【作者】 王菲；

【导师】 程焱；

【作者基本信息】 天津医科大学 ， 神经病学， 2008， 硕士

【摘要】 目的本课题通过应用重复经颅磁刺激,从行为学、形态学和分子生物学等不同层面综合研究重复经颅磁刺激对血管性痴呆大鼠学习记忆的影响,探讨其作用的分子机制,为血管性痴呆的临床治疗提供有价值的实验依据和方法。方法将Wistar大鼠随机分为以下四组:①正常对照组、②痴呆模型组、③重复经颅磁刺激低频治疗组(简称低频刺激组)、④重复经颅磁刺激高频治疗组(简称高频刺激组)。痴呆模型组、低频刺激组和高频刺激组均采用两血管阻断法(2VO)制备血管性痴呆模型。造模前、造模后和重复经颅磁刺激治疗后均进行Morris水迷宫行为学测试,包括定位航行试验和空间探索试验,计算全时程平均逃避潜伏期和游泳路程,计算在原平台象限的游泳时间和在原平台象限游泳路程占总游泳路程的百分比。低频刺激组大鼠接受频率0.5Hz重复经颅磁刺激(六个疗程,每疗程持续5天),高频刺激组大鼠接受频率5Hz重复经颅磁刺激(六个疗程,每疗程持续5天)。各组大鼠断头取脑,应用免疫组织化学方法检测海马CA1区BDNF、NMDAR1、SYN蛋白表达情况。应用HE染色,光镜观察各组大鼠海马CA1区细胞形态。应用透射电镜观察各组大鼠海马CA1区突触形态、数量等超微结构。结果1.Morris水迷宫测试结果大鼠造模后2天进行水迷宫定位航行试验,痴呆模型组、低频刺激组、高频刺激组大鼠的全时程平均逃避潜伏期和游泳路程比正常对照组延长(p＜0.05),空间探索试验中在原平台象限的游泳时间和在原平台象限的游泳路程占总路程百分比比正常对照组缩短(p＜0.05)。重复经颅磁刺激治疗6个疗程后,低频刺激组和高频刺激组大鼠全时程平均逃避潜伏期和游泳路程比痴呆模型组缩短(p＜0.05),低频刺激组与高频刺激组差异无统计学意义(p＞0.05),空间探索试验中在原平台象限的游泳时间和在原平台象限的游泳路程占总路程百分比比痴呆模型组延长(p＜0.05),低频刺激组与高频刺激组差异无统计学意义(p＞0.05)。2.免疫组化染色结果:痴呆模型组大鼠BDNF、NMDAR1、SYN蛋白表达量比正常对照组大鼠减低(p＜0.05),低频刺激组和高频刺激组BDNF、NMDAR1、SYN蛋白表达量比痴呆模型组增高(p＜0.05),低频刺激组与高频刺激组差异无统计学意义(p＞0.05)。3.光镜观察显示,痴呆模型组大鼠海马CA1区神经细胞层次数量明显减少,排列松散,细胞受损严重,大量萎缩,轮廓不清,细胞深染,出现核固缩,细胞器结构不清。低频刺激组和高频刺激组多数细胞排列较整齐,细胞形态较完好,仅见少量核固缩。4.电镜下观察,痴呆模型组突触数量虽多于正常组,但突触体积明显减小,形态改变,为长形或不规则形;高倍观,突触前区变形,突触小泡减少并聚集,线粒体凝聚,突触后区体积减小,突触前后膜增厚,融合,突触间隙消失,失去其正常形态。低频刺激组和高频刺激组的部分突触结构近于正常,部分突触前后膜略见增厚,但突触间隙可辨认。结论1.本研究从行为学、形态学及分子生物学等不同水平,研究了血管性痴呆的特点,通过对血管性痴呆大鼠海马CA1区BDNF、NMDAR1、SYN等几个蛋白的表达情况综合分析,结果表明BDNF、NMDAR、SYN蛋白表达的增加有利于学习记忆功能的恢复。2.重复经颅磁刺激对血管性痴呆认知功能障碍有恢复治疗作用,主要表现为影响了突触可塑性,其可能的机制为:(1)rTMS对突触后可塑性的影响:rTMS促进BDNF等神经营养因子的表达,进而可通过增加NMDA受体通道的开放频率,特异性地增强突触后致密物上NMDA亚单位的磷酸化。NMDA受体被激活时,使钙离子通透性增加,进入细胞内。钙离子可进一步激活细胞内钙离子依赖的酶,从而引起一系列生化过程,并且促进了NMDA受体依赖的长时程增强的产生,改变突触的传递效能,促使学习记忆功能的增强。(2)rTMS对突触前可塑性的影响:rTMS使突触前区内的SYN表达增加,具有促进突触再建和增强,提高突触效能的作用,促进神经损伤修复和功能重塑,对于血管性痴呆大鼠的学习记忆有恢复作用。3.本研究为血管性痴呆的临床治疗提供了新的线索,为临床应用重复经颅磁刺激治疗血管性痴呆提供了有价值的理论依据,同时为血管性痴呆的药物治疗提供了可能的新的作用靶点。更多还原

【Abstract】 ObjectiveVascular dementia（VaD）is one of main types of senile dementia.While the definite pathogenesis is still not clear.Repetitive transcranial magnetic stimulation （rTMS）plays an important role in the recovery of cerebral ischemia.We investigated the effect of repetitive transcranial magnetic stimulation on the ability of learning and memory and studied its molecular mechanism.This will provide therapy foundation in clinical rTMS application on vascular dementia and help to discover more effective therapy of vascular dementia.MethodsTwo-vessel occlusion（2VO）was employed to make vascular dementia rat models. Morris water-maze test was performed to study the ability of learning and memory in each group of rats.The expression of BDNF,NMDAR1,Synaptophysin（SYN）in hippocampal CA1 region were determined by immunohistochemistry assay.The ultrastucture changes of the synapses were observed with electron microscopy.1.Animals36 male Wistar rats weighting 290±20g were randomly divided into control group, vascular dementia group,vascular dementia with low frequency stimulation group and vascular dementia with high frequency stimulation group.2.Vascular dementia rat modelTwo-vessel occlusion（2VO）method was used to make vascular dementia animal model.The rats were performed fasting while could drink 8-12h before operation. Apply abdominal anesthesia with 10%chloral hydrate（0.3ml/100g body weight）. Fixed the rats lying on the back,selected median incision on abdomen,and performed blunt dissection to expose double-side common carotid.The artery was occlusion by 4 type line.In the control group,rats were not performed 2VO operation.3.Repetitive transcranial magnetic stimulationLow frequency rTMS group rats were given 0.5 Hz rTMS for six weeks.High frequency rTMS group rats were given 5 Hz rTMS for six weeks. 4.Morris water maze testBefore Morris water maze test,a free swimming trail was run in which the platform was removed so that all rats were adapted to temperature and environment of Morris water maze.The rats that had poor ability of swimming were ticked out of test.During the place navigation test,a submerged platform was placed in the Morris water maze and rats were studied to learn the location of the platform that could be used to escape from swimming.The rats were placed in the maze in different starting positions that were equally distributed around the perimeter of the maze.The trail was lasted for 4 days.Record the escape latencies in each group.In the 5^thday,a spatial probe test was given which consisted of a 120 seconds free swimming period without the platform.Times of crossing the former site of the removed hidden platform were calculated statistically.5.Immunohistochemistry assayThe expression of BDNF,NMDAR1,SYN in hippocampal CA1 region were detected with immunohistochemistry assay.6.Statistical analysisUse SPSS 13.0 software for statistical disposition.All results were shown by mean±standard deviation.Differences between three groups were evaluated using one-way ANOVA,and differences between two groups were evaluated using Student’s t test for statistical analysis.P＜0.05 was considered as the level of significance.Results1.Morris water maze test resultsThere was significant difference among four groups.The escape latencies in VaD group were statistically longer than those in control group significantly（p＜0.05）,and the escape latencies in the low frequency rTMS group and in the high frequency rTMS group were shorter than those in VaD group significantly（P＜0.05）.The time and distance of crossing the former site of the removed hidden platform in VaD group were less than those in control group（P＜0.05）,and the time and distance of crossing the former site of the removed hidden platform in the low frequency rTMS group and in the high frequency rTMS group were longer than those in VaD group significantly （P＜0.05）.Low frequency rTMS group had no statistical difference with high frequency rTMS group（P＞0.05）.2.The expression of BDNF,NMDAR1,SYN in hippocampus CA1 regionThe average expression of BDNF,NMDAR1,SYN protein in VaD group was lower than that in control group（P＜0.05）.The average expression of BDNF, NMDAR1,SYN protein expression in low frequency rTMS group and in the high frequency rTMS group were higher than that in VaD group（P＜0.05）.Low frequency rTMS group had no statistical difference with high frequency rTMS group（P＞0.05）.3.The morphology of nerve cell in hippocampus CA1 regionThe morphology of nerve cell in hippocampus CA1 reginon in VaD group disordered and damaged obviously compared with control group.The damaged changes of nerve cell morphology not only in low frequency rTMS group but also in high frequency rTMS group improved compared with VaD group.There was no statistical difference with low frequency rTMS group and high frequency rTMS group.4.The ultra-micro structure changes of the synapsesThe structure of synapses in VaD group disordered and damaged obviously compared with control group.The damaged changes of synapses structure not only in low frequency rTMS group but also in high frequency rTMS group improved compared with VaD group.There was no statistical difference with low frequency rTMS group and high frequency rTMS group.ConclusionsVascular dementia rats have deficiency in the ability of spatial learning and memory,rTMS can improve the ability of spatial learning and memory in vascular dementia rats.Our research proved that rTMS could increase the expression of BDNF, NMDAR1 and SYN protein,and influence the plasticity of synapses,thus in the end it improve the function of behavior,learning and memory.更多还原