THE ANATOMICAL RECORD 292:2002–2012 (2009) Ischemic Tolerance: The Mechanisms of Neuroprotective Strategy JAN LEHOTSKÝ,1* JOZEF BURDA,2 VIERA DANIELISOVÁ,2 MIROSLAV GOTTLIEB,2 PETER KAPLÁN,1 AND BEATA SANIOVÁ3 1 Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Centre of Excellence: Biomembranes, Martin, Slovakia 2 Institute of Neurobiology, Centre of Excellence, Košice, Slovakia 3 Jessenius Faculty of Medicine, Clinic of Anesthesiology and Intensive Medicine, Comenius University, Martin, Slovakia ABSTRACT The phenomenon of ischemic tolerance perfectly describes this quote ‘‘What does not kill you makes you stronger.’’ Ischemic pre- or postconditioning is actually the strongest known procedure to prevent or reverse neurodegeneration. It works speciﬁcally in sensitive vulnerable neuronal populations, which are represented by pyramidal neurons in the hippocampal CA1 region. However, tolerance is effective in other brain cell populations as well. Although, its nomenclature is ‘‘ischemic’’ tolerance, the tolerant phenotype can also be induced by other stimuli that lead to delayed neuronal death (intoxication). Moreover, the recent data have proven that this phenomenon is not limited to application of sublethal stimuli before the lethal stress but reversed arrangement of events, sublethal stress after lethal insult, is rather equally effective. A very important term is called ‘‘cross conditioning.’’ Cross conditioning is the capability of one stressor to induce tolerance against another. So, since pre- or postconditioners can be used plenty of harmful stimuli, hypo- or hyperthermia and some physiological compounds, such as norepinephrine, bradykinin. Delayed neuronal death is the slow development of postischemic neurodegeneration. This allows an opportunity for a great therapeutic window of 2–3 days to reverse the cellular death process. Moreover, it seems that the mechanisms of ischemic tolerance-delayed postconditioning could be used not only after ischemia but also in some other processes leading to C 2009 Wiley-Liss, Inc. apoptosis. Anat Rec, 292:2002–2012, 2009. V Key words: ischemic tolerance; neuroprotection; mechanisms INTRODUCTION The third leading cause of death and disability is ischemic stroke. Ischemic stroke arises in humans as a consequence of cardiac arrest or the stoppage of blood ﬂow to the brain, which can be due to embolic or thrombic occlusion of arteries. Both global or focal ischemia are very severe pathogenic events with multiple, parallel, and sequential pathogenesis. Global forebrain ischemia leads to selective cell death of vulnerable pyramidal neurons in the hippocampal CA1 region. It also leads to death of cerebral cortex neurons (layers 3, 5, and 6) and the dorsolateral striatum. When blood ﬂow decreases Grant sponsor: Ministry of Education (Slovak Republic); Grant numbers: VEGA 0049/09, VEGA 2/141/09; Grant sponsor: Ministry of Health (Slovak Republic); Grant numbers: EU COST B30, APVV LPP 0235-06, VVCE 0064-07, 55-UK-16/2007. *Correspondence to: Jan Lehotský, Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Malá Hora 4, 036 01 Martin, Slovakia. Fax: 421-434136770. E-mail: email@example.com J. Lehotský and J. Burda contributed equally to this article. Received 18 February 2009; Accepted 10 June 2009 DOI 10.1002/ar.20970 Published online in Wiley InterScience (www.interscience.wiley. com). 2003 ISCHEMIC TOLERANCE INDUCED BY PRE AND POSTCONDITIONING TABLE 1. Examples of anoxia-tolerant animals and natural neuroprotective stimuli in humans (Kirino, 2002; Dirnagl et al., 2003; Gidday, 2006; Obrenovitch, 2008) Inducing stimuli Innate fetal and neonatal tolerance High altitude Transient ischemic attacks Hibernation, hypoxia Winter dormancy Breath holding submersion Breath holding submersion Hibernation Humans during focal ischemia, the area known as ‘‘pneumbra’’ (surrounding necrotic core of ischemia) is perfused by collateral vessels. It also undergoes the apoptotic ﬁnal fate of neurons (Endres et al., 2008). Despite decades of intense research, no effective neuroprotective drugs are available yet to treat acute stroke or cardiac arrest. For this reason, recent attention has shifted to deﬁning the brain’s own evolutionarily conserved endogenous neuroprotective mechanisms, which occurs in ischemic tolerance (IT) or after ischemic preconditioning (IP). IT induced by several paradigms represents an important phenomenon of central nervous system (CNS) adaptation to sublethal short-term ischemia. This results in increased tolerance of CNS to lethal ischemia (Kirino, 2002, Dirnagl et al., 2003; Gidday, 2006). The molecular mechanisms underlying IT are not yet fully understood because of its extreme complexity, involving many signaling pathways and alterations in gene expression. Additionally, a metabolic depression has been suggested to play an important role in IT (Yenari et al., 2008). MECHANISMS OF IT Differences in intensity, duration, and frequency of speciﬁc inducer/stressor determine the spectrum of responses to noxious stimuli, that is, when the stimulus is too weak to induce any response, when it is sufﬁcient to serve as a tolerance trigger, or when it is too strong and harmful, resulting in apoptotic or necrotic damage. It is symptomatic that there are no clear boundaries between acquisition of tolerance and cellular apoptosis/ necrosis (Dirnagl et al., 2003). Rodent and cell culture models serve as a basis for the study of the tolerance phenomenon. To understand this, mother nature serves as a model. In nature, we ubiquitously ﬁnd adaptation to extreme environmental conditions, for example, hypoxic or anoxic tolerance. Hibernation is another example of inherent adaptation to extreme low-blood perfusion in animals. As such, IT can be conceived as an evolutionary conserved form of cerebral plasticity (Dirnagl et al., 2003; Gidday, 2006) (Table 1). It is not surprising that different animals have evolved different molecular strategies to cope with anoxia and severe metabolic stress. This leads to the trigger of the neuroprotective tolerance state. A number of common mechanisms with different relevance features can be recognized: i. depression of metabolic rate, ii. modulation of glycolytic enzymes, Animals Fetus and neonates Children, adults Adults Reptilia: snake, frog Pisces: carp, shark Reptilia: turtle Mammalia: seal Squirrel, bear iii. reduction of ion channel ﬂuxes, iv. suppression of neural activity, v. expression of chaperones and heat shock proteins (Hsp), vi. activation of antioxidant defense systems, vii. adaptation of blood rheology and others (for review, see Gidday, 2006; Obrenovitch, 2008). In the past decade, the phenomenon of IT has attracted immense interest in experimental and clinical ﬁelds as a potential and robust neuroprotective mechanism to the extreme metabolic stress, such as hypoxia/ anoxia/ischemia (Kirino, 2002; Dirnagl et al. 2003; Gidday, 2006; Perez-Pinzon, 2007; Obrenovitch, 2008). STIMULUS-I INDUCING TOLERANCE BY IP A large number of different activating mechanisms/ paradigms are used to induce IT, see Table 2. Although preischemic challenge such as: (i) global ischemia (Kitagawa et al., 1991), or (ii) focal ischemia (Stagliano et al., 1999), or (iii) cerebral hypoxia represents prototypical preconditiong stimuli, IT can be induced by exposing animals, cell cultures or tissue slices to various types of endogenous or exogenous stimuli. For example, cortical spreading depression (Kobayashi et al., 1995), chronic hypoxia/hypoperfusion (Sharp and Bernaudin, 2004), or hyperoxia or oxidative/nitrosative stress, hypothermia or hyperthermia, and heat shock (Nishio et al., 2000) and different pharmacological agents such as chemical preconditioning agents (e.g., metabolic inhibitors, lipopolysaccharides, inﬂammatory cytokines) or natural regulators (erythropoietin, hormones) have been used to induce IT. Brain cooling or therapeutic hypothermia may also be used as factors, which induce IT, see recent reviews (Gidday, 2006; Obrenovitch, 2008). The concept of IP was introduced in the late 80s in the heart by Murry et al. (1986) and later on in the brain by Schurr et al. (1986) and Kitagawa et al. (1991). Although the molecular mechanisms underlying IT induced by IP are not yet fully understood, two windows have been identiﬁed. One window represents very rapid and short-lasting posttranslational changes, and the second window develops slowly (over days) after initial insult. It develops as a robust and long-lasting transcriptional change involving de novo protein synthesis (Barone et al., 1998; Burda et al., 2003; Dirnagl et al., 2003; Meller et al., 2005). Most stressors, including preischemia/hypoxia, induce both rapid and delayed tolerance phenotypes (Gidday, 2006). Mechanisms that are prominent during the ﬁrst phases of acute ischemic insults 2004 LEHOTSKÝ ET AL. such as excitoxicity are presumed to be induced during rapid IT. In particular, elevation of adenosine and activation of adenosine receptor with the modulation of ATP TABLE 2. Examples of inducing stimuli leading to ischemic tolerant phenotype in different animals and in vitro models (Kirino, 2002; Dirnagl et al., 2003; Gidday, 2006; Obrenovitch, 2008) Inducing stimuli Sublethal preischemia/ preconditioning Sublethal postischemia/ postconditioning Remote ischemia Hypoxia or HIF modiﬁers Spreading depression/ excitoxicity pH/ion imbalance Hypoperfusion Epilepsy Hyperthermia/hypothermia Oxidative/nitrosative stress Anesthetics Hypoglycemia/metabolic inhibitors and dysfunction Lipopolysaccharides Immunization/inﬂammatory cytokines Exercise Erythropoietin Other factors (hormones, agonists/antagonists, toxins) Experimental models Gerbils, rats, mice, slices, cell cultures Rats, gerbils, mice Rats, mice Gerbils, rats, mice, slices, cell cultures Rats, mice Rats, mice, cell cultures Gerbils, rats, mice Mice Gerbils, rats, cell cultures Gerbils, rats, mice, cell culture Rats, mice, slices Gerbils, rats, mice, slices, cell cultures Rats Gerbils, rats, mice, cell cultures Gerbils, rats, mice, Gerbils, rats, mice, Gerbils, rats, mice, cell cultures sensitive Kþ channel are paralleled by the activation of protein kinase C and other kinases in rapid tolerance. A critical role for nitric oxide signaling pathways in IP and tolerance was also suggested (Nandagopal et al., 2001). As was recently shown by Meller et al. (2008), the selective ubiquitin–proteasome degradation of a cell deathassociated protein, Bcl-2-interacting mediator of cell death (Bim) with the reduced activation of programmed cell death-associated caspases (caspase 3) could play an important role in rapid tolerance to ischemia. As mentioned earlier, IT can be induced by various stimuli that are not necessarily ischemic or hypoxic (Fig. 1). Thus, the phenomenon of cross-tolerance implies that noxious stress can initiate cellular tolerance to subsequent stress that is different in nature from the ﬁrst one. Therefore, one stressor can promote cross-tolerance to another; however, the efﬁcacy of this tolerance may be more modest, and it appears to vary with the nature and intensity of the ﬁrst challenge. Additionally, the window of evolved IT may also be shifted. However, the nature of the stimulus may determine the speciﬁc protective or in worse meaning the reduced damage epiphenotype. SIGNALING PATHWAYS INVOLVED WITH IT HIF, hypoxia inducing factor. Numerous studies have investigated the overall signaling cascades and key molecules in various in vitro and in vivo models of IT. As mentioned earlier, most of the stressors induce both rapid and delayed tolerance. However, interrelated, multifactorial processes are responsible for adaptive changes leading to IT. As reviewed recently by Obrenovitch (2008), the features of IT partially resemble naturally occuring adaptive mechanisms, including at cellular levels (Fig. 2): Fig. 1. Time scale of injured phenomena induced by ischemic insult without previous preconditioning leading to ischemic/reperfusion injured phenotype. Cerebroprotection can be induced by different types of preconditioning or postconditioning stimuli (ischemic, immu- nological, pharmacological and anesthetic). Temporary deﬁned responses during therapeutical window may induce protective response with which subsequent ischemia serve as basis of the ischemic-tolerant phenotype. ISCHEMIC TOLERANCE INDUCED BY PRE AND POSTCONDITIONING 2005 Fig. 2. Overview of the cellular components that are activated by different pre- and postconditioning stimuli leading to ischemic tolerant phenotype. Akt, serine/threonine protein kinase; AP1, activating protein 1; BCl-2, B cell/lymphoma protein 2; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element binding protein; COX2, cyclooxygenase 2; ERK1,2, extracellular signal regulated kinase; GRP 78, glucose-regulated protein 78; HIF1, hypoxia inducing factor 1; HO-1, heme oxygenase 1; HSP 70, heat shock protein 70; ICAM, intercellular adhesion molecules; JAK-STAT, Janus thyrosine kinasesignal transducer and activator of transduction; JNK, c-jun terminal kinase; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; NOS, nitric oxide synthase; NMDA, N-methyl Daspartate; NFjB, nuclear factor kappa B; NGF, nerve growth factor; ORP 150, oxygen-regulated protein; p53, tumor suppressor p53; PI3K, phosphatidyl inositol 3-kinase; SOD, superoxiddismutase; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor. i. Alterations in energy cellular metabolism (metabolic depression). Although anoxic energy producing pathways cannot be increased during ischemia (limitation of substrate), preconditioning preserves mitochondrial function and membrane integrity (Dave et al., 2001; Racay et al., 2007). Additionally, IP induces upregulation of genes involved in energy metabolism. ii. Preservation of mitochondrial membrane potential and possibly the opening of ATP sensitive Kþ channels with protection against the deﬁcits affecting respiratory complexes and protecting mitochondrial oxidative phosphorylation and antioxidant enzymes such as superoxide dismutases (SODs), catalase, glutathione peroxidase, and thioredoxin system (Danielisova et al., 2006; Racay et al., 2007). iii. Amelioration of different aspects of neuronal excitotoxicity and latency of anoxic/ischemic depolarisation, which leads to a shift of excitatory to inhibitory neurotransmission, such as suppression of glutamate release, downregulation of glutamate AMPA and NMDA receptors and increases in GABA release and downregulation of the glial glutamate transporter isoforms (EAAT1 and EAAT2) and upregulation of GABAA receptors (Dave et al., 2005). Improved capacity to preserve cellular ionic and pH homeostasis by ion transport systems (Ca2þ-ATPase, Naþ, Kþ-ATPase and Naþ/Ca2þ exchanger) and transporters for metabolites. Expression of Hsps as one form of a universal feature of the cellular response to insult. Suppression of cell death and apoptotic mechanisms, such as reduction of cytochrome c release, inhibition of caspases and proapoptotic genes, and activation of survival pathways such as serine/threonine activated kinases (Akt) and extracellular signal-regulated kinases (ERK) and trophic factors. Activation of DNA repair and self-repair/plasticity mechanisms including increasing activity of growth/ neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). iv. v. vi. vii. 2006 LEHOTSKÝ ET AL. viii. Reduction of inﬂammatory response initiated by activation of Toll-like receptor and suppression of induced proinﬂammatory cytokines [(tumor necrosis factors (TNFs) and interleukins and activation of transcription nuclear factor jB (NFjB)]. ix. Cerebrovascular adaptation by vascular remodeling as a result of vascular endothelial growth factors (VEGF) activity, a key angiogenic regulator, and activation of erythropoietin and heme oxygenase 1 as targets of hypoxia inducing factor a1 (HIFa1). The preservation of the blood–brain barrier acts as a result of the attenuated activity of matrix metalloproteinases and cell adhesion molecules. For a comprehensive review of the signaling pathways activated by different types of IT, see the review by Gidday (2006). TRANSLATIONAL MACHINERY AND ANTIOXIDANT DEFENSE In 1991, our laboratory results (Burda et al., 1991) showed that graded postischemic reoxygenation could be used as a simple and effective neuroprotective tool diminishes secondary postischemic damage in nervous tissue. This also includes the newly synthesized proteins (Burda et al., 1991). Later, we have documented that duration of the sublethal ischemic episode (for 3–8 min) (IP) and the time that had elapsed between the second ischemia is important not only for the acquisition of IT (at least in the most vulnerable CA1 hippocampal sector), but it also determines the expression of the 72-kDa Hsp as a proposed molecular transducer of tolerance (Burda et al., 2003). In addition, the following articles presented evidence that the repression of global proteosynthesis induced during reperfusion of the ischemic brain is signiﬁcantly attenuated by IP. Detailed analysis of translational machinery showed that the translation attenuation occurs through multiple molecular targets. We have also shown that IP affects the activity of several translational initiation factors. Additionally, IP affects activities of the Akt, extracellular signal-regulated protein kinase phosphorylation, expression of glucose-regulated protein (GRP78) and growth arrest DNA damage-inducible protein 34 (GADD34) proteins. However, the role eEF2 (translational elongation factor) plays in different phosphorylation states during preconditioning-induced derepression of proteosynthesis was not proven in the hippocampal or cortical areas (Garcı́a et al., 2004a,b). Matrix-assisted laser desorption ionization time of ﬂight (MALDI TOF) and mass spectrometry (MS) were further used in animal models of IT. This was done to analyze complexes of serine/threonine phosphatase 1 (PP1) with the substrates. These substrates are presumably inﬂuenced by IT (Cid et al., 2007). As shown before, inhibition of the PP1 expression is associated with the early ischemic/reperfusion damage and PP1 inhibitors prevent protection evolved by preconditioning (Martin de la Vega et al., 2001; Horiguchi et al., 2002). Moreover, expression of the PP1c positive modulator and GADD34 is induced by IT (Garcı́a et al., 2004a). Ischemic/reperfusion injury altered the interaction of heat shock cognate 71 kDa-protein (HSC), creatine kinase B, and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) with both PP1a and PP1c, and the interaction of phosphodiesterase-6B, transitional ER ATPase, lamin-A, GRP78, c-enolase, neuroﬁlament-L, and ubiquitin ligase with PP1c. Remarkably, preconditioning prevented most of the ischemia-induced effects, which points to the potential role of PP1; it acts as a target of the endogenous protective mechanisms induced by IT. In addition, translational attenuation occurring rapidly during the ﬁrst minutes of reperfusion after ischemic episode is linked with the phosphorylation of the a subunit of eukaryotic translation initiation factor 2 (eIF2a). eIF2a is one of the substrates of protein phosphatase 1. As we have shown by microcystin chromatography and 2D ﬂuorescence difference gel electrophoresis (2D DIGE), the highest levels of phosphorylated eIF2a correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and heat shock cognate protein (HSC70) proteins. They returned to control values in the later reperfusion period. Remarkably, IT promoted a decrease in eIF2a phosphorylated levels. It also induced association of GADD34 with PP1c while preventing DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1a and PP1c isoforms were detected. GRP78 was only detected in PP1c immunoprecipitates. Thus, we suggest that PP1 through different signaling complexes with their interacting proteins, modulates the phosphorylation states of eIF2a, and affects tolerance acquisition with the important role of GADD34/PP1c complexes (Garcı́a-Bonilla et al., 2007). The effect of preconditioning was further studied on functional states of two substrates of Ca2þ-dependent cysteine protease calpain. These are protein p35 and eukaryotic translational elongation factor eEF4G (GarciaBonilla et al., 2006). Although the induced tolerance did not promote signiﬁcant changes in p35 and p25 levels, it stimulated a slight increase in calpastatin (calpain inhibitor) and eIF4G levels in the hippocampal subregions after 4 hr of reperfusion. Remarkably, the iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis reﬂects ischemic damage. Recirculation in rats was signiﬁcantly attenuated by IP. It also prevented neuronal destruction of the hippocampal CA1 region induced by a 20-min ischemia (Danielisova et al., 2004). In accordance with this evidence, we observed activity of the main antioxidant enzymes, SOD and catalase (CAT) in the hippocampus, striatum, and cortex. After induced IT by IP, SOD and CAT were signiﬁcantly increased after 5 hr, 1 and 2 days of reperfusion. The observed increased levels of antioxidant enzymes can be ascribed to the de novo proteosynthesis. However, in spite of upregulation, a large number of CA1 hippocampal neurons underwent neurodegeneration within 7 days after ischemia (Danielisova et al., 2005). CROSS-TALK BETWEEN CELL ORGANELLES Mechanisms that potentially caused protection against Ca2þ toxicity in IT were studied in CA1 hippocampal neurons by the analysis of the level of plasma membrane Ca2þ-ATPase (PMCA) in ischemia-tolerant gerbils. We showed in our laboratory that ischemic-reperfusion insult selectively decreases the level of PMCA1 isoform in hippocampal cells (Lehotsky et al., 1999). This ISCHEMIC TOLERANCE INDUCED BY PRE AND POSTCONDITIONING decrease can be ascribed to the activity of calpain proteases (Lehotsky et al., 2002). As was shown in another study by Garcia-Bonilla et al. (2006), IT increases activity of calpastatin, an inhibitor of calpain. This is in line with the results of Ohta et al. (1996). By histochemical methodology, Ohta determined that IP signiﬁcantly increased the Ca2þATPase level before the start of lethal ischemia. It remained at a higher level subsequently, compared with ischemia-nontolerant CA1 neurons. These results strongly resemble data on CA3 neurons, which are constitutively resistant to periods of ischemia. It also signiﬁes the role of Ca2þ-ATPase in the process of acquired tolerance. This view has been supported by the experiments of Kato et al. (2005), which indicates IT acquisition. PMCA is not essential; however, PMCA1 plays an important role in the enhancement of IT. A recent article of Sun et al. (2007) describing the effect of preconditioning on the heart identiﬁed that S-nitrosylation of Ca2þ-ATPase is involved in the protective role against ischemia. Brain ischemia/reperfusion (IRI) initiates a catastrophic cascade in which many subcellular organelles play an important role. In our own experiments, we have evaluated the effect of IP on ischemic brain injury. This was done by examining the endoplasmic reticulum (ER) stress response (UPR/unfolded protein response) on mRNA and protein levels of speciﬁc genes such as, ATF6, GRP78, and XBP and expression of the Golgi apparatus (secretory pathways) Ca2þATPases (SPCA1) after global ischemia/reperfusion. Ischemia/reperfusion initiated time-dependent differences in ER gene expression at both the mRNA and protein levels. ER gene expression is affected by preischemic treatment. The differences between naı̈ve ischemic and IP animals were detected in both the mRNA and protein levels of all products of ER stress response (Lehotsky et al., 2008, 2009; Urban et al., 2009). In addition, IP partially suppresses the extent of lipo- and protein oxidation in hippocampal membranes initiated by ischemia. IP also leads to partial recovery of the ischemia-induced depression of SPCA activity. At the molecular level, IP initiates an earlier cellular response to the injury by signiﬁcant elevation of mRNA expression and SPCA protein levels (Pavlikova et al., 2009). This suggests that preischemic treatment may exert a role in the attenuation of ER stress response and in the correlation of SPCA-Golgi function; speciﬁcally, in the neuroprotective phenomenon of acquired IT. Changes in the gene expression of the key proteins provide insight into ER stress pathways. It might suggest possible targets of future therapeutic interventions to enhance recovery after stroke (Lehotsky et al., 2009; Urban et al., 2009). Ischemic insult leads to severe disturbance in the energy metabolism and general mitochondrial functions, such as mitochondrial protein synthesis and inhibition of respiratory chain complexes. Mitochondrial dysfunction is considered one of the key events in ischemic neuronal cell death, because of its capacity of releasing apoptogenic proteins. As shown in this laboratory by Racay et al. (2007, 2008, 2009), IP affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Similar to global proteosynthesis, IP also attenuated the repression of mitochondrial translation induced during reperfusion time in hippocampus. However, the activity of respiratory complex IV was protected by IP only 2007 slightly and the activity of complex I remained depressed. Despite this, IP partially reduced changes in the integrity of the mitochondrial membrane, as shown by ﬂuorescent probe measurement. Apoptotic changes initiated by ischemic insult in the CA1 sector of the hippocampus were detected by the Fluoro-Jade C staining, detection of the genomic DNA breaks, translocation of apoptoptic proteins p53 and Bax. Interestingly, preconditioning abolished mitochondrial apoptosis initiation and translocation of p53 protein largely in the hippocampus but not in the cerebral cortex. The results indicate that both the initiation and execution of mitochondrial apoptosis can be abolished by preconditioning in the CA1 hippocampal sector (Racay et al., 2008, 2009). ISCHEMIC POSTCONDITIONING Ischemic postconditioning is a phenomenon that intermits interruptions of blood ﬂow in the early phase of reperfusion which can protect an organ from ischemia/ reperfusion injury. These brief periods of ischemia performed just at the time of reperfusion can reduce the volume of injured tissue. This ‘‘rapid’’ postconditioning is an effective protective treatment in the case of a heart infarct (Zhao et al., 2003; Galagudza et al., 2004; Kin et al., 2004), ischemia of liver (Sun et al., 2004), kidney (Liu et al., 2007), muscles (McAllister et al., 2008), skin (Moon et al., 2008) as well as intestinal mucosa (Santos et al., 2008). In the CNS, postconditioning increases the neuronal survival rate and decreases the size of an infarct area after transient global cerebral ischemia (Pateliya et al., 2008; Wang et al., 2008), focal ischemia (Zhao et al., 2006; Xing et al., 2008), spinal cord ischemic injury and is effective in a rat hippocampal slice model of cerebral ischemia, too (Scartabelli et al., 2008). Gao et al. (2008) found that the protective properties of postconditioning depend on the number of cycles and on the duration of each cycle of reperfusion and occlusion and the onset time of postconditioning; and that they are comparable to those of rapid preconditioning but not as robust as those of delayed preconditioning (Gao et al., 2008). Although this method is quite new, it largely resembles a 20-year-olds attempt to prevent postischemic burst of free oxygen radicals by ‘‘graded (controlled) reoxygenation’’ (Marsala et al., 1989; Burda et al., 1991, 1995). As usual in a novel neuroprotective approach, very little is known about the mechanism of postconditioning effects. Wang et al. (2008) provided the evidence that an appropriate ischemic postconditioning strategy had neuroprotective effects, which are associated with its ability to improve disturbed cerebral blood ﬂow (partial prevention of reactive hyperemia and subsequent hypoperfusion) and prevent cytochrome c translocation. An equal effect mediated by mitochondrial pore transition was suggested in a postconditioned rabbit heart (Argaud et al., 2005). Pignataro et al. (2008) documented the activation of the protein kinase Akt. Activation of the phosphoinositide 3-kinase-linked pathway has been described in postconditioned mice (Rehni and Singh, 2007). A new approach to adopt the postconditioning phenomenon is based on the idea to induce the mechanism of IT by application of drugs known as inducers of IT (O’Sullivan et al., 2007). Authors used diazoxide known to induce preconditioning through its effect as a 2008 LEHOTSKÝ ET AL. mitochondrial potassium ATP channel opener and succinate dehydrogenase inhibitor. This treatment resulted in signiﬁcantly increased Hsps HSP25 and HSP70 expression and induction of tolerance. mechanisms of IT-delayed postconditioning can be exploited not only after ischemia but also in some other processes leading to apoptosis. POSSIBLE CLINICAL APPLICATIONS OF IT DELAYED POSTCONDITIONING In clinical settings, where the interruption of reperfusion cannot be performed immediately at the end of ischemia, the therapeutic window is extremely short. This inspired Ren et al. (2008). He explored that if delayed postconditioning is conducted a few hours after reperfusion, it offers protection against stroke. In rats, after 3–6 hr of focal ischemia, he applied six cycles of 15 min occlusion/15 min release of the ipsilateral common carotid arteries which resulted in a robust reduction of the infarction size. Substantially different is the delayed protein synthesis dependent approach used in Burda’s laboratory. This method is based on discovery that the end effector of IT is activated by lethal ischemia itself (Burda et al., 2005) and full strength tolerance consists of the combination of events occurring after ﬁrst stress with those activated by the repeated stress (Burda et al., 2006). Delayed postconditioning applied 2 days after lethal ischemia exploits the fact that death of most sensitive pyramidal neurons in hippocampal CA1 region is a slow process lasting 2–3 days before the ﬁrst morphological changes can be observed (Kirino, 1982; Pulsinelli et al., 1982). Coincidentally, the process of IT maturation lasts the same time. Thus, application of the second sublethal stress 2 days after lethal stress, outruns irreversible changes leading to apoptosis like delayed neuronal death. All compounds commonly used as preconditioners (short ischemia, hypoxia, norepinephrine, 3-nitropropionic acid, bradykinin) would be effective if used as postconditioners too (Burda et al., 2005, 2006; Danielisova et al., 2006, 2008; Nemethova et al., 2008). With regard to the mechanism of postconditioning, a common feature of all compounds (conditioners) is the activation of SOD and catalase immediately after administration regardless of whether they are used as preconditioners (Danielisova et al., 2005) or postconditioners (Danielisova et al., 2006, 2008). If a postconditioner is used 2 days after the ﬁrst stress when the activity of antioxidant enzymes decreases, but the level of other possible protective proteins (e.g., HSP72) increases (Burda et al., 2003), postconditioner provokes de novo synthesis and increases the activity of endogenous antioxidant enzymes. However, application of the second stress does not exclude occurrence of another protecting compound with antiapoptotic/antidegenerative attributes. Paradigm of delayed postconditioning can be effectively utilized also after kainic acid intoxication (Burda et al., 2007). Number of degenerated CA1 hippocampal neurons, assessed by analysis of neuronal density by Fluoro Jade B-staining, exceeded 64% after 7 days after kainic acid injection. In rats treated 2 days after kainate by i.p. injection of norepinephrine or bradykinin, neurodegeneration in CA1 subﬁeld signiﬁcantly reduced to 6%. These results support the therapeutical value of postconditioning in neuronal protection. Monitoring the interval between ischemia and the ﬁrst signs of delayed neuronal death opens up a broad potential therapeutic window for reversal of cellular death process. Moreover, Since the seminal work of Murry et al. (1986) on the protective effect of preconditioning in the heart, this endogenous response has been reproduced in all species including humans and in a variety of organs. Other than the heart, these also include the kidneys, liver, and brain. In a clinical setting, when an acute myocardial infarction is preceded by preinfarction angina, the resulting infarction is milder. The heart has fewer cardiac arrhythmias and a better left ventricular function compared to hearts without preceding angina (Rezkalla and Kloner, 2007). This observation has promoted the therapeutic application of brief balloon inﬂations instead of longer coronary interventions. This allows protection of the heart (early preconditioning approach) and promotes the development of delayed preconditioning mimetic compounds such as nitro glycerin. Both strategies have been successfully tested in randomized controlled trials (Argaud et al., 2005; Jneid et al., 2005). Nevertheless, the demonstration of adaptation to ischemia in patients in the setting of coronary bypass surgery (Perrault et al., 1996) strongly suggests that IP occurs in humans. However, whether the myocardium in patients with acute myocardial infarct (MI) can be preconditioned independently of collateral ﬂow development still remains a controversial issue. Earlier studies (Ottani et al., 1995) suggested the possible occurrence of IP in patients with acute MI undergoing thrombolytic therapy. However, later studies (Tomoda and Aoki, 1999) questioned this beneﬁcial effect of preinfarction angina and instead suggested that earlier recanalization of occluded coronary arteries and myocardial reperfusion may be a more likely explanation for the limited infarction size seen in those patients. A more recent study (Takahashi et al., 2002) suggested that a protective effect of preinfarction angina is mediated by inhibition of microcirculatory damage after reperfusion. Thus, the available evidence in the clinical setting of IP indicates that cardiomyocytes are not effectively preconditioned in human subjects (Otani, 2008). Indeed, IT of the CNS has also been shown to have consistent and robust neuroprotective effects in the laboratory. Although the clinical importance of IT is obvious, the resulting protective manipulations have so far not yet been transferred into the clinical environment. In clinical settings, it might have practical use for cardiopulmonary bypass surgery, cardiac transplantation, and other types of brain surgeries; where necessary, periods of ischemia are provoked in healthy tissue and possibly also in the management of brain tumors and trauma. Importantly, the multimodal nature of tolerance described in experimental studies indicates that protective pathways are probably highly evolutionary conserved and that harnessing endogenous protecting pathways could potentially allow us to develop therapies that would be well tolerated and capable of reducing multiple types of injuries (Dirnagl et al., 2009). Even in safe strategies, an additional factor needs to be understood; especially, when the target population for preventive neuroprotection is usually elderly patients. ISCHEMIC TOLERANCE INDUCED BY PRE AND POSTCONDITIONING Protective stimuli might be speciﬁc to sex, dependent on age and affected by diet and clinical comorbidities. In addition, it is still unknown whether cumulative neuroprotection can be achieved by the combination of tolerance and pharmacological protection in the risk stroke patients and patients with transient ischemic attacks (Pignataro et al., 2009). In cardiology and nephrology, the tolerance phenomenon is under active clinical investigation with ongoing trials. In neurology, by contrast, only a few clinical studies have been conducted on this topic to test the safety and efﬁcacy of preconditioning strategies. In addition, a consensus has not yet been developed that transient ischemic attack (TIA) provides protection against stroke (Yenari et al., 2008). TIA is deﬁned as a brief episode of neurological dysfunction caused by focal brain ischemia without evidence of acute infarction. Several studies (clinical retrospective reports) support the hypothesis that induced neuroprotection is important in patients with TIA who have a subsequent ischemic stroke. In early 1999, Weih et al. (1999) analyzed 148 ischemic stroke patients with TIA (n ¼ 37) and without TIA (n ¼ 111) and showed that TIA was an independent predictor of mild stroke. Additional studies in humans are consistent with the view that acute stroke preceded by prodromal TIA results in fewer neurological deﬁcits than strokes do without a preceding TIA. Moncayo et al. (2000) reported that brief TIA episodes lasting 10–20 min experienced less than 1 week before infarction have a higher proportion of favorable outcome than those with TIA 1–4 weeks before stroke. Similarly, as shown by Castillo et al. (2003) in patients with ischemic stroke including atherothrombotic and cardioembolic infarcts, groups were signiﬁcantly lower in patients with relevant TIA. In a group of nonlacunar stroke with TIA, Arboix et al. (2004) demonstrated a signiﬁcantly higher favorable outcome with a history of stroke. Interestingly, in the group of lacunar stroke patients, the percentage with neurological deﬁcits was similar and suggested that the previous TIA has some distinctive clinical features. More recently, Schaller (2005) revealed that strokes with the prodromal TIA showing a similar clinical pictures (NIHSS scores) on admission had better outcome than those lacking prodromal TIA. The very recent article of Fu et al. (2008) documented no signiﬁcant effect of prodromal TIA on the outcome in a group of all ischemic stroke patients. However, when they subdivided patients according to the NIHSS difference scores on (i) prodromal TIA lasting up to 4 min; (ii) two prodromal TIA attacks and/or; (iii) prodromal TIA-stroke interval within 7 days separately, they conclusively showed that patients in subgroups 1 and 2 exhibited signiﬁcantly better outcomes. However, no signiﬁcant effect was found in subgroup three, although this TIA group did show better outcome when considering the NIHSS changes. The authors suggested that the neuroprotection conferred by the TIA may be associated with the duration and the frequency of the TIA, although the relationship between the TIA-stroke interval and prognosis is not clear. Based on the above ﬁndings, it seems that TIAs have a protective effect in patients with nonlacunar cerebral infarction when TIAs occur weeks before cerebral infarction and last for more than 10 min. On the other hand, Johnston (2004) failed to show correlation between the occurrence of TIA and reduced disability attributable to 2009 subsequent stroke in a cohort study of more than 1,707 patients with prodromal TIA within 1 day, 1–7 days, and 90 days of a stroke. The reasons for these discrepancies are unclear, but it is difﬁcult to control certain factors in clinical settings such as the duration and timing of TIAs relative to the occurrence of stroke. However, previous work leads to better insights. The spatial and temporal limitations of the protective window provided by preconditioning reminds us of the need to increase our understanding of the nature and consequences of TIAs at least because of limited duration of protection. The previous studies mentioned earlier and study of Schaller and Graf (2002) documented that several TIAs within the week prior to a stroke were strongly associated with a better outcome. Because nobody will prescribe a TIA for a patient prior to experiencing a stroke, the other important question is how to imitate TIA protective effect by introduction of small molecule therapies that regulate targets like Hsps that play a critical role in as we may learn to mimic the biochemical effects of a TIA (Obrenovitch, 2008). However, without a deﬁnite biomarker of TIA, the strong debate over TIA constitutes will continue. Although the recent EXPRESS studies suggested that up to 80% of strokes can be prevented by the emergent evaluation and treatment of patients with TIA (Rothwell et al., 2007, Luengo-Fernandez et al., 2009), the rapid response systems for TIA are just now being implemented. Several clinical trials are ongoing to test the safety and efﬁcacy of other precondioning strategies. 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