D-半乳糖对大鼠空间学习记忆行为与脑海马结构电生理以及突触形态学的影响(英文)

Influence of D-galactose on rat spatial learning memory behavior as well as cerebral hippocampal electrophysiology and synaptic morphology

背景:学习记忆障碍是衰老的主要表现之一,D-半乳糖致衰老模型是近年来常用的动物模型,长期D-半乳糖处理可引起动物神经细胞形态学的改变。目的:对D-半乳糖引起拟衰老改变时大鼠的空间学习记忆行为进行观察,并结合其体内诱导海马齿状回长时程增强以及海马CA3区突触形态学的变化予以探讨。设计:随机对照观察。单位:上海第二医科大学解剖学教研室,上海中医药大学生理学教研室。材料:实验于2000-08/2001-04在上海中医药大学生理学教研室完成。选取3月龄雄性Wistar大鼠22只,随机分正常组和D-半乳糖组,11只/组。D-半乳糖(上海化学试剂二厂),Morris水迷宫(上海中医药大学老年所提供,自制)。方法:正常组每天皮下注射生理盐水1mL,D-半乳糖组每天皮下注射D-半乳糖800mg/kg,连续给药6周。大鼠空间学习记忆行为以Morris水迷宫潜伏期作为判定标准;应用体内记录单脉冲刺激穿通纤维在海马齿状回诱发的群体电位,测量高频刺激前后单脉冲刺激诱发的电位幅值变化,将高频刺激前的记录作为基线值,进行组间比较;应用透射电镜结合图像分析对大鼠海马CA3区突触形态结构进行观察。水迷宫潜伏期采用重复测量设计的方差分析,长时程增强各时段群体电位峰值潜伏期的组间差异采用t检验,长时程增强的诱导率用χ2检验,用XY-540型生物图像处理系统对电镜突触照片进行测量,对所得数据进行t检验。主要观察指标:①主要结局:Morris水迷宫潜伏期测试成绩以及长时程增强诱导率、群体电位的变化。②次要结局:海马CA3区突触形态结构的变化。结果:实验纳入大鼠22只,水迷宫测试中无脱落,其后在电生理实验中正常组和D-半乳糖组各有1只死亡,最后每组随机抽取3只用于电镜观察。①两组Morris水迷宫潜伏期成绩的比较:与正常组比较,D-半乳糖组寻找水下平台潜伏期明显延长[(14.77±10.10),(51.36±12.45)s,P<0.05]。②高频刺激前正常组与D-半乳糖组海马齿状回诱发电位的比较:两组群体电位幅值及群体电位潜伏期均无明显差异[(1.05±0.47),(0.91±0.41)mV;(5.46±2.09),(5.38±2.26)ms;P>0.05]。③两组齿状回长时程增强诱导率比较:与正常组比较,高频刺激后D-半乳糖组明显降低(80%,20%,χ2=7.20,P<0.01)。④两组高频刺激后不同时间段的群体电位比值的比较:与正常组比较,D-半乳糖组于高频刺激后20,30,60min均明显降低(1.104±0.196,0.919±0.162;1.354±0.212,0.999±0.219;1.236±0.174,0.875±0.311;P<0.05)。⑤两组大鼠海马CA3区突触结构各参数的比较:与正常组比较,D-半乳糖组海马CA3区突触后致密物厚度变薄[(40.60±18.26),(26.35±8.15)nm,P<0.05],突触间隙宽度增大[(17.69±6.28),(26.95±5.67)nm,P<0.05],突触活性区长度缩短[(265.13±76.50),(229.13±90.68)nm,P<0.05]。结论:皮下注射D-半乳糖可损害大鼠的空间学习记忆能力,降低大鼠在体海马齿状回长时程增强诱导率,使长时程增强增幅降低,并可显著影响大鼠脑海马CA3区的突触超微结构。提示D-半乳糖对大鼠海马齿状回长时程增强诱导的抑制和对CA3区突触形态结构的影响,是其导致空间学习记忆行为障碍的基础。

BACKGROUND： Learning memory disorder is one of the major manifestations of aging. The model of aging induced by D-galactose is a commonly used animal model in recent years, and long-term D-galactose exposure may cause nerve cell morphological changes in animals. OBJETCIVE： To observe spatial learning memory behavior during D-galactose-induced aging process in order to further explore in vivo evoked long-term potentiation in hippocampus dentate gyms and synaptic morphological changes in hippocampal CA3 region. DESIGN： Randomized controlled observation. SETTING： Anatomical Teaching and Research Sectile, Shanghai Second Medical University; Department of Physiology, Shanghai Traditional Chinese Medicine University. MATERIALS： The experiment was carried out at the Physiological Laboratory of Shanghai Traditional Chinese Medicine University between August 2000 and April 2001. Totally 22 male Wistar rats of 3-month birth age were included and randomized into normal group and D-galactose group with 11 rats in each group. D-galactose was produced by Shanghai No. 2 Chemical Reagent Factory, Morris water maze was home-made by the Institute of Geriatrics, Shanghai Traditional Chinese Medicine university. METHODS： Rats were subjected to hypodermic injection of 1 mL normal saline every day in normal group, or D-galactose of 800 mg/kg daily for 6 consecutive weeks in D-galactose group. Rat spatial learning memory behavior was assessed by the latency of Morris water maze; hippocampal dentate gyrus community potentials evoked by monopulse stimulation on perforating fibers were recorded in vivo; meanwhile, the amplitude of monopulse evoked potentials was determined before and after high frequency stimulation, with the amplitude before high frequency stimulation taken as baseline. Transmission electromicroscope was applied in combination with imaging analysis to observe synaptic morphology and structure in rat hippocampal CA3 region. Water labyrinth latency was compared using the variance analysis of repetitive survey design, t-test was used to compare the differences of peak potential latency of community potentials at various time points after long-term potentiation. Moreover, inducing rate of long-term potentiation was compared by x^2-test, XY-540 type biological imaging processing system was used to analyze electromicroscopic pictures, and all available data were analyzed with t-test. MAIN OUTCOME MEASURES： ① Main outcomes： Changes of Morris water maze latency, as well as inducing rate of long-term potentiation and community potentials. ② Secondary outcomes： Synaptic morphological and structural changes in hippocampal CA3 region. RESULTS： Totally 22 rats were enrolled in this study, with no one lost during water labyrinth test, but one rat in both normal group and D-galactose group died during electrophysiological experiment. Finally 3 rats were randomly selected from each group for electromicroscopic observation. ① Comparison of the latency for Morris water maze： In contrast with that of normal group, latency for seeking submarine platform was obviously prolonged in D-galactose group [（14.77±10.10）, （51.36±12.45） s, P 〈 0.05]. ② Comparison of evoked potential in hippocampus dentate before high frequency stimulation： The two groups did not obviously differ in community potential amplitude and community potential latency [（1.05±0.47）, （0.91±0.41） mV; （5.46±2.09）, （5.38±2.26） ms; P 〉 0.05]. ③ Inducing rate of long-term potentiation in hippocampal dentate gyms： Compared to that of normal group, inducing rate in D-galactose group obviously reduced after high frequency stimulation （80%, 20%, x^2=7.20, P 〈 0.01）. ④ Comparison of community potential ratio at different time points after high frequency stimulation： Compared to that of normal group, it was notably reduced in D-galactose group at post-stimulation 20, 30, 60 minutes, respectively （1.104±0.196, 0.919±0.162; 1.354±0.212, 0.999±0.219; 1.236±0.174, 0.875±0.311; P 〈 0.05）. ⑤ Comparison of synaptic structural parameters in hippocampal CA3 region： Compared to that of normal group, postsynaptic dense bodies became thickened in hippocampal CA3 region of D-galactose group [（40.60±18.26）, （26.35±8.15） nm, P 〈 0.05], the synapse gap increased [（17.69±6.28）, （26.95±5.67） nm, P 〈 0.05] while synaptic active zone was shortened [（265.13±76.50）, （229.13±90.68） nm, P 〈 0.05]. CONCLUSION： Hypodermic injection of D-galactose does harm to rat spatial learning memory by reducing the long-term potentiation, inducing rate in rat hippocampal dentate gyms, attenuating the increase of long-term potentiation-evoked potential amplitude, and even remarkably changing the synaptic ultrastructure in rat hippocampal CA3 region. It suggests that D- galactose inhibits the long-term potentiation at hippocampal dentate gyms and affects synaptic structure in hippocampal CA3 region, which is consid- ered as the basis of spatial learning memory behavioral disorder.