I. INTRODUCTION

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A.  Defining Cell Death

1.  Cell death is an active process

This review is geared toward those working in the field of ischemic tissue damage and toward students and others who want to develop a critical overview of the area. The text carefully examines the evidence that certain chemical and ionic changes, protein and enzyme changes, and changes in key functions are involved in ischemic cell death. The long-term goal of this review is to make as cogent a model of ischemic cell death as is possible at present, which is done in the final section, and to highlight what is not known to excite rigorous, causally directed, experimental work. The latter is done throughout the text.

To study what causes cell death, cell death has to be recognized. However, there is no well-accepted definition of the point at which a cell dies (706, 1137). The most satisfactory definition of cell death, and the one that would be the most valuable to know therapeutically, is the point at which the cell becomes unable to recover its normal morphology and function even if all processes leading to dissoution are stopped pharmacologically (the point of no return). This would be analogous to cessation of breathing or cardiac function for death of the organism. At this stage, however, we do not know enough about the ischemic death process of a cell to be able to identify what the point is. Thus the only unequivocal definition of cell death is a morphological one, the elimination of the cell, which can arise either by cell disintegration or phagocytosis. These are both very clear end points, and they are often used in studying mechanisms of cell death. Before this there are certain metastable morphological states toward which dying cells tend that are almost certainly precursors of disintegration or phagocytosis (121, 814) and that are also commonly used as end points. In this review these are termed the end stages of the cell death process and are generally considered to represent dead cells, undergoing necrosis, autophagocytosis, or the end stages of apoptosis (Fig. 1, column 5). As defined in this way, cell death is an anatomical event that represents a state from which the cell cannot recover to even near normalcy. The state reflects major macromolecular change, and the study of cell death, at this stage, is the study of what leads to these drastic macromolecular changes.

View larger version (91K): [in this window] [in a new window]   Fig. 1. Pathways of ischemic cell death. This is an overview of processes involved in ischemic cell death that is consistent with organization of review. It illustrates major events that are hypothesized to contribute to cell death and also the extremely complex interactions between these events. This figure is not meant to be complete, but does include processes known or thought to be important at this stage. Column 5 lists 5 principal morphological forms taken by dying or dead cells after an ischemic insult. Determining how these end stages are reached is the ultimate goal of research on ischemic cell death. Column 4 lists 6 long-term functional or structural changes, all of which, except changes in membrane permeability, are known to occur as a result of ischemia. It is hypothesized that one or more of these account for one or more forms of ischemic cell death. As indicated by lack of 1:1 connections in figure, no direct causal sequence has been established between any of functional changes in this column and particular end stages shown in column 5. Potential relationships are discussed extensively in text (see sects. III and IV). Column 3 lists actions that are likely to cause long-term functional changes described in column 4. These are termed "perpetrators" because they are considered to be key damaging events in ischemic cell death. No direct effects of perpetrators on critical functional changes are insinuated in figure because none has been completely established. Many such effects are, however, reasonably well established and are discussed in text. Columns 2 and 1 show changes in many variables, initiated by original inhibition of electron transport, whose most important end result is considered to be activation of perpetrators, but which may also have more direct effects on cell damage, that are not shown. Major outputs of these changes, lumped together as initiators and activators, are changes shown in column 2, which activate perpetrators. Direct causal interactions between specific changes are shown for these earlier events because far more is known than about interactions at later stages of the process. However, all these interactions are not established with same degree of assurance, as discussed in text. These causal interactions are indicated by including changes within same toned horizontal band, as shown for columns 2 and 3. For example, both increased nitric oxide and free radicals lead to damaging actions of free radicals and peroxynitrite. They are also indicated by including causal changes within a box whose outline color is same as that of variable they are changing. For example, both increased calcium and gene activation contribute to formation of increased nitric oxide. This way of describing events indicates the very high level of interaction between different changes caused by ischemia, involving many positive-feedback loops. Interpreting the figure: as indicated above, colored boxes encompass different changes that cause change in variable represented by that color. For example, increased nitric oxide (peach color) results from increased calcium and gene activation. Colored horizontal arrowheads represent all events within box of that color. For example, red arrowhead (for ATP) represents loss of oxygen and increase in sodium, which are enclosed within red box associated with ATP (near bottom of figure). Using this notation allows the very large connectivity of system to be represented in a manageable way. For example, changes putatively contributing to increase in sodium, enclosed within green box at top of figure, are extremely numerous when "contents" of each arrowhead are considered. In addition, changes contributing to increase in cytosolic calcium, and hence calpain activity, are all those included in boxes and arrowheads in top gray band. Large number of positive-feedback cycles is readily seen. Depol, depolarization; pHi, intracellular pH; Nai, intracellular Na+; Cai, intracellular Ca2+; FFA, free fatty acids; PAF, platelet-activating factor; e Transport, electron transport.

Ultimately, it will be important to identify the point of cell death, defined above as the point of no return, because some agents might block the major morphological change but not block the irreversible damage to the cell. It seems reasonable that prolonged damage to the plasma membrane, mitochondria, cytoskeleton, or protein synthesis could each be a point of cell death defined as above, and section IV is devoted to evaluating their roles in ischemic cell death.

B.  Major Features of Ischemic Cell Death

1.  Three major modes of cell death

There appear to be at least three distinct modes of cell death that participate in ischemic cell death (149, 205, 706). Two of these, apoptosis and autophagocytotic cell death, clearly involve ordered physiological processes in that new structures are formed that are integral to the process, and the progress toward cell elimination is stereotyped. Furthermore, they are a part of normal cell function, during development and other times (205). There is some conflict as to whether autophagocytosis can be profound enough to cause cell death (149, 205, 835), but there are quite persuasive arguments by Clarke (205) that it is. These include the high percentage of neuronal area that is covered by autophagic vacuoles during some developmental cell death (205), as well as the ability of 3-methyladenine, a phosphatidylinositol kinase blocker (101) which prevents autophagocytosis, to completely prevent cell death in at least one case (149).

A third category of cell death, which has been termed necrosis or necrotic cell death, is fundamentally different from apoptosis and autophagocytotic cell death in that it only occurs after an exogenous insult; furthermore, no new structures are elaborated as part of the process. Thus it does not appear to be a programmed, or a normal, physiological process. Because the process is not yet well characterized, it is best defined now as cell death that is neither apoptotic nor autophagocytotic.

There are at least two quite different forms of this cell death; by far the most common is ischemic/homogenizing cell change, where the cell first shrinks dramatically and becomes very electron dense. Less common is edematous cell change, where the cell swells greatly and organelles lose their form. There is no satisfactory term for these modes of cell death. It is widely termed necrosis or necrotic cell death, but necrosis is really the "decay" process after the point of cell death and occurs in all forms of cell death (1137). However, for the sake of continuity, necrotic cell death and necrosis will be used in this review. At this stage, it is not completely known why a particular mode of death occurs in a given cell as a result of a particular ischemic insult. However, it is apparent that one insult can spawn more than one mode of death in the same population of cells and also that in many cases one cell will manifest signs of more than one mode of cell death (205, 716). Thus neurons have the potential for exhibiting all modes of death in response to an ischemic insult. The pathway taken must be a function of the nature of the insult, the cell type, age, and very probably the state of the cell at the time of the insult (716). This issue is considered in section III.

In addition to its multimodal nature, ischemic cell death is also characterized by a long delay between the insult and manifestation of major cell damage. This delay varies greatly, depending on the nature of the insult and the brain region being affected. In some cases it is as long as several days or even weeks (276, 582), whereas in others it is a few hours or less (742). It is clear that, all other factors being equal, the greater the energy deprivation, the less time it takes for damage to develop (579). The very prolonged delay is quite an extraordinary phenomenon; it shows that very short-lived, albeit profound, derangements of metabolic patterns can initiate the development of damage and cell death that takes place, between a day and 3 wk later. Remarkably, normal homeostatic mechanisms are unable to prevent this process. Thus lethal ischemic insults are those that move cells far enough from their steady state to eliminate the possibility of reattaining that steady state; sublethal insults produce early damage, but this is able to be recouped by the cell. One very important and unresolved issue is what the basis is for this "destabilization" of the cell. It is discussed theoretically in section IX.

C.  Overview of Ischemic Cell Death

1.  Stages of ischemic cell death

Ischemic cell death is initiated by changes that result directly from inhibition of oxidative phosphorylation and create an early maelstrom of activity. These changes include decreased pH, decreased ATP, initiation of free radical production by the mitochondrial chain, increased cell Na⁺ and membrane depolarization as a result of the loss of ATP substrate for the Na⁺-K⁺ pump. These lead to secondary changes in ion and chemical concentrations which, in concert with the initiating changes, result in activation of damaging processes. These damaging processes are termed "perpetrators" in this review and are characterized by their abilities to produce long-term changes in macromolecules. Activation of one or more of the perpetrators is considered the key step in necrotic aspects of ischemic cell death. The perpetrators include proteases, phospholipases, and free radical actions. The macromolecular changes they cause, and the ensuing functional changes, are considered to be the proximal causes of cell death. At present, the way that ischemic apoptosis is activated is not well enough understood to include the same perpetrators in that process with any certainty, although caspases certainly are involved. Thus the process of cell death has at least three major stages: an early development of ionic and chemical changes, a resulting activation of perpetrators, and a subsequent change in critical functions and structures that denote or lead to cell death by one of three different routes.

The overall process is extremely complex due to the large number of interactions between the variables, illustrated by Figure 1. Although incomplete, Figure 1 provides a good working description of the probable flow of damage and the very complex interactions between different changes.

Two very important limitations in our understanding are not apparent from Figure 1.

The first is the degree of certainty that the different changes shown are actually involved in ischemic cell death. It is fairly certain that most, or all, of the activators and perpetrators in Figure 1 are actually involved in ischemic cell death, although, as will be seen, experimental evidence is often surprisingly weak. Much less is known about how these changes lead to cell death (Fig. 1, columns 4 and 5). As will be seen, the identities of the critical functional changes, how they actually might lead to cell death, and the end stages are little known for any of the three modes of cell death.

The temporal aspect of cell death is also missing from Figure 1. End stages or cell disintegration develop anywhere from 6 h to several days after the ischemic insult. The basis for these time delays is not yet known. Gene activation and subsequent synthesis of damaging proteins such as cytokines, inducible cyclooxygenase-2 (COX-2), inducible nitric oxide (NO) synthase (iNOS) may be important. However, it is also likely that there are long-term low-level activations of perpetrators when the insult is relatively mild so that major changes in cell function occur only after prolonged periods. The factors that determine the temporal aspects of ischemic death are poorly understood at the moment.

Figure 1 provides a framework for the organization of the review. After the different model systems used for studying ischemic damage are considered (see sect. II), the main body of the review begins from the right-hand side of Figure 1 and essentially walks backwards; for example, section III describes the end stages of ischemic cell death. Knowing the downstream events makes it easier to evaluate the role of a particular change. For example, understanding that mitochondrial dysfunction may be a mediator of cell death (see sect. IV) provides a focus for examining the targets of free radical action. Despite the connectivity of the different major parts, each section is designed to be usefully read independently of its order of appearance and to fully examine the possible contribution of a different variable to damage.

The achievable goal of the review is to critically evaluate the involvement of different variables in ischemic cell death. This entails describing the changes in the variable that occur during and after ischemia, determining how they are likely to occur, critically assessing pharmacological and gene manipulation studies done to show that the change contributes to cell death, and then trying to understand the mechanism by which the change is contributing to cell death.

In section IX, a still speculative attempt is made to synthesize the different sections and describe probable sets of events that lead to cell death in the different experimental models. It is hoped that this will provide useful directions for future studies.

	II. SYSTEMS USED TO STUDY ISCHEMIA

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Many experimental models are used to study ischemic damage. Mechanisms of cell damage are determined by testing effects of different manipulations on the extent of cell death in a model. To meaningfully compare results between models, the essential differences between the ischemic insults and the way damage is assessed in these models needs to be appreciated. The factors influencing development of damage in the different models need to be known, and problems in interpreting measurements of cell death have to be recognized.

The three main classes of in vivo rodent models are global ischemia, focal ischemia, and hypoxia/ischemia. In the latter, vessel occlusion is combined with breathing an hypoxic mixture; this modified Levine preparation is now almost exclusively used in young animals. Larger mammals have been used to study ischemia (534), as have nonmammalian vertebrates (453), but this review will focus on what has been learned from rodent models. In vitro models that include exposure of neuronal, neuronal/glial or organotypic slice cultures, or the freshly isolated hippocampal brain slice, to anoxia or to anoxia in the absence of glucose (in vitro ischemia) have provided important information and are discussed when they provide insight into events in vivo. Other single-cell or synaptosomal models have been used very effectively (307, 340) but will not be considered because the metabolic states of these preparations may be quite different from in vivo (307).

The degree to which blood flow is reduced directly determines the changes in three variables that are almost undoubtedly the genesis of all future changes. Two of these, inhibition of electron transport and decreased ATP, appear to be directly related to the extent of flow reduction (see Table 1). The third variable, free radical production, is likely to be a more complex function of flow rate because it is activated both by electron transport inhibition and also by oxygen so that it will be maximal at blood flow values greater than zero. (The precise relationship between flow rate and free radical production has not been determined.) Other damaging changes such as decreased pH and leukocyte accumulation are more severe in the presence of residual blood flow. Thus the amount of damage need not be a direct function of the decrease in blood flow. Elevated blood glucose enhances damage in many cases despite maintaining ATP levels so that the fall in ATP is not a reliable indicator of the efficacy of an insult either. As a further complication, the nature of damage (e.g., extents of apoptosis vs. necrosis) may be a complex function of the amount of the ATP/electron transport decrease and free radical increase and thus may be a complex function of the flow rate during ischemia (194, 870). Thus, except for increased duration of an insult, which always enhances and accelerates damage, it is difficult to estimate, a priori, the potential impact of an insult based simply on the degree of ischemia and fall in ATP.

In vivo, distinct neuronal populations have very different vulnerabilities to ischemia, and these have been reviewed relatively recently (169). For example, 5-min global ischemia caused delayed death in almost all CA1 pyramidal cell neurons with no effects on other populations, whereas 20 min caused cell death in CA3 neurons but had almost no effect on dentate granule cells in the hippocampus (579, 583) or on interneurons in CA1 (322). The basis for differential vulnerability has not been determined, although, as will be seen, there are many differences between vulnerable and less vulnerable cells that might well account for it, as described in Table 5. In addition to these, differences in presynaptic events may be responsible for apparent selective vulnerabilities of postsynaptic neurons as will be seen in section VIB. The large range of selective vulnerabilities is quite an extraordinary phenomenon. It indicates large differences in metabolism between different cell types that presumably reflect their different physiological functions and lead, serendipitously, to differential vulnerabilities to ischemia. The phenomenon has been used to provide insight into mechanisms of damage and is discussed in that context throughout the review.

The Levine model, in which hypoxia is combined with unilateral carotid occlusion (644), adapted in the mid-1960s in landmark studies by Brown, Brierly, and co-workers (121, 129, 742), established the basis for systematic study of brain cell damage in rodents. Most in vivo models now rely on vessel occlusions that predominantly affect the forebrain, developed between the late 1970s and early 1980s (331, 366, 461). They are generally divided into two categories, global and focal ischemia, but a modified Levine preparation (hypoxia/ischemia) has some properties of both models.

The three most widely used global ischemia models are four-vessel occlusion (4-VO) and two-vessel occlusion (2-VO) combined with hypotension in the rat, and two-vessel occlusion in the gerbil. The importance of transgenic mice has spawned the use of the 2-VO in the mouse also, and the latter is now being systematically studied (588, 1074). Results to date are similar to those with rat and gerbil (588).

A) 4-VO IN RAT. The 4-VO in rat involves permanent coagulation of the vertebral arteries (which has no deleterious effects) and temporary ligation of the two common carotids. Ligation of temporal muscles reduces flow more reliably (926, 928). In Wistar rats, which show loss of righting reflex within 15 s, blood flow is reliably <3% of control values in hippocampus, striatum, and neocortex (931). The electroencephalogram (EEG) generally becomes isoelectric within 30-40 s (935), and spontaneous cortical activity is abolished within 1 min when skull temperature is maintained at 37°C (1233). No anesthetic is required during ischemia, and usually none is used.

B) 2-VO IN RAT. This involves ligation of the common carotid arteries only, along with a blood pressure reduction to ~50 mmHg, and has slightly more profound effects than 4-VO. Blood flows fall to ~1% in hippocampus, neocortex, and striatum (1055, 1056), and the EEG generally becomes isoelectric within 15-25 s. However, in the hands of one group, blood flow is only reduced to 15% of control levels (1089). This less-intense insult is more easily protected against, for example, by N-methyl-D-aspartate (NMDA) receptor blockade, and this has led to some confusion in the literature that will be dealt with in section VIC. Anesthetic is used during ischemia.

C) 2-VO IN GERBIL. This is induced by temporarily ligating the carotid arteries, with no reduction in blood pressure. Because there are no posterior communicating arteries in gerbils, this produces profound forebrain ischemia (583). Changes are similar to those in the rat models; blood flow in cortex is <1% and in hippocampus is ~4% of control values (548), whereas EEG failure occurs within 20 s (1088). Damage has been successfully dissociated from the occurrence of postischemic seizures that also characterize the insult in gerbils (493). Anesthetic is not used during ischemia.

D) COMPLETE VERSUS INCOMPLETE GLOBAL ISCHEMIA. Finally, there is complete global ischemia, generally achieved by neck-cuff (259, 297, 682), cardiac arrest (228, 294), or by ligating or compressing all arteries stemming from the heart (560, 915). Blood flow to the whole brain is zero or <1% in these models (297).

Vessel occlusion is often termed "incomplete ischemia" because of the residual blood flow, and there is controversy as to whether complete or incomplete ischemia is more damaging, with the thought being that the larger fall in pH or increase in free radicals during incomplete ischemia might enhance damage. This does not seem to be the case. Complete ischemia for 10 min in rats, even without maintenance of temperature, causes complete destruction of cells in the CA1 pyramidal cell layer after 7 days (226). This damage is certainly as severe as the incomplete global ischemia models (see sect. IID3).

Overall, gerbil vessel occlusion is the most studied model because the operation is simpler than the rat. However, there are many differences between the two species, both in terms of metabolic and genetic changes during and after ischemia, and the abilities of different agents to protect against damage. Thus the processes of cell death may be different; results from one species cannot necessarily be generalized.

Global insults are generally short, between 3 and 30 min, with 5-20 min being the usual exposure times. Longer insults become lethal, and these shorter insults are quite good models for reversible cardiac arrest pathology. Levels of ATP fall quite rapidly during this period (Table 1). By 2 min, levels are 15% or lower in gerbil, and also in rat during complete ischemia. Unfortunately, there are no good kinetic studies for the rat vessel occlusion models; by 10 min, ATP is 10% or less of normal values (Table 1), but the rate of decay, which may be important in determining extent of damage, is not known.

Levels of ATP in vulnerable cell regions recover quite well after global ischemia so that delayed damage develops in the face of ATP levels that are at least 70-80% of normal for most of the reperfusion period (162, 929). Tissue metabolic rate is generally depressed by 50% or more, for at least 6 h after the ischemia (291, 306, 606, 760). The basis for this is not known, and its consequences have not been determined, but they are likely to be important.

A) DEVELOPMENT OF DAMAGE. A cardinal feature of global ischemic insults is a substantial delay between the end of the short insult and cell death. This is generally between 12 h and several days. The delay depends on the cell population and the duration of the insult. This "delayed neuronal death" was first noted and documented in rat by Pulsinelli and Brierly (926, 927) and in gerbil by Kirino (580). It is most widely studied in the CA1 region of the hippocampus, but the striatum, parts of the CA4 region of hippocampus, and layers 2 and 5 in cerebral cortex also show the phenomenon in response to short insults.

Overall, gerbil hippocampus is slightly more sensitive than rat. With body temperature at 36°C, but brain temperature falling normally, 5 min of ischemia caused massive cell death in the hilus of the dentate gyrus (CA4) and the striatum within 24 h and complete cell loss in the CA1 region of the gerbil hippocampus within 3-4 days (223, 580). Ischemia for 3.5 min caused 75% cell death in CA1 after 7 days (847), whereas 2 min caused no measurable damage in that region (543). It did cause extensive damage in the somatostatin-containing cells in the hilar region (735).

When rat brain was maintained at 36-37°C, 6-10 min of 4-VO caused near-complete CA1 pyramidal cell death in 3 days (647, 681), and 10 min of 2-VO caused almost complete cell death in 7 days (210, 519, 819, 921). Ischemia for 5 min caused 50% loss (519) compared with complete cell loss in gerbil, and 2-min ischemia caused a (very minor) neuronal loss in CA1 and CA4 (1055). As with the gerbil, hilar and striatal neurons die much more rapidly than CA1 pyramidal cells (896, 997), although, interestingly, a greater insult duration was necessary to initiate the cell death in striatal neurons than in hippocampal neurons (997). This shows a dissociation between factors that initiate the damage and those involved in its development.

Damage from successive insults can be cumulative. When spaced 1 h apart, three insults that normally gave no damage individually caused major damage (543).

B) MORPHOLOGICAL CHANGES DURING DELAYED NEURONAL DEATH. There are four major sets of changes during development of the delayed death in gerbil. Most notable is a massive proliferation of stacks of endoplasmic reticulum (ER) that peaks after ~2 days; ER fragments accumulate, with lysosomes, around the nucleus (579, 581). After ~3 days, there is a regression of the proliferated ER and the formation of a large number of autophagosomes with a very large increase in the lysosomal proteases, cathepsins B, H, and L (834). The autophagic vacuoles may arise from the expanded ER as the two have been associated during developmental cell death (205). Thus a long-term program for autophagocytosis may have been activated by 5 min of ischemia.

There is also a gradual increase in the numbers of small dense bodies ~1 µm or less in diameter in both somata and dendrites, which largely localize under the plasmalemma. Throughout the 3 days, the dendrites show progressive swelling, microvacuolization, and disappearance of microtubules, beginning immediately after ischemia (1236). After early swelling, mitochondria remain normal until ischemic cell change (ICC) or edematous cell change (ECC) become apparent.

The delayed death in the rat appears very different, although it develops at about the same rate. The extent of ER proliferation is far less (251, 581, 897) and, perhaps relatedly, no autophagasomes have been explicitly identified. The small dense bodies are somewhat more prevalent than in the gerbil and may be undegraded proteins that participate in damage (251, 582), although there is no explicit evidence.

As the duration of the insult increases, and so presumably the extent of the biochemical changes, so does the rate of cell death increase (579, 897). The nature of the process may ultimately change; ER proliferation, for example, is absent in gerbils after a 30-min insult that causes cell death in 12 h (897).

At a certain point, these rather subtle changes are transformed into quite abrupt major changes in cell structure associated with the end stages of cell death. These are discussed in section III. The mechanisms of this dramatic change are not known; presumably accumulated changes become great enough to cause what is essentially a "phase change," as discussed by Gallyas et al. (350).

Almost all focal ischemic models, whether larger mammal such as cat (1077) or nonhuman primate (1092) or rodent, primarily involve occlusion of one middle cerebral artery (366). In some cases, the carotid artery is also ligated. The insult is thus differentiated from global ischemia in two very important ways. First, even at the core of the lesion, the blood flow is almost always higher than during global ischemia so that longer insults are required to get damage. Secondly, there is a significant gradation of ischemia from the core of the lesion to its outermost boundary, and hence there are different metabolic conditions within the affected site. Because of its duration, and heterogeneity, the insult is much more complex than global ischemia, but it is an invaluable model for stroke and is thus widely studied.

In permanent focal ischemia, the arterial blockage is maintained throughout an experiment, usually for 1 to several days, whereas in temporary focal models, vessels are blocked for up to 3 h, followed by prolonged reperfusion. Despite the big difference in insults, maximal lesion sizes are comparable in the two cases, and the progression of damage, in terms of the numbers of damaged neurons and the extent of that damage over 6-72 h, is remarkably similar (1283). Nevertheless, there are clear differences illustrated by effects of different protectants so that the mechanisms of cell death may well be different. As with global ischemia, mouse models have now been introduced (434, 588) but will not be discussed explicitly.

There are variations within each of the two basic classes of insult: temporary and permanent ischemia. Several different rat strains are used (one is a spontaneously hypertensive strain that has a less extensive collateral circulation during ischemia and is more easily damaged; Refs. 137, 222, 391). Different strains show minor quantitative differences in lesion locations and sizes but qualitative differences in responses to different protectants.

Also, different sites and techniques of artery occlusion are used, to try and obtain reproducibility of flow reduction and lesion size (462, 860). There are two principal occlusion sites. In proximal occlusion, the middle cerebral artery (MCA) is occluded close to its branching from the internal carotid, before the origin of the lenticulostriate arteries (860, 1104, 1105). Appropriate care can produce lesion sizes with coefficients of variation that are <20% (860). A newer and now widely used approach to proximal MCA occlusion is the insertion of a nylon suture into the carotid artery past the point at which the MCA branches so that the latter is occluded at its origin (79, 598, 655, 798). Used optimally, the coefficient of variation in lesion size is ~8% and of blood flow is 35% (79). A similarly small coefficient of variation in lesion size, along with a large lesion of ~250 mm³ at 24 h, was obtained with the silicone-coated suture in Fischer 344 rats (44). There are some potentially important artifacts with this widely used method. Flow following temporary ischemia is somewhat compromised by the partial occlusion of the carotid arteries with the filament (79). Also, even a temporary occlusion leads to a long-term increase in core temperature at ~1.5°C due to decreased circulation and damage to the hypothalamus, at least in Wistar rats (1241, 1297). Finally, there is quite extensive damage to small arteries in the ischemic field, with loss of endothelium and muscle damage (832). This occurs after normal insults (e.g., 6-h ischemia or 2-h ischemia and several hours of reperfusion). It has been suggested that this damage may affect subsequent neuronal cell death, for example, by exacerbating the leukocyte response in the period after ischemia (435). This is not at all proven but needs to be considered.

In distal MCA occlusion, flow to the basal ganglia is not blocked so that damage is only cortical. The occlusion can be made surgically, in which case the lesion is readily reversible (137) or, less invasively, by creating a (permanent) thrombus by laser irradiation when the artery is perfused with Rose Bengal (714). Distal MCA occlusion has to be combined with occlusion of the ipsilateral carotid artery to get adequate flow reduction (122, 189).

F.  Focal Ischemia: Core and Penumbra

1.  Blood flows

In both proximal and distal MCA occlusions, there are core ischemic regions, served primarily by the occluded MCA, where blood flow is reduced to <15%, and there are penumbral regions where flow is <40%. There are also extrapenumbral cortical regions in which flow is >40%. These approximate flow-based definitions of core (284, 816, 977, 1104) and of penumbra (48, 369, 462) as well as the extrapenumbral (or peri-infarct) region are generally agreed upon, although there is still much discussion. Using flow is a simple and physiologically meaningful way to differentiate the regions, which suffer fundamentally different insults as indicated by efficacies of protectants, and major morphological changes. Given a long enough temporary (or permanent) insult, both core and penumbra become infarcted (1299), whereas the extra penumbral zone only shows death of isolated neurons. The density of cell death is not great enough to cause infarct.

In proximal MCA occlusion, the core is in the lateral portion of the caudate putamen and overlying parietal/somatosensory cortex (106, 751, 798, 1104). Most other regions of neocortex and entorhinal cortex, as well as the medial caudate-putamen, constitute the penumbra (79, 106, 751, 798). In the distal MCA occlusion/carotid artery occlusion, the striatum is spared, and core and penumbra span the parietal cortex, with penumbra occupying the more inferior regions. There is significant variation in the extent of the different regions among Sprague-Dawley rats, spontaneously hypertensive rats, and Wistar rats (48, 122, 137, 714).

With development of on-line measurements of flow using positive emission tomography (PET) scans, and the use of more detailed time course studies using iodoantipyrene, there is emergent evidence that blood flow varies throughout the penumbral region and also decays with time, although the extent of decay varies widely in different models (79, 438, 798, 1297). The implications of this decay are currently being considered. Clearly, it intensifies the insult in that region over time, and so may be important in the development of penumbral damage (325). It is considered in greater depth in the above-referenced papers dedicated to flow measurements and analysis.

The core undergoes rapid anoxic depolarization within 1-3 min with a concomitant rise in extracellular K⁺ to ~70 mM, which may show some short intermittent returns to baseline (359, 818). There is a large decrease in extracellular Ca²⁺ within 1-2 min at flow levels that are present in the core (428, 429), signifying entry into the tissue. During the reperfusion period, after 2 h temporary focal ischemia, extracellular K⁺ returns to control levels for 6 h before rising slightly (to 5 mM) for the remainder of a 24-h recirculation period. Despite this return to near normalcy, the insult produces severe damage with a large infarct including all of the core (359).

Events in the penumbra are less drastic, although they still lead to infarct. There is EEG silence (1077) and very much reduced evoked transmission (977), but there is no permanent anoxic depolarization with concomitant ion changes during the ischemia. Presumably ATP levels, which are maintained at 50-70% of normal, do not fall enough to allow the anoxic depolarization. However, there are sporadic transient depolarizations (369, 759) that are here termed "intraischemic depolarizations" (462, 818). They arise differently from anoxic depolarizations. Anoxic depolarizations are independent of NMDA-type glutamate receptors (394, 611, 635, 934, 1231) and only very slightly (1231) or not at all (635) dependent on non-NMDA glutamate receptors. In contrast, the frequency of intraischemic depolarizations is markedly dependent on both classes of ionotropic glutamate receptors (47, 188, 483, 759). The depolarizations may actually originate from glutamate release in the infarct core. Nitric oxide synthase knockout mice show reduced frequencies along with less release of glutamate in the core of the lesion (1031). The depolarizations occur very abruptly, last between 3 and 5 min, and have the characteristic form of spreading depressions. They do not occur during reperfusion (818). As discussed in section IIH, they may well enhance damage.

Levels of ATP fall to ~25% of basal values in the core and remain there between 5 min and 4 h of ischemia (328, 329, 1084, 1201) (Table 1). Later measurements have not been reported. After damaging durations of temporary ischemia, core ATP levels return to about two-thirds normal, for at least 4 h (329), although damage is as great as in permanent ischemia. Oxygen levels return to normal (801), showing that compromised flow is not the basis for the damage that occurs during reoxygenation.

Levels of ATP in the penumbra average ~50-70% of normal during ischemia (Table 1). Glucose utilization is actually elevated in the penumbral region (48, 816, 1252) at early times but falls to ~50% normal, equal to the core, by 3.5 h (1252), suggesting significant compromise of cell function at this point. Unfortunately, there are no reports of oxygen utilization during the insult, which would allow a more complete assessment of the rate of energy metabolism. Although penumbral blood flow declines during the insult, it remains far higher than in the core; infarct develops despite this flow difference.

Levels of ATP during the reperfusion period after temporary ischemia do not seem to recover much from their ischemic values (489, 590), although further measurements are needed to confirm this conclusion. Glucose utilization in some penumbral tissue is reduced to well below normal during early reperfusion, and this is considered a reliable indicator of the tissue that will later become part of the infarction (79, 1299). Presumably, the low glucose metabolism indicates severe cell damage of some kind at this early stage.

Thus, as with global ischemia, the core of the focal lesion undergoes massive ion and metabolite changes that recover quite strongly after a temporary insult. Despite this recovery, it is almost impossible to prevent damage by any intervention during the reperfusion period. It has also proven extremely difficult to prevent core damage by any single intervention, even before the onset of ischemia, when ischemia lasts for 2-3 h. The penumbra can be rescued by many interventions. These differences suggest different mechanisms of damage in the two regions. As with global ischemia, postischemic energy metabolism appears to be compromised in both core and penumbra.

In contrast to ion and metabolite changes, a recent study demonstrated very little free radical change in the core as compared with penumbra (1060). Free radicals, as measured by hydroxylation of salicylate in microdialysates, increased early during ischemia in the penumbra, remained elevated throughout 3 h, and then became further elevated with the onset of reperfusion. In marked contrast, there was no increase in the core during 3 h of ischemia. There was an increase in the core after ~4 h of reperfusion. Thus the exposure of the core to free radicals during the main period of damage development must be considered minimal. The opposite is true of the penumbra.

Unlike global ischemia, which leads to neuronal cell death in isolated regions, focal ischemia produces a contiguous mass of damaged brain tissue termed the infarct. Rather than measuring damage by counting dead cells, as in global ischemia, damage is generally expressed as the volume of the infarct, for which there are standard measurement techniques (e.g., Ref. 860). This is a rough measurement in that the appearance of infarct is predicated on a large fraction of the cells in the region being damaged; otherwise, individual cell damage is noted.

The gold standard of infarct delineation is a pallid region when tissue is stained with hematoxylin and eosin or pure Nissl stains (860). The pallor results from vacuolization of the neuropil, including swelling of glia, presynaptic elements and dendritic elements, and the loss of Nissl staining in the neuropil (355). So identified, it signifies cell damage but not necessarily cell death. This is exemplified by a delayed reduction in infarct size in animals in which iNOS is blocked (799).

In permanent ischemia, the infarct is first seen after 3-12 h (76, 137, 276, 355, 359, 814, 860). The wide time range appears to reflect strain differences, with the early infarct delineation seen in Sprague-Dawley rats (860) and the later delineation seen in Wistar rats (355), possibly because the lesion is less uniform in the latter (351, 355). The infarct begins in the core but reaches close to its maximal size, which includes core and penumbra, 6-24 h after the onset of ischemia. The infarct continues to grow after 1 day, although at much slower rate, so that between 24 and 72 h it grows by ~30% in Wistar rats (355). Thus damage continues to increase in penumbral regions.

Different durations of temporary ischemia lead to graded involvement of different regions in the infarct as measured after 1-2 days reperfusion (752). Ten to twenty minutes produces scattered dead neurons in the core (653, 752), whereas 1 h leads to infarct in the core (354, 526, 752). Infarct in the penumbra develops fully when the temporary occlusion is 2-3 h (354, 526, 752). In this case, the size of the infarct is the same as it is in permanent ischemia (e.g., Refs. 752, 1283), although it develops slightly more slowly (1283). After ~7 days, there is almost complete loss of cellular elements in the infarcted region; this is termed pan-necrosis. As little as 30 min of temporary ischemia also leads to an infarct in the core region, but this only first appears after 3 days and takes 2-3 wk to mature to its full size (276).

In summary, development of the infarct is quite rapid, but it matures over several days, indicating a significant delayed component of damage in tissue where the insult intensity is relatively small. As indicated above, expansion of the infarct indicates expansion of the region in which the large preponderance of cells are damaged. The relationship of infarct to cell death is considered in section IIG.

Diffusion weighted magnetic resonance imaging (DWMRI) delineates the core of the (future) infarct as early as 1 h after the onset of ischemia, and at later times, DWMRI quite faithfully defines the developing infarct (151, 362). It is very likely that the altered signal intensity represents regions of tissue in which there is intracellular edema (nicely discussed in Ref. 362).

Staining frozen sections with 2,3,5-tetraphenyltetrazolium chloride (TTC) (359), which is colored red by electron transport in active mitochondria, defines a region in which mitochondrial function is severely compromised. Damage is not necessarily irreversible. The region is slightly smaller than the infarct size defined by hematoxylin and eosin (H and E) at early times but overlaps the H and E staining at 24 h (955).

Finally, the loss of mitogen-activated protein (MAP) 2 immunostaining, which is almost certainly a reflection of increased cytosolic Ca²⁺ and activation of calpain (Y. Zhang and P. Lipton, unpublished data) quite faithfully follows the development of the infarct as defined by H and E staining (233). Thus several markers of cell dysfunction parallel the expansion of the infarct as measured by loss of Nissl stain, indicating that the cellular elements that are poorly stained are metabolically compromised.

It is critical to relate the infarct to cell death. Individual cell damage within the infarct develops slowly, but more rapidly than delayed cell death in global ischemia, suggesting that the prolonged focal insults have more impact than the shorter, more intense global insults. The best descriptions of this cell damage have come from Garcia and co-workers (351, 355, 1283). Cell death, defined as eosinophilic shrunken neurons (advanced ischemic cell change) or neurons showing autophagocytotic morphology (178), and also neurons showing apoptotic morphology (178, 652) (see Fig. 1), first becomes significant at ~6-12 h in the core of the lesion, with a major increase between these two time points. At 12 and 24 h, ~80% of the neurons have suffered cell death. A similar although less extensively described time course is seen in spontaneously hypertensive rats after 80 min of ligation (202). Although development of cell death in the penumbra appears to lag development in the core, it is hard to be sure, from the data provided. A less-detailed time course study found that 2 days after 2-h focal ischemia, 55% of the cells in the core and penumbral infarcts were dead, whereas 30% were damaged but did not show signs of death; 15% appeared healthy. These may represent different cell types, but that was not discussed (656).

At least for the first 72 h, the rate and extent of developments of cell death caused by 2-h temporary ischemia are the same as they are for permanent ischemia (1283). Furthermore, the number of apoptotic cells in the penumbra is the same in the two models throughout this 72-h period (785).

For the early infarct (before pan-necrosis), on which most measurements are made, the implicit assumption is thus that the uniform pallor and other indicators within the infarct largely result from neurons so severely damaged that they will die or have already died. Overall, this is justified in that a large percentage of neurons within the infarct die after 24-72 h, at least in Wistar rats. Thus, if a treatment reduces the radius of the early infarct, it is very likely to be slowing down cell death in penumbral neurons. On the other hand, in at least one case, infarct size was reduced after 48 h when delayed protective treatments were applied (1278), indicating that the majority of cells at the borders of the infarct are not yet dead after 48 h (1278). Overall, it is most satisfactory to measure infarct size after 72 h, but this is often dificult to do.

In general, there may not be a precise 1:1 correspondence between infarct size and the amount of cell death. Infarct requires a certain percentage of the cells to die (as witnessed by the peri-infarct area that has cell death but no infarct), indicating that the infarct in a certain region may be abolished but that significant cell death may well remain. It would be much more useful for determining cell death mechanisms if cell counting as well as infarct size were used to assess cell death whenever possible. This being said, the infarct size provides a reliable semiquantitative measure of the extent of neuronal death and is far easier to measure than individual cell death.

The final stage of infarct development in focal ischemia is pan-necrosis, in which the neuronal death is accompanied by glial and vascular cell death and loss of cellular elements. This does not occur in most global models that lead to widespread neuronal death, although it does occur in extremely insulting conditions, such as low pH (522) or a prolonged very intense insult (259). It occurs about 1 wk after most focal insults. The basis for pan-necrosis is not clear, but for some reason, both the endothelial and glial cells become major targets, suffering early changes and eventual death (914). In hyperglycemic global ischemia, which leads to infarct, glial pH falls to 4.3 (607), a level that can kill these cells (608). No measurements were made of endothelial cell pH. It would be very useful to measure focal ischemic effects on glial (or endothelial cell) pH to see whether drastic lowerings might explain development of the infarcts in those conditions.

During prolonged hypoxia/ischemia, which also leads to infarct, there was an early activation of pinocytotic transport across the endothelial layer, which was quite specific to conditions in which infarct was developing (895, 898). This might be important, but it is not known whether it occurs in focal ischemia. Direct exposure of brain to free radical-generating systems leads to infarction-like changes (174), suggesting that prolonged production of free radicals may be leading to pan-necrosis, but there is no explicit evidence. Thus there are reasonable lines of investigation of the pan-necrotic mechanism. It does not seem to relate to neuronal death because it occurs so late, but it might.

The bases of the intraischemic depolarizations or spreading depression-like depolarizations (818) discussed previously are not completely known; they may well be the spread of increased extracellular K⁺ and glutamate from the core of the lesion (463, 818). Their frequencies are halved by either NMDA or DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) antagonists, indicating that glutamate-mediated depolarization contributes markedly. The evidence linking the development of damage in the penumbra to these depolarizations is extensive but is largely correlative (47, 151, 462, 463, 818). For example, factors that reduce the frequency of the intraischemic depolarizations, such as glutamate antagonists and hypothermia (462, 818), as well as blockade of iNOS (1031), reduce the size of the infarct that develops in the penumbral region. The most persuasive direct evidence that they are damaging is that artifactually increasing the numbers of depolarizations during ischemia, by about twofold, using either KCl (151) or electrical stimulation (47), increased the size of infarct, and peri-infarct damage, by 30-100%, respectively. In one case, this was measured 24 h after distal MCA occlusion and in the other 2 h after the onset of proximal MCA occlusion, using DWMRI (255). Thus the artifactual depolarizations do appear to be damaging in ischemic conditions, and this suggests that the endogenous depolarizations are also damaging. Unfortunately, it is very difficult to specifically target the latter with a drug, to more directly establish their importance.

The basis for the putative damaging effect of the depolarizations is not known; similar magnitude and duration depolarizations are not damaging in nonischemic tissues (818), although in those cases there is an increased blood flow that may help to maintain energy levels. One possibility is that they lower ATP levels because blood flow is reduced and that this is damaging. There is evidence, from an abstract published several years ago (1101), that the depolarizations are responsible for the reduction in ATP in the penumbra. However, ATP only falls to ~70%, and it is not clear at all that this would be damaging during the 5-min depolarizations. It would be very useful to measure the effects of the artifactually increased frequencies of intraischemic depolarizations on ATP levels to see if there were a correlation between the latter and the increased damage.

One of the most intriguing lines of study to develop over the past several years has been three lines of evidence that white blood cells contribute to focal ischemic damage after temporary insults (41, 204, 248, 431, 592). These are 1) the appearance of neutrophils in ischemic tissue at an appropriate time; 2) the protection afforded against damage by agents that should prevent the accumulation or activation of neutrophils and 3) known damaging actions of these cells when they accumulate in tissue (1200). Effects of various manipulations suggest that the neutrophils are important in temporary, but not permanent, focal ischemia (203, 775, 1285). The evidence is generally strong and is bolstered by the probable involvement of neutrophils in ischemic injury in other tissues (399, 933). However, there are important conflicting reports and alternate interpretations of data that need to be considered. Furthermore, it remains to be determined whether damage results from effects on blood flow or from direct toxic effects on the vasculature or the neurons.

There is an accumulation of neutrophils in infarcted tissue 24 h after permanent or temporary ischemia (63, 120, 542, 1244). At this time, neutrophils are present in the vasculature and have, in most cases, infiltrated the parenchyma (63, 353). A very notable exception to the latter is human stroke where neutrophils accumulate early in severe cases (9) but, in the one relevant study, were almost exclusively confined to the vessels even more than a day after the stroke (665).

If it helps cause neuronal damage, neutrophil accumulation should be present well before 24 h after ischemia, and this does seem to be the case. Polymorphonuclear leukocytes accumulate in blood vessels within 4 h of the onset of permanent (353, 592), or temporary (563) ischemia (the latter measurements made in baboons). Perhaps the most striking evidence correlating neutrophil accumulation with damage emerges from studies using PET scans on humans. Accumulation begins within 6 h of the stroke, and there is a striking positive correlation between the amount and duration of leukocyte accumulation and both the size of the infarct and the severity of neurological outcome (9).

An important issue is whether the neutrophils act after infiltration through the blood vessels. Significant infiltration into the parenchyma only begins 12 h after the onset of permanent occlusion, by which time there is major cell necrosis (353, 1283), but it is more rapid following temporary ischemia, the insult for which there is evidence that neutrophils are damaging. There was significant accumulation 6 h after 1- to 2-h occlusion in two different studies (202, 542, 1283), which increased by 50% at 12 h (1283). This time course is consistent with parenchymal neutrophils causing cell damage. However, in one of the studies, the location of the early accumulation was not described (202) and in the other the accumulation did not reach significance in cortex at 6 or 12 h (1283); cortex is the region in which damage is prevented by antileukocyte treatment. Thus there is no well-documented evidence that accumulation in the parenchymal tissue is likely to be causing damage.

In contrast to the large number of results showing timely accumulation of neutrophils in at least the vasculature, Bartus and co-workers (435) found that there was no significant neutrophil accumulation until 21 h after 2-h distal MCA occlusion occlusion despite the occurrence of infarct by 8 h. There is other evidence that neutrophil accumulation does not always accompany cell damage. Two very careful sets of studies on spontaneously hypertensive rats, using essentially identical methodologies, showed either a large neutrophil accumulation 6-12 h after 80 min of MCA occlusion in the penumbral tissue (63, 202) or no neutrophil accumulation except in severely damaged tissue after 60 min of MCA occlusion (642). In the latter, there was a major activation of microglia/macrophages ~6 h after the occlusion. Thus as yet unidentified factors may determine the nature of the inflammatory response to an insult.

It has been suggested that artifactual mechanical effects of the insult may actually cause the neutrophil accumulation (for example, filament occlusion or laser irradiation) (435), and this has not been ruled out by careful studies. However, neutrophils do accumulate in the vasculature after human stroke (9, 665), where there is clearly no mechanical artifact. At present, there is no simple explanation for the conflicting data on neutrophil accumulation in rodents. Certainly they suggest that these white blood cells may not always be important in damage.

Neutrophil accumulation in vessels requires interactions between several adhesion molecules. These include intracellular adhesion molecule (ICAM)-1 and E- and P-selectins on the endothelial cells, fibronectin and laminin in the extracellular matrix, and integrins and L-selectins on the white blood cells. There are excellent short reviews of the process (204, 964, 1216) and, indeed, antibodies and peptides that bind to these molecules profoundly inhibit the accumulation of neutrophils after transient or permanent ischemia (197, 1243, 1245, 1285, 1287).

Ischemia activates this adhesion system. On endothelial cells, ICAM is increased 2 h after 2-h MCA occlusion (542, 1286) and also after human stroke (665). E-selectin is upregulated some time between 4 and 24 h (427, 1287). On the neutrophils, the integrin complex CD11/CD18 may also be upregulated, although this is less certain (63).

There is reasonable but very incomplete evidence that one or both cytokines, interleukin (IL)-1 and tumor necrosis factor- (TNF-), which do upregulate ICAM (965), are mediators of this response. It seems likely that there is a cascade involving free radical generation, increased TNF- and IL-1, and the activation of the nuclear transcription factor NFB. (1186). The NFB upregulates more proximally than the cytokines and is probably a critical intermediary step. Other factors are also probably necessary (56). There may be important positive feedback between the cytokines and NFB and free radicals as they all are able to activate each other (1186), and it is not clear which of these is the initiating step.

Tumor necrosis factor- is increased within 30 min of the onset of focal ischemia in the circulation and in brain tissue (154, 636), as is IL-1 (1295). The only study directly assessing the importance of the cytokines showed that treatment with the IL-1 receptor antagonist reduced accumulation of neutrophils during permanent MCA occlusion (352). Less directly, but still significant, blockade of TNF- action with a recombinant receptor greatly enhanced the number of perfused microvessels at the end of 4-h permanent MCA occlusion (412).

The postulated interaction is supported by studies in cultured endothelial cells (1035). Hypoxia induced the formation of IL-1, the subsequent expression of ICAM and E-selectin, and increased adherence of leukocytes. Antagonizing IL-1 action attenuated leukocyte adherence by ~60% (unfortunately adhesion molecule changes were not explicitly measured). A similar effect was observed in microvascular cells from human brain (1068). Further studies on these cells show that activation of the transcription factor NFB is very probably critical to the expression of ICAM. Hypoxia and reoxygenation caused activation of NFB and, later, ICAM (464). Both these responses were blunted by the proteasome inhibitor n-tosyl-Phe-chloromethylketone (464), which blocks the activation of NFB. The ICAM upregulation was also blocked by the free radical scavenger pyrrolidine dithiocarbamate, and cerebral endothelial cells in culture produce abundant free radicals under these conditions (1226).

Although evidence for protective effects of blocking cytokine action is now abundant (see sect. VIIIE), this cannot be taken to show that leukocyte accumulation is damaging; blockade of cytokines reduces tissue damage even in permanent ischemia (352, 946), against which adhesion antagonists are ineffective (203, 775, 1285).

A very large number of studies show that conditions that reduce or completely block leukocyte accumulation greatly reduce infarct size. Infarct volume 24 h after 45- to 60-min ischemia was reduced by about two-thirds when circulating neutrophils in mice (215) or in rats (729) were completely depleted with an antineutrophil antibody. Inhibiting the activity of the endothelial adhesion molecule ICAM, either by creating null mice (215, 1062), or using monoclonal antibodies in rats (197, 730, 1285), strongly reduced neutrophil accumulation and reduced infarction size by 50% (rats) to 75% (mice). Slightly smaller reductions, ~33%, were produced in rats by antibodies to the neutrophil integrins CD10/CD18 (197, 730) and by peptides that interfere with fibronectin binding (1243, 1244) or laminin binding (1245). A peptide that binds to selectin (775) decreased infarct volume by >50% in spontaneously hypertensive rats. Many of these studies involved 45-min to 1-h exposures to ischemia, but in some, ischemia was 2 or 3 h so the protective effects are not restricted to short insults. These effects are quite dramatic; there is essentially no further development of infarct once reperfusion begins.

Importantly, leukocytes do not appear to be involved in damage during permanent focal ischemia, despite their accumulation in the tissue vasculature (120, 542). Several interventions against this insult, which are protective against temporary ischemic damage, were ineffective in preventing damage (203, 775, 1285).

Although these studies taken together are impressive indictments of neutrophils in ischemic damage, there is at least one exception, which has not been explained away. In the study where no neutrophil accumulation was measured, neutropenia had no protective effect (435). Because of this, it is important to consider alternate explanations for the effects of the antileukocyte accumulation studies.

Neutrophil depletion may be protective as a result of lowering blood levels of damaging cytokines, TNF- and IL-1, that can be produced by these cells (280). Measurements of the effects of neutropenia, or other protective treatments, on cytokine levels during ischemia would help interpret results. The adhesion molecules and basal laminar proteins may also allow critical damage to vasculature or neurons in ways other than via leukocyte accumulation. Indeed, there is a marked depletion of lamin and fibronectin that is thought to contribute to microvascular damage (416). Perhaps the laminin and fibronectin peptides that attenuate damage (1243, 1245) are acting by maintaining vascular integrity. Although these explanations are not particularly likely, it is important to ensure that they, or others, are not correct. This has not been done.

Leukocytes might induce damage by causing local vascular occlusion, or they might initiate toxic reactions, including free radical production by NADH oxidase, production of hypochlorous acid, or protease activation (504, 728, 1200). The latter two require prior free radical production.

A) CHEMICAL TOXICITY. Efforts to determine whether free radical production is important have not been conclusive. The one study in which effect of neutrophils on free radicals was actually measured, carried out by Kogure and co-workers (728), showed that neutropenia completely abolished the generation of free radicals in cortex after 60-min MCA occlusion. This very dramatic effect was seen by measuring formation of ascorbyl free radicals (728), a reliable semiquantitative method (143). Unfortunately, this is the only such report, and the only study in which this method has been used to measure free radical formation. Further experimental support for the conclusion is very necessary as it seems, a priori, unlikely that neurons and glia themselves do not produce free radicals during and after ischemia. If it is correct, it ascribes a very important role to neutrophils in damage, as free radical attenuation produces major reductions in infarct size. However, at this stage, it cannot be considered likely.

Neutrophils produce free radicals via activation of NADPH oxidase. Walder et al. (1185) studied transgenic mice lacking NADPH oxidase. Infarct size was reduced by 50%, suggesting that leukocyte NADPH oxidase mediated the damaging effect of the neutrophils. However, Walder et al. (1185) then found that selective elimination of either leukocyte or parenchymal NADPH oxidase did not reduce damage; both had to be eliminated. Thus elimination of neutrophil NADPH oxidase does not account for the protective effects of eliminating neutrophil accumulation; if it did, then eliminating the enzyme selectively in neutrophils would have prevented damage.

An hypothesis that is consistent with both sets of studies is that neutrophils are necessary for the generation of toxic levels of free radicals but themselves are not the major source of the free radicals. Thus large-scale free radical production would be dependent on leukocytes, as noted by Kogure and co-workers (728), but could be initiated by NADH oxidase in parenchymal tissue or in the leukocytes. The way in which leukocytes might lead to free radical generation is unknown. It might be by producing cytokines (280, 349) which then activate free radical and NO production by parenchymal tissue or by leukocytes themselves (177, 671). Certainly, the data of Walder et al. (1185) suggest a central role for NADH oxidase. Unfortunately this has not been explored further.

B) INDUCIBLE NOS. The inducible form of the enzyme, iNOS, begins to appear in neutrophils that have invaded the ischemic tissue between 1 and 2 days after the onset of permanent focal ischemia (480). Any role for this iNOS is very questionable because blocking neutrophil accumulation has no effect on permanent focal ischemic damage (1285). Thus the fact that aminoguanidine (AG), which inhibits iNOS, attenuated the increase in infarct size that occurred between 24 and 72 h (481) may well be due to inhibition of the iNOS induced in endothelial cells (479, 836). Alternatively, AG may be protective because it is inhibiting another enzyme, for example, polyamine oxidase, which is very damaging (208, 498). Although protection was eliminated by the NO precursor arginine (481), this does not necessarily show that inhibition of NOS by the AG was giving the protection. The excess NO production due to arginine might have overwhelmed the effects of inhibiting another damaging reaction.

Alternatives to free radical-mediated damage are damage mediated by hypochlorous acid produced by myeloperoxidase, or protease activation; they have not been explicitly tested.

C) BLOOD FLOW REDUCTION. The alternative to chemically mediated damage is that leukocytes block blood flow (no reflow). White cell accumulation does appear to attenuate blood flow in the early postischemic period (63, 775). However, evidence that this is damaging is not at all compelling because, as discussed in section IIK, there is no evidence that postischemic penumbral blood flow is limiting. One hour after 3-h ischemia, 40% of the small (<6 µm) capillaries appear to be plugged by leukocytes (247) but at many later time points after 2-h ischemia there was much less vessel plugging (~10%) in core or penumbral regions (1283). Indeed, plugging was far less than during permanent ischemia, where it was ~50% (353, 1283). Thus, if vessel plugging is important, it is surprising that antileukocyte adhesion paradigms do not reduce infarct size in permanent ischemia.

Hemispheric blood flow 24 h after ischemia was improved by antileukocyte treatment (e.g., Ref. 215). However, this may well reflect the attenuation of infarct size and hence more actively metabolizing tissue in the protected animals.

There is no evidence that leukocytes enhance damage after normal short global ischemic episodes. There was a delayed accumulation of leukocytes 3 h after a global insult, but only if ischemia was extended to 40 min or longer, far longer than the 10 min necessary to get major damage (20). When neutropenic rats were challenged with 15-min global ischemia, there was elevation of blood flow in the postischemic period relative to control rats, but there was no amelioration of damage to CA1 or CA3 neurons (1007).

On the other hand, there is proliferation of microglia in damaged regions within a day after short-term global ischemia, which is prolonged in regions that will go on to suffer damage (512). Intringuingly, when proliferation of the microglia is prevented by tetracycline derivatives, minocycline, or doxycycline, damage after 5-min ischemia in gerbil is attenuated by 75% (1265). Although this is consistent with an important role for microglia in damage, it does not prove it. Nevertheless, the fact that microglia are major sources of caspase-1 (ICE) by 2 days after ischemia (94) and so may contribute to IL-1 production and iNOS production (1265), indicate that these cells may play an important role in damage (512).

Unilateral carotid occlusion is combined with hypoxia. In adults, the oxygen is lowered to ~3% and leads to eventual infarction in the MCA territory. It is essentially a focal insult that is now used infrequently (895) because adult animals do not survive the prolonged hypoxia reliably enough (948). However, the technique has been very successfully adapted to neonates that can reliably survive for days after insults of up to 3.5 h, and it is considered a very good model for major forms of neonatal metabolic brain damage (1164). Oxygen is generally lowered to 8% (for review, see Refs. 948, 959, 1141). This is almost a pure hypoxic insult, because the hypoxic vasodilation brings blood flow up to near control levels on the ligated side (982), although lateral cortex and caudoputamen still show blood flow reductions (368).

Fifteen to thirty minutes of hypoxemia (3%) causes early ischemic cell change and delayed neuronal death in CA1-CA3, striatum, and layer 5 of cortex in adults, much like global ischemia (980). In young rats, exposures of 60 min or longer to 8% hypoxia cause delayed development of infarct providing ATP levels fall to between 50 and 70% of control values (1213), much like their values in focal ischemia.

K.  Postischemic Blood Flow and Damage

1.  Global ischemia

There are major changes in brain blood flow after global ischemia. It is important to know if they exacerbate damage because drugs used to study damage mechanisms often affect blood flow also.

There is a rapid hyperemia following vessel occlusion or complete ischemia, which lasts ~15 min, where blood flow may be as much as a two- to threefold higher than control levels (228, 515, 931). There is then almost (1180) always a marked hypoperfusion that lasts between 6 and 24 h, with blood flow between 30 and 50% of normal (228, 291, 297, 515, 632, 760, 931, 1070).

The literature strongly suggests that this postischemic hypoperfusion does not cause damage; rather, it appears to reflect a reduced metabolic rate in the postischemic tissue. The latter may reflect mitochondrial damage or reductions in ATP-utilizing processes. Whatever its basis, there is an excellent correlation between the lowered glucose utilization and the hypoperfusion after global ischemia in dog (291, 756) and rat (606, 931). pH, ATP, and lactate levels are about normal (228). Neutrophil depletion had no effect on the marked hypoperfusion in hippocampus and striatum, two very vulnerable structures (396), further indicating that the hypoperfusion in these vulnerable regions is metabolically driven. Neutrophil depletion did increase flow somewhat in nonvulnerable structures (396).

In contrast to the above studies, when ischemia is prolonged beyond the time required to cause major neuronal death there is a larger reduction in blood flow than in glucose metabolism or high-energy phosphate utilization, in tissues which will become damaged (367, 645, 646). In one of these cases, the region that suffered damage, the striatum, was the region with the largest dissociation between flow and metabolic rate. Although the mismatch may contribute to damage in extreme cases, shorter ischemic exposures are lethal in the absence of any mismatch between flow and metabolism.

There is no compelling evidence that reduced postischemic flow following temporary focal ischemia contributes to development of the infarct. Core regions have normal blood flow levels (137, 798), whereas flow in the penumbra is generally ~40-50% of normal during the 12 h after temporary focal ischemia, when damage is developing (184, 798). However, as with global ischemia, there is no evidence for a mismatch between flow and metabolism. In penumbral regions destined for infarct, glucose utilization is decreased by 50% or more in the first hour after ischemia, and recent detailed studies show a strong correlation between the decrease in blood flow and in glucose metabolism in the postischemic period (79, 1299). In the few measurements that have been made, ATP levels during this time are between 70 and 100% of their preischemic values, only a small reduction. One study showed a reasonable correlation between increased postischemic blood flow and reduction of early infarct size when prostaglandin synthesis was inhibited (184). However, there are many other potentially beneficial effects of blocking prostaglandin metabolism aside from vasodilation, for example, preventing free radical production. Thus the experiments do not establish the importance of postischemic blood flow, and the general preservation of metabolism-to-flow ratios argues against reduced flow as a cause of damage. This was also discussed in section III.

The role of vascular change, and resulting edema, will not be considered at any length in this review because it really is its own topic. Nevertheless, it is useful to have a general idea of what contribution vascular changes may make to damage. Certainly capillary endothelial cells are quite vulnerable to short periods of anoxia and reperfusion. Cultured brain endothelial cells are damaged in a free radical-dependent manner after 20 min of anoxia, as assessed by a large increase in lactate dehydrogenase (LDH) leakage (1226).

The blood-brain barrier becomes severalfold more permeable to poorly permeant small solutes such as inulin and sucrose 6 h after global ischemia but then returns to normal by ~24 h (922). There is little evidence of major uptake of protein into the brain during much of the postischemic period, except immediately after the insult. However, starting ~12-24 h before delayed cell death, there is a quite massive uptake of horseradish peroxidase or protein-bound Evans blue into the parenchyma (370). This may well result from activation of pinocytosis in the endothelial cells because there is no major extracellular edema and there are no reports of major cell damage to the endothelium. There is no indication as to whether the increased blood-brain barrier transport contributes to damage.

Focal ischemia is quite different. There is a major extracellular edema that develops early during the focal ischemic insult, largely due to activation of Na⁺ transport across the blood-brain barrier (91). Reperfusion and very prolonged permanent ischemia both cause much more dramatic changes. There is damage to the endothelial cell layer, along with extravasation of protein and protein-bound Evans blue, as early as 30 min after recirculation following 60-min ischemia. This does not occur during at least 6 h of permanent ischemia (527, 800, 1247). The dependence of this vascular damage on reperfusion is striking; it may result from enhanced free radical production (849), but this has not been proven. This endothelial transport change very probably contributes to edema also (1247).

The relationship of the vascular damage and extracellular edema to cell death has not been well studied. Some evidence indicates that at least early neuronal death occurs in the absence of vascular changes. In a study specifically directed at this question, there was significant ischemic cell change 6 h after 1-h focal ischemia in caudate putamen, but there was no evidence for extravasation of albumin, or leakage of the smaller molecule, aminobutyric acid from the circulation into the parenchyma of the caudate-putamen at that time (10).

On the other hand, recent studies suggest that movement of molecules from blood to brain across the "damaged" endothelium are important in the general development of neuronal damage. A matrix metalloproteinase, MMP-9, was upregulated in endothelial cells during the first 24 h of permanent ischemia, probably by cytokines, and when an antibody to this enzyme was administered systemically, it reduced infarct size after 24 h, by 30% (958). Both MMP-9 and other matrix metalloproteins act on the extracellular matrix associated with the vascular basement membrane, and it is reasonable to assume that protection by the antibody results from inhibition of basement membrane breakdown and maintenance of vascular integrity. Further studies are needed to show that vascular integrity is indeed maintained when the enzyme is inhibited. It would be of much interest to make a similar study in temporary ischemia, where vascular integrity seems to be most compromised.

Although there is no established causal relationship between edema and neuronal damage, there is a very strong correlation between infarct size and the amount of hemispheric edema when protective, or damaging, agents are used to alter cell death (e.g., Ref. 527). Indeed, edema is often used as a measure of damage. Effects of edema on blood flow might be important; for example, it may partially account for the progressive decrease in blood flow in the penumbra during occlusion, discussed above. However, at this stage there is no evidence that edema contributes directly to cell damage. Increased edema may simply reflect local blood-brain barrier damage caused by the damaged neurons, glia, and endothelial cells.

M.  Effects of Temperature on Damage

1.  Artifactual maintenance of temperature

There is little spontaneous change in brain or core temperature during focal ischemia except in the filament occlusion model discussed above, where temperature rises.

However, global ischemia is very different. Brain temperature in gerbils and rats drops spontaneously by 3-5°C (152, 762, 1241). Core temperature drops by about the same amount in rats, but actually rises somewhat in gerbils (211, 584, 628). Temperatures recover within ~1 min of reperfusion (152, 762), although in gerbils there is a short period of hyperthermia beginning ~15 min after ischemia and lasting ~45 min. It is 1.5°C in anesthetized and 2.5°C in unanesthetized animals (768). Because of its strong effect on damage, brain temperature should be known or regulated during and for at least 24 h after an insult (see below). This means maintaining the temperature of the brain at a known level during the insult, with a lamp or other device, and it means maintaining or knowing body temperature after the insult. These precautions eliminate the possibility that treatments will affect brain temperature during the insult via changes in flow or other variables and eliminate the possibility that they will affect core temperature, and hence brain temperature, after the insult. Many agents do the latter when the animal is not regulated. Brain temperature is most faithfully monitored in the temporalis muscle (152). However, when it is warmed by a lamp, the skull faithfully remains ~1-2°C below brain temperature (211, 762) and is easier to monitor. Although brain temperature is now generally well regulated during an insult (152, 211, 762), there is quite a lot of variability in the duration for which core temperature is maintained after an insult, and this seriously compromises some studies. These cases are pointed out in the review.

Table 2 shows the dramatic effects of different brain temperatures during or after global ischemia on the degree of postischemic damage.

Not shown in Table 2, damage is actually exacerbated when brain temperature is maintained at 39°C (258).

Effects of temperature are manifested for long periods after an insult. Hypothermia is protective when it occurs up to 12 h after 2-VO, and if temperature is elevated to 39°C even 24 h after mild (5-7 min) 2-VO, global ischemia damage is strongly enhanced (49). Thus even the delayed processes involved in cell death are remarkably temperature sensitive. Temperature differences during the insult do not just influence the rate of appearance of damage; they affect the final outcome. For example, hypothermic protection lasts for at least 1 mo in adults and 6 mo in young gerbils (219). Temperature thus strongly determines the damaging potential of insults.

Temperature is also important in focal ischemic damage. Mild hypothermia reduced infarct size by 35% during 12-h permanent MCA occlusion (53). Reducing temperature to 30°C for 1 h after 2-h temporary focal ischemia reduced infarct size, measured after a week, by 50% (1284). Hypothermia (34°C) was very protective against 3-h hypoxia/ischemia in young rats (1235).

These results show that at least one of the major processes involved in ischemic damage is extremely sensitive to temperature. Furthermore, the very long-lasting sensitivity discussed above indicates that a process occurring at least 24 h after the insult is also temperature sensitive. Unfortunately, as is discussed in several of the ensuing sections, a great many potential contributors to ischemic damage are very sensitive to temperature so that no particular mechanistic information is provided by the dependence (see Ref. 62 for a good discussion of this).

N.  In Vitro Models: Brain Slice

1.  Nature of insult

Brain slices, and particularly the hippocampal slice, have become widely used models for studying anoxic or ischemic damage (531, 673, 686, 695, 1008). In this insult the bathing solution is rapidly changed from O₂/CO₂ equilibrated to N₂/CO₂ equilibrated. When glucose is maintained in the anoxic buffer, the insult is termed anoxia (or hypoxia), and when glucose is omitted, the insult is termed in vitro ischemia or oxygen/glucose deprivation. Adenosine 5'-triphosphate falls less completely during in vitro ischemia than it does during global ischemia and falls more slowly in the presence of glucose (Table 1). Lowering temperature reduces the degree of damage (949).

Generally 5-7 min of in vitro ischemia at 36-37°C, or a period of ischemia extending 2-3 min beyond the anoxic depolarization, leads to rapid damage to various properties of CA1 pyramidal cells which lasts for the 8- to 14-h life of the slice. Somewhat longer exposures are necessary for damage to dentate granule cells so that slices do show the same ranking of selective vulnerability as in vivo tissue. However, differences are not nearly as dramatic because acute damage, rather than prolonged damage, is being measured (532), and acute damages are very similar in different hippocampal regions in vivo (348).

Properties that are damaged include synaptic transmission (531, 1008), protein synthesis (164, 936), maintenance of ATP levels (916), cytoskeletal integrity (1291), and neuronal morphology (936). Damage to all these occur within the first 30 min and persist throughout the reperfusion period (286, 348, 916). Changes in protein synthesis are similar to those observed in vivo, but the profound loss in (evoked) synaptic transmission and the very intense morphological damage and disruption of the cytoskeleton are generally not observed as early or as strongly in vivo, indicating that slices are more sensitive to ischemic damage than are cells in situ. Nevertheless, slices fulfill a very important role by facilitating analysis of mechanisms of early changes. Although it is always possible that these are different from in vivo, rates of change of ions, metabolites, and protein synthesis that have been measured so far are quite similar to those measured in vivo, suggesting that mechanisms are the same.

Shorter insults, which are stopped just when the anoxic depolarization occurs, lead to a more slowly developing damage, requiring ~12 h to be manifested. This is closer to the delayed neuronal death seen in vivo. To date, only damage to synaptic transmission has been studied in this way (1188), but the approach holds much promise for more sophisticated comparisons between slice and in vivo conditions.

O.  In Vitro Models: Cell Cultures

1.  Different models

Primary neuronal/glial cultures from cortex (237, 376), hippocampus (704, 1199), cerebellum, and hypothalamus (791) of embryo or perinatal rats and mice have been used extensively to study anoxic or ischemic damage since 1983 (966). Organotypic hippocampal slice cultures from perinatal rats were first used in about 1995 and are being used increasingly (1072, 1073). They promise to become more valuable models, particularly as they show delayed death (923, 1072, 1073). However, although they do show some selective vulnerability of CA1 versus CA3 versus dentate gyrus if the in vitro ischemia is kept relatively short (30 min) (65, 1072), it is not very dramatic. There is twice as much cell death in CA1 as CA3. Longer durations (60 min) produce larger and equal amounts of cell death in all hippocampal regions (947, 1072). Like the cultured cells, this system also requires prolonged in vitro ischemia to produce cell damage (1072).

In most studies the insult is in vitro ischemia, although there are several studies of "chemical" anoxia or "chemical" ischemia in which oxygen is maintained but mitochondrial inhibitors are used (1199). The latter is a poor model, and its results will not be considered here. Free radical generation is almost undoubtedly much higher than during anoxic conditions. Indeed, in liver cells, such damage is often markedly decreased by concurrent anoxia (115, 234).

Damage usually develops 8-24 h after 30- to 60-min in vitro ischemia and is generally monitored as a gross increase in membrane permeability to dyes or protein, in particular leak of LDH (791) or nonexclusion of trypan blue or propidium iodide (1073). Sometimes frank disappearance or disintegration of the cells is measured (376), and more recently apoptotic and necrotic changes are being monitored by standard methods of nuclear staining (405).

A major difference from damage in vivo is the very long duration of oxygen and glucose deprivation that is generally required to induce cell death. This is true for both primary cultures and organotypic cultures (376, 1072). Although 30- to 60-min deprivation is not long for focal insults, the degree of oxygen and glucose deprivation that cultures are exposed to is similar to that in global ischemia, where 5-10 min at 36°C produces profound delayed damage. There are several possible reasons for the difference. In the widely used model developed by Goldberg and Choi (376), ATP does not fall nearly as much as it does in vivo (Table 1) and, probably relatedly, the release of glutamate is very delayed (376). The small fall in ATP would certainly explain the requirement for the prolonged insult, because it approximates that in the focal ischemic penumbra (Table 1). Why the energy disturbance is less is not apparent, but an intriguing possibility is that the cells in culture may adopt protective mechanisms that reduce ATP turnover during hypoxia as has been nicely discussed for the general case by Hochachka et al. (453). This possibility is given added credibility by the fact that the ATP fall during chemical hypoxia is far greater than during actual anoxia (Table 1).

Another significant difference between cell cultures and in vivo tissue is that damage in primary cultures following the 30-45 min exposures is completely dependent on activation of NMDA receptors (376). Longer insults (90 min) produce an NMDA-independent form of damage that appears largely apoptotic (405). In organotypic cultures, NMDA antagonists provide only partial protection (5), and combined glutamate receptor antagonists are required to protect against 60-min in vitro ischemia (1073). In that sense, the organotypic cultures seem better models for in vivo events and at this stage seem a better route to take.

Although damage in cultures is easily studied, it is, of course, essential to verify any inferences by studies of in vivo tissue. Neuronal gene expression is likely to alter very significantly in culture; the anatomical relationships between cells are very different, and there are no vascular cells. The latter two objections do not pertain for organotypic cultures.

P.  Summary

1.  In vitro systems

The goal of research on ischemia is to understand what occurs in vivo. In vitro systems are able to provide clues, but for reasons discussed above, and others, conclusions cannot be simply extended to in vivo situations. The absence of blood flow as a variable, and the absence of blood vessels themselves in cultures, facilitates interpretations. However, by the same token, the absences eliminate what may be important components of the damage process. It must be borne in mind that all tissue types suffer ischemic damage, so to show that both neuronal cultures and in vivo brain tissue suffer delayed ischemic damage does not mean that the mechanisms are the same.

The composition of brain slices is closer to that of in vivo tissue, and there is little time for major genetic change. However, slices are in a compromised metabolic state, with low ATP values and elevated aerobic glycolysis (677), and are hypersensitive to ischemic insults. They show different morphologies from in vivo tissue, and the preparation of the slice itself induces transcription of some stress-related mRNA and immediate early genes, as well as accumulation of Fos and Jun immunoreactivities (1303). The consequences of this are not known but may alter ischemic sensitivity. Culture and slice work can be used as bases for later in vivo studies but the latter, eventually, must be done.

Global and focal ischemias are two very different insults. Global ischemia involves a short (usually 15 min or less) very intense insult in which ATP is severely lowered. It is characterized by a slow development of cell death during reperfusion, which shows great selectivity. There is no intimation of involvement of the vasculature, although there is a real possibility that microglia/macrophages play a role in the process. Overall, the insult is straightforward and quite uniform, but in the hands of one group, residual blood flow is much higher than the usual 1-3% (1089).

Focal ischemia is a far more complex insult, with several unresolved issues. There are many variations of the model including whether or not there is reperfusion (temporary vs. permanent), where the lesion is, the strain of rat, and the degree of temperature control. These lead to a very complex literature. One goal of this section has been to describe these variations well enough so that results obtained in different systems can be related to each other.

A) CORE AND PENUMBRA. Ionic and metabolic changes in the core and penumbra are dramatically different, and these probably account for the great disparity in how well the two regions can be protected by pharmacological interventions. The fall in ATP in the penumbra is quite small (levels do not fall much below 70% of control during development of damage in permanent ischemia), and it really is not clear that this fall per se is adequate to cause damage. For example, when damage was initiated by mitochondrial blockers, ATP fell to 25% for several hours, and the lesion developed over 1-2 days (736). A real possibility is that effects in the core add to the insult in the penumbra and are essential for damage. Examples are diffusion of K⁺ and glutamate into the region. The intraischemic depolarizations that may be important in development of damage in the penumbra may well be driven by ions and glutamate generated in the core. Core damage is terribly severe when ischemia is maintained for >1 h.

B) TEMPORARY VERSUS PERMANENT FOCAL ISCHEMIA. Development of damage after 2- to 3-h temporary ischemia and during permanent ischemia are very similar in many detailed respects. One explanation for this is that all the important events occur in the first 2-3 h so that reperfusion does not have important effects; there is no doubt that this is true to some extent. As an important example, ATP levels in the penumbra after temporary ischemia are about the same as during permanent ischemia; they do not appear to recover very well, indicating permanently compromised metabolism. This might help maintain the similarity between the two insults. However, there are important differences; free radical and NO generation are both enhanced during the first 40-60 min of reperfusion (622, 893). Vascular damage is much greater after temporary ischemia. The most glaring difference is the apparent importance of leukocytes in damage following temporary ischemia. In some way, these cells appear necessary to allow damage to continue to develop in the reperfusion period. They are not necessary for development of damage in permanent ischemia.

C) GLIAL AND VASCULAR RESPONSES. The astroglial responses to focal and global ischemia are very different from each other. Global ischemia is characterized by quite rapid glial hypertrophy, increased protein synthesis, and hyperplasia (642), whereas the more prolonged focal ischemia is characterized by quite rapid glial swelling and, eventually, cell death during formation of the infarct (355). Glial cell death in the core, and possibly penumbra, of the infarct might well result from combination of lowered pH and ischemia; combining pH 6.2 with respiratory chain inhibition kills glia cells in culture within several hours (1090). It is important to consider that glial damage may, in some way, be enhancing neuronal damage.

Roles for vascular changes and extracellular edema in development of damage in focal ischemia are not yet compelling, although evidence that a metalloprotease that attacks the basement membrane is likely to be important in damage raises the issue of the role of the vasculature. There is currently no resolution.

Of paramount importance is the question of how well these animal models replicate events during and after human stroke. This issue is outside the scope of this review. However, the difficulty to date in successfully applying principles learned from these models to human therapy trials suggests that there are added complexities in the human disease. These may include greater sensitivities to detrimental side effects of some drugs, such as MK-801 and its analogs (135, 299), or more complex processes of cell death that cannot be halted by only one pharmacological intervention.

	III. MORPHOLOGIES AND BIOCHEMISTRIES OF ISCHEMIC CELL DEATH

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As discussed in section I, there are at least three recognizable pathways of ischemic cell death: necrotic cell death, apoptotic cell death, and, very probably, autophagocytotic cell death.

The purpose of this section is to describe the end stages of these pathways and to understand why different pathways are taken in different cases. Another elusive goal is to understand the molecular changes that constitute these end stages.

Most of the morphological changes associated with ischemic cell death were defined by Spielmeyer in the early part of the 20th century (1063) and led to work in the 1960s and 1970s by Brown and Brierly (129, 130, 355) that quite rigorously defined different stages of cell damage including forms of cell death. The earlier work, as well as that of Brown and Brierly, has been nicely reviewed (121, 128, 392). There are three categories of necrotic cell death.

A) DESCRIPTION. The cytoplasm is very swollen. Some mitochondria are swollen with disrupted cristae, but others are rounded and slightly shrunken. Endoplasmic reticulum, the Golgi apparatus, and polysomes are no longer apparent as complete structures but remnants, often with attached ribosomes that exist and often accumulate close to the nucleus. Microtubules and other filamentous structures are absent, and the cytoplasm is almost clear. The plasma membrane is irregular and sometimes shows frank breaks, but this is very rare. The nucleus is normal except for irregular clumping of chromatin (520, 521, 942). These changes have some characteristics of what is termed peripheral chromatolysis (392, 897).

B) OCCURRENCE. This end stage is not seen very often. It is predominant after 3- to 4-h hypobaric/ischemia in young animals (490), in whom it is also produced by glutamate exposure (490, 852, 920). Agonists of NMDA induce it in striatum of adult brain (919). It occurs 90 min after 30- or 10-min global vessel-occlusion ischemia during hyperglycemic conditions (494, 522) and occurs in the final stages of delayed death in gerbil and rat global ischemia, where the existence of some "swollen cells" is described, along with other end-stage morphologies (579, 582, 897, 942) and where, in one case, electron micrographs show this morphology (897).

A) DESCRIPTION. This end stage contrasts markedly with ECC and is far more common. In its more exaggerated phase, it is termed ICC with incrustations (128). There is a major darkening and shrinkage of the nucleus and cytoplasm (128, 130, 494, 521, 896, 897), and the nucleolus often assumes a honeycomb appearance (319). The plasma membrane and the nuclear membrane are both very irregular, and the cell often assumes a triangular shape (in 2 dimensions). The cytoplasm generally contains many large (1-2 µm diameter) vacuoles, some of which are greatly swollen mitochondria with disrupted cristae (130), but most of which are swollen Golgi cisternae (896), ER (130), or unidentified. Many of the latter may well be autophagic vacuoles (128, 834). Normal ER and Golgi organization into stacks is disrupted. There are few, if any, polysomes, but there is a plethora of ribosomes. At least some microtubules are present. This end stage and its incidence are very elegantly described in a recent review (716).

Viewed in the light microscope, these cells are intensely acidophilic (eosinophilic), which is a defining characteristic of this morphological stage; they are also intensely argyrophilic (223, 437).

B) OCCURRENCE. This end stage is prominent in many ischemic models. The cells coexist with the edematous cells in CA1 at the end of delayed degeneration in rat and gerbil global ischemic models (579, 582, 897), where frank eosinophilia is very apparent (899). They are the dominant form in the quite rapid (within hours) death that occurs in CA4 after global ischemia in gerbil (579) and rat (896). During or after focal ischemia, cells showing this change emerge and remain for several days in the peri-infarct cortex (814). They are abundant in the penumbra for 1-2 days and in the core of the lesion for a relatively short time before the cells disintegrate. They are widespread in adult rats after 40 min sporadic hypoxia/ischemia (121) where they take ~30 min to 1 h to appear after 40-min hypoxia and where they persist for ~24 h. Thus they constitute a relatively stable state toward which cells tend following ischemia. Ischemic cell change may well require reperfusion to form, because it is not seen immediately after even prolonged complete ischemia (521). This has not been rigorously tested. If so, there are many possible reasons such as the requirement for free radicals or near-normal levels of ATP.

A) DESCRIPTION. The nucleus is somewhat shrunken and slightly darker than normal, often with a fragmented membrane. There is irregular chromatin clumping. The cytoplasm is slightly shrunken and appears fragmented with small vesicles and dense bodies (128, 351, 355), but still retains its delimited structure. Although there are some shrunken mitochondria, there are no other recognizable organelles. The cell membrane appears disrupted. Cells are much paler than normal cells in the electron microscope and are weakly eosinophilic, giving the cells their "ghostlike" appearance. Nuclear "integrity" is maintained longer than cytoplasmic integrity (128, 355). This is the most disrupted of the three morphologies.

B) OCCURRENCE. This stage is generally assumed to follow from ICC, and this seems likely based on location and timing (128, 351). It arises ~24 h after anoxia/ischemia (128) and ~6-12 h into permanent focal ischemia in rat, in the core of the lesion (351), and sometime before 48 h after 2-h focal ischemia in the penumbra (652). It has not been described after global ischemic insults, although reported analyses have not been very rigorous (579, 896, 897).

Although these end stages are considered to represent dead cells, this has not been rigorously established except for homogenizing cell change (HCC) which, in the electron microscope is clearly associated with badly fragmented cells. Metabolism and function have not been carefully measured in cells showing ICC or ECC, and although the morphology is very distorted, the possibility of recovery cannot be ruled out. However, after focal ischemia, the disappearance of neurons showing ICC semiquantitatively parallels the decrease in neuronal density in the peri-infarct region of focal lesions (814). Furthermore, although silver-stained ICC neurons were visualized in the infarct after 24 h, several days later only silver-stained terminals remained (484). These results certainly suggest degeneration of the ICC neurons.

B.  Apoptotic Cell Change

1.  General considerations

Apoptosis, in its purest form, is best considered as one of the processes by which cells die in response to normal physiological stimuli (205, 565, 1227). In this sense, it is a "programmed" physiological process. The question is the extent to which this program of changes has been coopted by ischemic cell death.

This would be quite simple to answer if the morphological changes and biochemical events of apoptosis were unambiguously defined. However, there is no overwhelming consensus on this at present. In the next section, a set of criteria that allow this to be done as well as possible are described and discussed.

A large number of reviews detail changes associated with apoptosis in different cell types and conditions (32, 1004). The following are reasonable minimal criteria for identifying apoptosis in ischemia. A key is differentiating it from ischemic cell change that has somewhat similar characteristics.

A) MORPHOLOGICAL CHANGES. The most unequivocal morphological markers are the formation of smoothly contoured spherical-, or lunar crescent-shaped masses of chromatin within the nucleus and the subsequent formation of apoptotic bodies that are cell membrane-bound structures containing cytoplasm and dark chromatin masses (32, 564, 652). This contrasts with ICC where the chromatin is irregularly clumped. Before formation of the apoptotic bodies, the cytoplasm (in electron microscopy) becomes darkened and pynknotic, as in ICC, but neither it nor the nucleus becomes nearly as dark as in ICC, so this is a reasonable way to identify apoptosis (716). The organelles are relatively normal (as distinct from the very swollen mitochondria and cisternae of ICC) (32, 564, 716), but there is often some vacuolization (564, 1006), so the presence of vacuoles cannot be used to identify ICC. Mitochondria usually only become visibly swollen and malformed in the end stages of apoptotic change (541, 919), and even then, changes are often very minor (564) compared with necrotic cell death (541, 564, 919). However, they do swell so that, as with vacuolization, swollen mitochondria cannot unequivocally be used to identify necrosis. However, cell shrinkage and darkening, in the absence of mitochondrial swelling and vacuoles, can be used to identify apoptosis. This, however, is not seen after ischemia, so the issue is not simple. Importantly, in contrast to ICC, cytoplasm does not become eosinophilic. This is a very useful way to distinguish the two processes.

B) BIOCHEMICAL CHANGES. Double-stranded breakdown of DNA into nucleosomal segments is a very strong criterion (111, 859) and is manifested as DNA laddering, with fragments being multiples of ~200 bp. This is not unambiguous identification of apoptosis because it is observed in populations that almost certainly have undergone necrotic cell death (1147). Glutamate toxicity in cultures (404) and in vivo (918) leads to prominent nucleosomal fragments of DNA at the same time as the cell population shows a distinctly necrotic morphology. In situ end labeling of DNA using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), a histochemical approach, is even less selective for apoptosis because it labels both nucleosomal and nonnucleosomal fragments (918); the latter are formed during necrotic cell death. Although not at all selective for nucleosomal cleavage, TUNEL is relatively selective for double-stranded breaks as distinct from singe-stranded nicks (82), so is somewhat discriminatory.

Although the biochemistry of apoptotic death is not completely understood, there are extremely useful identifying characteristics. The activation of caspases, particularly caspase-3 (719, 886), and inhibition of cell death by inhibition of these proteases (886, 1172, 1301) is the strongest criterion, although the ever-emerging complexity of the caspase system makes the situation somewhat uncertain (739). Caspases do not appear to be activated in necrotic death (e.g., Refs. 33, 387). There are several manifestations of caspase-3 activation that can be measured quite readily including cleavage of poly(ADP-ribose) polymerase (PARP) (318), cleavage of caspase itself (953), and characteristic cleavage of spectrin (811). Another important criterion is the release of cytochrome c from mitochondria (941), which activates caspase-3 by binding to the ced4 analog APAF3 and cleaving the procaspase (441). This now appears to be an almost universal step in apoptosis (591, 941); in some cases, another mitochondrial protein, apoptotic initiation factor, may play a role analogous to cytochrome c (1275). Neither of these changes has been noted in necrotic cell change to date. Another unique feature of apoptosis is the transfer of phosphatidylserine to the outer leaflet of the plasma membrane as part of the process by which the cell is targeted for phagocytosis. This can be monitored by binding of annexin V, which preferentially binds to this phospholipid in the presence of Ca²⁺ (1189).

Another frequently occurring feature of apoptotic death is an increase in the ratio of Bax or other proapoptotic members of the Bcl family to the antiapoptotic members of this family, Bcl-2 or Bcl-xL, (940). Thus there are a large number of unique characteristics of apoptosis that can be measured or observed.

Blockade of cell death by inhibition of protein synthesis is sometimes used as a criterion for apoptosis (194), but it is not satisfactory. Not only is apoptosis often immune to inhibition of protein synthesis (61, 718, 1125), but more importantly there is strong evidence for postischemic synthesis of two very damaging enzymes, COX-2 and iNOS (477, 479, 837). Indeed, in a study of effects of mild focal ischemia, cycloheximide was very protective but abolished both DNA laddering and DNA smearing, indicating strong effects on necrotic death as well as apoptotic (304).

Thus, at this stage, uniformly condensed chromatin and apoptotic bodies identified in the light microscope, a shrunken cytoplasm that is not eosinophilic, activation of caspases, and/or inhibition of cell death by caspase inhibitors, extracellular binding of annexin V, and release of cytochrome c into the cytoplasm are all strong indications that the apoptosis process has been activated and is making a major contribution to ischemic cell death.

With the use of these criteria as best as can be done at this time, there is now reasonable evidence that apoptosis contributes significantly to cell death in focal ischemia and anoxia/ischemia. Evidence for its role in global ischemia is much weaker, although aspects of the process almost undoubtedly contribute to cell death there also.

A) GLOBAL ISCHEMIA. I) Chromosomal and morphological changes. There are now many reports of both nucleosomal DNA laddering and TUNEL labeling in extracted tissue and sections of the CA1 region of hippocampus, following short durations of global ischemia in rats (443, 698) and in gerbils (568, 848, 951, 1016). These occur during the period that neuronal death is developing. When observed in detail, TUNEL staining occurs only over cells undergoing severe cell shrinkage and not in adjacent, healthy, cells (568, 834). However, as discussed, these chromosomal changes are not unambiguous identifications of apoptosis (899).

Morphological evidence for apoptosis is weak, despite the title of one publication claiming that delayed neuronal death is apoptosis (834). In that paper, light microscope staining was intense with TUNEL when the cells were dying, but there was no light microscope evidence for chromatin condensation or apoptotic bodies. Some (it is not stated how many) nuclei showed condensed chromatin, but it was not at all smooth edged when viewed in electron microscopy, and the shrunken dark cells showing the condensed chromatin were highly vacuolated and contained autophagosomes. In fact, the process seems far more akin to autophagocytotic cell death than apoptosis. Two studies of rat hippocampus, after quite mild 2-VO (251) and 4-VO (899) insults, failed to note any morphological signs of apoptosis. Only ischemic cell change was noted. This is also true in cat hippocampus after 10-min global ischemia and in Purkinje cells (716).

Apoptotic bodies probably have a short lifetime (~3 h), and it has been suggested that this might explain their absence (150). This seems unlikely because apoptotic cells are readily seen in focal ischemia, hypoxia/ischemia (see sect. IIIB), and also in a global ischemic-like insult to spinal cord (541).

II) Biochemical studies. There are no reported studies of cytochrome c localization changes. There is a series of studies on caspases and their inhibitors which suggest that these enzymes are involved in cell death and thus suggest a contribution from apoptotic processes.

CA1 neurons in the gerbil were almost completely protected against cell death by a broad-spectrum caspase inhibitor (451), with no effect on temperature. The protection persisted, albeit at a lower level, when the hippocampus was injected with IL-1 at levels that were adequate to enhance ischemic damage, indicating that protection did not result from inhibition of ICE (caspase-1) and subsequent reduction of IL-1. However, these studies did not specify the caspase that is involved. Less, but significant, protection was afforded in rat by the more specific caspase-3 inhibitor ZVAD-CHO; 4.5 µg injected into the ventricle saved ~40% of the CA1 neurons after 3 days and 20% of the neurons after 7 days (183). The specific ICE inhibitor YVAD had no effect. These protective effects are not terribly strong but do show an involvement of caspases.

Less direct, but important, evidence for caspase involvement is the upregulation of the enzyme. This occurs either by cleaving the existing procaspases (699) or by synthesizing a new enzyme that is then cleaved. Caspase-3-like mRNA is upregulated between 8 and 24 h after 15- to 30-min ischemia in rat, predominantly in CA1 pyramidal cells, and total caspase-3 protein is increased by 8 h (183, 826). Cleavage of the procaspase increased by 4 h after ischemia, indicating an early activation of the enzyme, although measurements of enzyme activity indicated a somewhat later activation. Caspase-3-like activity measured on artificial substrate was elevated ~10-fold in hippocampus between 8 and 24 h after ischemia, and PARP breakdown began sometime between 24 and 72 h after the insult (183). This timing is consistent with the appearance of active caspase-3 fragments at 24 h after 12-min global ischemia (1232). In gerbils, there was also cleavage of procaspase-3, but the timing was not ascertained (451).

Overall, these studies indicate that activation of caspase-3 occurs fairly early in development of damage, which normally takes 3-4 days. There are some timing details that are difficult to explain such as the fact that total caspase-3 was elevated slightly before the elevation in mRNA and that breakdown of a synthetic substrate by caspase was far earlier than the action on a natural substrate, PARP. These may reflect subtleties of the apoptotic process that are not yet apparent; the work is quite recent, and small discrepancies are likely to become resolved with time.

Although caspase-3 appears to be important in the rat, caspase 2 (Nedd-2) may be more important in gerbil. Nedd-2 mRNA was upregulated severalfold in CA1 and CA3 pyramidal neurons 3-6 h after 5-min ischemia in gerbil. By 12 h, it was down to baseline (572). There was no change in caspase-3 mRNA, nor was caspase-2 upregulated in rat (826). Thus there may be a real species difference here. No protein measurements have been reported.

There are further chemical changes that are suggestive of apoptosis; the proapoptotic proteins BAX (185, 420) and Bcl-Xs (264) are expressed in vulnerable CA1 cells after global ischemia before and during the time of maximal cell death.

III) Summary. Evidence is conflicting. Chemical evidence suggests a contribution of apoptotic processes to cell death after global ischemia insofar as there is DNA laddering, caspase activation that is reasonably timely, and some attenuation of damage by caspase inhibitors. BAX and BCl-Xs expression are also consistent with an apoptotic pathway. Certainly the attenuation of cell death by caspase blockers is not nearly as strong as it is when death is clearly via apoptosis, for example, in nerve growth factor (NGF) or serum withdrawal (739, 1106), but it is substantial.

In ischemia, the caspase inhibitor was protective when added 2 h after the insult (183), and it had to be injected within 12 h of the insult (451). This is early in the cell death process, which lasts ~3-4 days. Caspase activation is a very late step in many cases of apoptosis (963, 1106, 1302), generally being activated by mitochondrial release of cytochrome c (441), but this apparently is not the case in global ischemia, or if it is, then early caspase activation is also important. The implications of this are considered in section IVC.

Although biochemical evidence strongly indicates that apoptotic processes occur, morphological evidence does not support a standard apoptotic process. Most importantly, there is no evidence of spherical nuclear chromatin clumping/apoptotic bodies at the light microscope level, and these are a hallmark of classical apoptosis. Perhaps the caspase-activated endonuclease (301) is not able to act after ischemia. At the electron microscopic level, the cytoplasms and nuclei of cells appear too dark and too disorganized (e.g., Ref. 716). Furthermore, almost all cells are eosinophilic.

The most reasonable overall assessment of the situation is that caspases are very clearly activated and that their proteolytic actions contribute to the cell death. However, other important processes occur that bias the final morphology away from classical apoptosis.

B) FOCAL ISCHEMIA AND HYPOXIA/ISCHEMIA. I) Morphology. There is DNA laddering in the penumbra of permanent lesions after one to several hours (669, 670, 1124), 24 h after 2-h temporary occlusion (652), and several days after very short (30 min) temporary lesions (276, 304). Unlike in global ischemia, there is also quite strong morphological evidence for apoptotic changes in both focal ischemia and hypoxia/ischemia.

Two groups showed similar effects of temporary ischemia (178, 652). Apoptotic nuclei, showing condensed chromatin /apoptotic bodies, first appeared in the infarct core ~15-30 min after beginning ischemia (653) and were very apparent there within 30 min after 1- or 2-h temporary MCA occlusion (178, 652). After this time, they were continually present at high density in the periphery of the infarct (178, 652) for at least 2 days (652). They also appeared in regions of individual cell necrosis after milder insults (653). Thus apoptotic neurons appear to arise quite soon after a focal insult and to predominate either early or in regions where the insult is less severe. The ratio of apoptotic cells to necrotic (ghost) cells was 9:1 in the penumbra versus 1:1 in the core 4 h after 2-h temporary ischemia (178). One study compared apoptosis in temporary and permanent ischemia (785). The time course of development and the number of apoptotic cells were identical in the penumbras in the two insults; however, there were about one-half the number of apoptotic cells in the core area of the permanent lesion, probably reflecting the continuing profound energy deprivation (785).

Cells with spherical condensed chromatin structures are present in great numbers 24 h after permanent or 1-h temporary ischemia in mouse. In the penumbra, they constitute ~5-10% of the total number of cells (601, 786), indicating a major apoptotic component to cell death. TUNEL staining was almost exclusively over neurons as opposed to glia (304); no more than 10% of the TUNEL cells were colabeled with glial fibrillary acidic protein (652). This confirms the neuronal nature of the apoptotic cells.

Apoptotic death was convincingly demonstrated after 60 min of ischemia/hypoxia in newborn piglets (745). There was coincident strong basophilia of the nuclei, TUNEL labeling, and presence of apoptotic bodies and condensed cells in the cingulate gyrus 2 days after the mild insult, which reduced ATP levels to ~50%. A similar insult, applied to young rats, yielded cells with greatly increased labeling by annexin V 48 h after the ischemia (1189), also suggesting apoptotic change.

High-resolution study of the cell changes in the various models indicate that the morphology is often not that of classical apoptosis. The cells showing apoptotic nuclear morphologies are mostly shrunken, but some of the apoptotic nuclei are in distinctly swollen cells, with no possible apoptotic morphology (178). At the level of electron microscopy, the condensed chromatin in nuclei of cells that very probably had stained positive with TUNEL did not actually have the smooth appearance of chromatin in apoptotic cells and furthermore was associated with cells that were highly vacuolated and were often undergoing quite severe homogenizing-like cell change (155). This is a secondary necrosis, following major apoptotic changes.

Overall, these morphological studies strongly indicate nuclear changes associated with apoptosis. However, the cytoplasm often shows much more degeneration than is expected in classical apoptosis, including vacuolization and disintegration. More morphological studies would be very beneficial, along the lines of the elegant studies of Portera-Cailleau and co-workers on glutamate toxicity and global ischemia (716, 919).

In most studies, apoptosis tends to be favored in regions where insult intensity is less (penumbra rather than core of lesion, following shorter rather than longer insults, Ref. 178), but the apoptotic changes certainly exist in the lesion core.

II) Biochemical studies. Mutant mice in which ICE is knocked out (993) or rendered ineffective (421) have infarcts that are 50% the volume of controls after transient focal ischemia. However, the mutants have reduced build-up of IL-1 after ischemia (421), which could account for protection. The relatively specific caspase-3 inhibitor Z-DEVD.FMK reduced infarct size by 27% in a mouse model of 2-h ischemia followed by 18-h reperfusion (422). It was only effective if added within 1 h of the end of the ischemia, not when added at 2 h (694), again indicating that caspases play a role early in the cell death process.

Caspase-3-like activity was more important to overall cell death in a less severe model (30-min ischemia). Z-DEVD.FMK reduced infarct size by ~60% when added anytime before 6 h after the insult. It was ineffective added 18 h after the insult but partially protected when added at 12 h (304). An elegant more detailed time course study showed that caspase inhibition was effective only up to to 9 h in this model, and that corresponded to the point at which caspase-3 was activated (324). As with the effects of caspases in global ischemia, this is still early relative to the final stages of cell death, which do not occur until 3 days or more after ischemia in this model.

There are many caspases (11 at present), and the full contribution of caspase activation, as distinct from its role in IL synthesis, will require improved inhibitor specificity. Also, two more universal inhibitors, V-ICEinh and BAF, which prevent neuronal apoptosis that is unaffected by the inhibitors of caspase-3 and caspase-1 (877), will be of help in showing the extent of caspase involvement in ischemic cell death. It may be greater than indicated by the ~25% inhibition produced by caspase-3 inhibition with Z-DEVD.

Several studies have been carried out on caspase changes after one or more hours of focal ischemia. As in global ischemia, the increase in de novo synthesis of caspases appears rather leisurely, but there is an earlier activation of the procaspase-3 by cleavage. Upregulation of caspase-3 mRNA in MCA territory is delayed until 16-24 h after the onset of permanent focal ischemia, whereas caspase-2 mRNA is upregulated by 4-8 h, and is down to baseline by 16 h (42). Two hours of temporary focal ischemia in mouse (807) led to an increase in caspase-3 protein levels in the penumbra after 24-h recovery; there was no change in the focus. [There is a recent report in abstract form showing a far more rapid increase in caspase-3 mRNA in layers 2 and 5 cortical neurons in spontaneously hypertensive rats, beginning within 15 min of occlusion, with no change in caspase-2 mRNA (825).] There was an early increase in the 20-kDa caspase cleavage product, peaking between 1 and 4 h after the 2-h insult but actually beginning during the ischemia. This was accompanied by a manyfold increase in caspase-3-like activity, measured with an artificial substrate, which was maximal immediately after ischemia and decayed within ~6 h. This is consistent with the requirement for very early inhibition of caspase-3 to attenuate damage in this mouse model (422, 694). The very delayed synthesis of caspase-3, and even the less-delayed synthesis of caspase-2, is perhaps too late to be of great functional significance in focal ischemic damage, which is strongly manifested between 6 and 12 h, but this needs to be tested by future studies.

Caspase activation via movement of cytochrome c from the mitochondria into the cytosol is a cardinal feature of most apoptotic systems. There is good evidence that this occurs in a large number of cells during development of focal ischemic damage (342). Substantial intracellular redistribution of cytochrome c from mitochondria to cytosol was measured both by immunocytochemistry and by Western blots within 4 h of reperfusion after 90-min ischemia, and this increased over the next 24 h. This was selective in that cytochrome oxidase did not show this change. This time course is in keeping with the measured activations of caspase.

Hypoxia/ischemia in young rats produces large numbers of apoptotic-like cells. Caspase activity increases and is half-maximal by ~12 h (191). Neuronal death and brain volume loss were markedly attenuated by the nonspecific caspase inhibitor BAF when administered within 3 h of the insult (191), at levels which did not affect core temperature for at least 2 h.

Taken together, these studies suggest that apoptosis contributes to cell death during focal ischemia and hypoxia/ischemia and also that caspases are important. Somewhat surprisingly, neither caspase antagonists nor knockouts have been tested in permanent focal ischemia, although the presence of apoptotic profiles suggests that apoptosis is important. The relative importance of the apoptosis in temporary focal damage, as evaluated by the efficacy of the caspase inhibitor, decreased as the duration of the insult increased, and this is in line with the general notion that less severe insults favor apoptosis over necrotic cell death (111). The role of caspase synthesis, as distinct from its early activation by proteolysis, is not clear. The former occurs quite late; it may play a role in ongoing cell death, perhaps that which is activated by induced enzymes such as iNOS and/or inducible COX-2.

It is, of course, possible that caspases aside from caspase-3-like forms can perpetrate apoptosis so that the effect of z-DEVD.CHO may represent a lower limit on the amount of apoptosis that occurs.

A) OVERVIEW. Apoptosis can be induced by a large number of different stimuli, and many of the conditions that prevail during ischemia are capable of inducing apoptosis in one or more cell types. These conditions include free radical generation (380, 502, 938, 1207), NO production (830), reduced mitochondrial activity and membrane potential (503, 894, 1220, 1275), microtubule disaggregation (110), increased Ca²⁺ (535, 831), activation of calpain (811, 1065), increased levels of ceramide (619), and increased expression of a mutant P53 (654, 978). The broad-spectrum protein kinase inhibitor staurosporine causes apoptosis in many cell types, including neurons (700), and both protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) are severely downregulated for many hours after global and (for CaMKII) focal ischemia; PKC has not been measured in the latter (35, 418, 780).

B) BAX, TNF, CD-95L (FAS LIGAND), AND CYCLINS. More specific inducers of apoptosis are also changed appropriately by ischemia. This includes a decreased ratio of Bcl-2 to BAX (365), upregulation of cJun (415), and changes in the "cell death domain" receptor ligands. Tumor necrosis factor- is elevated after global ischemia (975, 1155) and has been shown to be damaging in focal ischemia (636, 746), where it is also elevated within 20 min (636). Importantly, reported in abstract form, the Fas ligand, or CD-95 ligand, was upregulated in the ischemic region for 24 h after 2- to 3-h temporary focal ischemia and, as with TNF-, this appeared to be very damaging. Lesion size at 3 days was much reduced by injected antibody to CD-95 and also in mice in which the Fas ligand was knocked out (722). Although TNF- has many effects in addition to initiating apoptosis, this is not generally true for the CD-95 system. Thus these data suggest it may play a critical role in the ischemic apoptosis. Such a role is consistent with the widespread upregulation of CD-95 in postmortem brains showing many different neurodegenerative diseases (246). It would be of interest to measure ischemic damage in FADD knock-out mice; if lesion size was reduced, it would strongly support the role of the CD-95 system (43).

The cyclins are thought to be instrumental in activating apoptosis in some systems, including NGF withdrawal from sympathetic neurons. Cyclin D1 levels increase after withdrawal, and apoptosis is effectively prevented by inhibition of the cyclin-dependent kinases (876). Cyclin D1 mRNA is increased selectively in CA1 and subiculum 48-72 h after 20-min global ischemia in the rat, and cyclin D1 was expressed in individual CA1 pyramidal neurons by 72 h, before their undergoing ischemic cell change or apoptosis (1117). Neither the mechanism nor significance of this recent finding has yet been determined, but it is reasonable to think the activation of the protein plays a part in apoptotic cell death.

C) DIRECT EFFECTS ON MITOCHONDRIA. Another mechanism for cytochrome c release (and hence activation of apoptosis) is suggested by the recent observation that the combination of mitochondrial membrane depolarization and elevated perimitochondrial Ca²⁺, conditions which occur during ischemia, cause release of cytochrome c from isolated mitochondria in the absence of the mitochondrial pore transition (24).

D) TIMING OF CASPASE ACTIVATION. The timing of caspase activation with respect to cell death remains something of an enigma. In both global and focal ischemia, apparent activation of caspase-3 occurs many hours and in fact days before signs of cell death are manifested. This is true when evaluated by appearance of fragments, by appearance of proteolytic activity, and by the time at which caspase-3 inhibitor has to be added to prevent apoptotic cell death. There are several systems where caspase activation is very early, including the death-domain ligand/receptor interactions and staurosporine (618). However, this is generally an activation of a "precursor" caspase such as caspase-1 (618) or caspase-8 (1004, 1186, 1219). It is hard to envision how caspase-3, with its multiplicity of downstream critical targets, would take days to kill cells (this can be compared with NGF withdrawal where nuclear condensation and cell death follows caspase-3 activation within 1 h or less). Understanding this time course should give important insight into the control of ischemic apoptosis.

In some cases apoptosis requires protein synthesis, whereas in others it does not. Whether or not it does so in ischemic death provides insight into the mechanism of its activation. Protein synthesis inhibition does ameliorate ischemic damage in many cases; however, damaging proteins such as iNOS and COX-2 are synthesized and may be the target of this protective action (as may temperature reduction; the studies are not generally well controlled for this). Thus the challenge is to demonstrate that inhibition of synthesis protects because it specifically prevents apoptosis. The most persuasive pertinent studies on in vivo ischemia are those in which mild transient focal ischemia (30-min distal MCA occlusion) was the insult and where artifactual inhibition of protein synthesis, which probably lasted for 12 h, almost completely protected against the damage (276, 304). The evidence that apoptosis was occurring was not strong in one of the studies, comprising only TUNEL labeling and DNA laddering, but it was stronger in the other where there was chromatin condensation also. There is a caveat. Apoptotic cell death following ischemia is very sensitive to small decreases in temperature, being abolished by a 2°C fall (292). Unfortunately, there were no controls for possible reduction of temperature by cycloheximide at the levels that were used in the studies (1 and 10 mg/kg), beyond 1 h after ischemia. The studies thus suggest, but do not prove, that protein synthesis is required for ischemia-induced apoptosis. A similar conclusion emerged from work on cell cultures where non-NMDA-mediated cell death after ischemia showed TUNEL labeling and was blocked by cycloheximide (405); here again, independent evidence for apoptosis was not terribly strong, although the cell death was inhibited by the general caspase inhibitor Z-VAD FMK (387). There is no possibility of a temperature artifact in culture. Although more careful studies would be very useful, testing a correlation between protein synthesis inhibition and identified apoptotic-like death with good temperature control, the current evidence indicates that ischemia-induced apoptotic death requires protein synthesis.

If protein synthesis is required, it might explain the absence of apoptosis following global ischemia, where protein synthesis is severely inhibited throughout the postischemic period (see sect. IVC).

If synthesis is required, then the question is what proteins are likely to be synthesized, and what is the trigger? The answers are not known. Caspase-3 expression increases in global and focal ischemia, but the timing of the increase is quite late for it to be playing a major role in apoptosis, as discussed above. Quite extensive work on other systems, and particularly on thymocytes, strongly suggests that the newly synthesized proteins are required to enable one or more steps that cause the release of cytochrome c from mitochondria and ultimate activation of caspases (710, 909). For example, thymocyte apoptosis initiated "physiologically," by cortisone requires protein synthesis. However, thymocyte apoptosis initiated by directly opening the mitochondrial transition pore does not (710). Clearly, there may be differences in different cell types. However, it is possible that one effect of ischemia is to trigger synthesis of proteins that allow eventual release of cytochrome c by acting on the mitochondrial membranes. A challenge is to find such proteins.

There is DNA laddering in all the ischemic models, although the laddering of DNA may proceed by a somewhat different mechanism than in normal apoptosis. The DNA fragments produced by either focal or global ischemia in adult rats, or by hypoxia-ischemia in young rats, have staggered ends, as distinct from blunt ends seen in classical apoptosis. The 3'-end is recessed ~8-10 bp (J. MacManus, personal communication). One possibility is that an additional endonuclease may be activated in cerebral ischemia, which acts after the classical apoptotic nuclease(s) (301). There is strong evidence for caspase activation and some attenuation of cell death by caspase inhibition in global and focal insults, which seems to be greater for milder insults, a relatively common property of the apoptosis/necrosis dichotomy. Further work on the very rapidly developing field of caspase biology will, hopefully, clarify the roles of these enzymes.

The nuclear morphology of apoptosis is apparent in focal ischemia. What is not yet clear is how completely processes in the cytoplasm, which are almost undoubtedly the keys to cell death, mimic classical apoptosis and also what percentage of the population is showing the apoptotic events (699). At present, it is clear that the cytoplasm in neurons showing apoptotic changes is generally highly vacuolated compared with normal apoptosis, although the latter do often show vacuolation. It is also clear that there are a large number of eosinophilic or ghost neurons and that some of these show apoptotic nuclei, indicating a very mixed cell death pathway. Future studies in which characteristics of apoptotic cell death, and ischemic cell death, are defined more clearly will be of great help in determining what is occurring. Part of this should include characterizing the presence of specifically apoptotic protein changes such as spectrin and F-actin (gelsolin) cleavages, as well as phosphatidylserine redistribution in the plasmalemma. A promising line of work that is developing is the careful comparison between properties of apoptotic death in neurons, induced for example by staurosporine, and ischemic cell death (700). This issue is considered in section IIIE.

C.  Autophagocytotic Cell Death

1.  Basic properties of autophagocytotic cell death

A third distinct form of cell death that may pertain during ischemia is autophagocytotic cell death. This was classified by Clarke as type II (apoptotic death is type I) cell death and has been noted in several cases during development of the nervous system (205). The possibility that this occurs has been suggested in some discussions (178), but it has not been studied at all rigorously. In fact, though, it may be quite prevalent.

Autophagosomes are fusion products of lysosomes with mono- or multilayered lipid membrane vesicles enclosing regions of cytoplasm (637), which often include organelles (371). Normal protein turnover by this mechanism occurs at ~1%/h in most cells, and it is thought to be the principal mechanism of steady-state protein breakdown (637, 1015). Its involvement as a principal player in death implies that it is strongly activated, and there are now a number of studies implicating autophagocytosis in cell death, indicating that it is indeed capable of killing cells when activated well beyond its normal rate (205).

The characteristic morphology of autophagocytotic cell death includes a condensed cytoplasm containing many large vacuoles, most of which are autophagosomes or proliferating lysosomes, and a nucleus in which chromatin is irregularly clumped (205). This is not very different from ischemic cell change, or from cytoplasmic morphologies described for some cases of apoptosis. The nucleus is generally less pynknotic and less dense than in ICC.

The rate of autophagocytosis is greatly increased in some cells that have undergone injury (371) and is approximately doubled in liver 24 h after 60-min ischemia (724). There is only one direct identification of autophagocytosis in brain ischemia, but it is quite persuasive (834). Structures that appear to be autophagosomes proliferate ~3 days after 5-min ischemia in gerbil CA1 pyramidal cells. These structures are bordered by multiple membranes, contain organelles or parts of organelles, and in addition contain cathepsin. They thus appear to be true autophagosomes (371). This is the only study in which autophagosomes were looked for specifically, and it is possible that they are peculiar to gerbil global ischemia, because that insult is characterized by a huge proliferation of ER-like membrane, which could be the precursor to the structures. On the other hand, based on appearance of cells, it has been suggested as the principal mode of cell death in rat global ischemia and as very important in focal ischemia (178), and there is a large increase in immunoreactivity of the cathepsin B precursor protein within 24 h of 12- to 15-min global ischemia in rat (448). Further work in which lysosomal enzymes are localized and subjected to inhibition and where autophagosomes are identified would be needed to establish that the process is important in ischemic cell death. There are now relatively specific inhibitors of cathepsins, such as 3-methyladenine, that appear to work in vivo (149).

There is almost no pertinent information on what ischemic change might trigger autophagy. Basal autophagy in hepatocytes is strongly inhibited by depleting ER stores of Ca²⁺ (383), indicating that ischemia-induced changes in ER could be important. Alternatively, because phosphorylation of the ribosomal protein S6 inhibits autophagy in liver (100), it is possible that dephosphorylation of this protein occurs in the postischemic period and so activates autophagocytosis.

Steady-state regulation of cell volume is not well understood. Shrinkage of sympathetic neurons after NGF withdrawal is strongly correlated with loss of protein (289, 333) so that the shrinkage could be explained by massive proteolysis. There are now several very specific cell-permeant inhibitors of proteasomal activity (749, 1033), and their effects on ICC would be of interest. Indeed, a proteasome inhibitor, PS-519, does strongly decrease the size of the striatal core lesion after temporary focal ischemia (902), although this might not be related to prevention of massive protein breakdown but, rather, to prevention of activation of the transcription factor NFB (56). Individual cells were not studied in this report. Cell volume might also be decreased by activation of K⁺ and Cl channels as occurs in regulatory volume decrease (1116). The latter is triggered by increased cytosolic Ca²⁺, and it is possible that it could be activated by this or other mechanisms.

Argyrophilia might also reflect proteolysis, since it is greatly enhanced by puromycin, which produces small peptide fragments (1091). On the other hand, Gallyas et al. (350) emphasize the all-or-none quality of argyrophilia (350) and speculate that there is a cooperative macromolecular-skeletal change that exposes protein side chains, causing the argyrophilia and the increased electron density.

Eosinophilia is partly due a loss of hematoxylin or Nissl staining, whose basis is not known, perhaps loss of ribosomes or of polysomes. There may also be an increase in eosin staining. This could be due to major changes in pK_a and/or structure of proteins (1137).

Each of these speculations could be addressed experimentally to help determine the macromolecular basis for ICC.

Cell shrinkage and increased electron density are also features of apoptosis, although eosinophilia and argyrophilia do not occur. As discussed above, a net decrease in protein synthesis likely accounts for cell shrinkage in at least one model of apoptosis; it has not been studied in others. Specific cell changes in apoptosis are not very well known. However, the central role played by caspases, and by calpain (909), has revealed a wealth of potential protein changes. In cytoplasm, fodrin/spectrin is a caspase-3-like target (811), as are proteins involved in actin stabilization such as gelsolin (605). Thus a fairly massive attack on the cytoskeleton is suggested, which may well account for the apoptotic death. Caspase-3 also cleaves the endogenous regulator of protein phosphatase 2A, activating that enzyme (987). This could strongly affect interactions between cytoskeletal proteins. Of these changes, only caspase-mediated spectrin degradation has been shown during ischemia, in cultures (810). Actin breakdown has not been noted (183).

E.  Relationships Between Different Forms of Cell Death

1.  ECC and ICC

Edematous cell change and ICC change appear to be independent pathways of cell death. Only ECC is seen in hyperglycemic global ischemia and prolonged hypoxia/ischemia in young animals, whereas ECC has not been noted in focal ischemia. However, the two morphologies are seen together in some cases, in particular at the end stage of global ischemia and after NMDA injection in striatum. In the latter case, a detailed study indicated that they developed along parallel paths for 12 h and that by 24 h both types of damage showed major disintegration (919), suggesting no crossover. It seems most likely that when cell damage reaches a certain point it can lead to one or the other pathways; the basis for the very dramatic bifurcation is not known.

Apoptotic changes and ICC are seen at the same time and in the same populations during focal ischemia, hypoxia/ischemia (292), and after glutamate injection into striatum (919). However, evidence suggests they are quite distinct modes of death.

In piglet hypoxia/ischemia, reducing temperature by 2°C eliminated apoptotic death but caused no apparent change in the numbers of necrotic neurons (292). This is very difficult to reconcile with a sequential process. In spinal cord ischemia, different populations of cells, segregated by lamina, show almost entirely necrotic cell death, or apoptotic cell death (541). Thus different populations subjected to the same insult appear to take one or the other pathways. When two different glutamate agonists were injected into striatum of the adult rat, the NMDA analog quinolinic acid caused exclusively necrotic cell death while kainic acid caused almost exclusively apoptotic-like death (919), again indicating two distinct pathways. The distinctiveness of the two pathways is further suggested by the major morphological differences, particularly the eosinophilia/argyrophilia of the necrotic pathway as well as the very different nuclear morphology (although a transition from condensed spherical chromatin to uniformly condensed chromatin does not, at least naively, seem to be prohibitive). A recent study in cultured neurons exposed to either staurosporine (apoptosis) or high levels of glutamate (considered to be necrosis) emphasized the difference in nuclear morphology; the very darkened pynknotic nucleus in necrosis verses the less pynknotic-punctate chromatin nucleus in apoptosis (700).

If the two pathways are, indeed, independent, then the critical question becomes why different cells within the same apparent population proceed by different pathways during certain insults. One possibility is that the cells are actually from different populations, which have not yet been recognized. Other possibilities include subtle differences such as densities of NMDA receptors because these have a tendency to force cells into a necrotic rather than an apoptotic mode in some systems. This was mentioned above and is seen in some culture systems where NMDA-mediated damage is necrotic and non-NMDA-mediated damage is apoptotic (405, 870).

A quite large number of studies show that increased insult intensity appears to favor necrosis as indicated by the fraction of apoptotic-like cells following severe and mild focal insults (compare Refs. 422 and 304), by the relative abundances of apoptotic and necrotic cells in the core and penumbra of focal lesions (178, 785), and by the fraction of cultured cells taking the two pathways as NMDA or NO levels are increased (111). A nice study in kidney tubule cells shows a very strong relationship between the percentage decrement in ATP by metabolic inhibition and the cell death pathway taken. Apoptosis proceeded when ATP levels were >25% and necrosis when levels were 15% (658). Thus certain cells, because of their state at the time of the insult, may tend to one or the other pathway because the insult's effective intensity is different. This is suggested by the apparently random sorting of cultured cortical neurons into necrotic or apoptotic-like pathways determined by whether or not there is permanent damage to the mitochondrial membrane potential when cells are exposed to NMDA (25).

The reason that different insult intensities do predispose to one or other pathways is not known. A simple idea is that when ATP levels, or other parameters such as Ca²⁺, change drastically they disrupt apoptotic pathways, thus biasing the process toward necrosis. As an example, if protein synthesis, or phosphorylation, is required for apoptosis and it is drastically inhibited, then apoptosis may not be allowed. There are many other such possibilities and, indeed, in lymphocytes (452) artifactual opening of the mitochondrial transition pore (MTP) causes apoptosis in a cell that is set up to carry out the process but causes necrosis if, for example, appropriate caspases are inhibited. Thus necrosis may be a fallback pathway if apoptosis is somehow not available!

The final stages of apoptosis after ischemia or excitotoxicity are very similar to those of necrotic cell death (699). Unlike the controlled phagocytosis in physiological apoptosis, there is a "secondary necrosis" at the final stages of apoptosis so that cell dissolution occurs, as shown in a careful study of excitotoxicity (919). This further complicates identification of apoptosis.

	IV. CRITICAL FUNCTIONAL AND STRUCTURAL CHANGES

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The basis for this section is the hypothesis that development of cell death and of the end stages results from long-term damage to key cell functions or structures. Cell functions and structures whose changes seem most likely to be the proximal cause of necrotic or apoptotic cell change (membrane damage, mitochondrial damage, inhibition of protein synthesis, and cytoskeletal damage) are examined to see if they play a critical role in ischemic cell death.

The hypothesis may be wrong. Cell death may result from continued activation of damaging biochemical processes (perpetrators) set in motion by the ischemic insult, with ultimate breakdown of the cell as a unit. Cell death may not be perpetrated by an intermediary functional defect in one or more key processes. Nevertheless, this section describes ischemic changes in major intracellular systems because they are deemed likely to be involved in cell death.

A.  Altered Membrane Transport Properties

1.  Membrane permeability

The question is whether there are large increases in membrane permeability to ions or metabolites that might lead to cell death. There is a paucity of data on this very important subject.

There are a great number of studies of ischemia in culture showing a correlation between the number of disintegrated or necrotic cells and membrane breakdown, measured as LDH release or uptake of vital stains including trypan blue, propidium iodide, and ethidium bromide (376, 791, 1072, 1156). However, there are almost no studies aimed at determining whether the membrane changes precede large-scale cellular changes. Propidium iodide does enter neurons in culture that look normal but are in the process of dying by necrosis, indicating that membrane leakiness may be a proximal cause of damage in cell culture (697). Unfortunately, this work in culture is the only study addressing the issue.

The mobility of a spin probe (5-NS) within the membrane bilayer of synaptosomes increased during the first hour after 10-min ischemia and then increased again transiently ~8 h later (411). This may have reflected increased disorder in the phospholipid bilayer. Unfortunately, the study has not been followed up.

Breaks were noted in the postsynaptic dendritic membrane during the first hour after 10-min complete compression ischemia in rat, and there was also a marked increase in neuronal permeability to horseradish peroxidase (256). Unfortunately, this finding was not pursued so that neither its reproducibility nor its significance are known.

Although not related to a specific function, the fact that the phosphatidylcholine precursor cytidine diphosphate choline (cyticholine or CDP-choline) is protective in ischemia indicates that membrane damage may be critical (516, 855). When administered intraperitoneally, CDP-choline very significantly ameliorates the neurological deficit days after 20- to 30-min 4-VO (516) and survival after focal ischemia (337), and it decreases infarct size either in combination with NMDA blockade (855) or when used after short insults (36). In temporary ischemia, the molecule has several effects (516); however, it does very clearly act as a precursor to phosphatidylcholine incorporation into cell membranes (516), and it dramatically decreases the net liberation of free fatty acids (FFA) during ischemia (268, 1133), presumably by activating resynthesis of phospholipid. The interpretation of the protective studies is far from unequivocal at this stage; however, they may indicate that loss of membrane integrity is contributing to the neuronal death.

In conclusion, despite a widespread assumption that gross plasma membrane changes are important in ischemic cell death, there is almost no evidence for this, particularly in vivo.

One to two days of ~50% inhibition of the Na⁺ pump with ouabain does cause an apoptotic-like death in cultures (713) so that if there is submaximal Na⁺ pump inhibition after ischemia it might well contribute to apoptosis. Also, profound pump inhibition might lead to cell swelling and ECC.

Reasonably short exposures to several different free radical species cause severe irreversible damage to the Na⁺ pump (187, 473, 1022) and also make it more susceptible to proteolytic attack (473). There is some evidence that free radical generation following various ischemic insults does inhibit the pump (382, 701, 866). Sixty minutes of 2-VO in gerbil followed by 15- to 30-min recirculation caused a 70% reduction in ATPase of frontal cortex and hippocampus, which was blocked by pretreatment with agents that should reduce free radicals (866). However, there was no pump inhibition after 15-min ischemia (866), a duration that invariably leads to delayed neuronal death. The latter suggests that pump inhibition does not play a role in delayed neuronal damage from global ischemic damage. However, it may well be important in focal damage, based on the effects of 60-min ischemia. Studies of this possibility have not been reported and would be of great interest.

Currently, there are no reports of long-term effects of ischemia on other plasma membrane transporters or channels that might cause significant ion or volume imbalance. Such studies would be very valuable.

B.  Mitochondrial Damage

1.  General background

Mitochondria are assuming an increasingly important role in hypotheses about both apoptotic and necrotic cell death (591, 617, 1250), but definitive demonstrations that they play roles in ischemic damage are still lacking. There are three general ways in which mitochondrial damage or change might make an important contribution to ischemic cell death.

1) Inhibition of mitochondrial oxidative phosphorylation or significant uncoupling will lower ATP levels, increase mitochondrial free radical production, and remove the Ca²⁺ buffering ability of the organelle. These effects are capable of causing grave damage. This could occur as a result of direct effects on the tricarboxylic acid cycle enzymes, on the mitochondrial electron transport/oxidative phosphorylation system or the lipid composition of the mitochondrial membrane, or as a result of long-term opening of the MTP.

2) In addition, opening of the MTP may produce damaging effects by releasing intramitochondrial molecules and ions, for example, a factor able to increase L-channel Ca²⁺ permeability as recently shown by Nowicky and Duchen (841).

3) Finally, release of cytochrome c and or at least one other protein from the intermembrane space is now recognized as a critical step in apoptosis in all systems in which it has been studied. Such release can be effected by opening of the MTP but is very frequently due to more direct effects on the integrity of the outer mitochondrial membrane, probably mediated by a member of the BCl-2/Bax protein family, such as Bar (309, 941).

Alterations in mitochondria therefore have enormous potential for causing severe cell damage. Despite this, and despite well-informed speculation (625), there is still a lack of evidence that convincingly implicates mitochondrial changes in either necrotic or apoptotic ischemic cell death. There is, though, a body of work that bears strongly on the issue.

In assessing this work, it is notable that isolating mitochondria from ischemic or damaged tissue may actually lead to mitochondrial damage (34). This is not usually considered.

A) EVIDENCE FOR DAMAGE. Mitochondria almost always undergo a transient swelling for a few hours after any form of ischemia (130, 742, 896, 897, 1002, 1236). The origin has not been studied but may be the reversible opening of the MTP due to free radical generation and Ca²⁺ accumulation (278, 410). This swelling has not been directly correlated with function, but it certainly appears that mitochondria are inhibited during this period.

Tissue metabolic rate, as measured by glucose utilization, is generally temporarily depressed by 50% or more, for at least 6 h after transient global ischemia (291, 606, 760, 931), and is depressed in the penumbra during and after focal insults (79, 1252, 1299). The depression after global ischemia does not occur in all regions but always occurs in vulnerable cells; in gerbil hippocampus, only CA1 is affected (760). Along with the decreased metabolic rate, ATP levels are reduced by 20-30% for several hours after 30-min 4-VO and 2-VO in the rat and 10-min complete ischemia in the dog (1070) and cat (1179). Furthermore, cytochrome complex aa₃ is hyperoxygenated in intact tissue after global ischemia, consistent with failure of adequate substrate supply to the respiratory chain (961). These data are consistent with, but do not establish, mitochondrial dysfunction. They could equally be explained by a failure of glucose metabolism.

Unfortunately, the totality of studies on isolated mitochondria show a great deal of variability, some of which may well result from difficulties in their isolation. However, overall they do indicate inhibition in the postischemic period. State 3 respiration of isolated mitochondria is generally severely depressed (by 50% or more) immediately after global ischemia (449, 450, 668, 1012, 1054) and, in most measurements, for at least 1 h afterward (449, 944, 1080), although in one study it recovered within an hour (1054). Respiration of penumbral (and focal) tissue mitochondria is inhibited by 40% for at least 4 h after transient focal ischemia of between 1 and 2 h (624, 625). The damage almost always affects the NADH-linked portion of the respiratory chain (complex I) (743).

B) MECHANISM OF DAMAGE. Lee and co-workers (1012) suggest that the basis for the early mitochondrial inhibition is accumulation of mitochondrial Ca²⁺ during, and possibly shortly after, the ischemia. The conclusion was based on its very rapid reversal when EGTA or ruthenium red (RuRed) were added to the isolated mitochondria (1012). This is consistent with the site of action being the MTP, which is known to be rapidly activated by increased cystosolic Ca²⁺ (278), due to accumulation in the matrix (410), and which rapidly closes when isolated mitochondria are exposed to a chelator (475). This being said, it is very probable that other processes are involved; for example, the preferential blockade of complex I and hyperoxidation of cytochrome aa₃ are not obvious consequences of the MPT. Calcium accumulation appears to preferentially inhibit complex I (475), which may explain the apparent involvement of this complex in inhibition.

Several electron microscope analyses show an accumulation of Ca²⁺ in mitochondria very soon after global ischemia, which persists for several hours (285, 596, 1051, 1165, 1181, 1272) (there are no equivalent studies in focal ischemia), observations that are at least consistent with Lee's hypothesis.

C) ROLE FOR NO? The basis for the prolonged Ca²⁺ accumulation is not known; however, there is interesting evidence implicating NO. Accumulation in vivo was strongly blocked when an inhibitor of NOS, N^G-nitro-L-arginine, was added intraventricularly before 5-min ischemia in the gerbil (596). Intracellularly generated NO, at levels that arise during and shortly after ischemia, strongly depolarizes mitochondria, inhibiting ATP production (127). Although not tested, it is possible that this results from large-scale Ca²⁺ accumulation and so accounts for the observed effect of blocking NOS. Both the above effects could be due to NO or to peroxynitrite (see sect. VA). In the latter case, they would also depend on free radical production.

If this is the case, and if Ca²⁺ accumulation is tied to damage, then free radical and NOS blockade should prevent the early mitochondrial damage. Although studies are not very extensive, some data are at least compatible with this. Blockade of FFA production with the platelet-activating factor (PAF) antagonist BN50739 prevented ~50% of the postischemic mitochondrial inhibition and several free radical scavengers attenuated the early decrease in ATP after global ischemia in the dog (360). Furthermore, swelling of mitochondria 1 h after 5-min ischemia in hippocampal slices was prevented by calmidazolium (316), which, among other things, prevents activation of NOS. Thus data are consistent with peroxynitrite-mediated uptake of Ca²⁺ by mitochondria and subsequent inhibition of function. A mechanism whereby NO or peroxynitrite leads to Ca²⁺ accumulation is not yet known.

There are several reports indicating delayed damage in isolated mitochondria, but again results are quite variable, and timing does not always clearly show that the mitochondrial damage precedes cell death (e.g., Ref. 786). Several hours after 11-min anoxia in cats there was a reduced tissue oxygen consumption that was strongly associated with inhibited isolated mitochondria (1180). State 3 respiration measured on isolated rat mitochondria was reinhibited by ~50% 5 h after 30-min 7-VO in the rat (1012). In the very sensitive striatum, mitochondria became permanently inhibited 30-90 min after 2-VO ischemia and 6 h after 4-VO (449, 944, 1054), and they were inhibited 48 h after the ischemia in CA1 (1054), but only by 20%. There was a delayed loss of the respiratory control ratio 2-4 h after temporary MCA occlusion, in mitochondria isolated from both the core and the penumbra (625) as well as a decrease in state 3 respiration (801). There was a large decrease in mitochondrial membrane potential, measured in situ, after 24 h of focal ischemia (786). All of these results, except the last, strongly indicate mitochondrial damage in reasonably late stages of damage development. The last result could reflect changes in dead or dying cells because there is a lot of cell damage by 24 h.

There are decreases in tissue ATP levels before or around the time of cell death in global ischemia (760, 929) and in the penumbra in focal ischemia (329). Levels after global ischemia fall by 25 and 40% in rat CA1 hippocampus and striatum, respectively (929), and probably further in gerbil at 24 h (432). It is not known, though, that they result from mitochondrial damage. For example, in one study, the falls in ATP after focal ischemia could be explained by the falls in total adenylates (329). Decreased levels could also result from inhibited glucose metabolism.

Overall, these studies suggest late, functionally important, mitochondrial damage. They are not strong enough to establish this unequivocally, and further careful studies are required. Studies of the status of the MTP and also of cytochrome c leakage would be very valuable.

Pharmacological studies suggest the involvement of free radicals. The delayed loss of energy charge, and ATP, following temporary focal ischemia was protected by the spin trap N-tert-butyl--phenylnitrone (BPN) (329), and the delayed mitochondrial dysfunction measured after 7-VO in the rat was largely prevented by ascorbate (1011), as was the mitochondrial inhibition several hours after transient focal ischemia (801). The latter was also protected by the calcineurin inhibitor FK-506 (801), possibly because it prevents activation of constitutive NOS (cNOS) and subsequent accumulation of peroxynitrite, although this has not been shown.

Somewhat less directly, Mn-superoxide dismutase knockout mice generated more free radicals during permanent focal ischemia, showed a larger lesion, and showed a much larger number of cells with a loss of mitochondrial membrane potential after 24 h (786), demonstrating that excess free radical production enhances mitochondrial damage. However, this result does not explicitly show that normal free radical production causes the damage. All these studies are suggestive; however, they do not rule out the possibility that mitochondrial damage is an indirect result of free radical damage to another process (e.g., Ca²⁺ homeostasis).

II) In vitro. Exogenously generated free radicals severely damage mitochondrial state 3 respiration, particularly in the presence of elevated Ca²⁺ (119), and these effects may be the basis for the in vivo effects described above. Very short (5 min) exposures of cultured neurons to peroxynitrite cause ~50% inhibition of cytochrome-c oxidase and succinate-cytochrome c reductase that matures over 24 h (107). This is a very profound effect that has the possibility of occurring in vivo and being very important.

Nitric oxide donors (e.g., S-nitroso glutathione) applied to isolated mitochondria, and to thymocytes, open the MTP, leading to membrane depolarization and subsequent increased superoxide generation by the mitochondria (458). Generation of peroxynitrite from superoxide and NO at rates calculated to be similar to those encountered in pathological conditions, causes Ca²⁺ release and depolarization of liver mitochondrial membranes, with opening of the transition pore (863).

B) EFFECTS OF CA²⁺. In at least one study, the timing of Ca²⁺ accumulation is consistent with its causing delayed inhibition; it increased in mitochondria of the striatum 6 h after 4-VO, shortly before this region began to degenerate (1272). There was a large increase in total cell Ca²⁺ beginning 24 h after global ischemia in the gerbil that may well reflect large scale Ca²⁺ accumulation by the mitochondria. There was a concomitant fall in ATP (432). Unfortunately, there are no other good studies of delayed changes in mitochondrial Ca²⁺.

There is quite a large body of evidence that Ca²⁺ accumulation by mitochondria is a major mechanism of mitochondrial damage and subsequent cell toxicity in response to glutamate, raising the possibility of a similar effect after ischemia. Glutamate-induced necrosis in several culture systems (25) is associated with large-scale accumulation of Ca²⁺ by mitochondria, subsequent mitochondrial depolarization (26, 994, 1206), and lowering of ATP/ADP, down to 30% of normal levels (142). The inhibitor of the mitochondrial Ca²⁺/2Na⁺ exchanger CGP-37157, which enhances mitochondrial Ca²⁺ accumulation and decreases cytosolic Ca²⁺ accumulation, enhances depolarization and damage, establishing quite nicely that the mitochondrial Ca²⁺ accumulation is causing the depolarization and damage (1206). There is other strong evidence implicating mitochondrial Ca²⁺ accumulation as a key factor in this necrotic event (829, 1071).

The basis for the Ca²⁺ accumulation in those cases is the glutamate-induced cytosolic Ca²⁺ accumulation, which reaches several micromolar (476). If this mechanism of mitochondrial damage is to occur after ischemia, some factor must elevate mitochondrial Ca²⁺ uptake. This could be either increased cytosolic Ca²⁺ or, perhaps, an action on a mitochondrial Ca²⁺ transport system. Although NO or peroxynitrite generation seems capable of such action (see above), and there is delayed production of iNOS that might contribute to damage in this way, there is no evidence for such a mechanism at present.

C) MITOCHONDRIAL TRANSCRIPTION. Mitochondrial transcription of cytochrome oxidase mRNA is damaged within ~3 h of 3.5-min global ischemia in the gerbil. There was a progressive loss in enzyme activity and mRNA, with both falling to 20% of normal after 2 days, well before major manifestations of cell death. This was confined to the CA1 region (2). This is consistent with the fairly dramatic delayed lowering of ATP that was measured qualitatively in CA1 of the gerbil (432). This phenomenon has not been well-studied in other models but clearly may be important. The mechanism by which transcription is damaged is not known.

D) OTHER POSSIBILITIES. Ceramide, whose concentration rises within 6 h of starting focal ischemia (619), has been shown to reversibly inhibit isolated mitochondria (398). The elevated ceramide concentration is maintained throughout the ischemia, so it may inhibit in situ though it seems unlikely it would account for inhibition of the isolated mitochondria, since it is readily reversed on washing (398).

Another possible basis for damage is that a large release of cytochrome c, the probable activator of caspases, compromises the respiratory chain.

E) SUMMARY. There are several reasonable mechanisms for delayed mitochondrial damage following ischemia. Evidence for free radicals is strong in that several free radical scavengers, or agents which should reduce their generation such as PAF inhibitors, attenuate mitochondrial damage, and also protect against ischemic damage (1025). Furthermore, free radicals and peroxynitrite severely damage mitochondria in vitro. Further studies with NOS inhibitors are essential to determine the role of NO and/or ONOO in vivo.

Mitochondrial Ca²⁺ accumulation also remains a viable possibility as a damaging agent and may certainly act along with free radical production. More evidence concerning mitochondrial Ca²⁺ changes that presage damage would be very useful. Inhibition of cytochrome oxidase synthesis and/or loss of cytochrome c are also very reasonable mechanisms of damage.

If Ca²⁺ or free radicals are the active agents, then the molecular target needs to be determined. Delayed damage is not caused by irreversible damage to any of the dehydrogenase complexes (862). Free radicals might be the agents acting on mitochondrial DNA to block transcription of cytochrome oxidase mRNA. Free radicals may oxidize cardiolipins, the principal mitochondrial lipid. This has not been measured in ischemia but does occur when the MTP is opened and mitochondria generate free radicals (452). Cardiolipin changes may well produce defects in the electron transport chain (872).

The mitochondrial pore transition represents a reasonable target for damage; it could be mediated by increased Ca²⁺ or by free radicals, and by both acting synergistically (410). It produces profound changes in cell function (89, 410). Although early evidence concerning the pore opening was conflicting, it now appears that it does play a role in, at least, focal ischemic damage. Cyclosporin A, which inhibits the MPT, was very protective in a global ischemic model (1145), but the drug also inhibits calcineurin, and FK-506, the calcineurin blocker, is also protective after global ischemia (272). FK0506 is also protective after temporary focal ischemia (627), but in this insult, a derivative of cyclosporin, methyl-valine cyclosporin A, which does not inhibit calcineurin, reduces the infarct size by >50% (528). This result implicates opening of the transition pore in, at least, focal ischemic damage, although it does not show whether it is early or late opening that is damaging.

A) LOWERED ATP LEVELS. The issue is whether and how maintaining small (20-30%) reductions in ATP for many hours, as occurs in the postischemic phase, will lead to cell death. Elegant and important in vivo studies by Beale, Greenemayre, and co-workers show that brain-injected mitochondrial inhibitors cause a delayed cell death. However, the long transient fall in ATP, of ~50-60% at 3 h, more closely parallels conditions during focal ischemia, so the studies are not necessarily germane (1001). More pertinent studies have been done in culture where chronic treatment with mitochondrial inhibitors leads to cell death within 8-24 h (870, 1276, 1277). In one of these, ATP levels were maintained at 70-80% of normal. Cell death developed in 4-8 h (870) and was preventable by glutamate receptor blockade (870, 1277). Death showed selective vulnerability (1277), and there were mixed populations of apoptotic and necrotic neurons at 48 h (870). Thus low-level mitochondrial inhibition produces both apoptotic and necrotic populations in a timely fashion, but in this case in culture. Although not tested, the assumption is that MTP did not open and that there was not an enhanced production of mitochondrial free radicals so that this represents a "pure" effect of lowered ATP. Even if this is not so the results indicate that mitochondrial inhibition, to a level which approximates the change in vivo, can lead to cell death in a timely fashion.

B) FREE RADICAL GENERATION. Although in vitro studies of mitochondria strongly indicate that their production of free radicals should be increased by ischemia (see sect. VA), the actual evidence for this is really limited to only one paper. It was shown, using the salicylate trap to measure OH production, that free radical generation during the 1 h after global ischemia was completely prevented by blocking the mitochondrial respiratory chain with rotenone (908). No effects on subsequent damage were measured, and further work is clearly required.

C) INDUCTION OF APOPTOSIS. It is now clear that mitochondria play a central role in most (if not all) cases of apoptosis (1275) by releasing cytochrome c (591, 941, 1250), and possibly other proteins (1275), from the intermembrane space, and it is clear that adequate cytochrome c can induce apoptotic changes, at least in part by activating caspases (617, 941). There are several pathways by which cytochrome c could be released including the MPT (452, 617, 894, 1274). A possible mechanism for release after ischemia is by direct interaction of BAX with the outer mitochondrial membrane (941, 1161). Overexpression of Bax induces cytochrome c release from mitochondria in several systems (220, 882, 963), and recombinant Bax causes release of cytochrome c from isolated mitochondria (514). Bax is indeed upregulated in vulnerable cell populations after ischemia (185, 420, 1306), and such a pathway would explain the apparent requirement for protein synthesis in ischemic apoptosis.

D) OTHER MECHANISMS. A compromised mitochondrial membrane potential due to inhibition of oxidative phosphorylation will compromise the Ca²⁺ uptake pathway in mitochondria and hence reduce the Ca²⁺ buffering that occurs during normal high-frequency neural activity (1202). This would lead, on average, to chronically elevated cytosolic Ca²⁺ levels and could thus be damaging.

Another possibility is that other heretofore unidentified proteins may be released from mitochondria once the transition pore is opened, and lead to necrotic changes, e.g., proteins which enhance Ca²⁺ channel opening (841).

If mitochondrial function is indeed compromised after ischemia, then evidence from cell cultures indicates that even the low-level inhibition that appears to exist at that time, evaluated by ATP concentrations, could cause apoptotic and necrotic cell death. Unfortunately, there are no pertinent studies in vivo, in which effects of prolonged mild inhibition of mitochondrial function have been tested. The studies of glutamate toxicity indicate that profound mitochondrial Ca²⁺ accumulation is a major factor leading to cell death, although the mechanisms are not well understood (829). There is, though, no particular reason to think this occurs before ischemic cell death.

Thus, although mitochondrial lesions seem to be strong candidates for sites of cell death, or severe damage leading to death (1044), there is no compelling evidence at the present time except the very recent data on the effects of methyl-valine cyclosporin A, presently in abstract form. Whether or not lesions in this organelle are important in cell death, this section has pointed out that there is much to be done in determining the nature of mitochondrial damage associated with ischemia.

Prolonged inhibition of protein synthesis, if uncompensated by a decrease in breakdown of protein, would clearly lead to massive changes in cell properties and cell death. Protein synthesis is a complex process that is critically dependent on energy charge (936), intracellular K⁺/Na⁺ (672), and the integrity and phosphorylation level of a large number of proteins and RNA species (1230). It is strongly and permanently inhibited by ischemia in vulnerable cells, and the question is whether or not this is responsible for any forms of ischemic cell death. The question has to be considered in light of the marked activation of synthesis of certain damaging proteins in those same areas in which global synthesis is inhibited, making it a complex but very intriguing issue.

Glial proliferation occurs after most global ischemic episodes, and the increased protein synthesis (which is seen as soon as 3 h after ischemia in the hippocampal slice, Ref. 936) can more than compensate for any decrease in neuronal synthesis (332). Thus autoradiographic measurements are necessary to properly assess changes in neuronal protein synthesis.

There is a profound brain-wide depression of protein synthesis in the period during and immediately after global ischemia that persists in most brain regions of many different species for 1-24 h depending on the model (218, 257, 348, 790, 1113, 1209). Over 12-48 h there is complete, or near complete, recovery in the regions where cells will recover from the insult. In contrast, regions in which the cells will die never regain normal levels of synthesis or in some cases regain and lose them again (102, 257, 348, 790, 1113). In gerbils, CA1 pyramidal layer synthesis fell to unmeasurably low values 12 h after 5-min ischemia and recovered no further; polysomes remained dissociated (581, 790, 1113). In another study of gerbils, synthesis in the CA1 region was ~30% of basal values after 24 h (348). In the monkey, synthesis in hippocampus showed a transient recovery 6 h after complete compression ischemia, but became strongly inhibited between 12 and 24 h afterward. Synthesis was depressed in core and penumbra during at least 12 h of focal ischemia (757, 758); there are no reports of protein synthesis after temporary focal ischemia. Protein synthesis in CA1 pyramidal cells is reduced to ~40% 3 h after ischemia in rat hippocampal slices (936).

Thus, overall, neuronal protein synthesis remains profoundly depressed in vulnerable areas after global ischemia and during permanent focal ischemia. It would be useful to know what occurs after temporary focal ischemia.

Protein synthesis is extremely sensitive to cell energy charge and ion contents (672, 936). However, the persistent postischemic inhibition of synthesis occurs despite return of energy charge to normal values in slices (936) and of ATP to normal levels in vivo (12). Ribosomes from ischemic tissue seem competent (243) so are unlikely to be the site of damage.

A) INITIATION FACTORS. At this stage, the only changes that have been positively implicated in the decreased synthesis are changes in the two initiation factors, eIF-2 and eIF-4, consistent with the major loss in polyribosomes after ischemia (104).

eIF-4G is a substrate for calpain, and there is a 30-50% loss in its immunoreactivity after 10- and 20-min decapitiation ischemia in rabbit (821). This is very likely due to calpain-mediated proteolysis because the 70% loss of immunoreactivity at the end of 20-min cardiac arrest is completely prevented by MDL-28170, the quite selective calpain antagonist (820). There is a prolonged inhibition of protein synthesis in vascular endothelial cells as a result of free radical attack on eIF-4 family-like proteins (513), showing that eIF-4 is also inactivated for a long time by free radicals. Thus it is a very likely target.

There is also evidence that eIF-2 is involved in the inhibition. Ternary initiation complex formation in tissue homogenates is decreased throughout the cerebrum 30 min after ischemia and is decreased in vulnerable regions only (striatum and CA1 of hippocampus) 6 h after 15-min 2-VO ischemia (146, 466). There is conflict as to the basis for this inhibition. A priori, phosphorylation of EIF-2a or dephosphorylation of guanine nucleotide exchange factor (GEF) are the most likely inhibitory points (466, 1230). One set of studies concluded that there was no increase in EIF-2a phosphorylation but demonstrated a decrease in GEF activity that appeared to be due to dephosphorylation of a site that was normally phosphorylated by a tyrosine kinase. The decreased activity lasted at least 6 h in striatum and CA1 of hippocampus (466, 470). There was a correlation between decreased phosphorylation of MAP kinase and cell damage/protein synthesis inhibition (468), and it was speculated that inhibition of synthesis resulted from the lack of neurotrophin upregulation in vulnerable populations and resulting dephosphorylation of GEF. Certainly neurotrophin withdrawal dramatically and rapidly lowers protein synthesis in dependent cultures (239).

With the use of somewhat more sensitive methodology than used previously (466), inhibition of synthesis at 30-min postischemia was actually associated with a 20-30% increase in phosphorylation of eIF-2 (146). Furthermore, there is a threefold increase in the eIF-2 kinase activity of neocortical brain homogenates 30 min after 30-min ischemia in the rat, suggesting activation of protein kinase R (PKR) and providing a basis for the phosphorylation (147). More recent work, in which the antibody for this phosphorylated form of the initiation factor was used, amplified this considerably (244). There was a 20-fold increase in the phosphorylated form of eIF-2a that was confined to vulnerable CA1 cells, and some CA3 cells, 10 min to 1 h after 10-min cardiac arrest in rats. At 4 h, the antibody to this form was still present in the vulnerable cells, and although a great deal had shifted to the nucleus (244), a significant amount remained in the cytoplasm (1078). Importantly, when insulin, which dephosphorylated eIF-2, was injected several hours after ischemia, protein synthesis was restored to its normal level, strongly suggesting that the eIF-2 phosphorylation was the basis for the inhibition of synthesis (1078).

There is at least one caveat, however. The absence of the phosphorylated form in dentate granule cells after 10-min reperfusion (244) is inconsistent with the very profound inhibition of synthesis that occurs even in nonvulnerable cell populations for the first 6-24 h (348). Furthermore, it is essential to explain the very prolonged inhibition in CA1 (days). It is not clear that phosphorylation of eIF-2 would be maintained that long. For example, glutamate activates eIF-2 phosphorylation and inhibits protein synthesis. However, the phosphorylation is transient; the eIF-2 dephosphorylates within 1-2 h of removing glutamate (712). Prolonged maintenance of GEF dephosphorylation, which may result from inhibited tyrosine phosphorylation, could be accounted for by chronically lowered brain-derived neutrotrophic factor (BDNF) (599), as occurs in vulnerable populations. Clearly, proteolysis of eIF-4 is likely to be an enduring effect. However, it is not established that this factor is adequately rate limiting in brain to account for the inhibition of synthesis. This could be tested fairly readily by measuring the effects of the cell-permeant calpain inhibitors on long-term inhibition of protein synthesis.

B) RNA LEVELS. Unlike protein synthesis, global RNA synthesis seems to be remarkably stable, showing no decrease in one study up to at least 4 h after 5-min ischemia in the gerbil (103). However, there is a decrease in total mRNA in CA1 3 h after 5-min global ischemia in the same species (723). Thus there may be activation of mRNA breakdown, which could account for loss of mRNA and inhibition of synthesis. This needs to be tested further.

N-methyl-D-aspartate antagonism, which blocks much of the increase in cell Ca²⁺ in focal ischemia, protected against inhibition of synthesis in that model (758). Inhibition of synthesis in the CA1 pyramidal cell layer of hippocampal slices does not occur if the rise in cytosolic Ca²⁺ is prevented by incubating slices in 0-Ca²⁺ and 200 µM ketamine during ischemia (936), a combination which almost completely suppresses the increase in cytosolic Ca²⁺ (1288). Combined NMDA and AMPA/kainate receptor blockade also attenuated the inhibition (164, 1171), and this combination largely prevents the increase in cytosolic Ca²⁺ during ischemia (1288). It has been suggested that Ca²⁺ is not involved in inhibition because in one study protein synthesis in brain slices was inhibited by ischemia even in the absence of extracellular Ca²⁺ (265). However, there is a large rise in cytosolic Ca²⁺ in that condition that was not explicitly recognized by the authors (769, 1288).

Although increased cytosolic Ca²⁺ certainly should activate calpain-mediated breakdown of eIF-4, it might also act on eIF-2. A-23187 leads to a prolonged 70% increase in EIF-2a phosphorylation in cultured neurons (11) as does glutamate (712). The Ca²⁺ might activate PKR, which phosphorylates eIF-2. The protein is regulated in a complex way (507) and might be activated by Ca²⁺-mediated effects.

B) FREE RADICAL GENERATION. The free radical-mediated inhibition of protein synthesis in vascular endothelial cells described above (513) indicates that the abundant free radical production in ischemia might cause the damage.

C) DEPLETION OF ER CA²⁺. It has been suggested (244) and previously theorized (880) that depletion of ER Ca²⁺ might inhibit synthesis after ischemia. This depletion is known to inhibit protein synthesis, by activating PKR and hence phosphorylating and inhibiting eIF-2a (925). It is not known whether this ER depletion occurs during ischemia, but there are reasons to think it might. Arachidonic acid, at levels that are present after ischemia (30 µM), causes depletion of Ca²⁺ from the ER and inhibition of protein synthesis in cells (244, 969). Also, Ca²⁺ uptake into isolated ER microsomes is inhibited after ischemia, although the mechanism is not known. If this occurs in vivo, it would certainly lower ER Ca²⁺ (878). Presently, though, there is no definitive evidence for a change in ER Ca²⁺ at all so the mechanism is purely speculative (879).

This is most crucial. There are strong correlations between inhibition of synthesis and eventual cell death (460). Although 2-min ischemia does not cause cell death, 3-min ischemia leads to persistent inhibition of protein synthesis in CA1 and to cell death in the gerbil (31). Ischemic tolerance in gerbil (see sect. VIIIF) is associated with restoration of protein synthesis in CA1 (348, 544); hypothermia protects against histological damage and also prevents delayed inhibition of protein synthesis in the 4-VO model in rat (1209) as do barbiturates (114). Unfortunately, such correlations are by no means unique to global protein synthesis and so cannot be thought to implicate the process in cell death.

There is a paucity of good evidence linking synthesis inhibition with damage. For example, there are no reports on effects of agents that prevent cell death, such as calpain inhibitors or free radical scavengers on protein synthesis after ischemia. It is important to know if they prevent the inhibition of protein synthesis before preventing cell death. Also, it would be very useful to have good pharmacological studies using inhibitors of protein synthesis to see whether partial inhibition would eventually cause cell death in normoxia. In this context, the existing data argue against a role for protein synthesis inhibition in the cell death. In particular, 2-min ischemia in the gerbil does not cause any delayed morphological damage in spite of strongly inhibiting protein synthesis in CA1 for between 5 and 24 h (348). The best test of the role of protein synthesis would be an injection of insulin soon after the global ischemia. As described above, this dephosphorylates eIF-2 and restores protein synthesis, without causing significant hypoglycemia. If it is very protective it would suggest protein synthesis inhibition is an important factor.

Although the studies of inhibition of global synthesis in normoxic or only mildly insulted brain tend to argue against a crucial role for overall inhibition of synthesis in cell death, studies of the role of protein synthesis in ischemia, described in section VIIIF, suggest otherwise. The inability to synthesize protective proteins after ischemia, shown by vulnerable cells, may well be the basis for their ultimate demise. Furthermore, several key proteins are inactivated by ischemic events (e.g., protein kinases), and the inability to resynthesize these proteins could be very detrimental to the cell. These effects of inhibited protein synthesis would only be manifested following very damaging insults and so would not be observed in the mild insult of 2-min ischemia.

There is very little known about rates of protein degradation after ischemia. The average turnover times of brain proteins are 3-5 days (390, 1020). If normal protein degradation is blocked after ischemia, it would reduce the impact of synthesis inhibition. Such a close coupling has been seen in cultured sympathetic neurons where a 50% inhibition of synthesis is matched by an ~50% inhibition of degradation rates, although this matching requires the presence of growth factors (333).

There is a marked decrease in ubiquitin immunoreactivity in the CA1 region after global ischemia (703), suggesting that a major degradation pathway is blocked, and this might well lessen the impact of protein synthesis inhibition. However, degradation rates really need to be measured.

During focal ischemia, blocking proteasome activity using PS-519 was extremely protective to the core of the lesion (902). One explanation for this is that protein degradation makes a major contribution to core damage, in focal ischemia. Indeed, the protein content within the core of a focal lesion is severely reduced. However, another possible explanation is that proteasome activity is damaging because it allows activation of NFB (56). Protein degradation after both global and focal ischemia needs to be measured to resolve this issue.

Two studies showed that fairly well-maintained inhibition of synthesis for 2 days was remarkably protective against global ischemic damage in rat (386) and gerbil (1027). Unfortunately, however, temperature regulation was very transient and, as acknowledged by the authors, the protection may well have resulted from hypothermia.

Inhibition of protein synthesis for ~12 h after the insult also attenuates damage after mild (30-min ischemia) (276, 304) or relatively mild (90-min ischemia) (275) transient focal ischemia. This is consistent with the large component of apoptotic cell death in these paradigms. In one of these studies (275), temperature was actually monitored for 2 days after the insult and was unaffected by the cycloheximide treatment, so temperature is almost undoubtedly not an artifact. While important for these mild insults, these results do not show that profound inhibition of synthesis for one or more days, as occurs after global ischemia, is not damaging. Synthesis was inhibited for only several hours after the insult.

The localization and time course of bulk protein synthesis inhibition is completely consistent with its involvement in cell death, as is the correlation between inhibition of synthesis and cell death in several artifactual paradigms. However, it is difficult to positively implicate inhibition of synthesis in the ischemic death, as has been suggested (460, 757), particularly for focal ischemia.

The bulk turnover times of proteins are so long that even profound inhibition of protein synthesis should not alter composition enough to cause cell death in the 12-24 h after focal ischemia. This is supported by the absence of any ill effects after 2-min ischemia in gerbil despite a profoundly depressed protein synthesis for between 5 and 24 h after the ischemia. Damage would be still less likely if turnover of protein was diminished by the ischemic insult.

These considerations are not germane to short global ischemic episodes, where death often requires 3-4 days. If synthesis is depressed for that long it might well affect function severely. Effects of prolonged pharmacological inhibition of protein synthesis would be very informative, along with measurements of protein turnover after ischemia.

Independently of the effects of long-term loss of protein, the global inhibition of synthesis after ischemia may be extremely important in death of vulnerable cell populations by preventing synthesis of protective proteins at critical times and preventing resynthesis of proteins that are inactivated by the immediate sequelae of ischemia.

It is clear that microtubule dissolution occurs in ECC (495, 521, 522) and may play a major role in allowing this form of damage. This has not been tested with microtubule stabilizing drugs such as taxol, and little is known about the importance of microtubule dissolution (1122, 1123, 1236, 1237).

In the gerbil, microtubules in the distal dendrites of the very vulnerable CA1/subiculum region disassemble immediately after 5-min ischemia; this disassembly reaches the proximal dendrites after 10 min (1237). Loss of MAP2 and tubulin antibody staining accompanies the microtubule breakdown (1237, 1242). The cells within CA1 that are somewhat less vulnerable require longer durations of ischemia to show this early dissolution. However, between 6 and 24 h after 5-min ischemia there is a major microtubule disassembly in proximal (and distal) apical dendrites of all CA1 pyramidal cells (348, 1236). Semiquantitative estimates show a 50% loss in microtubules at this time (348). In general, these changes are irreversible in cells that are destined to die. They also occur in nonvulnerable cells where they recover after 12-24 h (348). There is complete loss of MAP2 antibody staining in apical dendrites of the CA1 pyramidal cells at this time also (726).

Kinesin and cytoplasmic dynein immunoreactivities are greatly decreased in CA1 at 3 and 8 h after 5-min ischemia (29), before the major microtubule disruption, suggesting an early blockade of dendritic transport of most proteins (29). This could be very important, and the result will, hopefully, be pursued.

Microtubule changes following global ischemia in the rat are much less dramatic than in gerbil, even up to 24 h after 2-VO (251, 357, 581, 1127). However, microtubules do become disorganized between 24 and 48 h, well before cell death. It is not at all clear why the gerbil and rat behave so differently; there may be more stabilizing elements such as calpastatin in rat, but this is not known. There are no measurements to see whether motor proteins might be compromised, as they are in gerbil.

There are no reports concerning early microtubule changes in focal ischemia.

Known mechanisms of microtubule dissociation include MAP2 phosphorylation or proteolysis, dissociation of the putative microtubule-stabilizing protein STOP decreased GTP/GDP, or proteolysis of tubulin (708, 770). All of these are activated by Ca²⁺ with the probable exception of the decrease in GTP/GDP (273). MAP2 phosphorylation and dissociation from microtubules is also activated by PKC and other kinases that may be activated very early during ischemia (132, 459, 491). Thus there are many potential mechanisms for microtubule dissolution.

Recent studies from our laboratory (316, 1291) on rat hippocampal slices, where there was significant breakdown of microtubules in CA1 dendrites during 6-min ischemia, indicate that the breakdown is almost certainly mediated by Ca²⁺. It was greatly attenuated when slices were incubated in 0 Ca²⁺ buffer and ketamine, a combination that prevents 80% of the normal rise in cytosolic Ca²⁺ (1288), or lidocaine and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which similarly blocked the cytosolic Ca²⁺ rise. It was also blocked by inclusion of 25 µM calmidazolium, the calmodulin antagonist (316) (which did not attenuate the cytosolic Ca²⁺ increase), and by the calpain inhibitor MDL-27,180 (1291).

The mechanism of breakdown is not known, but it is not mediated by MAP2 proteolysis or by proteolysis of microtubule only stabilizing protein, STOP or tubulin (Zhang and Lipton, unpublished data). The involvement of calmodulin might be because it causes dissociation of STOP from the microtubules, which it does in vitro (912), but this has not been shown for ischemia. These considerations suggest that prolonged, or delayed, elevation of Ca²⁺ might be responsible for the profound microtubule dissociation in gerbil and the milder disorganization in rat, but there is no current evidence.

The microtubule-dissociating drug colchicine causes cell death in cultured cerebellar granule cells (110), as well as other cell types (659, 1140, 1193). Death appears to be apoptotic. Furthermore, photoactivated disruption of microtubules leads to loss of intracellular organelle motility and quite rapid cell death in cultured kidney epithelial cells (639). Thus there is reason to think that the ischemic breakdown of microtubules would contribute to the cell death. Unfortunately, there are no data that support (or contradict) the possibility.

There are data showing correlations between microtubule dissolution and ischemic cell death. Ischemic tolerance in gerbils is associated with the reestablishment of the microtubules in the CA1 pyramidal cells (348), and MAP2 breakdown is affected by temperature in the same way as cell death. Reducing temperature to 33°C during ischemia markedly decreased loss of MAP2 immunoreactivity, whereas increasing temperature to 39.7°C markedly enhanced it (293).

The most exciting study to see would be the effect of the microtubule-stabilizing drug taxol on development of ischemic cell death. Unfortunately, there are no such reports. Taxol does attenuate ischemic microtubule breakdown in the slice (Zhang and Lipton, unpublished data), so it may well provide useful information if used in vivo.

A) SPECTRIN AND ANKYRIN. Spectrin provides major linkages between the cell membrane and membrane-associated proteins, which in turn are involved in maintaining integrity and localization of integral membrane proteins (80, 81, 471). It is very susceptible to calpain (471) and to caspase-3-like proteases (810), which cleave it at different sites (810).

After 5-min ischemia in the gerbil there is a rapid (within 30 min) breakdown of spectrin by calpain (641, 952, 973) in many brain regions but oddly, perhaps, not in CA1 pyramidal cells of hippocampus (952). However, there is a delayed breakdown that is measurable in dendrites of CA1 pyramidal cells after 1 day, peaking at 2 days, and persisting until cell degeneration (952, 973, 1258). This suggests a delayed activation of calpain in CA1 that is probably very important and is discussed in section VB. There is breakdown at the core of a focal ischemic lesion after 3 h (455), but no substantial studies have been reported for focal ischemia.

These results are certainly consistent with a role for spectrin breakdown in the delayed cell death. However, arguing against this somewhat is that only a very small percentage of total spectrin in a region is broken down. Although several studies clearly show the appearance of spectrin breakdown product, there are no notable decreases in band intensities of native spectrin on Western blots (13, 455, 641, 952, 973), even in the core of the lesion after 3-h focal ischemia (455). Thus the percentage reduction is small. It may, however, be important.

Caspase-activated spectrin breakdown, which may well be an important component of apoptosis, has not been investigated in vivo, but it is prominent during in vitro ischemia in rat cortical cell cultures (810).

Ankyrin plays a major role in linking membrane proteins, such as Na⁺-K⁺-ATPase and Na⁺ channels to the cytoskeleton. Two major isoforms, ankyrin B and ankyrin R, are localized to membrane fractions including the crude synaptosomal fraction (426). Thirty minutes of global ischemia in rat, followed by 60-min reperfusion, caused a 20% loss of immunoreactivity of ankyrin B and breakdown of ankyrin R, possibly mediated by calpain. The breakdown was largely in the postischemic period, suggesting that reperfusion sensitizes the system. These changes could certainly lead to malfunction of the Na⁺ pump or other membrane proteins and so contribute to cell death. This may account for the observed inhibition of the Na⁺ pump after 60-min ischemia (866). Effects of shorter, but still lethal, durations of global ischemia need to be demonstrated, as do effects of focal ischemia.

B) OTHER CYTOSKELETAL PROTEINS. There is a 30% loss of immunoreactivity of a 200-kDa neurofilament subunit 1 day after either 5- or 8-min 2-VO in the rat (519); the role of the protein is not known.

	V. PERPETRATORS OF FUNCTIONAL OR STRUCTURAL DAMAGE

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This section examines biochemical processes that are likely to play a part in cell death by causing long-term changes in macromolecules. These will lead to the functional changes discussed in section IV or other effects related to cell death. They are considered perpetrators of damage.

A.  Free Radical and Peroxynitrite Actions

1.  Free radical, NO, and peroxynitrite changes during and after ischemia

The possible roles of free radicals, NO, and peroxynitrite in ischemic cell damage have been extensively reviewed (170, 985, 1043, 1046) since the early suggestions of Siesjo (1041). As discussed in section VA2, there are many ways in which free radicals (including NO) and peroxynitrite may be generated during ischemia. There is massive evidence from many systems that these species can cause cell damage. The question addressed in this section is the extent to which they are actually involved in ischemic cell death and the ways in which they are involved.

A) GLOBAL ISCHEMIA. I) Free radicals. Several studies show increased free radical formation during vessel occlusion. The electron-spin resonance signal increases over 10-fold during 15 min of 4-VO, measured on extracellular fluid collected in a cortical cup (906). There is a similar increase during this period in the striatum (1305) and in hippocampus (908) as measured with microprobes containing a spin trap or salicylate, respectively. There is also an increase in PBN spin adducts after 20-min 2-VO (976). There are some contradictory results. In one study there was no evidence of increased free radicals at the end of 10-min ischemia in gerbil (851), and no change in reduced glutathione was measured after 30 min of 4-VO in the rat (217). However, overall the evidence showing increases is persuasive.

The increase in free radicals becomes larger and unambiguous during the early reperfusion period (263, 851, 906, 908, 976, 1260, 1305). The persistence of the increase depends on the duration of the ischemia. After 10-min ischemia in rat, lucigen chemiluminescence levels fell from two times baseline to baseline after 40 min. However, after 15-min ischemia, the levels of 2,3-DHBA, a measure of free radical or peroxynitrite attack on microprobe salicylate, was still rising 60 min after the end of ischemia (908), and after 20-min ischemia, chemiluminescence levels remained at 150% baseline for at least 2 h. Thus durations of global ischemia that lead to profound delayed neuronal death cause free radical increases that are probably maintained for at least 2 h. Most interestingly, 24 h after 10-min global ischemia, there is a large elevation in superoxide that is restricted to the vulnerable CA1 pyramidal layer in the hippocampus (173). This was measured using hydroethidine fluorescence. The increase is approximately coincident with an upregulation of the enzyme COX-2 in CA1 pyramidal neurons. This occurs between 8 and 24 h after the ischemia (806) and is a very likely source of free radicals.

II) NO and peroxynitrite. There was an increase in NO, to ~11 µM, at the end of 15-min 2-VO global ischemia (1126), as measured with an NO-sensing electrode, and there was also an increase after 7-min 2-VO in hippocampus (850). No on-line measurements have been made after the end of global ischemia, so it is not known if the radical is continually generated. Peroxynitrite, which is generated from NO and superoxide, is specifically monitored by measuring formation of nitrosotyrosine. The latter is elevated 4 h after 30-min global ischemia in rats (330), but this is the only reported study. Further quite strong evidence for peroxynitrite formation, from the same study, is that blocking NOS with N^G-nitro-L-arginine (L-NNA) increased the net production of superoxide (330), suggesting the latter is normally metabolized to peroxynitrite. Thus insofar as measured, there is evidence for NO and peroxynitrite accumulation in global ischemia. At present, there is no detailed knowledge about the time course of peroxynitrate except that it occurs at some time during the first 4 h.

There may well be delayed production. Inducible NOS is greatly increased in the astroglia that border the CA1 pyramidal layer after 10-min 4-VO global ischemia (302, 303). The increase is first seen 1 day after the insult and becomes prominent 2 days later. The increase is confined to the CA1 region and is adjacent to pyramidal cells, which die at about this time. No measurements of nitrosotyrosine have been reported.

B) FOCAL ISCHEMIA. Early indirect measurements of thiobutyric acid reactants (TBAR) accumulation (777), of loss of scavengers such as -tocopherol, and of reduced forms of ascorbate and ubiquinones (577) strongly indicated that free radicals were produced within 30 min of initiating focal ischemia. These early studies were very important in establishing that there was production of free radicals. Moreover, the study in which depletion of scavengers was measured indicated that there was continual production of free radicals for at least 6-12 h of ischemia (577).

More sophisticated on-line measurements, using a cytochrome c electrode (312), or measuring lucigenen enhanced chemiluminescence in a cranial window (893), show marked increases in free radical formation in the peri-infarct penumbra (where flow levels are ~50%) during and after focal ischemia. During the ischemia, there were about twofold increases in indicator signal that occurred within 15 min of onset in one study (312) and within 100-min of onset in another (773). These persisted throughout 2-3 h of ischemia, indicating continual generation. Reperfusion after 1 or 2 h gave a large burst that lasted ~40 min and persisted throughout the remaining 2 h of the study at a steady level about twofold over baseline (312, 773).

Free radical production appears largely restricted to the penumbral region. When localized salicylate microprobes were used, continued generation was measured in the penumbra during 3 h of ischemia and for 6 h after ischemia. There was no production in the core through 3-h ischemia and until after 3-h reperfusion (1060). There is major damage by this time. This is an important study, suggesting that damage in the core of the lesion is unlikely to result from free radicals, although, as seen below, NO and peroxynitrite do appear to rise in the core. The absence of free radical generation in the core is somewhat surprising given the apparent generation of free radicals during global ischemia, where O₂ levels are lower than in the ischemic core, and confirming studies will be important.

Actual rates of superoxide generation within the ischemic region, measured by the cytochrome c electrode, were ~25 µM/min (312). This is undoubtedly much lower than actual values, because some free radicals would have been scavenged before measurement.

I) NO and peroxynitrite. Nitric oxide appears to rise during focal ischemia. There are three reports of a transient (20 min) increase (622, 707, 1293) to ~2 µM (707). There is a continual generation in the postischemic period (622), although it is somewhat smaller than the early increase during ischemia (~0.6 µM) (707, 1293). It lasts for at least 60 min.

The increase in NO was enhanced by superoxide dismutase (SOD) infusion, which competes for superoxide radicals (74), indicating that the NO is normally reacting with O₂ to produce peroxynitrite (622). Measurements of 3-nitrosotyrosine confirmed a substantial generation of peroxynitrite (347). There was about equal formation in core and penumbra at the end of 2 h of focal ischemia and approximate doubling of this in the penumbral region during 3 h of reperfusion. This was all blocked when NOS was inhibited by N^G-monomethyl-L-arginine (347). Levels of 3-nitrosotyrosine rose to ~1% of all tyrosine residues, far higher than in basal conditions when there is no measurable nitrosotyrosine, and 5 times the levels seen in Huntington's disease. This is a major increase.

The rise of nitrosotyrosine in the core appears to conflict with the lack of free radical generation in the core (1060). More studies are required to resolve this, but it is quite possible that the enhanced NO production combined with a normal steady-state superoxide production will allow formation of peroxynitrite (74).

C) INDUCED EXPRESSION OF NOS AND OF CYCLOOXYGENASE AFTER FOCAL ISCHEMIA. Two enzymes that are intimately related to free radical production are upregulated during the 6- to 24-h period after temporary focal ischemia, as they are after global ischemia. Both iNOS (479) and COX-2 (837) are upregulated in the penumbral region during this period. The former is in endothelial cells and invading neutrophils (479, 836), whereas the latter is in neurons (836). Cyclooxygenase-2 (prostaglandin H synthase) generates superoxide radicals (837). The upregulation of these enzymes might lead to NO and superoxide synthesis during the later phases of reperfusion. Although they are in different cell types, they might well react to form peroxynitrite because both NO and superoxide can diffuse for several hundred microns (74). Further evidence for communication between the two processes is that about one-half of the enhanced COX-2 activity, although not the enzyme upregulation, results from the action of the iNOS (836). This was determined both by pharmacological and knockout studies.

D) SUMMARY. The studies definitively show that free radical production is elevated during both global and focal ischemia, for at least 2 h after focal ischemia and very probably for at least 6-12 h. Levels are elevated 24 h after global ischemia in vulnerable cells. Nitric oxide production is elevated to the low micromolar range during global and focal ischemia; it is also elevated after focal ischemia; no measurements have been made after global ischemia. Peroxynitrite is produced during or after both focal and global ischemia. Superoxide levels of 25 µM and NO levels of 1-15 µM have been measured. They should (74), and apparently do, yield a high level of peroxynitrite formation.

Critical questions that remain include the time course of free radical generation throughout the postischemic period and further localization and quantitation.

Normally the rates of free radical production and elimination are equal, leading to a steady state that is presumably tolerated by the cell. Ischemia creates several conditions that could account for the increased net production of free radicals (336, 413, 488).

A) XANTHINE/HYPOXANTHINE OXIDATION. The breakdown of adenine nucleotides during ischemia leads to accumulation of hypoxanthine within 10 min (the earliest published measurement, Ref. 407), which is then metabolized by xanthine oxidase (XO) or xanthine dehydrogenate (XDH). Xanthine oxidase produces free radicals, but XDH does not. The basal level of XO in brain tissue is similar to that in other organs such as liver and intestine (92).

The reaction for generating superoxide is as follows
Accumulation of urate also results from the action of xanthine dehydrogenase (XD), and allopurinol, which inhibits the XO, also scavenges free radicals. Thus unequivocally demonstrating this mode of free radical production in the tissue is difficult.

At the moment, the evidence favors its importance in global ischemia (578) but not in focal ischemia (92). This is reasonable because changes in ATP and, probably, Ca²⁺, are larger during the global ischemic insult. The activity of XO, produced by proteolytic cleavage of XDH, was increased about fivefold 30 min after 15-min global ischemia in rat, and there was concomitant urate production (578). This is reasonable evidence that free radicals are being produced by XO. Although there is a large production of urate (tens of µM) during focal ischemia (92, 577, 1152), there is no particular evidence that it reflects XO activity rather than xanthine dehydrogenase activity. There is no protease-mediated conversion of XD into XO during focal ischemia, measured at 24 h (92), and, more importantly, allopurinol does not prevent edema at doses that almost completely block the urate production (92). Other free radical scavengers do protect against edema in the same condition. The results thus suggest that free radical formation does not result from this reaction in permanent focal ischemia. There are no data for temporary focal ischemia.

B) ACCUMULATION OF EICOSANOIDS. Oxidative metabolism of accumulated arachidonic acid (549, 1111) via the cyclooxygenase pathway (602, 620, 623) or via the lipoxygenase pathway (488, 623) leads to superoxide or OH· production. Phospholipid breakdown into arachidonic acid is then further activated by free radicals (45, 175).

There is a manyfold increase of FFA, including arachidonic acid, during global and focal ischemia (804, 1111, 1292), and arachidonic acid metabolites are still 7-30 times basal 30 min after global ischemia (1111). No measurements were made right after focal ischemia, but by 4 h, there was no elevation (1292). The metabolism of arachidonic acid must thus be considered a likely source of the free radicals during and soon after both global and focal ischemia. However, there are no pharmacological studies directly establishing this in brain. There are such studies in retina, where inhibition of arachidonic acid metabolism reduced lipid peroxide formation and retinal damage (179).

A major mechanism for eicosanoid accumulation is the ischemia-induced synthesis of COX-2 that occurs 6-24 h after both global (806) and focal (837) ischemia. Inhibitors of this enzyme, such as SC-58125 or NS-398, dramatically reduce the delayed accumulation of prostaglandin E₂ in both insults (806, 836). This COX-2-induced eicosanoid production occurs at 6-48 h and so constitutes a major source of delayed free radical production.

C) ALTERED MITOCHONDRIAL FUNCTION. Generation of free radicals by mitochondria has been well reviewed (740, 1142). The organelles normally generate free radicals at a rapid rate (117, 1142), but one which is handled by normally functioning cells. When the electron carriers become highly reduced, as much as 2% of the electron flow leads to direct single-electron reduction of oxygen and formation of superoxide (117, 336, 561). This very probably occurs during focal ischemia, and low-flow global ischemia when the members of the respiratory chain are in a relatively reduced form. Accumulation of Ca²⁺ (1143) and opening of the MTP (617, 710, 1275) are among other factors that may well occur at some point after ischemia, and which very probably cause mitochondrial free radical production by mechanisms that are not yet known (see sect. IVB).

Despite the great potential for mitochondria as a source of free radicals, there is only one study that actually demonstrates their involvement (908). Direct measurement of free radical concentration, using the salicylate trap, revealed a steady increase during 15-min 2-VO and for the following 1-h reperfusion. This was completely prevented when mitochondrial respiration was blocked by including rotenone in the dialysate. It was restored if the rotenone block was by-passed by succinate, further implicating mitochondrial electron transport as the source. The free radical production was also prevented by haloperidol, which blocks respiration at complex I (908). As the authors point out, these results do not exclude other possible sources of free radicals, particularly prostaglandin metabolism, because salicylate is a potent inhibitor of cyclooxygenase and so could have been blocking that pathway while measuring free radical production.

Free radical production from electron transport also occurred in cultured neurons exposed to NMDA (282). The authors suggested this was due to accumulation of Ca²⁺. Consistent with this idea, the combination of elevated Ca²⁺ (2.5 µM) and ADP potently activated OH· production in isolated mitochondria (287), with the OH· acting as a positive feedback to enhance OH· production. Although the mechanism was not determined, the conditions approximate conditions after ischemia and after glutamate application.

Knockouts of mitochondrial SOD (Mn-dependent SOD) enhance the extent of the lesion after focal ischemia (786), whereas overexpression of the same enzyme decreases infarct size (562). If this enzyme selectively inactivates mitochondrially generated O₂·, then mitochondria are definitely a source. However, this selectivity has not been established (74).

D) NEUTROPHIL ACCUMULATION AND ACTIVATION. As discussed in section III, these cells adhere to the vessel walls and invade the parenchyma to a limited extent during and after focal ischemia (488, 1046). Neutrophils oxidize NADPH to generate superoxide and thus are important potential free radical donors during and after focal ischemia. In addition, neutrophils synthesize iNOS 1-2 days after focal ischemia (480). Evidence as to their role in free radical production is conflicting as discussed in section III. Neutrophils may play a priming role in activation of free radical production. There is no evidence concerning the roles of neutrophils in global ischemia free radical production.

E) MECHANISMS OF NO AND PEROXYNITRITE GENERATION. Nitric oxide is generated by neuronal or endothelial NOS in an oxygen-dependent reaction that is activated by Ca²⁺/calmodulin in most neurons and endothelial cells (477, 996). The Ca²⁺/calmodulin directly activates the enzyme and may also remove a phosphorylation-induced block by activating calcineurin (235). Activation of NOS by the Ca²⁺/calmodulin system has not been explicitly demonstrated by measuring, directly or indirectly, effects of appropriate agents on NO production during ischemia. The conclusion that the Ca²⁺/calmodulin system is important is based on the importance of Ca²⁺ in NOS-mediated glutamate toxicity, as well as the ability of calmodulin inhibitors and calcineurin inhibitors to prevent such toxicity (235). The latter studies are not persuasive, however, because calcineurin inhibitors prevent NMDA toxicity in cerebellar cultures where NOS is not mediating the toxicity (26). Thus proving the involvement of calmodulin remains an issue.

Both neuronal (1294) and inducible (478) NOS are upregulated after focal ischemia, and iNOS is also upregulated in glia 1-2 days after global ischemia (303, 477). There is no explicit demonstration that these cause NO accumulation, but it must be considered very likely.

Peroxynitrite is generated by the reaction between superoxide and NO
It can be protonated to produce the very reactive peroxynitrous acid that dissociates into OH· and various nitrogen/oxygen species (74). The unprotonated form, which is about 75% of total at pH 7.4, is also very reactive, with a half-life in biological systems of ~1-2 s and long diffusion distances of ~100 µm. There is good evidence that both the acid and the anion participate fairly equally in most reactions (1013). One or other of the forms readily crosses cell membranes so that peroxynitrite, like superoxide, can act in cells other than those in which it is generated (74, 1162).

Direct evidence for peroxynitrite formation was described above in all forms of ischemia, as accumulation of nitrotyrosine. As well-argued by Beckman (74), the peroxynitrite formation occurs because the rate constant for this reaction is very high (6.7 × 10⁹ M¹ · s¹) so that NO can effectively compete with the very active SOD for superoxide when it rises to 1-2 µM, as it does in ischemia. Peroxynitrite formation is not expected in the absence of NO generation unless superoxide generation is high enough to effectively saturate the competing SOD reaction. Mass action would then allow significant formation of peroxynitrite. This may, in principle, occur endogenously and does appear to occur when superoxide is artifactually generated in cultured PC6 cells (562).

F) MONOAMINE ACCUMULATION. There is quite strong evidence that H₂O₂ is produced from accumulated catecholamines (51) via monoamine oxidase (MAO) in the 5 min after 15-min global ischemia in rat (1053). Measurements were indirect, but rapid oxidation of glutathione was blocked by MAO inhibitors, suggesting MAO activation of H₂O₂ production. This certainly makes sense given the large release of monoamines during ischemia. Blockade of MAO was not protective (1053), indicating that this early H₂O₂ production was not damaging.

G) INTERACTIONS BETWEEN DIFFERENT SPECIES: FORMATION OF OH FREE RADICAL. Superoxide and NO are usually the first free radicals formed, but superoxide is not very reactive. The formation of ONOO is likely to mediate much of its toxicity. In addition, though, the reaction products of superoxide, OH· or HO₂·, are very reactive and more likely to mediate toxic effects (413). Formation of OH· is strongly favored by the decreased pH that prevails during and after ischemia (75, 488) and also by free iron (413), as shown below in the Fenton reaction

Brain iron is ~63 µM/g dry tissue, a concentration of ~12 mM. Most of this is in heme enzymes or bound to ferritin. Iron appears to be delocalized from the proteins in the postischemic phase and possibly also during ischemia (1205), probably as a result of lowered pH (488) or superoxide-induced reduction of Fe³⁺ (692). One hour of anoxia in cortical homogenates tripled the amount of delocalized iron (118). The increase in free iron should greatly enhance OH· production, and iron conjugation has been used somewhat successfully to reduce ischemic damage (46, 1205), probably by blocking this reaction. Decreased pH is an important initiator of iron delocalization and hence OH· production.

In contrast to this scenario, it has been argued that the Fenton reaction is of little importance. The reactivity of OH· may paradoxically render it less harmful than other free radicals because it will oxidize many small organic molecules and so be effectively "inactivated" with regard to damaging important macromolecules (74). Furthermore, the superoxide-driven Fenton reaction is very slow, even in the presence of iron, and it has been argued that superoxide would be largely metabolized by SOD and also by NO to peroxynitrite (74). Although sound, these considerations have not been verified experimentally and may not be applicable; lowered pH does actually enhance damage (see sect. VID) arguing for the importance of the Fenton reaction. However, protonated ONOO may well be more damaging than ONOO itself, providing an alternate way by which lowered pH could enhance damage (1013). At this stage, the importance of the Fenton reaction needs to be established. The efficacy of iron chelation suggests it is important.

H) CONCLUSION. Free radicals including NO, and also peroxynitrite, certainly rise as a result of ischemia. Despite the multiplicity of possible pathways, the only explicit demonstration of a mechanism for free radical formation is via the mitochondrial redox chain during and for 45 min after global ischemia. Monoamine oxidase acting on released monoamines very probably releases H₂O₂, which could then be metabolized to OH· in the Fenton reaction. This may be an important source of early free radical production, but it does not appear to contribute to damage. Specific demonstrations of XO-induced free radical generation, arachidonic acid metabolite-induced free radical generation, calmodulin-induced NO production, and neutrophil-induced superoxide formation from NADPH are lacking at this time. The fact that inhibitors of iNOS and of COX-2, as well as iNOS knockouts, attenuate damage suggests strongly that synthesis of these two enzymes generates NO and superoxide 12 h to several days after ischemia, but no measurements have been made.

There is good evidence that ONOO is formed from increased NO and superoxide. Indirect evidence suggests that it can also be formed after activation of NOS alone, using basal levels of superoxide, or from superoxide generation alone.

Peroxynitrite, superoxide, and OH· represent three very active species. It is, in fact, quite difficult to determine which of these contributes to the measured elevations in free radicals described in section VA1 because most of the standard measuring techniques for free radicals also respond to peroxynitrite. Lucigenin fluorescence is quite specific to OH·, but PBN will also respond to peroxynitrite or its acid (1013) as will the hydroxylation of salicylate to DBA (809).

A) GLOBAL ISCHEMIA. Several free radical scavenger systems protect against global ischemia in the gerbil. -Tocopherol rescued ~75% of the cells that normally died 7 days after 5-min ischemia (424); the animals were on heating pads, but their temperatures were not monitored. A similar degree of protection was afforded by the spin trap PBN when injected for 2 days starting right after the 5-min ischemia (1270). The PBN had no measurable effect on body temperature, although at higher concentrations it did strongly lower temperature, so there is a small suspicion that temperature may be a factor even at the lower concentrations. When infused into ventricles just before the ischemia, human recombinant Cu/Zn SOD attenuated damage in CA1 (1157) as did the XO inhibitor and general free radical scavenger oxypurinol (40 mg/kg), when injected intravenously. Temperature was not regulated in this study (903), but subsequent studies in rat pups (8 days) showed that the very similar drug, allopurinol, at higher doses did not lower body temperature (865). Intraventricular SOD (1135, 1136) protected against damage from three successive 2-min ischemic episodes, and as predicted, if free radicals are involved, the combined administration of SOD and catalase was more effective than either enzyme alone (1135, 1136).

Free radicals also play a major role in damage in the rat. The membrane-permeant free radical scavenger U-101033E attenuated damage in CA1 by 50% after 20-min 2-VO; temperature was unaffected for at least 70 min after the ischemia (1059). This protection contrasts with earlier results with another free radical scavenger that was found not to penetrate the blood-brain barrier (72). The OH· scavenger MCI-186 reduced CA1 pyramidal cell damage by 70% 3 days after 10-min 4-VO in a well temperature-controlled study (1238). This reduction in damage was associated with about a 50% reduction in OH radicals in that region, as measured by the salicylate-DHBA method. Oral administration of a flavinoid antioxidant, ()-catechin, for 2 wk before and 1 wk after completely protected CA1 in gerbil hippocampus against 5-min ischemia (496). There was an associated 25% increase in the free radical scavenging activity of cortical homogenates. Temperature was not controlled in the study. Fivefold overexpression of the cytosolic Cu/Zn SOD reduced CA1 damage in rat by ~50% 3 days after 10-min 2-VO (173), further implicating free radicals in the damage. The implication is supported by the protective effect of the COX-2 inhibitor SC-58125. This reduces damage 14 days after ischemia by ~35%, even when given 1 h after ischemia (836).

B) FOCAL ISCHEMIA. I) Temporary ischemia. Even the blood-brain-barrier-impermeant scavenger U-74006F strongly protected against damage in temporary focal ischemia, probably because of the more extensive involvement of the vasculature in this insult. It almost completely blocked the twofold increase in TBAR that usually occurred 24 h after 3-h MCA occlusion and reduced infarct area by ~25% (1154). Temperature was well-regulated. Superoxide dismutase (30), SOD conjugated with polyethylene glycol (679), and SOD entrapped in liposomes (492) consistently reduced infarct volumes by 25-40% in well temperature-controlled focal ischemia studies. These studies have been pursued and confirmed in a very sophisticated series, principally by Chan and colleagues (573, 1246), using mouse mutants. Overexpression of Cu/Zn SOD by about threefold reduced the infarct volume in two different models of temporary focal ischemia in mouse. The penumbra was significantly preserved 24 h after 1-h cortical ischemia (573) and 3 h after a 3-h proximal occlusion (26%) (1246). In both cases, there was also preservation of scavenging systems (reduced glutathione and/or ascorbate), strongly indicating that the mutants were more effectively dissipating free radicals. Overexpression of the mitochondrial Mn SOD, at a level twice its normal value, reduced infarct volume by ~20% 24 h after 1-h focal ischemia (562). The similar effect of the two overexpression mutants suggests that mitochondrial and cytosolic SOD might both act on the same pool of superoxide.

Uric acid, a very effective scavenger of peroxynitrite and OH·, reduced the infarct measured 24 h after 2-h MCA occlusion by 75% when injected either before or immediately after the ischemia (1266). This dramatic effect, which extended to the striatal core of the infarct, suggests the importance of peroxynitrite in damage to that region. Inhibition of COX-2 to levels that almost completely block excess generation of prostaglandins decreases infarct size by ~30%, suggesting that delayed development of the infarct is free radical dependent (837).

II) Permanent ischemia. There are apparently conflicting data on effects during permanent lesions; however, they can be resolved. The mice in which overexpression of Cu/Zn SOD prevented the temporary focal ischemic damage described above were not at all protected when subjected to 24-h permanent focal ischemia (172), suggesting free radicals play no role. On the other hand, the spin trap PBN reduced infarct size by >50% at the end of 48-h transient or permanent MCA occlusion, even when administered 3-12 h after the onset of the ischemia (160, 1298). This discrepancy may be readily resolved. Permanent ischemia is protected by inhibiting NOS (see sect. VA), implying damage is mediated by peroxynitrite. This might have been formed from excessive NO production and residual superoxide even when superoxide production was attenuated in the mutants. On the other hand, PBN should scavenge free radicals produced as intermediates in reactions of peroxynitrite with targets such as amino acid side chains or scavenge peroxynitrite or peroxynitrous acid themselves (1013).

The time window for damaging free radical action of between 3 and 12 h after the onset of ischemia has been further refined in a nice study on a mixed permanent/temporary model of thrombotic occlusion (1099). A short-lasting OH· scavenger, EPC-k-1, whose brain turnover half time is ~1 h was very protective when administered at 3 h after the onset of ischemia, but not at 0 or 6 h. This study very nicely pinpoints the first damaging action of OH· to between 3 and 6 h after the start of ischemia.

Further evidence that free radicals are important in permanent focal ischemia comes from the effects of altered dietary vitamin E in a well temperature-controlled study. Forty-eight hours after proximal MCA occlusion, the lesion size was twice as large in rats that had been fed a vitamin E-deprived diet as compared with those on a vitamin E-supplemented diet. Lesion size correlated well with blood levels of vitamin E in the two populations (542). This is a dramatic effect, indicating the great importance of free radical scavenging in preventing permanent focal ischemic damage.

C) ENDOGENOUS SOD CHANGES IN VULNERABLE REGIONS. Given the apparent importance of free radical changes in damage, it is extremely interesting that levels of both forms of SOD decrease in the vulnerable CA1 and CA4 regions 1 day after 10-min global ischemia in gerbil (734). Levels are unchanged or elevated in CA3 and dentate gyrus, two regions which are not vulnerable. There is a similarly selective downregulation of the predominantly mitochondrial Mn SOD after 5- to 10-min 4-VO in rat (680). It is clear from other systems that downregulation of these enzymes by using antisense to Cu/Zn SOD hastens apoptotic cell death (393, 967) and also increases the size of infarcts resulting from focal ischemia (601, 786). Thus the enzyme decrease following ischemia might well contribute to the vulnerability of CA1 pyramidal cells. This is rendered more likely by the quite dramatic selectivity of the superoxide increase that occurs 24 h after a global insult in rat. Although COX-2, a likely perpetrator of the delayed rise, increases throughout the hippocampus (806), superoxide only rises in the CA1 pyramidal layer (173).

D) SUMMARY. Various factors that should reduce free radical buildup provide reasonable but not complete protection against global ischemia in rats and gerbils. Protection against temporary focal ischemic damage ranges from reasonable (25% reduction in infarct size) to very strong when uric acid or the spin trap PBN were used. PBN was also very protective against permanent focal ischemia. Damage from permanent ischemia was also very sensitive to dietary vitamin E, again indicating a dependence on free radicals. Unfortunately, none of these studies included measurements of free radical levels, so the degree of free radical suppression is not known. Studies in which actual free radical levels are measured and in which the full time course of protection is measured are needed to help determine the mechanism of free radical damage.

A) NO. Interpretive difficulties in early studies were caused by conflicting effects of endothelial NOS activation and neuronal NOS (nNOS) activation (230). It is now clear that nNOS activity is damaging during and after ischemia in all major models and that endothelial cell NOS activation is beneficial. Although more sophisticated measurements may later sort out subtleties, the current evidence for this conclusion is quite compelling (861).

I) Focal ischemia. In focal ischemia, relatively specific inhibitors of nNOS, when used at levels that do not decrease blood flow or prevent the vessel response to ACh, are strongly protective against permanent (1263) and transient (308, 1296) ischemia in well-controlled studies. ARL-17477 reduced the infarct volume measured 7 days after 2-h MCA occlusion by 55%, at a dose that had no effect on blood flow and 7-nitroindazole (7-NI), and another more long-lasting inhibitor, TRIM, decreased infarct by ~45% 24 h after 2-h MCA occlusion. 7-Nitroindazole decreased infarct volume by 25% after 24-h permanent ischemia (1263). The reduction may have been smaller than with ARL because the occlusion was permanent rather than transient or, more probably, because the lifetime of the 7-NI is only ~2 h. In one study (1296), the drug was fully effective when injected at the start of reperfusion. In addition to effects of these pharmacological agents, nNOS knockout mice showed 40% reductions in infarct size measured after 24- and 72-h permanent MCA occlusion (474) and measured 24 h after temporary ischemia (423).

Inducible NOS knockout mice showed about a 20% decrease in infarct size measured after 96 h, but not 24 h, of permanent focal ischemia (482). These studies are consistent with the delayed (24-48 h) increase in iNOS, in invading neutrophils. They indicate that the late progress of damage may be due to production of NO from this source. These studies are more persuasive than effects of the iNOS blocker aminoguanidine (799) because the latter also inhibits other potentially damaging enzymes.

Thus there is strong evidence that NOS is very important in focal ischemic damage.

II) Global ischemia. Neuronal NOS knockout mice showed greatly reduced damage 72 h after 10-min global ischemia (868). A normal ischemic effect of 85% cell loss was reduced to a 32% cell loss in the CA1 pyramidal cell layer. This is a substantial effect and argues for an important role of NO generation. Earlier pharmacological studies did not support a role for NO in global ischemic damage because they showed no protection against CA1 damage in either rat or cat (138, 585). However, when the specific inhibitor of nNOS 7-NI was used in well temperature-controlled studies, it reduced damage 7 days after 20-min 2-VO in the rat by ~35% (808). The level of inhibition of NO production is not known, but the result indicates that NO generation during the first 2 h of reperfusion contributes to damage. Greater protection may have been achieved if the 7-NI was injected before the ischemia as NO is generated during global ischemia.

III) Cell cultures. Very dramatic effects are manifested in cell cultures where delayed cell death after 60-min in vitro ischemia is almost completely blocked in cultures from mutant mice lacking nNOS (237) and also by NOS inhibition (704).

IV) Localization of nNOS. In the resting state, almost all nNOS in the forebrain is localized to interneurons (261, 956, 1159, 1198). It is possible that a few pyramidal cell neurons contain nNOS, but several studies, referenced above, show very clearly that most nNOS neurons in forebrain structures are interneurons. The interneuron processes with nNOS are very ramified among the parenchyma, with good evidence that nNOS-containing processes are very close to non-nNOS-containing cells (956) and so could readily affect them (236). Thus it seems likely that interneurons, which are generally much less vulnerable to ischemia, must receive the (presumed Ca²⁺) signal to enhance NO production, release it in the vicinity of other neurons and, in this way, help cause toxicity.

Added to this, there is a major and very rapid increase in the number of nNOS-positive neurons during focal ischemia that peaks within 4 h (1294). This no doubt reflects de novo synthesis of the enzyme in these neurons (as mRNA also goes up) and may well contribute to the damage. The cells that nNOS is in are not clear, although in one micrograph there was quite extensive involvement of what appear to be principal neurons (1294). The role of this process has not been pursued since the original publication.

B) PEROXYNITRITE. Peroxynitrite is currently considered to be the major mediator of NO toxicity (107, 231, 678, 1093). Evidence for this is only indirect because there are no reliably specific scavengers. Urate, which has been used in some cases (1095), and does react with peroxynitrite, is not necessarily specific (74) and is not very reactive; it fails to prevent oxidation of protein thiol groups (1162). On the other hand, its actual effects in smooth muscle are most consistent with a selective blockade of peroxynitrite action (1095), so the compound could become useful with more control studies.

The experimental evidence that peroxynitrite is important comprises a large number of studies showing that both NO and superoxide are necessary for damage, even in situations where the primary event is activation of NO synthesis. Other studies show the much greater potency of peroxynitrite than NO.

A very elegant study by Xia et al. (1228), in a nonneuronal culture, demonstrated that A-23187-induced cell death did not occur when NO production was activated in the absence of superoxide production, or vice versa, but did occur when both NO and superoxide were generated by the NOS. Cell death could be blocked either by inhibiting NOS or by scavenging superoxide. In motor neurons subjected to BDNF withdrawal, there was induction of nNOS and an increase in protein nitrosotyrosines. Cell death was prevented by blockers of NOS or by the cell-permeant free radical scavenger manganese TBAP (310). This again suggests that peroxynitrite formation is important. In a similar vein, toxicity caused by the NO donor Na⁺ nitroprusside, or by SIN-1, is very strongly attenuated by SOD (237), providing strong evidence that NO is toxic as a result of its conversion to ONOO.

A study in newborn piglets is probably the most direct demonstration that peroxynitrite formation is important in brain ischemia, although it does not directly demonstrate a role in cell death. During hypoxia in the piglets, there is an increase in PBN spin adducts, indicating increased free radicals or ONOO and also in lipid peroxidation, as revealed by conjugated dienes. These are both prevented by pretreatment with the NOS inhibitor L-NNA (843). This strongly suggests that peroxynitrite, formed from NO and existing superoxide, is the active agent in this case.

The fact that either free radical scavengers or NOS inhibition block global and focal damage is consistent with peroxynitrite being the active agent. Another explanation for these results is that NO and a superoxide-derived product are independently acting on a molecule or function and that both actions are necessary for ischemic damage. This seems less likely than the involvement of the one peroxynitrite species.

It is worth considering why peroxynitrite might be the damaging agent in so many cases. Although NO alone is not necessarily toxic, superoxide or its products, H₂O₂, or the OH· radical, certainly can be (630). One possibility is that ONOO attacks specific molecules that are critical. There is no particular precedent for this. For example, single-strand DNA breaks, which seem very important as activators of PARP (see below), are induced very rapidly by ONOO (1093), O₂ or OH· (1112), or H₂O₂ (999). It is more likely that the reason is quantitative rather than qualitative. If superoxide (and probably therefore OH· or H₂O₂) generation is fast enough, toxicity is likely to be independent of NO; if it is not, then NO and peroxynitrite formation will become important. This could be tested.

C) CONCLUSION. Overall, the studies are quite persuasive in ascribing a major role to free radical generation in ischemic cell death. A large number of agents that prevent free radical buildup, or NO buildup, strongly protect against global ischemia and against penumbral damage in permanent or temporary focal ischemia. In several of the studies, temperature has been well regulated. There are, though, critical details that need to be understood.

The degree of protection is variable, depending on the agents used to prevent accumulation. The basis for this is unknown. It could be that treatments have different efficacies on free radical or NO buildup; measurements are required to resolve this. It could be that different species (e.g., OH·, ONOO) have different efficacies in different systems and that they are differentially affected by inhibiting NO or superoxide accumulation. Details of buildup and targets are really necessary to get at this next level of understanding.

Free radicals affect the structures of both lipids and proteins. They interact strongly with unsaturated bonds in lipids, leading to a chain reaction formation of peroxides, hydroperoxides, and aldehydes (488, 561). They activate phospholipase (PL) A₂, possibly because the peroxidation makes the lipids better substrates (1163). Probably as a result of the formation of aldehydes, they markedly reduce membrane fluidity (186). Free radicals oxidize protein side chains, forming carbonyl groups (336, 1067) or disulfides, and can also act as reductants, causing SH formation from S-S bonds (336).

Quite short exposures (10-90 min) strongly inhibit several very important enzymes, including plasmalemma Na⁺ (300, 446, 684) and Ca²⁺ (1017) pumps, creatine kinase (1267, 1271), and several mitochondrial dehydrogenases (1290). In the case of the Na⁺-K⁺-ATPase, oxidation by free radicals makes the enzyme susceptible to calpain-mediated proteolysis (1307). Peroxynitrite is equally toxic, permanently inactivating the cytochrome b and aa₃ complexes of neuronal cultures 24 h after a short exposure to 500 µM concentrations; smaller concentrations are also effective (107). Free radicals and peroxynitrite also produce single-strand breaks in DNA (999, 1093).

Any or many of these effects may account for damaging effects of free radicals, but in no case yet has a specific molecular change been tied to free radicals, and also been shown to be instrumental in damage. The most clear-cut change is probably DNA cleavage, as discussed in section VA6.

Activation of the enzyme PARP (or PARS) appears to play a critical role in damage following transient focal ischemia. The basic findings, from at least four different groups, are that blockade of PARP using either of two different classes of drug (683, 1098), one of which is highly specific (683, 1280), and also using knockout mice (298, 305) greatly decreases infarct size measured by different staining techniques 24 h after beginning 90-min to 2-h transient ischemia. Decreases in infarct size range from ~45 to 85%, the latter of which, in the mouse, and the rat (1280) is the largest effect of any drug or knockout treatment yet noted. In at least one case, the drug GPI-6150 is effective when given 1 h after 2-h ischemia (1280). Only one group has studied permanent ischemia, and although data are not presented, there is said to be little elevation of polyadenine-ribosylated protein and little protection against infarct by inhibition of PARP (305). This is a dramatic difference between the two forms of ischemia. There are no reports of measurements in global ischemia.

Cortical cell cultures from Parp/ mice were completely protected against cell death after 60-min in vitro ischemia (298). Cell death following 5-min exposure of cultured cortical neurons to 0.5 mM NMDA is essentially completely blocked by the quite specific PARP inhibitor DPQ (298).

Two major issues are how the PARP is activated and what its activity may be doing to cause damage.

A) ACTIVATION OF PARP. Poly(ADP-ribose) polymerase is a nuclear enzyme that is strongly activated by single-stranded DNA breaks; there are no other known activators (88, 1093). It is extremely likely that these breaks are caused by peroxynitrite (1093), although free radicals such as superoxide or hydroxyl are also capable of doing this and almost surely do so following ischemia in heart muscle (1112). Hydrogen peroxide has the same effect (999).

Such single-strand breaks, detected by DNA polymerase I-mediated biotin-dATP nick translation (PANT) have been observed in both core and penumbra neurons early during reperfusion after 1-h focal ischemia (182). Production of the single-strand breaks seems to require reperfusion. There were no breaks at the end of 60-min focal ischemia, yet there were a sizable number after just 1 min of reperfusion (182). This requirement may derive from the large spikelike increase in free radical formation that occurs at the onset of reperfusion after 1-h MCA occlusion (893). The requirement for reperfusion is consistent with the lack of involvement of PARP in permanent ischemia damage. The very rapid production of single-strand DNA breaks that was observed is also consistent with the early appearance of poly(ADP-ribose), within 5 min of terminating 2-h focal ischemia (305). It would be very important to know if blockade of free radical or NO buildup diminished the number of single-strand breaks and also the extent of PARP activation, which can be measured (305).

The importance of peroxynitrite in the strand breaks is suggested by work in several model systems including thymocytes (983), smooth muscle cells, and macrophages (1094), where 25-50 µM peroxynitrite, probably acting in its protonated form, causes strand breaks and PARP activation, followed by cell death, with timely kinetics (1304). Nitric oxide is very ineffective in this process (1093).

B) MECHANISM OF PARP TOXICITY. This has not been well defined. The PARP-catalyzed reaction involves the cleavage of NAD, and one commonly considered basis for toxicity is depletion of NAD, with resultant inhibition of mitochondrial function. This would cause depletion of ATP and possibly the opening of the MTP. Single-stranded DNA breaks lead to this sequence (absent any measurements of MPT) in several systems, and ATP and NAD are almost completely depleted if there is enough strand breakage (88, 1094). Hydrogen peroxide very likely causes cell death by this mechanism in several cases (216, 999).

The evidence for such an energy metabolism-mediated cell death in temporary focal ischemia is not great. In general, effects of PARP inhibitors on ATP changes have not been measured. In one study, there was a 65% fall in NAD in the ischemic territory, and both PARP knockouts and a PARP inhibitor 3-aminobenzamide attenuated this fall by ~40% (305). This result is somewhat difficult to interpret because measurements were made at 24 h, by which time there were a lot of dead cells. Thus the cell saving by PARP inhibition could be responsible for the higher NAD levels. Measurements before significant cell death would be more useful. The other difficulty is that there is no evidence for dramatic falls in ATP in temporary focal ischemia. It is possible that the small reductions in ATP may be damaging over a long period, as discussed in section IVB. Effects of PARP inhibition on these ATP levels need to be measured. Alternatively, ATP may fall dramatically for a short period during reperfusion, and this may trigger damage.

Thus, at this stage, there is no well-established mechanism for the damaging effects of PARP after ischemia. Possibilities other than inhibited energy metabolism include additional effects of NAD(H) depletion and resultant metabolite changes, or effects on histones and other nuclear proteins as a result of the transient ADP ribosylation (it lasts ~3 h; Ref. 305). Alternatively, this ADP-ribosylation appears to be a critical step in apoptosis in many model systems (1049) and could certainly play such a role in ischemia.

A) NFB. There is accumulating evidence in many tissues, including brain, that free radical damage following ischemia is at least partially mediated by the cytosolically located transcription factor NFB. NFB is activated by the inactivation of an inhibitory protein, IkB, which occurs in two major steps. Phosphorylation is mediated by effects of free radicals (1000) and other pathways (56, 747, 1005) on IkB kinases (254). Subsequent to this, the phosphorylated IkB is broken down by the 20S proteasome and rendered inactive (56). Two active subunits of NFB are then translocated to the nucleus. NFB activates synthesis of many proteins including iNOS and COX-2 (1005); the latter has been particularly nicely demonstrated during hypoxia, where it was shown that the p65 unit is responsible (995). It also upregulates the CD-95 ligand (1173), which is upregulated after global ischemia. It may upregulate proapoptotic BCl-xshort after global ischemia, but this is speculative (264). Although the NFB pathway can be antiapoptotic in some conditions (1005, 1186), there is evidence that the overall pathway is damaging after ischemia. The evidence for this, and its mediation by free radicals, is much stronger in noncerebral tissues such as liver (1309), vascular endothelium (464), and heart (176) than it is in brain, but there is emerging evidence that it may be instrumental in the latter also.

NFB is activated and translocated to the nuclei of hippocampal neurons after 30-min 4-VO global ischemia in rats (206); this occurs in all neuronal populations 24 h after ischemia, but only in the soon-to-die CA1 population 72 h after the ischemia. The delayed translocation is blocked by the antioxidant LY-231617 (207), indicating that it is mediated by free radicals. However, somewhat surprisingly, the early translocation is not blocked by the antioxidant, suggesting another pathway is responsible for that process, probably a kinase cascade (640, 1005). This differs from other tissues such as liver (1309), cerebral blood vessels (464), and heart (176), where early postischemic activation of NFB is prevented by free radical scavengers. The antioxidant was very protective against global ischemic damage (207), consistent with, but certainly not proving, the involvement of NFB in damage.

NFB is activated in the core and penumbra 1 day after 90-min temporary focal ischemia of the cortex (984). The specific proteasome inhibitor PS-519 strongly attenuated damage measured 24 h after 2-h ischemia (902). As discussed previously, this might reflect damage via proteolysis. However, because the proteasome pathway is required for NFB activation, the result may reflect the importance of NFB in focal damage. If so, it shows that the ischemic core is most susceptible to damage via this system.

Aspirin and salicylate, which directly block activation of NFB, protect strongly against glutamate toxicity in cultures and seem to do so by blocking NFB. Effects are not mediated by reducing Ca²⁺ entry or by inhibiting prostaglandin metabolism and are strongly correlated with inhibition of the activation of NFB by 50 µM glutamate (395).

NFB is likely to be instrumental in upregulation of iNOS and COX-2, which are damaging in both focal and global ischemia, and also of cell adhesion molecules that are damaging in focal ischemia. However, it is not at all established that this is its mode of action. The near-complete blockade of glutamate toxicity, which is not thought to be mediated by either of the enzymes or adhesion molecules, certainly suggests alternate modes of action.

B) CD-95 LIGAND. The 45-kDa CD-95 ligand is upregulated after focal ischemia and is a reasonable candidate for initiation of apoptosis, as discussed previously. This ligand is upregulated in 12 h by H₂O₂ in cultured microglial cells, and the mRNA is upregulated after hypoxia/reoxygenation (no measurements were made of protein) (1173). In the former case, the upregulation coincides with activation of NFB; this and the fact that the CD-95 ligand gene has a putative NFB binding region suggests that the transcription factor may mediate activation of the CD-95 ligand (1173). If this mechanism also pertains after cerebral ischemia, it would be an important way by which free radicals mediate apoptotic cell death. Further work along these lines is necessary.

C) ROLE OF CYTOKINES. Cytokines that are upregulated early in ischemia and reperfusion, particularly TNF- and IL-1, are strong activators of NFB (1039). Thus they, along with free radicals generated from other sources, are potential activators of the transcription factor. The mechanism by which cytokines activate NFB is not fully known, but it does appear likely that free radicals generated by the cytokines mediate the effect because several free radical scavengers strongly attenuate the effect (381, 1000). However, other pathways activated by the cytokines, such as ceramide synthesis and MEKK kinase cascades (1005, 1039, 1186), may also be important. It is not currently known what is responsible for the ischemic upregulation of cytokines TNF- or IL-1 (747).

At this stage, the actual activators of NFB during and after ischemia are not known.

1) As described above, activation of PARP which probably, but not surely, results from free radical/peroxynitrite-mediated cleavage of DNA may well mediate a lethal effect of free radicals after temporary focal ischemia. The very large saving produced by uric acid (1266), which is a good peroxynitrite scavenger, is consistent with this because PARP inhibitors also greatly reduce the size of the lesion (1280).

2) Synthesis of CD-95 ligand via activation of NFB might be a mechanism by which free radicals cause apoptotic cell damage, as may be the increase in TNF-. However, the pathways by which the CD-95 ligand and TNF- are produced after focal ischemia have not yet been determined, so this is very speculative. Free radicals appear to initiate apoptosis in several systems, so there may also be other pathways by which they do so in ischemia.

3) Other known functional targets of free radicals that could well lead to cell death include increased plasmalemma permeabilities (201, 849), prolonged inhibition of protein synthesis in (vascular endothelial) cells via effects on eIF-4-like proteins (513), and major changes in mitochondrial function, including opening of the MTP, as discussed extensively in section IVB. There is, though, no evidence positively identifying the importance of these phenomena in ischemic cell death.

4) Effects of free radicals on Ca²⁺ homeostasis could be important. After 60-min focal ischemia, cytoplasmic Ca²⁺ remains elevated for at least 30 min. This elevation is prevented if ventricles are perfused with SOD at high levels (30), suggesting that free radicals affect some aspect(s) of Ca²⁺ homeostasis, possibly by elevating Ca²⁺ fluxes through existing or new pathways, or possibly by inhibiting Ca²⁺-ATPase. This could reflect the same underlying process as the apparent dependency of postglobal ischemic mitochondrial Ca²⁺ accumulation on NOS activation (1208).

5) Inhibiting NOS during focal ischemia prevents the normal fall in pH in the penumbra, suggesting that NO synthesis in some way causes the pH drop (943). The mechanism of this somewhat surprising effect is not clear, but if it does do this, it could certainly be a way that NO enhances damage (see sect. VID). One mechanism for this may be the effect described immediately below, where NO contributes to the release of glutamate in focal ischemia. Glutamate generally acidifies neurons (159).

6) Nitric oxide (possibly via peroxynitrite production) appears to play an important role in the accumulation of glutamate in the core of a focal lesion and the subsequent spread of glutamate to the penumbra and the resulting intraischemic depolarizations. Release of glutamate in the core, and subsequent intraischemic depolarizations, were greatly attenuated in nNOS knockout mice (1031). The mechanism may be the peroxynitrite-mediated inhibition of glutamate transporters (1132).

Of course, because of their wide-spread effects on different proteins, free radicals might alter protein or proteins whose importance has yet to be shown.

Exogenous free radicals are able to cause necrosis (111), or apoptosis (393, 502, 788, 789, 967, 1134), in cultured neurons or neuronal-like cells; at a much more extreme level, free radicals (largely OH·) generated by the xanthine/XO/Fe²⁺ system in the intact brain caused dramatic cellular degeneration within 2 h, with neurons appearing as in ischemic-cell change and the blood-brain barrier becoming highly permeable to Evans blue. Infarct developed within 24 h (174). Thus whether free radicals selectively promote apoptosis or necrosis after ischemia may well be a function of the level of free radical production, as seen in cultures (111).

When excess free radicals were produced as a result of / SOD null mutants in mice, the region that became more vulnerable showed a greater number of apoptotic neurons than other regions (601), suggesting free radicals caused apoptosis. However, this could have arisen from the fact that the insult intensity in the newly vulnerable region (the former peri-infarct zone) was low. It would be very useful to explicitly count the effect of free radical, or NO, on attenuation of apoptotic and necrotic neurons in regions that were protected by free radical scavengers or by SOD overexpression to determine whether free radicals preferentially induce one or the other forms of cell death. They certainly have the potential to induce both forms.

The data suggest that free radicals persist for well beyond 24 h after the start of the insult in temporary focal ischemia; they have been measured explicitly for at least 6 h in the penumbra and the core, and iNOS and COX-2 are induced between 6 and 24 h of reperfusion, suggesting there will be prolonged production of free radicals and peroxynitrite. The latter accumulates for at least 3 h in the penumbra; later measurements are not reported. Free radicals have not been measured more than 3 h after the start of permanent focal ischemia but may well persist. Thus free radicals persist in the tissue for many hours and possibly days. What is important is when these free radicals are actually damaging. One can envision their being important early, perhaps as a trigger, as occurs in sympathetic ganglia following NGF withdrawal (239), perhaps to set off synthesis of NFB and resulting downstream effects or to alter Ca²⁺ homeostasis. One can then envision them acting later, effecting damage of important molecules involved in ICC. They seem to play such a role in staurosporine-induced apoptosis in neurons so that delayed addition of scavengers is very effective (618). Unfortunately, there is little available information, since most inhibitors are added early in the insult and are often maintained. The duration of their efficacy is often not known (e.g., Ref. 207). In permanent focal ischemia, there is an important window during which inhibitors are effective, between 3 and 6 or 12 h after the ischemia. There may be others, later. No equivalent information exists for temporary ischemia.

Free radicals and NO are elevated early during and after global ischemia and, again, most drugs are added very early so the window of activity has not been determined. However, there is reasonably good evidence that a delayed activation of free radicals, 24-48 h after ischemia, plays a major role in the delayed damage in CA1. Inducible NOS is increased in CA1 glia between 24 and 48 h after the insult, and COX-2 is increased between 6 and 24 h. There is a large increase in superoxide in CA1, along with a reduction in SOD 24 h after the ischemia. Thus there is certainly a conspiracy to effectively produce a delayed increase in free radicals in the CA1 pyramidal region. One effect may be upregulation of NFB, which is greatly elevated in CA1 some time between 24 and 72 h after ischemia. Pharmacological evidence that all these changes are functionally important is not impressive, being confined to the efficacy of COX-2 inhibition which shows that the delayed production of free radicals by this enzyme contributes significantly to damage.

Overall, more data in which the time windows of efficacies of different agents are better ascertained, and in which the time they remain in the brain is measured, would help sort out the times at which free radicals are acting and thus help to determine what they are doing.

The most likely bases for prolonged maintenance are COX-2 and iNOS inductions and possible ongoing production by mitochondria. These possibilities can be tested.

B.  Ca²⁺-Dependent Proteases

1.  Inhibitor specificities

There is strong, albeit not very plentiful, evidence that the Ca²⁺-activated neutral cysteine protease calpain plays an extremely important role in focal ischemic damage and very possibly in global damage (66, 379, 750, 971). This evidence is based on cell-permeant inhibitors of calpain that have been developed in the last few years (66, 715) and relies on their specificity. Knockout animals have not yet been made. Inhibitor specificity is somewhat problematic because there are at least three currently known alternate proteolytic systems that might play a role in ischemia. These are caspases and cathepsins, both of which play or may well play roles in ischemic cell death, and also the 20S proteasome.

The current generation of cell-permeant intracellular protease inhibitors are aldehyde-linked di- or tripeptides in which the peptides mimic the substrate specificity of the protease (715). Amino acid sequences that most potently inhibit calpain contain two hydrophobic residues. Val-Phe (MDL-28170) and Leu-aminobutyrate (AK-275). These are the two inhibitors that have been shown to possess the greatest potency in focal ischemia (67, 715). A much less cell-permeant molecule, leupeptin, was very protective in global ischemia (641).

Caspases have very different substrate specificities from calpain, and caspase inhibitors have two or three hydrophic amino acids but they must be accompanied by an aspartate. It is thus considered extremely unlikely that the calpain inhibitors will inhibit caspases. This being said, there is only one reported study in which it was shown explicitly that E64 and antipain, inhibitors that are similar but not identical to MDL-28170 and AK-275, have no effect on caspase-1 (ICE) (1214). One indirect but quite strong piece of evidence that calpain inhibitors do not affect caspases is that MDL-28170 does not prevent apoptosis in thymocytes when the apoptosis is triggered by insults that do not require protein synthesis (1064). This is very likely a caspase-mediated event.

Some three or four hydrophobic amino acid sequences that effectively block calpain (inhibitory constant ~10⁷) also block proteasomal activity, although at 100-fold lower potency (749); potency may be greater in situ (1033). Thus, at the doses used in vivo (~10 mg/kg or 30 µM to get strong protection), it is possible that the calpain inhibitors are also blocking proteasomal activity. Neither of the dipeptides used against ischemia has been tested against proteasomal activity yet. Leupeptin may also inhibit proteasomes; it blocks the trypsinlike activity of proteasomes (but not the chymotrypsin-like activity) (408). This is an important issue because a specific proteasomal inhibitor has recently been found to provide strong protection in focal ischemia (902). This issue would be best resolved by examining effects of calpain inhibitors with widely different specificities for proteasomal action (749, 1033) or by using the peptide calpain inhibitors at very low doses.

The protease that is most likely to be confounding the results is cathepsin B which, although it is a serine protease, has a very similar substrate specificity to calpain (715) and is thus inhibited by MDL-28170, and by leupeptin with potencies that are similar to those for calpain (66, 657, 744, 1191). There is at least one cell-permeant inhibitor that is much more effective against cathepsin than against calpain, ZYA-CHN2 (224), but this has not yet been used in ischemia. This would be a good way to test whether the inhibitors are acting on cathepsins.

Another way to attain reliable calpain specificity has been to develop a cell-permeant inhibitor that blocks at the Ca²⁺ binding site of calpain. This has been done (1190), and although it is effective in cell culture (1064), it is unfortunately not effective in brain slices because of penetration problems (Lipton and Zhang, unpublished data) and has not been studied in vivo. It is likely to be ineffective there too.

Although quite high concentrations of Ca²⁺ are necessary to initially activate the isolated enzymes µ- and M-calpain (10 µM and 1 mM, respectively), these concentrations are lowered by binding to polyphosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PIP₂) at the cell membrane and by products of lecithin breakdown (37). The mean affinity constant for Ca²⁺ activation of µ-calpain is reduced from ~10 µM to ~100 nM, and the sensitivity of M-calpain is also increased (971, 972, 1087, 1240). The Ca²⁺ sensitivity is also greatly increased by a membrane protein that has been found in several tissues, including the erythrocyte and brain (979). Once activated by Ca²⁺, the isolated enzyme often undergoes autoproteolysis, and the smaller protein is active at normal cell levels of Ca²⁺ (622). Another mode of activation may include changes in calpastatin, but little is known about this. Thus calpains may well be activated by quite small increases in Ca²⁺.

Even without mechanisms of increasing its sensitivity, at least µ-calpain can be expected to be activated during ischemia as both in situ (1048) and in slices (397) cytosolic Ca²⁺ appears to rise into the tens of micromolar range.

An important unresolved issue is how the enzyme might be inactivated after ischemia.

The localization of calpain may well be relevant to selective vulnerability of neuronal populations. µ-Calpain is widely distributed throughout the neuron, including spines, dendrites, cell somata, and axons (892). In rabbit hippocampus, it is primarily localized to pyramidal neurons, whereas M-calpain, the less sensitive isozyme, is primarily localized to inhibitory interneurons (344). This is interesting because of the great resistance that interneurons show to ischemic damage. The localization of calpastatin is interesting in this regard also. Although it is present in most cells, it is at a far lower concentration in (vulnerable) CA1 pyramidal cells than (resistant) CA3 pyramidal cells (344).

A) GLOBAL ISCHEMIA. Many measurements of calpain activity are actually measurements of spectrin breakdown into a fragment that is characteristic of calpain action (although all proteases have not been tested). Early studies showed spectrin proteolysis 30 min after 10-min global ischemia in CA1 of gerbil that was inhibited by leupeptin (641). However, for reasons that remain somewhat of a mystery, more recent immunohistochemical studies have revealed that the protease activation is biphasic. Five minutes after ischemia there is spectrin breakdown in CA3 and in cortex, but not in CA1 of hippocampus (952). After 10-min ischemia, there is a small amount of proteolysis in CA1 (973), but not until 24 h after ischemia is there a large spectrin breakdown in CA1 (952, 973, 1258). The very delayed nature of calpain activation is difficult to understand because Ca²⁺ certainly seems to rise in CA1, and previous studies had demonstrated an early calpain increase. If this holds up, and it has so far been demonstrated by two different groups, it suggests that delayed calpain activation may be more important to global ischemic damage than early activation (which occurs in nonvulnerable neurons). This could be tested with appropriately timed inhibitor studies.

Finally, there is a very large increase (to 40%) in spectrin breakdown around the time of cell degeneration (1258). If this in fact precedes cell death, it indicates a very strong calpain activation at a critical time, but the detailed timing is not yet known.

A direct measure of calpain autolysis indicated activation in whole rabbit brain during the first 10 min of global ischemia (823).

B) FOCAL ISCHEMIA. There was a large increase in spectrin proteolysis during 3 h of focal ischemia in regions that later developed infarct. This had begun by the time of the first measurement, at 1 h. There was a delayed increase in the peri-infarct regions, where individual cell death generally occurs. That increase was noted after 21-h reperfusion and was as large as the increase in the infarcted regions (455). Because cell damage was developing in the lesion throughout the study, it is not clear whether the calpain activation preceded the damage, although the significant proteolysis by 1-3 h in core and penumbra certainly preceded major cell death.

C) BRAIN SLICES. In rat brain slices, where calpain inhibitor-sensitive MAP2 proteolysis and microtubule dissociations were used to monitor calpain activity, there was a clear calpain activation during the first 5 min of in vitro ischemia, with continued activity in the reperfusion period (1291).

In liver cells, calpain was activated twofold during anoxia, without an increase in intracellular Ca²⁺. Activation appeared to be dependent on PLA₂ breakdown (37, 38). This is consistent with the ability of phosphatidylcholine breakdown products to activate calpain (37).

In global ischemia, most of the early and delayed calpain upregulation is blocked by MK-801, suggesting that Ca²⁺ entering via NMDA receptors is important (952). This is consistent with either direct activation or activation via PLA₂ since the latter is Ca²⁺ dependent.

As far as tested, calpain inhibitors appear to reduce cell death very effectively. In the global gerbil model, leupeptin infusion into the ventricles prevented the early breakdown in spectrin and almost completely prevented cell death in CA1 7 days later (641). Temperature effects were not measured. This is the only reported study in global ischemia.

In focal ischemia, superfusing the cell-permeant calpain inhibitor AK-275 into cortical tissue at various times before during and after a temporary distal MCA/CCA occlusion reduced the size of the cortical infarct by 50-70% (67). The drug was very effective when administered 3 h after starting the ischemia but not 4 h after ischemia. This is a massive reduction in infarct volume and suggests that calpain plays a crucial role in the process, possibly even in damage to the core of the infarct. A similar level of protection was found against 3-h temporary focal ischemia where another dipeptide/aldehyde inhibitor, MDL-28170, reduced infarct size by ~70% when administered intravenously within 30 min of starting ischemia. The drug was still very effective when administered up to 6 h after starting the ischemia (3 h into the reperfusion) (715). In both studies, damage was only measured 24 h after occlusion so that very long-term protective effects were not tested. Temperature was quite well controlled in one study (67), where no effect of inhibitor on temperature was noted for at least 1 h after MCA occlusion, but was not discussed in the other study (715). The fact that the two inhibitors are very similar certainly suggests that temperature did not vary in that study either.

Cell death following in vitro ischemia in cerebrocortical cultures was attenuated by the cell-permeant calpain inhibitor that has almost no effect on cathepsins (1190), strongly implicating calpain in cell death in cultures. In conclusion, the effects of calpain inhibition on focal lesion size are more profound than all other ways of intervening. Unless these drugs are protecting by inhibiting cathepsin B activity, or proteasome activity, the results show a major contribution of calpain to cell damage in transient focal ischemia. There are, though, only very few studies, particularly in global ischemia.

A) SPECIFIC TARGETS. The enzyme attacks many cytoskeletal proteins, including MAP2 (900, 1291), spectrin, tubulin (971), and very probably ankyrin. It leads to loosening of the postsynaptic density (269) and participates in the breakdown of microtubules in an unknown way, as evaluated from effects of MDL-28170 and calpain inhibitor 1 (1291). Hence, dissolution of critical cytoskeletal elements may well be how it participates in cell death.

Opening of the MTP is another possible site of action. In hepatocytes, mitochondrial calpain was activated by increased intramitochondrial Ca²⁺ and by oxidative stress generated by tert-butyl hydroperoxide, leading to opening of the MTP (7).

Another possible site of action is eIF-4, which is broken down by calpain to one-third its normal level during 20-min global ischemia in the rabbit (820, 822). However, a role for global protein synthesis inhibition in focal ischemic death is quite unlikely, so this is probably not its mode of action. It may well be important in global ischemia.

Calpain may play a role in activating autophagocytosis. The activated form of µ-calpain was identified immunocytochemically on lysosomal membranes after global ischemia in gerbils (1240), before the formation of the autophagic vacuoles (834).

B) APOPTOSIS. Calpain appears to play a critical role in apoptosis in many cases, as judged both by its upregulation and the effects of calpain inhibitors. For example, T-cell apoptosis induced by anti-CD3 monoclonal antibody was blocked by calpain inhibitors, and there was a calpain blocker-sensitive breakdown of spectrin to the 150-kDa fragment during the process (720). This calpain-mediated cleavage of spectrin occurs after several different apoptotic stimuli in different types of cultured cells including hippocampal neurons (511, 720). There are many instances in which cell-permeant calpain inhibitors including calpeptin in neuroblastoma (78), MDL-28170 in cultured hippocampal cells (511), calpeptin and MDL-28170 in thymocytes and metamyelocytes (1065), and leupeptin in T cells (989), prevent apoptosis. One of the studies (1065) very nicely demonstrated the autoproteolysis of calpain I, showing calpain activity is turned on, using an antibody that was specific to the native form.

In thymocytes, calpain inhibition by the Ca²⁺ binding site-specific inhibitor PD-150656 produced a clear inhibition of all aspects of apoptosis when apoptosis required protein synthesis (for example, when induced by dexamethasone), but not when protein synthesis was unnecessary for apoptosis (1064), suggesting calpain is involved in a relatively early event in apoptosis.

Taken together, these results provide a picture of a strong involvement of calpain in apoptosis.

The role of calpain is not known and may be quite complex. It required protein synthesis to be activated in staurosporine-treated neuronal glial culture (909) where its inhibition not only prevented cell death but also DNA breakdown. A possible site of action is actin breakdown, a feature of apoptosis in most cell types. In chick neurons deprived of growth factor (1169), calpain inhibitors I and II blocked both apoptosis and actin proteolysis. The effect on actin is almost undoubtedly indirect. Prevalent forms of actin are not substrates for calpain (269, 971), whereas several actin-binding proteins are substrates (971).

C) A CAVEAT. Although this section has focused on damaging effects of calpain, the enzyme also appears to play an important role in remodeling dendritic structure after injury. In cultured neurons, NMDA induces reversible dendritic varicosities that result, at least in part, from disassembled microtubules. The reestablishment of normal dendritic structure was blocked by calpain inhibitors, implying that calpain helps in recovery from morphological damage (313). It is not clear if these effects relate to ischemia, but the results point out that calpain may be beneficial in reestablishing neuronal function during late phases of recovery. This is important when considering therapeutic aspects of the drug.