The Protective Effects Of Cerebral Ischemic Preconditioning On Cerebral Ischemia-reperfusion Injury And Its Mechanisms

PARTⅠStudy on the effects of cerebral ischemic preconditioning on cerebral ischemia-reperfusion injury and the relationships of the effects with the tumor necrosis factor-α(TNF-α) and inducible nitric oxide synthase (iNOS) in ratsObjective: To investigate the protective effects of cerebral ischemic preconditioning on cerebral ischemia-reperfusion injury and the relationships of the protective effects with the tumor necrosis factor-α(TNF-α) and inducible nitric oxide synthase (iNOS) in rats.Methods: The middle cerebral artery occlusion (MCAO) model of rat was made by using a thread to occlude the right middle cerebral artery. By this MCAO model, the cerebral ischemia-reperfusion with ischemic precondition group (IP+R group) rats were subjected 10 minutes cerebral ischemic preconditioning and 72 hours reperfusion, and then the rats were subjected MCAO for 90 minutes and reperfusion for 24 hours again. The neurological deficits and the volumes of cerebral infarction of rats were evaluated. The expressions of tumor necrosis factor-α(TNF-α) and inducible nitric oxide synthase (iNOS) in the right brain tissue were assayed by immunohistochemistry.Results: Compared with the ischemia-reperfusion with sham ischemic precondition group (R group) rats, the neurological deficits were improved significantly (P <0.01), and the volumes of cerebral infarctions were decreased significantly (P < 0.01), and the expressions of TNF-αand iNOS were decreased significantly in IP+R group rats(P<0.01).Conclusions: Cerebral ischemic preconditioning has protective effects on the cerebral ischemia-reperfusion injury in rats, and the protective mechanisms were probably from inhibiting the expressions of TNF-αand iNOS in cerebral ischemia-reperfusion tissue and thus alleviating inflammatory immunologic injury. PARTⅡThe cerebral ischemic preconditioning reduce the content of nitric oxide in cerebral tissue after ischemia-reperfusion of ratsSection A. Nitric oxide (NO) and cerebral ischemia-reperfusion injury Objective: To investigate the content change of the nitric oxide (NO) in cerebral tissue and the role of this change after cerebral ischemia-reperfusion in rats and the effect of medicine (Edaravone) intervention.Methods: The middle cerebral artery occlusion (MCAO) model of rat was made by using a thread to occlude the middle cerebral artery. The middle cerebral artery was reperfused after 40min cerebral ischemia. The ischemia-reperfusion with edaravone group rats were given edaravone and the ischemia-reperfusion group rats or the sham operation group rats were were given isovolumic isotonic Na chloride by intravenous injection at the reperfusion beginning. The neurological deficits of rats were estimated by Bederson method and the contents of NO were measured by nitrite reduction method after cerebral ischemia-reperfusion 24h.Results: (1) After cerebral ischemia-reperfusion 24h, the neurological deficits of the ischemia-reperfusion with edaravone group rats were less severe significantly than those of the ischemia-reperfusion group rats (P <0.01). (2) After 24h cerebral ischemia-reperfusion, the contents of NO in cerebral cortex and in hippocampal region of the ischemic and self-control side of ischemia-reperfusion group rats were significantly more than those of sham-operation group rats (P <0.01); the contents of NO in cerebral cortex and in hippocampal region of the ischemic and self-control side of the ischemia-reperfusion with edaravone group rats were significantly less than those of the ischemia-reperfusion group rats (P <0.01).Conclusions: The content of NO increase in cerebral tissue after cerebral ischemia-reperfusion in rats. NO maybe contribute to the cerebral ischemia-reperfusion injury of rats. Edaravone maybe has protection against cerebral ischemia-reperfusion injury in rats. The neuroprotective mechanism of edaravone maybe reduced the content of NO in cerebral tissue of rats.Section B. The effects of cerebral ischemic preconditioning on the content of nitric oxide in cerebral tissue after ischemia-reperfusion in ratsObjective: To investigate the protective effects of cerebral ischemic preconditioning on cerebral ischemia-reperfusion injury and the relationship between the protective effects and the cerebral nitric oxide (NO) in rats.Methods: The middle cerebral artery occlusion (MCAO) model of rat was made by using a thread to occlude the right middle cerebral artery. By this MCAO model, the ischemic preconditioning rats were subjected 10 minutes cerebral ischemic preconditioning and 72 hours reperfusion, and then the rats were subjected MCAO for 90 minutes and reperfusion for 24 hours again. The neurological deficits of rats were estimated by Bederson method. The cerebral contents of NO were measured by nitrite reduction method.Results: After 90min cerebral ischemia and 24h ischemia-reperfusion, the neurological deficits of the cerebral ischemia-reperfusion with ischemic precondition group (IP+R group) rats were less severe significantly than those of the ischemia-reperfusion with sham ischemic precondition group (R group) rats (P<0.05); the contents of NO in cerebral cortex and in hippocampal region of the ischemic and the self-control side in R group and IP+R group rats were significantly more than that of the sham ischemia-reperfusion with sham ischemic precondition group (S group) rats (P<0.01); the content of NO in cerebral cortex and in hippocampal region of the ischemic side and the self-control side in IP+R group rats were significantly less than those of the R group rats (P<0.01).Conclusions: Cerebral ischemic preconditioning maybe has protection against cerebral ischemia-reperfusion injury in rats. The neuroprotective mechanism of cerebral ischemic preconditioning maybe reduced the cerebral content of NO in rat. PARTⅢThe effects of 14-3-3 proteins on cerebral ischemic preconditioning-induced protection against cerebral ischemia-reperfusion injury and its mechanisms in ratsObjective: To investigate the effects of 14-3-3 proteins on cerebral ischemic preconditioning-induced protection against cerebral ischemia-reperfusion injury and its mechanisms in rats.Methods: Firstly, the distributions of 14-3-3 protein isoforms (β,η,γ,ε,θandζ) in hippocampal neurons with primary culture of rat were assayed by immunohistochemistry method. Secondly, the middle cerebral artery occlusion (MCAO) model of rat was made by using a thread to occlude the right middle cerebral artery. By this MCAO model, the ischemic preconditioning rats were subjected cerebral ischemic preconditioning for 10 minutes and reperfusion for 72 hours, and then the rats were subjected MCAO for 90 minutes and reperfusion for 24 hours again. Finally, the expression of 14-3-3 proteins in the hippocampus of ischemia-reperfusion sides in all groups rats were assayed by immunohistochemistry and western blot; the expressions of Bad, p-Bad and p-Akt in the hippocampus of ischemia-reperfusion sides in all groups rats were assayed by western blot; the apoptosis of the hippocampal neurons of ischemia-reperfusion sides in all groups rats were detected by TUNEL method.Results: The expression of all 14-3-3 protein isoforms (β,η,γ,ε,θandζ) were detected in hippocampal neurons with primary culture of rat. After each group rats were subjected the procedures of cerebral pre-ischemia for 10 min and reperfusion for 72 h and then cerebral ischemia for 90 min and reperfusion for 24 h, the immunohistochemistry of the hippocampus of ischemia-reperfusion side in rats shown that the expression of 14-3-3 proteins of hippocampal neurons of the cerebral ischemia-reperfusion with ischemic precondition group (IP+R group) was more significantly than that of the ischemia-reperfusion with sham ischemic precondition group (R group) and the sham ischemia-reperfusion with sham ischemic precondition group (S group) (P <0.01). Western blot of the hippocampus of ischemia-reperfusion side in rats showed: (1) The expression of 14-3-3 proteins of the IP+R group was more significantly than that of the R group and the S group (P <0.01), and the expression of 14-3-3 proteins of the R group was more significantly than that of the S group (P <0.01). (2) The expression of Bad of the R group was more significantly than that of the S group and the IP+R group (P <0.01), but the differences of the expressions of Bad between the S group and the IP+R group were not statistically significant (P = 0.065). (3) The expression of p-Bad of the IP+R group was more significantly than that of the R group and the S group (P <0.01), and the expression of p-Bad of the R group was more significantly than that of the S group (P <0.05). (4) The expression of p-Akt of the IP+R group was more significantly than that of the R group and the S group (P <0.01), and the expression of p-Akt of the R group was more significantly than that of the S group (P <0.05). (5) The proportionality of p-Bad/Bad of the IP+R group exceeded that of the R group and the S group, and the proportionality of p-Bad/Bad of the S group exceeded that of the R group.The number of TUNEL masc cell in hippocampus CA1 region of ischemia-reperfusion side in IP+R group rats was less significantly than that in R group rats (P<0.01).Conclusions: The expression of all 14-3-3 protein isoforms (β,η,γ,ε,θandζ) were detected in hippocampal neurons of rats. Cerebral ischemic preconditioning has protective effects on the cerebral ischemia-reperfusion injury in rats. The mechanisms of the protective effects include probably the up-regulated expression of 14-3-3 proteins, the activatin of PI3K/Akt signal transduction pathway and the sequentially up-regulated expression of the p-Akt and p-Bad induced by cerebral ischemic preconditioning in rat brain tissue of the ischemia-reperfusion; subsequently, the binding of 14-3-3 and p-Bad inhibit Bad-induced apoptosis and weaken cerebral ischemia-reperfusion injury in rats.