针刺足三里对MID模型大鼠海马cAMP、cGMP水平的影响

【作者】 宣雅波；

【导师】 张露芬；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 硕士

【摘要】 目的：采用栓子注入法制备多发梗塞性痴呆(MID)大鼠模型,观察针刺对MID大鼠行为学的影响,以及针刺对MID大鼠海马cAMP.cGMP水平的影响,从而探讨针刺改善学习记忆功能、治疗血管性痴呆方面可能的作用机制。方法：1.制备多发梗塞性痴呆(MID)大鼠模型：采用栓子注入法制备MID大鼠模型。取同种大鼠颈总动脉采血,制备直径100-200μm的血凝块备用。应用时每10mg血凝块加0.3ml生理盐水制成混悬液。儿科4.5号头皮针针头逆行穿刺进右颈外动脉,缓慢注入栓子盐水混悬液0.3ml,使栓子通过颈内动脉进入颅内至大脑各动脉,造成多灶性脑梗塞。假手术组手术方法同模型制作,只在注射时以生理盐水0.3ml代替栓子溶液。实验分为正常组、假手术组、模型组,其中模型组又分为模型对照组、模型针刺组、和模型非穴组。2.针刺处理：造模后第8天针刺。针刺组取双侧足三里穴行捻转补法各30s。非穴组取双侧肋下一个固定非穴点(肋下髂嵴上10mm),作为对照刺激点,用平补平泻捻转手法各30s。针刺组、非穴组于模型筛选后开始针刺,每日1次,治疗6d,休息1d,2周共治疗12次。模型组、假手术组和正常组进行与针刺组和非穴组相同时间、相同程度的捉抓刺激。3. Morris水迷宫隐蔽平台实验：实验在针刺结束后进行。实验前1d,先让动物在不含平台的水池中自由游泳2min,使其熟悉迷宫环境。实验共3d,实验时平台位置固定不变,平台中点离池壁40cm。以非平台象限中点的任一点作为入水点,记录大鼠从入水至找到平台的时间(逃避潜伏期),每天在3个入水点各训练1次。以3次潜伏期的算术均值作为这一天的成绩进行统计分析。4.采用放免法检测大鼠海马cAMP cGMP含量:Morris水迷宫隐蔽平台实验结束后检测各组大鼠海马cAMP、cGMP含量。结果：(1)游泳速度：各组内大鼠游泳速度无显著性差异(F=4.126,P>0.05)。各组间无显著性差异(F=0.854,P>0.05),各组与游泳速度之间不存在交互作用(F=1.424,P>0.05)。(2)逃避潜伏期：各组内大鼠逃避潜伏期存在显著性差异(F=6.846,P<0.05)。各组间存在显著性差异(F=12.990,P<0.05),各组与逃避潜伏期之间不存在交互作用(F=0.231,P>0.05)。与正常组比较,模型组逃避潜伏期显著延长(P<0.05)。与模型组比较,针刺组和非穴组逃避潜伏期显著缩短(P<0.05),但两组之间未见明显差异(P>0.05)。(3)海马cAMP、cGMP含量变化：与正常组比较,模型组cAMP含量明显下降(p<0.01)。与模型组比较,针刺组cAMP含量升高(p<0.01)。cGMP含量未见明显变化(P>0.05)。结论：1、本实验采用栓子注入法制备大鼠多发梗塞性痴呆(MID)模型,造模后第2d观察动物的活动情况,进行Bederson神经学分级评分：结果显示,造模后大鼠症状体征符合MID的临床特征及病理改变,模型制备成功。2、针刺可改善多发梗塞性痴呆大鼠的空间参考记忆。可能的机制是通过调控cAMP-PKA-CREB信号通路,进而达到改善大鼠学习记忆功能的作用。针刺穴位和针刺非穴之间未见明显差异。3、针刺穴位可调节中枢水平cAMP、cGMP的失平衡状态,其作用机制可能与以下途径有关,激活cAMP-PKA-CREB信号通路,改善脑血管扩张受抑及脑血管持续痉挛状态,减轻大鼠海马神经细胞的损害。综合上述得出结论,针刺可以调节多发梗塞性痴呆大鼠海马内的cAMP、 cGMP失平衡状态,并改善多发梗塞性痴呆大鼠空间参考记忆。其机制可能与调控cAMP-PKA-CREB信号通路有关。更多还原

【Abstract】 Objective:The multi-infarct dementia model was made by injection of emboli into the internal carotid artery. To observe the behavioral changes of MID rats, and hippocampus cAMP, cGMP content after acupuncture.Methods:1. preparation for multi-infarct dementia (MID) rat model:The multi-infarct dementia model was made by injection of emboli into the internal carotid artery. Take the same rats carotid arterial blood sampling, preparation diameter100-200μm blood clots. When application, every10mg blood clots with0.3ml normal saline made into suspension fluid. No4.5pediatric scalp needle, retrograde puncture right external carotid artery, slow injection embolus saline suspension liquid0.3ml,make embolus through the internal carotid artery intracranial arteries to the brain, cause multifocal cerebral infarction, sham operation group by the same method injection with saline0.3ml instead of embolus suspension fluid. Trials are divided into normal group,sham operation group,the model group, including control group, acupuncture group and placebo-acupuncture group.2. Acupuncture treatment:Acupuncture began after8days. Acupuncture group take double ST36acupuncture point30s.The placebo-acupuncture group take bilateral sides a fixed point (above iliac crest10mm), as a comparison exciting point, use normal reinforcement and normal reduction methods for twined technique each30s turn. Acupuncture group, placebo-acupuncture group began acupuncture after screening,1times a day, acupuncture6d, rest1d,3weeks of treatment18times. Model group, sham operation group and the normal group be gaved the same catch stimulation on time and degree with the acupuncture group and placebo-acupuncture group.3. Morris water maze hidden platform experiment:The multi-infarct dementia model was made by injection of emboli into the internal carotid artery. The dementia rats were randomly divided into3groups:model group, A normal group and a sham operation group were set up as contrast. Experiment after acupuncture. The day before the experiment, let animals swimming2min in the pool without platform, make its familiar maze environment. The experiment proceed3days altogether.The platform position is fixed and midpoint off the wall is40cm. Put the rat into water at the midpoint of quadrant with no platform, record the time from enter water to finding platform (escape incubation period), training3times every day in different entry point. With the three arithmetic means for the statistical analysis.4. After treatment, the hippocampus cAMP and cGMP contents of each group by radioimmunoassay were detected.Results:Swimming speed:The rat swimming speed has no significant difference (F=4.126, P>0.05). No significant differences between groups (F=0.854, P>0.05), each group and swimming speed does not exist between interaction (F=1.424, P>0.05).Escape latency period: in the rat escape incubation period there exist significant differences (F=6.846, P <0.05). There exist significant differences between the groups (F=12.990, P <0.05), and escape the incubation period does not exist between interaction (F=0.231, P>0.05). Compared with normal group, model group escape extended incubation period (P <0.05). Group compared with model, acupuncture group and placebo-acupuncture group escape was significantly shorten incubation period (P <0.05), but between the two groups did not see the difference obvious (P>0.05).Acupuncture influence to cAMP, cGMP content in the hippocampus: compared with normal group, cAMP content of model group decreased obviously (p <0.01). Group compared with model, acupuncture group cAMP content increased (p <0.01). Compared with normal group, model group cGMP content has not seen the abnormal changes obviously (P>0.05), acupuncture group also has not seen the obvious regulatory role (P>0.05).Conclusion:1. This experiment used injection of emboli into the internal carotid artery to make multi-infarct dementia (MID) rat model, made the observation of animals activities Bederson neurological classification rating after2days:results suggest that, the rats with symptoms and signs of MID clinical features and pathological change, model preparation success.2. Acupuncture may improve the multi-infarct dementia (MID) rat space reference memory. Possible mechanism is through regulation cAMP-PKA-CREB signaling pathways, and then to improve rats learning function and memory. Between acupuncture and placebo-acupuncture has not seen the obvious difference.3. Acupuncture may adjust the central level cAMP, cGMP imbalances state, and its mechanism may be relevant with the following approaches:activate the cAMP-PKA-CREB signaling pathways, improve inhibition to cerebrovascular expansion and cerebrovascular lasts convulsion state, reduce rat hippocampal nerve cell damage.A combination of the conclusion, Part of the mechanism that acupuncture improve multiple infarction rats dementia behavior related to regulation of the cAMP-PKA-CREB signaling pathways.更多还原