微管细胞骨架在小鼠脑缺血/再灌注超急性期海马CA1区组织中的表达及意义

Expression and significance of microtubule cytoskeleton in hippocampal CA1 region during hyperacute cerebral ischemia/reperfusion in mice

为探讨微管细胞骨架在小鼠脑缺血/再灌注(I/R)超急性期(6、12、24 h)海马CA1区神经元中的变化情况,初步阐明微管细胞骨架对早期脑缺血/再灌注损伤的影响,将96只8周龄健康雄性ICR小鼠随机分为假手术组(24只)和I/R组(72只), I/R组通过Longa改良线栓法制备小鼠左侧大脑中动脉栓塞模型,采用2,3,5-氯化三苯基四氮唑(TTC)染色评估各组小鼠脑缺血梗死体积,采用尼氏(Nissl)、Fluoro-Jade C (F-JC)和Neun染色检测各组小鼠海马CA1区神经元死亡情况,应用IF染色和Western blot试验检测小鼠海马CA1区微管细胞骨架相关蛋白α-tubulin、β-tubulin、acetyl-tubulin、β-tubulinⅢ和MAP2蛋白的表达水平。结果表明:与假手术组相比,I/R组在6、12、24 h时小鼠脑缺血梗死面积百分比明显增大(P<0.05),随再灌注时间和延长,脑缺血梗死面积百分比逐渐增大。Nissl、F-JC和Neun染色结果显示,I/R组在6、12、24 h时小鼠神经元随再灌注时间的延长,神经元损伤加重且存活数逐渐减少(P<0.05)。而免疫荧光染色结果表明,与假手术组相比,I/R组在6、12、24 h时小鼠海马CA1区微管细胞骨架相关蛋白β-tubulin、acetyl-tubulin、β-tubulinⅢ和MAP2表达水平明显下降(P<0.05)。综上,在小鼠I/R超急性期,微管细胞骨架相关蛋白随时间的延长,呈先缓慢后快速降解的趋势,且微管解聚进程与神经元死亡进程呈正相关,其微管细胞骨架参与早期I/R损伤进程。

To investigate the changes of microtubule cytoskeleton in hippocampal CA1 neurons in hyperacute phase(6, 12 and 24 h) of cerebral ischemia/reperfusion(I/R) in mice, and to clarify the effect of microtubule cytoskeleton on early cerebral ischemia/reperfusion injury. We used 96 healthy 8-week-old male ICR mice that were randomly divided into sham operation group(n=24) and I/R group(n=72). The left middle cerebral artery embolization model of mice in the I/R group was prepared by Longa modified thread embolization method, and the volume of cerebral ischemic infarction in various group was evaluated by 2,3,5-triphenyltetrazole chloride(TTC) staining. Nissl, Fluoro-Jade C(F-JC) and Neun staining were used to detect the death of neurons in hippocampal CA1 region. Immunofluorescence staining and Western blot were used to detect the expression levels ofα-tubulin,β-tubulin,acetyl-tubulin,β-tubulinⅢ and MAP2proteins in the hippocampal CA1region of mice.The results showed that the percentage of cerebral ischemic infarct area in I/R group increased significantly at 6,12 and 24h compared with that in sham operation group(P<0.05),and gradually increased with the extension of reperfusion time.The results of Nissl,F-JC and Neun staining showed that the damage to neurons was getting worse and the number of surviving neurons in I/R group decreased gradually with the extension of reperfusion time(P<0.05).Immunofluorescence staining results showed that the expression levels of microtubule cytoskeleton related proteinsβ-tubulin,acetyl-tubulin,β-tubulinⅢ and MAP2 in the hippocampal CA1 region of mice in the I/R group were significantly decreased at 6,12 and 24h compared with the sham operation group(P<0.05).In general,in the hyperacute phase of I/R in mice,microtubule cytoskeleton related proteins degrade slowly and then rapidly with the extension of time,and the process of microtubule depolymerization is positively correlated with the process of neuronal death.Its microtubule cytoskeleton is involved in the process of early I/R injury.