【作者】 卢英云；

【导师】 王翠兰；

【作者基本信息】 山东大学 ， 神经病学， 2008， 硕士

【摘要】 目的建立糖尿病大鼠合并急性脑缺血再灌注动物模型;探讨缺血后处理（I-Post）对糖尿病大鼠局灶性脑缺血再灌注损伤细胞凋亡、线粒体超微结构、自由基系统及ATP酶活性的影响;探讨I-Post对糖尿病合并急性脑缺血的保护机制。方法（1）采用腹腔内注射链脲霉素（streptozotocin,STZ）建立实验性大鼠糖尿病模型,在此基础上采用线栓法建立大鼠局灶性脑缺血再灌注模型。健康成年SD大鼠75只,STZ腹腔注射建立糖尿病（DM）大鼠模型,并随机分为空白对照组、假手术组、缺血再灌注组（I/R）、缺血后处理组（I-Post）。（2）缺血后处理方法:缺血90 min后完全复灌之前施行三次短暂缺血,将线栓拔出5mm,15s后再将线栓放入15s,即再灌注15s后缺血15s,反复3次。（3）再灌注6h后采用Longa 5分制评分标准对各组大鼠进行神经行为学评分（NDS）。（4）再灌注6h后采用TTC染色法测定脑梗死体积;光镜下观察缺血脑组织病理变化特点;应用TUNEL染色观察并计数脑组织缺血区阳性细胞数;电镜下观察线粒体超微结构;检测缺血侧脑组织线粒体中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）、Na⁺/K⁺-ATPase和Ca²⁺-ATPase含量的变化。（5）采用SPSS13.0统计软件,正态分布的计量资料以均数±标准差（（?）±s）表示,组间比较采用单因素方差分析;神经行为学评分以中位数表示,组间比较采用秩和检验,P＜0.05为差异有统计学意义。结果（1）糖尿病大鼠出现明显的多饮、多食、多尿等糖尿病的典型症状,大鼠血糖平均为（24.6±3.5）mmol/L。空白对照组和假手术组未见明显神经功能损害,NDS评分均为0分,I/R组和I-Post组均表现为不同程度的神经功能缺损,I/R组与I-Post组之间的神经行为学评分没有统计学意义（P＞0.05）。（2）鼠脑TTC染色结果非梗死区脑组织被染成红色,梗死区呈现苍白色,且苍白区与大脑中动脉供血分布区域一致,I/R组较I-Post组苍白区面积大（P＜0.05）。（3）细胞形态学观察空白对照组和假手术组未见明显的神经元损伤,I/R组显微镜下呈现典型的缺血性改变,表现为组织疏松、染色变浅、细胞空泡样变性,胞核固缩呈三角形,核仁消失。I-Post组梗死灶较I/R组明显减小,梗死中心区范围减小,周围可见形态基本正常的神经细胞。（4）原位细胞凋亡检测（TUNEL法）凋亡细胞的表现为神经元细胞体积缩小,细胞核固缩、染色质浓缩、边聚。空白对照组及假手术组仅可见少量散在的阳性细胞,I/R组可见梗死灶区有大量TUNEL阳性细胞,而I-Post组阳性细胞数明显减少（P＜0.01）。（5）脑线粒体的电镜观察空白对照组和假手术组可见基本正常的线粒体,I/R组可见神经元坏死,线粒体数量减少,明显肿胀、空泡化,嵴排列不规则,甚至断裂、溶解、消失。I-Post组可见部分线粒体空化,线粒体损伤较I/R组明显减轻。（6）脑线粒体ATP酶、MDA、SOD、GSH-PX的测定再灌注6h后I/R组脑线粒体的SOD、Na⁺-K⁺ -ATP酶、Ca²⁺-ATP酶及GSH活性出现显著降低（P＜0.01）,MDA含量明显增高;I-Post组的SOD、Na⁺-K⁺ -ATP酶、Ca²⁺-ATP酶和GSH活性与I-R组比较有不同程度的恢复（P＜0.05或0.01）,MDA含量明显降低（P＜0.05）。结论（1）I-Post可减轻缺血组织的再灌注损伤现象同样存在于脑组织;（2）I-Post对近期神经行为学的改善作用不明显;（3）I-Post可以抑制糖尿病大鼠脑神经细胞凋亡,减轻糖尿病大鼠局灶性脑缺血再灌注的病理损伤;（4）I-Post可以抑制糖尿病大鼠缺血脑组织线粒体超微结构的改变,提高线粒体ATPase活性,增强线粒体清除氧自由基的能力,减少脂质过氧化物的生成,从而起到线粒体保护,减轻缺血再灌注损伤的作用。更多还原

【Abstract】 ObjectiveTo establish an model of focal cerebral ischemia-reperfusion in streptozotocin-induced diabetic rats;To explore the effect of ischemic postconditioning（I-Post）on cortical neurons apoptosis in focal cerebral ischemia of diabetic rats;To investigate the effect of I-Post on the structure and function of mitochondria after focal cerebral ischemia-reperfusion in streptozotocin-induced diabetic rats;To debate the mechanism of I-Post in acute cerebral ischemia complicated with diabetes.Methods（1）The models of experimental diabetes rats were established by a single intraperitoneal injection of STZ（60 mg/kg body weight）,and the successful models were selected to establish the models of focal cerebral ischemia-reperfusion rats by Longa’s technique.Sixty-five rats were successfully made into diabetic models and randomly divided into 4 groups:1)control group: with no other intervention;2)Sham-operated group:the surgical procedure was identical to other groups,but the MCA was not ligated;3)I/R group:the brain was undergone ischemia for 90 min followed by reperfusion for 6h;4)I-Post group:rats were subjected to 3 cycles of 15s/15s reperfusion/reocclusion after 90 min of ischemia.（2）Postconditioning protocols:the brain was subjected to middle cerebral artery occlusion for 90 min,then treated for 15 sec,15 sec,15 sec by brief reperfusion consecutively,each was separated with occlusion for 15 sec,followed by a persistent reperfusion,which made the total reperfusion time the same as the other three groups.（3）The assessment standard of Longa’ 5 scores was applied to assess the neural function of the rats after cerebral ischemia-reperfusion.（4）TTC staining was applied to observe and note the formation of infarction zone;The pathologic characteristics of cerebral ischemic tissues were observed through microscope;The TUNEL was used to detect the change of TUNEL positive cells in every group;The ultrastructure of mitochondria was observed by electron microscope 6 hours later.Rat brain mitochondria were isolated in a medium of 0.01 mol/L sucrose,0.01mol/L Tris-HCl,0.0001mol/L EDTA-2Na, 0.8%Sodium Chloride,pH 7.4,by differential centrifugation of brain homogenates essentially as described previously.The level of malondialdehyde （MDA）,Na⁺,K⁺-ATPase,Ca²⁺-ATPase,Superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in mitochondria were measured 6 hours later, according to the commercial kit manual.（5）The data were expressed as mean±SD,the NDS was expressed as median.Data analysis was performed using the SPSS 13.0 statistical package programme.P＜0.05 was considered as statistically significant.Results（1）The diabetic rats appeared the diabetic symptoms such as eat more,drink more and urine more after the model was made.Plasma glucose in diabetic rats was routinely 24.6±3.5mmol/L.There was no significant difference in neurologic deficit scores between the I/R group and I-Post group（P＞0.05）.（2）The TTC staining results of the rat’s brain The brain tissues without infarction were stained as red,the infarction zone could not be stained and appeared as white and the white zones distributed in accordance with the distribution of the middle cerebral artery.There was a larger white areas in I/R than in I-Post after the same reperfusion time（P＜0.05）. （3）Observing the results of pathological sections stained by HE Under the optical microscopy,we found the ischemic changes in I/R group were more serious than I-Post group,which manifested the tissues became loose、the neurons nuclus atrophy,the staining became light,the cell morphous altered.But in the control and sham-operated groups,the tissue structure was clear,the nucleoli was distinct,the pathological changes was not remarkable.（4）TUNEL The apoptosis cells were characterized by body shrinkage, nuclear pyknosis,peripheral margination of chromatin crescents or chromatin clumps.Compared with I/R group,the apoptosis cells were less after reperfusion in I-Post group（P＜0.01）.（5）The ultrastructure of mitochondria Compared with control and sham-operated groups,the changes of the mitochondria of brain tissue in I/R group were obvious.The cristae of mitochondria were less and some of them became indistinct or disappeare;Some double membrane were not distinguishable clearly.The changes ultrastructure of mitochondria in cortex were significantly decreased in I-Post group compared with I/R group.（6）The level of malondialdehyde（MDA）,Na⁺,K⁺ -ATPase,Ca²⁺-ATPase, Superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in mitochondria The content of MDA in mitochondria was greatly higher in I/R group than it in control group and sham-operated group（P＜0.01）,Na⁺,K⁺ -ATPase,Ca²⁺-ATPase,SOD and GSH-Px activities in mitochondria were greatly higher in control group and sham-operated group than those in I/R group（all P＜0.01）.The content of MDA in mitochondria was markedly decreased and the activities of Na⁺,K⁺ -ATPase,Ca²⁺-ATPase,SOD and GSH-Px in mitochondria were increased in I-Post group compared with the I/R group（P＜0.05 or P＜0.01）.Conclusion（1）I-Post could protect brain against reperfusion injury as well;（2）I-Post could not attenuate behavioral deficits after focal cerebral ischemia in diabetic rats; （3）I-Post could reduce infarct size and the number of apoptotic cells of cortical neurons after focal cerebral ischemia-reperfusion in streptozotocininduced diabetic rats;（4）I-Post could protect diabetic rats against brain I/R injury.The brain protective effects of I-Post may be mediated,in part,by inhibiting the production of oxygen free radical,increasing antioxidation,ameliorating energy metabolism, and beneficially improving the integrity of structure and function of mitochondria in brain tissue.更多还原