【作者】 马莉；

【导师】 卢祖能；

【作者基本信息】 武汉大学 ， 神经内科， 2016， 博士

【摘要】 目的：探讨草酸艾司西酞普兰对急性脑缺血再灌注大鼠和慢性低灌注大鼠的神经保护作用,并初步探讨其可能的机制。方法：第一部分：取SD雄性大鼠60只,随机选择手术组大鼠,制作急性脑缺血再灌注即大脑中动脉闭塞/再灌注(MCAO/R)模型,假手术组不行血管闭塞和干预措施；手术组随机分为磷酸缓冲盐溶液(PBS)对照组和实验组,术后2h分别给予PBS和草酸艾司西酞普兰；然后采用改良神经功能缺损程度评分(mNSS)鉴定各组大鼠模型制备及评价神经功能缺损情况。在大鼠模型干预后2周,使用激光共聚焦三维血管成像观察各组大鼠缺血区微血管直径、密度和总面积；5-溴脱氧尿嘧啶核苷(BrdU)法检测各组大鼠缺血区颅内细胞的新生；TUNEL法检测各组大鼠局灶性神经细胞凋亡；透射电镜下观察各组大鼠的神经细胞形态；酶联免疫吸附法(ELISA)检测血浆内血管内皮生长因子(VEGF)的浓度。第二部分：取SD雄性大鼠60只,随机选择手术组大鼠制备慢性低灌注大鼠模型即双侧颈动脉闭塞(2-V0)模型,假手术组不行血管闭塞和干预措施；手术组随机分为PBS对照组和实验组,术后6周分别给与草酸艾司西酞普兰和PBS干预；在大鼠模型干预2周后,检测大鼠的空间学习记忆能力,采用的方法是行Morris水迷宫行为学检测；同时检测大鼠模型脑缺血区域BrdU标记的大鼠脑细胞增殖情况；并且采用激光共聚焦三维血管成像,检测缺血区脑组织的血管新生情况；ELISA检测血液VEGF浓度；采用免疫组化方法检测脑缺血区域VEGF表达的水平。结果：第一部分：(1)mNSS评分：干预后2周大鼠神经功能缺损明显改善,检测结果显示mNSS评分实验组较PBS组明显降低(P<0.05)。(2)新生神经细胞：BrdU法检测,各组大鼠缺血区脑组织内神经细胞的新生,实验组较对照组明显增加(P<0.05)。(3)血管增生：三维共聚焦血管成像检测,干预后2周,假手术组和PBS对照组血管直径明显大于实验组(P<0.05),假手术组和PBS组血管密度明显少于实验组(P<0.01),假手术组及PBS组微血管总面积明显小于实验组(P<0.05)。(4)神经细胞凋亡：TUNEL法检测各组大鼠局灶性神经细胞凋亡显示,实验组较对照组明显减少(P<0.05)。(5)神经细胞形态：透射电镜观察各组大鼠的神经细胞形态显示,对照组炎症水肿明显减轻。(6)VEGF浓度：ELISA法显示,实验组大鼠的血浆VEGF浓度显著高于假手术组、PBS组(P均<0.05)。第二部分：(1)空间学习记忆能力：Morris水迷宫大鼠的空间学习记忆能力检测结果显示,实验组逃避潜伏期明显短于对照组；而在空间探索试验中,实验组大鼠模型在原有平台所在的第一象限游泳时间明显长于对照组(P均<0.01)；提示实验组大鼠的空间学习记忆能力明显改善。(2)BrdU阳性细胞：实验组大鼠模型脑缺血区域中的BrdU阳性细胞数明显高于对照组,差异有显著性(P=0.003<0.01)。(3)毛细血管增生：激光共聚焦三维成像检测结果显示,实验组较对照组比较毛细血管内径显著性变短,大鼠同源组织缺血边界地区的分支点数目显著增加,实验组可以显著增加微血管总面积(P均<0.05)。(4)VEGF：与对照组相比,实验组血浆VEGF浓度(ELISA检测)和脑缺血区域VEGF蛋白表达的水平(免疫组化检测)显著增加。结论：草酸艾司西酞普兰可以明显减少急性脑缺血再灌注大鼠和慢性低灌注的神经功能损伤,发挥神经保护作用,推测其可能与VEGF介导的血管新生有关。更多还原

【Abstract】 Objective:The neuroprotective effects of escitalopram oxalate in rats with acute cerebral ischemia and chronic hypoperfusion and the possible mechanism were explored preliminarily.Methods:Part One:Focal middle cerebral artery occlusion model (MCAO) was prepared and given escitalopram oxalate (experimental group) or PBS (control group) after 2 hours. Two weeks after the operation, neurological deficit of rats in each group were evaluated by modified neurological severity score (mNSS) scale. The cell proloferation,three-dimensional vascular distribution, the number of apoptotic cells,cell morphological changes in ischemic area and the plasma vascular endothelial growth factor (VEGF) were detected to explore the possible mechanisms.Part Two:Chronic hypoperfusion (2-VO) model was prepared and given escitalopram oxalate (experimental group) or PBS (control group) after 6 weeks. Eight weeks after the operation, Morris water maze test was carried out to evaluate the learning and memory ability of the rats. The cell proliferation, three-dimensional vascular distribution in ischemic area and the plasma vascular endothelial growth factor (VEGF) were detected to explore the possible mechanisms.Results:Part One:(1)The mNSS in the experimental group was significantly lower than that in the control group after 2 weeks (P<0.05)(2) Cerebrovascular confocal detection results showed that the inside diameter of capillaries was significantly less in the experimental group than in the control group (P<0.05); the vascular density was significantly increased in the experimental group (P<0.01) and the total area of capillaries was also significantly increased in the experimental group as compared with the control group (P<0.05)(3) There was statistically significant difference in BrdU-positive cells in the ischemic brain tissue between the experimental group and the control group (P<0.05)(4)The number of apoptotic cells in the experimental group was significantly less than that in the control group (P<0.05)(5)The observation of transmission electron mictoscope showed that the infalmmatory edema in the experimental group was significantly reduced compare to the control group (P<0.05) (6) VEGF concentrations in the plasma and the ischemic area were higher in the experimental group than in the control group (P<0.05).Part Two:(1) Morris water maze test showed that the escape latency in the experimental group was significantly shorter than in the control group, while the first quadrant swimming time in the experimental group was significantly longer than the control group (both P<0.01).(2) Cerebrovascular confocal detection results showed that the inside diameter of capillaries was significantly less in the experimental group than in the control group; the vascular density was significantly increased in the experimental group and the total area of capillaries was also significantly increased in the experimental group as compared with the control group.(3) There was statistically significant difference in BrdU-positive cells in the ischemic brain tissue between the experimental group and the control group (P=0.003<0.01).(4) VEGF concentrations in the plasma and the ischemic area were higher in the experimental group than in the control group (P<0.05).Conclusion:It was concluded that escitalopram oxalate could significantly improve the neural functional recovery of the rats with acute and chronic cerebral ischemia probably by the VEGF-mediated angiogenesis.更多还原