| Objective:To observe the effects of cerebral ischemia and ischemic postconditioning on the ionic homeostasis in hippocampal neuronal microenvironment in tree shrews, to explore the variation and pathophysiological role of ASIC2a and SGK1, which belong to mechanosensitive molecules, during cerebral ischemia and ischemic postconditioning, and to reveal the molecular mechanisms and regulation aspects of endogenous neuroprotective effects.Methods:The thrombotic cortical ischemia model was induced by photochemical method in tree shrews. And at 4h after cerebral ischemia, the ischemic postconditioning was performed as following details:ipsilateral carotid artery was occluded and reperfusion for 5min alternatively as a circle, a totle of three cycles. At 4h,24h and 72h after ischemia, the regional cerebral blood flow (rCBF) of ischemic cortical area was detect by Laser Doppler Flowmetry and the changes of hippocampal microenvironment were tested by a single-pumped push-pull microdialysis system and an ion analyzer. Brain water content was measured, at the same time the pathological morphological evaluation of ischemic brain damage was performed by TTC staining, HE staining and the electronmicroscope ultramicrostructure observation. The changes of ASIC2a、SGK1'p-SGK(Thr256) protein expression in cortex and hippocampal of tree shrews were detected by Western blotting and immunohistochemical technique. The ASIC2a and SGK1mRNA expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Meanwhile, the reverse antagonistic model was established by the injection of SGK1 specific inhibitor GSK-650394 in the hippocampus. And the changes of the above indexes were observed in order to further explore the brain protective effect of SGK1 during ischemic postconditioning, and to discuss the relationship of SGK1 with ASIC2a.Results:At 4h,24h and 72h after cortical ischemia induced by photochemical technology, the rCBF of ischemic cortical area was significantly decreased in tree shrews, especially at 24h after ischemia. The ischemic postconditioning weakened the decreasing degree of rCBF in the ischemic cortex significantly, and compared with the ischemic group, the difference was significant (P<0.01). The hippocampal ionic microenvironment study showed that the pH and the concentrations of Na+、Ca2+ and Cl- decreased in the hippocampal microenvironment, while the concentrations of K+ increased after cortical ischemia in tree shrews. The changes were the most significant at 4h, the second at 24h and insignificant at 72h. The alterant direction of the pH and the concentrations of K+、Na+、Ca2+、Cl- in the postconditioning group was similar to the ischemia group, but the degree of turbulence was reduced significantly. The brain water content increased in cerebral cortex and hippocampal of tree shrews after cerebral ischemia, and the change was most obvious at 24h after ischemia. The brain water content of the cerebral cortex and hippocampal in postconditioning group was significantly reduced than ischemic group significantly at each time point after brain ischemia. The results of TTC staining showed that there was obvious infarction in the exposured cerebral cortex at 4h after photochemical reaction, and the infarct size was the maximum at 24h, and the infarct size was reduced at 72h. The infarct size of cerebral cortex was decreased in ischemic postconditioning than the ischemia group, especially at 24h and 72h after ischemia, the differences were significant. The results of HE staining showed that there was secondary neuronal injury in the ipsilateral hippocampus at 4h after cerebral cortical thrombosis induced by photochemical technology in tree shrew, reached a peak at 24h, and was still obvious at 72h. Ischemic lesion was significantly attenuated by ischemic postconditioning, while the tissue grade of hippocampal CA1 subfield was lower and the hippocampal neurons secondary damage significantly reduced at each time point after brain ischemia in postconditionng group. The observation of electronmicroscope displayed that neuron pyknosis decreased in cerebral cortex and hippocampus of tree shrews, the pathological changes of mitochondria and endoplasmic reticulum were alleviated significantly, and cell edema abated in postconditoning group. The results of Western blotting and immunohistochemistry staining showed that cerebral ischemia caused a transient increase of the ASIC2a、SGK1 and p-SGK(Thr256) protein expression in cortex and hippocampus. Compared with the ischemic group, postcondionting caused a further and more prolonged increase of the ASIC2a.SGK1 and p-SGK(Thr256) protein expression in cortex and hippocampus. At the same time, postcondionting caused a protective down-regulation of ASIC2a, SGK1 and p-SGK (Thr256) protein expression, called "adjusting period", at 24h after ischemia in the ischemic cortex, in order to avoid the disadvantageous effect of cell metabolism "overdraft". In addition, there was a correlation relationship between ASIC2a and SGK1 protein expression in the cortex and hippocampus of tree shrews during the cerebral ischemia and ischemic postconditioning. The results of RT-PCR also show that cerebral ischemia caused a transient increase of the ASIC2a and SGK1 mRNA expression in cortex and hippocampus. Compared with the ischemic group, postcondionting caused a further and more prolonged increase of the ASIC2a and SGK1 mRNA expression. The specific inhibitor of SGK1 called GSK-650394 was injected to the ipsilateral hippocampus through the brain stereotaxis system. Compared the GSK-650394 group with the DMSO group and the postconditioning group, the changes of rCBF, cortical infarct size and hippocampal neuronal density had no significant differences. However, compared with the DMSO group, the concentrations of Na+ of hippocampal microenvironment decreased significantly in the GSK-650394 group (P<0.05). The result of brain water content also showed that, in the GSK-650394 group, the effect of alleviating brain edema was less than the DMSO group and the ischemic postconditioning. The result of immunohistochemistry showed that GSK-650394 inhibited the expression of ASIC2a to a degree in cerebral cortex and hippocampus, because of the numbers of ASIC2a positive cells reducing and staining shallow. But the result of western blot and RT-PCR showed that the expression of ASIC2a was a slight decrease both in protein and in mRNA level, while the differences of ASIC2a expression were statistically insignificant, compared the GSK-650394 group with the DMSO group and the ischemic postconditioning group.Conclusions: ①The ischemic postconditioning has obvious neuroprotective effect because it can reduce cerebral cortical infarct size, increase hippocampal survival neuronal density and relieve the damage of cellular ultrastructure in cerebral cortex and hippocampus.②The disturbance of neuronal microenvironment, following the spreading of the ischemic microenvironment in ischemic core, may be an important reason for the hippocampal cells secondary injury. And ischemic postconditoning play a neuroprotective effect by increasing rCBF, alleviating the turbulence of hippocampal ionic microenvironment, reducing cerebral edema and other mechanisms.③Cerebral ischemia can induce a transient increase of the expression and activation of SGK1 and ASIC2a. Ischemic postconditioning may be more reasonable to adjust the expressive and activatory level of ASIC2a and SGK1, to strengthen the endogenous defense mechanism, for improving the ionic homeostasis in ischemic microenvironment and protecting vulnerable neurons.④During the cerebral ischemia and ischemic postconditioning period, there was a correlation relationship between ASIC2a and SGK1 protein expression. After SGKl inhibited by GSK-650394, the cerebroprotection effects of ischemic postconditioning did not be completely blocked, and it suggested that the mechanisms of endogenous neuroprotective effect was very complex in ischemic postconditioning, which involved in the comprehensive regulation among multiple cellular information molecules at different levels. |
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