The Journal of Neuroscience, August 1990, 70(8): 2493-2501

Feature Article

Cerebral : Some New Approaches and Unanswered Questions

Dennis W. Choi Department of Neurology, Stanford University Medical Center, Stanford, California 94305

The apoplexy of Hippocrates’ time remains with us today, un- 5, and 6, and cerebellar Purkinje (Brierley, 1976). More changed and untreated. We call this syndrome of acute brain sustained , compatible with the patient’s survival only damage “”; we know that it most commonly reflects lo- if localized, can produce infarction: a region of pannecrosis in- calized tissue hypoxia attributable to reduced blood flow (isch- volving neurons, glia, and endothelial cells. emia). Focal hypoxia-ischemia also occurs in such contexts as The special vulnerability of certain brain neurons to hypoxic- traumatic insults, or cerebral hemorrhages, while global hypox- ischemic injury was recognized by Vogt and Vogt (1937), who ia-ischemia occurs in , near-, and carbon hypothesized that it was explained by intrinsic neuronal prop- monoxide poisoning. The centuries since Thomas Willis, Jo- erties. Subsequent efforts to identify the parenchymal deter- hann Wepfer, and Giovanni Morgagni have brought precise minants of hypoxic neuronal injury focused on general meta- definition of cerebral vascular anatomy and the neurological bolic derangements: in particular, the gap between consequences of focal brain lesions, permitting full comprehen- demand and supply, and the resulting energy deficit. However, sion of functional deficits; we can prognosticate with sad ac- the simple idea that an energy deficit directly causes neuronal curacy. But the medical management of stroke patients in 1990 death conflicts with available data. Most neurons can survive is still the management of symptoms and associated conditions. periods of complete ischemia sufficient to reduce phosphocrea- Despite its status as a major worldwide cause of death and tine and ATP to negligible levels; and, paradoxically, incomplete disability, we are no more able than Hippocrates to treat cerebral ischemia causes more neuronal death than complete ischemia hypoxia itself. (Siesjo, 198 1). Furthermore, pharmacological reduction of ce- Nevertheless, hope for the development of effective therapy rebral metabolism by means of barbiturates does not consis- has endured, and in the last few years has been encouraged by tently protect against neuronal death in experimental or clinical the emergence of some promising strategies for reducing the (Safar, 1980; Nussmeier et al., 1986; but see brain’s intrinsic susceptibility to hypoxic insults. These tissue- Grotta, 1987). More recent evidence has suggested that the pa- level approaches, sometimes referred to as “parenchymal” ap- renchymal approach to the therapy of cerebral hypoxia can be proaches to distinguish them from other strategies aimed at improved by attending specifically to brain excitatory synaptic influencing blood how, are based on recent information sug- mechanisms. gesting that central neurotransmitter mechanisms, especially The pathogenesis of hypoxic neuronal injury was first linked those related to the excitatory neurotransmitter glutamate, may to synaptic transmission by Kass and Lipton (1982) and Roth- play an important role in the pathogenesis of hypoxic neuronal man (1983), who found that elevating extracellular magnesium death (Meldrum, 1985; Rothman and Olney, 1986; Choi, 1988b). reduced the vulnerability of hippocampal neurons in vitro to In this essay I will comment on the possibility of new therapies anoxic insult. Attention was focused on the role of glutamate- for cerebral hypoxia directed at glutamate-mediated injury mediated synaptic transmission by reports that glutamate an- mechanisms, and will briefly mention some other potential ap- tagonists reduced injury both in vitro (Rothman, 1984) and in proaches. vivo (Simon et al., 1984). Injury reduction has now been reported with glutamate antagonists [especially those effective against Glutamate and hypoxic neuronal injury N-methyl-D-aspartate (NMDA) receptors (see below)] in several The brain is critically dependent on its blood flow for a contin- models of focal brain ischemia, as well as in models of hypogly- uous supply of oxygen and glucose. The oscillations of the elec- cemia, prolonged , and mechanical trauma (Choi, 1988b, troencephalogram cease within seconds of cardiac arrest, and Albers et al., 1989). only a few minutes of severe ischemia can induce the selective Most likely, these protective effects of glutamate antagonists degeneration of certain neuronal populations, including pyram- reflect the blockade of neuronal death caused directly by glu- idal neurons in the CA1 region of the hippocampal formation, tamate overexposure. As discovered by Lucas and Newhouse striatal medium-sized neurons, neocortical neurons in layers 3, (1957), and Olney (1969), excessive exposure to glutamate or related excitatory amino acids can kill central neurons, a process I am grateful to G. Stem and J. Schwartz for helpful comments on the manu- Olney labeled “excitotoxicity.” Microdialysis measurements script. Supported by NIH grant NS26907, and by a grant from the American have indicated that extracellular levels of glutamate are in- Paralysis Association. creased during hypoxia (Benveniste et al., 1984; Globus et al., Correspondence should be addressed to Dennis W. Choi, Department of Neu- rology H-3 160, Stanford University Medical Center, Stanford, CA 94305. 1988), likely reaching levels sufficient to kill briefly exposed Copyright 0 1990 Society for Neuroscience 0270-6474/90/082493-09$03.00/O cultured neurons (Choi, 1988b). In addition, lesioning the glu- 2494 Choi * Cerebral Hypoxia tamate-mediated excitatory inputs to hippocampus reduces 1988a; Michaels and Rothman, 1990), perhaps because their hypoxia-induced selective neuronal loss (Johansen et al., 1986; channels carry Ca2+ as well (MacDermott et al., 1986). Gluta- Onodera et al., 1986). mate also activates a quisqualate-preferring metabotropic re- The hypothesis that glutamate neurotoxicity might contribute ceptor (Eccles and McGeer, 1979) which induces the hydrolysis to the pathogenesis of hypoxic-ischemic neuronal damage tied of phosphatidylinositol-4,5-bisphosphate (PIP,) to generate the together 2 previous ideas: (1) the idea of Van Harreveld (1959) second messenger’s inositol 1,4,5-trisphosphate (IP,) and diacyl- Hansen (1985) and others that the electrophysiological changes glycerol (Collingridge and Lester, 1989). accompanying hypoxia are similar to those accompanying Glutamate receptor overstimulation of neurons thus induces spreading depression, another phenomenon probably mediated a solute derangement consisting of an accumulation of intra- by NMDA receptors (Mody et al., 1987); and (2) the idea that cellular Ca2+, Nat, Cl-, water, IP,, and diacylglycerol, as well selective neuronal injury after hypoxic insult is an active process, as of a depletion of intracellular K+. Although this intracellular becoming apparent only after a delay of 48-72 hr (Kirino, 1982; image of glutamate receptor overstimulation is potentially Pulsinelli et al., 1982). Furthermore, the possibility that gluta- lethal, it precedes the occurrence of irreversibly lethal events. mate neurotoxicity might itself be mediated by an influx of Evidence that induction of the derangement can be uncoupled extracellular Ca2+ and the formation of free radicals (Choi, 1988b; from cell death is provided by the observation that virtually all also see below) may help account for the previously postulated cortical neurons destined to die after overexposure to glutamate participation of Ca2+ and free radicals in the pathogenesis of can be rescued by a 20 min incubation in Na+- and CaZ+-free hypoxic neuronal injury (Meldrum et al., 1985; Siesjo, 1989; solution (Hartley and Choi, 1989). Possibly, this treatment draws Siesjii and Bengtsson, 1989). Na+ and Ca2+ back out of neurons before irreversible harm has Several signaling systems other than those mediated by glu- occurred. tamate or related compounds may additionally influence hypox- Reducing hypoxic excitotoxic induction might be accom- ic neuronal injury. Stimulation of adenosine A, receptors re- plished most easily postsynaptically by antagonizing NMDA duces hypoxic neuronal injury both in vivo (Evans et al., 1988; receptors. Fortunately, NMDA receptors have many potential von Lubitz et al., 1988) and in vitro (Goldberg et al., 1988) antagonist target, sites, including: (1) the agonist-binding site whereas adenosine antagonists increase injury (Wieloch et al., itself; (2) the glycine-binding site; (3) a zinc-binding site; (4) 1985; Rudolphi et al., 1987). In forebrain ischemia, toxic lesions channel-blocking sites defined by phencyclidine, magnesium, or of the locus coeruleus aggravate hippocampal and cortical brain zinc; (5) a polyamine-binding site; and (6) regulatory sites sen- damage (Blomqvist et al., 1985; Davis et al., 1987), while sitive to changes in pH, phosphorylation, or oxidation (Collin- administration of a mixture of and noradrenaline gridge and Lester, 1989; Choi, 1990). Antagonist compounds ameliorate it (Koide et al., 1986). Finally, ablation of the sub- are available, most of them interacting at the agonist-binding stantia nigra attenuates striatal ischemic injury (Globus et al., site (Watkins and Olverman, 1987; Lehmann et al., 1988), the 1987). phencyclidine-binding site (Kemp et al., 1987), or the glycine- These effects are likely to be mediated in part by alterations binding site (Johnson and Ascher, 1987; Kemp et al., 1988). in the glutamate transmitter system, although other alterations, Although NMDA receptors may play a key role in the me- for example in blood flow or postsynaptic neuronal membrane diation of hypoxia-induced excitotoxicity, concurrent activation properties, may also participate. Adenosine in particular can of non-NMDA receptors or the metabotropic glutamate recep- inhibit glutamate release (Dolphin and Archer, 1983); norepi- tor may augment injury (Frandsen et al., 1989; also see below). nephrine may do the same (Dunlap and Fischbach, 198 1; Crow- Furthermore, certain neuronal subpopulations, such as cortical der and Bradford, 1987); and dopamine may inhibit electrical neurons containing the enzyme NADPH-diaphorase (Koh and stimulation-evoked glutamate uptake (Kerkerian et al., 1987). Choi, 1988) or parvalbumin-like immunoreactivity (Weiss et al., 1990), may be unusually vulnerable to non-NMDA receptor- Blockade of glutamate toxicity mediated injury. Antagonists for non-NMDA receptors have The hypothesis that excitotoxicity is an important cause of neu- been described (Sheardown et al., 1990). The pharmacology of ronal death in cerebral hypoxia-ischemia raises several possi- the metabotropic receptor is not well defined, but 2-amino-3- bilities for therapeutic intervention. Observations on cultured phosphonopropionate is a possible antagonist (Schoepp and neurons suggest that intense exposure to glutamate induces 2 Johnson, 1989). events: acute neuronal swelling dependent on extracellular Na+ Reduction of excitotoxic induction might also be accom- and Cl-, and delayed neuronal disintegration dependent on ex- plished presynaptically, by reducing glutamate release from axon tracellular Ca2+ (Choi, 1988a). By analogy to long-term poten- terminals, or by facilitating glutamate clearance from synaptic tiation, these events may reflect a sequence of 3 stages, each clefts. Attainment of the former goal of reducing glutamate re- perhaps amenable to specific therapeutic interference (Choi, lease will be aided by ascertaining the extent to which glutamate 1990). release in hypoxia-ischemia depends on glutamate synthesis, neuronal activity, Calf-dependent vesicular release, or reversal Stage I. Induction of carrier-mediated uptake. Currently promising approaches to This stage consists of the initial events leading to overstimu- the reduction of glutamate release stratagem include stimulation lation of glutamate receptors, and consequent immediate intra- of adenosine A, receptors (see above), indirect inhibition of cellular derangements. Glutamate activates both NMDA and glutamate synthesis with methionine sulfoximine (Swanson et non-NMDA (kainate and AMPA/quisqualate) type receptors, ai., 1990), and hypothermia (Busto et al., 1989). which are linked to Na+ and K+ channels (Watkins and Olver- man, 1987; Collingridge and Lester, 1989) and mediate acute Stage 2. ArnpliJication neuronal swelling. NMDA receptors are particularly important This stage consists of the postsynaptic cascades that augment in mediating subsequent delayed neuronal disintegration (Choi, the intensity of initial derangements, and promote the spread The Journal of Neuroscience, August 1990, IO(8) 2495

Table 1. Some enzymes possibly linking glutamate receptor activation to the lasting enhancement of excitatory synaptic efficacy and excitotoxicity

Enzyme Activators Possible result Protein kinase C Caz+, DAG increased glutamate release increased glutamate response increased VGCC conductance decreased afterhyperpolarization decreased Cl- conductance CaM kinase II Ca2+, CaM increased glutamate response increased glutamate release Calcineurin Ca2+ CaM decreased GABA, receptor-mediated response Calpain I Ca2+’ breakdown of cytoskeleton - remodeling of postsynaptic spines increased excitatory synaptic efficacy Phospholipase A, Ca*+ formation of arachidonic acid and metabolites - increased glutamate release decreased glutamate uptake increased glutamate response Abbreviations: DAG, diacylglycerol; VGCC, voltage-gated W+ channel; CaM, calmodulin; CaM kinase II, W+/calmodulin- dependent protein kinase II. References: Chin et al., 1985; Kaczmarek, 1987; Piomelli et al., 1987; Lynch et al., 1988; Bazan, 1989; Kennedy, 1989; Malenka et al., 1989; Melloni and Pontremoli, 1989; also see references listed in the text. of excitotoxicity to other neurons. The key derangement may (Rothman et al., 1987; Foster et al., 1988; Hartley and Choi, be a buildup of intracellular Ca2+; the role of elevations in Na+, 1989). IP,, and diacyglycerol may derive largely from their enhance- Protective strategies operative at the level of amplification ment of this accumulation. Three main events may occur. might include blockade of additional Ca2+ influx, blockade of First, the Caz+ initially entering through the NMDA receptor- Ca*+ release from intracellular stores, or interference with the gated channel may be augmented by other sources of Ca2+ influx specific mechanisms coupling glutamate receptor stimulation to (Choi, 1988a), including voltage-gated Ca2+ channels, reverse lasting enhancements of excitatory synaptic efficacy. A partic- operation of the Na+-Ca*+ exchanger (Nachsen et al., 1986), ularly attractive target for such blockage may be L-type voltage- some cation channels activated by Ca2+ (Partridge and Swan- gated Ca2+ channels, as clinical tests of dihydropyridine antag- dulla, 1988), channels activated by membrane stretch (Yang onists have shown evidence of protective efficacy in ischemia and Sachs, 1989), or membrane leak conductances. Further- (Grotta, 1987; Uematsu et al., 1989). Another promising target more, IP, will induce the release ofCa*+ from intracellular stores may be protein kinase C, which catalyzes phosphorylation of (Berridge and Irvine, 1989). many proteins and which has been implicated in both presyn- Second, stimulation of glutamate receptors and elevation of aptic and postsynaptic changes associated with long-term po- intracellular free Ca2+ activates several enzyme families-in- tentiation (see above). Protection against both glutamate tox- cluding C kinases, calmodulin-regulated enzymes, calpains, and icity and ischemic injury has been reported for gangliosides, phospholipases-which may orchestrate the long-term enhance- which can inhibit the membrane translocation of protein kinase ment of excitatory synaptic efficacy and circuit excitability (Ta- C (Favaron et al., 1988; Komatsumoto et al., 1988). Reduction ble 1). Specific alterations may include increased glutamate re- of ischemic brain injury has also been reported for other protein lease (Diaz-Guerra et al., 1988; Malinow et al., 1989; Williams kinase C inhibitors (Kogure, 1990). et al., 1989), decreased glutamate uptake (Yu et al., 1987; Bar- The benefits of methods for reducing excitotoxic amplification bour et al., 1989), potentiation of postsynaptic glutamate re- may depend on the magnitude of the initial NMDA receptor- ceptor-mediated responses (Kimura et al., 1985, Malenka et al., mediated Ca2+ influx. If the initial Ca*+ influx is large, reflecting 1989), increased Ca2+ current through voltage-gated channels intense activation of large numbers of NMDA receptors, then (Strong et al., 1987; Connor et al., 1988), and reduction of there may be little need for further amplification to reach toxic GABA, receptor function (Stelzer et al., 1988). The long-term levels of intracellular Ca2+ accumulation. A workable strategy enhancement of excitatory synaptic efficacy, and hence possibly in such cases might be direct reduction of effective Ca2+ accu- also of excitotoxicity, might also be promoted by early-imme- mulation: increasing the extrusion, sequestration, or binding of diate gene expression (Cole et al., 1989; Morgan and Curran, intracellular free Ca2+. For instance, injury of dentate hilar cells 1989; Szekely et al., 1989). induced by excessive perforant pathway stimulation can be re- Third, some combination of increased neuronal activity, tonic duced by the intracellular administration of a calcium chelator depolarization, Ca 2+ accumulation, and cell membrane damage (Scharfman and Schwartzkroin, 1989). may lead to a secondary efflux of endogenous glutamate stores and continued stimulation of glutamate receptors-a positive- Stage 3. Expression feedback loop that likely accounts for observations that NMDA This stage consists of the final events responsible for cell dis- antagonists applied after the end of exposure to toxic levels of integration, possibly triggered by high levels of intracellular Ca2+ glutamate or NMDA can still reduce resultant neuronal death (Cheung et al., 1986; Choi, 1988a; Siesja and Bengtsson, 1989). 2496 Choi - Cerebral Hypoxia

The activation of degradative enzymes and the generation of lem of methods aimed at antagonizing glutamate neurotoxicity free radicals may be particularly important factors in disinte- antagonism is the danger of interfering with normal excitatory gration. transmission. That problem is potentially greatest with ap- Neurons contain high levels of the protease calpain I, which proaches directed at interference with induction or amplifica- may undergo autoproteolytic activation in the presence of high tion, which are likely to be shared between excitotoxicity and concentrations of free Ca2+, and catalyze excessive proteolysis excitatory signaling. Thus, one must hope to achieve a man- (Melloni and Pontremoli, 1989). Hippocampal neurons exposed ageable separation between normal and pathological processes, to kainate or NMDA exhibit breakdown of spectrin and the at least over a term of brief therapy. Approaches directed at microtubule-associated protein, MAP2, which correlates well blocking the subsequent excitotoxic expression might avoid this with subsequent neuronal degeneration (Siman et al., 1989). problem, and might have an advantage when treatment is de- Intracellular free Ca2+ may also activate phospholipase A,, which layed and intracellular derangements have already occurred. can degrade membrane phospholipids, likely contributing to However, the induction stage of excitotoxicity is likely to be the catastrophic cell or organelle membrane failure, and generating most easily defined, once induction has taken place, events may arachidonic acid. Arachidonic acid may have potentiating ef- become increasingly dispersed and difficult to rein in with spe- fects on excitatory synaptic transmission (Table 1); its further cific therapies. It may turn out that the best result can be gained metabolism may produce oxygen-free radicals (see below) and with a multi-part approach: interfering with induction as much delay deleterious effects on tissue blood flow (see next section). as side effect tolerance will permit, and then blocking those Finally, Ca*+ -activated endonucleases can induce DNA frag- major components of amplification or expression that can be mentation, a process that occurs in programmed cell death (Ni- safely reached. cotera et al., 1989) and which may be the ultimate limit for The induction interference approach presently most advanced subsequent recovery. in development is the administration of NMDA antagonists, Ca2+ -induced formation of arachidonic acid can be expected but before clinical efficacy testing can take place, critical safety to enhance catabolic steps leading to the formation of substantial issues will have to be addressed. Dangers identified with specific amounts of oxygen free radicals (Chan et al., 1985; Siesjo, 1989). NMDA antagonists include excessive stimulation of cerebral Glutamate may also inhibit neuronal cystine uptake, leading to metabolic rate (Kurumaji et al., 1989), reversible neuronal vac- reduced glutathione synthesis and diminished capacity to scav- uolization (Olney et al., 1989), and behavioral disturbances (Koek enge free radicals (Murphy et al., 1989). An indication that free et al., 1988). In addition, the special role which NMDA receptors -induced damage, including lipid peroxidation, may be are postulated to play in synapse formation, neurotrophism, and a key mediator of glutamate neurotoxicity, is provided by the synaptic plasticity (Collingridge and Lester, 1989; Lipton and observation that glutamate-induced cortical neuronal damage Kater, 1989) may pose important constraints on the use of an- can be attenuated by 21-aminosteroids (Monyer et al., 1989) togonists that limit dose, duration of therapy, or application to which are novel free radical scavengers and lipid peroxidation the developing . inhibitors (Hall et al., 1987). The neuroprotective value of inhibiting various CaZ+-acti- Other therapeutic approaches vated catabolic enzymes warrants exploration. Some exogenous The strategy of reducing the intrinsic vulnerability of brain inhibitors are available; for example, the protease inhibitor, parenchyma to hypoxic-ischemic insult is based on a logical leupeptin, was found to reduce muscle injury caused by cholin- connection between 2 other complementary treatment strate- ergic overstimulation (Leonard and Salpeter, 1982). Alterna- gies, each now also gathering momentum. tively, it may be possible to enhance the function of endogenous inhibitory factors such as the calpain inhibitor, calpastatin (Mel- Strategy 1. Before brain injury occurs: improve blood flow loni and Pontremoli, 1989). This is a plausible frontal assault on initial pathophysiology, Reducing free radical-induced injury ought to be a straight- and the goal of most prior treatment efforts. The approach cer- forward task; quenching of free radicals can be accomplished tainly works in stroke prevention; reduction of arterial disease by either enzymatic or non-enzymatic agents, and indeed such by controlling , and the prophylactic use of anti- agents have been reported to attenuate hypoxic-ischemic neu- platelet agents such as aspirin, are proven methods of reducing ronal injury in several paradigms (Siesjd, 1989). Vitamin E pre- the incidence of stroke (Grotta, 1987). Whether improving blood treatment reduced injury in global ischemia (Yamamoto et al., flow will also be effective as an intervention after ischemia onset 1983); and administration ofthe free radical scavenger enzymes, is not yet established. The first attempts to open thrombosed superoxide dismutase and catalase, reduced infarct volume in carotid surgically many hours after occlusion were dis- focal ischemia (Liu et al., 1989). The 2 1-aminosteroid, U74006F, astrous because the restitution of arterial pressure into the in- improved outcome after transient carotid occlusion in gerbils farcted brain led to dangerous hemorrhages. But more encour- (Hall et al., 1988). Benefits of free radical scavengers could gb aging results have been obtained in recent studies using agents beyond reduction of excitotoxic expression, as free radical for- such as tissue plasminogen activator (tPA) to achieve throm- mation may mediate other forms of neuronal injury, for example bolysis shortly after vessel occlusion (Zivin et al., 1985; Levy glucose deprivation-induced injury of cultured superior cervical et al., 1989). It is possible that such agents will act synergistically ganglion neurons (Saez et al., 1987). with parenchymal approaches (Zivin, 1989) especially to the The greater challenge may not be in identifying effective meth- extent that improved drug access to ischemic tissue can be ob- ods for reducing excitotoxicity, but rather selecting the most tained. useful from the available possibilities. Two questions must be In addition, there has been growing recognition that the ac- answered for each possible method: how well does it work, and tivation of phospholipases and resulting phospholipid break- what are its associated adverse effects? As Costa and his col- down triggered by hypoxia-ischemia may have important del- leagues have emphasized (Favaron et al., 1988), a generic prob- eterious effects on subsequent blood flow. In the presence of The Journal of Neuroscience, August 1990, 70(E) 2497 oxygen, either during incomplete ischemia or after post-isch- therapeutic interventions. As more powerful interventions are emit restitution of blood flow (reperfusion), arachidonic acid is developed, it may be possible to restore normal function to cells metabolized to eicosanoids (including leukotrienes, prostaglan- with higher levels of injury. Ultimately, the barrier limiting the dins E,, F,,,, and H,, and thromboxane A,) which can potently possibility of recovery may be massive structural damage, es- induce vasoconstriction, brain edema formation, and blood cell pecially to cell membranes or genes. aggregation (Bazan, 1989; Hsu et al., 1989). Thus arachidonic acid metabolism may trigger a delayed period of tissue hypo- Why do NA4DA antagonists protect brain better against focal perfusion capable of augmenting the original ischemic insult. ischemia than against global ischemia? Recent studies have suggested that another result of phospho- Neuroprotective benefits of NMDA antagonists are, generally lipid breakdown, formation of platelet-activating factor (PAF), accepted for animal models of focal brain ischemia (Albers et may be a particularly important cause of this delayed post- al., 1989), but claims of benefits in global ischemia are contro- ischemic hypoperfusion (Braquet et al., 1989b). Pharmacolog- versial; in fact, it has been suggested that some early reported ical PAF antagonists, which can attenuate delayed post-ischemic successes might be explained by uncontrolled hypothermia hypoperfusion and other PAF-mediated events such as neutro- (Buchan and Pulsinelli, 1990). Key differences between the na- phi1 chemotaxis and free radical production, show promise as ture of focal and global ischemia may account for a greater treatments for ischemic insults in brain and other organs (Bra- involvement of NMDA receptor-mediated injury in the former. quet et al., 1989a). A strong candidate for such a difference is the ischemic “pen- Yet another method for therapeutically improving post-isch- umbra”- the transition zone of incomplete ischemia which sur- emit cerebral perfusion has been suggested by observations that rounds a region of focal ischemia, but which is absent in global electrical stimulation of axons in the cerebellar fastigial nucleus ischemia. Positron emission tomography has raised the possi- increases cortical blood flow but not glucose utilization (Chida bility that this penumbra can expand outward, recruiting brain et al., 1989). This maneuver can markedly reduce infarct volume tissue into the center zone of dense ischemia and resultant in- after occlusion of the middle cerebral in rats (D. Reis, farction (Hakim, 1987). Overstimulation of penumbral NMDA personal communication). receptors could be a key event in this outward expansion, me- diating a self-propagating cycle of ionic shunting, glutamate re- Strategy 2. After brain injury occurs: enhance functional lease, and excitotoxicity. Siesjij and Bengtsson (1989) have spec- recovery ulated that the state of incomplete energy depletion found in Once injury has occurred, it may still be possible to exert a the penumbra may specifically favor excitotoxicity, an attractive favorable influence on the ability of the acutely injured brain postulate which dovetails with recent information about the to achieve functional recovery. Transient treatment with am- glutamate system. There are several reasons why NMDA re- phetamine following frontal cortex ablation in rats or cats pro- ceptor-mediated injury might be greater in the penumbra than duces a lasting improvement in beam-walking ability (Feeney in the ischemic core (Fig. 1). et al., 1982; Sutton et al., 1989); conversely, transient treatment Greater glutamate e&x in the penumbra. ATP may be nec- with diazepam causes a lasting impairment of recovery of sen- essary for Ca2+ -dependent release of transmitter glutamate sory function in lesioned rats (Schallert et al., 1986). The days (Nicholls, 1989). Although glutamate efflux may also occur by following injury may be a period of critical regrowth and syn- carrier-mediated processes, the preservation of Ca2+-dependent aptic plasticity. Moreover, one can hope that progress in neu- release might enhance net efflux. ronal transplantation will make it possible in the long run to Greater NMDA receptor-channel complex phosphorylation in restore lost cells to the brain. the penumbra. NMDA-induced currents diminish by about half in whole cell recordings unless high-energy phosphates are pres- Many unanswered questions ent in the recording pipette, suggesting that phosphorylation Present investigations of the neurobiology of hypoxic neuronal may be needed to maintain the NMDA receptor-channel com- injury are therapeutically encouraging yet emphasize how little plex in its most active state (Mody et al., 1988). is known about the fundamental mechanisms. Prominent among A higher pH in the penumbra [about 6.7 compared with about unanswered questions are the following. 6.4 in the ischemic core (Hakim, 1987; Siesjii, 1988)J NMDA receptor-mediated currents in hippocampal neurons are atten- What is irreversible neuronal cell injury? uated at the lower end of this pH range (Morad et al., 1988), a Neuronal degeneration indisputably indicates irreversible in- finding confirmed in cultures of cortical neurons (Giffard et al., jury, and is an unambiguous endpoint suited to experimental 1990). In cell culture, reducing the pH to 6.4 decreasesglutamate investigation. However, the task of identifying hypoxic injury neurotoxicity, hypoxia-induced Wa2+ accumulation, and hyp- mechanisms would be facilitated by identification of more subtle oxic neuronal degeneration (Giffard et al., 1990; Tombaugh and indices of injury. It is still unclear at what stage specific pertur- Sapolsky, 1990). bations-for example, loss of high energy phosphate esters, dis- Availability of oxygen in the penumbra. Oxygen availability turbances of neurotransmitter metabolism, membrane break- accelerates the formation of superoxide and hydroxyl radicals, down, mitochondrial failure, or accumulations of intracellular and thus might enhance the expression stage of excitotoxic dam- Ca2+ -constitute irreversible injury. age. This mechanism is likely to explain the paradoxical finding Injury might be operationally defined as any abnormality that that incomplete ischemia can be more damaging than complete impairs cell function. Both structural damage and metabolic ischemia (see above). derangement can cause such impairments, and both can be re- Thus the conditions found in the penumbra may provide a versed, at least up to some point of no return that may reflect lethal singularity, at least with regard to NMDA receptor-me- a complex weighting of multiple pathological events. Exactly diated injury. The level of available metabolic energy may be where this critical point lies may depend on the availability of low enough to trigger excitotoxicity. (Indeed, partial impairment 2498 Choi l Cerebral Hypoxia

jury of isolated rat optic nerves is attenuated by removal of extracellular Ca*+ (Stys et al., 1990).

,:’ Unexplained, then, is the ability of NMDA antagonists to ,:’,..’ PENUMBRA “‘.,,..,,. reduce brain infarction-including non-neuronal cell loss-in ’ ‘:.., NORMAL the setting of focal brain ischemia. Perhaps the overstimulation of neuronal NMDA receptors somehow facilitates non-neuronal “z.: NL02 cell death by adversely altering the local environment. This i pH7 alteration could take the form of changes in ionic concentrations, ; NLATP generation of free radicals or toxic metabolites, or release of catabolic enzymes. As outlined above, phospholipases activated by NMDA receptor-mediated Ca*+ influx could enhance the formation of eicosanoids and PAF, leading to increased tissue ischemia. pH 6 . 7 ,,/ A particularly important alteration may be extracellular lactic ‘.._..__ , which may be promoted by NMDA receptor-induced “...,._.,,,,, 4 ATP _,,...... -~.““~” mitochondrial failure and energy depletion. Lactate acidosis has ” ...... _.._...... “’ been proposed as the direct cause of glia injury in infarction (Plum, 1983). Glia are more vulnerable to acid-induced injury Figure 1. Penumbra: zone of excitotoxicity? than neurons (Goldman et al., 1989); Norenberg et al. (1987) found that l-2 hr of exposure to pH 6 caused chromatin clump- ing and mitochondrial swelling. We have found that a pH of of energy-dependent Ca*+ pumps may be necessary for injury 6.4 for 8 hr kills large numbers of cultured cortical glia (R. to occur.) Yet available energy and pH may be high enough to Giffard, H. Monyer, and D. Choi, unpublished observations). maintain CaZ+ -dependent glutamate release and NMDA recep- Thus despite producing a beneficial reduction of NMDA recep- tor function, and enough oxygen may be available to accelerate tor-mediated neuronal loss, ischemic acidosis might potentiate free radical formation. Some penumbral neurons conceivably glial damage and foster infarction. might even span different energy zones, such that cell domains in zones with higher levels of energy might help maintain energy- Is reduction of neuronal death always desirable? dependent processes in lower energy zones, with deleterious While neuronal death is a convenient endpoint for the assess- consequences. ment of new treatment strategies, the ultimate goal of treatment In contrast, the conditions of global ischemia may only transit is functional benefit. Improved neuronal survival need not al- such a critical state, before settling into a state of total energy ways translate into functional benefit-for example, if a treat- depletion, low pH, and anoxia, where NMDA receptor-me- ment improved the survival of damaged neurons, led to the diated injury is blocked and cells die for other reasons (see formation ofincorrect synaptic connections, or disturbed critical below). Perhaps the length of time spent in this transitional state synaptic plasticity following injury. determines the extent to which NMDA receptors may contrib- Of all the problems that might theoretically arise with the ute to global ischemic injury. Ifglutamate is released, but NMDA therapeutic use of NMDA antagonist in cerebral ischemia, the receptors are inactivated by dephosphorylation or extracellular possibility of interference with functional recovery is probably acidosis, non-NMDA receptors could play a larger role in the most worrisome. Most other anticipated side effects are pathogenesis. It is noteworthy that the new non-NMDA antag- likely to be tolerable, especially if treatment duration can be onist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(~quinoxaline brief and hospital facilities are available. Demon- (NBQX) is reported to reduce global ischemic injury in the gerbil stration of functional benefit is a mandatory experimental hurdle (Sheardown et al., 1990). for any new treatment strategy. Although NMDA antagonists may have limited value in the treatment of global ischemia, they are beneficial in the treatment What is the predictive value of current experimental models of of another global insult-hypoglycemia (Wieloch, 1985). This cerebral ischemia? discrepancy could be explained by the arguments presented The development of stroke treatments is progressing apace, but above: hypoglycemia produces only incomplete energy deple- has not yet resulted in a proven clinical success. Until such tion, and perhaps, most importantly, it is not associated with success has been attained, the predictive value of specific ex- acidosis (Auer and SiesjB, 1988). perimental models will remain unknown. While the develop- ment of clinical therapy would be much enhanced by the avail- Why do glia die (and why do NMDA antagonists sometimes ability of experimental models capable of accurately predicting prevent this death)? treatment efficacy in patients, such perfect models may never NMDA receptor-mediated processes may help explain why neu- be found. rons are so vulnerable to hypoxic injury, but cannot directly In vitro model systems for the study of hypoxia obviously explain why glia or endothelial cells, which lack NMDA recep- lack many features relevant to stroke in vivo. Moreover, methods tors, also eventually succumb to hypoxic injury. Hypoxia may for inducing ischemia in animals may introduce substantial per- injure non-neuronal brain cells by mechanisms which qualita- turbations that do not reflect the pathophysiology of human tively resemble those underlying glutamate neurotoxicity, but stroke (Wiebers et al., 1990); and extensive experience with differ quantitatively in how rapidly excessive CaZ+ influx can rodent models has emphasized the importance of differences in occur without the conduit provided by NMDA receptor-gated species or experimental technique in determining outcome channels. Like glutamate neurotoxicity (see above), anoxic in- (Ginsberg and Busto, 1989). 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