中脑导水管周围灰质结构改变与癫痫发生发展的交互影响

发布时间：2017-12-27 06:20

  本文关键词：中脑导水管周围灰质结构改变与癫痫发生发展的交互影响　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 腹侧中脑导水管周围灰质 癫痫 vGLUT1 vGAT

【摘要】：目的通过匹罗卡品腹腔注射诱导大鼠癫痫模型,探索脑腹外侧中脑导水管周围灰质(the ventrolateral Periaqueductal Gray,vPAG)结构改变与癫痫发生发展的相互影响。方法将90只达到既定癫痫持续状态(Status Epilepticus,SE)行为学标准的大鼠随机分成6个亚组:12h、24h、72h、10d、20d和30d,另随机选择15只与试验组生物学属性相似的大鼠作为对照组。每个试验亚组在相应的时间点灌注取大鼠脑组织实施免疫双标荧光染色、HE染色、TUNEL凋亡染色和蛋白印迹(western blot,WB),用以分析vPAG区神经元的囊泡型谷氨酸转运体-1(Vesicular Glutamate Transporter1,vGLUT1)和囊泡型γ-氨基丁酸转运体(Vesicular GABA Transporter,vGAT)表达水平的变化。另外随机选择13只正常大鼠用于脑立体定向毁损手术,通过视频监控癫痫行为学以分析vPAG区神经元的毁损对自发性痫性发作(Spontaneous Recurrent Seizures,SRSs)的影响。试验中所有数据均以均数±标准误(M±SE)表示,单因素方差分析(One-way ANOVA)用于分析组织化学染色和WB数据,独立样本t检验用于分析癫痫发作行为学数据,以P0.05有统计学意义,P0.01有显著统计学意义。结果在达到SE后的最初72h内,vPAG区神经元的vGLUT1和vGAT表达水平以及平均TUNEL阳性细胞数均有明显的增高趋势,在SRSs慢性期,上述指标呈现出一个逐渐降低的趋势,然而直到实验期结束时的30d仍未恢复至对照组水平。在达到SE后的72h,vPAG区的vGLUT1和vGAT的平均荧光密度显著高于其他亚组(P0.01),试验组中vPAG区的平均TUNEL阳性细胞数较对照组明显增多(P0.01)。在SE后的72h时间点,vPAG区的vGLUT1和vGAT在WB中的标准化比值较对照组、12h、20d和30d均有有显著统计学差异(P0.01),此外,在SE后的24h,72h和10d时间点,vPAG区的vGLUT1和vGAT在WB中的标准化比值均较对照组有显著统计学差异(P0.01)。vPAG区受损的神经元在HE染色中表现为皱缩、深染的椭圆形细胞,TUNEL阳性细胞呈现出褐色或深棕色凝缩致密的细胞核。经过2周的癫痫行为学视频监测,毁损组中的大鼠痫性发作频率、持续时间及严重程度较对照组均有显著性差异(P0.01)。结论本研究表明癫痫的发生发展诱发了vPAG功能的失调和结构的改变,而vPAG结构的损坏也促进了癫痫的发生发展。
[Abstract]:Objective through the rat model of epilepsy induced by pilocarpine, explore the cerebral ventrolateral periaqueductal gray (the ventrolateral Periaqueductal Gray, vPAG) interaction structure changes and the occurrence and development of epilepsy. Methods 90 rats with Status Epilepticus (SE) behavior criteria were randomly divided into 6 subgroups: 12h, 24h, 72h, 10d, 20d and 30d, and 15 randomly selected rats with similar biological attributes were used as control group. Each test group at the corresponding time point perfused rat brain the immune double fluorescence staining, HE staining, TUNEL staining and Western blotting (western, blot, WB) is used to analyze vPAG neurons of vesicular glutamate transporter -1 (Vesicular Glutamate Transporter1, vGLUT1) and vesicular GABA butyrate transporter (Vesicular GABA Transporter, vGAT) expression. In addition, 13 normal rats were randomly selected for stereotactic brain surgery. The effect of neuronal damage in vPAG area on Spontaneous Recurrent Seizures (SRSs) was analyzed by video surveillance of epileptic behavior. All the data are mean standard error (M + SE), single factor analysis of variance (One-way ANOVA) for the analysis of histochemical and WB data, independent samples t test was used for the analysis of seizure behavior data, using P0.05 statistical significance, P0.01 has significant statistical significance. The results in the first 72h reached SE within vPAG neurons and vGLUT1 expression level of vGAT and the average number of TUNEL positive cells was significantly increased, in the chronic phase of SRSs, the index showed a decreasing trend, however, until the end of the experimental period 30d still did not recover to the level of the control group. After reaching SE, the average fluorescence density of 72h and vPAG in vGLUT1 and vGAT was significantly higher than that in other subgroups (P0.01). The number of TUNEL positive cells in vPAG group was significantly higher than that in control group (P0.01). At 72h time point after SE, vPAG and vGAT in the WB region of vGLUT1 in the standard group, 12h, 20d ratio and 30d were statistically significant compared with the control (P0.01), in addition, after SE, 24h, 72h and 10d time points, vPAG and vGAT in the area of the vGLUT1 standard in WB ratio was higher than the control group with significant difference (P0.01). VPAG area of damaged neurons in HE staining showed blebbing and deep staining oval cells, TUNEL positive cells showed brown or dark brown compact condensed nuclei. After 2 weeks of epileptic behavior video surveillance, the frequency, duration and severity of seizures in the lesioned group were significantly different from those in the control group (P0.01). Conclusion this study showed that the occurrence and development of epilepsy induced the dysfunction of vPAG and the change of structure, and the damage of vPAG structure also promoted the development of epilepsy.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1

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