慢性脑缺血对大鼠海马微血管构筑及胆碱乙酰基转移酶蛋白表达的影响

Effects of chronic cerebral ischemia on hippocampal microvascular architecture and ChAT expression in rats

目的:探讨慢性脑缺血致大鼠海马胆碱能神经元损伤的作用,初步阐明其作用机制。方法:选取90只健康成年雄性SD大鼠,随机分为假手术组(30只)和实验组(60只)。实验组大鼠采用结扎双侧颈总动脉的方法,建立慢性脑缺血模型;假手术组大鼠颈部正中切口,分离双侧颈总动脉,不做结扎。分别于术后第7、14和21天随机各取假手术组大鼠10只、实验组大鼠20只处死取材。免疫组织化学SP法观察海马神经元胆碱乙酰基转移酶(ChAT)蛋白表达水平的动态变化,单宁酸-氯化铁媒染法观察大鼠海马微血管构筑的动态变化,采用MiVnt图像分析系统定量分析免疫组织化学阳性结果、大鼠海马水平部的微血管密度(MVD)和微血管面积密度(MVA)。结果:实验组大鼠海马神经元ChAT的表达水平较假手术组大鼠明显降低(P<0.05);且随缺血时间的延长,ChAT的表达水平逐渐降低,实验组14d时大鼠ChAT的表达水平明显低于实验组7d时(P<0.05),实验组21d时明显低于实验组14和7d(P<0.05)。实验组各时间点大鼠海马的MVA和MVD均较假手术组明显降低(P<0.05)。随着造模时间的延长,MVA逐渐降低,实验组14和21d时大鼠的MVA明显低于实验组7d时(P<0.05);随着造模时间的延长,MVD也逐渐降低,实验组14和21d时的MVD明显低于实验组7d(P<0.05),实验组21d时明显低于实验组14d(P<0.05)。结论:慢性脑缺血可致大鼠海马MVA、MVD及ChAT蛋白的表达水平进行性下降,进而导致学习和记忆功能障碍逐渐加重,这可能是血管性痴呆发生的原因之一。

Objective To investigate the damage of hippocampal neurons induced by chronic cerebral ischemia in the rats,and to clarify its mechanism.Methods 90 healthy adult male SD rats were randomly divided into sham operation group （n=30 ） and experimental group （n=60 ）. The chronic cerebral ischemia rat models were established by permanently ligating the common carotid arteries on both sides of the rats in experimental group.The rats in sham operation group were established by incising the cervical median,only the common carotid arteries on both sides were separated without ligating. The rats in sham operation group and experimental group were respectively sacrificed at the 7th,14th and 21st day after operation.At each time point 10 rats in sham operation group and 20 rats in experimental group were selected and sacrificed.Immunohistochemistry staining was used to＆amp;nbsp;observe the dynamic changes of the expression levels of choline acetyltransferase（ChAT）in hippocampus neurons, and TA-FE method was used to observe the dynamic changes of hippocampal microvascualr architecture. MiVnt image analytical system was used to quantitatively analyze the immunohistochemistry result,the microvessel density （MVD）and micorvessel area density （MVA）of horizontal part of hippocampus in the rats. Results Compared with sham operation group,the ChAT expression levels in hippocampus neurons of the rats in experimental group at different time points were significantly decreased（P〈0.05）;and with the prolongation of time,the ChAT expression levels were gradually decreased;the ChAT expression level in 14-day experimental group was significantly lower than that in 7-day experimental group （P〈0.05 ）;the ChAT expression level in 21-day experimental group was significantly lower than those in 7-day and 14-day experimental groups（P〈0.05）.The MVD and MVA of hippocampus of the rats in experimental group at different time points were obviously decreased compared with sham operation group（P〈0.05）;the MVA was gradually decreased with the prolongation of time, and the MVA of hippocampus of the rats in 21-day and 14-day experimental groups were obviously decreased compared with 7-day experimental group（P〈0.05）;the MVD was gradually decreased with the prolongation of time,the MVD of hippocampus of the rats in 21-day and 14-day experimental groups was obviously decreased compared with 7-day experimental group（P〈0.05）;the MVD of hippocampus of the rats in 21-day experimental group was obviously decreased compared with 14-day experimental group（P〈0.05）.Conclusion Chronic cerebral ischemia can lead to the progressive decrease of the ChAT expression level,MVD and MVA of hippocampus of the rats to aggravate gradually the learning and memory dysfunction, which may be one of the reasons of vascular dementia.