Mechanisms of neuronal degeneration after ischemic stroke – Emerging targets for novel therapeutic strategies

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Drug Discovery Today: Disease Mechanisms

DRUG DISCOVERY

TODAY

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA

DISEASE Nervous system MECHANISMS

Mechanisms of neuronal degeneration after ischemic stroke – Emerging targets for novel therapeutic strategies Carsten Culmsee1,*, Josef Krieglstein2 1

Pharmazeutische Biologie-Biotechnologie, Department Pharmazie, Ludwig-Maximilians-Universita¨t, Butenandtstraße 5-13, Geba¨ude D, D-81377 Mu¨nchen, Germany 2 Institut fu¨r Pharmakologie und Toxikologie, Fachbereich Pharmazie, Philipps-Universita¨t Marburg, Germany

The mechanisms that trigger ischemic brain damage include a plethora of biochemical and cellular events, such as glutamate-mediated excitotoxicity, generation

Section Editors: Andrey Mazarati – UCLA, USA Claude Wasterlain – Department of Cardiology, UCLA, USA

of reactive oxygen species (ROS), DNA damage, regulation of pro-apoptotic factors and inflammatory responses involving endothelial cells, leukocytes and microglial cells. Here we summarize recent insights into mechanisms of ischemic neuronal death that are the basis for efficient stroke therapy in the future. Introduction The only causal therapy for ischemic stroke is reperfusion. Even if cerebral blood flow is re-established quickly enough to prevent immediate cell death, however, a large population of initially surviving neurons will die within the first few hours after reperfusion. According to current knowledge, major mechanisms involved in such delayed postischemic infarct development include energy failure, excitotoxicity, accumulation of reactive oxygen species (ROS) and apoptotic signaling as well as inflammatory processes [1]. ATP levels in neurons are rapidly decreased following the onset of ischemia, resulting in the impairment of membrane ionmotive ATPases, which in turn leads directly to membrane depolarization and activation of the N-methyl-D-aspartate (NMDA) subtype of synaptic glutamate receptor and voltage*Corresponding author: C. Culmsee ([email protected]) URL: http://www.cup.uni-muenchen.de/pb/aks/ewagner/ 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmec.2005.11.006

dependent calcium channels. Levels of ROS increase particularly during the reperfusion phase and include accumulation of superoxide anions, hydrogen peroxide, hydroxyl radical and peroxynitrite [2]. Mitochondrial dysfunction ensues as the result of oxidative stress, energy failure and disruption of cellular calcium homeostasis, and results in further production of ROS which damage cellular proteins, DNA and membrane lipids. Further, mitochondrial dysfunction and subsequent changes of mitochondrial ion permeability, release of apoptosis-inducing factor (AIF) and cytochrome c are involved in delayed ischemic cell death that displays prominent features of apoptosis-related mechanisms [3]. It is noteworthy that neuronal cell death after hypoxia/ischemia might display features of both apoptosis or necrosis and biochemical hallmarks of programmed cell death have been associated with the respective distinct morphological criteria in models of ischemic damage [1,4]. Emerging evidence suggests that on the cellular level ischemic injury can induce different subroutines of cell death including ‘pure’ apoptosis or necrosis, an apoptosis–necrosis continuum as well as a form of cell death designated as programmed necrosis [4–6]. Ischemic neuronal death involves regulation of Bcl-2 family members that either promote (Bax, Bad, truncated Bid) or prevent (Bcl-2, Bcl-xL) mitochondrial membrane www.drugdiscoverytoday.com

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Figure 1. Mechanisms of ischemic brain injury. After ischemia, the neurons suffer from a drastic reduction in their access to oxygen and glucose. Levels of ATP are rapidly decreased, resulting in impairment of ion motive ATPases, which in turn leads to membrane depolarization and activation of the N-methylD-aspartate (NMDA) subtype of glutamate receptors and voltage-dependent calcium channels. Membrane depolarization induces a burst release of glutamate and further calcium influx into the cells. The disruption of the calcium homeostasis is further accelerated through activation of acid-sensing calcium channels (ASIC) and after protease-mediated cleavage of plasma membrane Ca2+-ATPases (PMCA) and Na+–Ca2+ exchangers (NCX). In addition, enhanced formation of reactive oxygen species (ROS) and inflammatory reactions contribute to multiple pathways of cell death via apoptosis or necrosis involving mitochondrial dysfunction, activation of caspases, protein kinases or phosphatases, and damage of membrane lipids, structure proteins, enzymes or DNA. Many of the possible different subroutines of ischemic neuron death are connected, amplify one another in the form of vicious circles and ultimately lead to the irreversible infarction of brain tissue. [Ca2+]i: intracellular calcium concentration; DAG: diacyl-glycerol; PKC: protein kinase C; NOS: nitric oxide synthase.

permeability transition, activation of caspases and transcription factors such as NF-kB or the tumor suppressor p53 [7]. Cellular events that are involved in postischemic brain damage include microglial activation and invasion of leukocytes that contribute to increased cytokine levels, excessive NO production and other inflammatory responses in the injured brain tissue [8,9]. In addition, recent data indicated an involvement of vascular damage in pathological mechanisms after stroke, suggesting a crosstalk between damaged endothelium and neurons in the ischemic brain tissue [10,11]. These pathophysiological events (Fig. 1) that evolve after cerebral ischemia have been well documented in various cell culture and animal models of stroke, and in many cases 464

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cause–effect relationships have been established. Here we review the most recent advances towards the understanding of mechanisms involved in infarct development after ischemic stroke and the according novel therapeutic strategies that currently emerge (Table 1).

Ca2+ homeostasis Ischemic neuron death might result from various signaling pathways, that is, different subroutines of cell death ranging from established apoptotic signaling to primary or secondary necrosis. The cytotoxic accumulation of intracellular calcium has been well-established as a key step in ischemic neuronal cell death, and according to recent data Ca2+ might also be

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Table 1. Emerging targets for therapeutic strategies in ischemic stroke Strategic approach to target

Expected outcome of intervention at target

Therapies in trial

Stage in trial

Refs

Glutamate receptors

NMDAa receptor inhibition

Neuroprotection

NMDA-antagonists, memantine

Phase II

[12,13]

ASICb

ASIC-inhibitors, siRNA

Neuroprotection

Amiloride, psalmotoxin-1

Preclinical

[14]

Phase III, Phase II

[18–20]

Phase II

[21–24]

c

ROS

Antioxidants e

PARP-1

PARP-inhibitors

Neuroprotection

d

Edaravone, NXY-059 f

Neuroprotection

PJ-34 g

p53

p53 inhibitors

Neuroprotection

PFT

Preclinical

[23,25]

Bidh

Bid inhibitors

Neuroprotection

BI-6c9i

Preclinical

[27]

j

k

Bad

PI3K /Akt stimulation; PP2c inhibition

Neuroprotection

Growth factors, siRNA ;

Preclinical

[17,28,29]

PP2Cl

PP2c inhibition

Neuroprotection, endothelial protection

siRNA

Preclinical

[30,31]

Caspases, Calpains

Caspase/Calpain-Inhibition

Preservation of PMCAm/NCXn; neuroprotection

Caspase/-calpain-inhibitors

Preclinical

[4,5,7,33,35–38]

AIFo

AIF release inhibition; AIF-knockdown

Neuroprotection

PARP-inhibitors, Bid-inhibitors, siRNA

Preclinical

[3,21,26,27,40,41,43,44]

MMP-9p

MMP-2/MMP-9 inhibition

Neuroprotection, vascular integrity

MMP-inhibitors; SB-3CTq

Preclinical

[1,10,11,45,46]

iNOSr

iNOS-inhibition

Anti-inflammatory effects

Amino-guanidine

Preclinical

[8,9]

COX-2s

COX-2 inhibition

Anti-inflammatory effects

NS-398t

Preclinical

[8,9]

a

NMDA: N-methyl-D-aspartate. ASIC: acid-sensing ion channels. c ROS: reactive oxygen species. d NXY-059: disodium 2,4-disulfophenyl-N-tert-butylnitrone. e PARP: poly(ADP-ribose) polymerase. f PJ-34: PARP-inhibitor. g PFT: pifithrin-alpha. h Bid: BH3-interacting domain death agonist. i BI-6c9: Bid inhibitor. j PI3K: phosphoinositide-3-phosphate kinase. k siRNA: small interfering RNA. l PP2C: protein phosphatase-2C. m PMCA: plasma membrane calcium ATPase. n NCX: Natrium-calcium exchanger. o AIF: apoptosis inducing factor. p MMP: matrix metalloproteinase. q SB-3CT: thiirane gelatinase inhibitor. r iNOS: inducible nitric oxide synthase. s COX-2: cyclo-oxygenase-2. t NS-398: selective COX-2 inhibitor. b

the crucial link between the different postischemic apoptotic and necrotic processes [5]. Calcium overload can set off cell demise by activating proteases, lipases and DNases, change the balance of neuronal death from apoptosis to necrosis by depleting energy stores, or amplify other pathways of the ischemic cell death program. So far, it has been widely accepted that cytotoxic intracellular Ca2+ overload after ischemia is mainly mediated through stimulation of glutamate receptors, namely through NMDA receptors, Ca2+permeable AMPA/kainate receptors, metabotropic glutamate receptors and voltage-dependent Ca2+-channels [12]. Despite promising results in animal models of ischemia, clinical trials with NMDA receptor antagonists failed to improve the postischemic outcome in the past because of adverse effects such as neuropsychotic symptoms or hypertension

which occurred dose-dependently before neuroprotective plasma levels were achieved [13]. Recently, a new class of ion channel-coupled receptors has been identified to play a major role in postischemic disruption of Ca2+-homeostasis: acid-sensing ion channels (ASIC)-mediated calcium influx and neuronal cell death after lowering of the extracellular pH [14]. Because pH also rapidly drops in ischemic brain tissue, proton-activated ASIC could probably contribute to ischemia-induced calcium influx in neurons. Blocking ASIC by amiloride or psalmotoxin-1 (a toxin from the tarantula venom) or siRNA-mediated ASIC-knockdown prevented proton-induced neuronal death in vitro exposing ASIC as a new target for therapeutic approaches in stroke. Indeed, evidence from animal models of stroke supported the important role of ASIC in ischemic brain damage because ASIC-knockout or www.drugdiscoverytoday.com

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pharmacological inhibition of ASIC significantly reduced the infarct volume [14]. Notably, interference with ASICmediated calcium influx protected neurons from ischemic damage more potently than glutamate antagonism. In addition, the fatal accumulation of Ca2+ in apoptotic cells can result from the cleavage of ion pumps that under physiological conditions rapidly pump out Ca2+ to preserve a steady state cytosolic calcium. In neurons, isoforms of the plasma membrane Ca2+-ATPase (PMCA) have been identified to play an essential role in rectifying changes in intracellular Ca2+ in the long term [5]. In addition to the cleavage of PMCA2 and PMCA4, there is now evidence for the cleavage of the Na+–Ca2+ exchanger (NCX) in apoptotic neurons [15]. The latter Ca2+ ion pump effectively contributes to remove large amounts of calcium accumulated in the cytosol. After ischemic injury, activated caspases might eventually cleave these ion pumps which results in the disruption of calcium homeostasis that can finally switch apoptotic signalling to necrosis. Because ion homeostasis in general and Ca2+ homeostasis in particular are apparently vital for neuronal survival after ischemia, compounds which block ASIC or interfere with mechanisms involved in ion pump inactivation might emerge as potent neuroprotectants in stroke therapy (Table 1).

Oxidative stress There is compelling evidence that ROS accumulate to cytotoxic levels in brain tissue during ischemia and reperfusion [2]. Particularly devastating for neurons is membrane lipid peroxidation which results in the generation of toxic aldehydes such as 4-hydroxynonenal that impair the function of membrane ion motive ATPases and glucose and glutamate transporters, and thereby amplify disruption of cellular Ca2+ homeostasis [16]. In addition to membrane lipid or protein damage, ROS can mediate rapid DNA damage after stroke, which activates fatal apoptotic signaling through the tumor suppressor p53 and poly(ADP-ribose) polymerase (PARP) (Fig. 2) [2]. Experimental studies with transgenic animals either lacking or overexpressing elements of radical scavenging machinery of the cell, as for example Cu,Zn-superoxide dismutase (SOD) or Mn-SOD clearly demonstrated the essential role of this system for neuronal survival after ischemia [17]. Scavenging free radicals appeared therefore as a viable approach to neuroprotection. Many studies in animal models of stroke have confirmed neuroprotective effects of antioxidant nutrients such as Vitamin E, lipoic acid, green tea extract, ginkgo biloba extract, resveratrol or niacin [18]. In clinical trials, however, most antioxidants failed to produce consistent neuroprotective effects, partly because of their small therapeutic window. New hope is now generated by recent stroke trials that revealed beneficial effects of the free radical scavengers edaravone [19] or NXY-059 [20] (Table 1). 466

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DNA damage, activation of PARP and p53 Reactive oxygen species might induce early mechanisms of ischemic neuronal death through DNA damage. Oxidative DNA damage consists of highly specific chemical lesions such as hydroxyl radical-modified bases (8-hydroxyl-20 -deoxyguanosine) and DNA strand breaks. Such DNA damage is a prominent feature of ischemic brain injury and has been detected in neurons within the developing infarct very early, that is, within minutes after the insult and before internucleosomal DNA fragmentation that is characteristic of late stages of programmed cell death [21]. In addition, oxidative damage of proteins involved in cell cycle regulation or DNA repair might contribute to accumulating DNA damage and finally to activation of apoptotic signaling. DNA damageinduced apoptotic signaling (in neurons) involves, for example, activation of poly(ADP-ribose)polymerase-1 (PARP-1) and p53, and both factors have also been linked to cell death signaling in various models of ischemic stroke [22,23]. Upon activation by DNA strand nicks and breaks, PARP-1 builds up polymers of adenosine diphosphate ribose using NAD+ as a substrate. Poly(ADP-ribose) acceptors include, for example, DNA associated proteins and enzymes such as histones, topoisomerases and DNA ligase 2. In addition to the subsequent depletion of NAD+ and ATP that follows the massive NAD+ utilization by active PARP-1 the dysfunction of poly(ADP-ribosylated) enzymes might be involved in PARPmediated apoptosis signaling [22]. Recently, a role for apoptosis-inducing factor translocation from mitochondria to the nucleus has been identified as a downstream mechanism in PARP-1-dependent cell death, but how PARP-1 triggers AIF release and cell death is currently unknown [21]. A significant role for PARP activity in infarct development was demonstrated in PARP-1 knockout mice and animals treated with inhibitors of PARP-1 which were significantly protected from ischemic brain damage [22,24]. In addition, the tumor suppressor and transcription factor p53 was exposed as a promising target for stroke therapy, because p53 was rapidly upregulated in the ischemic brain tissue where it could mediate programmed cell death through transcription of pro-apoptotic Bcl-2 family members such as Bax, or the BH3-only proteins PUMA and NOXA, and subsequent mitochondrial damage and activation of caspases [23]. The p53 inhibitor pifithrin-alpha (PFT) prevented mitochondrial dysfunction, caspase activation and p53-dependent cell death in cultured neurons exposed to apoptotic stress and prevented ischemic brain damage in various models of stroke [23]. Of note, we demonstrated an extended therapeutic window of several hours after the ischemic insult for these PFT-mediated cerebroprotective effects, which has great clinical appeal [25]. In addition to the inhibition of p53, PFT might exert its neuroprotective effects through stimulation of survival signaling such as activation of the protein kinase Akt or enhanced NF-kB transcriptional activity [25].

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Figure 2. Mechanisms of programmed cell death in ischemic neurons. In ischemic neurons the cell death machinery is triggered through extracellular ligands of death receptors (e.g. Fas) or enhanced levels of intracellular calcium and reactive oxygen species (ROS). Mitochondrial dysfunction and subsequent acceleration of ROS formation and the release of pro-apoptotic mitochondrial factors are key steps towards the ischemic cell death program that ultimately leads to apoptosis, necrosis or an apoptosis–necrosis continuum. Such mitochondrial damage is induced by interaction of the pro-apoptotic Bcl-2 family proteins Bax, Bad or truncated Bid (tBid) with anti-apoptotic Bcl-2 or Bcl-xL at the mitochondrial membrane. Activation of these factors is either induced by calcineurin or protein-phosphatase 2c (PP2c)-mediated dephosphorylation of Bad and subsequent release from the 14-3-3 protein complex, caspase-8- or calpain-mediated enzymatic cleavage of Bid to tBid or enhanced synthesis of Bax after transactivation of p53. The transcription factor p53 as well as poly(ADP-ribose) polymerase (PARP) are activated after ROS- or peroxynitrite (ONOO
)-mediated DNA damage and play a pivotal role in ischemic brain damage through enhanced synthesis or activation of pro-apoptotic factors, respectively. Both, p53 and PARP might directly or indirectly enhance the release of cytochrome c (Cytc) and apoptosis-inducing factor (AIF) from mitochondria thereby stimulating caspase-dependent or caspase-independent cell death. Together with apoptotic protease activating factor 1 (APAf-1) and caspase-9 released Cytc forms the apoptosome that amplifies caspase-3 activity and caspase-dependent apoptosis. Mitochondrial AIF translocates to the nucleus where it induces large-scale DNA fragmentation and caspase-independent cell death.

Overall, DNA damage response mechanisms, and in particular activation of PARP-1 and p53 are early events after stroke and apparently play a pivotal role in initiating phases of the ischemia-induced cell death program (Fig. 2).

Mitochondria, caspases and caspase-independent mechanisms Mitochondrial damage has been considered as the ‘point of no return’ in the cell death cascade triggered in neurons after an ischemic insult [3]. Therefore, mechanisms upstream of mitochondrial dysfunction that are triggered early after an ischemic insult seem to be of particular interest for the development of neuroprotective stroke therapies. In particular, the regulation of Bcl-2 protein family members might be crucial for the maintenance of mitochondrial integrity and function thereby deciding the fate of a cell after severe stress

(Fig. 2, Table 1) [26]. Although earlier reports established a role for Bax and (truncated) Bid in ischemic neuron death [27], recent studies elucidated the activation of Bad in ischemic brain tissue [17,28]. The dephosphorylation of Bad is a crucial step towards full pro-apoptotic activity [29]. Although under physiological conditions phosphorylated Bad remains bound and inactive in a complex with the cytosolic 14-3-3 protein, dephosphorylated Bad is released from this complex to interact with the anti-apoptotic Bcl-xL (Fig. 2). Enhanced Bad phosphorylation by protein kinase A (PKA) or the phosphoinositide-3-phosphate kinase (PI3K)/ protein kinase B (Akt) pathway stabilizes the Bad/14-3-3 complex and prevents neuronal cell death. By contrast, dephosphorylation of Bad by protein phosphatases such as calcineurin (protein phosphatase 2B, PP2B) or protein phosphatase 2C (PP2C) might be a key step in the initiation of the www.drugdiscoverytoday.com

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cell death program in ischemic neurons [30]. Strikingly, recent data proposed PP2C-induced dephosphorylation of Bad as an underlying mechanism after induction of apoptosis by fatty acids that are part of low density lipoproteins (LDL) also in endothelial cells [30,31]. Overall, these results point at a pivotal role for Bad dephosphorylation for ischemic brain damage as well as in (fatty acid-induced) apoptosis in endothelial cells which might be relevant to the pathology of atherosclerosis [31]. PP2C might therefore be a promising target molecule for acute stroke treatment as well as for therapeutic approaches to attenuate atherosclerosis and thereby the risk of cardiovascular diseases and stroke. After an ischemic insult, mitochondrial dysfunction is a major cause for ATP depletion and further disruption of the intracellular calcium homeostasis [3]. In addition, damaged mitochondria release pro-apoptotic proteins such as cytochrome c, Smac/DIABLO or HtrA2/Omi which activate caspase-dependent apoptotic pathways [26,32]. Caspases are a family of cell death proteases that are considered as key executioners of the apoptotic cell death machinery, and activation of caspases is a well-established biochemical hallmark of programmed cell death [33]. After ischemia, death receptor-mediated signaling might rapidly activate caspase-1 or caspase-8, so-called initiator caspases, which then trigger the cell death cascade that ultimately leads to the activation of executioner caspases such as caspase-3 and caspase-9. Data from various models of cerebral ischemia provided strong evidence for the involvement of caspases in delayed neuronal death after cerebral ischemia [34,35]. Therefore, inhibition of caspases has been considered as a promising strategy to prevent apoptosis as well as (secondary) necrosis in ischemic neurons [4,35,36] (Table 1). Indeed, previous studies employing peptide caspase inhibitors or caspase knockout mice demonstrated a reduction of ischemic brain damage [36]. The therapeutic potential of currently available caspase inhibitors for stroke treatment, however, is rather limited because these peptides cannot pass the blood–brain barrier. Moreover, a considerable number of neurons still exposed pycnotic nuclei in the cortical penumbra region after transient focal ischemia in caspase-3 knockout mice [37,38], and caspase inhibitors also failed to prevent impairments in long-term potentiation after ischemia. Therefore, neuroprotection by caspase inhibition alone might not sufficiently preserve degeneration of axons and dendrites which are important for functional plasticity [39]. Additional key factors are probably involved in ischemic neuronal degeneration and death that need to be identified to develop effective new strategies for the treatment of stroke. Accumulating evidence now indeed demonstrates a substantial role of caspase-independent pathways downstream mitochondrial damage after ischemic brain injury [34,40,41]. Such pathways can be mediated, for example, through mitochondrial release of AIF or endonuclease G, which both 468

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translocate to the nucleus and induce DNA damage in a caspase-independent manner [26,27]. After oxygen glucose deprivation in vitro or after an ischemic insult in vivo AIF rapidly translocated to the nucleus of injured neurons and colocalized with DNA damage and apoptotic nuclear condensation [40]. Of note, mitochondrial release of AIF occurred several hours before cytochrome c release and caspase-3 activation, suggesting that AIF is in the first line of cell death signaling after ischemia. Although it is still unclear exactly how AIF exerts its apoptogenic function after mitochondrial release, the activation of PARP-1 has been identified as a key step in AIF-mediated apoptosis [21]. More recent data further suggested a potential involvement of calpains [42] and the pro-apoptotic bcl-2 family proteins Bax [43], BimEL [44] and tBid [41] for mitochondrial AIF release in neuronal apoptosis, indicating multiple routes of AIF release and nuclear translocation. The reduction of AIF protein levels in siRNA-treated cultured neurons or in harlequin (Hq) mutant mice resulted in a significant reduction of neuronal cell death in the respective experimental models of ischemia by approximately 50% [41]. These results expose AIF as a promising target for neuroprotective strategies in stroke therapy and other (neuro)degenerative diseases where ischemic cell death is prominent (Table 1).

Vascular damage after stroke Cerebrovascular dysregulation is a key feature in stroke pathology and, in particular, in hemorrhagic transformation after ischemia. Under physiological conditions the balance between energy demands owing to neural activity and substrate delivery through blood flow is tightly regulated by a functional unit of neurons, astrocytes and vascular cells [11]. After ischemic injury, the excessive activation of matrix metalloproteinases (MMP) contributes to the breakdown of extracellular matrix constituents which leads to a destruction of the microvasculature in the ischemic brain tissue [1,10]. In particular, the activation of MMP-9 has been exposed as a major cause for endothelial cell death, vascular dysfunction and hemorrhagic transformation after stroke [1,45]. Moreover, enhanced MMP-9 activity might contribute directly to neuronal apoptosis and ischemic brain damage. Inhibitors of MMPs, in particular MMP-9 inhibitors, could therefore be useful neuroprotectants in stroke therapy which also prevent hemorrhagic transformation [46] (Table 1). The protection of the neurovascular unit in addition to stimulation of angiogenesis and NO-mediated improvement of tissue perfusion emerge as hopeful strategies to preserve the delicate balance of blood supply and energy demand in microenvironment of the brain thereby reducing neurological deficits and neuron death after an ischemic insult [1,11].

Inflammatory reactions Cerebral ischemia triggers a plethora of inflammatory reactions that might progress for days after the onset of the insult

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and is initiated by cytokines, adhesion molecules, prostanoid mediators of inflammation and NO [8,9]. Intervention with such mechanisms has been proposed an attractive therapeutic strategy with a potentially wide therapeutic window in stroke treatment because ongoing inflammatory reactions have been predominantly associated with late stages of ischemic brain injury. In particular, strategies targeting interleukin-1b [47], intercellular adhesion molecule-1 (ICAM-1) [48] or inflammation-related enzymes such as iNOS or cyclooxygenase-2 (COX-2) [8,9] reduced experimental ischemic brain damage with an extended time window. Multiple lines of evidence demonstrated the involvement of postischemic inflammation in infarct development and, in particular, poor neurological outcome [8,9]. For example, ischemic brain damage was reduced after interference with leukocyte–endothelium interactions by depleting the number of circulating neutrophils by neutropenia-inducing treatments, by administration of antibodies blocking adhesion molecules, or by genetic knockout of ICAM-1 or p-selectin [8,48]. Inflammatory cells might contribute to the infarct development through adhesion to vessel endothelium thereby causing microvascular plugging and decreased blood flow. Further, excessive production and release of toxic mediators, in particular interleukin-1b, TNF-a or NO might aggravate ischemic tissue damage, and in particular worsen the behavioral outcome after stroke [9]. Whether leukocytes have to cross the blood–brain barrier to mediate such detrimental effects predominantly in late stages of the infarct development or whether pathological leukocyte– endothelial interactions already contribute to infarct development in early phases after cerebral ischemia is a matter of current investigations. Although clinical stroke trials addressing the efficacy of IL-1 receptor antagonist or antibodies targeting ICAM-1 failed to show a benefit in stroke patients, there is a strong rationale to further explore anti-inflammatory treatments in stroke therapy. In particular, there is strong evidence for an involvement of the enzymes iNOS and COX-2 as predominant inflammatory mediators, and promising preclinical data exist that demonstrate cerebroprotective effects of respective antagonists [8]. In particular, recent data indicate that COX-2 reaction products substantially stimulate iNOS expression and iNOS-mediated generation of cytotoxic NO. Such iNOS-derived NO was apparently required for the full expression of COX-2-induced neurotoxicity because COX-2 inhibition only attenuated ischemic brain damage in wild-type but not in iNOS-deficient mice. Notably, inhibitors of iNOS or COX-2 significantly reduced ischemic brain damage with an extended time window of several hours after onset of the insult [8], which strongly implicates these inflammatory enzymes as promising targets in stroke therapy (Table 1).

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Summary and outlook Around 30 years ago intensive research on stroke was started and developed rapidly. The first topics addressed in this field were the cellular calcium homeostasis, neuronal excitotoxicity and free radicals. Neuroprotective effects of voltagedependent calcium channel blockers, NMDA antagonists and radical scavengers were found in experimental models of cerebral ischemia, and some of these drugs were also tested in clinical studies. However, no convincing effects of these drugs could be demonstrated in stroke patients, or toxic sideeffects prevented their use. Despite these drawbacks, the scientists did not give up their search for stroke therapies. New research topics and drug targets were defined. Caspases and caspase-independent cell death, NO and NOS, the neurovascular unit, mitochondrial function, DNA damage and repair, inflammatory responses and last, but not least, reversible protein phosphorylation belong to these most promising topics. Thus, knowledge on the mechanisms of neuronal degeneration and protection has grown tremendously and there seems to be a good chance to establish efficacious stroke therapy during the next decade.

Acknowledgements We apologize to those authors whose work could not be cited and for restriction to selected mechanisms of cerebral ischemia owing to space limitations. A summary of novel therapeutic approaches and, in particular, regeneration (angiogenesis, neurogenesis) will appear in a separate review.

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