HZPPOCAMPUS, VOL. 2, NO. 3, PAGES 221-228, JULY 1992

COMMENTARY

The Mechanism of Cerebral Hypoxic-Ischemic Damage Avital Schurr and Benjamin M. Rigor Department of Anesthesiology, University of Louisville School of Medicine, Louisville, KY 40292 U.S.A.

ABSTRACT The four most prominent hypotheses on the cellular processes leading to hypoxic-ischemic neuronal damage or death are (1) the lactacidosis hypothesis, ( 2 )the calcium overload hypothesis, (3) the excitotoxic hypothesis, and (4) the oxygen-free radical hypothesis. The authors comment on the evidence in favor of and against each in an attempt to select the one hypothesis that best explains the mechanism of cerebral hypoxic-ischemic damage while withstanding the scrutiny of scientific testing. A major part of this inquiry is derived from in vitro studies that are suited to mechanistic exploration. l h e y conclude that the calcium overload hypothesis is the best qualified in this respect. It is important to note, however, that some of the other hypothetical mechanisms may play a secondary role in exacerbating neuronal damage by accelerating calcium influx and overload. Key words: cerebral hypoxia-ischemia, calcium influx, excitotoxicity, oxygen-free radicals, lactacidosis, neuronal damage

Significant milestones mark the research on cerebral hypoxia-ischemia (CHI) in the last two decades. Each of these milestones has been the focus of attention on the path to a better understanding of the possible mechanism(s) leading to hypoxic-ischemic neuronal damage. Thus, lactic acidosis, calcium influx and overload, formation of free radicals, and excitatory amino acid neurotransmitters’ toxicity have all been suggested as possible causes of neuronal damage, particularly in the hippocampus, following CHI. Without the availability of both in vivo and in vitro models of CHI, the advances made in this field undoubtedly would have been much slower. Moreover, this multiplicity of models allows us to scrutinize each hypothesis that attempts to explain the mechanisms of CHI-induced neuronal damage. Hence, only the hypothesis that can be applied to the majority of models will persist and possibly become the unifying theory for the cellular mechanism(s) underlying dysfunction-induced neuronal damage. Four postulated mechanisms have withstood such scrutiny to date: 1. Acidosis due to anaerobic accumulation of lactic acid is

the main reason for neuronal failure after an episode of CHI. 2. Calcium influx and its intracellular accumulation due to Correspondence and reprint requests to Dr. Avital Schurr, Department of Anesthesiology, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A.

failure of ionic pumps during and after CHI lead to neuronal damage. 3. Neurotoxicity of excitatory neurotransmitters that accumulate extracellularly during and after CHI is the primary cause of neuronal damage. 4. Formation of oxygen-free radicals following reperfusionl reoxygenation of hypoxic-ischemic tissue leads to damage of neuronal membranes. Although each of these proposed mechanisms could be solely responsible for irreversible hypoxic-ischemic neuronal damage, the possibility exists that two or more of them may work in concert. Furthermore, each of the four hypotheses has been generally accepted at one time and refuted at another. The lactacidosis hypothesis was very popular during the late 1970s; the postulated excitotoxic mechanism is the one most in favor today. Nevertheless, the above-mentioned hypotheses have already contributed significantly to the understanding of CHI and to the advancement of treatments that protect against and resuscitate from this brain condition. We will examine, evaluate, and comment on the four main mechanistic hypotheses of cerebral hypoxic-ischemic damage. Because we believe that the in vitr.0 approach is more advantageous than the in vivo in trying to find the answers to cellular and molecular mechanistic questions, we have placed greater emphasis on in vitro studies. We will not review the vast literature on the topic but refer the reader to some recent reviews (Siesjo, 1988a; Siesjo and Bengtsson, 1989; Meyer, 1989; Choi, 1988a; 1988b; Ginsberg et al., 1988; Garcia and Anderson, 1989).

221

222

HIPPOCAMPUS VOL. 2, NO. 3, JULY 1992

ACIDOSIS A N D CEREBRAL HYPOXICISCHEMIC DAMAGE

CALCIUM A N D CEREBRAL HYPOXICISCHEMIC DAMAGE

The tissue lactacidosis hypothesis, which was popular 815 years ago, has not gained substantial experimental support over the ensuing years. As early as 1954, Thorn and Heitmann suggested that rising lactic acid levels and lowered pH values may enhance cerebral ischemic damage. Later, other investigators attempted to explain their observations by suggesting the involvement of lactacidosis in ischemic brain damage (Lindenberg, 1963; Michenfelder and Sundt, 1971). However, this hypothesis received its main support from studies performed by Siesjo and his coworkers (Salford et al., 1973; Ljunggren et al., 1974; Salford and Siesjo, 1974; Siesjo, 1981; 1982; 1985; Siejo, 1988a). Myers and Yamaguchi (1977) showed that monkeys given infusions of glucose prior to experimentally induced cardiac arrest exhibited more severe brain damage than either fasted or saline-infused counterparts. Consequently, others were able to show similar results (Siemkowicz and Hansen, 1978; Welsh et al., 1980) and. in addition, a correlation between blood glucose levels before and brain lactate levels after hypoxia-ischemia has been demonstrated (Nordstrom and Siesjo, 1978; Welsh et al., 1980; Gardiner et al., 1982). Thus, the logical assumption that implicates lactic acid as the culprit in CHI-induced damage has been made. Although logical, the evidence supporting this assumption is mainly circumstantial. Direct, unequivocal evidence to tie lactacidosis with brain damage is lacking. Rather, an increasing number of studies indicate the lack of deleterious effect of lactcidosis after an episode of CHI (Schurr et al., 1988a; Natale and D'Alecy, 1989;Walz and Wuttke, 1989; Giffard et al., 1990; Tombaugh and Sapolsky, 1990; Waltz and Harold, 1990). Moreover, a beneficial effect of hyperglycemic levels of glucose (Schurr et al., 1987a; Dong et al., 1988; Grigg and Anderson, 1989), and elevated levels of lactic acid (Schurr et al., 1988a; Giffard et al., 1990; Tombaugh and Sapolsky, 1990; Boakye et al., 1991) on hypoxic-ischemic brain tissue or synaptosomes has been demonstrated. Recent studies indicate that under conditions of cerebral stimulation, intracerebral lactate increases to much higher levels than those of resting conditions (Fox and Raichle, 1986; Fox et al., 1988; Lear, 1990). In humans, under resting conditions, the mean whole-brain cerebral metabolic rate for oxygen (CMR-02) and the CMR for glucose (CMR-glu) were 1.50 and 0.37 kmol/min/100 g, respectively, or a molar ratio of 4.1:l (Fox et al., 1988). A similar ratio (CMR-O2 = 1.71 ~moUmin/100g; CMR-glu = 0.42 Fmol/min/100 g) was found in the visual cortex under resting conditions. However, upon visual stimulation, the CMR-glu rose a mean of 0.21 pmol/ min/100 g (51%) while the CMR-02 increased only a mean of 0.08 kmol/min/100 g (5%) to produce a molar ratio for the increases of only 0.4:l (Fox et al., 1988). These results and the demonstration that under normoxic conditions hippocampal slices are capable of utilizing lactate as a sole energy substrate for the maintenance of normal neuronal function (Schurr et al., 1988b) could change not only our views concerning CHI but also our basic concepts of brain energy metabolism. Thus, lactacidosis per se is probably not a major determinant in CHI (Siesjo, 1981).

A significant amount of information has accumulated over the last decade on the many roles of Ca2+,in both neuronal function and dysfunction. As early as 1977, a reduction in extracellular Ca2+concentration ([Ca2+],) was observed during anoxia in cerebral cortex (Nicholson et al., 1977), an observation that was confirmed in later studies on anoxic and toxic cell death (Schanne et al., 1979; Farber. 1981; Yanagahara and McCall, 1982; Hansen, 1985). Consequently, Siesjo postulated that the loss of calcium homeostasis underlies selective neuronal vulnerability in such cerebral conditions as ischemia, hypoglycemia, and status epilepticus (Siesjo, 1981). This hypothesis assumed that calcium influx in ischemic or hypoglycemic neurons occurs through voltagedependent channels that would open as a result of dwindling ATP reserves, failure of the Na+/K+ pump, and an ensuing membrane depolarization. The hypothesis of calcium-induced cell damage has been challenged by studies demonstrating cell death without a concomitant increase in intracellular calcium concentration ([Ca2+],). Rather, a diminution of ATP appeared to be the detrimental factor in studies on non-brain cells (Cheung et al., 1986; Lemasters, et al., 1987). Although calcium accumulation during a long in vivo ischemic episode occurred in all cells of brain tissue, some cells died while others did not (Hossmann et al., 1983; 1985). However, in vitro studies using brain slices support the view that an increase in [Ca2+],is a major detrimental factor in hypoxic-ischemic cell damage (Kass and Lipton, 1986; Schurr et al., 1987b; Schurr and Rigor, 1989; Schurr et al., 1990). Adding to the confusion is the limited success with calcium antagonists in protecting neurons against ischemic damage. Verapamil, the classic calcium antagonist, and flunarizine lack any protective properties against hypoxic damage in rat hippocampal slices (Schurr, unpublished data). A promising calcium antagonist, isradipine, reduced the infarct size of spontaneously hypertensive rats after occlusion of the middle cerebral artery by only 30-40% (Sauter et al., 1988). Similar results were obtained with nilvadipine, a new dihydropyridine-type calcium channel blocker (Takakura et al., 1991). The questions of whether calcium influx and overload are only partially responsible for ischemic neuronal death or whether other types of calcium channels are involved for which specific antagonists remain to be found have not yet been answered. Support for the latter possibility is rapidly mounting since the emergence of the excitotoxic hypothesis (see below) and the identification of multiple voltage-dependent calcium channels in neuronal tissue (Choi, 1988a; Kostyuk et al., 1989). Thus, it has been suggested, mainly by in vitro studies, that calcium influx causes neuronal damage when either cortical cell cultures or slices are exposed to excitatory amino acids (EAA), such as glutamate and aspartate, or to anoxic conditions (Choi, 1985; 1987; Garthwaite et al.. 1986; Rothman and Olney, 1986; Benveniste et al., 1988). Moreover, depleting hippocampal slices of Ca2' during their exposure to hypoxia completely protected them against the ensuing neuronal damage, protection seen even when the hypoxic episode was combined with exposure to EAA (Schurr et al., 1990; 1991a;

THE MECHANISM OF CEREBRAL HYPOXIC-ISCHEMIC DAMAGE / Schurr and Rigor

1991b; 1991~).Similar results were obtained when the hypoxic insult was replaced with a hypoglycemic one (Schurr et al., 1990; 1991a). Other recent studies have shown partial success in protecting brain tissue in vivo against hypoxicischemic or other cell injury by calcium antagonists such as (S)-emopamil (Ginsberg et al., 1991) and nimodipine, nicardipine, or isradipine (Bunnell et al., 1987; Grotta et al., 1988; Hadani et al., 1988; LeVere et al., 1989; Sauter et al., 1988; Sauter and Rudin, 1990; Greenberg et al., 1991; see also a review by Horowitz and Powell, 1989). In an immunocytochemical study, Leranth and Ribak (1991) showed that calcium-binding proteins are concentrated in the hippocampal CA2 region, which may explain this region’s high resistance to epileptic damage and other damage-causing insults such as hypoxia and ischemia. Using cultured astrocytes, MacVicar et al. (1991) clearly demonstrated the important role of Ltype calcium channels in modulating Ca” influx and [Ca”], and that these channels can be dynamically modulated by dihydropyridines such as nifedipine. In contrast, Lyden et al. (1988) found no benefit from lidoflazine, nirnodipine, or nicardipine in terms of neurologic functional outcome after focal ischemia. Stys and colleagues (1991) suggested that reverse operation of the Na+-Ca*+exchanger during anoxia is a critical mechanism of Ca2+ influx and subsequent injury in mammalian CNS white matter. Although delayed or secondary neuronal ischemic-hypoxic damage, for which the hippocampal CAI region is noted, could be considered the most important outcome of CHI, we believe that it is a direct result of the primary event, namely, calcium overload that immediately follows CHI. In cultures of proximal tubular epithelium from adult rats, Swann et al. (1991) showed that notable increases of [Ca”], precede both sublethal and lethal cellular changes. Prevention of the primary neuronal damage should prevent any delayed damage; CA2 neurons exhibit higher resistance to primary ischemichypoxic neuronal damage than CA1 neurons and, hence, are also more resistant to the delayed damage. The role of Caz+ in CHI damage appears to be more intricate as additional neuronal voltage-dependent and receptor-operated calcium channels are identified. The interrelationship between calcium and EAA neurotransmitters and their receptors is discussed later.

223

fined as any nocuous effect involving EAA, would be an acceptable definition to most investigators. In vitro experimentation on the possible mechanism of EAA toxicity has produced evidencc supporting at least two such mechanisms, both of which are believed to be ion-dependent. One mechanism involves the rapid degeneration (30 minutes) of neurons in either hippocampal cell cultures or kolated chick retinas in the presence of toxic concentrations of glutamate or any of the EAA (Olney, 1989). This type of excitotoxicity could be prevented by omitting either C1- or Na’ (cf. Stys et al., 1991) from the incubation media of the hippocampal cell cultures or the isolated retinas (Rothman, 1985; Olney et al., 1986; David et al., 1988). The other mechaniqm, which requires only a short exposure (5 minutes) of cultured neurons to glutamate is Ca“ -dependent and causes slow neuronal degeneration (Choi, 1985; 1987). Additional support for Ca2+-dependentexcitotoxicity ha5 been provided by studies with cerebellar (Garthwaite et al., 1986; Garthwaite and Garthwaite, 1986) and hippocampal (Schurr and Rigor. 1989; Schurr et a]., 1990) slices. The identification of EAA receptor subtypes, and especially of new EAA receptor agonists and antagonists, provided investigators of the excitotoxic mechanism with new and powerful tools. Of these, N-methyl-D-aspartate (NMDA) is probably the most important glutamate analogue synthesized (Curtis et al., 1961; Watkins, 1962). The glutamate receptor subtype. named the NMDA receptor. is believed to be the one responsible for hypoxic-ischemic neuronal damage and for other disease-induced neuronal death. Until recently, it was the most studied receptor subtype of at least five distinct ionotropic and metabotropic EAA receptor subtypes (Cotman and Monaghan, 1989; Monaghan et al., 1989; Olney, 1989). The involvement of the other EAA receptor subtypes in hypoxic-ischemic neuronal damage is less clear, although recent studies suggest that the kainate receptor subtype could be involved in neuronal damage due to CHI (Schurr, unpublished results). Another study points to this receptor subtype as a possible channel for Ca2+ entry (Gramsbergen and van der Sluijs-Gelling, 1991). The NMDA receptor is associated with a specific membrane ion channel; when activated by its agonist, the receptor allows Ca” influx as a result of passive diffusion down its concentration gradient (Mayer and Miller, 1990). This influx can be blocked by Mg2+ (Choi, 1988a; 1988b; Olney, 1989). Activation of this receptor requires the EXCITATORY A M I N O ACIDS A N D CEREBRAL coavailability of glycine, and its blockade can be achieved by HYPOXIC-ISCHEMIC DAMAGE either competitive NMDA antagonists, such as 2-amino-SThe early reports (Lucas and Newhouse, 1957; Olney, phosphonovalerate (APV) and 2-amino-7-phosphonoheptan1969a; 1969b) on neuronal death in retina and brain after sys- oate (APH), or noncompetitive antagonists, such as phentemic administration of monosodium glutamate were met with cyclidine, MK-801, dextrophan and ketamine, which act at great skepticism (Olney, 1989). Although these findings have the phencyclidine site, and Zn2+, which appears to act at a been reproduced many times since then, the excitotoxic hy- different site than the Mg2+ binding site (Choi, 1988b; Olney, pothesis did not flourish until the 1980s. Olney’s studies led 1989). him to formulate the hypothesis that EAA synaptic receptors The close linkage between the NMDA receptor and the mediate glutamate neurotoxicity (Olney, 1989). high conductance Ca2+channel has brought about an attempt The term excitotoxicity may be limited to cases of epilepsy. to merge the calcium-related neuronal damage and the exFor other brain disorders, such as ischemia, hypoxia, and citotoxic neuronal death hypotheses into one (Siesjo and hypoglycemia, the hypothesized involvement of EAA in the Bengtsson, 1989). Moreover, the excitotoxic hypotheses development of neuronal damage should not be limited to seems to fit mechanisms that are related to many other brain excitotoxicity ; a generalized term would be more useful and disorders, including hypoglycemia, spreading depression, probably more accurate. Thus, the term excitntnxicity , de- epilepsy, Parkinsonism, Huntington’s disease, Alzheimer’s

224

HIPPOCAMPUS VOL. 2, NO. 3, JULY 1992

disease, and aging (Choi, 1988b; 1989; Siesjo and Bengtsson, 1989; Olney, 1989; Henneberry et al., 1989). The question arises as to whether excitotoxins are the root of every known brain disorder. Assigning a central role for excitotoxins in cerebral damage resulting from such brain disorders may be premature and unwarranted. These words of caution are based mainly on limited in vitro studies: first, the report of Aitken et al. (1988) demonstrated a lack of protective effect of the NMDA antagonists APH and APV against hypoxic damage in hippocampal slices. Subsequently, we have found glutamate and aspartate to be innocuous at concentrations of up to 3 mM under normoxic conditions in rat hippocampal slices (Schurr and Rigor, 1989). Even NMDA did not affect normoxic neuronal function until its concentration was raised to 50-100 FM (Schurr and Rigor, 1989). Novelli et al. (1988) have shown a similar lack of detrimental effect by glutamate and NMDA on cultured cerebellar neurons under normoxic conditions in standard incubation medium. However, when energy supplies were compromised, over 95% of the cultured neurons died. In this and subsequent studies by the same investigators (Henneberry et al., 1989; Lysko et al., 1989), the toxic potency of glutamate was always higher than that of NMDA; hippocampal slices were much more sensitive to NMDA than to glutamate (Schurr and Rigor, 1989; Schurr et al., 1990; 1991a). The lack of a glutamate reuptake system in cultured cerebellar neurons could be the reason for their heightened sensitivity to glutamate, although during hypoxia this energy-dependent reuptake system should be inactive in the slice as well. Nevertheless, we have confirmed the dependence of the exctitoxicity of glutamate, aspartate, NMDA, and a score of other analogues on reduced energy levels in hippocampal slices (Schurr and Rigor, 1989; Schurr et al., 1990; 1991; 1992). However, there is disagreement concerning the ion linkages of the two model systems. The effect of NMDA agonists on cultured cerebellar neurons is Na+and Cl--dependent (Henneberry et al., 1989). Their effect on hippocampal slices is Ca2+-dependent (Schurr and Rigor, 1989; Schurr et al., 1990; 1991; 1992), yet Mg2+ can block excitotoxicity in both systems (Cox et al., 1989; Schurr et al., 1990; 1991; 1992). Several points that have emerged from these in vitro studies should be highlighted here: 1.

2.

3.

4.

5. The above four points are also true for hypoglycemic neuronal damage in vitro (Schurr et al., 1990, 1991; 1992).

FREE RADICALS A N D HYPOXIC-ISCHEMIC NEURONAL DAMAGE It is a common suggestion that membrane peroxidation by oxygen-free radicals and singlet oxygen could result from lack of oxygenation (ischemia) followed by reoxygenation (reperfusion). However, definitive evidence for this view of brain ischemic damage has proven elusive (Ginsberg et al., 1988). The severity of ischemicireperfusion damage depends on several factors, including the length of the ischemic episode (Floyd, 1990). The indications that tissue injury occurs almost exclusively during the reperfusion phase and is due to oxygen-free radical-mediated oxidative events (Granger et al., 1986; Floyd, 1990) are summarized in the following equations: A xanthine dehydrogenase xanthine

T

P

a xanthine

CHI (calpain’!)

xanthine oxidase

(1)

xanthine oxidase

(2)

+ 02-

(3)

uricacid

While ATP levels fall, xanthine accumulates during ischemia (equation 1). Calcium influx activates the protease calpain, which cleaves a peptide bond on xanthine dehydrogenase to convert it to xanthine oxidase (equation 2). Upon reperfusion and restoration of oxygen supply, xanthine oxidase catalyzes the conversion of xanthine to uric acid and the production of superoxide (equation 3 ) (Granger et al., 1986; Floyd, 1990). A recent study by Stark et al. (1989) demonstrated evidence against the role of calpain (equation 2). Although several studies have indicated a contribution by the above mechanism to the ischemic injury, tissues were found that completely lack xanthine oxidase activity either before or after ischemicheperfusion damage (Floyd, 1990). Hence, other oxidative mechanisms were sought to explain free radical damage. The fact that low-molecular-weight soluble iron increases postischemia, on the one hand, and the finding that desferoxamine, an iron chelator, protects against ischemicireperfusion damage, on the other, provided a basis for such a mechanism (Aust and White, 1985). Whatever the mechanism of free radHypoxic neuronal damage occurs even in the absence of ical formation, the prevailing notion places the polyunsatuEAA. Whatever is the amount of glutamate or aspartate rated fatty acids as the main target of peroxidation (Siesjo, secreted by hippocampal slices during hypoxia, it certainly 1981). The free radical hypothesis shares a common path with does not exceed 100 pM (an undamaging concentration of the excitotoxic hypothesis, namely its dependence on Ca2+, glutamate) and cannot account for the ensuing damage. since an increase in and accumulation of free fatty acids ocMoreover, this damage is not blocked by competitive curs due to phospholipase A2 activity, an enzyme known to NMDA antagonists. be activated by Ca2+ (Imaizumi et al., 1990; Sun, 1990). The Hypoxic neuronal damage, in both the absence and the main weakness of the free radical ischemiclreperfusion neupresence of EAA, can be abolished by omission of Ca2+ ronal damage hypothesis is in the elusiveness of its main playfrom the slices and the incubation medium. ers, the highly reactive oxygen-free radicals, which are very Similarly, elevated [Mg”] in the incubation medium abol- difficult to detect (Ginsberg et al., 1988). Is this the reason ishes hypoxic neuronal damage in both the presence and why in vitro studies of this hypothesis using cell cultures or the absence of EAA. brain slices are scarce? Future developments to improve free Hypoxic neuronal damage is enhanced by EAA, and this radical detection will help to determine how important this enhancement can be blocked by a competitive NMDA an- hypothesis is for the elucidation of the mechanism of CHItagonist. induced neuronal damage.

THE MECHANISM OF CEREBRAL HYPOXIC-ISCHEMIC DAMAGE / Schurr and Rigor

A WORKING HYPOTHESIS: IS IT TIME FOR A UNIFYING ONE? Siesjo has published more reviews, overviews, hypotheses, syntheses, and speculative articles on the topic of cerebral ischemia than any other investigator in this field (Siesjo, 1981; 1982; 1985; 1988a-1988c; Siesjo and Bengtsson, 1989; Siesjo et al., 1989). As the number of hypotheses on the mechanism of CHI-induced neuronal death increased, Siesjo centered his efforts on trying to unify them by inclusion of all existing information and postulates. These efforts may be perceived to indicate uncertainty as to which of the hypothesized mechanisms of cerebral ischemia is responsible for cell death. We must ask the question, Do we have sufficient information today to allow us t o choose from the available hypotheses? If the answer is yes, can we afford to remain undecided? The information available on the supposed causes of neuronal devastation following a CHI episode suggests that acidosis can be postulated to enhance both calcium-related damage and iron-catalyzed free radical damage (Siesjo, 1988a). However, acidosis does not occur, and free radicals are not formed during or after hypoglycemia, yet calcium influx and neuronal death d o result from this condition. Moreover, hypoglycemic neuronal damage in vitro could be aggravated by EAA via the acceleration of Ca2- influx (Schurr and Rigor, 1989; Schurr et al., 1990; 1991; 1992). The suggestion that EAA excitotoxicity is calcium-dependent (Choi, 1985; 1987; 1988a; 1988b; Garthwaite et al., 1986; Siesjo, 1988b; Siesjo and Bengtsson, 1989; Meyer, 1989; Siesjo et al., 1989) is probably better documented than the interrelationships between acidosis, oxygen free radicals, and calcium. Even the postulated fall in ATP levels as the cause of neuronal ischemic damage may not withstand the scrutiny imposed by recent findings (Kass et al., 1990). This study shows a lack of correlation between postanoxic recovery of neuronal function in thiopental-treated hippocampal slices and tissue levels of ATP during the anoxic period; untreated slices exhibited higher ATP levels during 3.5 minutes anoxic time and a lower percentage of recovery than thiopentaltreated slices. Our own studies (unpublished data) demonstrated that NMDA-treated hypoglycemic slices contained higher levels of ATP but showed much lower recovery rate upon return to normoglycemic conditions than NMDAtreated hypoglycemic slices depleted of calcium. Thus, despite very low levels of ATP and the presence of NMDA, calcium-depleted slices fully recovered from hypoglycemia, a phenomenon that signals the importance of calcium in neuronal metabolic perturbation and damage. The available evidence for the different possible causes of ischemic neuronal death should allow us t o separate and single out calcium influx and overload as its cause. Attempts to tie all the postulated causes together to formulate the mechanism of hypoxic-ischemic neuronal damage appear to hinder rather than advance our understanding and treatment. Hence, the calcium-related neuronal damage hypothesis (Siesjo and Bengtsson, 1989; Meyer, 1989; Siesjo, 1988b; Siesjo et al., 1989) should be advanced as the primary one. Neither excitatory amino acids, nor lactic acid, nor oxygen-free radicals alone could devastate neurons in the absence of calcium and thus should be considered only secondary factors. However,

225

ischemia-induced calcium influx and overload can take place without the presence of lactic acid, EAA, or free radicals. The mere existence of multiple Ca2' channels of both voltage-dependent and agonist receptor-mediated type o n the neuronal membrane should allow CaZ+ influx at an accelerated rate under hypoxic-ischemic conditions. Thus, EAA, which mediate only a fraction of these channels, and free radicals, either alone or in combination, may enhance calcium influx and overload t o exacerbate neuronal damage.

ACKNOWLEDGMENTS The authors wish to thank Patricia Bensinger and Maureen Schurr for assistance in the preparation of the manuscript. References Aitken, P. G., M. Balestrino, and G. G . Somjen (1988) NMDA antagonists: Lack of protective effect against hypoxic damage in CA1 region of hippocampal slices. Neurosci. Lett. 89:187-192. Aust, S . I).,and B. C. White (1985) lron chelation prevents tissue injury following ischemia. Adv. Free Radical Biol. and Med. 1:l17. Benveniste, H., M. B. Jorgensen, N. H. Diemer, and J . A . Hansen (1988) Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia. Acta Neurol. Scand. 78529-536. Boakye, P., E. J. White, and J. B. Clark (1991) Protection of ischaemic synaptosomes from calcium overload by addition of exogenous lactate. J . Neurochem. 57:88-94. Bunnell, 0. S., T. M. Louis. R. L. Saldanha, and A. E. Kopelman (1987) Protective action of the calcium antagonists, flunarizine and nimodipine, on cerebral ischemia. Med. Sci. Res. 15:15131514.

Cheung, J. Y., J. V. Bonventre, C. D. Malis, and A. Leaf (1986) Calcium and ischemic injury. N. Engl. J. Med. 314:16701676. Choi, D. W. (1985) Glutamate neurotoxicity in cortical cell culture is calcium dependent. Neurosci. Lett. 58:293-297. Choi, D. W. (1987) Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7:369-379. Choi, I). W. (1988a) Calcium-mediated neurotoxicity: Relationship to specific channel types and role in ischemic damage. Trends. Neurosci. I1:465-469. Choi, D. W. (1988b) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623-634. Choi, D. W., J. H. Weiss, J.-Y. Koh, C. W . Christine, and M. C. Kurth (1989) Glutamate neurotoxicity, calcium, and zinc. Ann. N.Y. Acad. Sci. 568:219-224. Cotman, C., and D. Monaghan (1989) Multiple excitatory amino acid receptor regulation of intracellular Ca*+. Ann. N.Y. Acad. Sci. 568: 138-148. Cox, J. A,, P. G. Lysko, and R. C. Henneberry (1989) Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: Role of the voltage-dependent magnesium block. Brain Res. 499:267-272. Curtis, D. R., J. W. Phillis, and J. C. Watkins (1961) Action ofaminoacids on the isolated hemisected spinal cord of the toad. Br. J. Pharmacol. 16:262-283. David, P., M. Lusky, and V. I. Teichberg (1988) Involvement of excitatory neurotransmitters in the damage produced in chick embryo retinas by anoxia and extracellular high potassium. Exp. Eye Res. 46:657-662. Dong, W.-Q., A. Schurr, K. H. Reid, C. B. Shields, and C. A. West

226

HIPPOCAMPUS VOL. 2, NO. 3, JULY 1992

(1988) The rat hippocampal slice preparation as an in vitro model of ischemia. Stroke 19:498-502. Farber, J. L. (1981) The role of calcium in cell death. Life Sci. 29: 1289-1295. Floyd, R. A. (1990) Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 4:2587-2597. Fox, P. T., and M. E. Raichle (1986) Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects. Proc. Natl. Acad. Sci. U.S.A. 83:1140-1144. Fox, P. T., M. E. Raichle, M. A. Mintun, and C. Dence (1988) Nonoxidative glucose consumption during focal physiologic neural activity. Science 241:462-464. Garcia, J. H., and M. L. Anderson (1989) Physiopathology of cerebral ischemia. Crit. Rev. Neurobiol. 4:303-324. Gardiner, A., M.-L. Smith, E. Kagstrom, E. Shohami, and B. K. Siesjo (1982) Influence of blood glucose concentration on brain lactate accumulation during severe hypoxia and subsequent recovery of brain energy metabolism. J. Cereb. Blood Flow Metab. 2:429-438. Garthwaite, G., and J. Garthwaite (1986) Neurotoxicity of excitatory amino acid receptor agonists in rat cerebellar slices: Dependence on calcium concentration. Neurosci. Lett. 66:193-198. Garthwaite, G., F. Hajos, and J. Garthwaite (1986) Ionic requirements for neurotoxic effects of excitatory amino acid analogues in rat cerebellar slices. Neuroscience 18:437-447. Giffard, R. G., H. Monyer, C. W. Christine, and D. W. Choi (1990) Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen-glucose deprivation neuronal injury in cortical cultures. Brain Res. 506:339-342. Ginsberg, M. D., B. D. Watson, R. Busto, S. Yoshida, R. Prado, H. Nakayama, M. Ikeda, W. D. Dietrich, and M. Y.-T. Globus (1988) Peroxidative damage to cell membranes following cerebral ischemia: A cause of ischemic brain injury? Neurochem. Pathol. 9:171-193. Ginsberg, M. D., B. Lin, E. Morikawa, W. D. Dietrich, R. Busto, and M. Y.-T. Globus (1991) Calcium antagonists in the treatment of experimental cerebral ischemia. Drug Res. 41:334-337. Gramsbergen, J. B. P., and A. J. vander Sluijs-Gelling(1991) Calcium accumulation in excitotoxic brain lesions: Time course and correlation with cell death. SOC.Neurosci. Abstr. 175. Granger, D. N., J. M. McCord, D. A. Parks, and M. E. Hollwarth (1986) Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 90:80-84. Greenberg, J. H., D. Uematsu, N. Araki, and M. Reivich (1991) Intracellular calcium and pathophysiological changes in cerebral ischemia. Drug Res. 41:324-332. Grigg, J. J., and E. G . Anderson (1989) Glucose and sulfonylureas modify different phases of the membrane potential change during hypoxia in rat hippocampal slices. Brain Res. 489:302-3 10. Grotta, J. C., L . C. Pettigrew, D. Rosenbaum, C. Reid, H. Rhoades, and D. McCandless (1988) Efficacy and mechanism of action of a calcium channel blocker after global cerebral ischemia in rats. Stroke 19:447-454. Hadani, M., W. Young, and E. S. Flamm (1988) Nicardipine reduces calcium accumulation and electrolyte derangements in regional cerebral ischemia in rats. Stroke 19:1125-1132. Hansen, A. J. (1985) Effect of anoxia on ion distribution in the brain. Physiol. Rev. 65:101-148. Henneberry, R. C., A. Novelli, J. A. Cox, and P. G. Lysko (1989) Neurotoxicity at the N-methyl-D-aspartate receptor in energycompromised neurons: A hypothesis for cell death in aging and disease. Ann. N.Y. Acad. Sci. 568:225-233. Hoffman, W. E., E. Braucher, D. A. Pelligrino, C. Thomas, R. F. Albrecht, and D. J. Miletich (1990) Brain lactate and neurologic

outcome following incomplete ischemia in fasted, nonfasted, and glucose-loaded rats. Anesthesiology 72: 1045-1050. Horowitz, J. D., and A. C. Powell (1989) Calcium antagonist drugs in the management of cardiovascular disease: Current status. Med. J. Aust 150:591-595. Hossmann, K.-A,, W. Paschen, and L. Csiba (1983) Relationship between calcium accumulation and recovery of cat brain after prolonged cerebral ischemia. J. Cereb. Blood Flow Metab. 3:346353. Hossmann, K.-A., B. Grosse Ophoff, R. Schmidt-Kastner, and U. Oschiles (1985) Mitochondria1 calcium sequestration in cortical and hippocampal neurons after prolonged ischemia of the cat brain. Acta Neuropathol. 68:230-238. Imaizumi, S . , H. Kinouchi, T. Tominaga, Y. Fujita, and T. Yoshimoto (1990) Free radicals and lipid changes in cerebral ischemia. In Cerebral Ischemia and Resuscitation, A. Schurr and B. M. Rigor, eds., pp. 151-164, CRC Press, Boca Raton, FL. Kass, 1. S., and P. Lipton (1986) Calcium and long-term transmission damage following anoxia in dentate gyrus and CAI regions of the rat hippocampal slice. J. Physiol. 378:313-334. Kass, I. S., J. E. Coltrell, and G. Chambers (1990) ATP does not correlate with recovery after anoxia and thiopental in the rat hippocampus. Anesthesiology 73:A673. Kostyuk, P., N. Akaike, Y. U . Osipchuk, A. Savchenko, a n d Y . A. Shuba (1989) Gating and permeation of different types of Ca channels. Ann. N.Y. Acad. Sci. 560:63-79. Lear, J. L. (1990) Glycolysis: Link between PET and proton MR spectroscopic studies of the brain. Radiology 174:328-330. Lemasters, J. J . , J. Diguiseppi, A. L. Nieminen, and B. Herman (1987) Blebbing, free CaZ+and mitochondria1 membrane potential preceding cell death in hepatocytes. Nature 32S:78-81. LeVere, T. E., T. Brugler, M. Sandin, and S. Gray-Silva (1989) Recovery of function after brain damage: Facilitation by the calcium entry blocker nimodipine. Behav. Neurosci. 103561-565. Lindenberg, R. (1963) Patterns of CNS vulnerability in acute hypoxemia including anesthesia accidents. In Selective Vulnerability of the Bruin to Hypoxerniu, J. Schade and W. McMenemy, eds., pp. 189-210, FA Davis, Philadelphia. Ljunggren, B., K. Norberg, and B. K. Siesjo (1974) Influence of tissue acidosis upon restitution of brain energy metabolism following total ischemia. Brain Res. 77: 173-186. Lucas, D. R., and J . P. Newhouse (1957) The toxic effect of sodium L-glutamate on the inner layers of the retina. Arch. Ophthalmol. 58:193-201. Lyden, P. D., J. A. Zivin, A. Kochhar, and V. Mazzarella (1988) Effects of calcium blockers on neurologic outcome after focal ischemia in rabbits. Stroke 19: 1020-1026. Lysko, P. G., J. A. Cox, M. A. Vigano, and R. C. Henneberry (1989) Excitatory amino acid at the N-methyl-D-aspartate receptor in cultured neurons: Pharmacological characterization. Brain Res. 499:258-266. MacVicar, B. A . , D. Hochman, M. J. Delay, and S . Weiss (1991) Modulation of intracellular Cat + in cultured astrocytes by influx through voltage-activated Ca+ channels. GLIA 4: 448-455. Mayer, L. M., and R. J. Miller (1990) Excitatory amino acid receptors, second messengers and regulation of intracellular Ca+ in mammalian neurons. Trends Pharmacol. Sci. 11:254-260. Meyer, F. B. (1989) Calcium, neuronal hyperexcitability and ischemic injury. Brain Res. Rev. 14:227-243. Michenfelder, J. D., and T. M. Sundt (1971) Cerebral ATP and lactate levels in the squirrel monkey following occlusion of the middle cerebral artery. Stroke 2:319-326. Monaghan, D. T., R. J . Bridges, and C. W. Cotman (1989) The excitatory amino acid receptors: Their classes, pharmacology, and +

THE MECHANISM OF CEREBRAL HYPOXIC-ISCHEMIC DAMAGE / Schurr and Rigor distinct properties in the function of the central nervous system. Annu. Rev. Pharmacol. Toxicol. 29:365-402. Myers, R. E., and S. Yamaguchi (1977) Nervous system effects of cardiac arrest in monkeys. Arch. Neurol. 34:65-74. Natale, J. E., and L. D'Alecy (1989) Protection from cerebral ischemia by brain cooling without reduced lactate accumulation in dogs. Stroke 20:770-777. Nicholson, C., G. T. Bruggencate, R. Steinberg, and H. Stockle (1977) Calcium modulation in brain extracellular microenvironment demonstrated with nonselective micropipette. Proc. Natl. Acad. Sci. U S A . 74:1287-1290. Nordstrom, C.-H., and B. K. Siesjo (1978) Effects of phenobarbital in cerebral ischemia. Part I: Cerebral energy metabolism during pronounced incomplete ischemia. Stroke 9:327-335. Novelli, A., J . A. Reilly, P. G. Lysko, and R. C. Henneberry (1988) Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res. 451 ~205-212. Olney, J . W. (1969a) Glutamate-induced retinal degeneration in neonatal mice: Electron microscopy of the acutely evolving lesion. J. Neuropathol. Exp. Neurol. 28:455-474. Olney, J. W. (1969b) Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate. Science 164:719721. Olney, J. W., M. T. Price, L . Samson, and J . Labruyere (1989) The role of specific ions in glutamate neurotoxicity. Neurosci. Lett. 65~65-71. Olney, J. W. (1989) Excitatory amino acids and neuropsychiatric disorders. Biol. Psychiatry 26505-525. Rothman, S. M. (1985) Excitatory amino acid neurotoxicity is produced by passive chloride influx. J. Neurosci. 6: 1483-1489. Rothman, S. M., and J. W. Olney (1986) Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19:105-111. Salford, L. G . , F. Plum, and B. K. Siesjo (1973) Graded hypoxiaoligemia in rat brain: I. Biochemical alterations and their implications. Arch. Neurol. 29:227-233. Salford, L. G., and B. K. Siesjo (1974) The influence of arterial hypoxia and unilateral carotid artery occlusion upon regional blood flow and metabolism in the rat brain. Acta Physiol. Scand. 92:130141. Sauter, A,, M. Rudin, and K.-H. Wiederhold (1988) Reduction of neuronal damage in irrevesible cerebral ischemia by calcium antagonists. Neurochem. Pathol. 9:211-236. Sauter, A., and M. Rudin (1990) Calcium antagonists for reduction of brain damage in stroke. J. Cardiovasc. Pharmacol. 15:S43s47. Schanne, F. A. X . , A. B. Kane, E. E. Young, and J. L. Farber (1979) Calcium dependence of toxic cell death: A final common pathway. Science 206:700-702. Schurr, A., C. A. West, K. H. Reid, M. T. Tseng, S . J. Reiss, and B. M. Rigor (1987a) Increased glucose improves recovery of neuronal function after cerebral hypoxia in vitro. Brain Res. 421:135139. Schurr, A., M.T. Tseng, and B. M. Rigor (1987b) Taurine improves the recovery of neuronal function following cerebral hypoxia: An in vitro study. Life Sci. 40:2059-2066. Schurr, A., W.-Q. Dong, K. H. Reid, C. A. West, and B. M. Rigor (1988a) Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro. Brain Res. 438:311-314. Schurr, A., C. A . West, and B. M. Rigor (1988b) Lactate-supported synaptic function in the rat hippocampal slice preparation. Science 240: 1326-1328. Schurr, A., and B. M. Rigor (1989) Cerebral ischemia revisited: New insights as revealed using in vitro brain slice preparations. Experientia 45:684-695.

227

Schurr, A,, P. Lipton, C. A. West, and B. M. Rigor (1990) The role of energy metabolism and divalent cations in the neurotoxicity of excitatory amino acids in vitro. In Pharmacdogy of Cerebral Ischemia, J. Krieglstein, ed., pp. 217-226, CRC Press, Boca Raton, FL . Schurr, A., C. A. West, and B. M. Rigor (1991a) Neurotoxicity of quinolinic acid and its derivatives in hypoxic rat hippocampal slices. Brain Res. 568: 199-204. Schurr, A., C . A. West, and B. M. Rigor (1991b) The excitotoxicity of heterocyclic dicarboxylic acids in rat hippocampal slices: structure-activity relationships. Brain Res. 571:145-148. Siemkowicz, E., and J . A. Hansen (1978) Clinical restitution following cerebral ischemia in hypo-, normo-, and hyperglycemic rats. Acta Neurol. Scand. 58:l-8. Siesjo, B. K. (1981) Cell damage in the brain: A speculative synthesis. J. Cereb. Blood Flow Metab. 1:155-185. Siesjo, B. K. (1982) Lactic acidosis in the brain: Occurrence, triggering mechanisms and pathophysiological importance. In Mefabolic Acidosis, pp. 77-100, Ciba Foundation Symposium 87, Pitman, Bath. Siesjo, B. K . (1985) Acid-base homeostasis in the brain: Physiology, chemistry and neurochemical pathology. In Progress in Brain Research, vol. 63, K . Kogure, K.-A. Hossmann, B. K. Siesjo, and P. A. Welsh, eds., pp. 121-154, Elsevier, Amsterdam. Siesjo, B. K. (1988a) Acidosis and ischemic brain damage. Neurochem. Pathol. 9:31-88. Siesjo, B. K . (1988b) Historical overview: Calcium, ischemia, and death of brain cells. Ann. NY Acad. Sci. 522:638-661. Siesjo, B. K . (1988~)Hypoglycemia, brain metabolism and brain damage. Diabete. Metab. Rev. 4:113-144. Siesjo, B. K., and F. Bengtsson (1989) Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: A unifying hypothesis. J. Cereb. Blood Flow Metab. 9:127-140. Siesjo, B. K., F. Bengtsson, W. Grampp, and S . Theander (1989) Calcium, excitotoxins, and neuronal death in the brain. Ann. N.Y. Acad. Sci. 568:234-251. Stark, K., P. Seubert, G. Lynch, and M. Baudry (1989) Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain). Biochem. Biophys. Res. Commun. 165:858-864. Stys, P. K., S. G. Waxman, and B. R. Ransom (1991) Na'-Caz+ exchanger mediates Ca2+ influx during anoxia in mammalian central nervous system white matter. Ann. Neurol. 30:375380. Sun, G. Y . (1990) Mechanisms for ischemia-induced release of free fatty acids in brain. In Cerebral Ischemia and Resuscifation, A. Schurr and B . M. Rigor, eds., pp. 123-135, CRC Press, Boca Raton, FL. Swann, J. D., M. W . Smith, P. C. Phelps, A. Maki, I. K. Berezesky, and B. F. Trump (1991) Oxidative injury induces influx-dependent changes in intracellular calcium homeostasis. Toxicol. Pathol. 19: 128- 137. Takakura, S., T. Susumu, H. Satoh, J. Mori, A. Shiino, and J. Handa (1991) Nilvadipine, a new calcium channel blocker, reduces ischemic brain injury in rats. Jpn. J. Pharmacol. 56547-550, Thorn, W., and R. Heitmann (1954) pH der gehirnrinde von kaninchen in situ wahrend perakuter totaler ischiamie, reiner anoxie und in der erholung. Pfluegers Arch. 258:501-510. Tombaugh, G. C., and R. M. Sapolsky (1990) Mild acidosis protects hippocampal neurons from injury induced by oxygen and glucose deprivation. Brain Res. 506:343-345. Walz, W., and W. A. Wuttke (1989) Resistance of astrocyte electrical membrane properties to acidosis changes in the presence of lactate. Brain Res. 504:82-86, 1989.

228

HIPPOCAMPUS VOL. 2, NO. 3, JULY 1992

Walz, W., and D. E. Harold (1990) Brain lactic acidosis and synaptic function. Can. J. Physiol. Pharmacol. 68:164-169. Watkins, J. C. (1962) The synthesis of some acidic amino acids possessing neuropharmacological activity. J. Med. and Pharm. Chem. 5: 1 187- 1198. Welsh, F. A , , M. D. Ginsberg, W. Rieder, and W. W. Budd (1980)

Deleterious effect of glucose pretreatment on recovery from diffuse cerebral ischemia in the cat. 11. Regional metabolite levels. Stroke 11:355-363. Yanagahara, T . , and J. T. McCall (1982) Ionic shift in cerebral ischemia. Life Sci. 30:1921-1925.