| In recent years,stroke has become a serious threat to the health of human and the quality of life,but the main therapeutic methods,thrombolysis therapy,could aggravate the brain damage.The cerebral ischemia-reperfusion(I/R)could cause severe neurological damage and lead to physical dysfunction or incomplete,what makes the protection of cerebral after ischemia-reperfusion becoming the focus of clinical research.As we know,mild hypothermia(32-35?)can reduce the injury after cerebral ischemia-reperfusion effectively,but the specific mechanism needs further more researches.It was found that the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)signaling pathway is associated with the anti-apoptotic action after cerebral ischemia-reperfusion,and can regulate the neural cells apoptosis,proliferation and differentiation.Glutamate,as an important excitatory neurotransmitter,is involved in excitatory neurotoxicity death due to hypoxic ischemic-reperfusion,while the effect of glutamate transporter-1(GLT-1)on decreasing of concentration of extracellular glutamate was significant.Objective: To observe the relationship between the expression of PI3K/Akt signaling pathway and GLT-1 in the hippocampus CA1 area of rats after the cerebral ischemia-reperfusion injury,and to provide new ideas and theoretical basis for clinical research and treatment.Methods:1 134 healthy adult male wistar rats were selected.2 The model of cerebral ischemia reperfusion was established by four vessel occlusion method.3 According to whether treat with mild hypothermia in rats or lateral ventricle injection GLT-1 antagonist dihydrokainate(DHK),PI3 K inhibitorLY294002(LY),saline(NS)or DMSO,the rats were randomly divided into control group(C group),sham group(S group),NI/R group,HI/R group,HI/R+DHK group,HI/R+LY group,HI/R+NS group,HI/R+DM group.C group has 8 rats,and the other groups have 18 rats.No treatment with C group;S group only exposed vertebral artery,and isolated bilateral carotid artery 2days later;In group HI/R,the vertebral artery was closed at the first operation,ater 2 days,the temperature of the hippocampus was cooled to(32-35?)by the methods of nasopharyngeal cooling,then occlusioned the bilateral common carotid artery for 8min,after that the reperfusion was performed,and continue the temperature for 2h,then recover naturally;The other groups were injected with the corresponding drug in the anterior ventricle of before the bilateral common carotid artery occlusioned,and the other treatments was same to HI/R group.During the operation should keep the anus temperature at37?.4 After the second operation for 7 days later,to take 8 rats in each group cut their head off for pathological analysis.5 The other rats were killed by decapitated after the second operation for8 h,and the left and right sides of the brain were used for immunohistochemistry and Western blot separately.6 Immunohistochemical staining was used to detect the expression level of Bax and Bcl-2 protein in the CA1 region to determine the level of apoptosis.The expression of PAkt and GLT-1 in the CA1 region was detected by immunohistochemistry and Western blot.Results:1 Neuropathological evaluationIt is showed that in the CA1 subfield of the C group,pyramidal neurons were arranged in order with 2-3 cell layers,the morphological of the cellswere rule and integrity,and the nucleus of the cells was full and distinct.Compared with C group,There was no delayed neuronal deathtaken place in the CA1 hippocampus neurons of S group,HI/R group,HI/R+NS group and HI/R+DM group,and no obvious change of neuronal density(P>0.05).Compared withsham group,the neurons of I/R group were almost all died(P < 0.05).HI/R+NS group compared with HI/R+DHK group,HI/R+DM group compared with HI/R+LY group,there were significantly death of neuron(P<0.05).2 The comparison of expression of Bcl-2,Bax in hippocampus CA1regionCompared with S group,the expression of Bcl-2,Bax protein was higher in the rest groups.Compared with I/R group,the expression of Bax protein was lower in HI/R group,HI/R+NS group and HI/R+DM group,but the expression of Bcl-2 was higher in these groups.Compared with HI/R group,the expression of Bax was higher in HI/R+DHK group and HI/R+LY group,the expression of Bcl-2 was lower in these groups.HI/R+NS group compared with HI/R+DHK group,HI/R+DM group compared with HI/R+LY group,the expression of Bax was higher in the HI/R+DHK group and HI/R+LY group,and the expression of Bcl-2 was lower in the both two groups.The differeces were statistically significant(P<0.05).3 The comparison of expression of P-Akt in hippocampus CA1 regionCompared with S group,the expression of PAkt protein was higher in the rest groups.Compared with I/R group,the expression of PAkt protein was higher in HI/R group,HI/R+NS group and HI/R+DM group.Compared with HI/R group,the expression of PAkt was lower in HI/R+LY group.Compare with HI/R+LY group,the expression of PAkt protein was higher in HI/R+DM group.Compare with HI/R group,the expression of the protein PAkt in HI/R+DHK group was higher.The differeces were statistically significant(P<0.05).4 The comparison of expression of GLT-1 in hippocampus CA1 regionCompared with S group,the expression of GLT-1 protein was higher in the rest groups.Compared with I/R group,the expression of GLT-1 protein was higher in HI/R group,HI/R+NS group and HI/R+DM group.Compared with HI/R+LY group,the expression of GLT-1 was higher in HI/R group,HI/R+DM group,HI/R+NS group.Compare with HI/R+DHK group,theexpression of GLT-1in HI/R group and HI/R+NS group has no statistical significance.Compare with HI/R+DHK group,the expression of GLT-1in HI/R+LY group is lower.The differeces were statistically significant(P<0.05).Conclusion:1 Cerebral mild hypothermia can reduce neuronal apoptosis / death after ischemia reperfusion.2 PI3K/Akt may play a positive role in upregulating the expression of GLT-1 by the methods of mild hypothermia during the cerebral ischemic-reperfusion. |
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