【作者】 李静；

【导师】 李燕；

【作者基本信息】 昆明医学院 ， 神经病学， 2011， 硕士

【摘要】 目的：采用线栓法制备较符合人类常见急性局灶性缺血性卒中类型的大鼠脑缺血／再灌注模型,探讨缺血后适应减轻大鼠脑缺血／再灌注损伤及对血脑屏障的保护作用,延迟再灌注的治疗时间窗。方法：本项实验分两个阶段进行。第一阶段(已完毕)：①寻找适宜脑缺血／再灌注时间窗,将SD大鼠随机分为4组,每组8只SD大鼠,分别为再灌注1h组、再灌注2h组、再灌注3h组、再灌注4h,对照组为永久梗死组。通过比较各实验组与永久梗死组之间神经功能、脑梗死体积,找出最佳的再灌注治疗时间窗。②在找到适宜的再灌注治疗时间窗后,根据这个时间窗来寻找脑缺血后适应治疗时间窗。对照组为脑缺血2h再灌注即刻给予后适应处理,实验组分为6组,每组8只SD大鼠,分别在脑缺血3h、3.5h、4h、4.5h、5.5h、12h、24h后再灌注即刻给予后适应处理。每只存活大鼠分别在术后1h、48h给予神经功能评分,48h神经功能评分完毕后立即处死大鼠,留取脑组织进行脑水肿、脑梗死体积计算。第二阶段：应用线栓法制备大鼠脑缺血／再灌注损伤模型。将15只成年健康雄性SD大鼠随机分成缺血2h再灌注组和缺血3.5h后适应组,每组分别为7只和8只；于大鼠脑缺血／再灌注24h时进行神经功能评分；24h后行TTC染色测定脑梗死体积。再将16只成年健康雄性SD大鼠随机分成缺血2h再灌注组和缺血3.5h后适应组,每组分别为8只；于大鼠脑缺血再灌注24h后应用免疫组化法定位半定量血脑屏障Ⅳ型胶原蛋白、ZO-1蛋白的表达水平。结果：(1)再灌注2h组的大鼠脑梗死体积明显小于再灌注3h组与永久梗死组,而3h及以后各组与永久梗死组的脑梗死体积无明显差异。缺血3.5h后适应组再灌注后1h和再灌注后48h三种神经功能评分,术后48h大鼠脑梗死体积、脑水肿程度与缺血2h后适应组无明显差异；(2)缺血3.5h后适应组大鼠脑梗死体积、术后24h Ludmila 12分、Zea Longa 5分、悬吊实验、旷场活动实验等神经功能评分、血脑屏障Ⅳ型胶原蛋白与ZO-1蛋白均较缺血2h再灌注组无明显差异(P>0.05)。(3)缺血2h再灌注组和缺血3.5h后适应组行斜坡实验神经功能评分不符合正态分布,采用秩和检验,P=0.017<0.05,两组间有显著差异。结论：(1)大鼠脑缺血再灌注最佳时间窗为2h。(2)缺血／再灌注即刻给予后适应处理能将再灌注时间窗延长至3.5h,为临床缺血性脑梗死溶栓治疗提供一种新的可能。(3)缺血3.5h后适应组对大鼠的Ludmila 12分神经功能评分、Zea Longa 5分神经功能评分、悬吊实验神经功能评分及旷场活动实验神经功能评分较缺血2h再灌注组无明显增加。(4)采用斜坡实验,缺血3.5h后适应组和缺血2h再灌注组的数据显示两组之间有显著差异。(5)缺血3.5h后适应组和缺血2h再灌注组相比,大鼠脑梗死体积无明显增加。(6)缺血3.5h后适应组脑梗死侧基底节ZO-1蛋白和Ⅳ型胶原蛋白阳性血管计数较缺血2h再灌注组相应部位的ZO-1和Ⅳ型胶原蛋白阳性血管数未见明显减少,提示缺血后适应能减轻(Ischemia/reperfusion,I/R)损伤,保护血脑屏障的完整性,从而利于延长再灌注治疗时间窗。更多还原

【Abstract】 Objective To study the effects of ischemic postconditioning on cerebral ischemia/reperfusion and blood-brain barrier in rats following middle cerebral artery occlusion (MCAO),approach the methods of chosing the suitable time windows to cerebral ischemia/reperfusion.Method The whole experiment was divided into 2 parts.The first stage①To study the time window of ischemia/reperfusion.Rats of MCAO were randomly divided into 4 experimental groups and 8 rats each one.Supplying respectively reperfusion at 1,2,3,4 hours after iscemia and named as 1-hour group,2-hour group,3-hour group,4-hour group, the control group was marked a group of permanent infarction.Each experiment group was compared for the differences of infarction volumes and neurological scores with the control group so as to find out the best time window of reperfusion.②To ascertain the time window of postconditioning at postischemia according the best time window of reperfusion.The control group was the one of postconditioning at 2 hour after ischemia.Experimental groups were divided into 7 groups and 8 rats for each. Supplying respectively reperfusion at 3,3.5,4.4.5,5.5,12,24 hours after iscemia.Neurological functional scores were tested at 1 hour and 48 hour after ischemia,and then their brain tissues were executed for weighing water content,calculating cerebral infarction volume.The second stage(The current one)Ischemia/reperfusion model in rats was performed with suture method.15 rats were randomly divided into two groups:Reperfusion group at 2h after middle cerebral artery occlusion (R-MCAO)(n=7), Postconditioning group by transient bilateral common carotid artery occlusion/referfusion at 3.5h after middle cerebral artery occlusion(P-MCAO)(n=8).Neurological functional deficits were evaluated at 24h after ischemia/reperfusion.Rats were sacrificed at 24h after the reperfusion,their brains were obtained for TTC staining examination.While 16 rats were randomly divided into two groups:Reperfusion group at 2h after middle cerebral artery occlusion (R-MCAO)(n=8), Postconditioning group by transient bilateral common carotid artery occlusion/referfusion at 3.5h after middle cerebral artery occlusion(P-MCAO)(n=8). At 24h after the reperfusion, their brains were obtained for detecting collagen-IV and ZO-1 expression by immunohistochemistry method.Result (1)The infarction volumes in 2-hour group obviously were smaller than the one of the 3-hour and the permanent infarction groups,meanwhile there was no marked differences between other groups(longer than 3-hours) and the group of permanent infarction.The infarction volumes.the extent of cerebral edema of the group of postconditioning in 3.5 hours after infarction did not increase comparing to the one of postconditioning in 2 hour after infarction,while there was no marked difference between the group of postconditioning of in 3.5 hour and the one in 2 hour in neurological functional scores not only after 1 hour reperfusion but also after 48 hours reperfusion(p> 0.05).(2)Neurological functional outcomes by Ludmila test,Zea Longa test,over hanging test and open fild test, cerebral infarct size,the expression of collagen-IV and ZO-1 were not down-regulated by ischemic postconditioning(p>0.05) in I/R 24h as well. (3)In inclined planet test,the rats of P-MCAO group had worse neurologicalseverity performance than that of MCAO group(p<0.05).Conclusion (1)Ascertaining initially therapeutic time window of ischemia-reperfusion in rats is 2h.(2)Seaching therapeutic time window of postconditioning at postischemia in rats is 3.5h.(3)Ischemic postconditioning group in 3.5h after MCAO does not reduce neurological functional outcomes by Ludmila test,Zea Longa test,over hanging test and open fild test than ischemic reperfision group in 2h after MCAO.(4)Ischemic postconditioning increases neurological severity scores by inclined planet test.(5)Ischemic postconditioning group in 3.5h after MCAO does not increase cerebral infarct size comparing to ischemic reperfision group in 2h after MCAO.(6)Ischemic postconditioning up-regulates collagen-Ⅳ,zo-1 of更多还原