【作者】 周扬；

【导师】 张惠； 武胜昔； 汪伟；

【作者基本信息】 第四军医大学 ， 麻醉学， 2009， 硕士

【摘要】 全身麻醉的发展已有160多年的历史,但全身麻醉的机理仍然不甚明了。尤其是全身麻醉的中枢机制,一直是麻醉学领域的研究重点和热点。近年来,异丙酚由于起效快、清除迅速和不良反应少等优点而被广泛应用于临床麻醉。但异丙酚作用于中枢神经系统的机制尚未完全阐明。离体和在体实验均表明异丙酚能增强GABA与GABAA受体的结合,增强抑制性突触后电流,从而达到麻醉的效果。但就其对突触前机制的研究甚少,异丙酚是否对GABA能神经传递的突触前神经元—GABA能神经元也有一定的作用将是本实验的重点。研究目的:观察异丙酚麻醉过程中谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠脑内Fos蛋白及GABA能神经元的活化情况,以了解突触前神经元在全麻机理中的可能作用,为阐明静脉麻醉药中枢神经系统的作用靶位及机制提供资料。研究方法:成年雄性GAD67-GFP基因敲入小鼠20只(第四军医大学基础部人体解剖学教研室提供)随机分为2组:C组(C Group):对照组,腹腔注射生理盐水;T组(T Group):实验组,又分为三个亚组T1(5 min),T2(30 min),T3(1 h)。采用腹腔注射异丙酚(130 mg/kg)麻醉,分别在注射后5 min,30 min,1 h进行行为学评分,按照评分标准打分,评价其麻醉状态。随后立即处死并取脑,制取脑薄片(片厚30μm),分套收集,应用免疫组织化学观察神经元活化标记物Fos在全脑的表达分布情况。用上述制备待用的脑薄片,进行GABA能神经元与Fos的免疫荧光双标染色。研究结果:1、行为学结果:在腹腔注射异丙酚后5 min、30min和1h时行为学评分分别为5 min时13分,30 min时14分,1 h时9分,表明5min和30min均能达到适度或深度麻醉效果,而1h时则处于过渡状态。2、对GAD67-GFP基因敲入小鼠脑内Fos蛋白表达的观察:与对照组相比,Fos在海马CA1、杏仁核(MeA)、丘脑室旁核(PV)、下丘脑室旁核(PaV)、齿状回(DG)、下丘脑腹内侧核(VMH)、中脑导水管周围灰质(PaG)的表达在三个时间点都有明显增加(P<0.05),在嗅球外丛状层(EPI)有表达但与对照组相比无明显差异(P>0.05)。3、对GAD67-GFP基因敲入小鼠脑内GABA和Fos蛋白共存表达的观察:腹腔注射异丙酚后5 min、30 min和1 h下丘脑腹内侧核有GABA与Fos共存,共存的细胞占该区域Fos阳性神经元分别为:T1:80.3%,T2:89.7%,T3:91.6%,C:24.9%。此外,在下丘脑室旁核也可见GABA与Fos共存,共存率为:T1:19.6%,T2:39.7%,T3:41.6%,C:8.6%。研究结论:静脉麻醉药异丙酚能诱导GAD67-GFP基因敲入小鼠脑内相关核团GABA神经元和Fos共存,并具有部位差异性。其中下丘脑腹内侧核GABA能神经元活化可能是异丙酚麻醉致意识消失的机制之一。更多还原

【Abstract】 General anesthesia has developed 160 years, but the mechanism underlying is still unknown, especially in central nervous system. The researches on mechanism of general anesthesia have always been the unode and focus.Poropfol has been used in induction and maintenance because of its pharmacological merits recent years. But the effect of poropfol on central nervous system is unknown. In vivo and ex vivo researches indicated that poropfol can enhance the binding of GABA and GABAA receptor. There are few research about the presynaptic mechanism. Whether poropfol has effect on presynaptic neurons is the focal point of this study.Objective: Emerging researches have focused on the participation ofγ-aminobutyric acid (GABA) neurons but there still lacks morphological evidence. To investigate the activation of GABAergic neurons during loss of consciousness induced by propofol in the GAD67-GFP knock-in mouse. Methods: 20 GAD67-GFP transgenic mice were used in the present study. The mice were grouped into two, control group and experimental group. Experimental group were grouped into three subsets, T1(5 min), T2(30 min), T3(1 h). Mice in experimental group were injected propofol i.p. 130 mg/kg, and saline was set as the negative control. Mice were graded by ethology while sacrificed at 5 min, 30 min and 1 h after injection, respectively. The whole brain was obtained and cut with a freezing microtome consecutively. Immunohistochemistry and double immuno-fluorescence were performed to observe the Fos expression and the coexistence of Fos and GABA in the whole brain.Results: 1 The scores of ethology were 13 at 5min, 14 at 30 min and 9 at 1h. It indicated that the mice were in the situation of moderate or deep anesthesia at 5 min and 30min, and various transition states at 1h. Expression of Fos protein increased remarkably in CA1, amygdaloid nucleus, thalamic paraventricular nucleus, hypothalamic paraventricular nucleus, hypothalamic ventromedialis nucleus, and periaqueductal gray compared with the control group at three time spots(p<0.05). 2 Neurons expressing Fos protein and GABA simultaneously could be observed in hypothalamic ventromedialis nucleus at 5 min, 30 min and 1 h, the proportion of which among Fos-positive neurons was 80.3%, 89.7% and 91.6%. The double staining neurons could also be seen in PaV, the proportion of which among Fos-positive neurons was T1:19.6%,T2:39.7%,T3:41.6%, C:8.6%。Conclusion: The activation of GABAergic neurons may be one of the mecha- nisms during loss of consciousness induced by propofol.更多还原