【摘要】：血管性癡呆(Vascular Dementia,VD)是繼阿爾茨海默病之后第二種最為常見的與年齡相關(guān)的神經(jīng)功能障礙。VD的主要癥狀是由于腦血管損傷誘發(fā)并累積腦組織損傷而導(dǎo)致的漸進(jìn)性認(rèn)知功能障礙。學(xué)習(xí)和記憶是各種認(rèn)知功能形成的基礎(chǔ),是研究VD認(rèn)知功能障礙的必選指標(biāo)。海馬是學(xué)習(xí)記憶的關(guān)鍵部位,尤其在空間學(xué)習(xí)記憶中起重要作用。哺乳類海馬主要分為CA1、CA3和齒狀回(dentate gyrus,DG),而DG作為信息進(jìn)入海馬的傳入點(diǎn),對空間信息進(jìn)行處理和編碼,并在空間學(xué)習(xí)記憶中起著至關(guān)重要的作用。雖然研究表明,海馬DG區(qū)在VD空間學(xué)習(xí)記憶損害中可能起重要作用,但對參與VD空間記憶損害過程的DG內(nèi)神經(jīng)遞質(zhì)及其作用目前尚未完全弄清楚。Y-氨基丁酸(γ-aminobutyricacid,GABA)作為腦內(nèi)主要的抑制性神經(jīng)遞質(zhì),廣泛存在于中樞神經(jīng)系統(tǒng)中,而海馬DG內(nèi)也存在大量的GABA能神經(jīng)元及其GABAA和GABAB受體。雖然研究表明,DG內(nèi)的GABA及其GABAA/GABAB受體參與調(diào)節(jié)突觸可塑性及某些學(xué)習(xí)記憶過程,但其在VD空間學(xué)習(xí)記憶損害中的作用未見報(bào)道。因此,本實(shí)驗(yàn)利用雙側(cè)頸總動(dòng)脈永久性結(jié)扎法制備VD模型,并應(yīng)用腦部微量透析法,高效液相色譜分析法,Morris水迷宮(Morris water maze,MWM),免疫組織化學(xué)法等實(shí)驗(yàn)方法探討海馬DG區(qū)GABA及其受體在血管性癡呆模型大鼠空間學(xué)習(xí)記憶損害中的作用及其部分機(jī)制。本實(shí)驗(yàn)的具體內(nèi)容如下:1.利用雙側(cè)頸總動(dòng)脈結(jié)扎方法,制備VD模型大鼠,應(yīng)用MWM觀察其空間學(xué)習(xí)記憶能力、利用腦部微量透析法和高效液相色譜法測定DG區(qū)細(xì)胞外液中的GABA含量、利用免疫組織化學(xué)染色法觀察DG區(qū)GABAA和GABAB受體表達(dá)。2.利用腦部微量注射法往海馬DG區(qū)注射GABAA和GABAB受體阻斷劑觀察其對VD模型大鼠空間學(xué)習(xí)記憶的影響。3.利用腦部微量注射法往海馬DG區(qū)注射GABAA和GABAB受體阻斷劑觀察其對VD模型大鼠DG區(qū)天冬氨酸(asparate,Asp)、谷氨酸(glutamate,Glu)、谷氨酰胺(glutamine,G1n)、甘氨酸(glycin,Gly)和�；撬�(taurine,Tau)含量的影響。4.觀察正常大鼠海馬DG內(nèi)的GABA在MWM實(shí)驗(yàn)過程中的變化。5.觀察正常大鼠海馬DG區(qū)給予GABAA和GABAB受體激動(dòng)劑對空間學(xué)習(xí)記憶能力的影響。本實(shí)驗(yàn)的結(jié)果如下:1.在4d的MWM定位航行實(shí)驗(yàn)中,大鼠找到站臺(tái)所需的逃避潛伏期均隨著訓(xùn)練天數(shù)的增加而逐漸縮短(均為P0.05),但VD模型組的逃避潛伏期明顯長于假手術(shù)組(P0.05);訓(xùn)練開始第5天進(jìn)行空間探索實(shí)驗(yàn),VD模型組穿越原平臺(tái)區(qū)的次數(shù)明顯少于假手術(shù)組(P0.05)。2.VD大鼠海馬DG區(qū)細(xì)胞外液中的GABA濃度為0.78±0.11μM,與假手術(shù)組(0.15±0.01μM)相比,GABA的濃度明顯增加(P0.05)。3.與假手術(shù)組相比,VD大鼠海馬DG區(qū)GABAA受體的表達(dá)沒有明顯變化(P0.05),而GABAB受體表達(dá)顯著減少(P0.05)。4.每天訓(xùn)練前往VD大鼠海馬DG區(qū)微量注射1μl的GABAA受體阻斷劑Bicuculline,不影響MWM實(shí)驗(yàn)中的逃避潛伏期和穿越原站臺(tái)區(qū)次數(shù)(均為P0.05)。5.每天訓(xùn)練前往VD大鼠海馬DG區(qū)微量注射1μl的GABAB受體阻斷劑Saclofen,明顯縮短MWM定位航行實(shí)驗(yàn)中的逃避潛伏期,并增加MWM空間探索實(shí)驗(yàn)中的穿越原站臺(tái)區(qū)的次數(shù)(均為P0.05)。6.假手術(shù)組大鼠DG區(qū)的Glu、G1n和Gly在Morris水迷宮訓(xùn)練過程中明顯增加(均為P0.05),而VD模型大鼠DG區(qū)的Glu和G1n雖有所增加,但增加明顯遲于假手術(shù)組,且DG區(qū)的Gly在訓(xùn)練過程中無明顯變化。7.每天訓(xùn)練前往VD大鼠海馬DG區(qū)微量注射lμ1的GABAA受體阻斷劑Bicuculline,對MWM實(shí)驗(yàn)中海馬DG內(nèi)的Glu、Gln和Gly含量沒有產(chǎn)生明顯的影響。8.每天訓(xùn)練前往VD大鼠海馬DG區(qū)微量注射1μl的GABAB受體阻斷劑Saclofen,可部分翻轉(zhuǎn)VD對Morris水迷宮訓(xùn)練過程中DG區(qū)Glu、G1n以及Gly變化的抑制作用。9.在4d的MWM定位航行實(shí)驗(yàn)中,正常大鼠的逃避潛伏期均隨訓(xùn)練天數(shù)的增加而逐漸縮短(P0.05),而訓(xùn)練第五天的空間探索實(shí)驗(yàn)中穿越原站臺(tái)區(qū)的次數(shù)為7.50±0.43 times/120s。其間,海馬DG區(qū)的GABA含量隨著訓(xùn)練天數(shù)的增加逐漸下降(P0.05)。10.每天訓(xùn)練前向海馬DG區(qū)微量注射1μl的GABAA受體激動(dòng)劑Muscimol或GABAB受體激動(dòng)劑Baclofen,30min后進(jìn)行MWM實(shí)驗(yàn)。在4d的定位航行實(shí)驗(yàn)中,對照組大鼠的逃避潛伏期隨著訓(xùn)練天數(shù)的增加而逐漸減少,但Muscimol組和Baclofen組大鼠的逃避潛伏期沒有出現(xiàn)明顯的變化,兩組的平均逃避潛伏期均明顯長于對照組(均為P0.05)。訓(xùn)練第五天的空間探索實(shí)驗(yàn)中,Baclofen組的穿越原站臺(tái)區(qū)次數(shù)明顯低于對照組(P0.05),而Muscimol組的穿越原站臺(tái)區(qū)次數(shù)雖然有所下降,但與對照組相比無顯著性差異(P0.05)。本實(shí)驗(yàn)的結(jié)論如下:1.VD模型大鼠空間記憶損害與海馬DG區(qū)的GABA濃度增加有關(guān)。2.VD模型大鼠海馬DG區(qū)內(nèi)增加的GABA主要通過GABAB受體參與其空間學(xué)習(xí)記憶的損害。3.海馬DG區(qū)的Glu、G1n以及Gly參與大鼠的空間學(xué)習(xí)記憶過程,而DG內(nèi)的GABAB受體可能通過抑制這些氨基酸類物質(zhì)的變化導(dǎo)致VD模型大鼠的空間學(xué)習(xí)記憶損害。
[Abstract]:Vascular Dementia (VD) is the second most common age-related neurological disorder after Alzheimer's disease. The main symptoms of VD are progressive cognitive impairment caused by cerebrovascular injury and cumulative brain tissue damage. Learning and memory are the basis of various cognitive functions and research. Hippocampus is the key part of learning and memory, especially plays an important role in spatial learning and memory. Mammalian hippocampus is mainly divided into CA1, CA3 and dentate gyrus (DG), and DG as the entry point of information into the hippocampus, processes and encodes spatial information, and plays a role in spatial learning and memory. Although studies have shown that the hippocampal DG region may play an important role in the spatial learning and memory impairment of VD, the neurotransmitters and their roles in DG involved in the spatial memory impairment of VD have not been fully understood. In the central nervous system, a large number of GABAergic neurons and their GABAA and GABAB receptors also exist in the hippocampus of DG. Although studies have shown that GABA and its GABAA/GABAB receptors in DG participate in regulating synaptic plasticity and some learning and memory processes, their roles in VD spatial learning and memory impairment have not been reported. VD model was established by permanent ligation of common artery. The effects of GABA and its receptors in hippocampal DG region on spatial learning and memory impairment in vascular dementia rats were studied by microdialysis, high performance liquid chromatography, Morris water maze (MWM) and immunohistochemistry. The specific contents of the experiment are as follows: 1. VD model rats were made by bilateral common carotid artery ligation. The spatial learning and memory abilities were observed by MWM. The GABA content in extracellular fluid of DG region was determined by brain microdialysis and high performance liquid chromatography. The expression of GABAA and GABAB receptors in DG region was observed by immunohistochemical staining. 2. The effects of GABAA and GABAB receptor blockers on spatial learning and memory of VD model rats were observed by intracerebral injection of GABAA and GABAB receptor blockers into the DG region of the hippocampus. The effects of glutamine, G1n, Gly and taurine (Tau) on spatial learning and memory were observed. 4. The changes of GABA in normal rat hippocampal DG during the MWM experiment were observed. 5. The effects of GABAA and GABAB receptor agonists on spatial learning and memory were observed in normal rat hippocampal DG. The results of this experiment were as follows: 1. The escape latency of VD model group was significantly longer than that of sham operation group (P 0.05), and the number of times that VD model group crossed the original platform area was significantly less than that of sham operation group (P 0.05). The concentration of GABA in extracellular fluid of hippocampal DG region was 0.78 [0.11] mu M. Compared with sham operation group, the concentration of GABA increased significantly (P 0.05). 3. Compared with sham operation group, the expression of GABAA receptor in hippocampal DG region of VD rats did not change significantly (P 0.05), but the expression of GABAB receptor decreased significantly (P 0.05). 4. Bicuculline, a 1-ml GABAA receptor blocker, did not affect the escape latency and the number of times crossing the original platform in the MWM experiment (both P 0.05). 5. The 1-ml Saclofen, a GABAB receptor blocker, was microinjected into the DG area of the hippocampus of VD rats every day, which significantly shortened the escape latency in the MWM navigation experiment and increased the spatial exploration of MWM. Glu, G1n and Gly in the DG area of sham-operated rats increased significantly during the training of Morris water maze (all P 0.05). Glu and G1n in the DG area of VD model rats increased slightly, but the increase was significantly later than that of sham-operated rats, and Gly in the DG area did not change significantly during the training. Microinjection of 1-mu-1 GABAA receptor blocker Bicuculline into the DG area of the hippocampus of VD rats had no significant effect on the contents of Glu, Gln and Gly in the DG area of the hippocampus in MWM experiment. The escape latency of normal rats was gradually shortened with the increase of training days (P 0.05), while the number of crossing the original platform was 7.50 (+ 0.43 times / 120 s) in the space exploration experiment on the fifth day of training. MWM test was performed 30 minutes after microinjection of 1 ml of GABAA receptor agonist Muscimol or GABAB receptor agonist Baclofen into the DG area of the hippocampus before training. The escape latency of the control group decreased gradually with the increase of training days, but the escape latency of the Muscimol and Baclofen groups decreased gradually. The average escape latency of the two groups was significantly longer than that of the control group (both P 0.05). In the space exploration experiment on the fifth day of training, the number of crossing the original platform area in the Baclofen group was significantly lower than that in the control group (P 0.05), while the number of crossing the original platform area in the Muscimol group decreased, but compared with the control group. There was no significant difference (P 0.05). The results of this study were as follows: 1. The spatial memory impairment of VD model rats was related to the increase of GABA concentration in hippocampal DG area. 2. The increased GABA in hippocampal DG area of VD model rats participated in the spatial learning and memory impairment mainly through GABAB receptors. 3. Glu, G1n and Gly in hippocampal DG area participated in the spatial learning and memory process of rats. GABAB receptors in DG may cause spatial learning and memory impairment in VD rats by inhibiting the changes of these amino acids.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R749.13

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