【作者】 刘超；

【导师】 闵苏；

【作者基本信息】 重庆医科大学 ， 麻醉学， 2012， 博士

【摘要】 第一部分不同电流和处置次数的电休克相应程度地调升嗅球切除抑郁模型大鼠海马内Glu的浓度目的：观察不同电量和不同处置次数ECT干预对嗅球切除抑郁模型大鼠海马Glu浓度的影响。方法：建立大鼠嗅球切除抑郁模型，采用随机单位组3×3析因设计：将每只大鼠视为1个单位，予以2个处理因素，即电量（3水平：25mA、50mA、75mA）和处置次数（3水平：3次ECT、6次ECT、9次ECT）的所有组合。72只嗅球切除抑郁模型大鼠随机分为9个实验组（n=8）。全部ECT处置结束24h内留取海马，高效液相色谱法（High Performance LiquidChromatography，HPLC）检测Glu在海马中的含量。结果： ECT引发海马Glu浓度升高，ECT的电量和处置次数均可影响该过程，两者作用叠加。结论： ECT导致海马Glu浓度升高，从而加剧海马Tau蛋白的磷酸化程度。第二部分兴奋性氨基酸受体拮抗剂减轻电休克诱发的WKY大鼠认知障碍和Tau蛋白的过度磷酸化目的：通过观察兴奋性氨基酸受体拮抗剂对ECT后WKY大鼠认知能力和Tau蛋白过度磷酸化的影响探讨兴奋性氨基酸受体拮抗剂对Tau蛋白过度磷酸化的调节及两者对抑郁大鼠认知能力的影响。方法：采用随机单位组析因设计：将每只大鼠视为1个单位，予2个处理因素，即ECT处置与否（2水平：无处置、施行一个疗程ECT）和兴奋性氨基酸受体拮抗剂使用与否（3水平：注射生理盐水、MK-801、DNQX）的所有组合。72只WKY大鼠随机分为6个实验组（n=8）。全部ECT处置结束24h内开始Morris水迷宫（Morris water maze，MWwM）检测认知能力，后留取海马。HPLC法检测Glu在海马中的含量；免疫组织化学（immunohistochemistry， IHC-SP）法和Western-lotting法检测Tau5（总Tau蛋白）、 p-PHF1Ser396/404、p-AT8Ser199/202、p-12E8Ser262在海马中的表达。结果：ECT和GluR拮抗剂均可造成大鼠认知障碍，即延长逃避潜伏期（escape latency，EL）并缩短空间探索时间（space exploration time，SET）；两者的影响呈相减效果，即ECT和Glu受体拮抗剂合用之后，其造成的大鼠认知障碍程度反而减轻。ECT可明显增加海马中Glu的浓度；GluR拮抗剂对海马中Glu的浓度没有明确影响。ECT和GluR拮抗剂对海马总Tau蛋白表达无明确影响。ECT可增加海马中磷酸化Tau蛋白的表达；GluR拮抗剂可减少海马中磷酸化Tau蛋白的表达；两者的影响呈相减效果，即GluR拮抗剂可使ECT造成的海马中磷酸化Tau蛋白的表达增加的幅度减缓。结论： NMDAR和AMPAR拮抗剂可以阻止“ECT导致海马Glu浓度升高，从而加剧海马Tau蛋白的磷酸化程度”这一过程，且两者效果相似；其具体机制与阻断兴奋性毒性有关。第三部分二异丙酚逆转嗅球切除抑郁模型大鼠电休克后的Tau蛋白过度磷酸化和认知障碍目的：通过观察二异丙酚对ECT后嗅球切除抑郁模型大鼠认知能力和Tau蛋白过度磷酸化的影响探讨兴奋性氨基酸受体拮抗剂对Tau蛋白过度磷酸化的调节及两者对抑郁大鼠认知能力的影响。方法：按随机单位组2×2析因设计设置2个干预因素，即ECT干预（2水平：无处置、施行一个疗程ECT）和二异丙酚干预（2水平：腹腔注射5ml生理盐水或5ml二异丙酚100mg/kg）的所有组合。选24W健康雄性Sprague-Dawley大鼠建立嗅球切除抑郁模型，将32只24W龄模型大鼠随机分为4个实验组（n=8）：I组（腹腔注射5ml二异丙酚100mg/kg）、II组（腹腔注射5ml二异丙酚100mg/kg+施行ECT1个疗程）、III组（腹腔注射5ml生理盐水）、IV组（腹腔注射5ml生理盐水+施行ECT1个疗程）。全部ECT处置结束24h内开始Morris水迷宫检测，之后留取海马组织.高效液相色谱法检测神经递质Glu在海马组织中的含量；免疫组化SP法和Western blotting法检测Tau-5（总Tau蛋白）、p-PHF1Ser396/404、p-AT8Ser199/202、p-12E8Ser262、GSK-3β1H8和PP-2A在海马组织神经元中的表达。结果：ECT和二异丙酚均可造成大鼠认知障碍，即延长EL并缩短SET；两者的影响呈相减效果。ECT可明显增加海马中神经递质Glu的浓度；二异丙酚可降低海马中神经递质Glu的浓度，且两者有相减效果。ECT和二异丙酚对海马总Tau蛋白和PP-2A蛋白的表达无明显影响。ECT可增加海马中磷酸化Tau蛋白和GSK-3β1H8蛋白的表达；二异丙酚可减少海马中磷酸化Tau蛋白和GSK-3β1H8蛋白的表达；两者的影响均呈相减效果。结论：ECT导致海马Glu浓度升高，通过上调GSK-3β1H8增加海马Tau蛋白的磷酸化程度导致认知功能障碍，而二异丙酚则可通过降低海马Glu浓度下调GSK-3β1H8的表达，从而减缓Tau蛋白的磷酸化程度以改善ECT后的认知功能。第四部分二异丙酚与NMDAR拮抗剂逆转嗅球切除抑郁模型大鼠电休克后认知障碍的神经心理学机制之比较目的：通过观察二异丙酚和对ECT后嗅球切除抑郁模型大鼠空间认知能力和Tau蛋白过度磷酸化的影响，探讨两者对Tau蛋白过度磷酸化的调节及两者对抑郁大鼠空间认知能力的影响，并比较其效果和作用途径，以期为改善认知障碍的神经心理学机制研究和临床干预性治疗提供实验依据。方法：采用随机单位组2×3析因设计：将每只大鼠视为1个单位，予以2个处理因素，即ECT（2水平：无处置、施行1疗程ECT）和药物（3水平：腹腔注射生理盐水、NMDAR拮抗剂MK-801、二异丙酚）的所有组合。48只嗅球切除抑郁模型大鼠随机分为6个实验组（n=8）。全部ECT结束24h内开始MWM检测，留取海马，HPLC法检测Glu在海马中的含量，IHC-SP法和Western-lotting法检测p-AT8Ser202和GSK-3β1H8在海马中的表达。结果：二异丙酚、NMDAR拮抗剂及ECT均可造成大鼠认知障碍，即延长EL并缩短SET；前两者效果相似且和ECT的影响呈相减效果。ECT可明显增加海马中Glu的浓度；二异丙酚可降低海马中Glu的浓度，且两者有相减效果；而NMDAR拮抗剂对海马中Glu的浓度无明显影响。ECT可增加海马中p-AT8Ser202及促进其磷酸化的GSK-3β1H8的表达；二异丙酚和NMDAR拮抗剂可减缓海马中p-AT8Ser202及促进其磷酸化的GSK-3β1H8表达上调；两者效果相似且和ECT的影响呈相减效果。结论：二异丙酚则可通过降低海马Glu浓度减缓Tau蛋白的磷酸化，从而改善大鼠ECT后的认知能力；GSK-3β是该信号通路的关键蛋白；二异丙酚改善电休克后的认知能力的效果优于NMDAR拮抗剂。第五部分二异丙酚、人参皂苷Rg-1和氯化锂逆转电休克后嗅球切除抑郁大鼠认知障碍的神经心理学机制之比较目的：观察二异丙酚与人参皂苷Rg-1和LiCl对ECT后嗅球切除抑郁模型大鼠认知能力、海马内Glu浓度和Tau蛋白磷酸化的影响，比较其药理机制，为临床治疗方案的制定提供新的理论依据和思路。方法：按随机单位组2×4析因设计，即ECT干预（2水平：无处置、施行1疗程ECT）和药物干预（4水平：海马CA1区微量注射二异丙酚、人参皂苷Rg-1、LiCl，20μg1μL-1）的所有组合。64只嗅球切除抑郁模型大鼠随机分为8个实验组（n=8）。全部ECT处置结束后开始MWM测试，取海马通过HPLC法检测Glu的含量。Western-blotting法检测Tau-5和p-AT8Ser202在海马中的表达。结果：二异丙酚和ECT造成认知障碍，而与ECT合用则会缓解ECT后的认知障碍；人参皂苷Rg-1和LiCl可改善ECT后的认知能力；ECT可明显增加海马中Glu浓度、二异丙酚和人参皂苷Rg-1在ECT前后均可减少Glu浓度；二异丙酚与人参皂苷Rg-1和LiCl可使ECT造成的海马中磷酸化Tau蛋白的表达增加的幅度减缓。结论：二异丙酚与人参皂苷Rg-1和LiCl均可缓解ECT后Tau蛋白过度磷酸化程度进而改善ECT后的认知能力；前两者与降低海马中Glu浓度有关。全文结论1ECT导致海马Glu浓度升高，Glu激动NMDAR和AMPAR，从而通过上调GSK-3β1H8增加海马Tau蛋白的磷酸化程度，从而导致认知障碍。2GluR拮抗剂、二异丙酚、人参皂苷Rg-1亦通过降低海马Glu浓度下调GSK-3β1H8的表达，从而减缓Tau蛋白的磷酸化程度以改善ECT后的认知能力；氯化锂缓解ECT后的Tau蛋白过度磷酸化与Glu无关。3人参皂苷Rg-1和氯化锂改善ECT后的认知能力的效果优于二异丙酚；二异丙酚改善ECT后认知能力的效果优于NMDAR拮抗剂；NMDAR拮抗剂与AMPAR拮抗剂改善ECT后的认知能力的效果相似。更多还原

【Abstract】 Protein Tau is a very unequal phosphoric microtubule associatedprotein, which affect the transport of substances in the axons of the neurons,whose phosphorylation is one of the key methods to regulate neuronalfunction. The hyperphosphorylation of protein Tau can damage thelearning and memory of rats. The impairment of learning-memory inducedby electroconvulsive shock in depressed rats is relevant to the functionfailure of glutamic acid signal system. The rise of glutamate which inducedby electroconvulsive shock in depressed rats can lead to the impairment oflearning-memory through up-regulate the hyperphosphorylation of proteinTau? Is this process related to the excitotoxicty of glutamate? Can theexcitatory amino acids receptor antagonists and propofolwhich play a roleas GluR antagonist inhibit this process? What are the similarities anddifferences about the effects and the neuropsychology mechanisms ofpropofol, ginsenoside Rg-1and lithium protects against the impairment oflearning-memory induced by electroconvulsive shock in depressed rats?The series of problems above attracted strong interest of researchers.Therefore, we engaged in this research in order to explore the mechanismof this process. PART ⅠThe electroconvulsive shock of different electric quantity and ofdifferent treatment number up-regulated the content of glutamate inthe hippocampus of the depression model rats whose olfactory bulbswere removedObjective This study explore the effect of the electroconvulsive shockof different electric current and different duration up-regulated the contentof glutamate in the hippocampus of depressed rats. Methods Thedepression model rats whose olfactory bulbs were removed wereestablished. As the analysis of variance of factorial design set up twointervention factors which are the electric quantity factor(three levels:25mA,50mA,75mA) and the treatment number factor(three level:3timeselectroconvulsive shock,3times electroconvulsive shock,9timeselectroconvulsive shock). Seventy-two adult depression model rats whoseolfactory bulbs were removed were randomly divided into six experimentalgroups (n=6, in each group). The hippocampus was removed within24hours after the course of electroconvulsive shock finished. Results Theelectroconvulsive shock can significantly up-regulated the content ofglutamate. The changes were correlated with the electric quantity and thetreatment number of the electroconvulsive shock. And both influencespresent additive effect. Conclusions Our results indicate that theelectroconvulsive shock up-regulated the content of glutamate in thehippocampus of rats.PART ⅡMK-801or CNQX reduces electroconvulsive shock-inducedimpairment of learning-memory and hyperphosphorylation of tau inWYK rats with depression-like behaviors Objective This study explore the reversion of the excitatory aminoacid receptor antagonists against the impairment of learning-memory andthe hyperphosphorylation of Protein Tau induced by electroconvulsiveshock in depressed rats. Methods As the analysis of variance of factorialdesign set up two intervention factors which are the electroconvulsiveshock (two levels: no disposition; A course of electroconvulsive shock) andthe excitatory amino acid receptor antagonists (three levels: iv Saline; ivNMDA receptor antagonist MK-801; iv AMPA receptor antagonistDNQX). Fourty-eight adult WYK rats were randomly divided into sixexperimental groups (n=8, in each group). The Morris water maze teststarted within1day after the course of electroconvulsive shock finished inorder to evaluate learning-memory. The hippocampus was removed fromrats within1day after the morris water maze test finished. Detect of thecontent of glutamate in the hippocampus of rats by High PerformanceLiquid Chromatography. Detect of the content of Protein Tau whichincludes Tau5(total protein Tau), p-PHF1Ser396/404, p-AT8Ser199/202,p-12E8Ser262in the hippocampus of rats with the methods ofimmunohistochemistry staining (SP) and Western blotting test. ResultsThe electroconvulsive shock and the glutamate ionic receptor blockers caninduce the impairment of learning-memory in depressed rats, extending theevasive latency time and shorten the space exploration time. And bothinfluences present subtract effect. The electroconvulsive shock cansignificantly up-regulated the content of glutamate in the hippocampus ofdepressed rats which was not affected by the glutamate ionic receptorblockers. The electroconvulsive shock and the glutamate ionic receptorblockers do not affect the total protein Tau in the hippocampus of rats. Theelectroconvulsive shock can up-regulated the hyperphosphorylation ofprotein Tau in the hippocampus of depressed rats which is can be reduced by the glutamate ionic receptor blockers. And both influences presentsubtract effect. Conclusions Our results indicate that the electroconvulsiveshock up-regulated the content of glutamate in the hippocampus ofdepressed rats, which up-regulated the hyperphosphorylation of protein Tauwhich can induce the impairment of learning-memory in depressed rats.PART ⅢPropofol protects against the impairment of learning-memory andreduces the hyperphosphorylation of protein Tau induced byelectroconvulsive shock in the depression model rats whose olfactorybulbs were removedObjective This study explore the reversion of the propofol against theimpairment of learning-memory and the hyperphosphorylation of proteinTau induced by electroconvulsive shock in depressed rats. Methods As theanalysis of variance of factorial design set up two intervention factorswhich are the electroconvulsive shock (two levels: no disposition; A courseof electroconvulsive shock) and the propofol (two levels:5ml Saline wasinjected peritoneally;5ml propofol was injected peritoneally by dosage of100mg/kg). Thirty-two the adult depression model rats whose olfactorybulbs were removed were randomly divided into four experimental groups(n=8, in each group). The Morris water maze test started within1day afterthe course of electroconvulsive shock finished in order to evaluatelearning-memory. The hippocampus was removed from rats within1dayafter the Morris water maze test finished. Detect of the content of glutamatein the hippocampus of rats by High Performance Liquid Chromatography.Detect of the content of Protein Tau which includes Tau-5(Total proteinTau), p-PHF1Ser396/404, p-AT8Ser199/202, p-12E8Ser262, GSK-3β1H8and PP-2Ain the hippocampus of rats with the methods of Western blotting test.Results The electroconvulsive shock and the propofol can induce the impairment of learning-memory in depressed rats, extending the evasivelatency time and shortening the space exploration time. And bothinfluences present subtract effect. The electroconvulsive shock cansignificantly up-regulated the content of glutamate in the hippocampus ofdepressed rats which was reduces by the propofol. And both influencespresent subtract effect. The electroconvulsive shock and the propofol doesnot affect the total protein Tau and protein PP-2A in the hippocampus ofrats. The electroconvulsive shock can up-regulated thehyperphosphorylation of protein Tau and the expression of GSK-3β1H8inthe hippocampus of depressed rats which is can be reduced by the propofol.And both influences present subtract effect. Conclusions Our resultsindicate that the electroconvulsive shock up-regulated the content ofglutamate in the hippocampus of depressed rats, which up-regulated thehyperphosphorylation of protein Tau through up-regulating the expressionof GSK-3β1H8, which can induce the impairment of learning-memory indepressed rats; and the propofol protects against the impairment oflearning-memory and reduce the hyperphosphorylation of protein Tauinduced by electroconvulsive shock in depressed rats through reducing theexpression of GSK-3β1H8and the content of glutamate in the hippocampus.PART ⅣComparison of the neuropsychology mechanisms of propofol andNMDA receptor antagonist protects against the impairment oflearning-memory induced by electroconvulsive shock in depressed ratsObjective This study explore the reversion of propofol and NMDAreceptor antagonist against the impairment of learning-memory and thehyperphosphorylation of protein Tau induced by electroconvulsive shock indepressed rats. Methods As the analysis of variance of factorial design setup two intervention factors which are the electroconvulsive shock (two levels: no disposition; a course of electroconvulsive shock) and the drugs(three levels: ip Saline; ip propofol; ip MK-801). Forty-eight adultdepression model rats whose olfactory bulbs were removed were randomlydivided into six experimental groups (n=8, in each group). The Morriswater maze test started within1day after the course of electroconvulsiveshock finished in order to evaluate learning-memory. The hippocampuswas removed from rats within1day after the Morris water maze testfinished. Detect of the content of glutamate in the hippocampus of rats byHigh Performance Liquid Chromatography. Detect of the expression ofprotein Tau which includes p-AT8Ser202and GSK-3β1H8in the hippocampusof rats with the methods of immunohistochemistry staining (SP) andWestern blotting test. Results The drugs (propofol and MK-801) and theelectroconvulsive shock can induce the impairment of learning-memory indepressed rats, extending the evasive latency time and shorten the spaceexploration time. And both influences present subtract effect. Theelectroconvulsive shock can significantly up-regulated the content ofglutamate, which was not affected by MK-801, in the hippocampus ofdepressed rats which was reduces by the propofol. And both influencespresent subtract effects. The electroconvulsive shock and the drugs do notaffect the total protein Tau in the hippocampus of rats. Theelectroconvulsive shock can up-regulated the hyperphosphorylation ofprotein Tau in the hippocampus of depressed rats which is can be reducedby propofol and MK-801. And the influences between theelectroconvulsive shock and the drugs present subtract effects. Our resultsindicate that the electroconvulsive shock up-regulated the content ofglutamate in the hippocampus of depressed rats, which up-regulated thehyperphosphorylation of protein Tau which can induce the impairment oflearning-memory in depressed rats; and propofol protects against the impairment of learning-memory and reduce the hyperphosphorylation ofprotein Tau induced by electroconvulsive shock in depressed rats. GSK-3βis the key protein in this signaling pathway. Conclusions Our resultsindicate that the electroconvulsive shock reduce the impairment oflearning-memory the hyperphosphorylation of protein Tau in depressed ratsthough up-regulated the content of glutamate.PART ⅤComparison of the neuropsychology mechanisms of propofol,ginsenoside Rg-1and lithium protects against the impairment oflearning-memory induced by electroconvulsive shock in depressed ratsObjective This study explore the reversion of propofol, ginsenosideRg-1, lithium against the impairment of learning-memory and thehyperphosphorylation of protein Tau induced by electroconvulsive shockand the concentration of glutamic acid in the hippocampus in depressed rats.Methods As the analysis of variance of factorial design set up twointervention factors which are the electroconvulsive shock groups (twolevels: no disposition; A course of electroconvulsive shock) and the drugintervention groups (four levels: Microinjection of propofol, ginsenosideRg-1and lithium,20μg1μl-1). Sixty-four adult depression model rats whoseolfactory bulbs were removed were randomly divided into eightexperimental groups (n=8, in each group). The Morris water maze teststarted within1day after the course of electroconvulsive shock finished inorder to evaluate learning-memory. Detect of the content of glutamate inthe hippocampus of rats by High Performance Liquid Chromatography.Detect of the content of protein Tau which includes Tau-5and p-AT8Ser202in the hippocampus of rats with the methods of Western blotting test.Results Propofol and the electroconvulsive shock can induce theimpairment of learning-memory in depressed rats. Ginsenoside Rg-1and lithium can protects against the impairment of learning-memory induced byelectroconvulsive shock in depressed rats. The electroconvulsive shock cansignificantly up-regulated the content of glutamate, which was reduces bypropofol. The electroconvulsive shock can up-regulated thehyperphosphorylation of protein Tau in the hippocampus of depressed ratswhich is can be reduced by these three medicines. Conclusions Our resultsindicate that propofol, ginsenoside Rg-1and lithium protects against theimpairment of learning-memory induced by electroconvulsive shock indepressed rats though reducing the hyperphosphorylation of protein Tau.The first two is not content with the glutamate in the hippocampus, withwhich lithium is not content.CONCLUSIONS OF THE TATOL PAPEROur results indicate that the electroconvulsive shock up-regulated thecontent of glutamate in the hippocampus of depressed rats, whichup-regulated the hyperphosphorylation of protein Tau throughup-regulating the expression of GSK-3β1H8, which can induce theimpairment of learning-memory in depressed rats. Propofol protects againstthe impairment of learning-memory and reduce the hyperphosphorylationof protein Tau induced by electroconvulsive shock in depressed ratsthrough reducing the expression of GSK-3β1H8and the content of glutamatein the hippocampus. GluR antagonists, ginsenoside Rg-1and lithiumprotects against the impairment of learning-memory induced byelectroconvulsive shock in depressed rats though reducing thehyperphosphorylation of protein Tau, too.更多还原