2009)). Interestingly, neuron-based treatments to improve functional recovery after stroke have not been successful, thus raising the demand for new, non-neuronal approaches (reviewed by Lo (2008)). One candidate is the inflammatory response, which has the potential to affect neuronal plasticity and organization of the circuit. Evidence for the involvement of BBB dysfunction in network reorganization following injury is indirect (for review see also Shlosberg et al. (2010)). BBB dysfunction may influence local network activity by inducing neuronal toxicity and death (thus forcing reorganization) or more directly by affecting synaptic rewiring. BBB dysfunction has been observed in several degenerative cerebral diseases including multiple sclerosis, Parkinson's and Alzheimer‘s (for reviews see Hawkins and Davis (2005); Abbott et al. (2006); Zlokovic (2008)). However, it is not clear whether BBB dysfunction in these conditions underlies neurodegeneration or reflects associated vascular damage. Opening the BBB in the rat cortex indeed results in reduced dendritic branching and a decrease in the number of neurons; this degenerative effect was found to develop several weeks after injury and may reflect an excitotoxic injury due to enhanced network excitability (see below (Tomkins et al., 2007)). BBB dysfunction could also affect network excitability and synaptic connectivity by inducing the transformation of astrocytes, resulting in compromised homeostasis of the extracellular environment (e.g., reduced buffering of extracellular potassium and glutamate see David et al.
- 94 - (2009)), and/or release of cytokines, which directly affect neuronal transmission (Riazi et al., 2008; Vezzani et al., 2008a, 2008b). In addition, BBB dysfunction, TGF- signaling, and stroke result in significant modulation of neuronal gene expression (Table 2). While the large number of neuronal genes altering their expression levels may predict increased (due to reduced expression of GABA receptors) or decreased (due to reduced expression of NMDA receptors) excitability, in vivo and in vitro electrophysiological recordings clearly demonstrate increased synchronicity and excitability, developing within the first week after stroke or BBB breakdown (Fig. 3).
In summary, we demonstrate that even a focal vascular injury may result in a wider cortical region showing long-lasting dysfunction of the BBB. The subsequent leak of BBB-impermeable serum proteins into the neuropil induces TGF- signaling and an inflammatory response that are associated with dysfunctional astrocytes and rewiring of the neuronal network. These complex dynamic interactions within the neurovascular unit are currently under study in several laboratories and promise to provide new therapeutic targets for the prevention and treatment of stroke. Our data point to BBB dysfunction as a potential key determinant for clinical complications observed following cerebral ischemia, including edema formation and increased intracranial pressure (due to increased brain water content; (Hofmeijer et al., 2009)); hemorrhagic transformation (due to malfunction of endothelial cells and activation of MMPs (see (Wang and Lo, 2003; Neumann-Haefelin et al., 2002)); seizures and epilepsy (due to astrocytic malfunction and network rewiring (see Kotila and Waltimo, 1992; Burn et al., 1997; Camilo and Goldstein, 2004)); and cognitive deterioration (due to neurodegeneration (see Ballard et al., 2003)). Future prospective studies including more accurate measurements of the spatio-temporal patterns of BBB permeability changes after stroke are required to determine whether these predictions hold true.
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- 104 - 3.4. The Axon Initial Segment of Layer 5 Pyramidal Neurons Following Stroke
Abstract
We examined changes in the axon initial segment in a photothrombotic model for cortical stroke. Current clamp recordings of action potentials in layer 5 pyramidal neurons from the peri- infarct zone revealed no change in AP properties: an intact first, presumably axonal upstroke component, a non-significant decrease in threshold, and a similar firing pattern in response to intracellular current injection. In an attempt to characterize the pattern of Na+ channel distribution in the axonal initial segment, we explored the spatial and temporal pattern of the Na+ influx into the AIS elicited by a single AP using a combination of patch-in-slice recordings and high-speed fluorescence imaging of the Na+-sensitive indicator SBFI. In neurons from control animals, Na+ influx was maximal in a region 18 ± 2 µm long, located at a distance of 8 ± 2 µm from the soma and 27 ± 4 µm from the edge of the myelin. Computer simulations indicated that maximal excitability with the minimum number of channels is achieved with the Na+ channel hotspot located at some optimal distance from the soma.
- 105 - Introduction Axons are neuronal processes that convey information over distances, acting as biological transmission lines. Ramon y Cajal was probably the first to postulate their function as carriers of the neuronal output signal (Cajal, 1911). The proximal axon, also known as the axon initial segment is the primary site of action potential initiation in cortical neurons (Stuart & Hausser, 1994; Stuart & Sakmann, 1994; Colbert & Johnston, 1996; Stuart et al., 1997a; Clark et al., 2005; Palmer & Stuart, 2006; Meeks & Mennerick, 2007; Shu et al., 2007).
The AIS has the lowest threshold for AP initiation due to several reasons: The AIS is thinner than the soma and therefore has a reduced membrane surface area per length, allowing reduced capacitative load for membrane currents, thus allowing faster depolarization and channel opening (Moore et al., 1983). Another contributing factor for the initial segments lower AP threshold is the higher density of Na+ channels available there. A computational study (Mainen et al., 1995) that assumed identical Na+ channel properties in all cellular compartments and homogenous channel density along the initial segment concluded that in order to achieve the lower axonal threshold, AIS Na+ channel density must be orders of magnitude higher in the soma. The high density of Na+ channels in the initial segment was also evident in ultrastructural studies observing dense undercoating (Palay et al., 1968). However, a more recent immunogold quantitative study has lowered these estimations. The current estimation is of a ratio of c.a. 30 soma-to-AIS for Nav1.6 (Lorincz & Nusser, 2010). Patch recording studies from the AIS and axonal blebs, however, suggest a similar density of channels in both regions (Colbert & Johnston, 1996; Colbert & Pan, 2002; Kole et al., 2008; Hu et al., 2009). Another approach for estimating channel density is sodium imaging. A recent study using fast sodium imaging estimates the soma-to-AIS ratio to be approximately three (Fleidervish et al., 2010) .
Assembly and maintenance of neuronal polarity and compartmental specialization requires anchoring of site-specific membrane proteins. It was found that formation of the AIS is intrinsically programmed, without a need for interactions with other cell types, as opposed to the formation of the nodes of Ranvier, which require glial-derived signals (Kaplan et al., 1997; Eshed et al., 2005; Ogawa & Rasband, 2008; Zonta et al., 2008; Feinberg et al., 2010). One protein, named AnkyrinG (AnkG/Ank3), is considered to be the master organizer of the AIS. It is restricted to the AIS and the nodes of Ranvier (Kordeli et al., 1995) and is able to bind sodium
- 106 - channels thru a cytoplasmic loop domain (Garrido et al., 2003; Lemaillet et al., 2003). Silencing of AnkG mRNA by shRNA resulted in complete dismantling of the AIS, which did not occur with shRNA for AIS CAMs, Na+ channels, and βIV spectrin (Hedstrom et al., 2008).
Studies have attributed different sodium channel isoforms with different roles. In pyramidal neurons, back-propagation is said to be mediated by the Nav1.2 isoform (Dulla & Huguenard, 2009; Hu et al., 2009), while Nav1.6 is said be the most important for spike initiation (Royeck et al., 2008). In accordance with current theories of spike initiation occurring at the distal AIS, Nav1.2 is situated proximal to the soma while Nav1.6 is more distal (Hu et al., 2009). In interneurons the Nav1.1 isoform was found to be more prevalent proximally but similar to the distribution in pyramidal neurons; distal sodium channels are mostly of the Nav1.6 isoform (Ogiwara et al., 2007; Van Wart et al., 2007; Duflocq et al., 2008; Lorincz & Nusser, 2008).
The AIS is postulated to be dynamic in nature working to fine-tune neuronal excitability. (Kuba et al., 2006) studied the chick auditory system to reveal different Na+ channel distributions in neurons responsive to different frequencies. In that study it was found that sodium channels were clustered within a short segment of the axon separated by a 20–50 m stretch of non-excitable membrane; in low-frequency responsive neurons they were clustered in a longer segment of the axon and closer to the soma. In another study (Grubb & Burrone, 2010), the long-term effect of excitation was studied in cultured neurons. It was found that neurons chronically exposed to either high-K+ containing extracellular fluid or subjected to light- activated excitation tended to shift the location of the AnkG segment to a more distal location (Grubb & Burrone, 2010), thereby supposedly lowering the firing threshold. Ca++ channels have also been shown to influence action potential shape and firing pattern (Bender & Trussell, 2009).
Disruption of the AIS has been observed in in vivo models for ischemia and in cultures following oxygen glucose deprivation (Schafer et al., 2009). Following ischemia, a rapid and robust degradation of AIS proteins alongside the disappearance of sodium channels occurred within hours. Protein loss was shown to be mediated by a calcium-activated degradation enzyme called calpain.
One of the early changes observed in the ischemic brain is dysfunction of the blood-brain barrier ((Mark & Davis, 2002). The BBB consists of a functional and anatomical barrier that acts
- 107 - to restrict or facilitate the flow of solutes in and out of the brain (for reviews see (Fenstermacher et al., 1988; Ballabh et al., 2004)). Recent studies have demonstrated the key role of BBB dysfunction in the pathogenesis of cortical dysfunction, epileptogenesis, and neurodegeneration (see chapter 3 and reviews by (Friedman et al., 2009; Shlosberg et al., 2010)). Extravasation of serum proteins (specifically albumin) as a result of BBB disruption has been suggested to underlie some of the secondary changes and was found to be mediated by activating transforming growth factor beta (TGF-) signaling (Ivens et al., 2007; Cacheaux et al., 2009; David et al., 2009).
The aim of this study was to search for changes in the AIS within the penumbra following acute stroke.
Figure 1: Nav channels are lost from the AIS following ischemic injury. A, B, After MCAO, contralateral (left) and ipsilateral (right) regions of rat cortex were immunolabeled for Nav channels (Pan-Nav, red), βIV spectrin (A, green) or ankG (B), and Hoechst to label nuclei (blue). The sections shown are from a brain collected 24 h after MCAO. Arrows indicate labeled AIS in layers 2/3 cortex. Scale bar, 50 μm. Reproduced from: Schafer et al. (2009) with permission from the Society for Neuroscience.
- 108 - Methods All procedures were approved by the Institutional Animal Care and Use Committee of the Ben-Gurion University of the Negev.
Microarrays. Total RNA from animals treated with DOC, TGF-β1, or with albumin was isolated from the somatosensory cortex, directly under the craniotomy area, using the TRIzol® reagent (Invitrogen, Carlsbad, CA), and prepared using the Affymetrix GeneChip one-cycle target labeling kit (Affymetrix, Santa Clara, CA). Biotinylated cRNA was then fragmented and hybridized to the GeneChip Rat Genome 230 2.0 Array according to manufacturer's protocols (Affymetrix Technical Manual). The array data were normalized using GCRMA (GC Robust Multi-Array Average) or RMA (Robust Multi-Array Average) analysis using the probe annotations distributed by Affymetrix (NetAffx). In addition, we compared these results to those obtained with annotations based on the Unigene build (Dai et al., 2005). The data from a sham- treated animal (24 h) was used to normalize the other arrays. To identify genes involved in axonal functions, we used GeneCards (http://www.genecards.org), querying for "axonal".
In vivo Stroke Model. Cortical stroke was induced in 3–4–week-old CD1 mice using a similar protocol established in rats and adapted to mice (Watson et al., 1985; Boquillon et al., 1992). In brief, mice were anesthetized using an intraperitoneal injection of a mixture of 6 mg/kg Xylazin (Rompun, Bayer, Germany) + 90–120 mg/kg Ketamin (Ketavet, Pharmacia and Upjohn, Germany). A thin cut was made to the skin over the left sensory-motor cortex and the skin folds were tucked to the sides to expose the skull. Using a small gauge needle (31G), 200 L of the photosensitive dye Rose-Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein, 5 mg/ml) dissolved in normal saline was injected into the tail vein. Immediately following injection, a green laser light (wavelength 532 nm, Laser 2000 CNI-532) illuminated the the exposed skull for 15 minutes at a distance of 25 cm (see Fig. 2). Mice were kept warm using a warming lamp until spontaneous movement was regained.
- 109 - Figure 2: Photothrombotic stroke and resulting BBB disruption. (a) Activation of the photosensitive dye Rose- Bengal (RBG) using laser light leads to a stroke formation. (b) 24 hr following stroke induction, Evans blue extravasation is evident in the stroke penumbra. (c) Imaging Evans blue in the stroke penubmbra.
Slice preparation and whole-cell recording. Experiments were conducted on slices prepared from either control CD1 mice or CD1 mice 24 hr following the aforementioned stroke procedure. On the morning of the experiment, mice received an intraperitoneal injection of 200 L of a saline solution containing 2 mg% Evans blue. Three hours following the injection, mice were anesthetized with isoflurane and decapitated. Stroke was confirmed by the visualization of extravasated Evans-blue within the brains parenchyma (see Methods). Recordings were made from slices submerged in a plastic chamber with a glass bottom. The slices were super-perfused with oxygenated artificial cerebrospinal fluid (ACSF) at room temperature. The ACSF contained
124mM NaCl, 2.5mM KCl, 2mM CaCl2, 2mM MgCl2, 1.25mM NaH2PO4, 26mM NaHCO3, and
10mM glucose; pH was 7.4 when bubbled with 95% O2/CO2.The chamber with submerged and superperfused slices was attached to a stage rigidly bolted to an air table and cells were viewed with a 10× or 60× water-immersion lens (Olympus) in an Olympus BX51WI microscope mounted on an X-Y translation stage. The peri-infarct area was readily identified by the presence of extravasated Evans blue surrounding an area that was devoid of neurons (see fig. 2). Neurons were considered in the peri-infarct area only if situated within the Evans blue stained area and within 200 m of the infarcted site as observed through Differential Interference Contrast
- 110 - microscopy (DIC). Recordings were made from L5 pyramidal cells in the peri-infarct area, the contralateral hemisphere, or cortices of control animals. Tight seals were made with the ―blow and seal‖ technique using video-enhanced differential interference contrast optics to visualize the cells. For current clamp experiments the pipette solution contained (in mM): 140 KCl, 2 MgCl2, 4 NaCl, 10 Hepes 10 (as K+ salt), at times supplemented with 2mM of sodium-binding benzofuran isophthalate (SBFI, Molecular Probes); pH was adjusted to 7.25 with KOH. Current clamp recordings were made with the Axoclamp-2A amplifier equipped with the enhanced capacitative compensation headstage; data were low-pass filtered at 30 kHz and digitized at 66 kHz–166 kHz. Maximal capacitance compensation and bridge correction was achieved using the amplifier‘s built in circuitry. Cells with an access resistance of more than 20 MΩ were not included for action potential shape measurements.
Dynamic sodium measurements. SBFI fluorescence was excited with a high intensity LED device with a peak illumination at 382 nm using a dichroic mirror = 409 nm and an emission filter at 510 (84) nm). For most experiments, changes in fluorescence were acquired using a back-illuminated 80 × 80 pixel cooled CCD camera (NeuroCCD-SMQ, RedShirt Imaging) controlled by the Neuroplex software (RedShirtImaging). Images were acquired at 500 frames per second. To improve signal-to-noise ratio of the traces, we typically averaged 20–40 trials. Illumination intensity during sodium measurements was reduced to the minimum possible to achieve a florescent signal without phototoxicity. Indicator bleaching was corrected by subtracting an equivalent trace without stimulation.
Computer Modeling. Numerical simulations were performed in the NEURON simulation + environment (Hines & Carnevale, 1997). Electrophysiological parameters and dynamic [Na ]i changes were studied in a simplified compartmental model that encompassed the fundamental morphological and electrical features of layer 5 pyramidal neurons. In the model, the 1.2–2 μm thick AIS extended over the first 40–50 μm of the axon in L5 pyramidal. The subsequent segment (length, 50 μm; diameter, 1.2 μm) was myelinated. The soma (length, 35 μm; diameter, 23 μm) gave rise to a single apical dendrite (length, 700 μm; diameter, 3.5 μm) and to two basal dendrites (length, 200 μm; diameter, 1.2 μm). For computational precision, all compartments were divided into many segments, with the length of individual segments usually less than 1 μm. 2 −2 The passive electrical properties Rm, Cm, and Ri were set to 15,000 Ω cm , 0.9 μF cm and 125
- 111 - Ω cm, respectively, uniformly throughout all compartments. Myelination was simulated by −2 reducing Cm to 0.02 μF cm and a ten-fold increase in membrane resistance. The resting membrane potential at the soma was set to −75 mV. All simulations were run with 10 μs time steps at a temperature of 20°C. The model incorporated a Hodgkin-Huxley–based Na+ conductance as previously described (Colbert & Pan, 2002). The activation time constant was + given by τm = k/(αm(Vm) + βm(Vm)); with a variable k. Na conductance was limited to an area 18 μm long that contained sodium channels at a density of 0.08 S/cm2 positioned variably along the + + AIS. The soma and dendrites lacked Na conductance. The model included Kv and Kv1-like K channels with kinetics and density as previously described (Kole et al., 2008). The K+ equilibrium potential was set to −85 mV. Diffusion of Na+ was modeled as the exchange of Na+ ions between adjacent neuronal compartments using the intrinsic protocols in NEURON assuming a diffusion coefficient of 0.6 μm2 ms−1(Kushmerick & Podolsky, 1969). The resting intracellular and the extracellular Na+ concentrations were set to 4mM and 151mM, respectively.
- 112 - Results
Blood-brain barrier induced changes in axon-related genes Considering recent evidence pointing to axonal changes as a possible mechanism for cortical pathologies (Schafer et al., 2009; Wimmer et al., 2010), we hypothesized that changes to the axon also accompany disruption of the BBB and are involved in the pathogenesis of post-insult epilepsy.
To test this hypothesis in our BBB disrupted model, we first looked for changes in mRNA expression for axon-related genes. We queried the on-line database GeneCards (www.genecards.org) for genes related to the term ―axonal‖, which yielded a list of 106 genes. Examining changes in mRNA levels for these 106 genes in our established BBB/Albumin/TGF- β1 animal models, revealed a similar pattern of expression changes among treatments (Fig. 3a). Particularly interesting was the downregulation of ankyrinG (ANK3), a key organizer of the axon initial segment (Kordeli et al., 1995).
Furthermore, querying our gene array data for changes in mRNA coding for sodium channels revealed that SCN1A (NaV1.1) is consistently downregulated together with the regulatory subunits SCN1B and SCN2B (Fig. 3b). Transcription of other genes including the main axonal Na+ channel alpha subunit (SCN8A, NaV1.6) did not change consistently.
- 113 - Figure 3: mRNA expression for axonal related genes under DOC, albumin, and TGF-β treatment. (a) altered mRNA expression for axon-related genes. (b) mRNA changes for sodium channels.
Action potential shape in peri-infarct area Taking together our gene array data, combined with recent studies observing the disappearance of ankyrinG and sodium channels from the AIS shortly after stroke (Schafer et al., 2009), we decided to test for physiological consequences of such changes in transcription in the BBB disrupted cortex within the stroke penumbra.
To this end, I established an animal model for stroke, using the photosensitive dye RBG in mice. Stroke in these animals is accompanied by BBB disruption as evident by the extravasation of Evans blue (see Methods section and Fig. 2).
As the upstroke of the AP is the result of sodium influx through voltage-gated sodium channels, we further hypothesized that the loss of the normally occurring, moderately high
- 114 - concentration of sodium channels in the AIS (Kole et al., 2008; Fleidervish et al., 2010) and/or change in their gating properties due to the loss of anchoring to the cytoskeleton will lead to changes in neuronal excitability and also affect the kinetics of the somatic APs‘ upstroke.
To examine initiation properties and the contribution of different neuronal compartments to the upstroke of the action potential, we recorded APs in Layer 5 pyramidal neurons in response to short, suprathreshold depolarizing currents. Cells in the peri-infarct area were similar in gross appearance to cells in the contralateral hemisphere. Apparent input resistance was similar (although due to low sample size significance cannot be calculated) between neurons in the peri- infarct area compared to that recorded in neurons from the contralateral hemisphere (144.5 ± 57 MΩ vs. 130 ± 37 MΩ, n=3 and 2, respectively, see Fig. 4a).
Previous studies have shown that the upstroke of somatic APs composed of at least two separable components, first observed in motor neurons by Coombs et al. (Coombs et al., 1957) and later also described in central neurons (Grace & Bunney, 1983; Colbert & Johnston, 1996; Shu et al., 2007). Spike upstroke shape is best appreciated by examining phase plots – plotting
to as recorded in the soma. The first component is thought to reflect axonal mediated current and occurs ~100–150 µs before somatic Na+ channel activation, which is believed to underlie the second component (Stuart et al., 1997b; Palmer & Stuart, 2006; Shu et al., 2007). Contrary to our hypothesis, rapid onset and biphasic waveform of pyramidal neuron APs were unaffected by stroke (Fig. 4b), indicating that either these features are not valid indicators of axonal AP initiation, that an intact cytoskeleton is not critically important for positioning the sodium channels, or that the cytoskeleton is not at all disrupted at this time point.
Interestingly, action potential threshold occurred at a slightly more hyperpolarized voltage in the peri-infarct area, at -44.9 ± 3.3 mV, compared with -39.1 ± 2.7 mV measured in the contralateral hemisphere (Fig. 4c). However, AP firing frequency in response to long (200 ms) current steps in peri-infarct neurons remained similar to that of controls, with a slope 88.3 ± 27.9 Hz/pA for peri-infarct vs. 74 ± 2.8 Hz/pA in the contralateral hemisphere (Fig. 4d).
- 115 - Figure 4: Neuronal properties following stroke. (a) Input resistance measured for neuron in the vicinity of the stroke vs. neurons in the contralateral hemisphere. (b)
plots of vs. for neurons in the peri-infarct area (black) and in the contralateral hemisphere (grey). (c) Action potential thresholds for neurons in the peri-infarct area and in the contralateral hemisphere. (d) Action potential firing frequency as a function of current injection (F/I).
Axonal sodium imaging in control mice In order to gain better spatial understanding of sodium channels within the axon initial segment and assess for changes in the spatiotemporal properties of sodium channels in the AIS following stroke, I used fast (500 Hz) sodium imaging in layer 5 pyramidal neurons to record sodium influx during APs.
In a representative control neuron, sodium transients (averaged, n = 40 trials) were elicited by action potentials provoked by brief somatic supra-threshold current step. Measuring ΔF transients which are proportional to Na+ flux (Fleidervish et al., 2010), reveals two types of axonal sodium signals (yellow arrows, Fig. 5a): in the proximal axon signals had a fast rise time (10–90% time c.a. 1 ms); at the more distal locations the fast rising signals were absent. Fast rising signals are consistent with local sodium influx through a membrane directly exposed to the extracellular space, while the slow rising transients in presumably myelinated sections of the
- 116 - axon reflect sodium diffusion from near-by areas. Measuring the peak amplitude of the ΔF transients along the axonal axis reveals a central plateau 18 ± 2 m long, located 8 ± 2 m from the cell body, followed by a decline in the signal (Fig. 5b).
Unfortunately, due to technical difficulties I did not gather enough Na+ imaging data to answer the question of whether the pattern of Na+ fluxes in the AIS of stroke animals differs from that in controls. From the data gathered thus far, however, it seems likely that the Na+ signals in neurons located near the infarction zone do not consistently differ from those recorded in the contralateral intact hemisphere.
Figure 5: Sodium influx in the axon initial segment. (a) Baseline fluorescence of an SBFI filled layer 5 pyramidal neuron (top). F represents sodium transients at locations in the soma and proximal axon (middle). Baseline fluorescence along axonal axis (bottom). (b) Peak F and F/F values along axis of axon.
Spike initiation and hotspot location In order to better understand the relationship between sodium entry hotspot location and spike initiation dynamics, we used a simplified, multi-compartmental computer model simulating the somatic, dendritic and axonal processes. We inserted a 15 m long segment with simulated sodium channels at distances between 5 and 120 m from the soma (Fig. 6a, top) and recorded voltage in axon in response to somatic current steps (Fig. 6a). With the sodium hotspot at a distance of 25 m from the soma, AP current threshold was lower by 200 pA than with the
- 117 - hotspot adjacent to the soma. Systematically varying the hotspot location revealed an optimal location with a decreased current threshold for AP initiation when the hotspot was c.a. 57 m from the soma (Fig. 6a, left). Furthermore, increasing sodium channel activation velocity
(modeled by decreasing activation τm) decreased the current threshold but did not change the hotspots‘ optimal location (Fig. 6a).
Figure 6: Location of the sodium influx hotspot influences spike properties. (a) Left, schematic diagram of a multi-compartmental model neuron with sodium channel hotspot (in red) showing action potentials evoked by somatic current steps with the sodium influx hotspot located 5 m or 25 m from the soma. Right, AP current threshold (defined as dV/dt2> 500 V∙s-2) at varying hotspot locations with different axonal sodium channel opening kinetics (τm scaling factors, k, see methods). (b) Current threshold for AP initiation at different hotspot locations with axon diameters of 0.5, 1.2, and 2.5 m. (c) Current threshold for AP initiation at different hotspot locations with axoplasmic resistances (Ra) of 25, 125, and 250 Ω∙cm.
- 118 - Discussion In this study, I examined the axon initial segment following stroke for changes that might underlie post-lesion neurological deficits and epileptogenesis. In our previously established epileptogenesis models using BBB disruption, albumin, and TGF-β1 we found a decrease in sodium channels and channel-targeting related genes. As these findings correlate with studies in animal stroke models, I established an in vivo cerebral stroke model in mice, using a photosensitive dye in order to examine the axon initial segment for changes in channel density. Electrophysiological recordings of action potentials in layer 5 neurons from the peri-infarct zone revealed an intact axonal component. In an attempt to more directly observe the fine structure of the initial segment Na+ fluxes, we explored the spatial and temporal pattern of the Na+ influx into the AIS elicited by a single AP using a combination of patch-in-slice recording and high-speed fluorescence imaging of the Na+-sensitive indicator SBFI. In neurons from control animals, we found Na+ influx to be maximal in a region 18 ± 2 µm in length, which was located at a distance of 8 ± 2 µm from the soma and 27 ± 4 µm from the edge of the myelin.
The observation that most sodium influx occurred at some distance from the soma prompted us to explore the physiological consequences of alterations in the location of the influx hotspot on neuronal excitability. Computer simulations indicated that for maximal excitability with the minimum number of channels there is an optimal location for the hotspot, always at some distance from the soma.
One of the key findings in our microarray results is the downregulation of SCN1A (Nav1.1). The SCN1A isoform is fairly specific to parvalbumin+ neurons, which are usually GABAergic interneurons with a chandelier or basket cells. Since we recorded only from morphologically identified pyramidal cells, changes in such cells may have been missed. Reduction in SCN1A has actually been observed in other models of epilepsy (Yu et al., 2006; Ogiwara et al., 2007; Martin et al., 2010). Examination of the AP shape and sodium channel distribution in interneurons in the peri-infarct areas would be a natural next step in this study.
Studies in in vitro expression systems revealed that AnkG negatively shifts the activation voltages of sodium channels (Shirahata et al., 2006), which could mean a positive shift with its
- 119 - disappearance following stroke. We did not observe such a change, which again, could be due to intact AIS.
Our results action potential analysis revealed an intact axonal component, which seemingly contradicts results of a study by Schafer et al. that detected a decrease in axonal channel immunostaining. This disagreement may result from the use of different stroke models – we used the non-invasive Rose-Bengal stroke model, while in the Schafer study the middle cerebral artery occlusion model (MCAo) was used. MCAo results in more extensive damage, and is also compounded with the effects of reperfusion. Another possible caveat is the use of immunostaining for Nav channels. As Nav channel immunostaining is notoriously non- reproducible and technically difficult, changes in the tissue following MCAo such as the inability to perfuse and fixate the tissue properly could have unsettled the staining procedure.
Another possibility is that the AIS component can also arise from more distal axonal components, which remain intact following stroke. Some studies advocate for AP initiation at the node of Ranvier (Colbert & Johnston, 1996; Clark et al., 2005), with a very small electrical distance separating the node and the AIS; somatic recordings are not useful in to rule out such changes. An alternative hypothesis is that with the loss of ankyrinG, sodium channels are re- distributed along the neuronal membrane, thereby retaining their kinetics, perhaps still generating the first component of the AP. Sodium imaging experiments should be able to better resolve the spatial distribution of AIS channels.
In this study, we explored the spatiotemporal properties of sodium influx during APs. In order to estimate sodium channel density with maximal spatial resolution, sodium signals were measured in response to a single action potential. This allowed us to examine the sodium signal within 2 ms of the peak of the spike, thus reducing the time allowed for sodium diffusion within the axon and allowing better spatial resolution. Using a train of APs would have elicited larger sodium influxes thus increasing signal-to-noise ratio, but could also distort the spatial distribution of our signal as several other factors would have to be accounted for, such as difference in sodium channel inactivation kinetics and increased diffusion time.
Deducing channel densities from changes in SBFI fluorescence is not straight forward. One obvious confounder would be the non-homogeneous dispersion of SBFI within the axonal
- 120 - compartment or changes in axonal fluorescence intensity due to its tortuous pathway, coming in and out of focus. We ruled out these confounder by observing that baseline fluorescence remains constant (Fig. 3c) along the axis of the axon (see also (Fleidervish et al., 2010)).
Another possible pitfall is changes in the shape of membrane AP within the AIS. Some studies indicate gradual changes to the spike shape in the AIS (Rudy & McBain, 2001; Vervaeke et al., 2006; Yue & Yaari, 2006; Kole et al., 2007; Shah et al., 2008), which, in turn, yield different patterns in sodium influx. An alternative explanation for our observed decrease in sodium influx in the distal AIS and the increasing density of Kv channels along the axon: Assuming most of the sodium influx reflects a component of the repolarization due to slow inactivation of channels, the increases in Kv channels would cause narrowing of the spike and less sodium influx.
Computer modeling revealed that neuronal excitability can be modulated by the relationship between the action potential initiation site and the interplay between synaptic potential attenuation and the capacitative sink of the soma. Simulations predict an optimal spot for sodium channels. This is in agreement with a study in the auditory system, showing that both threshold and spike timing depend on initiation site (Kuba et al., 2006).
Taken together, our data do not reveal changes in sodium channel distribution in the axon initial segment of layer 5 pyramidal neurons following stroke.
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- 126 - 4. Discussion and Conclusions
This thesis was based on previous studies from our laboratory indicating that vascular pathology and more specifically, dysfunction of the BBB is the initial event triggering the epileptogenic process thru extravasation of albumin into the brain parenchyma.
The key findings of my thesis include: 1. The discovery of a novel mechanism involved in epileptogenesis: TGF-βpathway signaling; 2. Establishing BBB disruption as a final common pathway for diverse cortical insults; 3. for the first time we described in detail the role of astrocytes in epileptogenesis, specifically, we described the physiological implications of "glial activation" which is accompanied by dysregulation of potassium and glutamate homeostasis; 4. No changes were observed in the distribution of sodium channels in the axon initial segment during the first 24 h following stroke; 5. First description of the spatial distribution of sodium channels in the axon initial segment.
Figure 4.1: Pathogenesis of BBB disruption mediated epileptogenesis.
- 127 - I believe that the use of multiple approaches and a wide variety of experimental methodologies – including gene expression, immunostaining, electrophysiological recordings and functional imaging in the same animal model and act to strengthen the overall conclusions of my thesis.
As with other pathophysiological studies, the study of epilepsy is held back by the lack of good animal models. Much of the epilepsy research done today is with status epilepticus based animal models, which inherently include massive and fairly diffuse cortical and subcortical damage (Sankar et al., 1998; Loscher, 2002). I believe that one of the strong points of my study is the use of a non-status epilepticus initiated, minimally invasive epilepsy model for the study of epileptogenesis. Another strong point is the examination of mechanisms and alterations during the earliest time points following insult. Most of my study has been conducted in the 24 h following the insult, probably limiting changes incurred by seizures themselves on the results. There is also a downside for the examination of such early time points, as acute changes may be transient in nature and not represent the actual epileptogenic changes.
My study confirms and strengthens a growing body of recent studies (Cornford & Oldendorf, 1986; Friedman et al., 1996; Tomkins et al., 2001; Seiffert et al., 2004; Ivens et al., 2007; Tomkins et al., 2007; van Vliet et al., 2007) indicating a key role for vascular pathology in epileptogenesis. Furthermore, it supports a more holistic approach towards the study neuropathology in general, and epilepsy in particular. Arguing these should not be investigated (and treated) solely as neuronal diseases but rather that neuronal dysfunction should rather be viewed as an endpoint or "symptom" of a preliminary change.
The period of epileptogenesis can perhaps serve also as a window of opportunity for the prevention of post insult epilepsy. Our current understanding of the disease has not led to the development of any clinically proven intervention able to alter the course of epileptogenesis. Perhaps thru the understanding of non-neuronal mechanisms involved in the development of the disease or thru we will be able to develop novel therapeutic tools.
No less important is being able to narrow down patient inclusion criteria for preliminary clinical studies for the evaluation of new drugs. Intelligent, data driven detection methods which will be able to predict which patients will benefit from astrocyte-protective, BBB repairing
- 128 - treatments can perhaps be achieved using new magnetic resonance imaging or even magnetic resonance spectroscopy, by detecting glutamate or potassium kinetics in-vivo. Deeper understanding of BBB kinetics can also dictate drug delivery regimens, enabling better control side effects of drugs.
An exciting discovery was that of the novel pathway for the development of epilepsy, the TGF-β pathway. Intervening with the activation of the TGF-β pathway following BBB disruption is a yet unforeseen approach for the prevention of epilepsy and could be a new drug target.
On a more general note, I it is perhaps interesting to note that in the many years of epilepsy research, many different mechanisms have been implicated in the pathogenesis of epilepsy, spanning almost every neuronal and glial target known to science. How could it be that so many different changes are capable of causing epilepsy? I believe that this can perhaps be explained by assuming the brain has an inherent, basic propensity towards rhythmic activity, which, in the normal brain is kept in check by tightly control mechanisms. In the process of epileptogenesis, any perturbation to the network that can alter the very delicate balance normally working to stop synchronization, can lead the brain to revert to the more basic state of synchronized activity. The study of epilepsy used to deal with the balance of excitation vs. inhibition, until recent years where it was determined that inhibition can also serve as synchronizing agent in the brain. Epileptogenesis is now discussed more in terms of synchronization and dis-synchronization. Perhaps thru recent advances in functional cellular level circuit mapping we could achieve better understanding at the mechanisms governing synchronization of both normal and ‗epileptic‘ circuits.
Another curios observation is the apparent increase in life time risk for epilepsy following brain insults. What can account for such a long lasting and perhaps ongoing changes in the brain? One can perhaps regard this finding as a clue to the dynamic nature of the brain. A possible explanation would involve the assumption of two separate mechanisms, one is the subtle, sub-threshold increase in the epileptogenic capacity of the injured brain and the other is the age dependent decrease in ‗seizure threshold‘.
- 129 - In conclusion, it is my belief that the insights gained by my work could lead to new means to diagnosis modalities to predict patients at risk and facilitate new approaches for the prevention and treatment of epilepsy.
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Watson, B.D., Dietrich, W.D., Busto, R., Wachtel, M.S. & Ginsberg, M.D. (1985) Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol, 17, 497-504.
Wimmer, V.C., Reid, C.A., Mitchell, S., Richards, K.L., Scaf, B.B., Leaw, B.T., Hill, E.L., Royeck, M., Horstmann, M.T., Cromer, B.A., Davies, P.J., Xu, R., Lerche, H., Berkovic, S.F., Beck, H. & Petrou, S. (2010) Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus. J Clin Invest, 120, 2661-2671.
Yu, F.H., Mantegazza, M., Westenbroek, R.E., Robbins, C.A., Kalume, F., Burton, K.A., Spain, W.J., McKnight, G.S., Scheuer, T. & Catterall, W.A. (2006) Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci, 9, 1142-1149.
Yue, C. & Yaari, Y. (2006) Axo-somatic and apical dendritic Kv7/M channels differentially regulate the intrinsic excitability of adult rat CA1 pyramidal cells. J Neurophysiol, 95, 3480-3495.
Zonta, B., Tait, S., Melrose, S., Anderson, H., Harroch, S., Higginson, J., Sherman, D.L. & Brophy, P.J. (2008) Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system. J Cell Biol, 181, 1169-1177.
- 139 - 6. List of Publications
1. YARON DAVID, Ilya Fleidervish and Alon Friedman. The axon initial segment in stroke. In preparation.
2. YARON DAVID& Ilya Fleidervish. 2011. Inhomegenous sodium influx in the axon initial segment. In preparation
3. Levy Nitzan, Yaron David, Itai Weissberg, Alon Friedman and Alon Monsengo. The role of TGF-β signaling in the glial response: Critical role for IL-6. In preparation
4. YARON DAVID, Karl Schoknecht (equal contributor) and Alon Friedman. 2011. Blood- Brain Barrier Dysfunction: A Target for the Prevention of Stroke Complications?Lancet Neurology, submitted.
5. YARON DAVID, Luisa P Flores, Sebastian Ivens, Uwe Heinemann, Daniela Kaufer and Alon Friedman. 2009. Astrocytic dysfunction in epileptogenesis: consequences of altered potassium and glutamate buffering? Journal of Neuroscience, 29(34):10588-10599.
6. Luisa P Cacheaux, Sebastian Ivens, YARON DAVID, Alexander J Lakhter, Guy Bar- Klein, Michael Shapira, Uwe Heinemann, Alon Friedman and Daniela Kaufer, 2009. Transcriptome profiling reveals TGF-β signaling involvement in epileptogenesis.Journal of Neuroscience, 29(28): 8927-8935.
7. Browne R.O., Ben Moyal-Segal L., Zumsteg D., DAVID YARON, Kofman O., Berger A., Soreq H., and Friedman A., Coding region paraoxonase polymorphisms dictate accentuated neuronal reactions in chronic, sub-threshold pesticide exposure.FASEB Journal. 20: 1733-1735
- 140 - המודל הצביע על כך שפגיעה בפינוי גלוטמאט תגרום לירידה בפעילות עצבית בקצבי הירי הגבוהים מ100- הרץ. בניגוד לכך, הפרעה בפינוי אשלגן הובילה להגדלת התגובה העצבית התלויה בקולטנים לגלוטמט מסוג NMDA והתרחשה בקצבי ירי של 01-01 הרץ. רישומים חשמליים בפרוסות מח איששו כי מתקבלת פעילות דמוית אפילפסיה בתדרי גירוי סביב 01 הרץ. מכך הסקנו הירידה בפינוי האשלגן החוץ תאי הינה ככל הנראה הגורם המכריע המוביל לשינויים בפעילות הרשת אשר מובילה לסינכרון היתר.
כנאמר, פריצה של מד"מ הינו ממצא נפוץ במח האיסכמי. על מנת לחקור את מעורבותו של מד"מ בגרימת סיבוכים הקשורים לשבץ איסכמי הקמנו מודל חולדות בו יצרנו מוקד איסכמי כשסביבו מתפתח איזור נרחב בו קיימת פגיעה במד"מ. בנוסף, השווינו שינויים בביטוי של אלפי גנים ממוחות של חולדות לאחר שבץ ולאחר פריצת מד"מ וביצענו רישומים חשמליים מהאזור הסמוך לאזור האיסכמי. אכן, מצאנו כי רבים מהשינויים הנצפים לאחר שבץ מתרחשים גם לאחר פריצת מד"מ. כמו כן, בדומה לנצפה לאחר פריצת מד"מ, פעילות רשת מסונכרנת הופיעה מספר ימים לאחר השבץ. בהתאם לשינויים בביטוי הגנים ועל סמך הרישומים החשמליים, אנו מציעים כי פריצה של מד"מ הינה גורם חשוב האחראי על סיבוכי שבץ ידועים כגון דימום תוך מוחי, התפתחות מחלת האפילפסיה וירידה בתפקוד הקוגניטיבי והנוירולוגי.
בפרק הרביעי של עבודתי בחנתי מהם השינויים המתרחשים בחלק הראשון של האקסון לאחר שבץ. ההיפותזה המנחה היתה קיומן של ראיות לשינויים בפיזור תעלות נתרן בחלק הראשון של האקסון – אותו חלק בו מתעורר לראשונה פוטנציאל הפעולה בתאי עצב. לצורך כך הקמתי מודל של שבץ בעכברים וביצעתי רישומים חשמליים מתאים בשכבה 0 של הקורטקס באזור שליד הפגיעה האיסכמית ובאונה הנגדית. רישומים של פוטנציאלי פעולה הצביעו על כך שחלקו של פוטנציאל הפעולה המתווך ע"י תעלות הנמצאות באקסון נותר על כנו כמו גם רמת העוררות של תאי העצב שנותרה דומה לרמתה באונה הלא מטופלת. לצורך בחינה מדויקת יותר של פיזור תעלות הנתרן, מיפיתי שינויים בריכוז הנתרן התוך תאי באמצעות דימות מהיר )500 הרץ( המשולב ברישום חשמלי מתא בודד של השינויים בנתרן הנגרמים עקב פוטנציאל פעולה יחיד. מצאתי כי כניסת הנתרן הינה מקסימלית באזור המרוחק במעט מגוף התא; באמצעות מודל ממוחשב בחנתי את חשיבותו של האזור הקרוב לגוף התא ואינו כולל תעלות נתרן לרמת העוררות של תאי העצב.
לסיכום, בעבודת המחקר שלי חקרתי שינויים המתרחשים מוקדם בתהליך התפתחות מחלת האפילפסיה. גיליתי כי בין תהליכים אלו בלטו שינויים באסטרוציטים שהחשובים בהם הינם הפעלה של מסלול TGF-β וירידה ביכולת פינויים את האשלגן החוץ תאי שהובהרו כהכרחיים להתפתחות מחלת האפילפסיה בעקבות פגיעה מוחית.
אני תקווה שהגילוים אותם השגתי במהלך עבודת המחקר שלי יאפשרו פיתוח של כלים חדשים לזיהוי של חולים בסיכון לפיתוח אפילפסיה ויתרמו לפיתוחם של כלים חדשים למניעה וטיפול במחלה.
מילות מפתח: מחסום דם-מח, אפילפסיה, אסטרוציטים, רישומים תוך תאיים, אשלגן, גלוטמט, האקסון הקריבני, דימות נתרן, TGF-β
- 141 - 7. תקציר מחלת האפילפסיה )הנקראת גם מחלת הכיפיון( היא מהשכיחות מבין מחלות מערכת העצבים המרכזית ומתאפיינת בהופעה התקפית של פרכוסים חוזרים. למרות הבנתנו ההולכת וגדלה את מחלת האפילפסיה, היא נותרה מחלה חשוכת מרפא הניתנת לשליטה באופן חלקי בלבד.
אפילפסיה מתפתחת לעתים עקב פגיעות מוחיות, למשל, פגיעה חבלתית, פגיעה באספקת הדם, זיהומים וגידולים. גם כיום, המנגנונים המדויקים העומדים בבסיס התהליך במהלכו המוח הבריא הופך לחולה נותרו עלומים ואין בידינו אמצעים למניעת התפתחות המחלה. מעניין לציין כי ברוב הפגיעות המובילות להתפתחות המחלה מתוארות גם פגיעות בכלי הדם ובמיוחד פגיעה בתפקוד מחסום דם-מוח )מד"מ(. מד"מ הינו מחסום מבני ותפקודי המאפשר למערכת העצבים המרכזית לשמור את הסביבה החוץ תאיתתחת פיקוח הדוק באמצעות מניעת כניסתם של מרכיבי דם שונים אל תוך רקמת המוח והוצאתם של מרכיבים אחרים.מחקרים בחיות שבוצעו במעבדתנו הראו כי הפרעה בתפקוד מד"מ מובילה באופן ישיר להופעת פעילות מוחית מסונכרנת ביתר, דמוית זו הנרשמת במחלת האפילפסיה.יתר על כן, הוכח כי השינוי בפעילות המוחית נגרם עקב חדירה אל תוך רקמת המח של חלבון הדם הנפוץ, אלבומין.
מטרתה העיקרית של עבודת המחקר שלי היתה להבין את המנגנונים העומדים בבסיס התפתחות מחלת האפליפסיה לאחרי פריצת מחסום דם-מח.
במסגרת עבודתי, מצאתי כי הפעלה ישירה של מסלול TGF-β ע"י TGF-β1 מובילה להתפתחות פעילות דמוית אפילפסיה, בדומה לפעילות שהופיעה לאחר חשיפה לאלבומין. יתר על-כן, מצאנו שאלבומין נקשר ישירות לקולטן ל-TGF-β ומפעיל אותו. ניתוח שינויים בביטוי כלל הגנים )באמצעות gene arrays( הצביע על שינויי שעתוק דומים הן עקב פריצת מחסום דם-מח והן עקב חשיפת המח לאלבומין או לTGF-β1-. קבוצות גנים שהראו שינויי ביטוי כללו בין השאר גנים השייכים למסלול TGF-β, גנים המזוהים עם הפעלה של אסטרוציטים )תאים המהווים חלק ניכר ממערכת העצבים המרכזית( ותהליכי דלקת. כמו כן נצפתה ירידה בביטויים של גנים המקודדים למעבירים בין-עצביים. בנוסף, הראתי כי חסימה של מסלול TGF-β מנעה את התפתחות הפעילות המסונכרנת, דמויית אפילפסיה לאחר חשיפת קליפת המח לאלבומין אוTGF- β1.
אחד הארועים הראשונים שנצפו עם פריצת מד"מ, הינו עליה ברמת ביטוי של חלבון ספציפי לאסטרוציטים הנקרא GFAP. במסגרת עבודתי מצאתי כי לעליה ברמת חלבון זה מתלווים שינויים ברמות ביטוי של גנים נוספים הידועים כיחודיים לאסטרוציטים, בינהם גנים האחראים על וויסות רמות האשלגן החוץ תאי והמתווך העצבי גלוטמט. על מנת להבין את השינויים הפיזיולוגיים המתרחשים בעקבות השינוי בביטוי הגנים, רשמתי זרמים חשמליים בתאי עצב הנובעים משיחרור גלוטמט ואכן מצאתי כי חלה ירידה ביכולת הפינוי של גלוטמט ע"י אסטרוציטים בחיות שנחשפו לאלבומין תוך מוחי. כמו כן, רישומים באמצעות אלקטרודות הרגישות לאשלגן הראו כי קיימת ירידה בפינוי אשלגן מהמרווח הבין תאי. על מנת להבין את משמעותם של שינויים אלו, דימיתי באמצעות מודל ממוחשב של תא עצב ואסטרוציט שינויים בפינוי גלוטמט ואשלגן.
- 142 - מנגנונים של תאי עצב ותאי גליה העומדים בבסיס מחלת האפילפסיה לאחר פריצת מחסום דם-מח
מחקר לשם מילוי חלקי של הדרישות לקראת תואר דוקטור לפילוסופיה
ע"י
דוד ירון
מוגש לסנאט של אוניברסיטת בן גוריון
בנגב
אישור המנחה______
אישור דיקן בית הספר ללימודי מחקר מתקדמים ע"ש קרייטמן ______
חשוון, ה'תשע"ב נובמבר 3122
באר-שבע
מנגנונים של תאי עצב ותאי גליה העומדים בבסיס מחלת האפילפסיה לאחר פריצת מחסום דם- מח
מחקר לשם מילוי חלקי של הדרישות לקראת תואר דוקטור לפילוסופיה
ע"י
דוד ירון
מוגש לסנאט של אוניברסיטת בן גוריון
בנגב
חשוון, ה'תשע"ב נובמבר 3122
באר-שבע