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目的利用大鼠双侧颈总动脉永久性结扎(permanent,bilateral common carotid artery occlusion,BCCA)方法制作慢性脑缺血模型,观察42 d与56 d两时间点在认知、情绪、组织状态的差异及小胶质细胞状态改变,在模型制作时间上为慢性脑缺血实验研究提供依据。方法雄性SD大鼠采用抽签法随机分为42 d-假手术组(42 d-Sham)、42 d-模型组(42 d-Model)、56 d-假手术组(56 d-Sham)及56 d-模型组(56 d-Model)。采用Morris水迷宫、新旧物体识别和旷场试验3种方法评价大鼠认知能力和情绪,HE染色观察大鼠海马组织状态改变,用脑组织冰冻切片Iba1免疫荧光染色观察各组海马及皮质小胶质细胞的活化状态。结果Morris水迷宫试验结果显示,与Sham组相比,Model组大鼠出现认知障碍,56 d较42 d下降更为显著,表现为逃避潜伏期延长,穿越平台次数减少。新旧物体识别试验结果表明,Model组大鼠认知能力下降,表现为新旧物体分辨指数下降,新物体探索时间减少,但42 d与56 d手术组及Sham组之间差异无统计学意义。旷场试验结果提示,Model组大鼠出现焦虑样行为,表现为直立次数和穿格次数明显增多。HE染色结果提示BCCA模型42 d及56 d在双侧海马均未出现明显的组织状态改变。免疫荧光结果显示,Iba1的表达在Model组与Sham组差异有统计学意义,Model组海马与皮质中小胶质细胞数量均较多,染色加深,胞体增大,突起增多、变短,呈“阿米巴样”。结论 BCCA法制作的大鼠慢性脑缺血模型42 d即出现认知能力下降,小胶质细胞出现活化改变,提示42 d即可作为慢性脑缺血动物实验模型制作的备选时间点。56 d较42 d认知能力下降更为显著,提示随时间延长,改变更为显著。但42 d及56 d均未出现HE染色可观察到的组织状态学改变。
OBJECTIVE: To establish a chronic cerebral ischemia model by bilateral common carotid artery occlusion (BCCA) in rats and observe the differences in cognitive, emotional and histological states between 42 and 56 d And microglia state changes in the model making time for the experimental study of chronic cerebral ischemia provide the basis. Methods Male SD rats were randomly divided into 42 d-sham group, 42 d-model group, 56 d-sham group and 56 d-model group (56 d-Model). Morris water maze, old and new object recognition and open-field test were used to evaluate the cognitive ability and emotion of rats. HE staining was used to observe the changes of hippocampal tissue in rats. Iba1 immunofluorescence staining was used to observe the changes of hippocampus and cortex Glial activation state. Results The results of Morris water maze test showed that compared with Sham group, Model rats showed cognitive impairment and decreased significantly at 56 days compared with 42 days. The results showed that the escape latency was prolonged and the number of crossing platform was decreased. The results of old and new object recognition test showed that the cognition ability of model rats decreased, showing that the resolution index of new and old objects decreased and the exploration time of new objects decreased, but there was no significant difference between 42 d and 56 d operation groups and Sham group. The results of the open-field test suggested that anxiety-like behavior occurred in Model rats, showing an obvious increase in the number of uprights and the number of wears. The results of HE staining suggested that there was no obvious change of tissue state in bilateral hippocampus at 42 d and 56 d after BCCA. The results of immunofluorescence showed that the expression of Iba1 was significantly different between Sham group and Model group. The numbers of microglia in hippocampus and cortex in model group were more than those in Sham group. The number of microglia was increased, the number of cytoplasm increased, the number of protuberance increased, Amoeba-like ". Conclusions The chronic cognitive impairment and microglial activation in rat models of chronic cerebral ischemia induced by BCCA were observed at day 42, suggesting that the model could be used as an alternative time point for experimental animal model of chronic cerebral ischemia after 42 days. 56 d than 42 d cognitive decline more significant, suggesting that over time, the change is more pronounced. However, on day 42 and day 56, no histological changes were observed in HE staining.