Experimental study - The role of mint1, a novel synaptic protein, following epileptic seizures in mice

目的：通过观察脑内薄荷糖的变化，了解癫痫发作后突触前功能的变化。方法：采用成年雄性Sprague Dawley鼠，体重200 ~ 250 g。所有动物均采用5 mg/kg甲基溴阿托品腹腔注射预处理。腹腔注射盐酸匹罗卡品320 ~ 350 mg/kg引起癫痫发作。监测癫痫发作活动，在抽搐状态癫痫发作3-4小时后，用10mg /kg地西泮终止癫痫发作。对照组大鼠只接受生理盐水治疗。只有在匹罗卡品治疗后表现出持续抽搐发作活动的动物才被用于以下实验。4 %多聚甲醛溶液于癫痫发作6周后2、4、6小时、1、2、3、4、7、14天灌注固定（每组n=2-3）。用振动原子仪对固定脑进行连续冠状切片（厚度为20 μm），并对mint1， munc18， syp2进行免疫细胞化学。以兔抗大鼠mint1、munc18、syp2多克隆抗体为一抗进行免疫染色，ABC法进行免疫染色。与相应的一抗反应后，使用Vectastain ABC Elite Kit进行免疫检测，然后使用0.05%二氨基联苯胺进行反应产物可视化。结果：在对照大鼠（n=3）海马中，邻内嗅皮层传入纤维和齿状回分子层颗粒细胞层苔藓纤维中均可见mint1的免疫反应性。海马组织中munc18的免疫反应性均匀染色。Syp2出现在苔藓纤维和齿状回中，在苔藓纤维中，mint1的免疫反应性在癫痫损伤后2 ~ 7天增加。另一方面，正常小鼠和癫痫小鼠在该区域对munc18和syp2的免疫反应性无显著差异。在齿状回中，mint1、munc18和syp2的免疫反应性在分子层受到极大的抑制。在第14天，它们显著分布于分子细胞层的内层。这些免疫反应性持续到初始癫痫状态后6周。评论：本研究表明，CA1锥体神经元的兴奋性神经元的短暂性突触前功能障碍从mint1开始延长，而苔藓纤维中的munc18和syp2标记分布直到7天。这表明mint1在癫痫发作后从内嗅皮层到海马的神经传递中起重要作用。少

Purpose : The aim of present study is to clarify the presynaptic function following epileptic seizures by estimating the change of mints in the brain.Methods : Adult male Sprague Dawley rate weighing 200-250 g were used. All animals were pretreatment with injections of atropine methylbromide 5 mg/kg intraperitoneally. Epileptic seizures were produced by intraperitoneal injection of pilocarpine hydrochloride 320-350 mg/kg. Seizure activity was monitored behaviorally, and after 3-4 hours of convulsive status epileptics, seizures were terminated with diazepam 10 mg/kg. Control rats were received saline only. Only animals that displayed continuous, convulsive seizure activity after pilocarpine treatment were used in the following experiments. Animals were perfusion-fixed with 4 % paraformaldehyde solution 2, 4, 6 hr, 1, 2, 3, 4, 7, 14 day 6 weeks after epileptic seizures (n=2-3 in each group). Serial coronal sections (20 μm in thickness) were obtained from the fixed brains with a vibratome … More and then subjected to immunocytochemistry for mint1, munc18, syp2. Immunostaining was performed with rabbit polyclonal antibody against rat mint1, munc18, syp2 as a primary antibody, followed by the ABC method. After reaction with respective primary antibodies, immunodetection was performed using the Vectastain ABC Elite Kit followed by reaction product visualization using 0.05 % diaminobenzidine.Results : In control rats (n=3) hippocampus, the immunoreactivity of mint1 was stained in the afferent fiber from the adjacent entorhinal cortex and mossy fiber from granule cell layer, in the molecular layer of the dentate gyres. The immunoreactivity of munc18 was uniformly stained in the hippocampus. Syp2 revealed in the mossy fiber and dentate gyros, In mossy fiber, the immunoreactivity of mint1 increased from 2 to 7 days after epileptic insult. On the other hand, there was no significant different of the immunoreactivity for munc18 and syp2 between control and epileptic mice in this area. In the dentate gyres, the immunoreactivity of mint1, munc18 and syp2 were greatly strained in the molecular layer. At the day 14, they marked distributed in the inner layer of molecular cell layer. These immunoreactivity continued until 6 weeks after initial epileptic status.Comments : The present study demonstrated that transient presynaptic dysfunction in the excitatory neurons to CA1 pyramidal neurons be prolonged since mint1, but not munc18 nor syp2 in the mossy fiber marked distributed until 7 days. These suggested that mint1 played the important role of neurotransmission from the entorhinal cortex to hippocampus following epileptic seizures. Less