复合应激所致大鼠PTSD的发生特点及病理机制研究

  本文关键词：复合应激所致大鼠PTSD的发生特点及病理机制研究，由笔耕文化传播整理发布。

        创伤后应激障碍(posttraumatic stress disorder，PTSD)是一组由于创伤等应激因素引起的持久慢性的精神心理障碍。引起PTSD的事件主要是经历或者目睹生命受到威胁、死亡，严重的伤害，恐吓以及伴随着强烈的害怕、恐惧或者无助的情感。PTSD患者常常表现为身体外观完好，但精神上遭受巨大痛苦，甚至失去正常社会功能，给患者家庭和社会造成巨大负担。目前由于严重自然灾害、战争、恐怖及其它突发应激事件等的频发，致使PTSD的发生呈现增高的趋势，而且因为临床尚无理想的防治方法，因此，PTSD已经日益引起各国政府及军队的高度重视，成为全球研究的热点。PTSD的研究起步较晚，但进展迅速，目前存在一些重要的基础性问题：1.作为PTSD实验研究的一种必不可少手段，动物模型的建立一直为研究者所重视，但目前仍无一个被广泛接受的动物模型。既往相关研究已经构建了几种的动物模型如较常用的连续单一应激(SPS)模型，但这些模型不同程度都在一些局限，迫切需要建立一种更有效、能更好模拟人类PTSD发生发展的动物模型。2. PTSD的发病机制研究取得了一些进展，但是仍存在许多问题，一些现有的研究结果存在相互矛盾的地方。其中脑的各个区域在PTSD发病中扮演什么角色仍有待深入探讨。海马与学习记忆密切相关，此外还是中枢神经系统(central nervous system,CNS)对应激调控的重要区域。研究表明，海马在PTSD的发生发展中可能起着重要作用，有报道在PTSD患者发现海马的体积变化，但其在PTSD发生发展中的具体作用特点及机制目前并不十分清楚，也亟待深入研究。3. PTSD的发生发展中，神经内分泌的变化起到至关重要的作用，有关下丘脑-垂体-肾上腺(hypothalamic–pituitary–adrenal，HPA)轴的研究报道较多，而谷氨酸/γ-氨基丁酸(Glu/GABA)的研究才刚刚起步并日益受到重视。谷氨酸是广泛存在于大脑的兴奋性神经递质，通过感觉传入形成意识和记忆。GABA是大脑中重要的抑制性神经递质。二者的平衡对于维护大脑的功能至关重要。在过度应激中，GABA下调，Glu过度兴奋，使患者海马神经元受损并导致创伤相关的记忆加强。推测：Glu/GABA及其受体的功能异常可能是PTSD重要的发病机制之一，但尚需进一步实验证实。为了解决这些问题，我们通过综合目前几种PTSD动物模型中的应激刺激的优点，来建立一种新的成年大鼠PTSD模型──复合应激模型，其具体建模方法为：将大鼠禁锢2小时，期间同时对大鼠足底施以不可逃避电刺激(4mA，60V)持续2秒，随机变化的间隔30-120秒。禁锢结束后，将大鼠取出进行力竭式游泳(水温20℃)，直到大鼠放弃挣扎沉底30秒钟视为力竭。造模结束后将大鼠放入笼中常规喂养，随意进食水。同时按常规方法建立SPS模型做阳性对照，用正常大鼠做阴性对照，实验SD大鼠经一周适应期后随机分为正常组、SPS模型组、复合应激模型组三组，由专门实验员在造模后4周内进行大体行为学观察，为了避免急性应激反应的影响选择应激后14天进行系统的行为、学习记忆功能和病理形态学的对比研究，并进一步探讨大鼠PTSD发生的重要脑区病理改变特征及其与海马内Glu/GABA递质和受体之间的关系。为今后PTSD发生机制和防治药物方法的深入研究提供的理论基础和实验依据。主要结果和结论如下：1.行为与学习记忆功能研究：采用定性和定量相结合的方法进行具体结果有：(1)大体行为：复合应激组拒俘反射评分与对照组比较有非常显著的增高(P＜0.01)，与SPS组比较有显著增高(P＜0.05)。提示模型组大鼠较正常大鼠攻击行为和逃避行为明显增加，以复合应激组增加更显著。(2)旷场实验：检测到复合应激组大鼠与对照组比较跨格次数和站立次数均有非常显著差异(P＜0.01)，复合应激组和SPS组间比较跨格次数和站立次数也有显著差异(P＜0.05)，修饰行为无显著差异，提示复合应激组大鼠活动度减少、焦虑状态更明显。(3)高架十字迷宫实验：检测到复合应激组与对照组比较OE%， OT%和OE+CE有非常显著差异(P＜0.01)，复合应激组和SPS组间比较OE%，OT%和OE+CE也有显著差异(P＜0.05)，向下探究次数和站立次数无显著差异，进一步表明复合应激组大鼠活动度减少、焦虑状态更明显。(4)水迷宫定位航行实验：复合应激组与对照组比较在实验第1、2天(即应激后第16、17天，下同)第1次实验的寻靶潜伏期无显著差异，定位航行实验第3、4天(即应激后第18、19天，下同)第1次寻靶潜伏期显著延长(P＜0.01)；复合应激组和SPS组间比较寻靶潜伏期第1、2天无显著差异，第3、4天潜伏期显著延长(P＜0.05)，提示模型组大鼠空间记忆受损，且复合应激组大鼠损害更明显。(5)水迷宫空间探索实验：复合应激组与对照组空间探索实验的进入目标象限次数和穿越平台位置次数均有非常显著差异(P＜0.01)，复合应激组和SPS组间比较也有显著差异(P＜0.05)，也提示复合应激组大鼠空间记忆受损更严重。综合以上结果表明：复合应激模型大鼠能更好地模拟出人类PTSD样临床表现，该模型是一种简便、有效而更理想的PTSD实验动物模型。2.病理形态学研究：采用HE染色、Nissl染色、Holmes嗜银染色重点对14d时三组大鼠脑皮层和海马进行了病理学观察，结果观察到：三组大鼠大脑皮层形态结果基本正常，尚未见到明显的神经元变性坏死。正常组海马结构各亚区神经细胞的分布均匀，DG区颗粒细胞密集分布，细胞突起少见；CA3区细胞胞体较大，在辐射层可见许多树突横断面；CA1区细胞胞体分布较规则，可见树突纵断面。应激组海马CA3和DG区细胞密度均较对照组减小，各区内可见一些染色极深且形态不规则的颗粒，，可能为坏死细胞核分解的碎片。CA3区细胞间隙加宽，DG区有团簇状浅染现象。Nissl染色观察，与正常组组相比，PTSD组细胞排列整齐，胞体呈圆形或菱形，胞体膨胀，胞膜不完整，可见尼氏体有不同程度的浅染，有少量尼氏体彻底消失。但SPS组和复合应激组光镜观察差异不明显。Holmes染色相对于对照组，PTSD组的海马神经元损伤较为明显，CA3区有神经纤维轴突断裂、变短、排列不整齐现象，SPS组和复合应激组无明显差异。上述结果说明：PTSD模型大鼠海马发生明显的神经细胞变性坏死，CA3和DG损伤更明显，而且复合应激组大鼠病理损害相对较重。3.病理发生机制研究：比较三组大鼠脑皮层和海马Glu/GABA受体表达的变化，结果观察到：(1)PTSD模型大鼠接受应激14d后，大鼠海马组织内Glu含量明显高于正常组，其中复合应激组明显高于SPS组(P＜0.05)；大鼠海马组织内GABA含量明显低于正常对照组，复合应激组和SPS组无明显差异。(2)PTSD模型大鼠海马组织内GluR2表达较正常对照组明显降低，复合应激组较SPS组降低更为显著(P＜0.05)。GABAARγ2亚单位表达降低，二者无显著差异。提示：Glu/GABA系统功能失调可能是大鼠PTSD发病机制之一。

    Posttraumatic stress disorder(PTSD) is a permanent chronic psychological dysfunctioncaused by stressor such as trauma. The main affairs leading to PTSD is life-threatening,death, severe injury, terrors and intensive fear or helpless feeling. Although body isWell-tended appearance, the patients of PTSD suffered torture psychiatrically, even lossnormal function and pose a huge burden on society. Currently, the occurrence of PTSDexhibited an increasing tendency due to severe natural disaster, war, terrors and otherstressing affairs. But there are not effective measures to prevent and cure the disease. Somany governments and militaries all over the world pay more attention to the disease and itbecome the hot-point of researching fields.The beginning research of of PTSD is late, but the progress is rapid. There areremained some basic questions to be solved in the current:1. As a necessary experimentaltool for PTSD research, the establishment of aminal models is interested by researchers.But there is still no one widely accepted animal models of PTSD. Several animals of PTSDhave been established in previous researches, such as single-prolonged stress(SPS) model.Because of the limit of animals models in some degree, it is urgent to create a valid modelsto mimic the occurrence and development of PTSD in human.2. The mechanism of PTSDoccurrence have been made some progresses, but still exists many questions to be clarified.For example, the effects of different region of brain in the happen of PTSD is worth to bediscuss deeply. At the same time, there are some conflicted aspects in current researchingresults. Hippocampus is closely relevent to learning and memory, besides it is an importantstress-regulated region in the central nervous system. Many researches indicated thathippocampus may play crucial effects in develop of PTSD. Some researchers found thechange of hippocampal volume in patients with PTSD, but the detailed characteristics andmechanism of which in PTSD developing process are not clear and need to be deeplystudied.3. The regulation of neuroendocrine system plays key roles in occurrence of PTSD. There are more reports about hypothalamic-pituitary-adrenal (HPA) axis, but the researchof glutamate (Glu)/gamma-aminobutyric acid (GABA) is emerging and attractingincreasingly. Glutamine is an excitory neurotransmitter generally existing in the brain,which can form consciousness and memory through sensory afferent. GABA is aninhibitory neurotransmitter. The balance of Glu/GABA is remarkably important formaintaining the function of brain. During excessive stress, GABA decreased and Gluexhibited hyper-excitable activity, which led to the injury of hippocampal neurons andstrengthen trauma-related memories. It is concluded that: the dysfunction of Glu/GABA andtheir receptors may be one of the considerable mechanisms of PTSD pathological process.There are still to be confirmed through experiments.In order to solve above questions, a new PTSD model–combined stress model, inadult rats was established through thinking of the advantages in current several PTSDanimal models. The method is as follows: First, the adult rats were restrained for2hoursaccompanying inevitable electrical stimulation (4mA,60V) lasting2seconds withrandom intervals of30-120seconds. After restraining, rats proceeded fatigue swimming(water temperature is20℃) up to sink into the bottom of water for30seconds. Aftermodeling, the rats were regularly feed in cages and freely drink water. SPS modelestablished according to regular method is for positive control and normal rats for negativecontrol. All rats for experiment were divided into normal group, SPS model group andcombined stress model group after one-week accommodation. Specialized laboratoryassistant observed the general behaviors with four weeks after modeling. To avoid theinfluence of acute stress reaction, the examination of system behavior, learning andmemory functions, pathological morphology were carried out at14day after stimiulation.Furtherly, the change of pathological characteristic in important region of brain related toPTSD development and the relationship of Glu/GABA transmitters and their receptors inhippocampus was discussed. Through above measurements, it can provide theoreticalfoundation and experimental proofs for thorough researches on mechanism and drugs forprevention and treatment PTSD in the future.Results and conlusions:1. Study on the behaviors, learning and memory: The results of qualitation andquantitation were as fellows:(1) Behaviors in general: Responses to the capture of combined stress group were higher than the results in control group(P＜0.01)，and alsoshowed higher than those in the SPS group(P＜0.05), which indicated that the rats incombined stress group was prone to attacking and escaping behaviors than the other twogroups.(2) Open field test: The rats in combined stress group showed significant differencein the crossing and standing behaviors，comparing to the control(P＜0.01)and SPS groups(P＜0.05)，while no difference in the grooming behaviors, it indicated that the rats incombined stress group showed less locomotors and more anxiety.(3) Elevated plus mazetest: There were clear differences in the OE%，OT%and OE+CE between the cominedstress group and control group(P＜0.01), and the results of OE%，OT%and OE+CEdisplayed different between the complex stress group and SPS group(P＜0.05)， nodifference were found in dipping head or standing, which showed the rats of comined stressgroup reduced the locomotors and explained more anxious in mood.(4) Water maze test:No difference were found on the first escape latency of the1st and2nd day on the test (16thand17th after stress), the escape latency was prolonged on the3rd and4th day(18th and19th after stress) between the comined stress group and normal group(P＜0.01); nosignificant difference was found between the comined stress group and SPS group, but thelatencies on3rd and4th day were increased remarkably indicating that the space memory ofrat PTSD model was damaged(P＜0.05), furthermore, the space memory in rat ofcomined stress group was injured more obviously.(5)Water maze exploration test: Therewere differences on the times of target quadrant and crossing platform in combined stressgroup than in normal group(P＜0.01), more significant changes were found in rats ofcomined stress group than those in SPS groups(P＜0.05), it indicated that the spacememory of rats in compined stress group was affected more severely. As a result, thecombined stress rats might mimic the clinical symptom of PTSD patients and the combinedstress model is an easy and ideal animal model for PTSD.2. Study on the pathomorphology：The pathomorphologies on cortex and hippocampusof rat in control, SPS and combined stress groups on the14th day after stress were observedwith H.E, Nissl and Holmes staining. Almost no clear changes were found in the cortex ofthree groups, no neuronal degeneration was found. In the control group, the neurons ofsubregion in hippocampus were well distributed, the granular cells of DG were in the closeset, no cell prominency were found; the cell bodies in the CA3were comparatively large and more cross sections of dendrite were evident in the stratum radiatum; the cell bodies inthe CA1region distributed regularly, and the cross sections of dendrite were seen. The celldensity of CA3and DG regions in the combined stress group decreased than control group,and the deeply dying and irregular particles of subregions might be the fragment of deadcell nucleus. Intercellular space of CA3region was widened, the DG region showedunderstain in cluster. As to the Nissl’s staning, the cells in PTSD group were in line and thecell bodies were round or in arris, the cell bodies were expanded, the cell membranes wereuncompleted, the Nissl’s bodies were understained in different degrees. In some cells, theNissl’s bodies completely disappeared, but no differences were found in the combinedstress and SPS groups. In the studies of Holmes staining, the hippocampus neurons inPTSD group damaged more clearly, the axonotmesis was found, and also the nerve fiberwere found short and lined untidily. No significant differences were found in SPS and CSgroups. These results indicated that the neuron degeneration took place in the hippocampusof PTSD model, and the changes in the CA3and DG regions were more obviously,furthermore, the pathological lesion in the rat of combined stress group was more severity.3. The study of the pathogenesis：The changes of receptors of Glu/GABA in the cortexand hippocampus among three groups. The results were as follows:(1)The concentration ofGlu in hippocampus of PTSD model increased more evidently than that in control group onthe14th day after stress, and the concentration of combined groups was higher than that ofSPS group(P＜0.05)；the content of GABA in hippocampus was lower than that in thecontrol group, but no significant difference was found between combined stress group andSPS group.(2)The concentration of GluR2expression of hippocampus in PTSD modelsdecreased compared with that in control group, the result in combined stress groupdecreased more obviously than that in SPS group(P＜0.05).The expression of GABAARγ2was down regulated，but there was no difference between SPS and combined stress groups.These results showed that dysfunction of Glu/GABA system may be one of the PTSDpathogenesis.

复合应激所致大鼠PTSD的发生特点及病理机制研究

英文缩写简写一览表5-7英文摘要7-10中文摘要11-14前言14-19    参考文献17-19第一部分 复合应激所致大鼠PTSD模型的建立与行为学研究19-30    材料与方法19-22    结果22-27    讨论27-28    小结28    参考文献28-30第二部分 复合应激所致大鼠PTSD的神经病理改变研究30-41    实验一 复合应激所致PTSD大鼠大脑皮层的病理改变30-35        材料与方法30-34        结果34        讨论34-35    实验二 复合应激所致PTSD大鼠海马的病理改变35-39        材料与方法35-36        结果36-37        讨论37-39    小结39    参考文献39-41第三部分 复合应激所致大鼠PTSD的病理机制研究41-52    实验一 复合应激所致PTSD大鼠脑区GluR2和GABAARγ2的表达变化41-45        材料与方法41-42        结果42-44        讨论44-45    实验二 复合应激所致PTSD大鼠脑组织的Glu和GABA含量变化45-50        材料与方法45-47        结果47-49        讨论49-50    小结50    参考文献50-52全文总结52-54致谢54-55图片55-73文献综述1 PTSD动物模型的研究现状73-80    参考文献78-80文献综述2 PTSD相关睡眠障碍研究的新进展80-88    参考文献86-88学习期间发表的文章88

  本文关键词：复合应激所致大鼠PTSD的发生特点及病理机制研究，由笔耕文化传播整理发布。