【作者】 张晓抒；

【导师】 杨运宽；

【作者基本信息】 成都中医药大学 ， 针灸推拿学， 2013， 博士

【摘要】 目的：研究针刺对阿尔茨海默病模型(快速老化小鼠)SAMP8的海马长时程增强(LTP)的影响,肯定针刺对AD模型海马学习记忆能力影响的电生理效应,同时观察海马神经元超微结构和钙调蛋白酶的变化,揭示针刺介导AD神经康复的相关机制。方法：1.确定不同品系小鼠LTP实验定位特点；2.建立了针刺实验条件下正常Balb/c小鼠模型,Balb/c小鼠体重20-30g。针刺“百会”、“涌泉”穴,每闩一次,7天一疗程,疗程间间隔2天。根据疗程将动物随机分为空白2周组、对照2周组、针刺2周组以及空白4周组、对照4周组、针刺4周组。治疗结束后,即刻将实验动物麻醉,观察针刺对正常Balb/c小鼠的LTP影响；3.选择9月龄SAMP8小鼠为AD动物模型,随机分为模型对照P8组和针刺P8组。雄性同源同月龄SAMR1小鼠为正常对照R1组。同样的针刺方法结束后,对小鼠海马进行在体LTP测试。以透射电镜观察动物海马CA1区神经元突触界面超微结构突触后致密带(post synaptic density,PSD)厚度、突触间隙宽度、突触界面曲率变化。以免疫组织化学的方法检测海马p-CaMK Ⅱ表达。结果：1.确定了不同品系小鼠LTP的电极定位调整方法和不同品系小鼠LTP实验的电极定位坐标特点；2.正常Balb/c小鼠不同疗程组的LTP结果：LTP引发率以对照4周组最低；空白2周组、对照2周组、针刺2周组三组之间的LTP的群峰电位(PS)和潜伏期幅度变化没有显著性差异：空白4周组、对照4周组与针刺4周组组间比较PS增幅有显著性差异(p<0.05),潜伏期没有差异；空白4周组和对照4周组的陌增幅比较有显著性差异(p<0.05),潜伏期降幅没有差异；窄自4周组和针刺4周组的Ps增幅和潜伏期没有显著性差异；对照4周组和针刺4周组的PS增幅变化有娃著性謦肄(p<0.05)：3. SAMP8与SAMR1的LTP结果：LTP引发率比较对照P8组和针刺P8组尢差异,正常Rl组较低；正常对照Rl组、模型对照p8组与针刺P8组组间比较PS增幅与潜伏期降幅有显著性差异(p<0.05);正常对照Rl针刺P8组比较,PS增幅和潜伏期降幅均有显著性差异(p<0.05)：模型对照P8组与针刺P8组比较,潜伏期降幅均有显著差异(P<0.05)；正常对照的Rl与模型对照P8组比较,PS增幅与潜伏期变化均无显著性差异；4.4疗程后,与正常对照Rl组比较,模型对照P8组小鼠海马CAl区神经元PSD变薄(p<0.05)；突触间隙增宽(p<0.05)：突触界面曲率下降(p<0.05)。与模型对照P8组比较,针刺P8组PSD增厚(p<0.05)；突触间隙宽度下降(p<0.05)：突触界面曲率增大(p<0.05);5.4疗程后,正常对照Rl组、模型对照P8组、针刺P8组的海马p-CaMK Ⅱ总量及亚区含量上匀未见差异(p>0.05);结论：1.4周的针刺束缚实验条件可能对正常小鼠的学习记忆能力产生非良性作用,针刺可以拮抗由不良刺激造成的学习记忆能力损伤；2.针刺可以提高SAMP8小鼠的LTP,提示增强突触可塑性刺改善SAMP8小鼠的学习记忆能力的机制之一;3.通过改善海马神经元的结构参数,进而提高神经元突触兴奋性传递功能,最终提高学习记忆能力,是针刺实现AD神经康复的机制之一。更多还原

【Abstract】 Objective:We chose senescence-accelerated mouse prone8(SAMP8) as Alzheimer’s Disease model and tested the hippocampal long-term potentiation (LTP) of these mice after a fixed period of acupuncture treatment.LTP result is to comfirm the electric physiological effects and active influences on learning and memory ability.At the same time, the changes of ultra microstructure and calmodulin in hippocampal nerve cells can reveal the mechanism of AD nerve recovery gained by acupuncture treatment.Subjects and Experimental Paradigm:1.Defining the specific locations of electrode needle for different kinds of mice;2.The influences on LTP of normal mice under the different course of acupuncture treatment is observed to estimate our experimental conditions.The Balb/c mice were chosen, weighed20to30grams.Acupuncture treatment included two points "Baihui acupoint (GV20)" and "Yongquan acupoint(KL1)" and puncturing once per day.The subjects differed by different courses.They are2-week with no measures,2-week with holding,2-week with acupuncture,4-week with no measures,4-week with holding, and4-week with acupuncture.The mice was anaesthetized after treatment and places on stereotaxic apparatus to test LTP;3. SAMP8mice aged9months were chosen as AD animal model.They were randomly divided into two groups, control group and acupuncture group.The same aged male homologous SAMR1mice were chosen as the normal group.The LTP test was applied after4weeks treatment.Transmission electron microscope combining with stereological method was used to observe and measure the synaptic ultrastructure in hippocampal neurons of CA1subregion.Western blot were used for the phosphorylation CaMK Ⅱ. Results:1.We fixed the specific locations of electrode needle for four kinds of mice;2.The analysis of LTP results in Blab/c mice demonstrate that (1)There exists no difference in PS and Latency between2-week groups;(2) there is a significant difference between4-week groups in terms of PS growth-rate(P<0.05);(3) There is a significant difference between4-week normal group and4-week control group in PS(P<0.05);(4)There is a significant difference between4-week control group and4-week acupuncture group in PS(P<0.05);(5) There exists no difference in Latency among three4-week groups;3.The comparison of LTP between SAMP8and SAMR1groups shows that:There are significant differences among three groups both in PS and Latency (P<0.05).The P8acupuncture group are better than R1normal group both in PS increases and Latency declines (P<0.05). The result of Latency declines of SAMP8acupuncture group is significantly higher than SAMP8control group (P<0.05). P8control group has no significant difference compared with R1normal group in PS and Latency;4. In comparison to the control group, the thickness of PSD (P<0.05) and the curvature of synaptic interface (P<0.05) decreased, the width of synaptic space increased (P<0.05) in hippocampal neurons of CA1area in the model group, while the thickness of PSD(P<0.05) and the curvature of synaptic interface(P<0.05) was increased, the width of synaptic space decreased (P<0.05)5.There were no significant differences in the total quantity of phosphorylated CaMK Ⅱ in hippocampus among the three groups (P>0.05)Conclusion:Our data reveal that 1. There might be a correlation between LTP of normal mice and the bound under the condition of acupuncture.But acupuncture treatment has rivalry effect on deficiency of memory and study ability.2. The acupuncture treatment can improve the memory and study ability of AD mice by changes related with synaptic plasticity.3. The influences from acupuncture might attribute to the improvements in nerve structures which also means the more active synaptic transmission, which implied the mechanism that acupuncture therapy can contribute to nerve recovery in AD diseases.更多还原