大鼠局灶脑缺血预处理对脑功能保护的实验研究

【作者】 王金春；

【导师】 宋利春；

【作者基本信息】 中国医科大学 ， 神经病学， 2003， 硕士

【摘要】 目的 建立脑缺血耐受模型，研究凋亡相关基因Bcl-2、Bax和缺血耐受的关系，探讨局灶脑缺血预处理是否通过抗凋亡机制产生缺血耐受作用。 方法 1)动物模型的建立和分组：选择体重为240mg～270mg的健康雄性Wistar大鼠(购于中国医科大学附属二院动物部)56只，参照改良Haruo Magasawa法建立大脑中动脉缺血模型。动物随机分为4组：缺血组(16只)，予缺血1小时后再灌注；预处理组(12只)，予缺血10分钟；预处理缺血组(16只)，予缺血10分钟后再灌注，3天后再次缺血1小时；假手术组(12只)，不阻断血流。每组分为两部分，一部分大鼠断头处死，行TTC染色，用数码相机摄像后，利用图像分析仪测定梗死面积，然后根据公式计算梗死体积；另一部分大鼠经心脏4％多聚甲醛灌注固定处死，石蜡包埋，按6μm切片，切片贴附于预处理的载玻片上，用前脱蜡至水，行免疫组化等染色。(2)TTC和HE染色：观察大鼠脑梗死的体积及组织病理学改变。(3)POD法染色：观察神经细胞凋亡情况。(4)免疫组化染色：观察Bcl-2、Bax蛋白表达的变化。5)数据处理：所有实验数据以均值±标准差(±s)表示，统计分析用t检验。6)试剂：TTC染色、Bcl-2和Bax免疫组化染色、凋亡细胞POD染色试剂盒均购于武汉博士德公司。 结果 1)TTC染色：假手术组和缺血预处理组无梗死灶，缺血组梗死灶位于缺血侧领叶、顶叶皮质、尾状壳、壳核区，预处理缺血组的梗死体积（64d4．74nun勺较缺血组＊ ig．79nun勺明显减小仲＜0．01人2）HE染色：假手术组和预处理组均未见明显病理改变，缺血组的项体积与17’n染色显示相同。3）原位凋亡细胞测定（POD法X预处理组和假手术组仅可见0－3个散在的凋亡细胞出现于皮质、济既体及基底节区。缺血组梗死灶周围的半暗带内可见大量神经元胶质细胞及部分血管内皮细胞发生凋亡。预处理缺血组半暗带内的凋亡细胞数较缺血组明显减少（P＜o．01人4）Bcl－2及Bax蛋白表达：假手术组及各实验组非缺血侧的大脑半球无或仅有少量化一2弱阳性表达。与假手术组相比，预处理组缺血侧大脑半球化一2阳性细胞数明显增加O＜0．ofX缺血组在缺血侧梗死周边区h－2蛋白表达也增加（P＜0．of人与缺血组比较，预处理缺血组优一2蛋白表达显著升高河＜0．of人Bax蛋白在各组表达无显著差异。 结 论 ＊局灶脑缺血预处理后大鼠的同侧脑内产生缺血耐受，具体表现为：产生化一2蛋白的表达，并且协一2蛋白表达上调；于再次缺血后，其梗死面积大大减小，半暗带内凋亡细胞明显减少。局灶脑缺血预处理不影响Bax蛋白的表达。 2）局灶脑缺血预处理后产生的h－2蛋白表达是一个主动的过程，与缺血耐受的产生相一致。 3）局灶脑缺血预处理可以通过调节细胞内凋亡抑制基因BCI－2的表达诱导抗凋亡，从而启动内源性保护机制。更多还原

【Abstract】 PrfaceStroke is a major cause of death and disability in the elderly. A phenomenon of " ischemic tolerance" in animals previously subjected to short ischemic episodes, rendering the more tolerant to subsequent occurrences of persistent cerebral ischemica. " Preconditioning " is a phenomenon in which brief exposure to ischemia makes brain less vulnerable to damage after longer ischemia, ischemic tolerance has drawn considerable attention as it provides a way to study the mechanisms of endogenous neuroprotection. The mechanism of " ischemic tolerance" is not well understood. Evidence supporting apoptotic modulation in cerebral ischemia includes the slow progressive nature of selective neuronal cell death in the hippocampus following global ischemia. The upregulation of apoptosis - promoter genes may be indicative of a tendency toward cell death. The overexpression of apoptosis - suppressor genes in brain may reflect a mechanism compatible with endogenous neuroprotection.To further investigate the potential role of Bel - 2 and Bax as an effecter of ischemic tolerance in brain. We used the intraluminal suture technique to precondition the striatum against subsequent focal ischemia and studied the expression of Bel - 2 and Bax protein in the region of tolerant stratum.Experimental Data1. subjectsMale Wistar rats weighing 240 to 270g were, used in all experiments and were allowed free access to food and water before and after all procedures, animals were randomly divided four group: the sham -operated group ( SO group), the ischemic preconditioning group (IP group),the ischemic preconditioning-cerebral ischemic group(IPCI group) and cerebral ischemic group (CI group) .2. ReagentTTC,Bcl -2,Bax and POD kits were supplied by WuHan Bostor experimental Co.Experimental Methods1. Animal methodThe rats were anesthetized with. 10% Chloral hydrate (350mg/ kg). The bifurcation of the common carotid artery (CCA) was exposed and external carotid artery ligated with suture. The internal carotid artery (ICA) was isolated and separated from the vagus nerve. The origin of the middle cerebral artery (MCA) was occluded by introducing a 3 -0 monofilament nylon suture (its tip rounded by heating) into the ICA lumen, through the CCA, the suture was gently advanced 17 to 20mm past CCA bifurcation, then the CCA was ligated and the wound closed.The sham - operated group underwent the same surgical procedure,anesthesia, the suture was inserted and advanced for a distanceof 5mm beyond the ICA/ECA bifurcation. The CI group underwent 60 minutes MCA occlusion. Two - 5 - minutes MCA occlusion separated by 5 minutes reperfusion was used to induce precondition. 3 days later, the IPCI group were again challenged with 60 minutes of focal ischemia.2.TTCBrain were rapidly removed and sectioned coronally at 2 - mm intervals. The sections were immersed in 2% 2,3,5 - triphenyltetrazoli-um hydrochloride (TTC) in saline for 30 minutes at 37 C and then transferred to 4% paraformaldehyde. Six sections were analyzed for infarction size by using a computerized imaging analysis system. Infarction volume was calculated by summing the infarction areas of all sections and multiplying by the slice thickness.3. Perfusion Fixation EmbedmentAfter animals were anesthetized, they were perfused transcardial-ly with saline (100ml) and 4% paraformaldehyde (100ml). Brain were removed and postfixed in the same fixative for 24 hour, then were embedded in pafaffin. And 6-m-thick coronal sections were taken at - 0. 3mm from the bragma. Some section were stained with cresyl violet, and some section were for immunocytochemistry4. immunocytochemistrythe sections were deparafinized , then heated and boiled by microwave. Nonspecific activity was blocked with normal goat serum. Brain sections then were incubated at 37 for 2 hour with rabbit monoclonal antibody against mouse Bel - 2 or Bax, and then incubated 20 minutes with goat anti-rabbit immunconjugate. The section for Bcl-2 or Bax staining were counterstained wit更多还原