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Life, 52: 247–254, 2001 c 2001 IUBMB Copyright ° 1521-6543/01 $12.00 + .00 IUBMB

Critical Review Mitochondrial Involvement in Acute Neurodegeneration Tadeusz Wieloch Laboratory for Experimental Brain Research, Lund University Hospital, Lund, Sweden

INTRODUCTION Acute neuronal degeneration occurs when brain tissue isexposed for a prolonged period of cerebral blood  ow below a certain critical level, as is seen in man following stroke, brain trauma, and cardiac failure (1). Also hypoxia will lead to cell death, particularly in neonates. A decrease in blood glucose, hypoglycemia, caused by hepatic failure or an overdose of insulin leads to selective neuronal death (2). In any one of these conditions, failure of mitochondrial function is a central event leading to cell death and is preceded by insufŽ cient ATP formation to meet cellular needs, an aberrant calcium homeostasis and release of mitochondrial factors that directly cause cell death. Because mitochondrial involvement in ischaemic brain damage has been recognised for some time, and because mitochondria are the key players in the activation of apoptosis, a question often posed is whether ischaemic and hypoglycemic brain injury is apoptosis or necrosis. As often, when deŽ nitions of processes such as apoptosis that cannot be deŽ ned by precise mechanisms are discussed, con icting results emerge. There are reports supporting the notion that ischaemic cell death occurs in an apoptotic fashion, yet others demonstrate clear necrotic morphology. Yet others suggest that a continuum between the two events or propose that ischaemic neuronal death has unique features. If we adopt the deŽ nitions of apoptosis and necrosis as presented by Kerr and coworkers (3), necrosis is thought to be a passive process. Solutes are equilibrated across cell membranes; cells and organelles swell and rupture leading to in ammation. Also, many cells die, causing regions of degenerated tissue. In apoptosis, physiological signals outside or inside cells activate processes that will cause the demise of the cell, which will be rapidly eliminated by phagocytosis. In apoptosis, cells die in a regulated fashion, with preserved mitochondrial integrity, because apoptosis requires energy. The morphological criteria Accepted 14 September 2001. Address correspondence to Tadeusz Wieloch, Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, BMC A13, 221 84 Lund, Sweden. Fax: C46462220615 . E-mail: Tadeusz.wieloch@ expbr.lu.se

of apoptosis encompass cell shrinkage, chromatin condensation and fragmentation, membrane blebbing, and the disintegration of the cell into membrane-enclosed apoptotic bodies (3). In the case of ischaemic and hypoglycemic brain damage, it has to be understood that the cell death is indeed a very dynamic process, and proceeds with variable speed and intensity, depending on the duration and severity of the insults. Today, it is also clear that the morphological changes of ischaemic neuronal death have both features of apoptosis and necrosis. For example, following ischaemia and hypoglycemia, cells shrink rather than swell indicative of apoptosis, but then, and importantly, no blebbing is observed and no apoptotic bodies are formed. Moreover, round globules of chromatin are formed, suggestive of apoptotic bodies. However, several detailed studies have demonstrated that the chromatin is not surrounded by a membrane, and that this process takes place once gross degradation of cell membranes has occurred (4). In some instances, ischaemic cell death may take up to several days to develop. In these cells the plasma membrane is intact and mitochondrial structure is normal in the early postischemic stages. This would be a hallmark of apoptosis, but then DNA fragmentation occurs once cell membranes are dissolved, and these cells die in groups with prominent in ammation, strongly suggesting a necrotic process. The granule cells of the dentate gyrus are particularly sensitive to insulin-induced hypoglycemia, and die within 3 h after transient hypoglycemic insult. During and following hypoglycemia, cells shrink, suggesting an apoptotic process, while mitochondria swell, indicative of necrosis. Later, chromatin is condensed and an oligonucleosomal ladder can be observed when DNA is analysed by agarose gel electrophoresis. Still, the condensed and globular chromatin is found in cells where cell membrane is completely dissolved. Ischaemic cell death has been proposed to be a continuum of changes between necrosis and apoptosis (5) where the level of ATP in the cell is one critical factor (6). Clearly, if we adopt the morphological criteria of apoptosis and necrosis as stated before, cell death following transient global (7–9) or focal (4) ischaemia or insulin induced-hypoglycemia in the adult rodent does not have the 247

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morphological characteristics of classical apoptosis or necrosis. Cell death due to cerebral ischaemia and hypoglycemia is evidently associated with its own particular morphological characteristics. As with many other cellular events, such as exocytosis, cell division, and indeed apoptosis that occur concomitant with certain morphological characteristics, the particular morphological changes associated with ischaemic and hypoglycemic cell death have biochemical explanations. From the aspects of cell morphology, these reactions are apparently different from those occurring in classical apoptosis and necrosis, and in addition vary depending on the density and duration of insults and the cell type affected. In this review, the involvement of mitochondria in this process are highlighted. Ischemia and hypoglycemia activate multiple reactive processes including metabolism, cell repair, plasticity, and cell degeneration. If the insult is sufŽ ciently severe, repair processes will be hampered and degenerative processes activated passing a threshold of damage that is not compatible with survival and cells will die. This simplistic model of cell death in the CNS suggests that death occurs as a result of a cooperative and integrated effect of different mechanisms acting in concert, as a sandwich of reactions where individual detrimental processes are “the slices.” The mitochondria-mediated processes can be envisaged as one such “sandwich slice.”

Mitochondria and ATP Synthesis Cerebral ischaemia and hypoglycemia can be readily studied in animal models. Stroke is mimicked by a permanent or transient occlusion of the middle cerebral artery, whereas global ischaemia caused by cardiac arrest is induced by occlusion of carotid and vertebral arteries. A well-characterised model of severe hypoglycemia induced by an overdose of insulin has been described (2). Ischaemic and hypoglycemic damage is a threshold phenomenon. If global ischaemia is of relatively short duration (5– 20 min), recovery of cell metabolism takes place upon reperfusion. Still, selective neuronal damage develops in discrete brain areas, albeit at different periods of reperfusion (10). Depending on the magnitude of the decrease in cerebral blood  ow, 5 to 60 min of ischaemia is required for the induction of cell death. Similarly, at least 10 min of severe hypoglycemia (deŽ ned as brain isoelectricity) is required for cell death to occur. Following middle cerebral artery occlusion (MCAO), ischaemia of variable degree is induced depending on vascular architecture, animal species and strains (11). A decrease to below 10 to 20% of control levels inevitably leads to tissue infarction, while following a decrease to 20 to 40% of control levels, the supply of oxygen and nutrients will support energy production for some time, but if prolonged (2–4 h), tissue infarction ensues (12). This underperfused tissue, also called ‘the penumbra’ (13), is at risk of becoming included in the core of the brain infarct (14). Because the tissue in the penumbra can be salvaged by pharmacological

interventions, this area is of particular importance for drug development against stroke. The brain mitochondria consume approximately 20% of the oxygen transported by the blood, although the brain only comprises approximately 2% of the body weight. Normally, mainly glucose oxidation supports mitochondrial ATP production (Fig. 1). The ATP is to a large extent consumed by the plasma membrane NaC /KC ATPase, which maintain a high extracellular and low intracellular sodium concentration, and a low extracellular and high intracellular potassium concentration, a gradient utilised for interneuronal and intracellular signalling. The gradient is also used by membrane transporters to translocate transmitters, particularly glutamate, nutrients, ions, and cellular components between different cellular compartments. Following global ischaemia, mitochondrial ATP synthesis is inhibited and available ATP is rapidly consumed (Fig. 1). The blood glucose and small amounts of glycogen and high energy phosphate compounds such as phosphocreatine and adenosine phosphates, can keep up neuronal function for 1 to 2 min (1). Within 2 min, the plasma membrane is depolarised, releasing potassium into the extracellular space and allowing sodium and calcium to enter the cell, resulting in equilibration of the ionic gradients across the neuronal plasma membrane (1). Persistent membrane depolarisation occurs in global ischaemia and in the ischaemic core following MCAO, and neurons in a depolarised state succumb due to activation of catabolic reactions. On the other hand, in the penumbra the residual blood  ow will supply neurons with glucose keeping up mitochondrial ATP production, and maintaining membrane pumps partially activated. This will be re ected in transient membrane depolarisations (15). Transient membrane depolarisations per se are not lethal if sufŽ cient energy is present for the neurons to activate ion pumping and support cell protection and repair. (16). However, in the penumbra the mitochondria ATP production is limited (17), and prolonged membrane depolarisations of neurons will progressively activate the cell death processes, and the infarct will expand. Evidently, mitochondrial function and ATP synthesis is crucial for the maintenance of plasma membrane integrity, and critical for the prevention of tissue damage.

Mitochondria and Calcium The brain extracellular calcium concentration is around 1.2 mM, which is 10,000-fol d higher than the intracellular concentration. This gradient is upheld by the low membrane permeability for calcium, and efŽ cient calcium extruding mechanisms. Of importance for ischaemic and hypoglycemic brain damage are the NMDA receptors, which gate calcium ions, and can cause cell death when overactivated (18). Driven by a negative potential inside the mitochondrial matrix, mitochondria have a high capacity to accumulate calcium ions. The accumulated calcium can be released through a sodium/calcium exchange mechanism (19) or by opening of the mitochondrial permeability transition pore (mtPTP) (20).

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The loss of mitochondrial membrane potential during ischaemia and hypoglycemia will affect mitochondrial calcium homeostasis. A partial or complete inhibition of ATP synthesis will depress or inhibit the ATPases. The resulting increase in intracellular sodium levels will reverse the NaC /Ca2C exchanger leading to in ux of calcium down its concentration gradient. Calcium will also enter the cytosol through the voltage and receptor operated calcium channels. As long as the mitochondrial proton gradient can be upheld, the elevated cytosolic calcium will be taken up by the mitochondria, but eventually the accumulated calcium will activate mtPTP releasing stored calcium. In addition, sodium entry into the cell will release mitochondrial calcium through the mitochondrial NaC /Ca2C exchanger. During ischaemia the intracellular calcium concentrations will increase to as high as 50 to 100 ¹M and will activate many if not all calcium-dependent processes (1), including proteases and lipases that contribute to the development of ischaemic and hypoglycemic cell death.

Figure 1. The mitochondrial ATP production is utilised by the cell to form ionic gradients across plasma membrane, and to allow for transport of solutes and transmitters (top). Glucose oxidation leads to activation of the electron transport chain (ETC) and the formation of the proton gradient across the inner mitochondrial membrane (mito), used by the ATP synthase (ATPsynth) to form ATP. ATP is utilised in the formation of the sodium and potassium gradient (by the Na/K ATPase) across the plasma membrane, which can be used in the activation of (NaCh)-voltage operated sodium channel; (CaCh)-voltage and receptor operated calcium channel, and for transporting ions (Na/Ca) and transmitters (Tr). Ischaemia or hypoglycemia leads to inhibition of ATP formation (below) and inhibition of ATPases, leading to an increase in cellular sodium and calcium concentrations as well as in release of neurotransmitters from intracellular pools.

Mitochondria, Oxidants, and Free Radicals Free radicals are formed in the penumbra during ischaemia/ reperfusion. Of particular relevance for ischaemic cell death are nitric oxide (NO), the superoxide anion (O?¡ 2 ), and the hydroxyl radical (OH¡ ). Nitric oxide reacts with superoxide to form the highly reactive oxidant peroxynitrate (ONOO¡ ) and superoxide dismutase forms the oxidant hydrogen peroxide from superoxide (21). If free radical formation is overwhelming and override the antioxidant defence of the cell, the oxidants will react with macromolecules leading to loss of membrane integrity, cytoskeletal damage, and breaks and mutations in the DNA, contributing to cell death (22). Free radicals may also trigger apoptosis by activating mitochondrial permeability transition (23), and by promoting the release of cytochrome c (24). The mitochondria generate the largest amounts of superoxide in neurons. The electron transport chain constantly leaks electrons that react with molecular oxygen, forming superoxide anion radicals. In the penumbra during MCAO ischaemia, hypoxia/ischaemia partially inhibits the electron transport chain, which becomes reduced, favouring electron transfer from complex I and II to molecular oxygen (25). The importance of free radicals in ischaemic brain damage has been demonstrated by the treatment with free radical scavengers such as nitrones that effectively decrease infarct size after MCAO (26). In defence against the formation of superoxide, high levels of glutathione and manganese superoxide dismutase (MnSOD) are speciŽ cally found in mitochondria. The importance of mitochondrial superoxide in cell death was demonstrated in MNSOD ¡/¡ mice. In these mice, transient occlusion of the MCA led to increased infarct size (24). We can conclude here that in the penumbra tissue of brains subjected to focal ischaemia, mitochondrial dysfunction leads to glutamate and calcium toxicity as well as free radical damage.

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Mitochondria and Apoptosis Mitochondrial involvement in apoptosis has been extensively studied during recent years (27, 20). Mitochondrial apoptosis encompasses a signalling process whereby calcium and free radicals as well as changes in levels, location, and phosphorylation of Bcl-like proteins stimulate the release of factors (cytochrome c, Smac/DAIBLO, AIF), residing in the space between the inner and outer mitochondrial membranes, and that subsequently lead to the activation of executioners of apoptosis (proteases or DNAses). The release of cytochrome c from mitochondria is a central process in mitochondria-induced apoptosis. Once released, cytochrome c forms a complex with APAF-1 and procaspase-9, which becomes activated by auto-proteolysis, and the complex then activates procaspase-3 to caspase-3. The inhibitors of apoptosis proteins (IAPs), NAIP and XIAP, prevent activation of this potentially lethal process at multiple points. The mechanism whereby cytochrome c is released is not completely understood. It occurs by either the formation of a pore in the outer membrane large enough to allow passage of cytochrome c, or by physical disruption of the outer membrane, as is discussed next (28). The Bcl-2-like proteins regulate the release of cytochrome c from the mitochondrial intermembrane space. This class of proteins comprises those that promote (Bax, Bak, Bcl-x-s) or prevent (Bcl-2, Bcl-x-l, Bcl-w) apoptosis. Several hypotheses for cytochrome c release have been proposed. A pore can be formed by oligomerization of Bax and/or Bak (28), or by the interaction of Bax with the voltage-dependent anion channel (VDAC), leading to the formation of a Bax-VDAC pore (29). The formation of either pore is prevented by the antiapoptotic Bcl-2-like proteins, which form heterodimers with Bax-like proteins. Bad, Bim, and Bid translocate to the mitochondria during apoptosis. Bim is bound to dyenin and actin, and can be released following dissolution of the cytoskeleton. Bid is cleaved by caspase-8 following TNF/Fas receptor activation, and the truncated Bid translocates to the mitochondrial membrane, enhancing cytochrome c release (27). Bad is bound to the 14-3-3 protein in its phosphorylated form, but is dephosphorylated by calcineurin and released from 14-3-3 and bound to mitochondria, activating apoptosis. In addition, Bax can be induced by p53. Alternatively, cytochrome c can be released by opening of the mitochondrial permeability transition pore (mtPTP), which is a protein complex spanning the inner and outer mitochondrial membranes (30). The main constituents of the pore are considered to be the adenine nucleotide transferase (ANT) and VDAC. The mtPTP is opened by the binding of cyclophilin D (CypD), a mitochondrial matrix speciŽ c cyclophilin, to ANT (31). In the opened state, the outer membrane can open (rupture) due to osmotic swelling, leading to the release of cytochrome c and AIF, while the inner membrane is intact. This allows some ATP synthesis to continue, which may be sufŽ cient to activate downstream apoptotic cascades. Depending on the severity of ATP depletion, mtPTP can induce classical apoptosis or necrosis (6, 32). Cyclosporin A (CsA), an inhibitor of calcineurin,

and its analogue MeValCsA, which does not inhibit calcineurin, both prevent the binding of CypD to ANT, and are inhibitors of mtPTP and cell death (31). MtPTP is activated under depolarising conditions and in the presence of high levels of calcium ions as well as oxidants (31), that is, factors generated during ischaemia and hypoglycemia. Are these biochemical apoptosis events (Fig. 2) relevant in ischaemic and hypoglycemic brain damage? Some recent reviews have dealt extensively with this issue (33). For example, in mice overexpressing the Bcl-2 and Bcl-x-l genes, respectively, infarct size decreases following MCAO (34). Also, transfection of the Bcl-2 gene into cortex following ischaemia also diminishes cell death (36), whereas an increased infarct size is seen in Bcl-2 ¡/¡ mice (35). Dyenin is degraded following ischaemia (37) that may result in release of Bim. Also, Bax is translocated to mitochondria following ischaemia (38). There is a clear

Figure 2. Events that may take place during mitochondriainduced neuronal death, following cerebral ischaemia and hypoglycemia. During ischaemia calcium ions enter cells and the mitochondria, activating mitochondrial pores such as mtPTP. Also reactive oxygen species (ROS) and oxidants are formed that stimulate pore opening. Pore opening leads to release of mitochondrial calcium into the cytosol and to calcium toxicity. Pore opening also enhances ROS formation and free radical damage. Following ischaemia the proapoptotic protein Bax is translocated to membranes and release of cytochrome c (cyt c) from the mitochondria is stimulated. Bax oligomerisation is further stimulated by translocation of dephosphorylated Bad to the mitochondria. When released into the cytosol, cytochrome c binds to apoptosomal proteins that activate caspase-3. Caspase-3 activation leads to cell death by degradation of DNA repair enzymes, degradation of the cytoskeleton, and activation of endonucleases. Caspase-3 can be activated by caspase-8. Calpains can activate cathepsins that degrade Bid, thereby promoting apoptosis.

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release of cytochrome c during and following global and focal ischaemia (39, 40). Neuroprotection has been seen following overexpression of XIAP and NAIP (41). Following ischaemia and hypoglycemia, there is an imbalance in cell signalling affecting protein phosphorylation, which affects the phosphorylation states of Bcl-2/Bax proteins (42). Glutamate receptor activation contributes to ischaemic and hypoglycemic cell death, and GABA and adenosine receptors are protective, implying second messenger and protein kinase/ phosphatase systems in the cell death process. Also, the mitogenactivated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the p38 mitogen-activated protein kinases (p38s) are activated following ischaemia and hypoglycemia. Inhibition of the ERK activator MEK-1 by PD98059 (43) and inhibition of p38 by SB 203580 provide neuroprotection in a model of focal ischaemia (44). Neurotrophins protect against ischaemic cell death (45), possibly by activating adaptor protein interactions, leading to kinase activation, including the phosphatidylinositol-3-kinase (PI(3) kinase) pathway. PI(3) kinase activates the AKT kinase that phosphorylates the pro-apoptotic Bad (46), thereby preventing mitochondria-induced apoptosis. Following ischaemia AKT phosphorylation is diminished, suggesting that neurotrophin signalling and Bad phosphorylation may be depressed after ischaemia (47). Calcineurin is a calcium calmodulin-dependent serine/ threonine phosphatase that has been shown to induce cell death

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by dephosphorylating Bad (48), or prevention of Bcl-2 binding (49). Calcineurin is inhibited by the immunosupressive compound CsA and by FK506. Both FK506 and CsA can decrease infarct size in the rat when given up to 3 h into reperfusion following MCAO (50, 51), while following global ischaemia in the rat only CsA is neuroprotective (52). The protective effect of CsA and FK506 treatment could therefore be due to Bad dephosphorylation or preventing Bcl-2 binding to cell membranes. Alternatively, as will be discussed next, CsA also may be protective by inhibiting mitochondrial permeability transition (53, 54). Indeed, activation of mtPTP contributes to ischaemic cell death because MeValCsA, a selective blocker of the mtPTP, diminishes infarct size following MCAO (54). The effect of CsA and MeValCsA in ischaemia could therefore be to prevent detrimental cytochrome c and calcium release through the mtPTP and the activation of downstream apoptotic cascades and calciumdependent toxic mechanisms, respectively. Perhaps the most compelling evidence on the involvement of mtPTP in acute neurodegeneration in vivo comes from studies on the effects of insulin-induced hypoglycemia (2). During hypoglycemic isoelectricity when membranes are depolarised due to energy shortage, calcium and sodium ions  ood the cell interior, and dendrites and mitochondria are dramatically swollen, (Fig. 3). In animals treated with CsA prior to hypoglycemia, dendrites are swollen to the same degree as in vehicle-treated animals, yet mitochondria appear to be condensed with normal cristae (53). CsA provides full protection against damage,

Figure 3. Activation of mitochondrial permeability transition in vivo. An electron micrograph of the distal dendrites of the dentate gyrus granule cells in control animals showing a dense neuropil with condensed mitochondria (arrowheads). During insulin-induced hypoglycemia with 30-min isoelectricity (Iso), dendrites swell (star) as do mitochondria. Following treatment with cyclosporin A (50 mg/kg i.p.) (IsoCCsA) prior to membrane depolarisation, isoelectric brains display mitochondria that look similar to those in control brain, yet dendrites remain swollen.

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strongly suggesting that mtPTP activation is responsible for the cell death induction. The calcineurin pathways should not be of crucial importance because FK506 did not provide neuroprotection. Of interest is the fact that cellular calcium levels during hypoglycemia increase similarly in CsA- and vehicle-treated animals. Activation of Proteases. There is ample evidence showing that events downstream from mitochondrial release ofcytochrome c are activated during ischaemia. In the brain, the presence of caspases in neurites of cultured neurons, and the degradation of receptors by caspases, suggest that caspases may also play a role in neurotransmission and in neuronal plasticity (55). Caspase-3 is also an executioner of neuronal apoptosis and is activated during and following ischaemia. Inhibition of caspase-3 can diminish delayed neuronal death in the rat following global ischaemia (56) and can decrease damage in focal ischaemia (57). Following mild focal ischaemia, protection was attained when inhibition was introduced as late as 6 h after MCAO. Also, caspase-9 is released from mitochondria during ischaemia (58). Moreover, procaspase-8 is found in cortical neurons and is activated after MCAO (59). Mice with a defective FAS expression show smaller infarcts following MCAO than wild-type mice (60). This suggests that caspase-8-mediated cell death may occur in the penumbra. Caspase-12 can degrade Bclx-l and induce apoptosis. However, deletion of the caspase-12 gene does not diminish neuronal or astrocytic death following MACO ischaemia (61), and caspase-12 may therefore not be essential for ischaemic neuronal death. The cathepsins participate in the turnover of cellular proteins and organelles, but have recently been implicated in apoptosis through a process that involves cleavage of Bid (62). Activation of cathepsins by calpains during ischaemia has been demonstrated (63), and cathepsin inhibitors can diminish ischaemic damage in primates (64). DNA Fragmentation. In addition to cytochrome c, caspase2, -3, and -9, mitochondria harbour proteins that actively participate in the killing process, such as apoptosis-inducing factor (AIF) (65), that cleaves DNA producing fragments of high molecular weight. Also, caspase-3 activates the caspaseactivated DNase (CAD) by proteolysis of ICAD. DNA fragmentation in ischaemic damage has been the subject of ample discussions, see (33). Of importance in this context is that DNA is degraded differently from what is seen in classical apoptosis mediated by CAD (66). This suggests that other DNases are activated following ischaemia. It is also evident that DNA fragmentation occurs late after ischaemia and is seen after frank neuronal degeneration (67). Following cellular DNA damage, p53 is activated promoting DNA repair or inducing apoptosis, by activating either of three classes of genes: the cyclin-dependent kinase (CDK) inhibitor p21; DNA repair genes such as gadd45; and apoptosis genes such as bax, fas, and death receptor 5 (68, 69). Mice with the p53 gene deleted have larger infarcts following MCAO (70), and p53 is markedly expressed in resistant neurons following

global ischaemia (71). The data suggest that the DNA repair promoting property of p53 is more important following brain ischaemia than its promotion of apoptosis. CONCLUSIONS The mitochondria are important in neuronal function and survival by supplying energy to the cell, regulating calcium homeostasis, and by controlling the activation of apoptosis. Also, by virtue of their high oxygen consumption, mitochondria produce large amounts of free radicals that are normally scavenged by cellular defence mechanisms. During ischaemia and hypoglycemia these homeostatic mechanisms fail, and mitochondria contribute to cell death by enhancing calcium toxicity, free radical damage, proteolysis, and DNA fragmentation. The relative contribution of the different detrimental mechanisms to cell death depends on the density and duration of ischaemia and hypoglycemia, and will be o by morphological changes that are unique for the particular insult. ACKNOWLEDGEMENTS The author thanks Maj-Lis Smith and Gustav Mattiasson for comments and suggestions. Supported by the Swedish Medical Research Council 8644. REFERENCES 1. Siesjo, B. K. (1992) Pathophysiology and treatment of focal cerebral ischaemia. Part I: Pathophysiology. J. Neurosurg. 77, 169–184. 2. Auer, R. N., Olsson, Y., and Siesjo, B. K. (1984) Hypoglycemi c brain injury in the rat. Correlation of density of brain damage with the EEG isoelectric time: a quantitative study. Diabetes 33, 1090–1098. 3. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: a basic biological phenomeno n with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. 4. van Lookeren Campagne, M., and Gill, R. (1996) Ultrastructural morphological changes are not characteristic of apoptotic cell death following focal cerebral ischaemia in the rat. Neurosci. Lett. 213, 111–114. 5. Martin, L. J., Al-Abdulla, N. A., Brambrink, A. M., Kirsch, J. R., Sieber, F. E., and Portera-Cailliau, C. (1998) Neurodegeneratio n in excitotoxicity, global cerebral ischaemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis. Brain Res. Bull. 46, 281–309. 6. Nicotera, P., Leist, M., and Manzo, L. (1999) Neuronal cell death: a demise with different shapes. Trends Pharmacol. Sci. 20, 46–51. 7. Colbourne, F., Sutherland, G. R., and Auer, R. N. (1999) Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischaemia. J. Neurosci. 19, 4200–4210. 8. Deshpande, J., Bergstedt, K., Linden, T., Kalimo, H., and Wieloch, T. (1992) Ultrastructural changes in the hippocampa l CA1 region following transient cerebral ischaemia: evidence against programmed cell death. Exp. Brain Res. 88, 91–105. 9. Petito, C. K., Torres-Munoz, J., Roberts, B., Olarte, J. P., Nowak, T. S., and Pulsinelli, W. A. (1997) DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischaemia in the rat. J. Cereb. Blood Flow Metab. 17, 967–976. 10. Smith, M. L., Auer, R. N., and Siesjo, B. K. (1984) The density and distribution of ischaemic brain injury in the rat following 2–10 min of forebrain ischaemia. Acta Neuropatho l (Berl) 64, 319–332. 11. Ginsberg, M. D., and Busto, R. (1989) Rodent models of cerebral ischaemia. Stroke 20, 1627–1642.

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