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Ischemia and Ischemic Tolerance in the Brain: an Overview
NeuroToxicology 25 (2004) 895–904
Review
Ischemia and Ischemic Tolerance in the Brain: an Overview Daniel Zemke1, Jeremy L. Smith2, Mathew J. Reeves3, Arshad Majid1,* 1
Department of Neurology and Ophthalmology, Michigan State University, East Lansing, MI 48824, USA Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA 3 Department of Epidemiology, Michigan State University, East Lansing, MI 48824, USA 2
Received 4 December 2003; accepted 18 March 2004 Available online 6 May 2004
Abstract Stroke is the third leading cause of death and the leading cause of adult disability in the United States. This review outlines the pathways that lead to cell death following stroke, and also summarizes the current literature on the phenomenon of ischemic tolerance. Ischemic tolerance is an endogenous neuroprotective mechanism by which neurons are protected from the deleterious effects of brain ischemia that occur during and after stroke. A better understanding of the processes that lead to cell death after stroke and endogenous neuroprotective mechanisms like ischemic tolerance could help in the development of new treatment strategies for this devastating neurological disease. # 2004 Elsevier Inc. All rights reserved.
Keywords: Stroke; Ischemia; Tolerance; Preconditioning
INTRODUCTION The brain is one of the most sensitive organs to injury in the entire body. A constant flow of blood to the brain is essential in delivering oxygen and glucose to neurons. If this flow is disrupted for even a short period of time, the result is cell damage or death. Neurons are rarely replaced once they have died, therefore the damage to affected regions may be permanent. There are many causes of blood flow disruption to the brain, and these are collectively referred to as stroke. Causes of stroke can be divided into two main categories, hemorrhage and ischemia (Caplan, 1993). Hemorrhage describes a rupture of a blood vessel within the brain, which leads to leakage of blood into the brain cavity and causes damage to the brain. In ischemia, damage to the brain is caused by a reduction or complete blockage of blood flow to parts of the brain, resulting in glucose and oxygen deficiency. The three main natural causes * Corresponding author. Tel.: þ1-517-432-6247; fax: þ1-517-432-9414. E-mail address: [email protected] (A. Majid).
of ischemia are thrombosis, embolism, and systemic decrease in blood perfusion (Caplan, 1993). Two animal models of cerebral ischemia are generally used in brain ischemia studies. Blood vessel occlusion by thrombosis or embolism can lead to a severe restriction or complete blockage of blood flow to the brain. The resulting condition is known as focal ischemia, as only a portion of the brain is affected. The size and position of the affected area depends on which vessel is occluded. The condition is generally temporary, and blood flow is restored when the occluding material is dislodged. Global ischemia, on the other hand, affects the entire brain, and often is the result of a systemic decrease in blood flow. Causes may include low blood pressure or a reduction in blood volume. The effect can be temporary, or may be longer lasting if caused by a chronic condition. Numerous animal models of cerebral ischemia have been developed, simulating both focal and global ischemia. The ischemia induced in these models may be either temporary or permanent. Even when the type of ischemia is the same in two models, the methods by which that ischemia is induced may differ significantly, and could lead to
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.03.009
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Fig. 1. Methods of experimentally inducing ischemic tolerance. Some methods, such as blood vessel occlusion, sleep deprivation, and spreading depression, can only be used with animal subjects. Other methods can also be used to induce tolerance in cultured cell lines. The possibility exists that additional methods remain to be discovered.
different results. This may make it difficult to compare the results from different studies. Recently, a significant amount of attention has been focused on the phenomenon of ischemic tolerance, also known as ischemic preconditioning. Ischemic tolerance refers to a state in which cells are resistant to the damaging effects of periods of ischemia, resulting in reduced cell death. Ischemic tolerance was first identified in the heart (Murry et al., 1986), but was subsequently found to occur in the brain as well (Kitagawa et al., 1991). Tolerance to ischemia in the brain can be induced by a number of different mechanisms, as depicted in Fig. 1. Many of these mechanisms are potentially damaging to the brain, but when administered at a low level that is insufficient to cause permanent damage, can stimulate protective responses that reduce the damage caused by more severe ischemic events. Natural causes of ischemia such as thrombosis or embolism can be simulated by occlusion of one of the major blood vessels supplying the brain. Typical sites of occlusion in rodent models are the common carotid artery (Plaschke et al., 2000) and the middle cerebral artery (Mackay et al., 2002). With occlusion of a single vessel, the result is focal ischemia. Global ischemia can be produced by occlusion of both carotid arteries. Another common method to precondition neural tissue is hypoxia, or exposure to reduced atmospheric oxygen concentration (Miller et al., 2001). In some cases, reduced atmospheric pressure has been used in combination with reduced oxygen concentration (Romanovskii et al., 2001). Hypoxia can be used to precondition cultured neurons in vitro, in a technique known as oxygen–glucose deprivation (Bruer et al.,
1997). Interestingly, conditions of high atmospheric pressure and oxygen concentration have also been found to have protective effects (Prass et al., 2000). Other preconditioning methods may not actually cause ischemia, but rather stimulate some of the same pathways. A variety of chemical agents have been used to initiate protection, often by interfering in the action of major proteins involved in neuronal damage. Examples include inhibitors of succinic dehydrogenase (Kuroiwa et al., 2000), glutamate receptor agonists (Lam et al., 1998), and hormone analogs (Urayama et al., 2002). Tolerance to ischemic insults can also be produced by cortical spreading depression (Yanamoto et al., 1998), sleep deprivation (Hsu et al., 2003), dietary restriction (Yu and Mattson, 1999), and both hyperthermia and hypothermia (Dirnagl et al., 2003). It has been found that there are two threshold values for cerebral blood flow. Reduction of blood flow below the first threshold results in electrical failure within neurons, and further reduction of flow below the second threshold leads to the failure of metabolism and ion pumps (Astrup et al., 1981). Cells below the second threshold are destined to die. Cells between the two thresholds are electrically silent, but maintain a low level of metabolic activity and can be stable for hours. If full blood flow is restored within a reasonable amount of time, they may recover with no apparent damage. Longer periods of ischemia, however, will result in their death. Cells between the thresholds therefore have a variable fate, and constitute what is known as the ischemic penumbra (Astrup et al., 1981). It is the cells within the penumbra that receive the most benefit from ischemic preconditioning, as their chances of survival are increased.
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DAMAGE AND CELL DEATH DURING ISCHEMIA Failure of normal cellular processes during ischemia results in the accumulation of a number of neurotoxic substances. A significant amount of damage to neurons is caused by excessive amounts of the excitatory amino acid glutamate. The release of glutamate at the synapse is increased in response to an ischemic event, while uptake is decreased (Caplan, 1993). This is a consequence of electrical failure of the cell, and is often termed excitotoxicity. Excess glutamate activates a variety of glutamate receptors, opening ion channels that allow potassium ions to exit the cell and sodium and calcium ions to enter. The influx of sodium ions causes water to enter the cell, leading to edema. Influx of calcium activates a number of calcium-dependent signal pathways, which may be either beneficial or detrimental. Oxygen deficiency during ischemia leads to the production of additional toxic substances. Mitochondria are unable to perform oxidative phosphorylation under conditions of low oxygen concentration and switch to the anaerobic mechanism of lactate fermentation, leading to the accumulation of lactic acid to toxic levels (Caplan, 1993). Reactive oxygen species are also produced under conditions of low oxygen, and can react with and destroy organelles and the plasma membrane. A damaged membrane results in the inability of the cell to control ion flux, leading to mitochondrial failure. The antioxidant Edaravone has shown promising results in reducing infarct size in initial trials (Kogure, 2002). Melatonin is another potentially useful antioxidant for treatment, and is non-toxic even at high doses (Cheung, 2003). Reactive oxygen species, as well as calcium influx and other factors, can induce permeabilization of the mitochondrial membrane (Mattson and Kroemer, 2003). Molecules normally confined to the mitochondria, such as cytochrome c, are released into the cytoplasm, where they may bind and inactivate inhibitors of caspases. Enzymes that destroy DNA may also be released. Both of these factors are involved in the process of apoptosis, which leads to cell death. Strategies targeting the process of apoptosis are being developed that may offer protection through the use of proteins that inhibit caspase activity (Onteniente et al., 2003). A number of protective mechanisms are initiated in the body in response to the damaging processes of ischemia. The oxygen extraction fraction, the percentage of the oxygen available in the blood that is delivered to the tissues, is increased in ischemic
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regions (Schaller and Graf, 2002). This may allow sufficient levels of oxygen to be provided even under conditions of reduced blood flow. A lack of oxygen also triggers the activation of the hypoxic response pathway through the hypoxia-inducible factor 1 (HIF1) transcription factor (Dirnagl et al., 2003). Glucose transport and glycolysis are increased, which may allow the mitochondria to sustain low levels of metabolism. Some of the body’s responses, however, may have both beneficial and detrimental effects. Damaged tissue stimulates the process of inflammation, resulting in increased blood flow to the area and the release of cytokines to attract leukocytes (del Zoppo et al., 2001). Although an increase in blood flow helps to alleviate the ischemia and delivers essential glucose and oxygen, it may also deliver more calcium, which further stimulates damaging mechanisms. Leukocytes attracted to the damaged areas react by releasing more cytokines. Cytokine levels may accumulate to the point that they become toxic, thereby increasing tissue damage. A number of reviews have been published debating the advantages and disadvantages of inflammation in relation to ischemia and stroke (del Zoppo et al., 2001; Feuerstein and Wang, 2001; Xi et al., 2003).
IMPORTANT MOLECULES IN ISCHEMIC TOLERANCE Although glutamate release and glutamate receptor activation are responsible for much of the cell damage due to excitotoxicity, they might also be important for the establishment of protection following preconditioning. Exposure of cortical cell cultures to low levels of glutamate or NMDA to induce NMDA receptor activation has been found to have a preconditioning effect (Grabb and Choi, 1999). Additionally, preconditioning by oxygen–glucose deprivation was blocked if an NMDA antagonist was applied. Unfortunately, although NMDA receptor antagonists have been shown to be neuroprotective in animal models, many have been found to have undesirable side effects in humans that make them unsuitable for therapeutic use (Lees, 1997). One promising alternative is treatment with magnesium, which not only blocks NMDA receptors, but also blocks calcium channels, is an antagonist of glutamate release, and helps maintain cerebral blood flow (Muir, 2002). Other glutamate receptor subtypes besides the NMDA receptor might also be involved in ischemic cell damage. The expression of AMPA receptor subunits is decreased in preconditioned tissues (Chazot et al., 2002; Deng et al., 2003). These subunits
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are linked to increased calcium permeability that may lead to cell damage. Expression of neurotoxic metabotropic glutamate receptor subtypes is also reduced following preconditioning (Sommer et al., 2000). Preconditioning with cortical spreading depression has been shown to result in the downregulation of glutamate transporters on glial cells, but not in neurons (Douen et al., 2000). Although these transporters are normally involved in glutamate uptake, it has been suggested that the influx of sodium that occurs during excitotoxicity may cause their reversal and result in additional glutamate release. Downregulating these transporters may therefore contribute to ischemic tolerance. The response to calcium influx is also important for the development of protection against ischemia. Calcium influx has been linked to the production of reactive oxygen species leading to cell damage (Kristia´ n and Siesjo¨ , 1998). Calcium is also responsible for the initiation of a number of signaling cascades, however. The cyclic AMP responsive element binding protein (CREB) is required for the induction of ischemic tolerance (Hara et al., 2003). CREB is a transcription factor that activates a number of genes in response to intracellular calcium. Phosphorylation of CREB is increased for several days following ischemia in dentate granule cells, which are relatively resistant to cell damage (Hu et al., 1999). Preconditioning results in enhanced CREB phosphorylation within the ischemic penumbra (Nakajima et al., 2002). The amount of intracellular calcium, and therefore the amount of gene activation by CREB, is regulated by NMDA receptor activation, which further implicates these receptors as important players in the preconditioning process. NMDA receptor activation also results in the production of nitric oxide (Nandagopal et al., 2001). The activity of nitric oxide synthase (NOS) and the total amount of nitric oxide present in the brain are increased following exposure to hypoxia (Lu and Liu, 2001). After repeated exposures involved in preconditioning, however, their levels are significantly decreased. Nitric oxide is a signaling molecule, and activation of Ras by nitric oxide with the subsequent activation of Erk is required for protection from oxygen–glucose deprivation (Gonzalez-Zulueta et al., 2000). One potential target of Erk is CREB, which has been previously mentioned (Nandagopal et al., 2001). Nitric oxide is also a free radical, however, and may react with and damage important molecules within cells. Nitric oxide can combine with superoxide to form peroxynitrite, which causes single strand breaks in DNA (Love,
1999). The resulting activation of DNA repair enzymes consumes vital energy needed for other processes. Nitric oxide may also be involved in the induction of apoptosis. Treatment with nitric oxide scavengers and NOS inhibitors reduces the death of cerebral endothelial cells following oxygen–glucose deprivation (Xu et al., 2000). On the other hand, inhibition of NOS results in increased adherence of leukocytes to endothelial cells in cerebral blood vessels (Gidday et al., 1998). The presence of activated leukocytes has been implicated in cell damage in cerebral ischemia. There are also differences in the effects produced by different forms of NOS. The neuronal (nNOS) and inducible (iNOS) forms lead to brain damage, but the endothelial (eNOS) form causes vasodilation and increases blood flow, and therefore is considered to be beneficial (Bolanos and Almeida, 1999). Permeabilization of the mitochondrial membrane as a result of reactive oxygen species and calcium influx is proposed to be mediated by the pro-apoptotic proteins Bax and Bak, which may oligomerize to form pores (Mattson and Kroemer, 2003). Cytochrome c released from the mitochondria binds Apaf-1 in the cytoplasm, resulting in the recruitment and activation of caspase-9 (Wang, 2001). Caspase-9 further activates caspase-3, one of the main enzymes involved in apoptosis. In some studies, ischemic preconditioning results in a reduction of caspase-3 activation, and a subsequent reduction in neuron cell death by apoptosis (Cantagrel et al., 2003; Qi et al., 2001). However, there is also evidence to suggest that activation of caspase-3 can occur without cell damage, and that this activation may be necessary for ischemic tolerance (McLaughlin et al., 2003). One important factor in apoptosis is the amount of ATP present within the cell. ATP is required for nuclear condensation and DNA degradation in the final stages of apoptosis (Leist et al., 1997). Depletion of ATP forces cells to switch to death by necrosis rather than apoptosis. Apoptosis, or programmed cell death, involves condensation of the cytoplasm, fragmentation of DNA, and breakdown of the cellular structures into membrane bound bodies that are phagocytosed and digested by macrophages (Yuan et al., 2003). In contrast, necrosis is characterized by swelling of the cell and its organelles, resulting in cell rupture and release of cellular materials into the surrounding area. Apoptosis is more highly regulated than necrosis, and its processes are more clearly understood, providing the opportunity for methods of intervention that may prevent cell death. The use of anti-apoptotic agents, therefore, can potentially salvage cells within the penumbra, which have a low level of metabolism that is sufficient
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to prevent death by necrosis. The anti-apoptotic gene Bcl2 is of particular interest in this regard, and transfer of this gene via liposomes or viral vectors has been shown to be neuroprotective (Cao et al., 2002; Lawrence et al., 1997). Other factors involved in the development of ischemic tolerance are still being discovered. The transcription factor NFkB is activated and translocated in response to sublethal ischemia (Blondeau et al., 2001). Inhibition of NFkB activity blocked neuroprotection by preconditioning. The expression of heat shock proteins increases following ischemia and preconditioning (Currie et al., 2000; Pringle et al., 1999; Tanaka et al., 2002). It has been suggested that certain heat shock proteins might be involved in protection against ischemic insult. Adenosine receptors may also be involved, and antagonists to these receptors block ischemic tolerance (Hiraide et al., 2001). Countless other genes and their protein products have been found to be upregulated or downregulated in response to ischemic preconditioning.
CHEMICAL TREATMENT AND ISCHEMIC TOLERANCE A number of chemical treatments have been found that can lead to the development of ischemic tolerance. Often these involve causing small amounts of damage to induce the body’s protective processes. One way to precondition tissues against ischemic damage is by inducing inflammation. One way to induce inflammation is with the protease thrombin. Preconditioning with low doses of thrombin is known to induce protection against a subsequent ischemic insult (Masada et al., 2000). At higher concentrations, however, the effects are deleterious (Striggow et al., 2000). Low concentrations of thrombin produce a spike in intracellular calcium concentrations but high concentrations caused a sustained elevation of calcium, which may be the determining factor between beneficial and harmful effects. Thrombin treatment results in the activation of Erk, which may explain the neuroprotective effects of low doses (Xi et al., 2001). Inhibitors or antagonists of normal biological processes can also be used to induce tolerance against ischemia. Ischemic damage to cultured neurons can be reduced by treatment with 3-nitropropionic acid, a compound that blocks the respiratory chain by inhibiting succinate dehydrogenase (Weih et al., 1999). This compound has also been used to induce protection in vivo in gerbils (Kuroiwa et al., 2000). Haloperidol,
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which blocks mitochondria at a different step, also has protective effects (Riepe and Ludolph, 1997). Lipopolysaccharide, a component of bacterial endotoxin, can also be used to precondition neural tissue (Puisieux et al., 2000). Not all preconditioning treatments involve the use of low doses, however. The polyamine spermine is neurotoxic at low levels (Virgili et al., 1999). In contrast, high concentrations have been found to be neuroprotective, possibly by blocking NMDA receptor channels (Ferchmin et al., 2000). Although chemical treatments artificially stimulate protection and may not accurately reflect natural mechanisms of ischemia, the results can often be quite similar.
IMPORTANT FACTORS IN ISCHEMIC PRECONDITIONING The protection provided by ischemic preconditioning appears to occur in two distinct phases. The early, or rapid, phase is established within minutes and may last for several hours (Dirnagl et al., 2003; Schaller and Graf, 2002). Protection has been observed when an ischemic insult is administered as little as 30 min following preconditioning (Pe´ rez-Pinzo´ n et al., 1997). It is thought that changes in cell metabolism may account for the immediate response. The activation of existing proteins and upregulation of normal processes such as the oxygen extraction fraction are probably involved as well. The late, or delayed, phase of protection requires days to develop and provides lasting protection. It has been found that protein synthesis is suppressed in certain parts of the brain in the early stages following an ischemic insult (Kato et al., 1995). After 1–2 days, protein synthesis has recovered in preconditioned animals, but not in controls. In other regions of the brain, protein synthesis is suppressed only in non-preconditioned animals. If the protein synthesis inhibitor cycloheximide is administered prior to preconditioning, no protection is observed (Barone et al., 1998). Therefore, establishment of the late phase appears to require gene expression and protein synthesis. Microarray studies have been used to examine the changes in gene expression that occur following hypoxia (Bernaudin et al., 2002). Changes in expression were immediate for some genes, but delayed for others. Some genes showed a change in expression at only a single time point, indicating a transient change, whereas others had a continued change in expression across multiple time points. Development of a properly designed preconditioning protocol is essential for the study of ischemic tolerance. First, the preconditioning treatment must be severe
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enough to initiate a response, but not so severe as to cause permanent damage. In a study of ischemia in gerbils, for example, it was found that at least 2 min of ischemia was required in order to induce protection against a more severe insult 2 days later (Kitagawa et al., 1991). Second, the subsequent insult must be planned to occur at a time point at which tolerance has been established. In one study, protection was not observed until 2 days after preconditioning, and had disappeared by 7 days after preconditioning (Chen et al., 1996). The delay in forming protection likely reflects the need for protein synthesis during the late phase, and its disappearance indicates that the protective effect is only temporary. Third, if the subsequent ischemic insult is too severe, the amount of protection provided by preconditioning will not be enough to prevent permanent damage. In the initial study of ischemic preconditioning in the heart, the preconditioning regimen used provided protection against a subsequent 40 min insult, but not a 3 h insult (Murry et al., 1986). Of course, differences in species, method of inducing ischemia, and other factors may influence the development of tolerance, therefore it is necessary to optimize the preconditioning protocol to best match the system under study. There is evidence to suggest that genetic variation can have an effect on the severity of damage produced by ischemia. In a study of three common mouse strains, it was found that one of the strains had significantly larger infarcts than the other two strains following permanent cerebral ischemia (Majid et al., 2000). Different responses to preconditioning have also been observed in spontaneously hypertensive rat strains. Rats from a stroke-prone strain had a lower response to ischemic preconditioning and a larger infarct size than rats that were not prone to stroke (Purcell et al., 2003). Transgenic mice expressing a portion of the mutant human gene involved in Huntington’s disease show protection against cerebral ischemia (Schiefer et al., 2002). These studies emphasize the need to determine differences in gene expression between inbred strains and identify which differently expressed products might be involved in the induction of tolerance.
THE SEARCH FOR NEW THERAPEUTIC TARGETS Molecules and proteins that are important for the development of ischemic tolerance in the brain are potential targets for the development of new treatments for ischemia and stroke. One strategy to identify such
targets is to look at factors that have already been determined to be important for tolerance in other tissues. As mentioned previously, ischemic tolerance was first identified in the heart, and has been studied more extensively in the heart than in the brain. It is likely that some of the processes essential for the development of tolerance are the same in both tissues. Newly identified targets in the heart should therefore be considered as being potentially important in the brain, and vice versa. Another strategy to identify new targets is to look at molecules and proteins that interact with currently known targets. If one component of a particular pathway is known to be important for the development of tolerance, the other components of that pathway might be important as well. Of course, there may be novel factors involved in tolerance that cannot be detected by these strategies and that will require broader studies. Often, potential targets are first identified by the observation of changes in gene expression level, protein level, or protein activity following ischemia or the preconditioning treatment. If certain genes are suspected to be involved in tolerance, changes in their expression levels can be individually assessed. Alternately, the entire mRNA population of a tissue can be screened to detect all genes with changed expression, which allows the identification of novel targets. Microarrays are one way to accomplish such broad screening. Changes in gene expression must then be confirmed to cause a corresponding change in protein level. Changes in protein levels can also be analyzed directly without first looking at gene expression levels. Individual proteins can be studied, or broader techniques such as two-dimensional electrophoresis can be used to screen the entire protein population and identify novel targets. There is also the possibility that a change in protein activity is involved, rather than a change in gene expression or protein level. Potential targets must then be tested to determine if they are important for the development of tolerance. Genes can be modified so that they are constitutively expressed or their expression is blocked. Mutants or gene knockouts can also be generated. Proteins can be inhibited or activated with various compounds, or they can be overexpressed. The effect of these manipulations on protection against ischemic damage in the brain will determine if the targets have therapeutic potential.
SUMMARY The response to ischemia in the brain involves a complex interaction between many different processes,
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Fig. 2. Overview of the some of the processes involved in damage and protection of the brain following ischemia. Lines with arrowheads indicate positive interactions. Lines with flat ends indicate inhibitory interactions.
and is still not completely understood. Fig. 2 summarizes some of the processes currently known to occur following cerebral ischemia. In the presence of low oxygen, cell metabolism results in the production of damaging reactive oxygen species and reduced ATP production by lactate fermentation. Excess glutamate release stimulates calcium influx and nitric oxide production by nNOS/iNOS that, together with reactive oxygen species, damage cellular structures and lead to inflammation and cell death by apoptosis or necrosis, depending on ATP levels. In response, inflammation and the production of nitric oxide by eNOS
stimulate increased blood flow to the area. Nitric oxide production by nNOS/iNOS and inflammation result in the activation of Erk, which, together with calcium influx, activates CREB and initiates pathways that inhibit apoptosis. Erythropoietin is activated by the hypoxia response pathway, and may lead to the inhibition of apoptosis as well. The hypoxic response pathway also stimulates increased glucose transport and the formation of new blood cells and blood vessels. The responses to cerebral ischemia can be both beneficial and harmful at the same time, and the survival of neurons within ischemic regions depends
Table 1 Beneficial and detrimental effects of processes and molecules involved in ischemia Factor
Benefits
Detriments
Glutamate
Low levels have preconditioning effect through NMDA receptor activation Activates CREB, leading to activation of gene pathways leading to tolerance Increased blood flow delivers more oxygen and glucose to affected area Signaling molecule leading to Erk activation. Production by eNOS stimulates increase in blood flow Low levels of caspase-3 may be required for ischemic tolerance
High levels lead to imbalance of ion flux due to receptor overstimulation High concentrations participate in imbalance of ion flux and mitochondrial membrane permeabilization Cell damage caused by increased delivery of calcium and inflammatory cells secreting cytokines Free radical can damage cell structures. Reacts to form peroxynitrite, which does more damage Caspase activation leads to initiation of apoptotic mechanisms
Calcium Inflammation Nitric oxide Caspase activity
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critically on this delicate balance. Table 1 lists the beneficial and detrimental aspects of some of the major players in ischemia and ischemic tolerance. In many cases, it seems that even harmful substances may be beneficial when present in low doses. Ischemic preconditioning, by providing a moderate level of protection against cell damage, can alter this balance so that a larger proportion of cells in the affected regions will survive. Although the mechanisms behind the formation of protection are still largely unknown, significant progress has been made toward identifying some of the major molecules involved. Once the processes involved in preconditioning are more fully understood, the potential benefits for prevention and treatment of brain damage due to ischemia are substantial. Additionally, many of the damaging processes involved in stroke also play roles in other neurological conditions. It is possible that these conditions might benefit from preconditioning as well.
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Review
Ischemia and Ischemic Tolerance in the Brain: an Overview Daniel Zemke1, Jeremy L. Smith2, Mathew J. Reeves3, Arshad Majid1,* 1
Department of Neurology and Ophthalmology, Michigan State University, East Lansing, MI 48824, USA Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA 3 Department of Epidemiology, Michigan State University, East Lansing, MI 48824, USA 2
Received 4 December 2003; accepted 18 March 2004 Available online 6 May 2004
Abstract Stroke is the third leading cause of death and the leading cause of adult disability in the United States. This review outlines the pathways that lead to cell death following stroke, and also summarizes the current literature on the phenomenon of ischemic tolerance. Ischemic tolerance is an endogenous neuroprotective mechanism by which neurons are protected from the deleterious effects of brain ischemia that occur during and after stroke. A better understanding of the processes that lead to cell death after stroke and endogenous neuroprotective mechanisms like ischemic tolerance could help in the development of new treatment strategies for this devastating neurological disease. # 2004 Elsevier Inc. All rights reserved.
Keywords: Stroke; Ischemia; Tolerance; Preconditioning
INTRODUCTION The brain is one of the most sensitive organs to injury in the entire body. A constant flow of blood to the brain is essential in delivering oxygen and glucose to neurons. If this flow is disrupted for even a short period of time, the result is cell damage or death. Neurons are rarely replaced once they have died, therefore the damage to affected regions may be permanent. There are many causes of blood flow disruption to the brain, and these are collectively referred to as stroke. Causes of stroke can be divided into two main categories, hemorrhage and ischemia (Caplan, 1993). Hemorrhage describes a rupture of a blood vessel within the brain, which leads to leakage of blood into the brain cavity and causes damage to the brain. In ischemia, damage to the brain is caused by a reduction or complete blockage of blood flow to parts of the brain, resulting in glucose and oxygen deficiency. The three main natural causes * Corresponding author. Tel.: þ1-517-432-6247; fax: þ1-517-432-9414. E-mail address: [email protected] (A. Majid).
of ischemia are thrombosis, embolism, and systemic decrease in blood perfusion (Caplan, 1993). Two animal models of cerebral ischemia are generally used in brain ischemia studies. Blood vessel occlusion by thrombosis or embolism can lead to a severe restriction or complete blockage of blood flow to the brain. The resulting condition is known as focal ischemia, as only a portion of the brain is affected. The size and position of the affected area depends on which vessel is occluded. The condition is generally temporary, and blood flow is restored when the occluding material is dislodged. Global ischemia, on the other hand, affects the entire brain, and often is the result of a systemic decrease in blood flow. Causes may include low blood pressure or a reduction in blood volume. The effect can be temporary, or may be longer lasting if caused by a chronic condition. Numerous animal models of cerebral ischemia have been developed, simulating both focal and global ischemia. The ischemia induced in these models may be either temporary or permanent. Even when the type of ischemia is the same in two models, the methods by which that ischemia is induced may differ significantly, and could lead to
0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.03.009
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Fig. 1. Methods of experimentally inducing ischemic tolerance. Some methods, such as blood vessel occlusion, sleep deprivation, and spreading depression, can only be used with animal subjects. Other methods can also be used to induce tolerance in cultured cell lines. The possibility exists that additional methods remain to be discovered.
different results. This may make it difficult to compare the results from different studies. Recently, a significant amount of attention has been focused on the phenomenon of ischemic tolerance, also known as ischemic preconditioning. Ischemic tolerance refers to a state in which cells are resistant to the damaging effects of periods of ischemia, resulting in reduced cell death. Ischemic tolerance was first identified in the heart (Murry et al., 1986), but was subsequently found to occur in the brain as well (Kitagawa et al., 1991). Tolerance to ischemia in the brain can be induced by a number of different mechanisms, as depicted in Fig. 1. Many of these mechanisms are potentially damaging to the brain, but when administered at a low level that is insufficient to cause permanent damage, can stimulate protective responses that reduce the damage caused by more severe ischemic events. Natural causes of ischemia such as thrombosis or embolism can be simulated by occlusion of one of the major blood vessels supplying the brain. Typical sites of occlusion in rodent models are the common carotid artery (Plaschke et al., 2000) and the middle cerebral artery (Mackay et al., 2002). With occlusion of a single vessel, the result is focal ischemia. Global ischemia can be produced by occlusion of both carotid arteries. Another common method to precondition neural tissue is hypoxia, or exposure to reduced atmospheric oxygen concentration (Miller et al., 2001). In some cases, reduced atmospheric pressure has been used in combination with reduced oxygen concentration (Romanovskii et al., 2001). Hypoxia can be used to precondition cultured neurons in vitro, in a technique known as oxygen–glucose deprivation (Bruer et al.,
1997). Interestingly, conditions of high atmospheric pressure and oxygen concentration have also been found to have protective effects (Prass et al., 2000). Other preconditioning methods may not actually cause ischemia, but rather stimulate some of the same pathways. A variety of chemical agents have been used to initiate protection, often by interfering in the action of major proteins involved in neuronal damage. Examples include inhibitors of succinic dehydrogenase (Kuroiwa et al., 2000), glutamate receptor agonists (Lam et al., 1998), and hormone analogs (Urayama et al., 2002). Tolerance to ischemic insults can also be produced by cortical spreading depression (Yanamoto et al., 1998), sleep deprivation (Hsu et al., 2003), dietary restriction (Yu and Mattson, 1999), and both hyperthermia and hypothermia (Dirnagl et al., 2003). It has been found that there are two threshold values for cerebral blood flow. Reduction of blood flow below the first threshold results in electrical failure within neurons, and further reduction of flow below the second threshold leads to the failure of metabolism and ion pumps (Astrup et al., 1981). Cells below the second threshold are destined to die. Cells between the two thresholds are electrically silent, but maintain a low level of metabolic activity and can be stable for hours. If full blood flow is restored within a reasonable amount of time, they may recover with no apparent damage. Longer periods of ischemia, however, will result in their death. Cells between the thresholds therefore have a variable fate, and constitute what is known as the ischemic penumbra (Astrup et al., 1981). It is the cells within the penumbra that receive the most benefit from ischemic preconditioning, as their chances of survival are increased.
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DAMAGE AND CELL DEATH DURING ISCHEMIA Failure of normal cellular processes during ischemia results in the accumulation of a number of neurotoxic substances. A significant amount of damage to neurons is caused by excessive amounts of the excitatory amino acid glutamate. The release of glutamate at the synapse is increased in response to an ischemic event, while uptake is decreased (Caplan, 1993). This is a consequence of electrical failure of the cell, and is often termed excitotoxicity. Excess glutamate activates a variety of glutamate receptors, opening ion channels that allow potassium ions to exit the cell and sodium and calcium ions to enter. The influx of sodium ions causes water to enter the cell, leading to edema. Influx of calcium activates a number of calcium-dependent signal pathways, which may be either beneficial or detrimental. Oxygen deficiency during ischemia leads to the production of additional toxic substances. Mitochondria are unable to perform oxidative phosphorylation under conditions of low oxygen concentration and switch to the anaerobic mechanism of lactate fermentation, leading to the accumulation of lactic acid to toxic levels (Caplan, 1993). Reactive oxygen species are also produced under conditions of low oxygen, and can react with and destroy organelles and the plasma membrane. A damaged membrane results in the inability of the cell to control ion flux, leading to mitochondrial failure. The antioxidant Edaravone has shown promising results in reducing infarct size in initial trials (Kogure, 2002). Melatonin is another potentially useful antioxidant for treatment, and is non-toxic even at high doses (Cheung, 2003). Reactive oxygen species, as well as calcium influx and other factors, can induce permeabilization of the mitochondrial membrane (Mattson and Kroemer, 2003). Molecules normally confined to the mitochondria, such as cytochrome c, are released into the cytoplasm, where they may bind and inactivate inhibitors of caspases. Enzymes that destroy DNA may also be released. Both of these factors are involved in the process of apoptosis, which leads to cell death. Strategies targeting the process of apoptosis are being developed that may offer protection through the use of proteins that inhibit caspase activity (Onteniente et al., 2003). A number of protective mechanisms are initiated in the body in response to the damaging processes of ischemia. The oxygen extraction fraction, the percentage of the oxygen available in the blood that is delivered to the tissues, is increased in ischemic
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regions (Schaller and Graf, 2002). This may allow sufficient levels of oxygen to be provided even under conditions of reduced blood flow. A lack of oxygen also triggers the activation of the hypoxic response pathway through the hypoxia-inducible factor 1 (HIF1) transcription factor (Dirnagl et al., 2003). Glucose transport and glycolysis are increased, which may allow the mitochondria to sustain low levels of metabolism. Some of the body’s responses, however, may have both beneficial and detrimental effects. Damaged tissue stimulates the process of inflammation, resulting in increased blood flow to the area and the release of cytokines to attract leukocytes (del Zoppo et al., 2001). Although an increase in blood flow helps to alleviate the ischemia and delivers essential glucose and oxygen, it may also deliver more calcium, which further stimulates damaging mechanisms. Leukocytes attracted to the damaged areas react by releasing more cytokines. Cytokine levels may accumulate to the point that they become toxic, thereby increasing tissue damage. A number of reviews have been published debating the advantages and disadvantages of inflammation in relation to ischemia and stroke (del Zoppo et al., 2001; Feuerstein and Wang, 2001; Xi et al., 2003).
IMPORTANT MOLECULES IN ISCHEMIC TOLERANCE Although glutamate release and glutamate receptor activation are responsible for much of the cell damage due to excitotoxicity, they might also be important for the establishment of protection following preconditioning. Exposure of cortical cell cultures to low levels of glutamate or NMDA to induce NMDA receptor activation has been found to have a preconditioning effect (Grabb and Choi, 1999). Additionally, preconditioning by oxygen–glucose deprivation was blocked if an NMDA antagonist was applied. Unfortunately, although NMDA receptor antagonists have been shown to be neuroprotective in animal models, many have been found to have undesirable side effects in humans that make them unsuitable for therapeutic use (Lees, 1997). One promising alternative is treatment with magnesium, which not only blocks NMDA receptors, but also blocks calcium channels, is an antagonist of glutamate release, and helps maintain cerebral blood flow (Muir, 2002). Other glutamate receptor subtypes besides the NMDA receptor might also be involved in ischemic cell damage. The expression of AMPA receptor subunits is decreased in preconditioned tissues (Chazot et al., 2002; Deng et al., 2003). These subunits
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are linked to increased calcium permeability that may lead to cell damage. Expression of neurotoxic metabotropic glutamate receptor subtypes is also reduced following preconditioning (Sommer et al., 2000). Preconditioning with cortical spreading depression has been shown to result in the downregulation of glutamate transporters on glial cells, but not in neurons (Douen et al., 2000). Although these transporters are normally involved in glutamate uptake, it has been suggested that the influx of sodium that occurs during excitotoxicity may cause their reversal and result in additional glutamate release. Downregulating these transporters may therefore contribute to ischemic tolerance. The response to calcium influx is also important for the development of protection against ischemia. Calcium influx has been linked to the production of reactive oxygen species leading to cell damage (Kristia´ n and Siesjo¨ , 1998). Calcium is also responsible for the initiation of a number of signaling cascades, however. The cyclic AMP responsive element binding protein (CREB) is required for the induction of ischemic tolerance (Hara et al., 2003). CREB is a transcription factor that activates a number of genes in response to intracellular calcium. Phosphorylation of CREB is increased for several days following ischemia in dentate granule cells, which are relatively resistant to cell damage (Hu et al., 1999). Preconditioning results in enhanced CREB phosphorylation within the ischemic penumbra (Nakajima et al., 2002). The amount of intracellular calcium, and therefore the amount of gene activation by CREB, is regulated by NMDA receptor activation, which further implicates these receptors as important players in the preconditioning process. NMDA receptor activation also results in the production of nitric oxide (Nandagopal et al., 2001). The activity of nitric oxide synthase (NOS) and the total amount of nitric oxide present in the brain are increased following exposure to hypoxia (Lu and Liu, 2001). After repeated exposures involved in preconditioning, however, their levels are significantly decreased. Nitric oxide is a signaling molecule, and activation of Ras by nitric oxide with the subsequent activation of Erk is required for protection from oxygen–glucose deprivation (Gonzalez-Zulueta et al., 2000). One potential target of Erk is CREB, which has been previously mentioned (Nandagopal et al., 2001). Nitric oxide is also a free radical, however, and may react with and damage important molecules within cells. Nitric oxide can combine with superoxide to form peroxynitrite, which causes single strand breaks in DNA (Love,
1999). The resulting activation of DNA repair enzymes consumes vital energy needed for other processes. Nitric oxide may also be involved in the induction of apoptosis. Treatment with nitric oxide scavengers and NOS inhibitors reduces the death of cerebral endothelial cells following oxygen–glucose deprivation (Xu et al., 2000). On the other hand, inhibition of NOS results in increased adherence of leukocytes to endothelial cells in cerebral blood vessels (Gidday et al., 1998). The presence of activated leukocytes has been implicated in cell damage in cerebral ischemia. There are also differences in the effects produced by different forms of NOS. The neuronal (nNOS) and inducible (iNOS) forms lead to brain damage, but the endothelial (eNOS) form causes vasodilation and increases blood flow, and therefore is considered to be beneficial (Bolanos and Almeida, 1999). Permeabilization of the mitochondrial membrane as a result of reactive oxygen species and calcium influx is proposed to be mediated by the pro-apoptotic proteins Bax and Bak, which may oligomerize to form pores (Mattson and Kroemer, 2003). Cytochrome c released from the mitochondria binds Apaf-1 in the cytoplasm, resulting in the recruitment and activation of caspase-9 (Wang, 2001). Caspase-9 further activates caspase-3, one of the main enzymes involved in apoptosis. In some studies, ischemic preconditioning results in a reduction of caspase-3 activation, and a subsequent reduction in neuron cell death by apoptosis (Cantagrel et al., 2003; Qi et al., 2001). However, there is also evidence to suggest that activation of caspase-3 can occur without cell damage, and that this activation may be necessary for ischemic tolerance (McLaughlin et al., 2003). One important factor in apoptosis is the amount of ATP present within the cell. ATP is required for nuclear condensation and DNA degradation in the final stages of apoptosis (Leist et al., 1997). Depletion of ATP forces cells to switch to death by necrosis rather than apoptosis. Apoptosis, or programmed cell death, involves condensation of the cytoplasm, fragmentation of DNA, and breakdown of the cellular structures into membrane bound bodies that are phagocytosed and digested by macrophages (Yuan et al., 2003). In contrast, necrosis is characterized by swelling of the cell and its organelles, resulting in cell rupture and release of cellular materials into the surrounding area. Apoptosis is more highly regulated than necrosis, and its processes are more clearly understood, providing the opportunity for methods of intervention that may prevent cell death. The use of anti-apoptotic agents, therefore, can potentially salvage cells within the penumbra, which have a low level of metabolism that is sufficient
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to prevent death by necrosis. The anti-apoptotic gene Bcl2 is of particular interest in this regard, and transfer of this gene via liposomes or viral vectors has been shown to be neuroprotective (Cao et al., 2002; Lawrence et al., 1997). Other factors involved in the development of ischemic tolerance are still being discovered. The transcription factor NFkB is activated and translocated in response to sublethal ischemia (Blondeau et al., 2001). Inhibition of NFkB activity blocked neuroprotection by preconditioning. The expression of heat shock proteins increases following ischemia and preconditioning (Currie et al., 2000; Pringle et al., 1999; Tanaka et al., 2002). It has been suggested that certain heat shock proteins might be involved in protection against ischemic insult. Adenosine receptors may also be involved, and antagonists to these receptors block ischemic tolerance (Hiraide et al., 2001). Countless other genes and their protein products have been found to be upregulated or downregulated in response to ischemic preconditioning.
CHEMICAL TREATMENT AND ISCHEMIC TOLERANCE A number of chemical treatments have been found that can lead to the development of ischemic tolerance. Often these involve causing small amounts of damage to induce the body’s protective processes. One way to precondition tissues against ischemic damage is by inducing inflammation. One way to induce inflammation is with the protease thrombin. Preconditioning with low doses of thrombin is known to induce protection against a subsequent ischemic insult (Masada et al., 2000). At higher concentrations, however, the effects are deleterious (Striggow et al., 2000). Low concentrations of thrombin produce a spike in intracellular calcium concentrations but high concentrations caused a sustained elevation of calcium, which may be the determining factor between beneficial and harmful effects. Thrombin treatment results in the activation of Erk, which may explain the neuroprotective effects of low doses (Xi et al., 2001). Inhibitors or antagonists of normal biological processes can also be used to induce tolerance against ischemia. Ischemic damage to cultured neurons can be reduced by treatment with 3-nitropropionic acid, a compound that blocks the respiratory chain by inhibiting succinate dehydrogenase (Weih et al., 1999). This compound has also been used to induce protection in vivo in gerbils (Kuroiwa et al., 2000). Haloperidol,
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which blocks mitochondria at a different step, also has protective effects (Riepe and Ludolph, 1997). Lipopolysaccharide, a component of bacterial endotoxin, can also be used to precondition neural tissue (Puisieux et al., 2000). Not all preconditioning treatments involve the use of low doses, however. The polyamine spermine is neurotoxic at low levels (Virgili et al., 1999). In contrast, high concentrations have been found to be neuroprotective, possibly by blocking NMDA receptor channels (Ferchmin et al., 2000). Although chemical treatments artificially stimulate protection and may not accurately reflect natural mechanisms of ischemia, the results can often be quite similar.
IMPORTANT FACTORS IN ISCHEMIC PRECONDITIONING The protection provided by ischemic preconditioning appears to occur in two distinct phases. The early, or rapid, phase is established within minutes and may last for several hours (Dirnagl et al., 2003; Schaller and Graf, 2002). Protection has been observed when an ischemic insult is administered as little as 30 min following preconditioning (Pe´ rez-Pinzo´ n et al., 1997). It is thought that changes in cell metabolism may account for the immediate response. The activation of existing proteins and upregulation of normal processes such as the oxygen extraction fraction are probably involved as well. The late, or delayed, phase of protection requires days to develop and provides lasting protection. It has been found that protein synthesis is suppressed in certain parts of the brain in the early stages following an ischemic insult (Kato et al., 1995). After 1–2 days, protein synthesis has recovered in preconditioned animals, but not in controls. In other regions of the brain, protein synthesis is suppressed only in non-preconditioned animals. If the protein synthesis inhibitor cycloheximide is administered prior to preconditioning, no protection is observed (Barone et al., 1998). Therefore, establishment of the late phase appears to require gene expression and protein synthesis. Microarray studies have been used to examine the changes in gene expression that occur following hypoxia (Bernaudin et al., 2002). Changes in expression were immediate for some genes, but delayed for others. Some genes showed a change in expression at only a single time point, indicating a transient change, whereas others had a continued change in expression across multiple time points. Development of a properly designed preconditioning protocol is essential for the study of ischemic tolerance. First, the preconditioning treatment must be severe
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enough to initiate a response, but not so severe as to cause permanent damage. In a study of ischemia in gerbils, for example, it was found that at least 2 min of ischemia was required in order to induce protection against a more severe insult 2 days later (Kitagawa et al., 1991). Second, the subsequent insult must be planned to occur at a time point at which tolerance has been established. In one study, protection was not observed until 2 days after preconditioning, and had disappeared by 7 days after preconditioning (Chen et al., 1996). The delay in forming protection likely reflects the need for protein synthesis during the late phase, and its disappearance indicates that the protective effect is only temporary. Third, if the subsequent ischemic insult is too severe, the amount of protection provided by preconditioning will not be enough to prevent permanent damage. In the initial study of ischemic preconditioning in the heart, the preconditioning regimen used provided protection against a subsequent 40 min insult, but not a 3 h insult (Murry et al., 1986). Of course, differences in species, method of inducing ischemia, and other factors may influence the development of tolerance, therefore it is necessary to optimize the preconditioning protocol to best match the system under study. There is evidence to suggest that genetic variation can have an effect on the severity of damage produced by ischemia. In a study of three common mouse strains, it was found that one of the strains had significantly larger infarcts than the other two strains following permanent cerebral ischemia (Majid et al., 2000). Different responses to preconditioning have also been observed in spontaneously hypertensive rat strains. Rats from a stroke-prone strain had a lower response to ischemic preconditioning and a larger infarct size than rats that were not prone to stroke (Purcell et al., 2003). Transgenic mice expressing a portion of the mutant human gene involved in Huntington’s disease show protection against cerebral ischemia (Schiefer et al., 2002). These studies emphasize the need to determine differences in gene expression between inbred strains and identify which differently expressed products might be involved in the induction of tolerance.
THE SEARCH FOR NEW THERAPEUTIC TARGETS Molecules and proteins that are important for the development of ischemic tolerance in the brain are potential targets for the development of new treatments for ischemia and stroke. One strategy to identify such
targets is to look at factors that have already been determined to be important for tolerance in other tissues. As mentioned previously, ischemic tolerance was first identified in the heart, and has been studied more extensively in the heart than in the brain. It is likely that some of the processes essential for the development of tolerance are the same in both tissues. Newly identified targets in the heart should therefore be considered as being potentially important in the brain, and vice versa. Another strategy to identify new targets is to look at molecules and proteins that interact with currently known targets. If one component of a particular pathway is known to be important for the development of tolerance, the other components of that pathway might be important as well. Of course, there may be novel factors involved in tolerance that cannot be detected by these strategies and that will require broader studies. Often, potential targets are first identified by the observation of changes in gene expression level, protein level, or protein activity following ischemia or the preconditioning treatment. If certain genes are suspected to be involved in tolerance, changes in their expression levels can be individually assessed. Alternately, the entire mRNA population of a tissue can be screened to detect all genes with changed expression, which allows the identification of novel targets. Microarrays are one way to accomplish such broad screening. Changes in gene expression must then be confirmed to cause a corresponding change in protein level. Changes in protein levels can also be analyzed directly without first looking at gene expression levels. Individual proteins can be studied, or broader techniques such as two-dimensional electrophoresis can be used to screen the entire protein population and identify novel targets. There is also the possibility that a change in protein activity is involved, rather than a change in gene expression or protein level. Potential targets must then be tested to determine if they are important for the development of tolerance. Genes can be modified so that they are constitutively expressed or their expression is blocked. Mutants or gene knockouts can also be generated. Proteins can be inhibited or activated with various compounds, or they can be overexpressed. The effect of these manipulations on protection against ischemic damage in the brain will determine if the targets have therapeutic potential.
SUMMARY The response to ischemia in the brain involves a complex interaction between many different processes,
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Fig. 2. Overview of the some of the processes involved in damage and protection of the brain following ischemia. Lines with arrowheads indicate positive interactions. Lines with flat ends indicate inhibitory interactions.
and is still not completely understood. Fig. 2 summarizes some of the processes currently known to occur following cerebral ischemia. In the presence of low oxygen, cell metabolism results in the production of damaging reactive oxygen species and reduced ATP production by lactate fermentation. Excess glutamate release stimulates calcium influx and nitric oxide production by nNOS/iNOS that, together with reactive oxygen species, damage cellular structures and lead to inflammation and cell death by apoptosis or necrosis, depending on ATP levels. In response, inflammation and the production of nitric oxide by eNOS
stimulate increased blood flow to the area. Nitric oxide production by nNOS/iNOS and inflammation result in the activation of Erk, which, together with calcium influx, activates CREB and initiates pathways that inhibit apoptosis. Erythropoietin is activated by the hypoxia response pathway, and may lead to the inhibition of apoptosis as well. The hypoxic response pathway also stimulates increased glucose transport and the formation of new blood cells and blood vessels. The responses to cerebral ischemia can be both beneficial and harmful at the same time, and the survival of neurons within ischemic regions depends
Table 1 Beneficial and detrimental effects of processes and molecules involved in ischemia Factor
Benefits
Detriments
Glutamate
Low levels have preconditioning effect through NMDA receptor activation Activates CREB, leading to activation of gene pathways leading to tolerance Increased blood flow delivers more oxygen and glucose to affected area Signaling molecule leading to Erk activation. Production by eNOS stimulates increase in blood flow Low levels of caspase-3 may be required for ischemic tolerance
High levels lead to imbalance of ion flux due to receptor overstimulation High concentrations participate in imbalance of ion flux and mitochondrial membrane permeabilization Cell damage caused by increased delivery of calcium and inflammatory cells secreting cytokines Free radical can damage cell structures. Reacts to form peroxynitrite, which does more damage Caspase activation leads to initiation of apoptotic mechanisms
Calcium Inflammation Nitric oxide Caspase activity
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critically on this delicate balance. Table 1 lists the beneficial and detrimental aspects of some of the major players in ischemia and ischemic tolerance. In many cases, it seems that even harmful substances may be beneficial when present in low doses. Ischemic preconditioning, by providing a moderate level of protection against cell damage, can alter this balance so that a larger proportion of cells in the affected regions will survive. Although the mechanisms behind the formation of protection are still largely unknown, significant progress has been made toward identifying some of the major molecules involved. Once the processes involved in preconditioning are more fully understood, the potential benefits for prevention and treatment of brain damage due to ischemia are substantial. Additionally, many of the damaging processes involved in stroke also play roles in other neurological conditions. It is possible that these conditions might benefit from preconditioning as well.
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