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A “radical” view of cerebral ischemic injury
Progress in NeurobiologyVol. 42, pp. 441 to 448, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0301-0082/94/$26.00
Pergamon
MINI REVIEW A " R A D I C A L " V I E W OF C E R E B R A L I S C H E M I C I N J U R Y JOrlN W. PHILLIS Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, Michigan 40201, U.S.A.
CONTENTS 1. Introduction 2. Reactive oxygen species (ROS) and lipid peroxidation 2.1. Lipid peroxidation 2.2. Enzymes protecting against ROS 2.3. Sources of ROS 2.3.1. Ubisemiquinone radical 2.3.2. Purine catabolism 2.3.3. Arachidonic acid metabolism 2.3.4. Neutrophils 2.3.5. Glutamate receptor agonists 2.3.6. Nitric oxide 3. ROS and cerebral ischemia/reperfusion injury 3.1. Detection of free radicals in brain 3.2. Pharmacological evidence of free radical involvement 3.2.1. Cerebroprotection with PBN and its effect on ROS formation 3.2.2. Oxypurinol, a xanthine oxidase inhibitor 4. Aging, cerebral ischemia and ROS 5. Conclusions Acknowledgements References
1. INTRODUCTION The free radical hypothesis of cerebral ischemia/ reperfusion injury was formulated to provide a conceptual basis for the evaluation of the sequence of events leading to delayed neuronal death in brains subjected to a period of iscbemia, such as that occurring during a cardiac arrest or stroke, followed by reperfusion. The free radical hypothesis of tissue iscbemia/reperfusion injury, as exemplified by the work of McCord and his coworkers (Parks et al., 1982), was first applied to cerebral ischemic injury over a decade ago (Flamm et al., 1978; Demopoulos et al., 1980). The brain appears to be particularly vulnerable to oxidative damage. It contains relatively high concentrations of readily peroxidizable fatty acids. Various regions of the brain are highly enriched in iron, which possesses an ability to catalyze the production of damaging oxygen free radical species. Furthermore, the brain is relatively poorly endowed with protective antioxidant enzymes or antioxidant compounds. Free radicals are believed to initiate damage to many cellular elements such as lipids, proteins and nucleic acids. The damage to membrane lipids results not only in changes in fluidity and permeability of the cell membranes, but also in
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the function of phospholipid-dependent proteins. The consequences of these alterations in cellular function are potentially lethal. The intent of this work is to present a brief review of the evidence supporting the free radical hypothesis of cerebral ischemia/ reperfusion injury.
2. REACTIVE OXYGEN SPECIES (ROS) AND LIPID PEROXIDATION Oxygen radicals, also known as reactive oxygen species (ROS), arise in sequential fashion from molecular oxygen by successive single-electron reduction reactions (Halliwell and Gutteridge, 1985) ROS are themselves chemically reactive because in order to regain thermodynamic stability they attempt to remove electrons or hydrogen atoms from neighboring molecules, thus radicalizing these molecules in turn, creating a so-called "cascade" effect. The initial oxygen radical in the sequence is the superoxide amino radical (O~-), followed by the perhydroxyl radical (HO2), hydrogen peroxide (H202) and the extremely reactive hydroxyl radical (OH), which is capable of reacting with almost every type of molecule found in living cells, including sugars, proteins, amino acids,
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DNA bases and organic acids including polyunsaturated fatty acids (Halliwell and Gutteridge, 1985; Hailiwell, 1992). A role of ROS in ischemia/reperfusion injury was suggested by the observed reduction in peripheral tissue damage when animals were treated with scavengers of these agents (McCord, 1985; Granger et al., 1986) and they have since been implicated in the pathogenesis of ischemic injury to brain (Flamm et al., 1978; Demopoulos et al., 1980; Kontos, 1985). Indeed, recent advances in understanding the fundamental mechanisms of post-ischemic injury have suggested that most tissue injury is associated with the admission of oxygen to the tissue at the time of reperfusion (Downe3 1988; Ernster, 1988) and the generation of ROS (McCord, 1986).
2. I. LIPID PEROXIDATION An important consequence of ROS formation is lipid peroxidation by O H or other radicals; a reaction in which the double bonds in the fatty acid side chains are rearranged. Their large numbers of unsaturated fatty acids make cell membranes particularly susceptible to a chain reaction of lipid peroxidation, with resultant changes in fluidity and permeability, which can profoundly affect the functioning of the membrane. Lipid peroxidation can also inhibit the function of membrane bound receptors and enzymes. Lipid peroxides are chemically unstable and can degrade into other chemically reactive species, such as aldehydes, including malondialdehyde. Lipid peroxidation is an important phenomenon primarily because it is a chain reaction, in which an initiating free radical can precipitate the destruction of numerous adjacent molecules. Peroxidation of an unsaturated fatty acid results from the removal of a methylene hydrogen atom, whereupon the resultant fatty acid radical converts to a conjugated diene. This moiety is itself peroxidized by the addition of O: to yield a peroxy radical which can initiate a new cycle by repeating the hydrogen atom sequestration from another polyunsaturated fatty acid molecule, yielding a lipid hydroperoxide and another lipid alkyl radical. In the presence of a transition metal, the lipid hydroperoxide can be converted into a lipid alkoxyl radical, which can initiate further lipid peroxidation, with further expansion of the chain reaction. The initiating hydroxyl radical arises from the decomposition of hydrogen peroxide or lipid hydroperoxides by metal ions (Fenton reaction), of which iron is undoubtedly the most important (Aust and White, 1985). Termination of the chain reaction occurs when the propagating radicals are neutralized by organic scavenger compounds (Vitamin E, ascorbic acid) or by enzymic reduction of the lipid hydroperoxides and hydrogen peroxide by glutathione peroxidase, which prevents their degradation to hydroxyl, peroxy or alkoxy radicals. Tissue lactacidosis can dramatically enhance the formation of free radicals and lipid peroxidation in brain tissue by its mediation of an increased dissociation of catalytic iron from proteins of the transferrin type (Rehncrona et al., 1989).
2.2. ENZYMESPROTECTINGAGAINSTROS
Specific enzymes have evolved to deal with the ROS. Superoxide dismutase catalyzes the conversion of the superoxide anion radical into hydrogen peroxide. Because it is non-polar, unlike O2, hydrogen peroxide readily diffuses across cell membranes. In the presence of ferrous iron, hydrogen peroxide is converted into the highly reactive O H radical (Fenton reaction). Because iron is bound in the ferric form in vivo, a complementary reaction is required to generate ferrous iron and this is mediated by the superoxide anion. The net iron-catalyzed, superoxide dependent, conversion of H20: to O H is known as the Haber-Weiss reaction. Hydrogen peroxide is removed by two enzymes. Glutathione peroxidase catalyzes the reduction of H202 (or fatty hydroperoxides) to water (or hydroxy fatty acids) whilst converting reduced glutathione (GSH) into oxidized glutathione (GSSG). Catalase decomposes hydrogen peroxide to oxygen and water. The central nervous system is relatively poorly endowed with superoxide dismutase, glutathione peroxidase and catalase, which may account, in part, for its susceptibility to ischemic injury. 2.3. SOURCESOF ROS Reactive oxygen radicals may be generated by a number of pathways (Kontos, 1989; Ikeda and Long, 1990; Schmidley, 1990) including those described in the following sections. 2.3. i. Ubisemiquinone radical Oxidation of ubisemiquinone radical, the diffusable electron transfer agent in the mitochondriai respiratory chain, yields 02. This pathway is operative during regular metabolism yielding small amounts of ROS which can be eliminated by the cell's defences, but its enhancement during reoxygenation is potentiated by the reducing conditions present during ischemia. 2.3.2. Purine catabolism During ischemia ATP is utilized, but cannot be resyntbesized and as a result of the cessation of mitochondrial oxidative phosphorylation its metabolites adenosine, inosine, hypoxanthine and xanthine accumulate (Fig. 1). In the normoxic brain, hypoxanthine is metabolized by xanthine dehydrogenase (XDH) to xanthine and ultimately to uric acid. With the onset of ischemia, the rise in intracellular calcium results in a rapid, proteolytic conversion of xanthine dehydrogenase to xanthine oxidase (XO), which uses molecular oxygen instead of the nueleotide radical of NAD as its electron acceptor and catalyzes the production of O f during reperfusion. XDH/XO is present in the brains of several species, including man (Wajner and Harkness, 1989), predominantly in the X D H form. Rat brain XO is concentrated in the capillary endotbelium (Betz, 1985) and during ischvmia there is a substantial conversion of XDH to XO (Kinuta et al., 1989). Bovine brain endothelial
FREE RADICALS AND BRAIN INJURY
and acid releatsc CExcitotoxic amino acids")
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ATP depl~ion Adenosine
1
Failme of plasma membrane Na+ and Ca:z+ pumps I
• Ca2+ in cytoplasm
i
Inosin¢
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hospholipase activation Free fatty acids
Cyclooxygenase Lipoxygenase
l.O.. Dehyed hypoperfusion
x. i.
+3
Uric acid + 02-"
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Secondary ischemia Cellular membrane desm~tion ~'~
FzG. I. Proposed pathways for events linking cerebral isehemia to neuronal death.
cells are able to generate superoxide anion formation by a xanthine oxidase-dependent mechanism (Terada et al., 1991) and anoxia/reoxygenation-induced hydroxyl radical formation occurs in isolated rat brain microvessels (Grammas et al., 1993).
cyclins, thromboxanes and leukotrienes. Cyclooxygenase catalyzes the addition of two molecules of oxygen to an unsaturated fatty acid to produce prostaglandin G, which is rapidly peroxidized to prostaglandin H with the concomitant production of O~-' (Kuehl and Ekgan, 1980). Hydroxyl radicals can be generated by the lipoxygenase pathway which produces ieukotrienes.
2.3.3. Arachidonic acid metabolism Arachidonic acid, generated as a result of membrane lipolysis occurring during ischemia, can serve as a precursor for ROS formation. Calcium entry into isehemic neurons activates phospholipases including phospholipase A2, which cleaves a fatty acyl chain from the b position of phospholipids to produce arachidonic acid, the levels of which rise rapidly during ischemia (Yasuda et al., 1985; Abe et aL, 1987). Arachidonic acid may be metabolized by either cyclooxygenase or lipoxygenase (Fig. !), producing a variety of vasoactive substances including prostaglandins, prosta-
2.3.4. Neutrophils Neutrophils are another potential source of ROS. Phagocytosis stimulates O~- synthesis by NADPH oxidase during the respiratory burst. Neutrophils and perhaps microglia, responding to tissue injury may inappropriately destroy adjacent tissues, resulting in infarct extension. Hailenbeck et ai. (1986) have observed neutrophil accumulation in the brain regions with low blood flow during the early post-ischemic
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period, where they may contribute to cerebral delayed hypoperfusion. However, treatment with antineutrophil serum at the time of reperfusion is not associated with improved post-ischemic blood flow in rats (Grogaard et al., 1989) or improved neurologic recovery in dogs (Schott et al., 1989). It is therefore unlikely that neutrophils contribute significantly to ROS formation during ischemia and in the immediate post-ischemic period. 2.3.5. G l u t a m a t e receptor agonists Oxidative stress is induced by glutamate receptor agonists. Cerebral ischemia is accompanied by massive increases in the extracellular concentration of the excitatory amino acids glutamate and aspartate (Benveniste et al., 1984; Hagberg et al., 1985; Globus et aL, 1988; Simpson et al., 1992). Glutamate, aspartate and their analogs in excessive concentration are neurotoxic and may cause neuronal death by elevating intracellular calcium levels. Bondy and Lee (1993) have recently demonstrated that agonists specific for each of the three major glutamate ionotropic receptor sites (NMDA, kainate, AMPA) enhanced the rate of ROS generation in cerebrocortical microsacs (synaptoneurosomal fraction), whilst an agonist for the metabotropic receptor (ACPD) was inactive. Evidence for a ROS generating action of kainate had previously emerged from a study which demonstrated that inhibition of the formation of XO from XDH or of the action of XO by allopurinol, or the addition of free radical scavengers, protected mouse cerebellar neurons in microwell culture from kainate-induced neurodegeneration (Dykens et al., 1987). These results appear to directly implicate ROS in the neurotoxic actions of glutamate receptor agonists. The interconnectedness of excitotoxic amino acid release and free radical generation has been emphasized by Pellegrini-Giampietro et al. (1990), who showed that inhibition of ROS formation reduced excitotoxic amino acid release from "ischemic" rat hippocampal slices and suggested that free radical formation and glutamate release are mutually related and cooperate in precipitating ischemic neuronal death. Studies on ischemic brain edema (Oh and Betz, 1991) have also implicated a causal or cooperative relationship between the excitatory amino acids and ROS. 2.3.6. Nitric oxide Nitric oxide, the recently discovered endotheliallyderived relaxing factor and putative neurotransmitter, is itself a free radical with both neuroprotective and neurodegenerative effects. NO - mediated neurotoxicity is engendered, at least in part, by its reaction with superoxide anion, apparently leading to the formation of peroxynitrite ( O N O O - ) (Lipton et al., 1993). In contrast, its neuroprotective effects may result from a downregulation of N M D A receptor activity by a reaction with thiol groups of the receptors redox modulating site (Lipton et al., 1993). The enzyme responsible for nitric oxide formation, NO synthase, catalyzes its formation from L-arginine by a Ca 2÷/calmodulin dependent mechanism. Thus an increase in intracellular Ca 2÷ can activate the enzyme. As glutamate neurotoxicity mediated by
N M D A receptors involves Ca -~~ entry into cells, the possible participation of NO in neuronal injury induced by ischemia has been hypothesized. Dawson et al. (1991) have demonstrated that NO synthase inhibitors prevent neurotoxicity elicited by N M D A and related amino acids in rat primary cortical cultures. Protection against cerebral ischemic injury in vivo has also been reported (Nowicki et al., 1991; Buisson et al., 1992). The nitric oxide donor Snitroso-N-acetylpenicillamine increase extracellular levels of glutamate and aspartate in the rat medulla oblongata (Lawrence and Jarrott, 1993). Ischemiaevoked rat striatal glutamate release was significantly reduced by an NO synthase inhibitor (L-NAME) (Buisson et al., 1993) indicating that NO may contribute to the excitotoxic process by facilitating ischemiaevoked glutamate overflow. However, in another study L-NAME failed to reduce ischemia-evoked glutamate release in the rat hippocampus (Zhang et al., 1993). In unpublished experiments conducted in my laboratory, L-NAME significantly depressed the initial rate of rise in extracellutar glutamate and aspartate levels during a 20 min period of ischemia but did not reduce the total release (release during ischemia and reperfusion) of these amino acids.
3. ROS AND CEREBRAL ISCHEMIA/REPERFUSION INJURY 3.1 DETECTIONOF FREE RADICALSIN BRAIN The direct detection of unstable ROS in cerebral tissue has been difficult to achieve as most radicals are short lived and produced in trace amounts. Most studies on free radical involvement have utilized free radical scavengers or inhibitors of synthesizing enzymes to infer a role of ROS in tissue damage. Superoxide anion has been detected indirectly by its reaction with nitro blue tetrazolium to yield an insoluble blue formazan precipitate which can be quantified spectrophotometrically (Nelson et al., 1992). Hydroxyl radical formation in vivo has been assayed by HPLC in terms of 2,5-dihydroxybenzoate, assumed to result from the hydroxylation of infused salicylate (Cao et al., 1988; Boisvert, 1992). Direct evidence for ROS formation during ischemia/reperfusion has been obtained with the application of spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. Short lived free radicals are "trapped" with nitrone or nitroso spin-trapping agents upon which the resulting adduct gives rise to a stable nitroxide free radical, which can be measured with high specificity and sensitivity by EPR. Using these techniques, free radicals were detected by EPR in CSF from the brains of pigs subjected to global cerebral ischemia/reperfusion with ~-phenyl-t-butylnitrone (PBN) as a spin probe (Lange et al., 1990). Zini et al. (1992) were unable to detect any adducts in PBN-containing striata! microdialysates from rats subjected to global ischemia followed by reperfusion, but did observe a free radical adduct when pyridyl-N-oxide-t-butylnitrone (POBN) was used as the trapping agent. The spin adduct occurred during ischemia and early reperfusion, but not under basal conditions.
FREE RADICALSAND BRAININJURY
Hydroxyl radical production /n vivo, following reperfusion of ischemic rat cerebral cortex has been assayed by electron paramagnetic resonance with POBN administered both topically and systemically (Sen and Phillis, 1993; Phillis and Sen, 1993). Using the cortical cup technique, it was possible to collect serial samples of POBN-containing aCSF prior to ischemia, during ischemia and following reperfusion. Experiments were conducted both with and without the chelating agent DETAPAC in the aCSF with identical results. Pre-ischemic samples did not elicit an EPR signal. During and following a 30 min period of ischemia (four vessel occlusion), OH. radical adduct signals (characterized by six line EPR spectra) were detected in the superfusate samples. The signals reached their maximal amplitude during the initial 20 min of reperfusion and were no longer detectable after 90 min of reperfusion. 3.2. PHARMACOLOGICALEVIDENCEOF FREE RADICAL INVOLVEMENT
There have been numerous reports describing cerebroprotective actions of a variety of oxygen free radical scavengers including superoxide dismutase (especially the more stable polyethylene glycol-conjugated SOD), catalase, dimethylsulfoxide, dimethylthiourea, mannitol, Vitamin E, ascorbic acid, iazaroids such as U74006F (tirilazad mesylate) and of inhibitors of xanthine oxidase such as allopurinol or oxypurinol (see Ikeda and Long, 1990; Schmidley, 1990; Halliwell, 1992). Deferoxamine, a chelator of ferric iron, when administered may also reduce cerebral ischemic injury (Halliwell, 1992). Although the results with these interventions are not always consistent and the pharmacological actions of the agents may not be completely understood, the findings are generally supportive of an involvement of ROS in ischemia/ reperfusion injury. In spite of its potential significance as a free radical generating pathway, there is relatively little information in the literature on cerebroprotective effects of inhibitors of arachidonic acid metabolism to prostaglandins, prostacyclins, thromboxanes and leukotrienes. Acetyl salicylic acid has reportedly beneficial effects in human stroke patients (Schror, 1986) and ibuprofen improves survival and neurologic outcome after resuscitation of dogs from cardiac arrest (Kuhn et al., 1986). All of the evidence provided by these studies, however, is indirect and therefore inconclusive. More specific evidence for a role of free radicals has come from experiments with the spin-trap agent ct-phenyl-t-butylnitrone (PBN) and the xanthine oxidase inhibitor, oxypurinol. 3.2.1. Cerebroprotection with P B N and its effect on R O S formation
Protection against ischemia/reperfusion injury with free radical trapping agents such as PBN and 5,5dimethyI-L-pyroline-L-oxidase (DMPO) was initially reported in cardiac studies (Tosaki et al., 1990; Bolli, 1991; Bradamante et al., 1992). The rationale for using these agents was that they would react with free radicals to interrupt the cascade of reactions which ultimately results in membrane lipid peroxidation.
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PBN administered either prior to, or 30 min after a 5 min period of bilateral carotid occlusion in gerbils, prevented the increase in locomotor activity observed in saline injected control ischemic animals and significantly reduced the damage to hippocampal CAI pyramidal cells observed 5 days post-ischemia. These effects were no longer apparent when PBN was administered 2 hr post-ischemia (Phillis and CloughHelfman, 1990a; Clough-Helfman and Phillis, 1991). Yue et al. (1992) reported that PBN attenuated both forebrain edema and hippocampal CA 1 neuronal loss in ischemic gerbils and that it protected rat cerebellar neurons in primary culture from glutamate-induced neurotoxicity. PBN administration diminishes the increase in oxidized protein and the loss of glutamine synthetase activity that accompanies both ischemia/reperfusion injury and aging in the gerbil brain (Carney et al., 1991). Chronic treatment with PBN also results in a recovery of the loss in temporal and spatial memories in the aged gerbil brain, implying that there is an age related increase in the vulnerability of brain tissues to oxidation which can be reversed by free radical trapping compounds (Carney and Floyd, 1991). The neuroprotective effects of PBN in temperature stabilized rats subjected to middle cerebral artery (MCA) occlusion have recently been evaluated (Phillis and Cao, 1994). PBN, administered i.p. at 100 mg/kg in repeated doses significantly attenuated both cortical infarct volume and cerebral edema measured at 48 hr, even when the initial drug administration was delayed until 12 hr post-MCA occlusion. These reductions in histopathological ratings were accompanied by significant reductions in neurological deficit scores. Interestingly, the neurological deficit scores obtained at 48 hr were lower than those at 24 hr, suggesting that an effective repair process had already been initiated. These results appear to provide dramatic evidence for a continuing role of ROS in the extension of neuronal losses into the penumbral region of cerebral infarcts. Confirmation that the cerebroprotective actions of PBN could be a result of its ability to prevent hydroxyl radical formation was obtained in EPR experiments in which systemic administration of PBN greatly attenuated the amplitude of the radical adduct signal and a combination of systemic and topical (100 mM) PBN virtually abolished the signal (Sen and Phillis, 1993). PBN differs from POBN in being substantially more lipophilic and having a different intracellular distribution (Cheng et al., 1993), including access to the nuclear and mitochondrial fractions of cells (Cova et al., 1992). It is likely therefore that PBN achieves an adequate concentration in the mitochondria to interrupt the ROS cascade at its site of initiation. Otherwise H_,O2 or OH. formed intracellularly would diffuse across the plasma membrane to be trapped as an OH. radical adduct by POBN in the extracellular space. In this regard, it may be significant that DMPO, another hydrophilic spin-trap agent has proven to be less effective than PBN in myocardial ischemia/reperfusion injury (Bradamante et al., 1993). POBN and DMPO also failed to protect isolated rat atria against adriamycin-induced cardiotoxicity, whereas PBN was effective (Monti et al., 1991). The authors attributed the protective ability of
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PBN to its lipophilicity and ability to cross cell membranes. 3.2.2. Oxypurinol, a xanthine oxidase inhibitor Oxypurinol was tested for cerebroprotective activity in the gerbil and rat MCA occlusion models of cerebral ischemia. When administered either prior to or 30 min post-ischemia, oxypurinol (40 mg/kg) significantly reduced both the hypermotility and the extent of CA1 hippocampal pyramidal cell damage observed in comparison to that in control gerbils (Phillis and Clough-Helfman, 1990b). Its cerebroprotective actions were not secondary to reductions in body temperature as measured with intraperitoneally implanted telemetry probes. Oxypurinol was also protective when administered either prior to, or 1 hr post-ischemia, in the rat MCA occlusion model. It attenuated the degree of neurological deficit observed in control animals and significantly reduced both infarct volume and the extent of brain swelling (Phillis and Lin, 1991; Lin and Phillis, 1992). Protection against ischemic injury was not observed when oxypurinol administration was delayed for 5 hr post stroke. Body temperature measurements with telemetry probes confirmed that the cerebroprotective effects of oxypurinol were not a result of hypothermia. In a related study, the xanthine oxidase inhibitor, oxypurinol, was administered prior to the onset of ischemia to POBN treated animals. Oxypurinol greatly attenuated the appearance of OH- radical adduct in cerebral cortical superfusates during ischemia and reperfusion confirming that xanthine oxidase is likely to be involved in ROS formation (Phillis and Sen, 1993). It may be significant that xanthine oxidase is localized in capillary endothelial cells, as these will inevitably be an important site of free radical formation. The resultant destruction of the blood-brain barrier may be a major contributor to the edema formation which occurs in stroked brains.
4. AGING, CEREBRAL ISCHEMIA AND ROS The studies of Framingham and Whitehall have shown age to be one of the most important risk factors for brain infarction and its mortality (Hubert et al., 1983; Fuller et al., 1983). It is therefore important to take age-related changes into account when considering abnormalities with respect to brain ischemia due to disturbances in macro- or micro-circulations. Reports have been published on age-related changes in rat brain for peroxidative potential (Sawada and Carlson, 1987; Devasagayam, 1989)and the activities of superoxide dismutase, catalase and glutathione peroxidase (Victorica et al., 1984; Scarpa et al,, 1987; Barja de Quiroga et al., 1990; Semsei et al., 1991). Succinate concentrations in ischemic brain tissue of aged rats are elevated and may contribute to an acidosis (Hoyer and Krier, 1986). Increased levels of iron associated with aging have been reported in rat brain (Floyd et al., 1984). There is an
increase in the formation of free radicals with advancing age (Harman, 1981; Leibovitz and Siegel, 1980) which may account for the observation that mortality rates are higher in older gerbils subjected to ischemia (Floyd, 1990). The evidence therefore suggests that free radical generation during cerebral ischemia in aged rats may be significantly more pronounced than in younger animals and could account for the greater severity of ischemia/reperfusion injury in the aging brain.
5. CONCLUSIONS Cardiac arrest and stroke are two leading causes of death and when patients survive it is usually with some degree of neurological impairment. A massive release of the excitotoxic amino acids, glutamate and aspartate, occurs during ischemia, precipitating the entry of calcium into cells. Elevated intracellular calcium levels in turn activate calcium-dependent enzymes, including phospholipases, proteases and nucleases, resulting in the conversion of xanthine dehydrogenase to the free radical generating xanthine oxidase and the formation of free fatty acids, including arachidonic acid, as a consequence of membrane lipolysis. Arachidonic acid can in turn act as a substrate for cyclooxygenases and lipoxygenases in another series of free radical producing reactions. Free radicals, in particular the highly reactive hydroxyl radical, can initiate a chain reaction of lipid peroxidation, with extensive destruction of plasma and mitochondrial membranes with a failure of cellular homeostasis and metabolism. Administration of thrombolytic enzymes, including tissue plasminogen activator, to restore cerebral blood flow in stroke victims may actually precipitate the phenomenon known as "reperfusion injury", thought to be a consequence of oxygen free radical generation. Currently available evidence strongly implicates ROS in the chain of events leading to ischemia/reperfusion injury and suggests promising new avenues for therapeutic intervention in this condition. by awards from the U.S.P.H.S. and the American Heart Association. I am grateful to Dr M. H. O'Regan for his helpful comments on the manuscript. Acknowledgements---Supported
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FgEE RADICALS AND BRA~N INJURY BOlSV~T, D. P. (1992) In vivo generation of hydroxyl radicals during glutamate exposure. In: The Role of Neurotrammitters in Brain Injury, pp. 361-366. Eds. M. GLOBus and W. D. DIETRICH. Plenum: New York. BOLLLR. (1991) Oxygen-derived free radicals and myocardial reperfusion injury: an overview. Cardiovasc. Drugs Ther. 5, 249-268. Botany, S. C. and I.~, D. K. (1993) Oxidative stress induced by glutamate receptor agonists. Brain Res. 610, 229-233. BR^DAM^WrE, S., Mown, E., PARACCm~I, L., LAZZA~, E. and PtCCIN~I, F. (1992) Protective activity of the spin trap tert-butyl~,-phenyl nitrone (PBN) in reperfused rat heart. J. molec, cell. Cardiol. 24, 375-386. BRADAMANTE, S., JOTTLA., PARACCHIN1,L. and MONT1,E. (1993) The hydrophilic spin trap, 5,5-dimethyl-l-pyrroline-l-oxide, does not protect the rat heart from reperfusion injury. Eur. J. Pharmac. 234, 113-116. BuIssON,A., PLOTKIN~E,M. and BOULU,R. G. (1992) The neuroprotectire effect of a nitric oxide inhibitor in a rat model of focal cerebral ischemia. Br. J. Pharmac. 106, 766-767. BUISSON,A., MARGAILL,I., CALLEBERT,J., PLOTKINE,M. and BOULU, R. G. (1993) Mechanisms involved in the neuroprotective activity of a nitric oxide synthase inhibitor during focal cerebral ischemia. J. Neurochem. 61, 690-696. CAO,W., CARNEY,J. M., DUCHON,A., FLOYD,R. A. and ~ O N , M. (1988) Oxygen free radical involvement in ischemia and reperfusion injury to brain. Neurasci. Lett. 88, 233-238. CARKe~',J. M. and FLOYD,R. A. (1991) Protection against oxidative damage to CNS by a-phenyl-tert-butylnitrone (PBN) and other spin-trapping agentw--a novel series of nonlipid free radical scavengers. J. molec. Neurosci. 3, 47-57. CARNEY,J. M., STARKE-PO/ED,P. E., OLIVER,C. N., LANDUM,R. W., CHL~O, M. S., WU, J. F. and FLOYD, R. A. (1991) Reversal of age-related increases in brain protein oxidation, decrease in enzyme activity and loss in temporal and spatial memory by chronic administration of the spin-trapping compound N-tert-butyl-aphenylnitrone. Proc. ham. Acad. Sci. U.S.A. 88, 3633-3636. CRENG, H. Y., Ltu, T., FEUEgST~IN,G. and BAROI,~, F. C. (1993) Distribution of spin trapping compounds in rat blood and brain-in vivo microdialysis determination. Free Rad. Biol. Med. 14, 243-250. CLOUGn-HELFMAN, C. and PHmLIS, J. W. (1991) The free radical trapping agent N-tert-butyl-a-phenylnitrone (PBN) attenuates cerebral ischemic injury in gerbils. Free Rad. Res. Commun. 15, 177-186. COVA,D., DEANGELIS,L., MONTLE. and Pxccns'r~LF. (1992) Subccllular distribution of two spin trapping agents in rat he.art--possible explanation for their different protective effects against doxorubicin-induced cardiotoxicity. Free Rad. Res. Commun. 15, 353-36O. DAWSON,V. L., DAWSON,T. M., LONDON,E. D., BREDT,D. S. and SNYDER,S. H. (1991) Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. nam. Acad. Sci. U.S.A. 88, 6368-6371. DEMOVOULOS, H. D., FLAMM, E. S., PIETROVeGRO, D. D. and SELIGMAN, M. L. (1980) The free radical pathology and the microcirculation in the major central nervous system disorders. Acta physiol, scand. 492 (Suppl.), 91-119. DEVASAGAYAM,T. P. A. (1989) Decreased peroxidativc potential in rat brain microsomal fractions during ageing. Neurasci. Lett. 103, 92-96. DOWNEY,J. M. (1988) Ischmia-reperfusion injury: Current research status. Free Rad. Res. Commun. 5, 185-208. DVKENS,J. A., STERN, A. and Tg~KER, E. (1987) Mechanism of kainatc toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury. J. Neurochem. 49, 1222-1228. ERNSTER,L. (I 988) Biochemistry of reoxygenation injury. Crit. Care Med. 16, 947-953. FLAMM,E. S., DEMOPOULOS,H. B,, SELmMAN,M. L., POSEn,R. G. and RANOSHO~,J. (1978) Free radicals in cerebral ischemia. Stroke 9, 445-447. FLOYD,R. A. (1990) Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 4, 2587-2597. FLOYD, R. A., Z~L~KA, M. M. and HARMON,H. J. (1984) Possible involvement of iron and oxygen free radicals in aspects of aging in brain. In: Free Radicals in Molecular Biology, Aging and Disease, pp. 143-161. Eds. D. ARMSTRONG,R. S. SOt-IAL,R. G. CUTt~g and T. F. SLATEg. Raven: New York.
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FULLER,J. H., SmPLEY,M. J., ROSE,G., JAn~rr, R. J. and KEEN,H. (1983) Mortality from coronary heart disease and stroke in relation to degree of glycaemia: the Whitehall study. Br. Med, J. 287, 867-870. GLOBU$, M. Y.-T., BUSTO, R., D~rRIcH, W. D., MARnNEZ, E., VALDES,1. and G r N s ~ o , M. D. 0988) Effect of ischemia on the in vivo release of striatal dopamine, glutamate and y-aminobutyric acid studied by intracerebral microdialysis. J. Neurochem. 51, 1455-1464. GRAMMAS, P,, Lxu, G.-J., WOOD, K. and FLOYD, R. A. (1993) Anoxia/reoxygenation induces hydroxyl free radical formation in brain microvessels. Free Rad. Biol. Med. 14, 553-557. GR^NG~Z, D. N., HOLLWAgTH, M. E. and PARKS, D. A. (1986) Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta physiol, scand. 126 (Suppl. 548), 47-63. GRoG~m~, B., SeHug~z, L., GwmN, B. and Ammos, K. E. (1989) Delayed hypoperfusion after incomplete forebrain ischemia in the rat. The role of polymorphonuclear leukocytes. J. cereb. Blood Flow Metab. 9, 500-505. HAGBERG,H., LEHMANN,A., SANDBERG,M., NYSTROM,B., JACOBSON, I. and HAMBERGER,A. (1985) Ischemia-induced shifts of inhibitory and excitatory amino acids from intra- to extracellular compartments. J. eereb. Blood Flow Metab. 5, 413--419. HALLENBECK,J. M., DUTKA,A. J., TANISHIMA,T., KOCHANEK,P. M., KUMAROO, K. K., THOMPSON,C. B., OBKENOVlTCH,T. P. and CONGAS, T. J. (1986) Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during early postischemic period. Stroke 17, 246-253. HALUWELL, B. (1992) Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609-1623. H^LLIW~LL,B. and GUTTERIDGE,J. M. C. (1985) Oxygen radicals and the nervous system. Trends Neurosci. 8, 22-26. HARMAN,D. (1981) The aging process. Proc. hath. Acad. Sci. U.S.A. 78, 7124-7128. HILLERED, L., HALLSTROM,A., SEGERSVARD,S., PERSSON, L. and UNGERSTEDT,U. (1989) Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. 3'. cereb. Blood Flow Metab. 9, 607-616. HoY~z, S. and KRmR, C. (1986) lschemia and the ageing brain. Studies on glucose and energy metabolism in rat cerebral cortex. Neurobiol. Aging 7, 23-29. HUn~ZT,H. B., FEINLEm,M., MCNAMARA,P. M. and CAS~LLI,W. P. (1983) Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham heart study. Circulation 67, 968-977. IKEDA,Y. and LONG,D. M. (1990) The molecular basis of brain injury and brain edema: the role of oxygen free radicals. Neurosurgery 27, 1-11. K1NUTA,Y., KIKUCHI,H., ISHIKAWA,M., K1MURA,M. and ITOKAWA, Y. (1989) Lipid peroxidation in focal cerebral ischcmia. J. Neurosurg. 71, 421-429. KONTOS,H. A. (1985) Oxygen radicals in cerebral vascular injury. Circ. Res. 57, 508-515. KO~TOS,H. A. (1989) Oxygen radicals in CNS damage. Chem. biol. Interact. 72, 229-255. KUEHL, F. A. and EKGAN,R. A. (1980) Prostaglandins, arachidonic acid and inflammation. Science 210, 978-984. KUHN, J. E., STEIMLE,C. N., ZELENOCK,G. B. and D'AL~cY, L. G. (1986) Ibuprofen improves survival and neurologic outcome after resuscitation from cardiac arrest. Resuscitation 14, 199-212. LANGE,D. G., KIRSCH,J. R., HELFAER,M. A. and TRAYSTMAN,R. J. (1990) A continuous in vivo model of ischemia/reperfusion induced free radical production in the CSF of pig using spintrapping agents and EPR techniques. Free Rad. Biol. Med. 9 (Suppl. 1), 97. LAWRENCE, A. J. and JARROTT, B. (1993) Nitric oxide increases interstitial excitatory amino acid release in the rat dorsomedial medulla oblongata. Neurosci. Lett. 151, 126-129. LEmOVtTZ, B. E. and SIEGEL, B. V. (1980) Aspects of free radical reactions in biological systems: Aging. J. Gerom. 35, 45-56. LIN, Y. and PHILLIS,J. W. (1992) Deoxycoformycin and oxypurinol: Protection against focal ischemic brain injury in the rat. Bruin Res. 571, 272-280. LIPTON, S. A., CHOI, Y.-B., PAN, Z.-H., LEI, S. Z., CHEN,Z.-H. V., SUCHER,N. J., LOSCALZO,J., StNGEL,D. J. and STAMLER,J. S. (1993)
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Pergamon
MINI REVIEW A " R A D I C A L " V I E W OF C E R E B R A L I S C H E M I C I N J U R Y JOrlN W. PHILLIS Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, Michigan 40201, U.S.A.
CONTENTS 1. Introduction 2. Reactive oxygen species (ROS) and lipid peroxidation 2.1. Lipid peroxidation 2.2. Enzymes protecting against ROS 2.3. Sources of ROS 2.3.1. Ubisemiquinone radical 2.3.2. Purine catabolism 2.3.3. Arachidonic acid metabolism 2.3.4. Neutrophils 2.3.5. Glutamate receptor agonists 2.3.6. Nitric oxide 3. ROS and cerebral ischemia/reperfusion injury 3.1. Detection of free radicals in brain 3.2. Pharmacological evidence of free radical involvement 3.2.1. Cerebroprotection with PBN and its effect on ROS formation 3.2.2. Oxypurinol, a xanthine oxidase inhibitor 4. Aging, cerebral ischemia and ROS 5. Conclusions Acknowledgements References
1. INTRODUCTION The free radical hypothesis of cerebral ischemia/ reperfusion injury was formulated to provide a conceptual basis for the evaluation of the sequence of events leading to delayed neuronal death in brains subjected to a period of iscbemia, such as that occurring during a cardiac arrest or stroke, followed by reperfusion. The free radical hypothesis of tissue iscbemia/reperfusion injury, as exemplified by the work of McCord and his coworkers (Parks et al., 1982), was first applied to cerebral ischemic injury over a decade ago (Flamm et al., 1978; Demopoulos et al., 1980). The brain appears to be particularly vulnerable to oxidative damage. It contains relatively high concentrations of readily peroxidizable fatty acids. Various regions of the brain are highly enriched in iron, which possesses an ability to catalyze the production of damaging oxygen free radical species. Furthermore, the brain is relatively poorly endowed with protective antioxidant enzymes or antioxidant compounds. Free radicals are believed to initiate damage to many cellular elements such as lipids, proteins and nucleic acids. The damage to membrane lipids results not only in changes in fluidity and permeability of the cell membranes, but also in
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the function of phospholipid-dependent proteins. The consequences of these alterations in cellular function are potentially lethal. The intent of this work is to present a brief review of the evidence supporting the free radical hypothesis of cerebral ischemia/ reperfusion injury.
2. REACTIVE OXYGEN SPECIES (ROS) AND LIPID PEROXIDATION Oxygen radicals, also known as reactive oxygen species (ROS), arise in sequential fashion from molecular oxygen by successive single-electron reduction reactions (Halliwell and Gutteridge, 1985) ROS are themselves chemically reactive because in order to regain thermodynamic stability they attempt to remove electrons or hydrogen atoms from neighboring molecules, thus radicalizing these molecules in turn, creating a so-called "cascade" effect. The initial oxygen radical in the sequence is the superoxide amino radical (O~-), followed by the perhydroxyl radical (HO2), hydrogen peroxide (H202) and the extremely reactive hydroxyl radical (OH), which is capable of reacting with almost every type of molecule found in living cells, including sugars, proteins, amino acids,
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DNA bases and organic acids including polyunsaturated fatty acids (Halliwell and Gutteridge, 1985; Hailiwell, 1992). A role of ROS in ischemia/reperfusion injury was suggested by the observed reduction in peripheral tissue damage when animals were treated with scavengers of these agents (McCord, 1985; Granger et al., 1986) and they have since been implicated in the pathogenesis of ischemic injury to brain (Flamm et al., 1978; Demopoulos et al., 1980; Kontos, 1985). Indeed, recent advances in understanding the fundamental mechanisms of post-ischemic injury have suggested that most tissue injury is associated with the admission of oxygen to the tissue at the time of reperfusion (Downe3 1988; Ernster, 1988) and the generation of ROS (McCord, 1986).
2. I. LIPID PEROXIDATION An important consequence of ROS formation is lipid peroxidation by O H or other radicals; a reaction in which the double bonds in the fatty acid side chains are rearranged. Their large numbers of unsaturated fatty acids make cell membranes particularly susceptible to a chain reaction of lipid peroxidation, with resultant changes in fluidity and permeability, which can profoundly affect the functioning of the membrane. Lipid peroxidation can also inhibit the function of membrane bound receptors and enzymes. Lipid peroxides are chemically unstable and can degrade into other chemically reactive species, such as aldehydes, including malondialdehyde. Lipid peroxidation is an important phenomenon primarily because it is a chain reaction, in which an initiating free radical can precipitate the destruction of numerous adjacent molecules. Peroxidation of an unsaturated fatty acid results from the removal of a methylene hydrogen atom, whereupon the resultant fatty acid radical converts to a conjugated diene. This moiety is itself peroxidized by the addition of O: to yield a peroxy radical which can initiate a new cycle by repeating the hydrogen atom sequestration from another polyunsaturated fatty acid molecule, yielding a lipid hydroperoxide and another lipid alkyl radical. In the presence of a transition metal, the lipid hydroperoxide can be converted into a lipid alkoxyl radical, which can initiate further lipid peroxidation, with further expansion of the chain reaction. The initiating hydroxyl radical arises from the decomposition of hydrogen peroxide or lipid hydroperoxides by metal ions (Fenton reaction), of which iron is undoubtedly the most important (Aust and White, 1985). Termination of the chain reaction occurs when the propagating radicals are neutralized by organic scavenger compounds (Vitamin E, ascorbic acid) or by enzymic reduction of the lipid hydroperoxides and hydrogen peroxide by glutathione peroxidase, which prevents their degradation to hydroxyl, peroxy or alkoxy radicals. Tissue lactacidosis can dramatically enhance the formation of free radicals and lipid peroxidation in brain tissue by its mediation of an increased dissociation of catalytic iron from proteins of the transferrin type (Rehncrona et al., 1989).
2.2. ENZYMESPROTECTINGAGAINSTROS
Specific enzymes have evolved to deal with the ROS. Superoxide dismutase catalyzes the conversion of the superoxide anion radical into hydrogen peroxide. Because it is non-polar, unlike O2, hydrogen peroxide readily diffuses across cell membranes. In the presence of ferrous iron, hydrogen peroxide is converted into the highly reactive O H radical (Fenton reaction). Because iron is bound in the ferric form in vivo, a complementary reaction is required to generate ferrous iron and this is mediated by the superoxide anion. The net iron-catalyzed, superoxide dependent, conversion of H20: to O H is known as the Haber-Weiss reaction. Hydrogen peroxide is removed by two enzymes. Glutathione peroxidase catalyzes the reduction of H202 (or fatty hydroperoxides) to water (or hydroxy fatty acids) whilst converting reduced glutathione (GSH) into oxidized glutathione (GSSG). Catalase decomposes hydrogen peroxide to oxygen and water. The central nervous system is relatively poorly endowed with superoxide dismutase, glutathione peroxidase and catalase, which may account, in part, for its susceptibility to ischemic injury. 2.3. SOURCESOF ROS Reactive oxygen radicals may be generated by a number of pathways (Kontos, 1989; Ikeda and Long, 1990; Schmidley, 1990) including those described in the following sections. 2.3. i. Ubisemiquinone radical Oxidation of ubisemiquinone radical, the diffusable electron transfer agent in the mitochondriai respiratory chain, yields 02. This pathway is operative during regular metabolism yielding small amounts of ROS which can be eliminated by the cell's defences, but its enhancement during reoxygenation is potentiated by the reducing conditions present during ischemia. 2.3.2. Purine catabolism During ischemia ATP is utilized, but cannot be resyntbesized and as a result of the cessation of mitochondrial oxidative phosphorylation its metabolites adenosine, inosine, hypoxanthine and xanthine accumulate (Fig. 1). In the normoxic brain, hypoxanthine is metabolized by xanthine dehydrogenase (XDH) to xanthine and ultimately to uric acid. With the onset of ischemia, the rise in intracellular calcium results in a rapid, proteolytic conversion of xanthine dehydrogenase to xanthine oxidase (XO), which uses molecular oxygen instead of the nueleotide radical of NAD as its electron acceptor and catalyzes the production of O f during reperfusion. XDH/XO is present in the brains of several species, including man (Wajner and Harkness, 1989), predominantly in the X D H form. Rat brain XO is concentrated in the capillary endotbelium (Betz, 1985) and during ischvmia there is a substantial conversion of XDH to XO (Kinuta et al., 1989). Bovine brain endothelial
FREE RADICALS AND BRAIN INJURY
and acid releatsc CExcitotoxic amino acids")
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ATP depl~ion Adenosine
1
Failme of plasma membrane Na+ and Ca:z+ pumps I
• Ca2+ in cytoplasm
i
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hospholipase activation Free fatty acids
Cyclooxygenase Lipoxygenase
l.O.. Dehyed hypoperfusion
x. i.
+3
Uric acid + 02-"
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Secondary ischemia Cellular membrane desm~tion ~'~
FzG. I. Proposed pathways for events linking cerebral isehemia to neuronal death.
cells are able to generate superoxide anion formation by a xanthine oxidase-dependent mechanism (Terada et al., 1991) and anoxia/reoxygenation-induced hydroxyl radical formation occurs in isolated rat brain microvessels (Grammas et al., 1993).
cyclins, thromboxanes and leukotrienes. Cyclooxygenase catalyzes the addition of two molecules of oxygen to an unsaturated fatty acid to produce prostaglandin G, which is rapidly peroxidized to prostaglandin H with the concomitant production of O~-' (Kuehl and Ekgan, 1980). Hydroxyl radicals can be generated by the lipoxygenase pathway which produces ieukotrienes.
2.3.3. Arachidonic acid metabolism Arachidonic acid, generated as a result of membrane lipolysis occurring during ischemia, can serve as a precursor for ROS formation. Calcium entry into isehemic neurons activates phospholipases including phospholipase A2, which cleaves a fatty acyl chain from the b position of phospholipids to produce arachidonic acid, the levels of which rise rapidly during ischemia (Yasuda et al., 1985; Abe et aL, 1987). Arachidonic acid may be metabolized by either cyclooxygenase or lipoxygenase (Fig. !), producing a variety of vasoactive substances including prostaglandins, prosta-
2.3.4. Neutrophils Neutrophils are another potential source of ROS. Phagocytosis stimulates O~- synthesis by NADPH oxidase during the respiratory burst. Neutrophils and perhaps microglia, responding to tissue injury may inappropriately destroy adjacent tissues, resulting in infarct extension. Hailenbeck et ai. (1986) have observed neutrophil accumulation in the brain regions with low blood flow during the early post-ischemic
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period, where they may contribute to cerebral delayed hypoperfusion. However, treatment with antineutrophil serum at the time of reperfusion is not associated with improved post-ischemic blood flow in rats (Grogaard et al., 1989) or improved neurologic recovery in dogs (Schott et al., 1989). It is therefore unlikely that neutrophils contribute significantly to ROS formation during ischemia and in the immediate post-ischemic period. 2.3.5. G l u t a m a t e receptor agonists Oxidative stress is induced by glutamate receptor agonists. Cerebral ischemia is accompanied by massive increases in the extracellular concentration of the excitatory amino acids glutamate and aspartate (Benveniste et al., 1984; Hagberg et al., 1985; Globus et aL, 1988; Simpson et al., 1992). Glutamate, aspartate and their analogs in excessive concentration are neurotoxic and may cause neuronal death by elevating intracellular calcium levels. Bondy and Lee (1993) have recently demonstrated that agonists specific for each of the three major glutamate ionotropic receptor sites (NMDA, kainate, AMPA) enhanced the rate of ROS generation in cerebrocortical microsacs (synaptoneurosomal fraction), whilst an agonist for the metabotropic receptor (ACPD) was inactive. Evidence for a ROS generating action of kainate had previously emerged from a study which demonstrated that inhibition of the formation of XO from XDH or of the action of XO by allopurinol, or the addition of free radical scavengers, protected mouse cerebellar neurons in microwell culture from kainate-induced neurodegeneration (Dykens et al., 1987). These results appear to directly implicate ROS in the neurotoxic actions of glutamate receptor agonists. The interconnectedness of excitotoxic amino acid release and free radical generation has been emphasized by Pellegrini-Giampietro et al. (1990), who showed that inhibition of ROS formation reduced excitotoxic amino acid release from "ischemic" rat hippocampal slices and suggested that free radical formation and glutamate release are mutually related and cooperate in precipitating ischemic neuronal death. Studies on ischemic brain edema (Oh and Betz, 1991) have also implicated a causal or cooperative relationship between the excitatory amino acids and ROS. 2.3.6. Nitric oxide Nitric oxide, the recently discovered endotheliallyderived relaxing factor and putative neurotransmitter, is itself a free radical with both neuroprotective and neurodegenerative effects. NO - mediated neurotoxicity is engendered, at least in part, by its reaction with superoxide anion, apparently leading to the formation of peroxynitrite ( O N O O - ) (Lipton et al., 1993). In contrast, its neuroprotective effects may result from a downregulation of N M D A receptor activity by a reaction with thiol groups of the receptors redox modulating site (Lipton et al., 1993). The enzyme responsible for nitric oxide formation, NO synthase, catalyzes its formation from L-arginine by a Ca 2÷/calmodulin dependent mechanism. Thus an increase in intracellular Ca 2÷ can activate the enzyme. As glutamate neurotoxicity mediated by
N M D A receptors involves Ca -~~ entry into cells, the possible participation of NO in neuronal injury induced by ischemia has been hypothesized. Dawson et al. (1991) have demonstrated that NO synthase inhibitors prevent neurotoxicity elicited by N M D A and related amino acids in rat primary cortical cultures. Protection against cerebral ischemic injury in vivo has also been reported (Nowicki et al., 1991; Buisson et al., 1992). The nitric oxide donor Snitroso-N-acetylpenicillamine increase extracellular levels of glutamate and aspartate in the rat medulla oblongata (Lawrence and Jarrott, 1993). Ischemiaevoked rat striatal glutamate release was significantly reduced by an NO synthase inhibitor (L-NAME) (Buisson et al., 1993) indicating that NO may contribute to the excitotoxic process by facilitating ischemiaevoked glutamate overflow. However, in another study L-NAME failed to reduce ischemia-evoked glutamate release in the rat hippocampus (Zhang et al., 1993). In unpublished experiments conducted in my laboratory, L-NAME significantly depressed the initial rate of rise in extracellutar glutamate and aspartate levels during a 20 min period of ischemia but did not reduce the total release (release during ischemia and reperfusion) of these amino acids.
3. ROS AND CEREBRAL ISCHEMIA/REPERFUSION INJURY 3.1 DETECTIONOF FREE RADICALSIN BRAIN The direct detection of unstable ROS in cerebral tissue has been difficult to achieve as most radicals are short lived and produced in trace amounts. Most studies on free radical involvement have utilized free radical scavengers or inhibitors of synthesizing enzymes to infer a role of ROS in tissue damage. Superoxide anion has been detected indirectly by its reaction with nitro blue tetrazolium to yield an insoluble blue formazan precipitate which can be quantified spectrophotometrically (Nelson et al., 1992). Hydroxyl radical formation in vivo has been assayed by HPLC in terms of 2,5-dihydroxybenzoate, assumed to result from the hydroxylation of infused salicylate (Cao et al., 1988; Boisvert, 1992). Direct evidence for ROS formation during ischemia/reperfusion has been obtained with the application of spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. Short lived free radicals are "trapped" with nitrone or nitroso spin-trapping agents upon which the resulting adduct gives rise to a stable nitroxide free radical, which can be measured with high specificity and sensitivity by EPR. Using these techniques, free radicals were detected by EPR in CSF from the brains of pigs subjected to global cerebral ischemia/reperfusion with ~-phenyl-t-butylnitrone (PBN) as a spin probe (Lange et al., 1990). Zini et al. (1992) were unable to detect any adducts in PBN-containing striata! microdialysates from rats subjected to global ischemia followed by reperfusion, but did observe a free radical adduct when pyridyl-N-oxide-t-butylnitrone (POBN) was used as the trapping agent. The spin adduct occurred during ischemia and early reperfusion, but not under basal conditions.
FREE RADICALSAND BRAININJURY
Hydroxyl radical production /n vivo, following reperfusion of ischemic rat cerebral cortex has been assayed by electron paramagnetic resonance with POBN administered both topically and systemically (Sen and Phillis, 1993; Phillis and Sen, 1993). Using the cortical cup technique, it was possible to collect serial samples of POBN-containing aCSF prior to ischemia, during ischemia and following reperfusion. Experiments were conducted both with and without the chelating agent DETAPAC in the aCSF with identical results. Pre-ischemic samples did not elicit an EPR signal. During and following a 30 min period of ischemia (four vessel occlusion), OH. radical adduct signals (characterized by six line EPR spectra) were detected in the superfusate samples. The signals reached their maximal amplitude during the initial 20 min of reperfusion and were no longer detectable after 90 min of reperfusion. 3.2. PHARMACOLOGICALEVIDENCEOF FREE RADICAL INVOLVEMENT
There have been numerous reports describing cerebroprotective actions of a variety of oxygen free radical scavengers including superoxide dismutase (especially the more stable polyethylene glycol-conjugated SOD), catalase, dimethylsulfoxide, dimethylthiourea, mannitol, Vitamin E, ascorbic acid, iazaroids such as U74006F (tirilazad mesylate) and of inhibitors of xanthine oxidase such as allopurinol or oxypurinol (see Ikeda and Long, 1990; Schmidley, 1990; Halliwell, 1992). Deferoxamine, a chelator of ferric iron, when administered may also reduce cerebral ischemic injury (Halliwell, 1992). Although the results with these interventions are not always consistent and the pharmacological actions of the agents may not be completely understood, the findings are generally supportive of an involvement of ROS in ischemia/ reperfusion injury. In spite of its potential significance as a free radical generating pathway, there is relatively little information in the literature on cerebroprotective effects of inhibitors of arachidonic acid metabolism to prostaglandins, prostacyclins, thromboxanes and leukotrienes. Acetyl salicylic acid has reportedly beneficial effects in human stroke patients (Schror, 1986) and ibuprofen improves survival and neurologic outcome after resuscitation of dogs from cardiac arrest (Kuhn et al., 1986). All of the evidence provided by these studies, however, is indirect and therefore inconclusive. More specific evidence for a role of free radicals has come from experiments with the spin-trap agent ct-phenyl-t-butylnitrone (PBN) and the xanthine oxidase inhibitor, oxypurinol. 3.2.1. Cerebroprotection with P B N and its effect on R O S formation
Protection against ischemia/reperfusion injury with free radical trapping agents such as PBN and 5,5dimethyI-L-pyroline-L-oxidase (DMPO) was initially reported in cardiac studies (Tosaki et al., 1990; Bolli, 1991; Bradamante et al., 1992). The rationale for using these agents was that they would react with free radicals to interrupt the cascade of reactions which ultimately results in membrane lipid peroxidation.
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PBN administered either prior to, or 30 min after a 5 min period of bilateral carotid occlusion in gerbils, prevented the increase in locomotor activity observed in saline injected control ischemic animals and significantly reduced the damage to hippocampal CAI pyramidal cells observed 5 days post-ischemia. These effects were no longer apparent when PBN was administered 2 hr post-ischemia (Phillis and CloughHelfman, 1990a; Clough-Helfman and Phillis, 1991). Yue et al. (1992) reported that PBN attenuated both forebrain edema and hippocampal CA 1 neuronal loss in ischemic gerbils and that it protected rat cerebellar neurons in primary culture from glutamate-induced neurotoxicity. PBN administration diminishes the increase in oxidized protein and the loss of glutamine synthetase activity that accompanies both ischemia/reperfusion injury and aging in the gerbil brain (Carney et al., 1991). Chronic treatment with PBN also results in a recovery of the loss in temporal and spatial memories in the aged gerbil brain, implying that there is an age related increase in the vulnerability of brain tissues to oxidation which can be reversed by free radical trapping compounds (Carney and Floyd, 1991). The neuroprotective effects of PBN in temperature stabilized rats subjected to middle cerebral artery (MCA) occlusion have recently been evaluated (Phillis and Cao, 1994). PBN, administered i.p. at 100 mg/kg in repeated doses significantly attenuated both cortical infarct volume and cerebral edema measured at 48 hr, even when the initial drug administration was delayed until 12 hr post-MCA occlusion. These reductions in histopathological ratings were accompanied by significant reductions in neurological deficit scores. Interestingly, the neurological deficit scores obtained at 48 hr were lower than those at 24 hr, suggesting that an effective repair process had already been initiated. These results appear to provide dramatic evidence for a continuing role of ROS in the extension of neuronal losses into the penumbral region of cerebral infarcts. Confirmation that the cerebroprotective actions of PBN could be a result of its ability to prevent hydroxyl radical formation was obtained in EPR experiments in which systemic administration of PBN greatly attenuated the amplitude of the radical adduct signal and a combination of systemic and topical (100 mM) PBN virtually abolished the signal (Sen and Phillis, 1993). PBN differs from POBN in being substantially more lipophilic and having a different intracellular distribution (Cheng et al., 1993), including access to the nuclear and mitochondrial fractions of cells (Cova et al., 1992). It is likely therefore that PBN achieves an adequate concentration in the mitochondria to interrupt the ROS cascade at its site of initiation. Otherwise H_,O2 or OH. formed intracellularly would diffuse across the plasma membrane to be trapped as an OH. radical adduct by POBN in the extracellular space. In this regard, it may be significant that DMPO, another hydrophilic spin-trap agent has proven to be less effective than PBN in myocardial ischemia/reperfusion injury (Bradamante et al., 1993). POBN and DMPO also failed to protect isolated rat atria against adriamycin-induced cardiotoxicity, whereas PBN was effective (Monti et al., 1991). The authors attributed the protective ability of
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PBN to its lipophilicity and ability to cross cell membranes. 3.2.2. Oxypurinol, a xanthine oxidase inhibitor Oxypurinol was tested for cerebroprotective activity in the gerbil and rat MCA occlusion models of cerebral ischemia. When administered either prior to or 30 min post-ischemia, oxypurinol (40 mg/kg) significantly reduced both the hypermotility and the extent of CA1 hippocampal pyramidal cell damage observed in comparison to that in control gerbils (Phillis and Clough-Helfman, 1990b). Its cerebroprotective actions were not secondary to reductions in body temperature as measured with intraperitoneally implanted telemetry probes. Oxypurinol was also protective when administered either prior to, or 1 hr post-ischemia, in the rat MCA occlusion model. It attenuated the degree of neurological deficit observed in control animals and significantly reduced both infarct volume and the extent of brain swelling (Phillis and Lin, 1991; Lin and Phillis, 1992). Protection against ischemic injury was not observed when oxypurinol administration was delayed for 5 hr post stroke. Body temperature measurements with telemetry probes confirmed that the cerebroprotective effects of oxypurinol were not a result of hypothermia. In a related study, the xanthine oxidase inhibitor, oxypurinol, was administered prior to the onset of ischemia to POBN treated animals. Oxypurinol greatly attenuated the appearance of OH- radical adduct in cerebral cortical superfusates during ischemia and reperfusion confirming that xanthine oxidase is likely to be involved in ROS formation (Phillis and Sen, 1993). It may be significant that xanthine oxidase is localized in capillary endothelial cells, as these will inevitably be an important site of free radical formation. The resultant destruction of the blood-brain barrier may be a major contributor to the edema formation which occurs in stroked brains.
4. AGING, CEREBRAL ISCHEMIA AND ROS The studies of Framingham and Whitehall have shown age to be one of the most important risk factors for brain infarction and its mortality (Hubert et al., 1983; Fuller et al., 1983). It is therefore important to take age-related changes into account when considering abnormalities with respect to brain ischemia due to disturbances in macro- or micro-circulations. Reports have been published on age-related changes in rat brain for peroxidative potential (Sawada and Carlson, 1987; Devasagayam, 1989)and the activities of superoxide dismutase, catalase and glutathione peroxidase (Victorica et al., 1984; Scarpa et al,, 1987; Barja de Quiroga et al., 1990; Semsei et al., 1991). Succinate concentrations in ischemic brain tissue of aged rats are elevated and may contribute to an acidosis (Hoyer and Krier, 1986). Increased levels of iron associated with aging have been reported in rat brain (Floyd et al., 1984). There is an
increase in the formation of free radicals with advancing age (Harman, 1981; Leibovitz and Siegel, 1980) which may account for the observation that mortality rates are higher in older gerbils subjected to ischemia (Floyd, 1990). The evidence therefore suggests that free radical generation during cerebral ischemia in aged rats may be significantly more pronounced than in younger animals and could account for the greater severity of ischemia/reperfusion injury in the aging brain.
5. CONCLUSIONS Cardiac arrest and stroke are two leading causes of death and when patients survive it is usually with some degree of neurological impairment. A massive release of the excitotoxic amino acids, glutamate and aspartate, occurs during ischemia, precipitating the entry of calcium into cells. Elevated intracellular calcium levels in turn activate calcium-dependent enzymes, including phospholipases, proteases and nucleases, resulting in the conversion of xanthine dehydrogenase to the free radical generating xanthine oxidase and the formation of free fatty acids, including arachidonic acid, as a consequence of membrane lipolysis. Arachidonic acid can in turn act as a substrate for cyclooxygenases and lipoxygenases in another series of free radical producing reactions. Free radicals, in particular the highly reactive hydroxyl radical, can initiate a chain reaction of lipid peroxidation, with extensive destruction of plasma and mitochondrial membranes with a failure of cellular homeostasis and metabolism. Administration of thrombolytic enzymes, including tissue plasminogen activator, to restore cerebral blood flow in stroke victims may actually precipitate the phenomenon known as "reperfusion injury", thought to be a consequence of oxygen free radical generation. Currently available evidence strongly implicates ROS in the chain of events leading to ischemia/reperfusion injury and suggests promising new avenues for therapeutic intervention in this condition. by awards from the U.S.P.H.S. and the American Heart Association. I am grateful to Dr M. H. O'Regan for his helpful comments on the manuscript. Acknowledgements---Supported
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