Pathobiology of ischaemic stroke: an integrated view

REVIEW Pathobiology of ischaemic stroke: an integrated view Ulrich Dirnagl, Costantino Iadecola and Michael A. Moskowitz Brain injury following transient or permanent focal cerebral ischaemia (stroke) develops from a complex series of pathophysiological events that evolve in time and space. In this article, the relevance of excitotoxicity, peri-infarct depolarizations, inflammation and apoptosis to delayed mechanisms of damage within the peri-infarct zone or ischaemic penumbra are discussed. While focusing on potentially new avenues of treatment, the issue of why many clinical stroke trials have so far proved disappointing is addressed.This article provides a framework that can be used to generate testable hypotheses and treatment strategies that are linked to the appearance of specific pathophysiological events within the ischaemic brain. Trends Neurosci. (1999) 22, 391–397

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SCHAEMIC STROKE results from a transient or permanent reduction in cerebral blood flow that is restricted to the territory of a major brain artery. The reduction in flow is, in most cases, caused by the occlusion of a cerebral artery either by an embolus or by local thrombosis. With an incidence of approximately 250–400 in 100 000 and a mortality rate of around 30%, stroke remains the third leading cause of death in industrialized countries. In the USA alone, four-million survivors are coping with its debilitating consequences1. Major breakthroughs in stroke pathophysiology have prompted a world-wide campaign to enlighten patients about the early symptoms of stroke (the ‘brain attack’ campaign). In conjunction with these efforts, positive results were reported for the first time in a major clinical trial2. At the same time, however, numerous drug trials have reported disappointing results, suggesting that in stroke patients, neuroprotection might not be as straightforward as it is in experimental animals. Despite these disappointments (see Box 1), it is likely that successful treatment of the ischaemic brain will be achieved in the near future, as injury mechanisms established using in vitro and in vivo models are relevant to mechanisms seen in humans. In this article, evidence will be presented that ischaemic brain injury results from a complex sequence of pathophysiological events that evolve over time and space. The major pathogenic mechanisms of this cascade include excitotoxicity, periinfarct depolarizations, inflammation and programmed cell death (Fig. 1).

Energy failure and excitotoxicity Brain tissue has a relatively high consumption of oxygen and glucose, and depends almost exclusively on oxidative phosphorylation for energy production. Focal impairment of cerebral blood flow restricts the delivery of substrates, particularly oxygen and glucose, and impairs the energetics required to maintain ionic gradients3. With energy depletion, membrane potential is lost and neurones and glia depolarize4. Consequently, somatodendritic as well as presynaptic voltage-dependent Ca21 channels become activated and excitatory amino acids are released into the extracellular space (Fig. 2). At the same time, the energy-dependent processes, such as 0166-2236/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.

presynaptic reuptake of excitatory amino acids, are impeded, which further increases the accumulation of glutamate in the extracellular space. Activation of NMDA receptors and metabotropic glutamate receptors contribute to Ca21 overload5, the latter via phospholipase C and Ins(1,4,5)P3 signalling. As a result of glutamatemediated overactivation, Na1 and Cl2 enter the neurones via channels for monovalent ions (for example, the AMPA receptor–channel). Water follows passively, as the influx of Na1 and Cl– is much larger than the efflux of K1. The ensuing oedema can affect the perfusion of regions surrounding the core of the perfusion deficit negatively, and also have remote effects that are produced via increased intracranial pressure, vascular compression and herniation. Brain oedema, which gives rise to the earliest markers for the ensuing pathophysiology, studied with MRI and computed tomography, is one of the major determinants of whether the patient survives beyond the first few hours after stroke. An increase in the universal second messenger, Ca21, is thought to initiate a series of cytoplasmatic and nuclear events that impact the development of tissue damage profoundly, such as activation of proteolytic enzymes that degrade cytoskeletal proteins, for example, actin and spectrin6, as well as extracellular matrix proteins, such as laminin7. Activation of phospholipase A2 and cyclooxygenase generates free-radical species that overwhelm endogenous scavenging mechanisms, producing lipid peroxidation and membrane damage. The important role of oxygen free-radicals in cell damage associated with stroke is underscored by the fact that even delayed treatment with free-radical scavengers can be effective in experimental focal cerebral ischemia (for example, see Ref. 8). In addition, the overproduction of radical-scavenging enzymes protects against stroke (for example, see Refs 9,10) and animals that are deficient in radical-scavenging enzymes are more susceptible to cerebral ischaemic damage11,12. Oxygen free-radicals also serve as important signalling molecules that trigger inflammation and apoptosis (see below). Nitric oxide (NO) synthesized by the Ca21-dependent enzyme, neuronal nitric-oxide synthase (NOS) reacts with a superoxide anion to form the highly reactive species, peroxynitrite, that promotes tissue damage13,14. By contrast, PII: S0166-2236(99)01401-0

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Ulrich Dirnagl is at the Dept of Neurology, Charité Hospital, 10098 Berlin, Germany, Costantino Iadecola is at the Laboratory for Cerebrovascular Biology and Stroke, Dept of Neurology, University of Minnesota Medical School, Minneapolis, MN 55455, USA, and Michael A. Moskowitz is at the Stroke Research Laboratory, Harvard Medical School, Massachusetts General Hospital, and Neurosurgical Service and Dept of Neurology, Harvard Medical School and Massachusetts General Hospital, Charlestown, MA 02129, USA.

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Box 1. Why have so many clinical stroke trials failed? Despite the large number of therapeutic interventions that decrease damage in experimental animals, a number of clinical trials have produced negative results when testing the same agents (see, for example, Refs a,b). How can we explain this apparent discrepancy between bench and bedside studies? (See also Refs c–f.) Timing In some failed clinical trials, treatments were administered outside the temporal window of efficacy for the drug being testeda. While in animal models the onset of ischaemia and reperfusion can be precisely defined, in humans this is not always possible. For example, the onset of symptoms might not coincide with the onset of cerebral ischaemia, or there might be a delay before the patients become aware of these symptoms as in stroke at night or stroke syndromes characterized by unawareness of deficits. Thus, it is difficult to define accurately the time window in which a certain drug might be effective in each patient. This problem is compounded by the difficulty in transporting patients into the hospital quickly. In order to address these problems, several steps have to be taken. First, the ongoing efforts to educate the public about early symptoms and early treatment should continue. Second, imaging strategies (such as MRI-based approaches) should be used to assess the stage of evolution of brain injury in each patientg. Third, treatment approaches are needed to address injury mechanisms in the late stages of cerebral ischaemia, for example, post-ischaemic inflammation and apoptosis. Age and associated illnesses Most experimental studies are conducted on healthy, young animals under rigorously controlled laboratory conditions. However, the typical stroke patient is elderly with numerous risk factors and complicating diseases (for example, diabetes, hypertension and heart diseases). Therefore, emphasis should be placed on developing animal models that reflect the substrate in which human cerebral ischaemia occurs more accurately. Morphological and functional differences between the brain of humans and animals Although the basic biology of cerebral ischaemia is comparable between different species, there are important species differences in brain structure, function and vascular anatomy. Cerebral energy metabolism and, thus, blood flow in mammals is inversely related to their body weight. For example, in the rat, glucose and oxygen metabolism, as well as blood flow, are three times as high as in humans. Neuronal and glial densities are also quantitatively different in various mammals. Furthermore, the human brain is gyrated and larger. There are also large differences in the gross cerebral vascular anatomyh. Some rodents do not have a complete circle of Willis (gerbils), while

others can have even more effective collaterals between large cerebral vessels than humans (for example, rats). Accordingly, the size, anatomical distribution and temporal evolution of the infarct differ among speciesi. The penumbra zone, although well established in rodents, is less well characterized in humans and might be considerably smaller. The effect of neuroprotective treatments is also species dependent and rodents seem to be more amenable to neuroprotection than higher mammals. Therefore, greater emphasis should be placed on studying experimental stroke and neuroprotection in species that are phylogenetically closer to humans. Evaluation of efficacy In experimental animals, infarct size is measured quantitatively in order to evaluate treatment after injury and is rarely determined beyond seven days after ischaemic insult. By contrast, functional scores (National Institute of Health stroke scale, Barthel index, etc.) are typically used in humans and are assessed three or six months after stroke. These scores are of great importance because they reflect the extent of neurological deficits. However, functional scores are less amenable to statistical evaluation and might be less sensitive than markers of infarct volume or histopathology. Therefore, objectives of ischaemic damage should be developed and tested soon after ischaemic insult and at a later stage [for example, diffusion-weighted and flow-sensitive imaging, functional and spectroscopical imagingg, PET (Ref. j), etc.]. Plasma concentration of drugs and side-effects Most anti-excitotoxic compounds cause psychomimetic or cardiovascular side-effects, or bothk. This severely limits the tolerated dose such that levels in humans reach only a fifth of the effective concentrations observed in rodent models. It is, therefore, important to design drugs with better safety profiles and more-favourable pharmacokinetics with fewer side-effects. References a b c d e f g h

The American Nimodipine Study Group (1992) Stroke 23, 3–8 Scott, P. et al. (1996) Stroke 27, 1453–1458 Del Zoppo, G.J. (1995) J. Intern. Med. 237, 79–88 Del Zoppo, G.J. (1998) Neurology 51, S59–S61 Grotta, J. (1995) J. Intern. Med. 237, 89–94 Adams, R.J. et al. (1995) Stroke 26, 2216–2218 Koroshetz, W.J. (1996) Ann. Neurol. 39, 283–284 Luginbühl, H. (1966) in Cerebral Vascular Diseases. Fifth Conference (Millikan, C.H., Siekert, R.G. and Whisnant, J.P., eds), pp. 3–27, Grune and Stratton i Tagaya, M. et al. (1997) Stroke 28, 1245–1254 j Heiss, W.D. and Podreka, I. (1993) Cerebrovasc. Brain Metab. Rev. 5, 235–263 k Lees, K.R. (1997) Neurology 49, S66–S69

increased production of NO by endothelial cells can protect the tissue by improving the microcirculation15. Mitochondria, which are an important source of reactive oxygen species, are impaired by free-radicalmediated disruption of the inner mitochondrial membrane and the oxidation of proteins that mediate electron transport, H1 extrusion and ATP production16. The mitochondrial membrane becomes leaky, partly owing to the formation of a mitochondrial permeability transition pore, which promotes mitochondrial swelling, the cessation of ATP production and an oxygen free-radical burst17. Cytochrome C is released from mitochondria18 and provides a trigger for apoptosis (see below). The haemodynamic, metabolic and ionic changes described above do not affect the ischaemic territory homogeneously. In the centre or core of the perfusion deficit (Fig. 3), cerebral blood flow is 20% below normal19. Permanent and anoxic depolarization develops minutes after the onset of ischaemia. Cells are killed rapidly by lipolysis, proteolysis, the disaggregation of microtubules that follows total bioenergetic failure and the ensuing breakdown of ion homeostasis20. Between 392

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this lethally damaged core and the normal brain lies the penumbra, an area of constrained blood flow with partially preserved energy metabolism19,21,22. Given time and without treatment, the penumbra can progress to infarction owing to ongoing excitotoxicity (see above) or to secondary deleterious phenomena, such as spreading depolarization, post-ischaemic inflammation and apoptosis (see below). It is, thus, evident that the prime goal of neuroprotection is to salvage the ischaemic penumbra. Although there is ample evidence that the penumbra exists in human stroke patients (see, for example, Refs 23,24), the extent and temporal dynamics of this area are less well defined: it might be smaller and exist for a shorter time period in humans25.

Glutamate receptors: the gateway to excitotoxicity The evidence presented above indicates that activation of glutamate receptors, through the attendant failure of ion homeostasis and increase in intracellular Ca21 concentration, is a major factor involved in initiating ischaemic cell death. A straightforward therapeutic approach, therefore, is to block the receptors that are

Excitotoxicity Peri-infarct depolarizations

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Fig. 1. Putative cascade of damaging events in focal cerebral ischaemia. Very early after the onset of the focal perfusion deficit, excitotoxic mechanisms can damage neurones and glia lethally. In addition, excitotoxicity triggers a number of events that can further contribute to the demise of the tissue. Such events include peri-infarct depolarizations and the more-delayed mechanisms of inflammation and programmed cell death. The x-axis reflects the evolution of the cascade over time, while the y-axis aims to illustrate the impact of each element of the cascade on final outcome.

frequency of several events per hour and can be recorded for at least six to eight hours. As the number of depolarizations increases, the infarcts grow larger30. Drugs that reduce the number of depolarizations decrease infarct size31. As NMDA- as well as AMPA-receptor antagonists block peri-infarct depolarizations, some have attributed their neuroprotective potential to this effect. However, peri-infarct depolarizations have so far escaped detection by electrophysiological or functional imaging methods in humans, so that their relevance to human stroke pathophysiology remains unclear.

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activated by glutamate26. The NMDA receptor controls an ion channel that is permeable to Ca21, Na1 and K1. Antagonists at this receptor demonstrate robust neuroprotection when given before or at the time of occlusion of the middle cerebral artery (MCA) in models of permanent or temporary ischaemia. There is general agreement that the therapeutic time window of effectiveness of NMDA-receptor antagonists in these models is narrow, closing at around one or two hours after occlusion of the artery. The AMPA receptor gates Na1 and K1 conductances, and Na1 influx via the AMPA or kainate receptor induces Ca21 influx indirectly by depolarizing the resting membrane potential and subsequently alleviating the Mg21 block of the NMDA receptor. AMPAreceptor-antagonist treatment affords robust neuroprotection in several rodent models of focal cerebral ischaemia, which potentially exceeds the time window during which NMDA-receptor antagonists are effective27. Comparatively little is known about the role of metabotropic glutamate receptors in focal cerebral ischaemia; these do not gate ion channels but act via G proteins. There is growing evidence, however, that activation of group-II and group-III metabotropic glutamate receptors is neuroprotective28. It should be remembered that glutamate has an important physiological role as a neurotransmitter in the brain. Although blocking glutamate receptors protects against excitotoxicity, it can also have serious unwanted effects, such as psychotomimesis, respiratory depression or cardiovascular dysregulation. Some of the failures of clinical trials that have used glutamate-receptor antagonists (see Box 1) might be explained by these side-effects. By targeting subunits of glutamate receptors that are specifically upregulated in ischemia, more-selective drugs could be developed in the future that are clinically safer. Excitotoxicity is well established as an important trigger and executioner of tissue damage in focal cerebral ischaemia. Excitotoxic mechanisms can cause acute cell death (necrosis) but can also initiate molecular events that lead to a delayed type of cell death, apoptosis (see below). In addition, the intracellular signalling pathways activated during excitotoxicity trigger the expression of genes that initiate post-ischaemic inflammation, another pathogenic process that contributes to ischaemic injury. Thus, excitotoxicity is a prime target for stroke therapy. The therapeutic efficacy of strategies that inhibit excitotoxicity in vivo and in vitro, which, at present, focus on the inhibition of specific glutamate receptors, provides evidence for the pathophysiological role of excitotoxicity, while raising the hope that clinically relevant strategies can be developed that counteract this key pathophysiological entity.

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Peri-infarct depolarizations As outlined above, ischaemic neurones and glia depolarize owing to the shortage of energy supply, and the release of K1 and glutamate. In the core region of the affected brain tissue (Fig. 3), cells can undergo an anoxic depolarization and never repolarize. In penumbral regions (where some perfusion is preserved) cells can repolarize, but at the expense of further energy consumption. The same cells can depolarize again in response to increasing glutamate or K1 levels, or both, which accumulate in the extracellular space. Repetitive depolarizations, so-called ‘peri-infarct depolarizations’ occur29. Peri-infarct depolarizations have been demonstrated in mice, rat and cat stroke models, occur with a

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Fig. 2. Simplified overview of pathophysiological mechanisms in the focally ischaemic brain. Energy failure leads to the depolarization of neurones. Activation of specific glutamate receptors dramatically increases intracellular Ca21, Na1, Cl2 levels while K1 is released into the extracellular space. Diffusion of glutamate (Glu) and K1 in the extracellular space can propagate a series of spreading waves of depolarization (peri-infarct depolarizations). Water shifts to the intracellular space via osmotic gradients and cells swell (oedema). The universal intracellular messenger Ca21 overactivates numerous enzyme systems (proteases, lipases, endonucleases, etc.). Free radicals are generated, which damage membranes (lipolysis), mitochondria and DNA, in turn triggering caspase-mediated cell death (apoptosis). Free radicals also induce the formation of inflammatory mediators, which activate microglia and lead to the invasion of blood-borne inflammatory cells (leukocyte infiltration) via upregulation of endothelial adhesion molecules.

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of proinflammatory genes by inducing the synthesis of transcription factors, including nuclear factor-kB (Ref. 32), hypoxia inducible factor 1 (Ref. 33), interferon regulatory factor 1 (Ref. 34) and STAT3 (Ref. 35). Thus, mediators of inflammation, such as platelet-activating factor, tumour necrosis factor a (TNFa) and interleukin 1b (IL-1b), are produced by injured brain cells36. Consequently, the expression of adhesion molecules on the endothelial cell surface is induced37–40, including intercellular adhesion molecule 1 (ICAM-1), P-selectins and E-selectins41–43. Adhesion molecules interact with complementary surface receptors on neutrophils. The neutrophils, in turn, adhere to the endothelium, cross the vascular wall and enter the brain parenchyma. Macrophages and monocytes follow neutrophils, migrating into the ischaemic brain and becoming the predominant cells five to seven days after ischaemia (see Ref. 13 for

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Fig. 4. Inducible nitric-oxide synthase (iNOS) and cyclooxygenase 2 (COX2) immunoreactivity in the human brain following ischaemic stroke. Patients died 1–2 days after suffering an ischaemic stroke in the territory of the middle cerebral artery. After fixation, blocks of ischaemic cortex were paraffin embedded and sectioned (thickness 4 mm). (A) COX2-positive neurones at the border of the ischaemic lesion. (B) COX2-positive endothelial cells (arrowhead) and neutrophils (arrow) in the ischaemic lesion. (C) iNOS immunoreactivity in the wall of cerebral blood vessels. (D) iNOS positive neutrophils (arrow) in the ischaemic lesion. Scale bar, 50 mm.

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Fig. 3. The ischaemic penumbra. A brain region of low perfusion in which cells have lost their membrane potential terminally (‘core’) is surrounded by an area in which intermediate perfusion prevails (‘penumbra’) and cells depolarize intermittently (‘peri-infarct depolarization’). Note that from the onset of the focal perfusion deficit, the core and penumbra are dynamic in space and time (see also Fig. 6). Perfusion thresholds exist below which certain biochemical functions are impeded (colour-coded scale). For a detailed discussion on such thresholds in focal cerebral ischaemia see Ref. 19.

a review). Chemokines, for example, interleukin 8 and monocyte chemoattractant protein 1, are produced in the injured brain and guide the migration of bloodborne inflammatory cells towards their target44,45. Resident brain cells are also involved in the inflammatory response. Four to six hours after ischaemia, astrocytes become hypertrophic, while microglial cells retract their processes and assume an ameboid morphology that is typical of activated microglia46. Twenty-four hours after MCA occlusion the microglial reaction is well developed in the ischaemic brain, particularly in the penumbra. There is increasing evidence that post-ischaemic inflammation contributes to ischaemic brain injury (for a review see Ref. 47). Cerebral ischaemic damage is reduced: (1) when the infiltration of neutrophils is prevented by induction of systemic neutropenia; (2) when adhesion molecules or their receptors are blocked by neutralizing antibodies and in mice with deletion of Icam1 (Ref. 48); (3) when the action of crucial inflammatory mediators, such as IL-1, is blocked49; and (4) in mice with a deletion of the gene encoding interferon regulatory factor 1, a transcription factor that coordinates the expression of inflammation-related genes34. Post-ischaemic inflammation could contribute to ischaemic damage by many mechanisms. Whereas microvascular obstruction by neutrophils can worsen the degree of ischaemia50, production of toxic mediators by activated inflammatory cells and injured neurones also has important consequences. In rodent models of cerebral ischaemia, as in patients with stroke, infiltrating neutrophils produce inducible NOS (iNOS), an enzyme that produces toxic amounts of NO (Ref. 51; Fig. 4). The pathogenic potential of NO produced by iNOS is underscored by the observations that pharmacological inhibition of iNOS reduces ischaemic brain injury and that iNOS null mice have a reduction in ischaemic damage13,52. The delayed nature of the protection exerted by iNOS inhibition or gene deletion is consistent with the hypothesis that ischaemic injury evolves over several days53,54. In addition, ischaemic neurones express cyclooxygenase 2 (COX2) (Fig. 4), an enzyme that mediates ischaemic injury by producing superoxide and toxic prostanoids55. Ischaemic neurones also produce TNFa, a cytokine that can exacerbate ischaemic injury56. However, its role remains uncertain because studies in mice with a deletion of TNFa receptors suggest that this cytokine can be beneficial to the injured brain, owing to its induction of antioxidant enzymes57. Activated microglia have also the potential to produce neurotoxins, including NO, reactive oxygen species and toxic prostanoids46. In addition, the inflammatory reaction might also be linked to apoptosis because antibodies against adhesion molecules attenuate post-ischaemic inflammation and reduce apoptotic cell death in the ischaemic

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brain58. However, in the brain, as in other organs, inflammatory cells participate in tissue remodelling and reconstruction following injury, processes that take place days to weeks after ischaemic injury. However, the role of inflammatory cells in tissue remodelling following ischemia has not been defined. Post-ischaemic inflammation is, therefore, a promising target for therapeutic intervention in ischaemic stroke. Treatments that are aimed at downregulating the neutrophilic infiltration, as well as drugs inhibiting enzymes that produce toxic mediators, such as iNOS and COX2, could be viable strategies for targeting the late stages of injury. Furthermore, the potential beneficial effects of inflammation in tissue repair and remodelling need to be considered when developing treatment strategies. A recent clinical trial using anti-ICAM-1 antibodies did not show that they had any effect59. However, this study has not yet been published in full and the reasons for its failure remain to be determined. Irrespective of the outcome of this ICAM-1 trial, however, there is a strong rationale for using anti-inflammatory therapy in ischaemic stroke.

Apoptosis: suicide in the penumbra? Brain cells that are compromised by excessive glutamate-receptor activation, Ca21 overload, oxygen radicals or by mitochondrial and DNA damage can die by necrosis or apoptosis. The decision in part, depends on the nature and intensity of the stimulus, the type of cell, and the stage it has reached in its life-cycle or development60. Necrosis is the predominant mechanism that follows acute, permanent vascular occlusion, whereas in milder injury, cell suicide becomes unmasked and death resembles apoptosis, particularly within the ischaemic penumbra (Fig. 3). The genes for caspases as well as genes that suppress (for example, Bcl2, Iap) or augment [Bax, Trp53 (formerly p53)] cell death are expressed at higher levels and activated in both the early and late stages of ischaemia, and genetic manipulations or drugs that block caspasefamily members61 or enhance the actions of BCL2 (Ref. 62) confer resistance to ischaemic injury. Caspases are aspartate-specific cysteine proteases and exist as zymogens in cells. Among the 12 that have been identified, caspases 1 and 3 seem to have a pivotal role in ischaemia-mediated apoptosis. How do the caspases promote ischaemic cell death? Activated caspases are protein-cleaving enzymes and thereby modify crucial homeostasis and repair proteins that, in turn, disassemble and kill cells. More than 30 proteins can be cleaved, including the DNA-repairing enzyme poly (ADP-ribose) polymerase (PARP) and the cytoskeletal protein, gelsolin, in addition to presenilins, huntingtin protein and other caspases. Although little is known about upstream initiating steps in ischaemic tissues, both receptor- and non-receptor-driven mechanisms promote caspase activation in non-neural systems63. It is known from related models that caspases become activated when cytochrome C, released from mitochondria, activates an apoptosome complex [apoptosis-activating factor (APAF1) plus pro-caspase 9] in the presence of dATP (Ref. 64). Cytochrome C can enter the cytosol from its location on the external side of the inner mitochondrial membrane. The formation of the apoptosome complex, which is suppressed by BCL2L1 (formerly known as bcl-xl), promotes clipping and activation of caspase 3 (Ref. 64).

Fig. 5. Evolution of apoptosis after mild experimental focal cerebral ischemia. Time-dependent changes in the appearance of terminaldeoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL)positive cells (dark, round) and glial fibrillary acidic protein (GFAP, ramified, brown–purple) staining after reperfusion following 30 min of middle cerebral artery occlusion in the mouse. Tissue sections (6 mm) were obtained at 24 h (A), 48 h (B), 72 h (C) and 7 days (D). Numbers of TUNEL-positive cells are low at 24 h, increased at 72 h and decreased at 7 days. The number of GFAP-positive glial cells increases over time, but GFAP-positive cells do not show TUNEL staining. Scale bar, 50 mm. For further details see Ref. 67.

Neurones appear to be particularly susceptible to caspase-mediated cell death following focal cerebral ischaemia, where terminal-deoxynucleotidyl-transferasemediated dUTP nick end labelling (TUNEL)-positive staining, oligonucleosomal DNA laddering, and caspase3 and caspase-1 processing65,66 were described (see Fig. 5). Mice that possess a dominant negative isoform of caspase 1 (Refs 68,69) and caspase-1 knockout mice are resistant to ischaemic injury69. Caspase-3 cleavage precedes the onset of DNA laddering by several hours, as does the cleavage of caspase 1 and its substrate pro-IL-1b. If injury is mild and cell death is delayed, caspase activation is also delayed and a greater proportion of cells die by a caspasedependent mechanism. After mild (30 min) reversible MCA occlusion, cytochrome-C release and caspase processing are detected at 6 and 9 h, respectively, and cell death becomes prominent between 24 and 72 h (Fig. 5). In some models, the caspase-cleavage fragment is found in approximately 40–50% of TUNEL-positive cells, which allows for other caspase-dependent mechanisms of cell death. Other caspase-family members might be important in the late stages of cell death as ischaemia increases levels of mRNA encoding caspases 1, 2, 3 and 8. Caspase activity is blocked in vivo and in vitro by administering small peptides that bind covalently and TINS Vol. 22, No. 9, 1999

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penumbra (Fig. 6; Ref. 54). As collateral perfusion develops, brain function can be restored within the penumbra23. In addition, the structural lesion solidifies over time and might recruit parts of the ischaemic penumbra into infarction. Thus, symptoms can regress while the lesion actually expands. After days to weeks, neurological deficits reflect the size and location of the structural lesion more closely. Recovery of function from this time point is best explained by plasticity and tissue reorganization.

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Fig. 6. Regression of the functional neurological deficit while the structural lesion grows. Early in the course of stroke, clinical symptoms mostly reflect an impairment of function (green) but not necessarily a structural lesion (blue). Over time, some areas either spontaneously, or because of therapy, recover function, which explains why symptoms in patients can regress while the structural lesion actually grows.

irreversibly to the catalytic pocket after alkylating the cysteine residue70. Caspase inhibitors not only attenuate the volume of dead tissue in focal ischaemia but also decrease neurological deficit65, which thereby reflects the possible functional preservation of ischaemic neuronal tissue. In milder ischaemia, the inhibitors are particularly effective as they reduce tissue injury synergistically when injected with MK801, an NMDA-receptor antagonist, or with growth factors, such as fibroblast growth factor. Moreover, unlike NMDA-receptor antagonists, caspase inhibitors reduce injury when injected many hours after brief ischaemia71. A single intracerebroventricular injection of zDEVD.FMK, a relatively selective caspase-3 inhibitor, is effective if given 9 h after 30 min of reversible ischaemia71 and when administered after 3 h in a neonatal hypoxic–ischaemic model72. Cell protection persisted for at least 22 days in one study. Some investigators challenge whether ischaemic cells become apoptotic primarily because the typical electronmicroscopic features are infrequently found, except in neonatal brain cells. Moreover, there are no compelling published data that make an argument for or against apoptosis in human stroke tissue at the present time. Nevertheless, the data summarized above from experimental animals, combined with evidence that caspase inhibition protects in other types of experimental brain injury, such as head trauma73, suggest a potential therapeutic application for synthetic inhibitors targeted at one or more caspase-family members.

Is there evidence for delayed damage in stroke patients? Recent experimental evidence suggests that cerebral ischaemic damage evolves at a slower pace than was believed previously. Thus, neurones at the border of the ischaemic territory can survive for many hours or even days after the ischaemic insult53. Similarly, studies using MRI have suggested that the progression of ischaemic damage is also delayed in stroke patients54. Such delayed progression of brain damage might lead to neurological deficits increasing with time in patients with ischaemic stroke. However, this is clearly not the case because symptoms tend to improve during the first week after the insult74. This clinical observation has argued against the importance of delayed mechanisms of ischaemic damage, including inflammation and apoptosis in humans. However, it is known that initially, the neurological deficits reflect injury to the core as well as the 396

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There are a number of crucial questions that remain unanswered, both at the bench and at the bedside. For example, do protocols that combine two or more treatment strategies, for example, reperfusion plus hypothermia plus anti-excitotoxic agent(s), etc., provide added protection? Does preventing apoptosis include the risk of saving neurones without function, or of impairing physiological mechanisms that suppress inflammation? Do peri-infarct depolarizations occur in humans? Do inflammation and apoptosis contribute to ischaemic damage in stroke patients significantly? How can we determine the stage of evolution of the ischaemic process in stroke patients accurately? Do new MRI (Ref. 75) or optical-imaging methods76 hold the key to pathophysiologically based therapy and to the quantitative assessment of tissue outcome in stroke trials?

Concluding remarks Tissue damage following cerebral ischaemia results from the interaction of complex pathophysiological processes such as excitotoxicity, peri-infarct depolarizations, inflammation as well as apoptosis. All four are potential targets for therapy. Stroke is caused, at least initially, by a disorder of blood flow in the brain and successful attempts to establish reperfusion early can reduce the magnitude and extent of tissue injury. It is, therefore, very likely that, in the future, interventions will combine strategies that enhance both early reperfusion and neuroprotection. Crucial issues remain unresolved regarding the translation of preclinical developments to the bedside. Failed clinical trials need not revive an attitude of therapeutic nihilism. Rather they provide an opportunity to decide whether failure is caused by the choice of drug and its pharmacokinetics, its pre-clinical evaluation, or clinicaltrial design and analysis, as well as to reflect on the appropriateness of the therapeutic target. Selected references 1 Taylor, T.N. et al. (1996) Stroke 27, 1459–1466 2 The National Institue of Neurological Disorders and Stroke rt-PA Stroke Study Group (1995) New Engl. J. Med. 333, 1581–1587 3 Martin, R.L., Lloyd, H.G. and Cowan, A.I. (1994) Trends Neurosci. 17, 251–257 4 Katsura, K., Kristian, T. and Siesjo, B.K. (1994) Biochem. Soc. Trans. 22, 991–996 5 Nehls, D.G., Park, C.K. and McCulloch, J. (1989) J. Cereb. Blood Flow Metab. 9, S376–S376 6 Furukawa, K. et al. (1997) J. Neurosci. 17, 8178–8186 7 Chen, Z.L. and Strickland, S. (1997) Cell 91, 917–925 8 Zhao, Q. et al. (1994) Acta Physiol. Scand. 152, 349–350 9 Weisbrot-Lefkowitz, M. et al. (1998) Mol. Brain Res. 53, 333–338 10 Yang, G. et al. (1994) Stroke 25, 165–170 11 Kondo, T. et al. (1997) J. Neurosci. 17, 4180–4189 12 Murakami, K. et al. (1998) J. Neurosci 18, 205–213 13 Iadecola, C. (1997) Trends Neurosci. 20, 132–139 14 Beckman, J.S. and Koppenol, W.H. (1996) Am. J. Physiol. 271, C1424–C1437 15 Huang, Z. et al. (1994) Science 265, 1883–1885 16 Dugan, L.L. and Choi, D.W. (1994) Ann. Neurol 35, S17–S21

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K.M.A., Caplan, L.R., Reis, D.J., Siesjö, B.K. and Weir, B., eds), pp. 117–124, Academic Press Feuerstein, G.Z., Wang, X. and Barone, F.C. (1998) in Cerebrovascular Disease: Pathophysiology, Diagnosis and Management (Ginsberg, M. and Bogousslavsky, J., eds), pp. 507–531, Blackwell Science Connolly, E.S., Jr et al. (1996) J. Clin. Invest. 97, 209–216 Loddick, S.A. and Rothwell, N.J. (1996) J. Cereb. Blood Flow Metab. 16, 932–940 Del Zoppo, G.J. et al. (1991) Stroke 22, 1276–1283 Forster, C. et al. (1999) Acta Neuropathol. 97, 215–220 Iadecola, C. et al. (1997) J. Neurosci. 17, 9157–9164 Dereski, M.O. et al. (1993) Acta Neuropathol. 85, 327–333 Baird, A.E. et al. (1997) Ann. Neurol. 41, 581–589 Nogawa, S. et al. (1997) J. Neurosci. 17, 2746–2755 Barone, F.C. et al. (1997) Stroke 28, 1233–1244 Bruce, A.J. et al. (1996) Nat. Med. 2, 788–794 Chopp, M. et al. (1996) J. Cereb. Blood Flow Metab. 16, 578–584 The Enlimomab Acute Stroke Trial Investigators (1997) Neurology 48, A270 Leist, M. and Nicotera, P. (1998) Exp. Cell Res. 239, 183–201 Thornberry, N.A. and Lazebnik, Y. (1998) Science 281, 1312–1316 Adams, J.M. and Cory, S. (1998) Science 281, 1322–1325 Bergeron, L. and Yuan, J. (1998) Curr. Opin. Neurobiol. 8, 55–63 Green, D.R. and Reed, J.C. (1998) Science 281, 1309–1312 Hara, H. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2007–2012 Namura, S. et al. (1998) J. Neurosci. 18, 3659–3668 Endres, M. et al. (1998) J. Cereb. Blood Flow Metab. 18, 238–247 Friedlander, R.M. et al. (1997) J. Exp. Med. 185, 933–940 Schielke, G.P. et al. (1998) J. Cereb. Blood Flow Metab. 18, 180–185 Nicholson, D.W. and Thornberry, N.A. (1997) Trends Biochem. Sci. 22, 299–306 Fink, K. et al. (1998) J. Cereb. Blood Flow Metab. 18, 1071–1076 Cheng, Y. et al. (1998) J. Clin. Invest. 101, 1992–1999 Yakovlev, A.G. et al. (1997) J. Neurosci 17, 7415–7424 Jorgensen, H.S. et al. (1995) Arch. Phys. Med. Rehabil. 76, 406–412 Koroshetz, W.J. (1996) Ann. Neurol. 39, 283–284 Villringer, A. and Chance, B. (1997) Trends Neurosci. 20, 435–442

Acknowledgements U.D. is supported by grants from Deutsche Forschungsgemeinschaft, The Hermann and Lilly Schilling Foundation and Deutsche Schlaganfallstiftung, C.I. is supported by grants from the National Institute of Health (NS34179, NS35806, NS37853) and M.A.M. is principal investigator on National Institute of Health Indepartmental Stroke Program Project (NS 10828-23).

Complement anaphylatoxin receptors on neurons: new tricks for old receptors? Serge Nataf, Philip F. Stahel, Nathalie Davoust and Scott R. Barnum Activation of the complement system has been reported in a variety of inflammatory diseases and neurodegenerative processes of the CNS. Recent evidence indicates that complement proteins and receptors are synthesized on or by glial cells and, surprisingly, neurons. Among these proteins are the receptors for the chemotactic and anaphylactic peptides,C5a and C3a,which are the mostpotent mediators of complement inflammatory functions.The functions of glial-cell C3a and C5a receptors (C3aR and C5aR) appear to be similar to immune-cell C3aRs and C5aRs. However, little is known about the roles these receptors might have on neurons. Indeed, when compared with glial cells, neurons display a distinct pattern of C3aR and C5aR expression, in either the normal or the inflamed CNS.These findings suggest unique functions for these receptors on neurons. Trends Neurosci. (1999) 22, 397–402

T

HE COMPLEMENT SYSTEM has an important role in innate and specific immune responses with functions that include the opsonization of invading pathogens for phagocytosis and clearance; cytolysis, causing increases in vascular permeability, which lead to edema and the recruitment of phagocytic cells; the augmentation of the acute-phase response; and the activation of B cells1. This broad array of host defense responses is largely mediated by ligand–receptor interactions between the proteolytic fragments derived from C3 and C5, which serve as the ligands, and the unique 0166-2236/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.

receptors for each fragment. Specificity of the responses is controlled by the tissue- and cell-specific synthesis of the receptors and local activation of complement proteins. The most potent inflammatory molecules generated on activation of complement are C3a and C5a, two small peptides (~9–11 kDa) that are highly pleiotropic in function and produce their effects at picomolar to nanomolar concentrations. Of these peptides, C5a induces the widest range of responses, which include chemotaxis of all the myeloid cell lineages; degranulation of mast cells and basophils (leading to the release PII: S0166-2236(98)01390-3

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Serge Nataf, Nathalie Davoust and Scott R. Barnum are at the Dept of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA, and Philip Stahel is in the Division of Trauma Surgery and in the Division of Research, Dept of Surgery, University Hospital, CH-8091 Zürich, Switzerland.

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