γ-氨基丁酸A受體在大鼠急性腦缺血中的變化

1、-氨基丁酸A受體在大鼠急性腦缺血中的變化         11-01-16 16:07:00     編輯：studa20                        作者：孫智敏張茂林張俊清薛繼嬌【摘要】  目的：觀察大鼠大腦皮質(zhì)-氨基

2、丁酸A受體(-aminobutyric acid A receptors,GABAAR)在腦缺血不同時段的表達(dá)及其動態(tài)變化的規(guī)律，探討其在急性腦缺血中變化的意義。方法：65只雄性Wistar大鼠隨機(jī)分為對照組，腦缺血6 h組、12 h組、24 h組、3 d組、7 d組、14 d組(均為手術(shù)組)，假手術(shù)組;采用大鼠大腦中動脈栓塞(MCAO)模型，至規(guī)定時間點(diǎn)取大腦組織，行HE染色，用光學(xué)顯微鏡觀察神經(jīng)元損傷程度;用免疫組織化學(xué)技術(shù)判斷神經(jīng)元陽性表達(dá)的強(qiáng)弱;用形態(tài)分析系統(tǒng)檢測各組GABAAR表達(dá)的陽性細(xì)胞數(shù)。結(jié)果：各手術(shù)組GABAAR表達(dá)均低于假手術(shù)組及對照組，與對照組相比，缺血6 h其表達(dá)既有降

3、低，至12 h降至最低。而假手術(shù)組與對照組相比無統(tǒng)計學(xué)意義。結(jié)論：急性腦缺血可引起GABAAR表達(dá)下調(diào)，致使其對興奮性氨基酸毒性的抑制作用減弱，這種下調(diào)和減弱程度與腦缺血的嚴(yán)重程度相一致，提示GABAAR在缺血性腦損傷中有神經(jīng)保護(hù)作用,可能減緩了缺血性腦血管病的進(jìn)程，同時GABAAR作為腦內(nèi)最主要的一種抑制性氨基酸神經(jīng)遞質(zhì)受體,除通過其配體門控性Cl-通道介導(dǎo)的效應(yīng)對抗“興奮毒性”作用外,GABAAR本身也因缺血所致理化因素的改變而發(fā)生變化。 【關(guān)鍵詞】  腦缺血;-氨基丁酸A受體;大鼠AbstractObjecive: To observe the expression of GA

4、BAAR and the dynamic regulation in the cortex of rats with focal cerebral ischemia in different phases and to investigate its significance in acute ischemia. Methods: Forty-eight male Wistar rats were randomly divided into 13 groups: The normal control, sham operation and ischemic operation group(su

5、bdivided into 6,12,24 hour and 3,7,14 days) by middle cerebral artery occlusion model. Brain specimens of the rats from each group were obtained for HE staining to observe the damage of neurons with light microscope and to judge the positive expression of neurons in immunohistochemistry technique, a

6、nd then to determine the positive cell number expressed by GABAAR in each group with morphologic analysis system. Results: Compared with the normal control group and the sham operation group, the expression of GABAAR in each ischemia operation group was decreased 6h after ischemia and reached the bo

7、ttom within 12 hours after ischemia. However, there was no significant difference between the normal control group and sham operation group. Conclusion: Acute cerebral ischemia can cause down-regulated expression of GABAAR and weaken its inhibitory action on glutamate excitotoxicity, which is accord

8、ant with the degree of cerebral ischemia injury, which suggests that GABAAR plays a role of nerve protection in cerebral ischemia injury and may slow down the progression of ischemic cerebrovascular disease, and that GABAAR, a major inhibitory neurotransmitter receptor of glutamate, undergoes some c

9、hanges because of the physicochemical factors brought about by ischemia as it inhibits excitotoxicity through the action mediated by ligand gated chloride channel.Key wordsCerebral ischemia; Gamma-aminobutyric acid A receptors; Rats腦缺血時,由于各類氨基酸大量釋放,興奮性氨基(exicitatoryamino acids,EAA)與抑制性氨基酸(inhibitory

10、 amino acids,IAA)間平衡被破壞,是導(dǎo)致神經(jīng)元急性和遲發(fā)性“興奮性毒性”損傷的關(guān)鍵因素。GABAA受體(GABAAR)作為腦內(nèi)最主要的一種抑制性氨基酸神經(jīng)遞質(zhì)受體,除通過其配體門控性Cl-通道介導(dǎo)的效應(yīng),對抗興奮性氨基酸的興奮性毒作用外,GABAAR本身也因缺血所致理化因素的改變而發(fā)生變化。為了研究GABAA受體在腦缺血中的作用機(jī)制，我們在MCAO模型基礎(chǔ)上，動態(tài)研究GABAAR在缺血性腦血管病中表達(dá)的變化，期待從分子水平揭示GABAAR在腦缺血中的作用機(jī)制。1材料與方法1.1實(shí)驗(yàn)動物及材料健康Wistar雄性大鼠65只，體重260310 g,由內(nèi)蒙古大學(xué)動物試驗(yàn)中心提供。鼠抗兔

11、GABAAR多克隆抗體購于美國麥莎公司;即用型SABC免疫組化染色試劑盒、抗體稀釋液、DAB顯色試劑盒、PBS緩沖液、枸櫞酸鹽緩沖液、中性樹膠均購于武漢博士德公司。1.2實(shí)驗(yàn)方法1.3圖像分析采用江蘇省捷達(dá)科技公司的形態(tài)分析系統(tǒng)進(jìn)行圖像分析，每張切片隨機(jī)選出10個單位面積，測量免疫組化染色的陽性細(xì)胞數(shù)。1.4統(tǒng)計學(xué)分析采用SPSS 10.0統(tǒng)計軟件，各組數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，兩組間數(shù)據(jù)比較采用t檢驗(yàn)，并進(jìn)行方差齊性檢驗(yàn)，P<0.05為差異有統(tǒng)計學(xué)意義。2結(jié)果2.1神經(jīng)細(xì)胞病理形態(tài)學(xué)改變光鏡觀察對照組、假手術(shù)組神經(jīng)細(xì)胞形態(tài)結(jié)構(gòu)正常、核圓淡染、核仁明顯，未見組織壞死，見圖1;手術(shù)組缺血灶內(nèi)可見血管和神經(jīng)元周圍膠質(zhì)成分腫脹，神經(jīng)元呈三角形或桿狀，胞核偏位并深染，有的甚至裂解、消失，見圖2;腦缺血時間越長，細(xì)胞變化越明顯，缺血重的組織內(nèi)甚至可見液化性壞死灶，灶內(nèi)神經(jīng)元?dú)埓孑^少甚至缺失。2.2免疫組化結(jié)果免疫組化陽性細(xì)胞信號呈棕黃色，在正常組織內(nèi)GABAAR主要位于細(xì)胞膜和胞漿內(nèi)，見圖3;缺血后大腦皮質(zhì)內(nèi)GABAAR陽性神經(jīng)元顯著減少，有些神經(jīng)元胞漿內(nèi)著色亦變淡，見圖4。與相應(yīng)時間點(diǎn)HE染色結(jié)果比較，發(fā)現(xiàn)GABAAR陽性神經(jīng)細(xì)胞減少以缺血灶及其周圍部位最為顯著，在一些缺血嚴(yán)重的神經(jīng)組織內(nèi)看