痴呆模型鼠脑海马突触素改变及其与学习记忆的关系

背景学习记忆与中枢神经系统突触的结构和功能的可塑性密切相关.目的探讨痴呆模型鼠海马的突触素改变及其与学习记忆的关系,为老年性痴呆的发病过程提供实验依据.设计随机对照实验.单位广州医学院人体解剖学教研室.材料实验于2003-02/2004-05在广州医学院人体解剖学教研室完成.选取成年SD大鼠20只,随机分为正常组和损伤组,10只/组.方法损伤组大鼠切断左侧穹窿海马伞,建立基底前脑-海马胆碱能通路受损的痴呆模型.造模4周后,用三等分Y型迷宫检测两组大鼠的学习记忆能力.大鼠学习成绩以连续10次测试中有9次达到正确反应(2 min以内逃避电击并第一时间到达安全区)时所需的训练次数来表示,记录每只大鼠达到学会标准所需的训练次数,训练次数少说明学习能力强.训练结束24 h后测试记忆能力,以10次训练中的正确反应次数代表记忆保持能力的优劣,正确反应次数少则记忆能力低.然后用免疫组织化学染色和图像分析技术等方法对大鼠海马突触素进行定量分析,以实际吸光度值进行比较,以避免染色过程中的非特异性染色导致的误差.主要观察指标①两组大鼠行为测试中学会逃避电击并第一时间到达安全区所需的训练次数.②两组大鼠海马结构突触素免疫反应物检测结果.③两组大鼠损伤侧海马CA1区和齿状回分子层突触素免疫反应物的实际吸光度值.结果实验纳入大鼠20只,全部进入结果分析.①两组大鼠学会逃避电击并第一时间到达安全区所需的训练次数损伤组学习能力达到标准所需的训练次数明显多于正常组(98.40±4.51),(62.21±2.43)次,P＜0.01;而记忆能力达到标准所需的训练次数则明显低于正常组(3.82±0.64),(8.81±0.32)次,P＜0.01.②两组大鼠海马结构突触素免疫反应物检测结果海马结构突触素免疫反应物呈板层分布,神经元胞体、胶质细胞、血管及白质不被染色.神经毡内免疫反应产物呈持异性颗粒状或点状.海马CA1区以多形层和辐射层染色较深,腔隙分子层次之,锥体层和颗粒层细胞的轮廓边缘有点状标记.齿状回染色较浅,突触素免疫反应物分布稀疏.③两组大鼠损伤侧海马CA1区和齿状回分子层突触素免疫反应物的实际吸光度值与正常组相比,损伤组海马CA1区多形层、辐射层、腔隙分子层和齿状回分子层均明显降(80.34±4.33,43.40±2.57;78.25±5.48,40.21±3.2230.01±4.05,12.37±1.78;60.50±3.80,26.63±2.36;P均＜0.01).经相关分析表明,大鼠穹窿海马损伤后学习记忆能力与海马CA1区多形层、辐射层、腔隙分子层和齿状回分子层突触素免疫反应物的实际吸光度值呈显著正相关(多形层r=0.739,P＜0.01;辐射层r=0.624,P＜0.05;腔隙分子层r=0.892,P＜0.01;齿状回分子层r=0.853,P＜0.01).结论痴呆模型鼠损伤侧海马CA1区多形层、辐射层、腔隙分子层和齿状回分子层突触素免疫反应物的吸光度明显下降,并与其学习记忆能力呈显著正相关.提示海马突触素含量减少与学习记忆能力存在密切关系.

Changes of hippocampal synaptophysin in rat model of dementia and the relationship of the changes with learning and memory

BACKGROUND: Learning and memory are closely relevant to the plasticity of structure and function of synapse in central nervous system.OBJECTIVE: To investigate the changes of hippocampal synaptophysin in rat model of dementia and the relationship of the changes with learning and memory so as to provide experimental evidences for the pathogenic course of senile dementia.DESIGN: Randomized controlled trial.SETTING: Teaching & Research Section of Anthropotomy, Guangzhou Medical College.MATERIALS: The experiment was completed in Teaching & Research Section of Anthropotomy of Guangzhou Medical College from February 2003 to May 2004. A total of 20 adult SD rats were selected and randomly divided into normal group and injury group with 10 in each group.METHODS: Left fimbria-fornix transection was performed on rats in injury group in order to establish rat models of dementia with fundus forebrain-damaged hippocampal cholinergic pathway. Four weeks later,detection of learning and memory ability was carried out on rats in each group with trisection Y-maze. Learning results was shown by training frequency of 9 out of 10 times of continuously correct responses (escaping to safe area after electric shock at once in 2 minutes). Frequencies.of responses required reaching the standard were recorded. Low frequency indicated good learning ability. The detection was repeated after 24 hours to observe memory. Memory was shown by correct response rate out of 10 times of training. Low rate indicated poor memory. After that,quantitative analysis was made on hippocampal synaptophysin of rats with the method of immunohistochemical staining and image analysis technique. Comparison of the actual absorbance value was conducted in order to eliminate the errors caused by non-specific staining during the process of staining.detected results of immnoreactant of hippocampal synaptophysin of rats in physin in injured side of hippocampal CA1 and dentate gyrus molecular layer of rats in each group.RESULTS: All the 20 rats in the experiment were admitted in result electric shock at once: Training frequencies of learning required for reaching the standard in injury group were significant higher than those in normal group (98.40±4.51) and (62.21±2.43) times, P ＜ 0.01; training frequencies of memory required for reaching the standard is significantly lower than those in normal group (3.82±0.64) and (8.81±0.32) times, P ＜ 0.01.rats: Immunoreactant of hippocampal snaptophysin was distributed in lamellar pattern while neuron bodies, glial cells, vessels and white matters could not be stained. Immunoreactant within nervous felt was presented in specific granular or spot shape. In hippocampal CA1, polymorphic layer and radiant layer were stained darker than the other parts, the staining of lacunar molecular layer was second to them in darkness, and spot markers could be found on the edges of cells in pyramidal layer and granular layer.Dentate gyrus was stained lighter than the other parts, and immunoreactant immunoreactant of synaptophysin in injured side of hippocampal CA1 and dentate gyrus molecular layer of rats in each group: Compared with normal group, the value significantly decreased in polymorphic layer, radiant layer,lacunar molecular layer and dentate gyrus molecular layer of hippocampal CA1 in injury group (80.34±4.33, 43.40±2.57; 78.25±5.48, 40.21±3.22;30.01±4.05, 12.37±1.78; 60.50±3.80, 26.63±2.36; P ＜ 0.01). As shown by correlative analysis, learning of rats with fimbria-fornix injury was in a significant positive correlation with actual absorbance value of synaptophysin in polymorphic layer, radiant layer, lacunar molecular layer and dentate molecular layer of hippocampal CA1 (polymorphic layer: r=0.739,P ＜ 0.01; radiant layer:. r=0.624, P ＜ 0.05; lacunar molecular layer:. r-=0.892,P ＜ 0.01; dentate molecular layer: r=0.853, P ＜ 0.01).CONCLUSION: The absorbance of immunoreactant of synaptophysin in polymorphic layer, radiant layer, lacunar molecular layer and dentate gyrus molecular layer of injured side of hippocampal CA1 of rat model of dementia decreases remarkably. The absorbance is in a significant positive correlation with learning and memory ability. It indicates that the decrease of hippocampal synaptophysin is closely correlated with learning and memory ability.