| Objective:The author will use the method of observing limb ischemic postconditioning,to observe the expression change of caspase-3 and p-Akt after focal cerebral ischemia/reperfusion in rats with hyperlipidemia.In order to discuss the limb ischemic postconditioning of the neuroprotective effect and its possible mechanism of ischemia reperfusion lesion in hyperlipidemic rats with focal cerebral ischemia/reperfusion.Methods:To establish hyperlipidemic rats model: select SD rats 100,weighing 160~180g,were randomly divided into 2 groups: normal diet group 10,high fat diet group was 90.Cardiac blood sampling was carried out in rats after 4 weeks,analyzer detect rats serum lipid indexes: total cholesterol(TC),triglyceride(TG),high density cholesterol(HDL-C),low density cholesterol(LDL-C).Using random number table,the successful model of 90 hyperlipidemia rats were randomly divided into 3 groups: sham operation group(30 rats),ischemia/reperfusion group(30 rats),limb ischemic postconditioning group(30 rats).Each group was given rats middle cerebral artery occlusion reperfusion after 2 h,according to the time of reperfusion,the model group was observed at five time point which were 6h,12 h,24h,48 h,72h.And 5 rats in each sub group.In addition,each group of 48 h reperfusion time point,additional 5 rats were stained withTTC staining method to measure the volume of cerebral infarction.Adopted the modified suture method making middle cerebral artery ischemia/reperfusion(MCAO)model,in the realization of reperfusion immediately giving the LIP group tourniquet bundling of both lower limbs of rat and the bilateral femoral artery ischemia 10 min,reperfusion for 10 min,repeated 3 cycles,the sham operation group did not plug line,double lower limbs do not bind tournique.Through the nervous functional defects score to determine whether the success of the model,and the successful model of the various time points of the nerve function defect assessment.Brain tissue pathological changes were observed by HE stain,TTC staining method to measure the volume of cerebral infarction,dynamic expression of p-Akt and caspase-3 were observed by immunohistochemical method in rats of differen groups.TUNEL method was used to measure the degree of apoptosis in different time points after operation.Result: l.Changes of the neurologic deficits score: Compared with group Sham,the neurological deficit scores of I/R group and LIP group at 6 h~72 h were significantly lower(P<0.05);compared with the I/R group,the neurological deficit scores of the LIP group at 12 h ~ 72 h were significantly increased(P<0.05);within the same group,I/R group and LIP group after 6 h,12 h,72 h compared with 24 h time points were statistically significant differences(P<0.05).2.Pathological changes of brain: HE staining under light microscope observation,the neurons in the brain of Sham group were arranged in order,the morphology and structure were intact,the nucleus was complete,there was noedema and inflammatory cell infiltration;the I/R group showed a typical ischemic change,the number of neurons in the cerebral cortex of ischemic side was significantly decreased,the cell nucleus was contracted and the nucleus disappeared,bubble like change,a large number of inflammatory cell infiltration;compared with the I/R group,LIP group at the same time point of ischemia,the number of nerve cells degeneration necrosis cell edema and vacuole like changes have been reduced.3.Cerebral infarction volume measurement:The volume of cerebral infarction was measured at 48 h after operation,There was no cerebral infarction in the Sham group(0.00±0.00 mm3),compared with the Sham group,the I/R group had obvious cerebral infarction(174.60±3.85 mm3)(P<0.05);Compared with group I/R,the infarct volume of LIP group(104.20±2.59 mm3)was significantly decreased(P<0.05).4.Expression changes of p-Akt positive cell: The p-Akt cells were occasionally observed in cerebral cortex area in the sham group,p-Akt positive cells began to increase from reperfusion 6 h,12 h further increased,24 h reached the peak,48 h~72 h gradually decreased in I/R group and LIP group;compared with the Sham group,the difference was statistically significant(P<0.05),compared with I/R group,the expression of p-Akt cells which in 6 h~72 h was increased obviously in cerebral LIP group(P<0.05);within the same group,I/R group and LIP group each time point compared with 24 h time points were statistically significant differences(P<0.05).5.Expression changes of caspase-3 positive cell: The caspase-3 cells were occasionally observed in cerebral cortex area inthe sham group,caspase-3 positive cells began to increase from reperfusion 6 h,12 h~24 h further increased,48 h reached the peak,72 h gradually decreased in I/R group and LIP group;compared with the Sham group,the difference was statistically significant(P<0.05),compared with I/R group,the expression of caspase-3 cells which in 12 h~72 h was increased obviously in cerebral LIP group(P<0.05);within the same group,I/R group and LIP group each time point compared with 48 h time points were statistically significant differences(P<0.05).6.The degree of ischemic brain tissue cell apoptosis: Expression changes of apoptosis positive cell: The apoptosis cells were occasionally observed in brain tissue in the sham group,apoptosis positive cells began to increase from reperfusion 6 h,12 h~24 h further increased,48 h reached the peak,72 h gradually decreased in I/R group and LIP group;compared with the Sham group,the difference was statistically significant(P<0.05),compared with I/R group,the expression of caspase-3 cells which in 12 h~72 h was increased obviously in cerebral LIP group(P<0.05);within the same group,I/R group and LIP group each time point compared with 48 h time points were statistically significant differences(P<0.05).Conclusion:1.Establishing the model of hyperlipidemia rats that were free to eat can fully simulate the evolution process of human disease,on the basis of the application of improved line plug method to establish middle cerebral artery ischemia/reperfusion rat model,and the human brain ischemia/reperfusion of pathophysiological changes,is the study of excellent animal model of cerebral ischemia/reperfusion injury.2.Limb ischemia postconditioning can reduce hyperlipidemia rat focal cerebral ischemia/reperfusion injury,improve nerve function defect situation,reduced cerebral infarction volume,reduce the nerve cell apoptosis.3.Limb ischemia postconditioning can increase p-Akt expression,inhibition of caspase-3expression,thus inhibiting hyperlipidemia caused by ischemia/reperfusion injury in rats of nerve cell apoptosis.4.Limb ischemia postconditioning in hyperlipidemia rats ischemia/reperfusion injury of brain protection,its mechanism may be related to the PI3K/Akt signal pathway activation,inhibition of nerve cell apoptosis. |
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