An Experimental Study On The HSP70 Expression And Neuroprotective Mechanism Of Ischemical Preconditioning-induced Ischemic Tolerance

PartⅠProtective effects of focal ischemical preconditioning and HSP70 expression on middle cerebral artery occlusion in ischemic tolerance ratsObjective To investigate protective effect of a short duration focal ischemic preconditioning(PC) and the HSP70 expression after PC on the subsequent permanet middle cerebral artery occlusion( MCAO) in rat brain.Methods Temporary MCAO (20 minutes) was used for PC , various periods of reperfusion (ie, 12 hours and 1,3 5, 7, and 14days) were allowed after PC and before permanent MCAO (PMCAO) (n=5 per group) establish ischemic tolerance (IT) compared with non-PC (sham-surgery) rats (n=5 per group) by Longa's . Infarct size, neurological deficits were measured after 24hours PMCAO by the triphenyltetrazoliumchloride(TTC) and neurological examination respectively. The hitological damage was evaluated by HE staining. The expression of HSP70 was analyzed by in situ hybridization, RT-PCR and immunohistochemistry, Western blotting at 12hours, 1, 3, 5, 7, and 14 adys after PC or non-PC(sham-surgery) .Results Hemispheric infarct was significantly (P<0.01) reduced only if PC was performed 1 , 3, 5 and 7 days before PMCAO compared with non-PC (sham-surgery) rats. PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). PC produced no brain injury but did significantly (P<0.01) increased HSP70 gene at 12hours,1 , 3 , and 5days and protein (P<0.05) at 1 , 3 , 5 , and 7days respectively compared with non-PC (sham-surgery) rats.Conclusion The results suggest that PC is a powerful inducer of ischemic brain tolerance as reflected preservation of brain tissue and motor function. The protective effects occur at the PC brain site 1 to 7days after PC. Meanwhile ,we found that PC increase HSP70 gene expression (12hours to 5 day) and HSP70 protein expression (1 to 7 day) after PC, PartⅡExpression of HSP70 protein following focal ischemic preconditioning and neural apoptosis on middle cerebral artery occlusion in ischemic tolerance ratsObjective To systematically evaluate the importance of protein synthesis in ischemic preconditioning( PC )-induced ischemic tolerance ( IT ) and neural apoptosis in ratsMethods Temporary middle cerebral artery occlusion(MCAO) by Longa (20 minutes) was used for PC (ischemic precondioning). 1day of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) compared with non-PC rats (n=5 per group). Infarct size, neurological deficits were measured 24hours after PMCAO by the triphenyltetrazoliumchloride(TTC) and neurological examination respectively. Samples of brain were taken 24hours after PC for the determination of HSP70 expression by Western blot analysis and the apoptotic neural cells were detected 24hours after PMCAO by TdT-mediated dUTP nick end labeling (TUNEL) techenique and flow cytometry . The effects of the protein synthesis inhibitor (cycloheximide administered just before PC or administered long after PC but just before PMCAO on IT ) were also determined (n=5 per group).Results Hemispheric infarct 24hours after PMCAO was significantly(P<0.01) reduced only if PC was performed 1day, and PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). The number of TUNEL positive cells and the apoptotic cell percentages of rat neocortex by flow cytometry were also significantly (P<0.05) reduced only if PC was performed. Cycloheximide eliminated (P<0.05) ischemic PC-induced IT effects on brain injury , neurological deficits and the number of TUNEL positive cells apoptotic and cell percentages if administered before PC but not if administered long after PC but before PMCAO. PC produced no brain injury but did increased HSP70 protein at 1 day after PC . Cycloheximide eliminated (P<0.05) the effects if administered just before PC but not if administered long after PC but just before PMCAO on IT.Conclusions PC induces IT that is dependent on de novo protein HSP70 synthesis which suppress the neural apoptosis following severe ischemia.PartⅢMechanism of HSP70 protein following focal ischemical preconditioning suppresing the neural apoptosis following severe ischemia in ischemic tolerance ratsObjective To explore the mechanism of HSP70 protein following focal ischemical preconditioning suppresing the neural apoptosis following severe ischemia.Methods Temporary middle cerebral artery occlusion(MCAO) by Longa (20 minutes) was used for PC (ischemic precondioning). 1d of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) compared with non-PC (sham-surgery) rats (n=5 per group). Samples of brain were taken 24hours after PMCAO for the determination of c-fos, bcl-2 and caspase-3 expression by immunohistochemistry and Western blot analysis. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC on IT ) were also determined (n=5 per group).Results The expression of c-fos and bcl-2 protein significantly (P<0.05) increased 24hours after PMCAO if PC was performed 1day compared with non-PC. PC rats also exhibited a significant (P<0.05) reduction in caspase-3 protein 24hours after PMCAO. Cycloheximide eliminated (P<0.05) the effects if administered just before PC on ITConclusion HSP70 suppresing the neural apoptosis following severe ischemia on IT is associated with upregulation of the expression of c-fos and bcl-2 protein and reduction of caspase-3 protein in cells in ischemic region. PartⅣNon-lethal oxygen-glucose deprivation-induced to lerance in cultured rat cortical neurones and critical role of HSP70Objective To explore the protective effect of non-lethal oxygen-glucose deprivation(OGD)-induced tolerance in cultured rat cortical neurones and changes in expression of HSP70 after non-lethal OGDMethods Primary cultures of rat cortical neurones were exposed to non-lethal oxygen–glucose deprivation (OGD), i.e. ischaemic preconditioning(PC), for 20 min. At a designated period after the PC stimulus (12, 24, 48, or 72 hours), the cultures were again deprived of lethal OGD for 50 min to establish ischemic tolerance (IT) compared with non-PC. Neuronal injury was assessed by measurement of lactate dehydrogenase (LDH) efflux into the medium 24 hours after lethal OGD. The expression of HSP70 was analyzed by RT-PCR and immunohistochemistry, Western blotting at 12, 24, 48, and 72hours after PC or non-PC(control). In addition, cell death was confirmed in some experiments by the trypan blue exclusion assay. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC on IT ) were also determined.Results All PC stimuli resulted in a significant(P<0.05) decrease in LDH activity and cell death 24h after OGD if PC was performed 24hours or 48hours. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 12 hours or increased to 72 hours . PC produced no cell injury but did increased HSP70 protein.Western blot analysis and immunohistochemistry demonstrated that there was significant(P<0.01) difference between HSP70 protein levels at PC 24hours or 48hours but was no significant difference at PC 12 hours or 72 hours and very significant (P<0.05) between HSP70 gene levels at PC 12hours, 24hours or 48hours but was no significant difference at PC 72 hours compared with non-PC respectively.These effects were abolished (P<0.05) by the protein synthesis inhibitor cycloheximide if administered just before PC on IT.Conclusion A non-lethal oxygen–glucose deprivation (OGD) induces ischaemic tolerance in the cultured neurons. The protective effects occur at 24 to 48hours after PC. Meanwhile, we found that the protective effects is dependent de novo protein synthesis HSP70 in the present model system.PartV The protective effect and mechanism of PC suppresing the neural apoptosis on non-lethal oxygen-glucose deprivation-induced tolerance in cultured rat cortical neuronesObjective To explore the protective effect and mechanism of PC suppresing the neural apoptosis following lethal oxygen-glucose deprivation -induced tolerance in cultured rat cortical neurones.Methods Primary cultures of rat cortical neurones were exposed to non-lethal oxygen–glucose deprivation (OGD), i.e. ischaemic preconditioning(PC), for 20 min. At 24 hours after the PC stimulus , the cultures were again deprived of lethal OGD for 50 min to establish ischemic tolerance (IT) compared with non-PC. The apoptotic neural cells were detected 24 hours after lethal OGD by TdT-mediated dUTP nick end labeling techenique (TUNEL),acridine orange staining and flow cytometry .The bcl-2 protein expression,and caspase -3 protein expression were confirmed respectively by immunohistochemistry and Western blot analysis 24 hours after lethal OGD stimuli. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC or administered long after PC but just before lethal OGD on IT ) were also determined.Results The number of TUNEL positive cells and the acridine Orange staining positive cells were significantly (P<0.05) reduced and the apoptotic cells percentages of rat neocortex by flow cytometry were also significantly (P<0.05) reduced only 24 hours after lethal OGD if PC was performed . The bcl-2 protein were significantly (P<0.05) increased 24 hours after lethal OGD compared with non-PC(control) , PC also exhibited a significant (P<0.05) reduction in caspase-3 protein 24 hours after lethal OGD .Conclusion The non-lethal OGD for PC may supprese the neural apoptosis following lethal OGD on IT, which is associated with upregulation of the expression of bcl-2 protein and reduction of caspase-3 protein in the present model system.