Adaptive Medicine 4(1): 20-26, 2012 DOI: 10.4247/AM.2012.

ABB020

Review

Mechanisms in Adaptation to Brain Ischemia by Ischemic Preconditioning

Yajun Liu 1 and Lianbi Chen 2
1 2

Institute of Microcirculation, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005 Institute of Physiology, School of Medicine, Shandong University, Jinan 250012, Shandong, Peoples Republic of China

During the last few years, the mechanisms of adaptation to brain ischemic preconditioning (IPC) of cerebral ischemia/reperfusion have been studied and various molecules and a number of molecular regulation pathways were proposed to participate in IPC. Better understanding of mechanisms in IPC may provide novel therapeutic interventions for preventive treatment of cerebral ischemia. Since mitochondria are the most important intracellular energy source and are crucial for the cells survival in the occurrence of ischemia, so more attention should be paid to the investigation of mitochondria related mechanisms of IPC. In this review, we will first survey the mitochondria related mechanisms including mitochondrial apoptotic pathway, uncoupling protein 2 (UCP 2 ), mitochondria deprived reactive oxygen species (ROS), and mitochondrial ATPsensitive K + channels (MitoKATP) in postischemic neuroprotection in brain. Then, to be more comprehensive, the mitochondria non-related mechanisms such as N-methyl-D-aspartate (NMDA) and Gammaaminobutyric acid (GABA) receptors, adenosine, adenosine A1 receptor and K + channels as well as nitric oxide will also be reviewed. Key Words: cerebral ischemic preconditioning, cerebral ischemia/reperfusion, mitochondria, NMDA receptors, adenosine, nitric oxide

Introduction
Cerebrovascular disease (stroke) is one of the leading causes of death in the world. In the last 20 years, there have been a large number of studies focusing on the investigation of neuroprotective strategies against cerebral ischemia/reperfusion. In mammals, ischemia induces the brain to become isoelectric within few minutes and is followed by the loss of ion gradients (depolarization) if ischemia continues. As ion gradients are lost, neurons release the excitatory neurotransmitter glutamate which induces and am-

plifies irreversible pathologies during ischemia. The mechanisms leading to neuronal cell death after cerebral ischemia included either apoptosis (programmed cell death), necrosis or a mixture of these processes (29). Different physiological adaptations for ischemia resistance have been described in the brain, which is referred to as preconditioning. This state is reached when a sub-threshold noxious stimulus is applied to the brain and activates the protective pathways which can help to reduce the neuronal damage caused by severe ischemic episodes. Preconditioning stimuli include ischemia, hypoxia, exposure to anesthetic inhalants (4, 17, 62), ethanol (57), cortical spreading depression (21), hypothermia, hyperthermia (35, 39), and some chemical agents such as scutellarin and magnesium sulfate (16, 47). Ischemic preconditioning (IPC) was first identified in the heart by Murry et al. (33), and was subsequently found to occur in the brain (19). The protective effects of preconditioning can be established at two distinct temporal phases: one relatively short-lived phase arises within minutes of exposure to the preconditioning stimulus and then disappears after 1-4 h (acute phase, early phase, or classical preconditioning) (44); the other one occurs 12-24 h delay by the appearance of a longer-lived (24-72 h) and often more powerful phase of tolerance to ischemia that is referred to as the second window of protection (late phase, or delayed preconditioning) (19). IPC induces both phases of ischemic tolerance. The acute phase is most likely due to rapid posttranslational modifications of proteins (32), whereas the late phase is dependent on de novo protein synthesis (18). Unraveling the basic mechanisms of IPC promises to significantly influence the therapeutic approaches to acute and chronic cerebrovascular accidents such as stroke. The purpose of this review is to provide a brief

Corresponding author: Professor Lianbi Chen, Institute of Physiology, School of Medicine, Shandong University, Jinan 250012, Shandong, Peoples Republic of China. Tel: +86-531-89901186, Fax: +86-531-88382502, E-mail: clb@sdu.edu.cn Received: December 3, 2011; Revised: March 2, 2012; Accepted: March 3, 2012. 2012 by The Society of Adaptive Science in Taiwan and Airiti Press Inc. ISSN : 2076-944X. http://www.sast.org.tw

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summary of the mitochondria related mechanisms and mitochondria non-related mechanisms in postischemic neuroprotection in the brain by IPC.

Mitochondria-Related Mechanisms in Neuroprotection Induced by IPC

Mitochondrion, a specific organelle, is the most important intracellular energy source, and has received special attention as a key player in cell death pathways. As is well known, ATP is generated by the mitochondrial respiratory chain located within the mitochondrial inner membrane. In tissues with high energy consumption, like brain and heart, mitochondria comprise nearly half of the cell volume and produce 80%-90% of the ATP (56). It is not surprising that the depletion of ATP due to cerebral ischemia leads to the death of neurons and causes the clinical syndromes affecting mainly the nervous system. It has been reported that a hyper-oxidation of mitochondrial respiratory chain components has been observed (42, 43) following cerebral ischemia. Previous studies suggested that this hyper-oxidation may result from loss of electron carriers from mitochondria, such as cytochrome c. The release of cytochrome c has been reported to be linked to apoptosis (20, 59), which leads to cell death and neuronal dysfunction in the brain. The Proinhibition of Mitochondrial Apoptotic Pathway in IPC It has been demonstrated that IPC protected mitochondria against delayed cell death induced by cerebral ischemia/reperfusion (8, 40). Studies from our lab reported that IPC alleviated the apoptosis of hippocampal neurons induced by cerebral ischemia reperfusion in a rat global ischemia model by the inhibition of cytochrome c release from mitochondria (26). Caspase-9 is a downstream apoptotic protein activated by released cytochrome c. It activates caspase-3 which is the final protein in the mitochondrial apoptotic pathway. It has also been found by our group that IPC reduced the release of caspase-9 and then protected neurons from apoptosis through prohibiting the mitochondrial apoptotic pathway (27). Upstream of the cytochrome c-dependent mitochondrial pathway of apoptosis is Bcl-2, which can inhibit cytochrome c translocation, thereby blocking caspase activation and apoptosis (20). Bcl-2 resides in the mitochondrial outer membranes and controls the permissibility of the mitochondria membrane. Apoptosis can be induced if the permeability increases (38). In a family of Bcl-2 proteins, Bcl-2 and Bcl-xl suppress apoptosis whereas Bax promotes it. Therefore, it draws the attention that the Bcl-2 family

proteins determine whether neurons undergo survival or apoptosis. The evidence from our lab demonstrated that IPC inhibited apoptosis and protected the hippocampal neurons against ischemia/reperfusion injury. We found that in the IPC group, both mRNA and protein expression of Bcl-2 are up regulated whereas the mRNA and protein expression of Bax are down regulated in hippocampal neurons (28). Shimizu et al. reported that the IPC induced protective effect of Bcl-2 to the transient focal ischemia was diminished after the administration of Bcl-2 antisense oligodeoxynucleotides into the cerebral ventricle (52). Another study indicated that the release of cytochrome c was very low when the isolated mitochondria were incubated with Bcl-2, however incubation of mitochondria with calpaintruncated Bcl-2 induced substantial or almost complete release of cytochrome c (12). Role of Mitochondrial Uncoupling Proteins in IPC Uncoupling proteins (UCPs) are located in the mitochondrial inner membrane. The physiological roles of UCPs include control of cellular energy balance as well as the protection against oxidative stress. Among the five mammalian UCPs, only UCP2 is expressed in significant levels in several brain regions (48, 53). Its role appears to be dissipating the proton electrochemical gradient through the mitochondrial inner membrane (11) and mildly uncoupling the respiratory chain. By this means, it improves the efficiency of electron transfer between complexes I and IV, and reduces reactive oxygen species (ROS) production, especially superoxide, by the respiratory complexes (10). UCP 2 may contribute to neuroprotection by inhibiting the production of ROS from mitochondria. Our experimental data in a rat global ischemia/ reperfusion model demonstrated that IPC increased UCP 2 expression in both mRNA and protein levels in hippocampal CA1 neurons (23). Other evidences that UCP 2 expression is induced by IPC provide a potential mechanism for mitochondrial adaptation to brain ischemia following IPC (9, 31). Role of Mitochondria Deprived ROS in IPC ROS is generated from multiple sources of the cell. Mitochondrion is a major site of ROS formation. Complex I and complex III are the two major regions in the mitochondrial electron transport chain where ROS are produced. ROS produced by mitochondria mainly includes superoxide and hydrogen peroxide. In the occurrence of ischemia/reperfusion, ROS formation from mitochondria is greatly increased, which promotes lipid, protein, and DNA oxidation that affects normal cell physiological activity and

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eventually leads to neuronal demise (apoptosis or necrosis). Dave et al. demonstrated that IPC protects hippocampus against delayed neuronal cell death by protecting mitochondrial oxidative phosphorylation (8). In an in vitro model of hypoxic preconditioning, the cortical neuron death induced by oxygen glucose deprivation (OGD) was reduced in the presence of hypoxic preconditioning (1). Furthermore, there was an increased activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase, and a decreased concentration of superoxide and hydrogen peroxide after OGD in the IPC group. Accumulated evidences support that the small production of ROS induced by mild ischemia could modify cell-signaling proteins and that these modifications have functional consequences which may be involved in ischemic tolerance. In gerbil brain, the administration of diethyldithiocarbamate, which inhibits superoxide dismutase and elicits ROS production, was neuroprotective when the global ischemia was induced 2-4 days later (37). The study from our group reported that treatment with superoxide dismutase at the time in a preconditioning ischemia greatly attenuated the increase in UCP 2 staining at 72 h, implying a role of oxygen radicals in UCP 2 induction, which has been recognized to be neuroprotective (23). The studies in heart demonstrated that an ROS-dependent activation of PKC is responsible for the protective effect of IPC in heart (36, 61). Furthermore, PKC was also demonstrated as a key player in the ROS induced induction of brain ischemic tolerance (41). Role of Mitochondria ATP-Sensitive K+ Channels (MitoKATP) in IPC MitoKATP are located in the mitochondrial inner membrane, which can promote K+ entry into the mitochondrial matrix, depolarize mitochondrial membrane potential, promote the rate of the electron transport chain and thus increase ATP production (49). On the other hand, mitoKATP activation triggers mild uncoupling, which prevents ROS release by the respiratory chain (3). It has been reported that BMS191095, a mitoKATP opener protects the brain against focal ischemic injury in rats by reducing ROS production (30). The activation of mitoKATP has also been found to protect neurons against ischemia-induced death by inhibiting apoptosis in which the suppression of Bax translocation and cytochrome c release are involved (22). Mayanagi et al. demonstrated that co-treatment of 5-HD, a mitoKATP blocker, reversed the protective effect of BMS-191095 in rat brain in vivo (30). In addition, apart from IPCs postischemic

neuroprotection, ghrelin and 3'-Methoxypuerarin (3'MOP) were found protecting hippocampal neurons against cerebral I/R induced injury in rat brain in our previous work (24, 25). Ghrelin is an acylpeptide gastric hormone and an endogenous ligand for growth hormone secretagogue (GHS) receptor 1a (GHS-R 1a). The functions of ghrelin include stimulation of growth hormone (GH) secretion, gastric motility and acid secretion, increasing energy metabolism, influence on pancreatic endocrine or other endocrine activities, control of behavior, and hemodynamic actions. 3'-MOP is an isoflavone extracted from radix puerariae and has been reported to have antioxidant and vasodilating effects. In our studies, the results showed that both ghrelin and 3'MOP can reduce I/R induced hippocampal neuron loss by inhibiting the occurrence of apoptosis. Thus, we speculate that although pharmaceutical agents may also exert neuroprotective functions to ischemia/ reperfusion, the involved endogenous mechanisms might be the same.

Mitochondria Non-Related Mechanisms in Neuroprotection by IPC

Excitatory/Inhibitory Neurotransmitters in IPC Glutamate is a major excitatory neurotransmitter in the mammalian brain. The activation of the NMDA glutamate receptors is generally believed to be responsible for the neuronal damage caused by excitotoxicity. However, it appears that the sub toxic concentrations of NMDA are protective for glutamate-mediated excitotoxicity. Using a gerbil cerebral ischemic model, it has been shown that the NMDA receptor antagonist significantly attenuated ischemia tolerance, suggesting that mild NMDA receptor activation is involved in IPC (5). In addition, in an in vitro model, the NMDA receptor activation induced by the exposure of cortical cell cultures to low levels of glutamate or NMDA has a preconditioning effect (14). The underlying mechanisms include the rapid adaptation of the voltage dependent calcium flux (51) and the rapid release of brain-derived neurotrophic factor (BDNF). BDNF activates its cognate receptor, receptor tyrosine kinase B (TrkB), which activates Nuclear Factor kappaB (NF-kappaB), a transcription factor involved in neuroprotection. In addition, there are other key mediators which are involved in synaptic NMDA receptordependent neuroprotection including phosphatidylinositol 3-kinase (PI3K), Akt, and glycogen synthase kinase 3-beta (55). However, these mediators can only be induced by low doses of NMDA via the action potential-dependent route (55). GABA is an inhibitory neurotransmitter in the brain. Sommer et al. found that the increased ligand

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Mitochondria apoptotic pathway + Mitochondria related mechanisms UCP2

Bcl-2

Bax ROS

+ +

Mitochondria deprived ROS

PKC ATP ROS BDNF +

+ Mito KATP

Mechanisms of IPC

NF-kappaB

NMDA NMDA receptors Mitochondria non-related mechanisms

+ +

PI3K Akt

GABA GABA receptors

Adenosine adenosine A1 receptor K+ channels +

NO

P 21(Ras)

Fig. 1. Schematic diagram of mitochondrial and non-mitochondrial mechanisms of IPC.

binding to GABA A receptors between 30 min and 48 h of recirculation was associated with ischaemic tolerance in the preconditioned gerbil hippocampus (54). Dave et al. reported that GABAB receptor activation was also demonstrated to be neuroprotective during ischemia or early reperfusion (7). They also found a robust release of GABA induced by IPC in rats after lethal ischemia. The above results suggest that the balance between excitotoxity and inhibition in the brain might be changed by preconditioning to such a way that proinhibitory forces might be more prominent in the induction of ischemic tolerance. Adenosine, Adenosine A1 Rreceptor and K+ Channels in IPC Adenosine is an endogenous neuroprotectant that can inhibit the release of excitatory amino acids. The binding of adenosine A1 receptor activates the internal cell signaling pathway in which G protein, phospholipase C, and protein kinase C are involved. Adenosine also opens the ATP-sensitive K + channels of plasma membrane, and induces the out flux of K + and the depolarization of the plasma membrane. Heurteaux

et al. demonstrated the essential roles of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in IPC (15). A series of reports have shown that preconditioning is dependent on the activation of adenosine A1 receptors (45, 46) and the ischemic tolerance phenomenon is abolished after the administration of adenosine receptor A1 antagonist (34). Activation of the K + ATP channels has also been demonstrated to play a role in IPC. Schulz et al. found that blockade of the K +ATP channel abolis hed

the protection afforded by adenosine and R-PIA (an adenosine A1 receptor agonist) (50). Futhermore,
activation of K +ATP channel with bimakalin (agonist) during a preconditioning ischemic episode reduced the time necessary to produce preconditioning in dogs (60). Nitric Oxide (NO) in IPC There is a large body of evidence demonstrated that NO and iNOS induction are critical for induction of ischemic tolerance. It has been reported that the inhibition of nitric oxide synthase (NOS) and scavenging of NO during preconditioning significantly attenuated the IPC induced neuronal tolerance, and

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neither the endothelial NOS nor the neuronal NOS knockout mice were protected by the rapid ischemic preconditioning (2, 6). There are several mechanisms responsible for NO-induced tolerance. Gonzalez et al. showed that p21 (Ras) activation induced by preconditioning is NMDA-dependent and NO-dependent, which leads to the downstream activation of Raf kinase, mitogen-activated protein kinases, and extracellular regulated kinase (13). In addition, the down regulation of glutamate transporter GLT-1 might be involved in NO mediated IPC (58).

Conclusion
There are already more than enough evidences which demonstrated the mechanisms of brain adaptation to ischemic injury from IPC (Fig. 1). However, the internal mechanisms of ischemic tolerance are complicated. Most researchers agree that there is more than one cell type or one mechanism which is exclusively responsible for endogenous neuroprotection. Instead, a network action of different cellular and molecular pathways is involved, in which many kinds of molecules and cell signal pathways are activated and they interact with each other to reduce the inflammatory response, adapt energy metabolism, and enhance neuroprotection. However, IPC promises practical usefulness of protective strategies for the vascular neurosurgery and the cerebral vascular diseases. Possible practical and key targets in the post ischemic neuroprotection induced by IPC should be systematically studied in order to contribute to the clinical application in cerebral vascular diseases.

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