大鼠急性脑缺血再灌注后血脑屏障通透性的超微结构与血清S100B蛋白水平的动态变化

1、大鼠急性脑缺血再灌注后血脑屏障通透性的超微结构与血清S100B蛋白水平的动态变化 07-08-21 11:12:00 编辑：studa20 作者：吴松笛，万琪，夏峰，耿晓英，刘学东，王津存【关键词】 血脑屏障Dynamic changes of bloodbrain barrier permeability and S100B protein level in serum after acute cerebral ischemia/reperfusion in rats【Abstract】 AIM: To investigate the dynamic changes of the blood

2、brain barrier（BBB） permeability and the S100B protein level in serum after acute cerebral ischemia/reperfusion in rats. METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was made by the thread occlusion method; the permeability of BBB was investigated by transmission elect

3、ron microscope using lanthanum nitrate as the tracer; the serum S100B protein concentrations were determined by ELISA at 0.5, 2, 6, 12, 24 and 48 h reperfusion after 2 h cerebral ischemia. RESULTS: Electron microscopy revealed that lanthanum granules appeared around the tight junction and in the end

4、otheliocytes at 0.5 h areperfusion, indicating the increase of BBB permeability. More phagotrophic lanthanum granules were found in the endotheliocytes that showed significant vacuolization at 2 and 6 h. The tight junction was opened and the astrocytes were separated from the basement membrane of th

5、e capillaries. The necrosis of both neurons and astrocytes with dissolved nucleus and the disruption of the capillary basement membrane were found at 12 and 24 h. The necrosis and disaggregation of many neurons and astrocytes were evident at 48 h. A lot of lanthanum granules were shown outside the b

6、rain vessels while many endotheliocytes were broken. The serum S100B protein level increased significantly only in rats after cerebral ischemia/reperfusion injury, but not in the shamoperation and the nonoperation control rats (P0.01). The serum concentration of S100B protein started to increase at

7、0.5 h of reperfusion. It kept at a high level until a peak appeared at 24 h. The serum S100B protein was maintained at a high level constitutively even at 48 h. CONCLUSION: The dynamic change of the S100B protein level in serum after ischemia/reperfusion reflects the change of the permeability of BB

8、B. S100B can therefore be used as a peripheral biochemical marker to assess the permeability of BBB.【Keywords】 bloodbrain barrier; brain ischemia; reperfusion; S100B protein【摘要】 目的： 研究大鼠急性脑缺血再灌注后血脑屏障通透性的超微结构与血清S100B蛋白水平的动态变化. 方法： 采用线栓法制作大鼠大脑中动脉阻塞再灌注动物模型；硝酸镧示踪电镜观察血脑屏障通透性；采用ELISA方法检测血清S100B蛋白，观察其分别在脑缺

9、血2 h再灌注0.5， 2， 6， 12， 24， 48 h时的变化. 结果： 硝酸镧示踪电镜结果表明，再灌注0.5 h，镧颗粒出现在紧密连接处；再灌注2 h和6 h，血管内皮细胞空泡化明显，星型胶质细胞的足突与毛细血管基底膜开始分离；再灌注12 h和24 h，许多神经细胞和胶质细胞变性坏死，细胞核溶解，毛细血管基底膜的连续性受到破坏；再灌注48 h，大量的神经细胞和胶质细胞坏死、崩解，多数血管内皮细胞形态不完整. 大鼠缺血再灌注后各组血清S100B蛋白水平与假手术组及对照组相比都明显升高，差异有显著性意义(P0.01). 再灌注0.5 h，血清S100B蛋白开始增高；再灌注2 h和6 h，血

10、清S100B蛋白处较高水平；再灌注12 h和24 h，血清S100B蛋白明显增高；再灌注48 h，血清S100B蛋白仍处较高水平. 结论： 脑缺血再灌注后血脑屏障通透性发生显著变化，血清S100B蛋白水平在脑缺血再灌注后的动态改变可较好的反映血脑屏障通透性的变化. 【关键词】 血脑屏障；脑缺血；再灌注；S100B蛋白0引言脑缺血再灌注损伤使血脑屏障(bloodbrain barrier, BBB)的结构和功能发生了改变，这种神经组织结构和功能的变化是缺血后病理生理改变的重要环节，也是影响药物治疗效果的关键因素之一. Nicola等1综合分析多项研究结果后发现，S100B蛋白在血脑屏障损伤后的外

11、周血中有明显的时间变化规律，并且与血脑屏障开放程度相关，提出S100B蛋白可作为反映血脑屏障损伤的外周生化标志物的理论. 为此，我们采用硝酸镧示踪电镜观察大鼠缺血再灌注后不同时间点血脑屏障通透性的改变和脑组织超微形态学变化；同时用ELISA方法检测血清S100B蛋白的动态变化，希望通过观察脑缺血再灌注后大鼠血清S100B蛋白与血脑屏障通透性的改变，来了解缺血再灌注后血脑屏障的动态变化过程.1材料和方法1.1材料成年健康雄性SD大鼠48只，体质量250300 g，由第四军医大学实验动物研究中心提供. 利用随机数字表将大鼠分为手术组(36只)、假手术组(6只)和对照组(6只)，其中手术组根据再灌注

12、时间不同分为脑缺血2 h再灌注0.5，2，6，12，24，48 h组，每组6只. JEM2000EX透射电镜（日本电子），检测大鼠血清S100B蛋白的ELISA试剂盒（第四军医大学生理学教研室）.1.2方法1.2.1动物模型的建立参照Longa线栓改良法2，制作大脑中动脉阻塞再灌注（MCAO/R）模型. 用10 g/L戊巴比妥钠(40 mg/kg)麻醉大鼠，取颈正中切口，暴露右侧颈总动脉、颈内动脉和颈外动脉，结扎颈外动脉、颈总动脉，于颈总动脉分叉下方剪一小口，将预先包被硅胶的4.0单丝尼龙缝线经该切口顺颈总动脉插入颈内动脉约(181) mm，将大脑中动脉起始部阻塞，2 h后将栓线拔出，恢复再灌

13、注. 术后左侧上肢屈曲、行走左转或左侧跌倒者；开颅取脑时无蛛网膜下腔出血者视为手术成功. 假手术组条件同上，但不线栓仅结扎右侧颈外动脉和颈总动脉.1.2.2血脑屏障通透性的超微结构参照文献3的方法. 取电镜标本方法：手术组大鼠在各固定的再灌注时间点、假手术组在结扎动脉后2 h，分别用10 g/L戊巴比妥钠(40 mg/kg)麻醉后，经左心室灌注生理盐水60 mL，然后再灌注镧醛固定液50 mL，迅速开颅取脑. 将整脑自视交叉向后冠状切成约1 mm厚脑片，分别于缺血侧及缺血对侧相应部位皮质及底节区各取2小块脑组织，约1.0 mm1.0 mm1.0 mm，镧醛固定液固定组织. 电镜标本由第四军医大学电镜中心制备完成后，透射电镜观察. 1.2.3S100B