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黄体酮对戊四唑致痫大鼠海马内GABA和MAP2影响的形态学研究

Effects of Progesterone on GABA and MAP2 in the Hippocampus of PTZ-induced Epileptic Rats

【作者】 于胜波

【导师】 隋鸿锦;

【作者基本信息】 大连医科大学 , 人体解剖与组织胚胎学, 2003, 硕士

【摘要】 动物实验和临床研究表明,黄体酮(progesterone,P)具有对抗癫痫发作的作用,但其机制尚不完全清楚。近年来,越来越多的研究表明,P对癫痫活动的影响是通过影响神经元的兴奋性来实现的。有学者认为,边缘脑区-特别是海马内存在性类固醇激素的结合位点,是孕激素作用的靶部位之一,海马是癫痫活动产生和散播的关键性脑区,其内部回路兴奋性的改变与癫痫活动密切相关。已有研究表明,孕激素对海马结构存在有结构和功能上的影响。本实验研究了P对戊四唑(pentylenetetrazol,PTZ)致痫的发作情况和海马神经元的影响,在形态学上探讨孕激素抗癫痫的可能机制。实验中采用成年去卵巢(ovariectomized,OVX)雌性SD大鼠,随机分成3组,即空白对照组(OVX+NS+NS)、实验对照组(OVX+NS+PTZ)和实验给药组(OVX+P+PTZ)。OVX+NS+NS、OVX+NS+PTZ组腹腔注射NS,OVX+P+PTZ组腹腔注射P,每天一次,三天后OVX+NS+PTZ组和OVX+P+PTZ组腹腔注射PTZ致痫,观察并记录行为学改变,NS+NS组注射NS。取背侧海马部位最大的脑片,进行免疫组化实验。光镜观察并计算机辅助分析海马齿状回门区内γ-氨基丁酸(GABA)免疫阳性反应细胞数、齿状回和CA1区内微管相关蛋白2(MAP2)免疫反应的变化。结果:(1)行为学观察:OVX+P+PTZ组大鼠均未出现癫痫发作,而OVX+NS+PTZ组大鼠均出现了Ⅳ级以上发作。表明P能对抗PTZ的致痫作用,进一步证实了孕激素具有抗癫痫作用。(2)GABA免疫反应的变化:OVX+NS+NS、OVX+NS+PTZ和OVX+P+PTZ三组间齿状回门区内GABA免疫阳性细胞数存在显著差别(P<0.01)。与OVX+NS+NS组(25.2±4.52)比较,NS+PTZ组的GABA免疫阳性细胞数(18.7±2.29)明显减少(P<0.05),而P+PTZ组的GABA免疫阳性细胞数(31.3±5.96)明显增加(P<0.05)。这说明P可对抗PTZ致痫所致的门区GABA能中间神经元的损伤,并提示,P具有上调GABA能系统的作用。(3)MAP2免疫反应的变化:在光镜下观察发现,齿状回分子层、门区和CAI区内OVX:+ NS+NS、OVX王NS+PTZ、OVX+P+P TZ三组间M冉卫2免疫反应存在差别。与OVX+NS+NS组比较,OVX+NS+P TZ组MAPZ免疫阳性反应减弱,而OVX+P+P TZ组则增强。结果表明,P可上调神经元内MAPZ的免疫活性,因而MAPZ可能参与了抗瘫痛机制。

【Abstract】 Laboratory and clinical studies suggested that progesterone reduced epileptic seizure activities. The mechanisms underlying this effect are not known. But there are increasing clinical and experimental evidences, which showed that hormones, in particular sex steroid hormones, influence neuronal excitability and other brain functions. Furthermore, limbic regions, especially hippocampus where is the target region of progesterone exists binding sites for sex steroids. Hippocampus is a key brain structure in the generation and propagation of seizure activities. The excitability of hippacampal circuit is associated with seizure activities. There are some experimental data .which showed that progesterone might affect structure and function of the hippocampus. The present study determined the effects of progesterone on pentylenetetrazol(PTZ)-induced seizures and neurons of the hippocampus in ovariectomized femal rats and helped to elucidate the mechanisms of progesterone against epilepsy seizure susceptibility.Ovariectomized (OVX) adult female Sprague-Dayley rats were divided randomly into three groups. Two control groups were designed. One group of OVX rats were injected matched doses of vehicle, normal saline(NS), intraperitoneally(i.p.) ( OVX+NS+NS group). The second one received matched doses vehicle given 0.5 h prior to PTZ treatment(OVX+NS+PTZ group). The progesterone-treated group of OVX rats were injected progesterone once per day for three days given 0.5 h prior to PTZ treatment(OVX+P+PTZ group). Convulsive responses were scored. Slices(40 μ m thick) were cut coronally using a vibratome. The slices with largest dorsal hippocampus were used for immunohistochemistry of γ -aminobutyric acid (GABA) and microtubule associated protein 2 (MAP2), respectively. Results of immunohistochemistry were analyzed by light microscope and computer analysis system.Pretreatment with the progesterone completely protected OVX females against PTZ-induced seizures, while OVX+NS+PTZ group of rats all exhibited stage 4 or 5 seizures. This suggested that P treatment significantly raised the thresholds of PTZ-induced seizures and had a significant anticonvulsive effect in adult female rats. GABA immunohistochemistry showed that the number of GABA positive neurons were significantly decreased in Hilus of NS+PTZ group as compared with OVX+NS+NS group(P<0.05). However, in OVX+P+PTZ group , the number of GABA positive neurons were significantly increased than those of OVX+NS+NS group(P<0.05) and OVX+NS+PTZ group(P<0.01). MAP2 immunohistochemistry showed that MAP-IR were decreased in Hilus, molecular layer of dendate gyrus and CA1 of OVX+NS+PTZ group as compared with NS+NS group. However, compared with those of NS+NS group and NS+PTZ group, MAP-IR were increased in OVX+P+PTZ group. These results supplied evidences that P might enhance MAP2 . This might be one of mechanisms of P’s anticonvulsant effects and protective action.

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