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Neuroprotective Strategies in Animal and in Vitro Models of Neuronal Damage: Ischemia and Stroke

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Cell death following cerebral ischemia is mediated by a complex pathophysiologic interaction of different mechanisms. In this Chapter we will outline the basic principles as well as introduce in vitro and in vivo models of cerebral ischemia. Mechanistically, excitotoxicity, peri-infarct depolarization, inflammation and apoptosis seem to be the most relevant mediators of damage and are promising targets for neuroprotective strategies.

Epidemiological Data

The incidence of stroke in the Federal Republic of Germany (a population of 81 million people) is approximately 250-400/100,000. Hence, an estimated total of 200,000 strokes occur per year. Stroke mortality amounts—irrespective of all therapeutic efforts—still are 25 to 30%. Stroke is the third leading cause of death in the industrialized world. For example, in Germany there are two million stroke victims alive (for comparison, four million in the United States of America). Stroke is the leading cause of disability; its direct and indirect costs amount to approximately 30 to 40 billion US$ per year.

Introduction

The metabolism of the brain depends exclusively on oxygen and glucose and adds up to the consumption of 75 l molecular oxygen and 120 g glucose per day. In comparison to other organs this situation is unique. Albeit the weight of the brain accounts for only 2% of the total body weight, the brain claims 20% of total body perfusion and 20% of total oxygen consumption. Even short durations of reduced brain perfusion leading to lack of oxygen and energy metabolites may lead to irreversible structural damage. Variable “ischemia thresholds” that are region- and cell type-specific determine whether a respective cell will survive the insult or die (see also Table 1).

Table 1. Perfusion thresholds during cerebral ischemia.

Table 1

Perfusion thresholds during cerebral ischemia.

Global vs. Focal Ischemia

In general, there are two mechanistically distinct modes of cerebral ischemia, i.e., global and focal ischemia, respectively. Global ischemia in man develops after transient circulatory arrest with resuscitation or after near-drowning (Table 2). Consequently, absolute cerebral blood flow falls off from 0.8 ml/g/min to zero within seconds. Loss of consciousness follows after approximately 10 seconds. EEG activity ceases after 30 to 40 secs, and a few minutes of global ischemia lead to irreversible cellular damage that evolves over days. The typical histological picture following global ischemic insults is described by delayed neuronal death sparing glial cells (sometimes even associated with astrogliosis). As a general rule, under normothermic conditions, 10 min of global ischemia are lethal in man. In the United States approximately 500,000 people/year die because of circulatory arrest leading to global ischemia.

Focal ischemia follows transient or permanent flow reduction in the territory of a cerebral artery. Typically, flow reduction is due to embolic or thrombotic vessel occlusion. In contrast to the situation after global ischemia, focal ischemia is characterized by the formation of a so-called “ischemic penumbra”. The penumbra is defined as the ischemia border-zone which is (still) metabolically active but functionally silent. While absolute regional blood flow in the ischemic core is diminished to levels <0.1 ml/g/min, blood flow in the penumbra typically remains at 0.2-0.4 ml/g/min. The typical histological picture following focal ischemia is a pan-necrosis that includes all cell types in the brain (neurons, astrocytes, oligodendrocytes, endothelial cells).

Animal Models of Cerebral Ischemia

Although stroke has been studied in many species (for example rabbits, dogs, cats, and baboons), rats and mice are the most widely investigated.1 Mice are especially useful because of the availability of unique strains that can be genetically engineered to over- or underexpress targeted genes.2-4 Several well-established models are available to study global ischemia. In the so-called “four-vessel occlusion model” (4VO), flow in both carotid arteries and vertebral arteries is blocked for a specified time period (“Pulsinelli-Brierley”-model).6 In the two-vessel occlusion model (2VO) which is also referred to as “severe forebrain ischemia”, only the carotid arteries are temporarily occluded, sometimes along with mild hypotension.7 In these models, injury develops selectively in cells most vulnerable to ischemic damage such as in the CA1 sector in hippocampus, medium-sized neurons in the striatum, and Purkinje cells in cerebellum. Neurons are more susceptible than glial cells, and die over hours to days after the insult; hence the term “delayed neuronal death”. Experimental focal ischemia is most commonly studied during permanent or transient occlusion of a middle cerebral artery (MCA).8 - 10 Proximal MCA occlusion can be induced by an intraluminal suture (so-called filament model) or with a vascular clip and causes injury to cortex and deep structures (striatum). Distal MCA occlusion (the so-called “Brint-model”) is usually produced by placing a vascular clip on a pial vessel or by cautery.11 The occlusion typically spares striatum and primarily involves the neocortex. Pan-necrosis develops in the territory supplied by the respective artery with glial and endothelial cell death. If recirculation is established early (2 hrs or less) outcome is better (transient MCA occlusion).12 In some ways, the reperfused brain imitates restoration of blood flow after spontaneous lysis of a thrombo-embolic clot in humans, even though reperfusion after clot lysis is certainly more complex than an on/off phenomenon as modelled by placement and retraction of an intravascular filament. During reperfusion, free radical production and NO generation are especially pronounced and contribute to “reperfusion injury”.13 After longer times of ischemia, reperfusion is incomplete due to microvascular occlusion which has been termed the “no-reflow phenomenon” some thirty years ago.14 Because oxygen-free radicals and NO promote apoptotic cell death, transient ischemia models have become especially useful to investigate cell death in vivo15,16 which may particularly apply for models of “mild ischemia”.17-19 In these models apoptosis is prominent after 30 min MCA occlusion followed by longer reperfusion times (several days).19,20 The pattern of cell death is reminiscent of global ischemia in that it is both selective for neurons and delayed. Mild ischemia models may be similar to transient ischemic attacks in man. In fact, changes in T1/T2-weighted MR imaging 7 days after 15 min MCAo occlusion in rats resemble those 7 to 10 days after transient ischemic attacks (TIA's) in patients with known cardiogenic embolism. However, selective neuronal death has not yet been convincingly documented following TIA's in humans.

In Vitro Models of Cerebral Ischemia

To study the effects of “cerebral ischemia” in post-mitotic neurons in vitro, the so-called “oxygen-glucose deprivation” (OGD) model is commonly used. Primary cultures of post-mitotic neurons from different regions of the brain (such as cortex, striatum, septum, hippocampus, etc.) can be established from rat or mouse embryos (day 16 to 18). After several days in vitro (10 to 14 days) these post-mitotic cells can be exposed to a combined deprivation of oxygen and glucose. Depending on the length and severity of the insult cell death develops and can be quantified on a morphological, biochemical, or molecular basis.21,22 Other possibilities to study ischemic damage in vitro include the studies of brain slices, particularly the hippocampal slice. Usually, the bathing solution is changed from a mixture of oxygen/carbon dioxide to nitrogen/carbon dioxide in the absence (hence “ischemia”) or presence (hence “anoxia”) of glucose. Generally, 5-7 min of ischemia lead to profound cell loss in the CA1 region. Shorter insults lead to a more slowly evolving damage, requiring approximately 12 h to be manifested. This type of cell death in many ways resembles “delayed neuronal death” seen in vivo.

Importance of Physiologic Parameters for Stroke Outcome

Both animal and clinical studies have proven that changes in physiologic parameters early after stroke onset influence ischemia outcome. Of great importance are changes in brain and body temperature, partial pressures of oxygen and carbon dioxide, mean arterial blood pressure and glucose metabolism which may have direct impact on the treatment of stroke patients.

Temperature: Fever is a frequent complication following stroke, and increases in body and brain temperature are associated with poor stroke outcome. Experimental studies unequivocally demonstrate that hypothermia reduces lesion volume while hyperthermia increases cell death following global ischemia (Table 3) and lesion volume after focal ischemia. Reducing temperature to 30°C for 1 h after 2 h transient occlusion of the middle cerebral artery reduced lesion volume by 50%. This has direct implications for the treatment of acute stroke victims. Even slight elevations of body temperature (>37.5°C) should be normalized by physical and pharmacological means. Current studies investigate acute treatment protocols using hypothermia.

Table 3. Effects of temperature on damage following global cerebral ischemia.

Table 3

Effects of temperature on damage following global cerebral ischemia.

Oxygen and carbon dioxide: Both hypoxia as well as hypercapnia have an adverse effect on stroke outcome in animal models of cerebral ischemia. Theoretically, hypercapnia has advantageous effects by inducing vasodilation. Perfusion in the penumbra, however, is decreased during hypercapnia possibly due to a “steal” phenomenon.

Mean arterial blood pressure: Cerebral autoregulation is severely impeded during cerebral ischemia. Hence, brain perfusion is directly dependent on mean arterial blood pressure. Recent experimental studies have indeed demonstrated that blood pressure lowering increases infarct size in spontaneously hypertensive rats.23 As a consequence, blood pressure in patients should be maintained at relatively high levels to improve brain perfusion and thus outcome.

Glucose: Albeit loss of glucose is one of the initiators of cell death after cerebral ischemia, elevated levels of blood glucose are nevertheless deleterious following focal ischemia. Accumulation of lactate by anaerobic glycolysis leads to acidosis with adverse effects on ischemic tissue.

Pathophysiological Cascades Following Cerebral Ischemia

Loss of oxygen and energy depletion initiates a self-promoting cascade of pathophysiologic events that evolve over minutes, hours, days, and even weeks. In this Chapter we will follow a putative sequence of events that can be differentiated in (1) excitotoxicity, (2) peri-infarct depolarization, (3) inflammation, and (4) apoptosis. Obviously, this is an oversimplification and both mechanistically and in terms of temporal evolution there is substantial overlap.24

Excitotoxicity and Energy Depletion

As mentioned above brain tissue has an high energy demand. Moreover, to generate energy metabolites the brain is exclusively dependent on oxidative phosphorylation. Following focal ischemia there is profound loss of both oxygen and glucose. Within minutes energy-dependent ion channels become activated which leads to the loss of the membrane potential.25 Both neuronal and non-neuronal cells become depolarized and voltage-dependent Ca-channels are activated. Depolarization also induces release of neurotransmitters such as excitatory amino acids (glutamate) from presynaptic axon terminals into the synaptic cleft.26 Since re-uptake mechanisms have failed, these excitatory amino acids subsequently accumulate in the synaptic cleft and induce activation of ligand-gated Ca-permeable channels (such as of the NMDA and AMPA subtype) as well as metabotropic Ca-channels.

Subsequently, there is influx of other extracellular ions (Na, Cl) that accumulate intracellulary and lead to cell swelling and intracellular (“cytotoxic”) edema. This intracellular edema can be visualized by magnetic resonance tomography using diffusion weighted imaging (DWI) techniques, providing the most sensitive non-invasive modality for detecting ischemia-induced tissue injury. Using perfusion weighted imaging (PWI), it is possible to compare regions of compromised blood flow (PWI) with already lesioned tissue (DWI). The difference between both volumes (“mismatch”) might correspond to salvageable “penumbra” tissue. The latter hypothesis is currently being tested in a number of clinical trials.

The most important trigger for all subsequent events leading to cellular disruption and cell death is the intracellular increase of calcium ions: Ca acts as a universal second and third messenger and triggers via enzyme induction multiple cytoplasmic and nuclear cascades: Ca activates a number of important enzymes: (1) proteolytic enzymes (such as calpain, gelsolin etc.) with subsequent degradation of the cytoskeleton and structural proteins (e.g., actin, laminin, spectrin, microtubuli-associated proteins);27-29 (2) xanthine oxidase and phospholipases (such as phospholipase A2) which leads to membrane degradation, (3) cyclooxygenase leading to free radical generation, (4) neuronal type nitric oxide synthase (nNOS) leading to the production of nitric oxide (NO) and NO-derived radical peroxynitrite by the reaction with superoxide.30,31

Nitric oxide plays a critical role during cerebral ischemia. Importantly, NO may exert beneficial as well a deleterious effects depending on the time-point and compartment of its production. Hence, analysis of the role of NO during cerebral ischemia has led to much confusion in the literature and was described as a “double-edged sword”. NO is synthetized from L-arginine and oxygen by NO synthases (NOS). There are two constitutive isoforms, neuronal (type I) and endothelial (type III), which are calcium/calmodulin-dependent, and one inducible isoform (iNOS, type II). Small quanta of NO synthetized by constitutive NOS regulate a wide variety of physiological functions such as blood pressure, vascular tone, permeability and neurotransmission.32 iNOS can be induced in microglia, astrocytes, endothelium, and vascular smooth muscle. Once expressed, it is continuously active, irrespective of intracellular calcium levels and leads to high output NO synthesis leading to cytotoxicity and inflammatory actions. Like iNOS, nNOS can also generate high amounts of NO and cause cytotoxicity under pathophysiological conditions, due to the above mentioned intracellular rise of Ca.21 Although nNOS-positive neurons comprise only 1–2% of all neurons they possess extensive branching. Of note, the nNOS-positive neurons themselves are surprisingly resistant against injury. Cortical levels of NO increase strikingly from approximately 10 nM to 2 μM within a few minutes after MCA occlusion. Animals lacking nNOS expression (nNOS knockout animals) have 38% smaller cerebral infarcts following permanent MCAo than control mice, unequivocally demonstrating the deleterious role of nNOS-derived NO during cerebral ischemia. NO neurotoxicity is mediated most likely by peroxynitrite formed by the reaction of NO with superoxide, a complex that rapidly decomposes into hydroxyl radicals, which are a highly reactive species.

An attractive downstream candidate for mediating NO-induced neurotoxicity is activation of the nuclear enyzme poly(ADP-ribose)polymerase (PARP). Formation of single-stranded DNA nicks (for example by peroxynitrite) is an obligatory stimulus of PARP leading to the formation of poly-ADP-ribose chains and depletion of its intracellular substrate NAD.33,34 PARP is activated within minutes following cerebral ischemia and reperfusion35 and inhibition of PARP activation or deletion of the PARP gene confers protection after ischemia.35,36 Consistent with the notion that PARP is a perpetrator in NO-mediated neurotoxicity nNOS knockout animals had strikingly reduced levels of PARP activation within ischemic tissue.37

On the other hand, NO produced by the endothelial NOS isoform may exert protective effects via augmentation of cerebral blood flow, inhibition of platelet aggregation and leukocyte activation. Animals lacking expression of the endothelial NOS subtype (Type III) have been generated (eNOS knockout mice ).38 Expectedly, these animals have elevated arterial blood pressure. Moreover, eNOS knockout animals develop enlarged cerebral infarcts following middle cerebral artery occlusion, demonstrating a protective role of type III NOS during cerebral ischemia. The susceptibility of eNOS mutants to ischemic injury may be due to their diminished capacity to adapt to reduced perfusion pressure (i.e.dilate) at the margins of an ischemic lesion. This coupled to enhanced platelet and neutrophil adhesion, renders eNOS mutants susceptible to injury. Consistent with this notion, blocking NOS activity by nitro-L-arginine administration increased infarct size in nNOS knockout animals, presumably due to inhibition of the constitutively expressed eNOS isoform.39In conclusion, genetic evidence suggests that two NOS isoforms, i.e., nNOS and iNOS, contribute to ischemic injury, most likely due to the generation of NO-derived radicals, while NO generated by another NOS isoform, i.e., eNOS, is protective by its effect on blood flow and platelet aggregation. Pharmacologic approaches for acute treatment should be directed at selective inhibition of nNOS and iNOS isoforms while eNOS activity should be augmented. Recently, statins (HMG-CoA reductase inhibitors) were shown to selectively upregulate eNOS activity and NO production after chronic administration. Mice treated for 14 days with statins had increased eNOS message and activity, augmented cerebral blood flow and decreased markers of platelet activation, and had significantly smaller stroke sizes after MCAo.40,41

Another important source for the generation of reactive oxygen species (ROS) are mitochondria, which may themselves be damaged by ROS initiating a vicious cycle.42,43 Specifically, the inner mitochondrial membrane is severely damaged by the generation of radicals and also by formation of a so-called “mitochondrial permeability transition pore” (MPT), which leads to organ swelling, generation of additional ROS and finally cessation of ATP production. This may be coupled to the specific or unspecific release of cytochrome c into the cytosol which has been recognized as a specific upstream trigger for the initiation of the proteolytic caspase cascade leading to apoptotic cell death (Fig. 1).44

Figure 1. Putative cascade of damaging events in focal cerebral ischemia.

Figure 1

Putative cascade of damaging events in focal cerebral ischemia.

Hemodynamic, metabolic, and ionic phenomena are not homogeneous in different ischemic brain regions. In the center or core of the ischemic region, cerebral blood flow is less than 20% of normal levels. In this region, permanent and anoxic depolarizations develop minutes after onset of ischemia. Subsequently, cells are killed rapidly by lipolysis, proteolysis and disruption of the cytoskeleton leading to loss of energy and ion homeostasis.45 Between this core region and normal, non-ischemic tissue lies the so-called penumbra, an ischemic border-zone which is functionally silent but metabolically (still) active. In theory, the whole penumbra region can be salvaged by timely therapeutic intervention. Over time, however, and without treatment the “core grows on the cost of the penumbra”, i.e., the penumbra can progress to infarction by mechanisms described in this Chapter.46-48Hence, the goal of all therapeutic interventions is to protect the ischemic penumbra region. Albeit there is good evidence for the existence of a penumbra during human stroke,49,50 its size and pathophysiologic relevance remains unclear.51

Glutamate Receptors and Excitotoxicity

Activation of glutamate receptors and subsequent calcium influx leading to increased intra-cellular calcium levels ([Ca]i) may be the most important early trigger of subsequent cell death after cerebral ischemia. Consequently, pharmacological inhibition of these receptors is an attractive treatment approach for stroke. Three different ligand-gated glutamate receptors can be differentiated based on their respective pharmacological affinities:(1) N-methyl-D-aspartate (NMDA) receptor, (2) α-amino-3 hydroxy-5 methyl-4- isoxazolone-proprionic acid (AMPA) receptor, and (3) kainate receptor. In addition, there are metabotropic G-protein coupled receptors.

NMDA receptors are permeable for Ca, Na and K ions. Following NMDA-receptor activation three stages inducing neurotoxicity can be differentiated: (1) changes in the intracellular milieu (induction), (2) exponential increase in [Ca]i (amplification), (3) neuronal degeneration (expression). There are several pharmacologically distinct receptor antagonists available: NMDA receptor antagonists (such as MK-801) inhibit almost completely intracellular Ca-influx in primary neurons following “oxygen-glucose-deprivationin vitro. NMDA receptor blockage using competitive and non-competitive receptor antagonists confers robust neuroprotection in animal model of focal (but not global) cerebral ischemia. Administration of NMDA receptor antagonists is neuroprotective only within a very limited “window of opportunity” (minutes to a few hours), however. Moreover, severe adverse effects of NMDA receptor blocker were demonstrated including exogenous psychosis and specific cell loss in the limbic system.52,53 Currently, site-specific antagonists (for example for the glycine site) are being tested as a possible alternative.

AMPA receptors mediate Na and K currents. Na-influx via AMPA as well as kainate receptors leads in turn to secondary influx of Ca-ions. Pharmacological blockade of these receptors via specific inhibitors confers significant reduction of infarct volume in animal stroke models. Of note, the treatment window is somewhat better than with NMDA receptor antagonists.54 In contrast to the above listed ligand-gated glutamate receptors, the role of metabotropic glutamate receptors during cerebral ischemia has so far remained unclear. Some studies suggest a protective role for some classes of metabotropic receptors.55

Tissue Acidosis

Following oxygen deprivation glucose metabolism is changed to anaerobic glycolysis leading to lactate accumulation and tissue acidosis. For a long time acidosis was recognized as a central pathomechanism of tissue injury following stroke. For example, neurons in culture die rapidly when exposed to low pH. This has been explained by increased radical production and other mechanisms at low pH which may mediate damage. Recent observations, however, have shed new light on the dogma that acidosis is always dismal after cerebral ischemia. For example, NMDA receptors become inactivated at low pH levels protecting neurons from further Ca-influx.

Protein Synthesis and Early Gene Expression

Even slight reductions of regional cerebral blood flow compromise total protein synthesis. In spite of these inhibitory effects on general protein synthesis there are some genes and gene products that are upregulated both on a transcriptional and translational level. Among these are the so-called “immediate early genes” whose gene products act as transcription factors. Via expression of these early genes both protective and destructive cascades are initiated.

Peri-infarct Depolarization

Loss of energy and glutamate release induce depolarization of neurons and non-neuronal cells. In the core of the ischemic lesion this may induce so-called anoxic depolarization: Due to lack of energy equivalent the cells are unable to repolarize and are destined to die. In the border-zone (“penumbra”) of the lesion, however, cells are able to re-polarize, but at the expense of further energy depletion. Waves of de- and re-polarization may establish which are called peri-infarct depolarizations.56, 57 Evidence of this phenomenon has been provided in several animal models and a number of observations argue that it may have pathophysiological relevance. (1) Peri-infarct depolarizations (PIDs) may occur several times per hour and can be detected up to 6-8 hours after ischemia onset. (2) Lesion volume and cell loss correspond to the number of PIDs.58 (3) Reduction of PIDs by therapeutic intervention reduces infarct volume.59 (4) Induction of additional PIDs by topical KCl administration further increases lesion volume. Albeit experimental evidence for PIDs as a relevant pathomechanism after stroke is convincing, so far evidence for the occurrence of PIDs after stroke in man is lacking.60

Inflammation

Expression of proinflammatory genes, such as NF-κB, hypoxia-inducible factor and interferon 1b is triggered early after onset of ischemia.61-63 These proinflammatory genes may initiate the production of several mediators of inflammation including platelet activation factor and tumor necrosis factor. In turn, these factors themselves initiate a cascade of events that includes the expression of adhesion molecules of endothelial cells (intercellular and vascular adhesion molecules; ICAM and VCAM as well as different selectins). Adhesion molecules mediate rolling, adhesion (“sticking”) and finally migration of leukocytes through the vascular wall.64-66 In a first wave neutrophils invade the brain which is followed by macrophages and monocytes. The latter express typical chemokines (e.g., interleukin-8 and monocyte chemoattractant protein-1) leading to a vicious cycle.

The x-axis reflects the evolution of the cascade over time, while the y-axis illustrates the impact of each element of the cascade on final outcome (adapted from Dirnagl et al, TINS 22:391-397). However, not only blood-born cells mediate inflammatory responses after ischemia but also brain-derived immunocompetent cells such as microglia. Microglial cells constitute the primary immunoeffector cells of the brain and amount to up to 20% of total brain cell number. Within hours after onset of an insult these cells become activated microglia. The question, however, whether inflammation after cerebral ischemia is “good or bad” has not unequivocally been resolved although the literature is in favor of a destructive effect. For example, (1) induction of neutropenia, (2) inhibition of several inflammatory mediators (such as Il-1b) or (3) adhesion molecules significantly protects from cell death and improves outcome after stroke.67,68

There are a plethora of molecular and biological mechanisms that contribute to inflammation-mediated cellular damage: The cerebral microcirculation becomes severely compromised by leukocyte plugging of small vessels. Neurons and macrophages may induce toxic enzymes such as inducible NO synthase (iNOS) or cyclooxygenase (COX). iNOS is produced by invading neutrophils which may lead to increased NO production. With the use of pharmacological inhibition or genetic deletion (iNOS knockout mouse) it has been unequivocally demonstrated that iNOS exerts neurotoxic effects during cerebral ischemia. Of note, the pathomechanism of iNOS induced cytotoxicity is a delayed one: iNOS becomes expressed only 24 h after the onset of the insult and administration of iNOS inhibitors is neuroprotective even when administered one day after stroke onset.63,69,70 COX-2 on the other hand is produced by neurons. It amplifies ischemic damage by production of superoxide anions and toxic prostanoids.71 The role of yet another inflammatory mediator, TNF-alpha, during cerebral ischemia has so far remained controversial.72,73

Apoptosis

The term “apoptosis” was first introduced by Kerr et al in 1972.74 Hallmarks of apoptosis include chromatin condensation, nuclear segmentation, cytoplasmic shrinkage, blebbing, and formation of apoptotic bodies (See Table 4). The possibility that ischemic brain cells may also die by apoptotic mechanisms was not proposed until 1993.75-77 In fact, ischemic cell death was considered the classical prototype for necrotic cell death based mainly on morphological criteria. Necrosis describes swollen cells with disruption of subcellular organelles and is also called “accidental cell death”.78 (see also Table 4). Now convincing biochemical, histochemical, molecular and genetic data favor apoptosis (or a closely related mechanism) as an additional mechanism of cell death in models of cerebral ischemia. There are numerous reports demonstrating oligonucleosomal DNA fragmentation either by gel electrophoresis (“DNA laddering”) or by the TUNEL in situ technique (“terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling”).75,77,79-82 Following focal ischemia apoptosis is predominant in the ischemic border-zone.75,83,84Following more severe insults TUNEL-positive cells appear earlier than after mild insults.19 DNA laddering and TUNEL staining, however, are not pathognomonic for apoptosis. Most neuropathologists accept electron microscopy criteria as the gold standard. Van Lookeren Campagne and Gill85 found 1996 no ultrastructural evidence for apoptosis after MCA occlusion in the rat.

Table 4. Apoptosis vs. necrosis.

Table 4

Apoptosis vs. necrosis.

It is generally accepted that new protein synthesis is needed for the initiation of apoptosis.74 In fact, the protein synthesis inhibitor cycloheximide protects cells and reduces injury after focal ischemia.18,19,75 In general, cycloheximide suppresses synthesis of all proteins, including those that protect cells. Hence, is has remained elusive by inhibition of which downstream products neuroprotection of cycloheximide is mediated.

Molecular Mechanisms

During development, apoptosis is triggered by evolutionally highly conserved signals, and is regulated by a balance between death promoting and death inhibiting factors.86-89 In the nematode Caenorhabditis elegans the death promoting ced-3 and ced-4 genes and the death inhibiting gene ced-9 were identified.90-93 As mammalian homologues for the ced-3 gene the so-called caspases (c: cystein proteinase; -aspase: cleavage after aspartate residues) were identified (formerly also known as interleukin-1β family).94,95 These proteases consist of at least 12 familiy members and are important executioners of apoptosis. The most important caspase for apoptosis execution is caspase-3. Caspase-3, as all caspases, is activated by proteolytic cleavage (posttranslational modification) and in turn cleaves itself multiple (>30) substrates. Known caspase-3 substrates are for example endonuclease (CAD plus inhibitor ICAD), lamin, spectrin, huntingtin, gelsolin, poly(ADP-ribose)polymerase (PARP, see above). Cleavage of these caspase substrates is thought to mediate the downstream events during apoptosis (beyond the point of “no-return”). Until recently, it had remained unclear how caspase-3 is activated during cerebral ischemia. Generally, two pathways exist: type I apoptosis via Fas/TNF-receptors, activation of intracellular death recepors and activation of caspase-8; type II apoptosis via release of cytochrome c from mitochondria, formation of the so-called “apoptosome” (which is a complex of cytochrome c, apoptosis activation factor and pro-caspase-9).96 Hence, after their respective cleavage either caspase-8 (type I) or caspase-9 (type II) activate caspase-3. Recent data favours the idea that caspase-3 is activated during cerebral ischemia predominantly via release of cytochrome c into the cytosol.28

Experimental Evidence for Caspase-Mediated Cell Death Following Cerebral Ischemia

Both caspase-1 and -3 become activated in the ischemic territory following focal cerebral ischemia,19,97an event that precedes markers of morphological damage and TUNEL staining by hours. Animals with deletion of caspase-1 or caspase-3 have smaller ischemic lesions.98-100Following a general rule that severe insults lead to necrotic cell death while after milder insults apoptosis is prominent, caspase activation was analyzed following short durations of MCA occlusion. Following 30 min MCAo caspase-3 was not activated until 9-12 hours after ischemia onset, while after 2 h MCAo it was active within minutes after reperfusion. More than 50% of all neurons within the ischemic tissue double stain for activated caspase-3 and TUNEL.20,97

Caspase Inhibition Protects From Cerebral Ischemia

Small oligopeptides (tri- or tetrapeptides) that mimic the cleavage site of a protease substrate can be used as relatively selective caspase inhibitors.101,102For example YVAD.cmk (acetyl-Tyr-Val-Ala-Asp.chloromethylketone) is relatively selective for caspase-1 (Ki 0.76 nM) while zDEVD.fmk (N-benzyloxycarbonal.Asp-Glu-Val-Asp-fluoromethylketone) is more selective for caspase-3 (Ki <0.1 nM).103,104 Intracerebroventricular administration of these inhibitors blocks the activation of capase-1 and -3, respectively, and significantly protects from injury following middle cerebral artery occlusion and global ischemia.19,20,100,105,106Following mild insults the treatment window was extended to 9 hr after the onset of ischemia.19,20Interestingly, caspase inhibitors act synergistically with anti-excitotoxic drugs such as the glutamate receptor blocker MK-801.107 Hence, it seems feasible that both anti-excitotoxic and anti-apoptotic drugs may be given in a “neuroprotective cocktail”; low doses may limit potential toxicity from either drug alone. Of note, non-peptide caspase inhibitors are being developed that can be administered systemically.

Conclusion

Cell death following cerebral ischemia is mediated by a complex pathophysiologic interaction of different mechanisms. As outlined in this Chapter, excitotoxicity, peri-infarct depolarization, inflammation, and apoptosis seem to be the most relevant mediators and are promising targets for neuroprotective intervention. After all, however, stroke is a vascular disease. Reperfusion of an occluded artery (e.g., by rt-PA) is still the most important early intervention and should be combined with neuroprotective strategies.

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