Pathophysiology and treatment of focal cerebral ischemia. Part II: Mechanisms of damage and treatment

Transcription

1 J Neurosurg 77: , 1992 Review Article Pathophysiology and treatment of focal cerebral ischemia Part II: Mechanisms of damage and treatment Bo K. SIESJ6, M.D. Laboratory for Experimental Brain Research, Experimental Research Center, Lurid University Hospital, Lund, Sweden ~" The mechanisms that give rise to ischemic brain damage have not been definitively determined, but considerable evidence exists that three major factors are involved: increases in the intercellular cytosolic calcium concentration (Ca++~), acidosis, and production of free radicals. A nonphysiological rise in Ca++~ due to a disturbed pump/leak relationship for calcium is believed to cause cell damage by overactivation of lipases and proteases and possibly also of endonucleases, and by alterations of protein phosphorylation, which secondarily affects protein synthesis and genome expression. The severity of this disturbance depends on the density of ischemia. In complete or near-complete ischemia of the cardiac arrest type, pump activity has ceased and the calcium leak is enhanced by the massive release of excitatory amino acids. As a result, multiple calcium channels are opened. This is probably the scenario in the focus of an ischemic lesion due to middle cerebral artery occlusion. Such ischemic tissues can be salvaged only by recirculation, and any brain damage incurred is delayed, suggesting that the calcium transient gives rise to sustained changes in membrane function and metabolism. If the ischemia is less dense, as in the penumbral zone of a focal ischemic lesion, pump failure may be moderate and the leak may be only slightly or intermittently enhanced. These differences in the pump/leak relationship for calcium explain why calcium and glutamate antagonists may lack effect on the cardiac arrest type of ischemia, while decreasing infarct size in focal isehemia. The adverse effects of acidosis may be exerted by several mechanisms. When the ischemia is sustained, acidosis may promote edema formation by inducing Na + and Cl- accumulation via coupled Na+/H + and C1-/ HCO3- exchange; however, it may also prevent recovery of mitochondrial metabolism and resumption of H + extrusion. If the ischemia is transient, pronounced intraischemic acidosis triggers delayed damage characterized by gross edema and seizures. Possibly, this is a result of free-radical formation. If the ischemia is moderate, as in the penumbral zone of a focal ischemic lesion, the effect of acidosis is controversial. In fact, enhanced glucolysis may then be beneficial. Although free radicals have long been assumed to be mediators of ischemic cell death, it is only recently that more substantial evidence of their participation has been produced. It now seems likely that one major target of free radicals is the microvasculature, and that free radicals and other mediators of inflammatory reactions (such as platelet-activating factor) aggravate the isehemic lesion by causing microvascular dysfunction and blood-brain barrier disruption. Solid experimental evidence exists that the infarct resulting from middle cerebral artery occlusion can be reduced by glutamate antagonists, by several calcium antagonists, and by some drugs acting on Ca ++ and Na influx. In addition, published reports hint that qualitatively similar results are obtained with drugs whose sole or main effect is to scavenge free radicals. Thus, there is substantial experimental evidence that the ischemic lesions due to middle cerebral artery occlusion can be ameliorated by drugs, sometimes dramatically; however, the therapeutic window seems small, maximally 3 to 6 hours. This suggests that if these therapeutic principles are to be successfully applied to the clinical situation, patient management must change. KEY WORDS 9 cerebral ischemia 9 penumbra 9 reperfnsion 9 brain injury 9 acidosis 9 free radical T HIS is the second part of a review devoted to the pathophysiology and treatment of focal cerebral ischemia. Part I t58 discussed the pathophysiol- J. Neurosurg. / Volume 77/September, 1992 ogy of focal ischemic lesions and the major metabolic events triggered by ischemia. In the present article, the discussion focuses on cellular and molecular mecha- 337

2 B. K. Siesj6 Definitions of Abbreviations AA = arachidonic acid ADP = adenosine diphosphate AMPA = amino-3-hydroxy-5-methyl-4-isoazole propionic acid AOCC = agonist-operated calcium channel ATP = adenosine triphosphate Ca++i = intracellular Ca ++ CPP = 4-(3-phosphoro-propyl)-2-piperazine- 2-carboxylic acid DAG = diacylglyceride DHP = dihydropyridine EAA = excitatory amino acid 5-HETE = 5-hydroxy-eicosatetraenoic acid 5-HPETE = 5-hydroperoxy-eicosatetraenoic acid IP3 = inositoltriphosphate MK-801 = dizocilpine mrna = messenger ribonucleic acid NBQX = 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline NMDA = N-methyl-D-aspartate PAF = platelet-activating factor PCP = phencyclidine PCr = phosphocreatine PIP2 = phesphatidylinositol biphosphate pk = ionization constant PKC = protein kinase C PLA2 = phospholipase A2 PLC = phospholipase C VSCC = voltage-sensitive calcium channel nisms of ischemic damage, and treatment of focal ischemia. Cellular and Molecular Mechanisms of Ischemic Brain Damage Recently, knowledge about the mechanisms of ischemic brain damage has increased considerably. Three major molecular events, or cascades of events, are at present the focus of interest. These are the results triggered by calcium overload, by excessive acidosis, and by enhanced production of free radicals. Although I will discuss each in turn, I wish to emphasize that none works in isolation. For example, most if not all effects elicited by a rise in intraeellular Ca 247 (Ca++~) result also from energy failure per se. Furthermore, since calcium triggers several reactions leading to the production of free radicals, the question arises whether the damage is calcium- or free radical-related. Nonetheless, it appears profitable to discuss these three mechanisms separately. This is because brain damage also occurs under conditions in which acidosis is not present (hypoglycemia) or in which free-radical damage may be only a minor component (such as in hypoglycemia and status epilepticus). Oversimplification is a frequently encountered trap; however, since we need a conceptual framework for the discussion, I will make two assumptions. First, selective neuronal vulnerability -- a nonvascular brain lesion observed in hypoglycemic coma, in epileptic seizures, and following brief periods of ischemia -- is calciumrelated. Second, pan-necrosis (infarction) is related to acidosis and free-radical production, and the vascular lesions in stroke are the result of inflammatory reactions which involve calcium, free radicals, and lipid mediators. Calcium-Related Damage. Proteins, Lipids, and Gene Expression Calcium fulfills important functions in cell-to-cell communication and plays the role of a second messenger in stimulus-response-metabolism coupling.l~'~2'85' lz~,~27 Calcium also elicits certain physiological responses, such as long-term potentiation, which probably represent the physiological counterpart of memory formation and storage 1~176 and it modulates neurite extension and the formation of synapses during development. ~~ It is generally believed, though, that an excessive rise in Ca++i triggers untoward reactions which can lead to cell death. 36'38'39'132'147'157A55 A decade ago, it was suggested that neuronal necrosis in hypoxia/ischemia, hypoglycemia, and status epilepticus is due to an uncontrolled rise in Ca++~, and that the phenomenon of selective neuronal necrosis can be explained by a high density of voltage-sensitive calcium channels (VSCC's) in the dendritic domains of vulnerable neurons. 154 The hypothesis of calcium-related neuronal necrosis is still viable; however, it now seems more likely that a pathogenetically important part of the calcium influx occurs via agonist-operated calcium channels (AOCC's), notably those gated by glutamate and related excitatory amino acids (EAA's). 38-4~176 The hypothesis of calcium-related (excitotoxic) cell death has recently been extended to encompass Alzheimer's disease and other neurodegenerative disorders, and calcium may even be involved in so-called "programmed cell death. "1~176 The molecular mechanisms whereby a nonphysiological rise in Ca++j may damage cells have been discussed extensively. 38"1~ An overview of the cascades elicited by adenosine triphosphate (ATP) failure, depolarization, transmitter release, and rise in Ca++j is given in Fig. 1. An excessive rise in Ca+ represents a nonphysiological stimulus which causes overaetivalion of lipases, proteases, and endonucleases and which, via activation of protein kinases, may alter the functions of receptors, membrane channels, and ion translocases by phosphorylation. One of the key events leading to acute cell damage may be activation of proteases which break down components of the cytoskeleton and sever the anchorage between the plasma membrane and the cytoskeleton. ~32"~43'a65 No doubt, protease inhibitors could become important therapeutic tools in the future; 94 but at present there are no inhibitors available that can rapidly penetrate the blood-brain barrier, and no data yet exist to suggest that they are useful in stroke. Another cascade, probably of equal pathogenetic importance, is that triggered by phospholipases. Since the adverse effects of such activation are at present more Neurosurg. / Volume 77/September, 1992

3 Treatment of focal ischemia FIG. 1. Diagram illustrating the primary effects of depolarization/receptor activation and the secondary effects of raised intracellular calcium (Ca++i). The scheme suggests that the major adverse effects of a nonphysiological rise in Ca++i encompass proteolysis, protein phosphorylation, and lipolysis, the latter involving formation of diacylglycerides (DAG's) and activation of protein kinase C (PKC). FFA = free fatty acid; LPL = lysophospholipid. (Reproduced from Siesj6 B: The role of calcium in cell death, in Price D, Aguayo A, Thoenen H (eds): Neurodegenerative Disorders: Mechanisms and Prospects for Therapy. London: John Wiley & Sons, 1991, pp with permission.) FIG. 2. Diagram illustrating principal pathways leading to production of lipid mediators following stimulation of phospholipases A2 or C. For explanation see text and definitions of abbreviations table. PG = prostaglandin; TX = thromboxane; LT = leukotriene. amenable to treatment, I will discuss them in some more detail. As mentioned in Part I of this review, I58 energy failure, influx/release of calcium, and receptor activation lead to the hydrolysis of phospholipids, with phospholipase A2 (PLA2) giving rise to lysophospholipids and free fatty acids, including arachidonic acid (AA), and with phospholipase C (PLC), when acting on phosphatidylinositol biphosphate (PIPz), catalyzing the formation of diacylglycerides (DAG's), another source of AA, and inositoltriphosphate (IP3). As discussed in early review articles, 9,154,195 lysophospholipids and free fatty acids, particularly the polyunsaturated ones, are assumed to have adverse effects of their own by acting as membrane detergents and ionophores. Results reported at that time also shed further light on the pathogenetic role of AA, and suggested that it acted as a precursor of free radicals. 32'9~ The role of AA in causing vasomotor problems and leakage of molecules across the blood-brain barrier has been confirmed, ls2 but the mediation of free radicals has been questioned. 181 It is becoming increasingly clear that many of the effects attributed to AA are in fact exerted by its oxygenated metabolites. These metabolites, arising as a result of cyclo-oxygenase and lipoxygenase activity, are now known to act as second messengers, modulating synaptic activity by local or by transsynaptic effectsj For example, it is assumed that AA, formed postsynaptically as a result of PLA2 activity, gives rise to metabolites that modify presynaptic membrane events, perhaps of the sustained type which constitute longterm potentiation, t~176 We will describe four lipolytic events that may have adverse effects. These effects encompass overactivation of the cydo-oxygenase and lipoxygenase pathways, production of platelet-activating factor (PAF), and activation of protein kinases. Figure 2 summarizes some of the major reactions involved. We envisage that PLC acts on PIP2 to yield IP3 and DAG, and that PLA2, acting on ethanolamine phosphoglycerides and plasmalogens and on choline phosphoglycerides, catalyzes the formation of lysophospholipids and AA. Degradation of DAG by di- and monoacylglyceride lipases yields additional AA. A separate PLA2-dependent pathway has come into focus during recent years, one leading to the production of PAF. 8'22'93'95'168 When a PLA2 acts on alkyl-2-acyl-snglycerophosphocholine, a minor component of the phosphatidylcholine fraction of cell membranes, lyso- PAF and AA are formed. Although lyso-paf is biologically inactive, catalysis by an acetyltransferase leads to the formation of PAF, which is proinflammatory and leads to activation of platelets as well as activation and adherence of leukocytes to endothelial cells. Binding sites exist for PAF in brain tissues, and since PAF antagonists reduce the amount of free fatty acids accumulated during seizures and ischemia, PAF may stimulate receptors coupled to a PLA2. s If this is so, one can envision that PAF acts as an amplification factor in the AA cascade. Brain tissues contain cyclo-oxygenase and lipoxygenase enzyme complexes and metabolize AA to the eicosanoids shown in Fig '185a96 Since production is limited by the supply of substrate, accumulation of AA during ischemia triggers a spurt of production of prostaglandins, prostacyclin, and thromboxanes 15'59'~45 as well as of leukotrienes, s6,~'4,ij7 Since oxidation of AA J. Neurosurg. / Volume 77/September,

4 B. K. Siesj6 by cyclo-oxygenase and lipoxygenase requires oxygen, such production is usually considered to be a reperfusion phenomenon. However, since some of the enzymes involved have relatively low Michaelis constant values for O2, oxidative metabolites of AA may well accumulate in a partly ischemic tissue. ~7.J45 All 5-1ipoxygenase products are biologically active, as are probably many metabolites arising as a result of 11-, 12-, and 15-1ipoxygenase activityj 85 The primary product of the 5-1ipoxygenase pathway (5-hydroperoxy-eicosatetraenoic acid (5-HPETE)) gives rise to 5- hydroxy-eicosatetraenoic acid (5-HETE), which has a powerful chemoattractant effect on polymorphonuclear leukocytes, and to a series of important compounds called leukotrienes. Leukotriene B4 is known as a very important chemoattractant for polymorphonuclear leukocytes, while leukotrienes C4, D4, and E,, collectively named "slow-reacting substance of anaphylaxis," constrict vessels and increase vascular permeabilityj 4~ Therefore, it is tempting to speculate that leukotrienes are involved in the breakdown of blood-brain barrier function and in edema formation. 7,~9'j ~4 However, such effects may be less pronounced in the brain than in other tissues. ~~ Even if this is so, the leukotrienes probably qualify as mediators of inflammatory reactions at the blood-endothelial cell interface. In the cerebrovascular field, most of the interest in cyclo-oxygenase products has been centered on thromboxane A2 and prostacyclin. This is because the former is a vasoconstrictor and promotes aggregation and the latter is a vasodilator and antiaggregatory. However, the prostaglandins (D2, Ez, and F2,) may well turn out to be important modulators of membrane and synaptic functions. 49 An imbalance between thromboxane A2 and prostacyclin could obviously disturb microcirculation in an energetically perturbed tissue, and such an imbalance may well arise during an AA cascade. Thus, although both thromboxane synthetase and prostacydin synthetase are likely to be stimulated, the latter enzyme is known to be inactivated by free radicals. In other words, ischemia could lead to relative overactivity of thromboxane, favoring vasoconstriction and platelet aggregation. The precedent for this emanates from results of studies on global or multifocal ischemia. Pioneering work by Kochanek and collaborators 87,88 showed that, at least following long periods of ischemia, a combination ofindomethacin, prostacyclin, and heparin improved reflow. A clear amelioration of postischemic hypoperfusion by a thromboxane synthetase inhibitor (1-benzal-imidazole) was recently shown by Pettigrew, et al., ~35 who induced 30 minutes of forebrain ischemia in rats. Their results suggest that a combination of thromboxane A2 antagonists and prostacyclin could favorably affect blood flow in a moderately ischemic tissue. A recent study demonstrated that, at least in gerbils, pretreatment with three different cyclo-oxygenase blockers (but not with a thromboxane synthetase inhibitor) markedly ameliorated the delayed damage affecting the CA1 region.~9 If confirmed, the data demonstrate an effect similar to that reported for amino-3-hydroxy- 5-methyl-4-isoazole propionic acid (AMPA) receptor blockers (see below). It is tempting to conclude that cyclooxygenase melabolites serve as messengers, which mediate an up-regulation of synaptic efficacy. Figure 2 reminds us that PLC activation leads to an accumulation of IP3 and DAG. Since IP3 formation and other mechanisms raise Ca+*i, conditions are at hand for the activation and membrane translocation of protein kinase C (PKC) (see Fig. 1). Such activation may cause sustained changes in membrane function by phosphorylation of receptors or ion channels. Connor, et al, 42 found that standing Ca 247 gradients could be induced in dendrites of CA1 neurons by the application of glutamate or N-methyl-D-aspartate (NMDA), particularly if the applications followed a priming or conditioning stimulus. Since the sustained rise in Ca+ was prevented by sphingosine, a PKC inhibitor, a likely mechanism was up-regulation of calcium channels by phosphorylation. Similar mechanisms may be responsible for physiological phenomena such as long-term potentiation ~~176 and, if involving nonphysiological stimuli, perhaps for pathology as well. t~ Manev, et al., 1~ exposing neurons in culture to toxic concentrations of glutamate, concluded that the key pathophysiological event was activation and membrane translocation of PKC. 4 In support, the cells were protected by gangliosides which act as PKC antagonists. J83 It is tempting to postulate that a similar mechanism is responsible for delayed neuronal death following transient ischemia, particularly since staurosporine, another PKC inhibitor, ameliorates ischemic damage in gerbils. TM However, although PKC is translocated to membranes following transient ischemia, evidence exists that the enzyme is deactivated, t94 Perhaps the putative up-regulation of synaptic function following a transient insult is due to deactivation of protein kinases. ~93 In fact, one may even conceive of changes in gene expression, with marked alteration of protein metabolism. Calcium is known to trigger expression of proto-oncogenes such as c-los and c-junj 16 It is known that messenger ribonucleic acids (mrna's) for these genes accumulate after transient ischemia,84.~3~ and it has recently been shown that both ischemia and hypoglycemia induce or repress mrna's for several growth factors. 97 Clearly, the result of a massive rise in Ca++i could be marked alterations of membrane function, if not molecular havoc. Given the fact that an abnormal rise in Ca++~ can trigger a host of adverse reactions, how can we conceptually explain the rapid death of cells in the focus and the presence of a penumbral zone where cells are at risk but can be salvaged by drugs that antagonize calcium influx? A reasonable working hypothesis is that cell death is only incurred if Ca++~ rises above a minimum critical value for a minimum period of time. If this is so, what matters is the degree of alterations of the pump/leak relationship at the levels of the plasma and intracellular membranes. 340 J. Neurosurg. / Volume 77 / September, 1992

5 Treatment of focal ischemia It may facilitate our discussion if we consider calcium transients in dense (or complete) ischemia, in hypoglycemic coma, during induced spreading depression in normal tissue, and in the penumbral zone of a stroke lesion (the latter events are assumed on the basis of recorded K + transients). As Fig. 3 shows, the extracellular Ca transients (solid lines) look similar in amplitude under all conditions. Yet for equal durations, dense ischemia causes more damage than hypoglycemic coma and spreading depressions cause none, even if repeatedly elicited over 4 to 5 hours. ~24 Clearly, factors other than the mere shift of calcium from extra- to intracellular fluids come into play. The decisive factor may be the magnitude and the duration of the rise in Ca++~ (assumed levels are given by the dotted lines). Thus, in dense ischemia the intracellular binding of calcium may be compromised (due to acidosis), and so certainly is its sequestration into endoplasmic reticulum and mitochondria in the energy-depleted and oxygendeprived cells. Although Ca++~ levels rise in hypoglycemic coma as well, 179 the rise may be less pronounced since about one-third to one-fourth of the ATP content remains, oxygen supply is upheld, and acidosis is absent. ~s6 Finally, in spreading depression neither the ATP production nor the oxygen supply is compromised, and the calcium that enters could be quickly bound and sequestered and subsequently extruded. Figure 3 upper left depicts events in complete or dense ischemia. The situation is probably similar in the focus of a stroke lesion. 178 Thus, at least in the focus one can expect to find rapid failure of ion homeostasis with calcium entry via multiple pathways. This probably explains why middle cerebral artery occlusion of only 30 minutes' duration yields infarction in the caudoputamen (see above). Events in the penumbral zone are different since, at least initially, changes in energy state are less dramatic and ionic transients are absent or of short duration. However, the penumbra is at risk since, due to the reduced blood flow, energy metabolism is compromised in the sense that it cannot adequately cope with metabolic stress. It was observed by Harris, el a/., TM that ischemic tissues that had not yet transgressed the threshold for gross membrane failure could show spontaneous ionic transients of the type seen in spreading depression. More pronounced, but also more variable, increases in extracellular K + sometimes progressing to overt loss of ion homeostasis were reported by Strong, eta/. t72:74 Additional results were later obtained in rats by Nedergaard and Astrup, ~z2 who noted irregularly occurring K transients in the penumbra of a stroke lesion due to middle cerebral artery occlusion. I have assumed that the spreading depression-like K + transients observed represent Ca ++ transients of a type depicted by the lowermost curve in Fig. 3. On the basis of these results one can formulate a working hypothesis which postulates that the gradual recruitment of damaged cells in the penumbra is due to irregularly occurring waves of depolarization, accom- FIG. 3. Schematic diagrams illustrating changes in extracellular Ca ++ during complete ischemia, hypoglycemic coma, spreading depression (SD), and SD-like depolarization waves in the penumbral zone of a stroke lesion (solid line), as well as presumed changes in intracellular Ca ++ (dotted line). panied by calcium transients. ~-'2'trj:3 Since calcium transients accompanying spreading depressions in the normal brain do not lead to brain damage, one would then have to postulate that spreading depression-like calcium transients act differently in an energy-compromised tissue of which the cells have a reduced capacity to extrude or sequester calcium (Fig. 3). However, it remains to be shown that spreading depressions or spreading depression-like transients are elicited in the penumbral zone of a stroke lesion in man. Acidosis-Induced Damage It has been shown beyond doubt that preischemic hyperglycemia worsens damage due to transient global or forebrain ischemia, probably by enhancing production of lactic acid during ischemia." 8. l ZS. ~ 3~. ~46. ~52-~54 Cardinal features in this exaggerated brain damage are gross edema and postischemie seizures, and more rapidly maturing brain lesions. Since preischemic hyperglycemia can convert selective neuronal necrosis into pannecrosis, it qualifies as one factor predisposing to infarction, possibly by causing damage to glial cells and/ or vascular endothelium as well. Following brief periods of ischemia in hyperglycemic subjects, edema formation and gross parenchymal lesions can appear after a delay of many hours. 146"166'186 Similar adverse effects of hyperglycemia were obtained in cats subjected to 2 hours of middle cerebral artery occlusion, followed by 1 hour of reperfusion) 84 In rats, hyperglycemia due to glucose infusion or to streptozotocin-induced diabetes increased the size of the infarction due to brief transient middle cerebral artery occlusion.'z~ This is the predicted result since, in that species, flow in the focus is low and events during reperfusion should mimic those following global or forebrain ischemia. The influence of hyperglycemia on infarct size following permanent middle cerebral artery occlusion is more ambiguous. In fact, some workers have noted a J. Neurosurg. / Volume 77/September,

6 B. K. Siesj6 worsening of the outcome, 44 others a reduction in infarct size, 62"199 and still others no change. ~23 Nedergaard and Diemer ~23 found that, whereas neither glucose infusion nor acute (2 days) streptozotocin-induced diabetes altered infarct size from that obtained in normally starved animals, infarct size was reduced in hypoglycemic subjects and increased in animals with chronic diabetes (4 months), the latter result probably reflecting vascular changes interfering with collateral blood supply. In hyperglycemic subjects, the border between infarcted and noninfarcted tissue was sharp, and the infarcted area showed total necrosis without proliferating capillaries. This suggests that acidosis is responsible for destruction of microvessels, thereby inducing pannecrosis. In contrast, hypoglycemic subjects showed an indistinct infarct border with many proliferating capillaries and a wide perifocal rim of selective neuronal necrosis, mainly localized to layer 2 of the neocortex. It can be tentatively proposed that perifocal selective neuronal necrosis occurs in hypoglycemic subjects because hypoglycemia predisposes for spreading depression-like depolarization waves, with repeated release of EAA's leading to excitotoxic nerve cell damage. 29 This interpretation is in line with the observation that hyperglycemia inhibits the spreading depression-like tran- sients. ~ 22 How then can the variable effects of hyperglycemia in permanent middle cerebral artery occlusion be explained? It is easy to envision a scenario where hyperglycemia aggravates damage and increases infarct size. Thus, if the energy failure in the penumbra is sufficiently dense to cause neuronal necrosis, it may also be dense enough to trigger exaggerated acidosis should hyperglycemia increase glucose delivery. Energy failure and excessive acidosis will then contribute to give total tissue destruction (in reversible ischemia the result will be a shortening of the revival time). It is more difficult to envision how hyperglycemia could improve conditions. There are two possible explanations, equally speculative: 1) by increasing substrate supply, hyperglycemia facilitates ATP synthesis without causing acidosis of a degree that wrecks the machinery; or 2) if not too pronounced, the acidosis is beneficial. It has been known for many years that low extracellular ph retards calcium flux via VSCC's and 3Na+/Ca ++ exchange in muscle, 79 and recent results suggest that calcium influx through NMDA-gated chan- nels is reduced or blocked when extracellular ph falls. 6~ It is not unlikely that this is beneficial. However, it remains to be explained why a blockade of cellular calcium influx should prevent pan-necrosis from developing but allow selective neuronal necrosis (a putative calcium-related lesion) to occur. Provided that acidosis is dense enough to aggravate tissue damage, by what mechanisms could that occur? Figure 4 illustrates four mechanisms that could interact to worsen damage in severely acidotic tissue, and prevent or retard recovery during reoxygenation. These encompass edema formation, inhibition of mitochon- FIG. 4. Diagrams showing putative effects of a raised [H (reduced ph). The following adverse effects of acidosis can be envisioned. A: Acidosis may activate an Na + antiporter which, if coupled to a passive CI-,/HCO3- antiporter, could shuttle Na + and C1- into the cell, with osmotically obligated water. B: A reduction in extracellular ph could outcompete Na + at the external site of the Na+/H + antiporter, thereby retarding (or preventing) H + extrusion from acidotic cells. C: Acidosis is known to retard oxidative phosphorylation in isolated mitochondria. This suggests that low ph curtails adenosine triphosphate (ATP) production. ADP = adenosine diphosphate; P~ = intracellular phosphorus. D: At low ph values, the lactate oxidase form of the lactate dehydrogenase complex may be blocked, retarding oxidation of lactate accumulated during ischemia. NADH = reduced form of nicotinamide-adenine dinucleotide. drial respiration, inhibition of lactate oxidation, and inhibition of H + extrusion.153'163 The acidosis-dependent edema mechanism shown in Fig. 4 is a speculative one, based on the simultaneous presence of Na+/H and CI-/HCO3- antiporters in cells (for example glia), m Coupled antiporters of this type exist in other cells, and seem to fulfill tasks of regulating cell volume. 3~ However, if cellular acidosis activates the Na + exchanger and H leaks back via the C1-/ HCO3- antiporter, a vicious circle is created which causes accumulation of Na and CI-, with osmotically obligated water. In other words, the cell would try to regulate intracellular ph at the expense of its own volume regulation. Clearly, if this were an important edema mechanism, blockers of the antiporters would have a positive effect. Diuretics acting in this way could become important tools in the treatment of edema. 43 At least in vitro, low ph retards or blocks adenosine diphosphate (ADP)-stimulated oxygen consumption in mitochondria, suggesting that very little ATP could be formed if the ph value falls toward Clearly, prompt restitution of mitochondrial function requires that intracellular ph is increased. This occurs by oxidation of accumulated lactate- and by accelerated Na+/H + exchange. Unfortunately, both events are probably inhibited at low ph values. At least in some tissues the affinity of the lactate dehydrogenase for lactate- is low at reduced ph simply because an essential imidazolium 342 J. Neurosurg. / Volume 77/September, 1992

7 Treatment of focal ischemia group in the enzyme molecule is titrated.]63 Thus, lactate- oxidation may be accelerated first when intracellular ph increases. This usually occurs by Na+/H + exchange. However, since this exchanger operates very slowly at low extracellular ph values, 67'82 the extrusion of H + is also slow until extracellular ph has increased. Clearly, the severely acidotic tissue is at risk even when circulation is restored. One recognizes the need for prompt recirculation with well-oxygenated blood. Evidently, all these mechanisms would operate in the acute phase when the acidosis is still present and they could then prevent recovery of a normal energy state and restitution of a normal intracellular ph. A different question is how transient acidosis, particularly if excessive, can lead to delayed damage. The solution to this problem may be found in the generation of free radicals during and/or following ischemia. Free Radical-Induced Damage It has been speculated for over two decades that free radicals contribute to cell death in a variety of tissues and under a variety of conditions that are accompanied by enhanced free-radical production. 54'56'73 In fact, the free-radical hypothesis is one of the most elaborate proposals developed to explain how a common factor can cause cell death in conditions as disparate as hyperoxic stress, aging, cancer, and ischemia. It is now almost 15 years since Demopoulos, et al., 45 proposed that free radicals contribute to brain lesions in stroke (see also Flamm, et al?~). It was subsequently underscored that ischemia-induced free-radical damage in brain 7~'~28'~54'~89 and other tissues 25"~~ is most likely to occur if ischemia is followed by recirculation; the hypothesis was soon extended to encompass brain and spinal cord trauma. 69,89 A large part of the information contained in this literature has been reviewed recently. ~61,~88 Parallel lines of research have defined corresponding hypotheses for ischemic damage in tissues such as the intestine and the heart. 91"19~ The principles underlying the free-radical hypothesis of ischemic damage are straightforward. First, since free radicals are formed in all aerobic cells and since they are inherently toxic, the cell must possess an appropriate defense system. This is both enzymatic and nonenzymatic. In fact, the major reason why cells contain certain vitamins and their analogs (for example, a- tocopherol and ascorbic acid) is that these function as free-radical scavengers. Furthermore, the sole function of some enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, is to metabolize free radicals or compounds which are precursors to free radicals. Second, several conditions predictably give rise to enhanced free-radical production. These encompass hyperoxia, which enhances the production of oxygen radicals, and the anaerobic-aerobic transition, which accompanies ischemia with recireulation. Thus, oxygen radicals and other radicals are formed when reduced compound (accumulated during ischemia) is reoxidized. Since the production of free radicals is propor- FIG. 5. Free radical reactions which may lead to the production of. OH. XO = xanthine oxidase; NOS = nitric oxide synthetase. NADP = nicolinamide-adenine dinucleotide phosphate; NADPH = reduced form of NADP. tional to oxygen tension, the spurt of free-radical production during recirculation may also be related to the increase in po2 that accompanies reactive hyperemia. We may therefore consider ischemia with recirculation as one form of oxidative stress. However, since freeradical production can occur at relatively low oxygen tensions and can be triggered by the accumulation of reduced compounds, recirculation is not a requirement for free-radical production to occur. It is thus conceivable that the "reductive stress" of partial isehemia leads to free-radical damage. ~59 Although the functions of the mitochondrial cytochrome a-a3 enable it to accept a package of four electrons without the formation of free radicals, oxygen has the tendency to accept one electron at a time, leading to the formation of superoxide radicals (. 02-) and H Such univalent reduction occurs at more proximal steps in the mitochondrial respiratory chain, in extramitochondrial reactions, and that in which hypoxanthine and xanthine are oxidized to uric acid by xanthine oxidase. We will take the latter reaction as an example of enzymatic formation of H202 and -O2-. The reaction is a likely source of free radicals during ischemia/recirculation (Fig. 5). This is because hypoxanthine and xanthine accumulate during ischemia, and because a rise in Ca++~ may activate proteases which convert xanthine dehydrogenase to xanthine oxidase.1~ There are no enzymatic reactions known leading to the formation of hydroxyl radicals (. OH). This is fortunate because.oh is extremely toxic, indiscriminantly attacking neighboring lipid, protein, and deoxyribonucleic acid molecules. However, 9 OH is produced J. Neurosurg. / Volume 77 / September,

8 B. K. Siesj6 if.oz- and H202 react in the so-called "Haber-Weiss reaction." This reaction is inherently slow unless catalyzed by iron according to the reactions constituting the so-called "iron-catalyzed Haber-Weiss reaction. ''72 A likely scenario is as follows. Iron, which is normally tightly bound by proteins such as ferritin and transferrin, can be released from ferritin by and reduced flavins, and from transferrin by acidosis. The enhanced production of free radicals at low ph levels has been clearly shown in vitro, as has the participation of iron? 38'16~ When chelated by nonprotein compounds, iron is pro-oxidant and may trigger the formation of toxic species such as 9 OH (and ferryl). Since 9 OH reacts at the site of production, and since and particularly H202 are diffusible, free-radical damage is to a large extent site-specific and occurs wherever catalytic iron is produced. One can envision that the diffusion of iron or of free radicals released from dead and disintegrating cells could lead to damage to neighboring cells. According to this scheme, free-radical damage requires two events to occur: 02- and H202 are formed in increased amounts, and protein-bound iron is decompartmentalized. This puts the focus on cellular iron metabolism, a poorly known subject] 97 However, another source of toxic free-radical species has recently been identified, one that could preferentially affect endothelial cells.~~ The endothelium-derived relaxing factor, now known to be identical to nitric oxide (NO-), is synthetized from the amino acid L-arginine by an NO. synthase which is activated by calcium with reduced nicotinamide-adenine dinucleotide phosphate as a cofactor. 5s Nitric oxide diffuses into the smooth muscle and causes vascular relaxation by triggering the production of cyclic guanosine monophosphate. Evidence exists that NO. is inactivated by.02- formed by endothelial cells, and that this is a physiological event. However, enhanced production of.02- may trigger endothelial damage, particularly if iron is decompartmentalized.n Another scheme is that proposed by Beckman, et al., ~~ who suggested that. 02- and NO. decompose to form the toxic peroxynitrate ion (NO3-). This ion is stable at alkaline values of the ionization constant (pk) but, since the conjugate acid has a pk of 6.6 (*C), it decomposes rapidly at acid ph levels to yield 9 OH (and NO2). Thus, if increased amounts of 02- are formed and if the NO synthase is stimulated by Ca ++, toxic free-radical species could be formed, giving rise to endothelial cell damage. In spite of the elaborate theoretical background, it has been difficult to define the conditions under which free radicals are formed, or indeed to establish if any formation observed is what causes ischemic damage. This has remained a controversial issue and numerous articles have been published, supporting or questioning the importance of free radical-mediated damagej 59 It seems that the controversy is now beginning to be resolved. Thus, Patt, et al., TM showed that 3 to 6 hours of unilateral carotid artery occlusion in gerbils, with reperfusion, was accompanied by enhanced H202 forma- tion, edema, and neurological damage which could be alleviated by drugs and procedures whose only known action is to quench free radicals or to prevent their formation. When these and other results were compared, it seemed likely that free radicals contribute to the damage incurred during sustained ischemia, particulady if followed by recirculation. 159'~88 This would explain why many unsuccessful attempts have been made to assess free-radical damage after relatively brief periods of ischemia, and to ameliorate brain damage to established flee-radical scavengers? Exceptions to this rule exist, since one group has obtained strong evidence that free radicals are formed after a brief ischemic period in gerbils. 13~ Species or methodological differences cannot be excluded. It has been postulated that the production of free radicals is likely to occur not only during sustained ischemia, but also under conditions of hyperthermia and excessive acidosis. 159 These are the conditions under which infarction predominates over selective neuronal necrosis. Therefore, it is tempting to assume that free radicals somehow trigger pan-necrotic lesions. Conceivably, this occurs because free radicals primarily affect microvessels, with an ensuing loss of barrier functions. There is circumstantial evidence supporting this contention. Thus, microvessels may well contain most of the tissue levels of xanthine oxidase, ~3'8] and they are exposed not only to the highest oxygen tensions but also to neutrophils, a source of.o2- and other free radicals]o.7~ Furthermore, microvascular permeability is increased and edema is induced if a mixture of xanthine, hypoxanthine, and xanthine oxidase is injected into the tissue. 33 Treatment of Focal Ischemia Current knowledge of the pathophysiology of focal ischemic damage and of the cellular and molecular mechanisms involved suggests that amelioration of ischemic lesions could be achieved by agents that improve the pump/leak relationship for calcium, by those reducing cellular acidosis, or by those that abort production of free radicals or scavenge those formed. The latter class of compounds probably encompasses iron chelators. There is also compelling evidence that agents that antagonize the action of PAF and other lipid mediators may act similarly to free-radical scavengers by suppressing inflammatory reactions threatening the patency of the microvessels. Glutamate and calcium antagonists, as well as freeradical scavengers, have given encouraging results in experimental ischemia. However, inconsistencies exist and the issue has been clouded by the fact that negative results were obtained in several well-controlled studies. On analysis, most of these discrepancies can be explained by differences in effect between experimental models, notably between transient global or forebrain ischemia of the cardiac arrest type, and permanent focal ischemia of the stroke type. 149'161"162 We recall that, in terms of density of ischemia, the focus of a stroke 344 J. Neurosurg. / Volume 77/September, 1992

9 Treatment of focal ischemia lesion resembles the whole brain in global ischemia or the forebrain in forebrain ischemia. The discrepancies in results can be summarized as follows. Brain damage due to brief transient ischemia of the global/forebrain type is usually not ameliorated by drugs that reduce cellular calcium influx or scavenge free radicals. Such compounds also fail to ameliorate the lesions affecting the focus of a stroke lesion. The only pharmacological compounds that have hitherto been shown to ameliorate lesions due to transient forebrain ischemia markedly and consistently are AMPA receptor blockers. There are single reports that cyclooxygenase inhibitors act similarly, and that a PKC inhibitor (staurosporine) has a significant effect. Some calcium antagonists, notably those that act on Na channels as well (like fiunarizine), are moderately efficacious. 47 The situation is different in experimental stroke. Thus, competitive and noncompetitive NMDA antagonists, the AMPA-receptor blocker 2,3-dihydroxy- 6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), some calcium/sodium antagonists, and several free-radical scavengers have all been shown to reduce infarct size by salvaging penumbral tissues. These differences in results will be taken into consideration in the discussion to follow. Drugs Improving the Pump/Leak Relationship for Calcium Since enhanced influx of calcium is assumed to be a major cause of cell damage, much interest has been focused on drugs that reduce calcium influx through VSCC's and AOCC's. It should be recalled, though, that any rise in Ca++i is determined both by the leak and by the pump. Thus, although it seems advantageous to reduce the leak, it would be equally important to improve pump function by enhancing production of ATP (for example, by raising cerebral blood flow). These principles will be highlighted when the effects of glutamate and calcium antagonists are considered. In the future, it is likely that drugs will be developed that reduce the release of EAA's from nerve endings. Such drugs may either reduce presynaptic calcium influx through the N type of calcium channels or act by hyperpolarizing the plasma membrane (for example, by raising K + or C1- conductances). At present, though, interest is mainly focused on calcium influx through postsynaptic channels. These are either gated by EAA's or by voltage changes. Pharmacology of Glutamate Antagonists. In order to discuss the pharmacology of glutamate antagonists we need a more elaborate description of the NMDA receptor complex. Figure 6 shows that, apart from the main recognition site for glutamate, the NMDA receptor also contains a modulatory site at which glycine acts, reportedly by increasing channel opening frequency3 3 This site may be saturated at physiological glycine concentrations, but inhibition of glycine action should reduce NMDA receptor activity. A separate modulatory site exists for polyamines such as spermine FIG. 6. Diagram showing modulators of the N-methyl-Daspartate (NMDA) receptor complex. A: The modulatory sites that set the stage for NMDA/glutamate activation of the receptor, or reinforce the activation, are depicted. B: Sites that are antagonistic or inhibitory are shown. These include sites at which competitive and noncompetitive NMDA antagonists work. See text for abbreviations. and spermidine, which potentiate NMDA action. 37 Although the schematic figure does not indicate so, polyamines may work from the inside of the membrane. It should also be recalled that activation of the receptor requires phosphorylation (the presence of ATP). Negative modulation of receptor activity is achieved by Zn 247 and by H and, as discussed above, Mg 247 blocks the channel in a voltage-dependent manner. The action of zinc is of physiological interest since zinc seems to be coreleased, with transmitter, from the Shaffer collateral endings exciting CA I pyramidal cells in the hip- Ix)campus. The pharmacology of glutamate antagonists has been worked out in some detail. 37'5~176 Although there has been a paucity of efficacious AMPA receptor antagonists, some nonspecific glutamate antagonists (such as kynurenate) and dipeptides (such as D-glutamylglycine) act on both AMPA and NMDA receptors. Recently, more potent antagonists of non-nmda receptors have been devdoped) 44 These encompass quinoxaline diones such as 6-cyano-7-nitro-quinoxaline- 2,3-dione and NBQX. Activation of NMDA receptors can be attenuated by competitive and noncompetitive antagonists. Phosphonates such as 2-amino-5-phosphonovalerate (APV) and 2-amino-7-phosphonoheptanoate (APH) block the glutamate recognition site. Newly developed compounds, which are less polar and thus more readily penetrate the blood-brain barrier, encompass 4-(3-phosphoro-propyl)-2-piperazine-2-carboxylic acid (CPP), or its unsaturated analog CPP-ene, and cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid (CGS 19755). Noncompetitive NMDA antagonists include phencyclidine (PCP), ketamine, and dizocilpine (MK-801). Such compounds block the NMDA-linked ion channel, all probably acting at the PCP site. Since these compounds are lipid-soluble they readily penetrate the blood-brain barrier. They show use dependency (they block the channel more efficiently when the membrane is depolarized). Although this property makes them J. Neurosurg. / Volume 77/September,

10 B. K. Siesjo particularly efficacious in low-flow situations, their effect may be attenuated when depolarization is massive. Excitation at glutamatergic synapses can also be blocked or attenuated at the sites where glycine, zinc, and polyamines work. 37 Kynurenate seems to block glutamatergic transmission at the glycine recognition site. Polyamine antagonism has also been suggested for the anti-ischemic effect of ifenprodyl and its derivative SL '66 Further development may produce drugs of even greater specificity, perhaps also those acting at the zinc and H + recognition sites. Therapeutic Effects of Glutamate Antagonists. Although initial attempts to ameliorate damage due to global or forebrain ischemia were encouraging, subsequent studies clearly showed that neither noncompetitive nor competitive NMDA antagonists were efficacious, z6"27'52'125 For some time, this paucity of effect hampered the interpretation of encouraging results obtained in stroke. It was subsequently proposed that NMDA antagonists are predictably not efficacious in dense ischemia because, if cells are massively depolarized and lack an ATP source, calcium can enter by multiple pathways; under those circumstances the blockade of only one entry pathway is not likely to prevent a precipitous rise in Ca++i. ~ For reasons that are not yet clear, an AMPA receptor blocker (NBQX) has been shown to ameliorate markedly brain damage due to forebrain ischemia in gerbils ~44 and rats. 2s,~25 Since the drug was efficacious even when given 1 to 3 hours after the ischemic transient, ~44 it seems likely that the final damage after dense, brief ischemia is significantly influenced by postischemic metabolic events, perhaps encompassing an increased calcium cycling across metabolically perturbed membranes. 46,~49 Possibly, an AMPA receptor blocker may be beneficial in this situation because it blocks fast excitation and depolarization secondary to AMPA receptor activation, a necessary upstream event triggering calcium influx by the multiple mechanisms available. ~49 The principle is explained in Fig. 7. One can imagine that drugs that block fast excitation by T channels or block other Na + channels than those gated by AMPA receptors may also ameliorate the lesions, simply because they would decrease depolarizations of postsynaptic membranes. The hypothesis of increased postischemic calcium cycling 46 implies that there is either an enhanced release of EAA's presynaptically or an up-regulation of receptors of ion channels postsynaptieally. A postischemic accentuation of stimulus-induced calcium transients has been demonstrated, 6 but the response elements have not been identified. Whether it is presynaptic or not, one can envision the therapeutic advantage of drugs that could block Ca ++ flux by presynaptic N channels or that prevent or diminish presynaptic (or postsynaptic) depolarization. If the ameliorating effects of PKC inhibitors and cyclo-oxygenase inhibitors on damage due to forebrain ischemia can be confirmed, phosphorylation seems a likely mechanism and prostaglan- FIG. 7. Schematic diagram showing the sequence of events in depolarization-triggered calcium influx in postischemic tissue. The diagram suggests that drugs acting "upstream" by blocking Na + influx via AMPA (amino-3-hydroxy-5-methyl- 4-isoazole propionic acid) receptor-coupled or other channels could be more efficacious than those acting "downstream" by blocking calcium influx by only one of the available routes. VSCC = voltage-sensitive calcium channel; AOCC = agonistoperated calcium channel; K/A = kainate/ampa. dins would enter as possible mediators of a change in synaptic efficacy. Focal ischemia due to middle cerebral artery occlusion is a different entity. It is in this type of ischemia that NMDA antagonists have been found efficacious. Amelioration of ischemic damage, with reduction of infarct size to 40% to 70% of control, has been obtained in cats, rabbits, rats, and mice. 27'37 The effect is observed with both noncompetitive and competitive antagonists. The former, MK-801, was found efficacious even when given 1 to 2 hours after middle cerebral artery occlusion. This was not the case with the less permeable competitive ones, probably because it takes a long time to build up a sufficient concentration in poorly perfused tissue. 34 Typically, NMDA antagonists salvage penumbral tissues without affecting the densely isehemic focus. Experiments with long recovery periods have shown that repeated doses have to be given, suggesting that receptor occupancy must be achieved for some time (for example, for 1 day). 48,65 Although NMDA antagonists may have some salutory effects on blood flow in the penumbra, 27 most of their effects are probably exerted on calcium influx via NMDA-gated channels. The NMDA antagonists are also efficacious in reducing neuronal damage in hypoglycemic coma, a condition with a relative rather than absolute deterioration of energy state. 136'191A92 It seems likely that when some ATP production persists, the calcium pumps may be able to balance the leaks if the latter are prevented from increasing or are reduced. As discussed above, the penumbral zone may be challenged by repeated calcium transients which are triggered by spreading depression-like depolarizations. In this situation of moderate or repeated depolarization, the NMDA-gated cation channels may carry the brunt of the calcium load, explaining the efficacy of NMDA 346 J. Neurosurg. / Volume 77/September, 1992

11 Treatment of focal ischemia antagonists. However, since this hypothesis assumes that the triggering event is Na influx and depolarization, one would expect that drugs blocking AMPA receptors would be at least as efficacious. This assumption was borne out by recent experiments showing that NBQX clearly reduced infarct size following 2 hours of middle cerebral artery occlusion in rats, even when given 90 minutes after vessel occlusion. 28 Pharmacology of Calcium Antagonists A majority of brain neurons in primary culture contain channels that are activated by depolarization and are blocked by calcium antagonists of dihydropyfidine or other types. ~3 Such VSCC's have been extensively studied in a variety of tissues and their pharmacology is relatively well known. 63"t 1.3,t69,177 In peripheral neurons, like dorsal root ganglion cells, most calcium entry blockers are on the L type of calcium channel, with relatively little effect on the N and T types. 177 The pharmacology of VSCC's in central neurons seems different from that in the dorsal root ganglion cells. For example, in some freshly prepared neurons the T, N, and L types of VSCC were all sensitive to nicardipine and, at least in some cells studied, flunarizine was the most potent inhibitor of the T type of calcium channel, followed by nicardipine) The N type of channel, which is at least in part involved in transmitter release, is blocked by 00-conotoxins. 139 Therapeutic Effects of Calcium Antagonists. Like NMDA antagonists, most calcium blockers tested have been poorly efficacious in ameliorating damage due to global or forebrain ischemia. Some effect was reported for nimodipine in monkeys subjected to global ischemia m and for nicardipine in rats with forebrain ischemia, 5 a seemingly more robust effect being obtained with flunarizine in rats. ~46 This lack of effect of most antagonists is not surprising (see above). It is interesting, though, that two of those showing efficacy (flunarizine and nicardipine) have effects on T channels, which may be involved in fast excitation. Possibly, such T channel blockers act to some extent like AMPA receptor antagonists, in that they reduce poslsynaptic excitation and depolarization during recovery from a transient insult. It is less clear if and under what circumstances calcium antagonists ameliorate damage due to focal ischemia. Early studies with a dihydropyridine such as nimodipine showed that the drug somewhat reduced infarct size when given before ~ t5 but not when given 5 minutes after middle cerebral artery occlusion? 4 These results were obtained by the same group of workers who subsequently demonstrated a clear effect of NMDA antagonists (see above). However, pretreatment with nimodipine, given in repeated doses, reduced infarct size in a model of neocortical infarction in rats. s~ Amelioration of stroke lesions was also reported in cats and, in these experiments, in vivo fluorescence measurements suggested that the drug reduced the rise in Ca+ during the measurements.179 Further results supporting an effect of nimodipine are those showing a reduction in the severity of the acidosis in the ischemic tissue.68.~ ~z This is an intriguing result which possibly reflects an improved mitochondrial function (see below). Three other calcium antagonists have been reported to ameliorate lesions markedly due to permanent middle cerebral artery occlusion. Isradipine, another dihydropyridine antagonist, was found to reduce infarct volume to about 50% of control when given before or up to 3 hours after middle cerebral artery occlusion in spontaneously hypertensive rats.~42 Interestingly, a single large dose of this antagonist (15 mg/kg) given 12 hours prior to middle cerebral artery occlusion significantly reduced infarct volume. ~42 This group of workers, using an identical stroke model, also compared isradipine to nimodipine, darodipine, nicardipine, and nitrendipine. The effect of nimodipine and nitrendipine was smaller while the other two calcium antagonists tested were inefficacious. Unfortunately, isradipine given in a lower dose had no effect on the stroke size in another modelj ~ A calcium antagonist, S-emopamil, which also has serotonin antagonistic properties, ameliorated damage due to permanent middle cerebral artery occlusion in rats. L2~ Like isradipine, when S- emopamil was given to spontaneously hypertensive rats, it reduced infarct size to the same extent as NMDA antagonists have been reported to do. An even more pronounced effect was recently reported in cats with RS-87476, a novel Na*-Ca channel modulator. 92 In general, although some of these effects are encouraging, these results are less consistent than those obtained with glutamate antagonists. It seems highly justified that future studies be performed with accurate determination of pharmacokinetics and bioavailability. It is not clear how these drugs work or if they work. Calcium antagonists of the isradipine type are very vasoactive, and increased cerebral blood flow in perifocal areas has been reported in some jh'~42 but not in alp 8~ studies. If cerebral blood flow increases, one can envision enhanced activities of ion pumps; however, calcium antagonists are likely to reduce leak fluxes of calcium as well. Thus, although calcium-related damage in vitro is assumed to be mainly mediated by glutamateinduced calcium influx, more prolonged depolarizations yield cell necrosis which can be ameliorated by classic calcium antagonists. Furthermore, ifca* in the ischernic ceils is reduced at constant cerebral blood flow levels/tm an effect on calcium channels must be invoked. Drugs Reducing Acidosis Acidosis occurs because ischemia leads to a mismatch between glycolysis and oxidative phosphorylation. The main cause must be lack of oxygen, hindering pyruvate oxidation (see Fig. 7 in Part I of this review158). However, two other mechanisms can be hypothesized. One would be mitochondrial dysfunction due to inhibition of pyruvate dehydrogenase, an enzyme the activity of which is regulated by phosphorylation/dephosphorylation, and the other would be the diversion of respiratory J. Neurosurg. / Volume 77/September,

12 B. K. Siesj6 energy toward sequestration of calcium, rather than production of ATP. It is probably futile to attempt reducing acidosis by the administration of bases since large amounts would have to be given. However, one can envision a therapy directed toward each of the three mechanisms leading to acidosis. Thus, an improvement in blood flow and/ or cellular oxygenation would tend to direct pyruvate toward oxidation in the citric acid cycle rather than toward reduction to lactate. It is obvious that an increase in blood flow or in blood oxygen tension would achieve this, but it is equally obvious that the effects may be transient and may carry some risks. For example, an increase in flow secondary to a rise in blood pressure could worsen edema, and hyperoxia carries the risk of inducing oxidative damage. A more substantial improvement of mitochondrial function may be achieved with calcium antagonists since they could have the dual function of increasing flow and reducing Ca++~. Any of these effects would tend to reduce acidosis, an increase in flow by increasing p02 and a decrease in Ca++i by diverting respiratory energy toward ATP production rather than Ca ++ sequestration. At least in theory, pyruvate dehydrogenase activity could be enhanced by drugs of the type represented by dichloroacetate, which has been tested in other types of ischemia.'8 It remains to be shown that they act in focal ischemia. Free-Radical Scavengers and Antagonists of Lipid Mediators It is only during the last 3 to 4 years that free radicals have been definitively established as modulators of ischemic brain damage. This is partly because new methods have become available for detecting formation of free-radical production, some of which are based in spin traps introduced into the tissue, 2~176 and partly because efficacious scavengers of free radicals have become available. These encompass not only the classic ones, such as a-tocopherol, allopurinol, and dimethylthiourea, but also the series of 21-aminosteroids ("lazaroids"), which are compounds that lack glucocorticoid activity but act as free-radical scavengers and, at least to some extent, as iron chelators. 24 The effects of the 21-aminosteroids have been most clearly documented on models involving trauma, intracranial bleeding, and circulation shock. 23,69 This caused Watson ~88 to speculate that disruption of tissue structure and decompartmentalization of initiators and substrates are prerequisites for a substantial therapeutic effect. As stated, our own group has postulated that a freeradical component first becomes prominent when the ischemia is of long duration and that the main target is the microvasculature. '48,'5'.'6~ Inherent in this working hypothesis is that selective neuronal vulnerability following a brief ischemic period of hypoglycemic coma is due to other adverse factors, such as the loss of neuronal calcium homeostasis. As reported in a very recent publication, 53 this hypothesis is based on the difficulty of reproducing published results on protein oxidation, as well as on the amelioration of damage by scavengers. However, we are aware of the fact that contradictory results exist, particularly in gerbils, 7~176 and that drugs acting in vitro to quench induced freeradical formation have been reported to ameliorate ischemic damage due to forebrain ischemia of 30 minutes' duration in rats. 4~ The results that most strongly suggest that free radicals generated during ischemia contribute to the aggravation of the tissue lesion are those pertaining to long periods of ischemia, with or without recirculation, and those demonstrating affectation of microvessels. The results of Patt, et al., j34 established that in gerbils exposed to 3 to 6 hours of unilateral carotid artery ligation followed by reperfusion, neurological deficit, edema, and H202 production were reduced by established freeradical scavengers such as dimethylthiourea and allopurinol. It is now clear that such scavengers can also reduce infarct size following permanent middle cerebral artery occlusion. Working in rats, Martz, et al., Jo4 found that both dimethylthiourea and allopurinol reduced infarct size by about 30%. A comparable effect was obtained when polyethylene glycol-conjugated superoxide dismutase and eatalase were given to rats subjected to distal middle cerebral artery occlusion. 78,98 It is also of considerable interest that transgenic mice overexpressing superoxide dismutase show less edema and smaller infarcts when they are subjected to focal ischemia. 3~ Results of this type have also focused interest on microvascular dysfunction, such as edema. As already remarked, in the experiments of Patt, et al., dimethylthiourea and allopurinol ameliorated the edema. One group has reported that the 2 l-aminosteroid U74006F reduces perifocal edema; ~98 however, this effect seems to be shared with other free-radical scavengersj Recently, evidence has accumulated that PAF, produced during ischemia, adversely affects recovery following short-lasting transient ischemia in gerbils. Thus, PAF antagonists improved recovery of neurological function and mitochondrial respiration, and reduced the free fatty acid concentration. '7''7~ Similar results were obtained in rats, since a PAF antagonist ameliorated CA1 damage following 10 minutes of forebrain ischemia. ~29 Additional models in which PAF antagonists have been reported to ameliorate damage include 14 minutes of ischemia in the isolated canine brain, 61 laser-induced microvascular obstruction, 55 and transient spinal cord ischemia. 96 Although these results suggest that PAF antagonists ameliorate tissue damage in a variety of ischemic models, the major target of such antagonists may be the microvessels.'50 Thus, like free radicals, PAF may be a mediator of ischemic damage only when recirculation is compromised following brief ischemia, or when flow is reduced over long periods. In support of this contention, PAF antagonists have been shown to reduce infarct size following middle cerebral artery occlusionj 6 Since a major lesion leading to infarction may be an inflammatory reaction at the blood-endothelial cell in- 348 J. Neurosurg. / Volume 77 / September, 1992

13 Treatment of focal ischemia terface, involving activation of leukocytes and thrombocytes and their adhesion to the vessel wall, 7~~ such drugs hold great promise for the future. Conclusions Experimental studies conducted during the last two decades have contributed greatly to our understanding of the pathophysiology of stroke lesions, and those conducted during the last 4 to 5 years have, for the first time, defined therapeutic principles that promise to improve clinical treatment of patients with focal ischemia. The following main facts emerge: 1. It is useful to consider a stroke lesion as consisting of a densely ischemic focus surrounded by better perfused tissue. This perifocal penumbral tissue is viable for 1 hour or more but, if not prevented from doing so, will eventually be recruited in the infarction process. 2. The factors that are responsible for the extension of the infarct into the penumbral zone may encompass irregularly occurring depolarizations, giving rise to calcium transients as well as to enhanced production of free radicals. The former mechanism may be largely responsible for the perifocal neuronal necrosis and the latter for the pan-necrotic destruction of all cells (for infarction). In this process, acidosis plays an ambiguous but probably important role. 3. When given before or within 1 to 2 hours following middle cerebral artery occlusion, NMDA antagonists reduce the final infarct size by a maximum of 50%, probably by reducing depolarization-coupled influx of calcium into cells. Blockers of AMPA receptors also hold great promise. At least in certain models, some calcium antagonists produce a comparable ameliorating effect, possibly by improving blood flow to the underperfused penumbra. 4. Established free-radical scavengers such as dimethylthiourea and allopurinol reduce infarct size following permanent middle cerebral artery occlusion, and probably prevent some of the reactions set in motion by reperfusion. A likely target for the free radicals formed is the endothelial cell which may receive the brunt of the free radical-mediated tissue damage during long periods of ischemia, whether followed by reperfusion or not. Evidence is now emerging that PAF plays a similar role in promoting inflammatory reactions involving platelets and leukocytes, thereby compromising microcirculation. Platelet-activating factor antagonists hold promise for the future. The role of eyclooxygenase and lipoxygenase metabolites for vascular and cellular alterations in transient forebrain ischemia is compelling, but sufficient data are not at hand to judge whether blockers of this pathway will be important in focal ischemia. 5. Recent experimental research has provided some challenging therapeutic avenues but, unfortunately, the window for successful treatment seems small. Thus, if these treatment strategies are going to become clinically important, patients must come under treatment much sooner than present routines allow. Acknowledgments The author thanks Katarina M~inson for devoted and skilled secretarial work, and Birgit Olsson for valuable help with the illustrations. References 1. Abe K, Yuki S, Kogure K: Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 19: , Agardh CD, Zhang H, Smith ML, etal: Free radical production and ischemic brain damage: influence of postischemie oxygen tension, fat J Dev Neurosci 9: , Akaike N, Kostyuk PG, Osipchuk YV: Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J Physiol (Load) 412: , Alkon DL, Rasmussen H: A spatial-temporal model of cell activation. Science 239: , Alps B J, Hass WK: The potential beneficial effect of nicardipine in a rat model of transient forebrain ischemia. Neurology 37: , Andin6 P, Jacobson I, Hagberg H: Calcium uptake evoked by electrical stimulation is enhanced postischemically and precedes delayed neuronal death in CA l of rat hippocampus: involvement of N-methyl-D-aspartate receptors. J Cereb Blood Flow Metab 8: , Ban M, Tonal T, Kohno T, et al: A flavonoid inhibitor of 5-1ipoxygenase inhibits leukotfiene production following ischemia in gerbil brain. Stroke 20: , Bazan N, Squinto S, Braquet P, et al: Platelet-activating factor and polyunsaturated fatty acids in cerebral ischemia or convulsions: intracellular PAF-binding sites and activation of a FOS/JUN/AP- 1 transcriptional signaling system. Lipids 26: , Bazan NG: Free arachidonic acid and other lipids in the nervous system during early ischemia and after electroshock. Adv Exp Med Biol 72: , Beckman JS, Beckman TW, Chen J, et al: Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Aead Sci USA 87: , Berridge M J: Inositol triphosphate and diacylglycerol as second messengers. Biochem J 220: , Berridge MJ: Inositol triphosphate and diacylglycerol: two interacting second messengers. Anna Rev Biochem 56: , Betz AL: Identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries. J Neurocbem 44: , Bevan S, Wood JN: Arachidonic-acid metabolites as second messengers. Nature 328:20, Bhakoo KK, Crockard HA, Lascelles PT: Regional studies of changes in brain fatty acid following experimental ischemia and reperfusion in the gerbil. J Neurochem 43: , Bielenberg G, Wagner G: PAF antagonists reduce infarct size in focal ischemia in the rat brain, in Krieglstein J, Oberpichler H (eds): Pharmacology of Cerebral Isehemia. Stuttgart: Wissenschaflliche Verlagsgesellschaft, 1990, pp Birkle DL, Kurian P, Braquet P, et al: Platelet-activating factor antagonist BN52021 decreases accumulation of free fatty polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock. J Neurothem 51: , 1988 J. Neurosurg. / Volume 77/September,

14 B. K. Siesj6 18. Biros MH, Dimlich RVW, Barsan WG: Postinsult treatment of ischemia-induced cerebral lactic acidosis in the rat. Ann Emerg Med 15: , Black KL, Hoff JT: Leukotrienes increase blood-brain barrier permeability following intraparenchymal injections in rats. Ann Neurol 18: , Boisvert DP: In vivo assessment of hydroxyl free radical production in the brain. J Cereb Blood Flow Metab 11 (Suppl):637, Bolli R, Jeroudi MO, Patel BS, et al: Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Evidence that myocardial "stunning" is a manifestation of reperfusion injury. Circ Res 65: , Braquet P, Touqui L, Shen TY, et al: Perspectives in platelet-activating factor research. Pharmacol Rev 39: , Braughler JM, Hall ED: Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. 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