本文關(guān)鍵詞：復合應(yīng)激所致大鼠PTSD的發(fā)生特點及病理機制研究，由筆耕文化傳播整理發(fā)布。

        創(chuàng)傷后應(yīng)激障礙(posttraumatic stress disorder，PTSD)是一組由于創(chuàng)傷等應(yīng)激因素引起的持久慢性的精神心理障礙。引起PTSD的事件主要是經(jīng)歷或者目睹生命受到威脅、死亡，嚴重的傷害，恐嚇以及伴隨著強烈的害怕、恐懼或者無助的情感。PTSD患者常常表現(xiàn)為身體外觀完好，但精神上遭受巨大痛苦，甚至失去正常社會功能，給患者家庭和社會造成巨大負擔。目前由于嚴重自然災(zāi)害、戰(zhàn)爭、恐怖及其它突發(fā)應(yīng)激事件等的頻發(fā)，致使PTSD的發(fā)生呈現(xiàn)增高的趨勢，而且因為臨床尚無理想的防治方法，因此，PTSD已經(jīng)日益引起各國政府及軍隊的高度重視，成為全球研究的熱點。PTSD的研究起步較晚，但進展迅速，目前存在一些重要的基礎(chǔ)性問題：1.作為PTSD實驗研究的一種必不可少手段，動物模型的建立一直為研究者所重視，但目前仍無一個被廣泛接受的動物模型。既往相關(guān)研究已經(jīng)構(gòu)建了幾種的動物模型如較常用的連續(xù)單一應(yīng)激(SPS)模型，但這些模型不同程度都在一些局限，迫切需要建立一種更有效、能更好模擬人類PTSD發(fā)生發(fā)展的動物模型。2. PTSD的發(fā)病機制研究取得了一些進展，但是仍存在許多問題，一些現(xiàn)有的研究結(jié)果存在相互矛盾的地方。其中腦的各個區(qū)域在PTSD發(fā)病中扮演什么角色仍有待深入探討。海馬與學習記憶密切相關(guān)，此外還是中樞神經(jīng)系統(tǒng)(central nervous system,CNS)對應(yīng)激調(diào)控的重要區(qū)域。研究表明，海馬在PTSD的發(fā)生發(fā)展中可能起著重要作用，有報道在PTSD患者發(fā)現(xiàn)海馬的體積變化，但其在PTSD發(fā)生發(fā)展中的具體作用特點及機制目前并不十分清楚，也亟待深入研究。3. PTSD的發(fā)生發(fā)展中，神經(jīng)內(nèi)分泌的變化起到至關(guān)重要的作用，有關(guān)下丘腦-垂體-腎上腺(hypothalamic–pituitary–adrenal，HPA)軸的研究報道較多，而谷氨酸/γ-氨基丁酸(Glu/GABA)的研究才剛剛起步并日益受到重視。谷氨酸是廣泛存在于大腦的興奮性神經(jīng)遞質(zhì)，通過感覺傳入形成意識和記憶。GABA是大腦中重要的抑制性神經(jīng)遞質(zhì)。二者的平衡對于維護大腦的功能至關(guān)重要。在過度應(yīng)激中，GABA下調(diào)，Glu過度興奮，使患者海馬神經(jīng)元受損并導致創(chuàng)傷相關(guān)的記憶加強。推測：Glu/GABA及其受體的功能異�？赡苁荘TSD重要的發(fā)病機制之一，但尚需進一步實驗證實。為了解決這些問題，我們通過綜合目前幾種PTSD動物模型中的應(yīng)激刺激的優(yōu)點，來建立一種新的成年大鼠PTSD模型──復合應(yīng)激模型，其具體建模方法為：將大鼠禁錮2小時，期間同時對大鼠足底施以不可逃避電刺激(4mA，60V)持續(xù)2秒，隨機變化的間隔30-120秒。禁錮結(jié)束后，將大鼠取出進行力竭式游泳(水溫20℃)，直到大鼠放棄掙扎沉底30秒鐘視為力竭。造模結(jié)束后將大鼠放入籠中常規(guī)喂養(yǎng)，隨意進食水。同時按常規(guī)方法建立SPS模型做陽性對照，用正常大鼠做陰性對照，實驗SD大鼠經(jīng)一周適應(yīng)期后隨機分為正常組、SPS模型組、復合應(yīng)激模型組三組，由專門實驗員在造模后4周內(nèi)進行大體行為學觀察，為了避免急性應(yīng)激反應(yīng)的影響選擇應(yīng)激后14天進行系統(tǒng)的行為、學習記憶功能和病理形態(tài)學的對比研究，并進一步探討大鼠PTSD發(fā)生的重要腦區(qū)病理改變特征及其與海馬內(nèi)Glu/GABA遞質(zhì)和受體之間的關(guān)系。為今后PTSD發(fā)生機制和防治藥物方法的深入研究提供的理論基礎(chǔ)和實驗依據(jù)。主要結(jié)果和結(jié)論如下：1.行為與學習記憶功能研究：采用定性和定量相結(jié)合的方法進行具體結(jié)果有：(1)大體行為：復合應(yīng)激組拒俘反射評分與對照組比較有非常顯著的增高(P＜0.01)，與SPS組比較有顯著增高(P＜0.05)。提示模型組大鼠較正常大鼠攻擊行為和逃避行為明顯增加，以復合應(yīng)激組增加更顯著。(2)曠場實驗：檢測到復合應(yīng)激組大鼠與對照組比較跨格次數(shù)和站立次數(shù)均有非常顯著差異(P＜0.01)，復合應(yīng)激組和SPS組間比較跨格次數(shù)和站立次數(shù)也有顯著差異(P＜0.05)，修飾行為無顯著差異，提示復合應(yīng)激組大鼠活動度減少、焦慮狀態(tài)更明顯。(3)高架十字迷宮實驗：檢測到復合應(yīng)激組與對照組比較OE%， OT%和OE+CE有非常顯著差異(P＜0.01)，復合應(yīng)激組和SPS組間比較OE%，OT%和OE+CE也有顯著差異(P＜0.05)，向下探究次數(shù)和站立次數(shù)無顯著差異，進一步表明復合應(yīng)激組大鼠活動度減少、焦慮狀態(tài)更明顯。(4)水迷宮定位航行實驗：復合應(yīng)激組與對照組比較在實驗第1、2天(即應(yīng)激后第16、17天，下同)第1次實驗的尋靶潛伏期無顯著差異，定位航行實驗第3、4天(即應(yīng)激后第18、19天，下同)第1次尋靶潛伏期顯著延長(P＜0.01)；復合應(yīng)激組和SPS組間比較尋靶潛伏期第1、2天無顯著差異，第3、4天潛伏期顯著延長(P＜0.05)，提示模型組大鼠空間記憶受損，且復合應(yīng)激組大鼠損害更明顯。(5)水迷宮空間探索實驗：復合應(yīng)激組與對照組空間探索實驗的進入目標象限次數(shù)和穿越平臺位置次數(shù)均有非常顯著差異(P＜0.01)，復合應(yīng)激組和SPS組間比較也有顯著差異(P＜0.05)，也提示復合應(yīng)激組大鼠空間記憶受損更嚴重。綜合以上結(jié)果表明：復合應(yīng)激模型大鼠能更好地模擬出人類PTSD樣臨床表現(xiàn)，該模型是一種簡便、有效而更理想的PTSD實驗動物模型。2.病理形態(tài)學研究：采用HE染色、Nissl染色、Holmes嗜銀染色重點對14d時三組大鼠腦皮層和海馬進行了病理學觀察，結(jié)果觀察到：三組大鼠大腦皮層形態(tài)結(jié)果基本正常，尚未見到明顯的神經(jīng)元變性壞死。正常組海馬結(jié)構(gòu)各亞區(qū)神經(jīng)細胞的分布均勻，DG區(qū)顆粒細胞密集分布，細胞突起少見；CA3區(qū)細胞胞體較大，在輻射層可見許多樹突橫斷面；CA1區(qū)細胞胞體分布較規(guī)則，可見樹突縱斷面。應(yīng)激組海馬CA3和DG區(qū)細胞密度均較對照組減小，各區(qū)內(nèi)可見一些染色極深且形態(tài)不規(guī)則的顆粒，，可能為壞死細胞核分解的碎片。CA3區(qū)細胞間隙加寬，DG區(qū)有團簇狀淺染現(xiàn)象。Nissl染色觀察，與正常組組相比，PTSD組細胞排列整齊，胞體呈圓形或菱形，胞體膨脹，胞膜不完整，可見尼氏體有不同程度的淺染，有少量尼氏體徹底消失。但SPS組和復合應(yīng)激組光鏡觀察差異不明顯。Holmes染色相對于對照組，PTSD組的海馬神經(jīng)元損傷較為明顯，CA3區(qū)有神經(jīng)纖維軸突斷裂、變短、排列不整齊現(xiàn)象，SPS組和復合應(yīng)激組無明顯差異。上述結(jié)果說明：PTSD模型大鼠海馬發(fā)生明顯的神經(jīng)細胞變性壞死，CA3和DG損傷更明顯，而且復合應(yīng)激組大鼠病理損害相對較重。3.病理發(fā)生機制研究：比較三組大鼠腦皮層和海馬Glu/GABA受體表達的變化，結(jié)果觀察到：(1)PTSD模型大鼠接受應(yīng)激14d后，大鼠海馬組織內(nèi)Glu含量明顯高于正常組，其中復合應(yīng)激組明顯高于SPS組(P＜0.05)；大鼠海馬組織內(nèi)GABA含量明顯低于正常對照組，復合應(yīng)激組和SPS組無明顯差異。(2)PTSD模型大鼠海馬組織內(nèi)GluR2表達較正常對照組明顯降低，復合應(yīng)激組較SPS組降低更為顯著(P＜0.05)。GABAARγ2亞單位表達降低，二者無顯著差異。提示：Glu/GABA系統(tǒng)功能失調(diào)可能是大鼠PTSD發(fā)病機制之一。

    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.

復合應(yīng)激所致大鼠PTSD的發(fā)生特點及病理機制研究

英文縮寫簡寫一覽表5-7英文摘要7-10中文摘要11-14前言14-19    參考文獻17-19第一部分 復合應(yīng)激所致大鼠PTSD模型的建立與行為學研究19-30    材料與方法19-22    結(jié)果22-27    討論27-28    小結(jié)28    參考文獻28-30第二部分 復合應(yīng)激所致大鼠PTSD的神經(jīng)病理改變研究30-41    實驗一 復合應(yīng)激所致PTSD大鼠大腦皮層的病理改變30-35        材料與方法30-34        結(jié)果34        討論34-35    實驗二 復合應(yīng)激所致PTSD大鼠海馬的病理改變35-39        材料與方法35-36        結(jié)果36-37        討論37-39    小結(jié)39    參考文獻39-41第三部分 復合應(yīng)激所致大鼠PTSD的病理機制研究41-52    實驗一 復合應(yīng)激所致PTSD大鼠腦區(qū)GluR2和GABAARγ2的表達變化41-45        材料與方法41-42        結(jié)果42-44        討論44-45    實驗二 復合應(yīng)激所致PTSD大鼠腦組織的Glu和GABA含量變化45-50        材料與方法45-47        結(jié)果47-49        討論49-50    小結(jié)50    參考文獻50-52全文總結(jié)52-54致謝54-55圖片55-73文獻綜述1 PTSD動物模型的研究現(xiàn)狀73-80    參考文獻78-80文獻綜述2 PTSD相關(guān)睡眠障礙研究的新進展80-88    參考文獻86-88學習期間發(fā)表的文章88

  本文關(guān)鍵詞：復合應(yīng)激所致大鼠PTSD的發(fā)生特點及病理機制研究，由筆耕文化傳播整理發(fā)布。