輕度低溫對腦缺血再灌注后細(xì)胞凋亡的影響

1、    輕度低溫對腦缺血再灌注后細(xì)胞凋亡的影響        摘要：目的:觀察輕度低溫對腦缺血再灌注后細(xì)胞凋亡的影響。方法: 沙鼠40只,隨機(jī)分假手術(shù)組、缺血組和輕度低溫(3233)2h組、6h組、12h組共5組。夾閉雙側(cè)頸總動脈20min再灌注72h觀察細(xì)胞凋亡。結(jié)果:FCM顯示缺血組海馬區(qū)凋亡細(xì)胞9.3%,輕度低溫2h、6h、12h組分別4.6%、2.6%、1.7%。HE顯示缺血組海馬CA1區(qū)損傷細(xì)胞為26.4%,低溫12h下降為4.4%。結(jié)論:輕度低溫能抑制腦缺血后凋亡

2、及壞死細(xì)胞。關(guān)鍵詞：腦缺血再灌注細(xì)胞凋亡輕度低溫The effects of mild hypothermia on apoptosis following cerebral ischemia and reperfusion in gerbilsLiu XuepingWang FengyanLeng Zhenpuet al（Depatrment of Neurology,Shandong Provincial Hospital,Jinan,250021）Abstract：Objective:To observe the protective effects of mild hypothermi

3、a on the apoptosis of hippocampal neurons after cerebral ischemia and reperfusion injury.Methods:40 gerbils were divided into five groups:shamoperated,ischemia and mild hypothermia 2h,6h and 12h,respectively bilateral common carotid arteries and were occluded for 20 min and reperfusion for 72h,apopt

4、osis was determined by FCM and HE dye.Results:The percentage of apoptotic cells was 9.7% in ischemia group,mild hypothermia 2h,6h,12h,could reduced the apoptotic cells (4.6%,2.6%,1.7%) respectively.The number of abnormal neurons (26.4%). was higher in ischemia group In contrast,the number of abnorma

5、l neurons were reduced lower (14.8%,8.3%,4.4%) in the mild hypothermia (2h,6h,12h).Conclusion:Mild hypothermia Could reduced apoptosis and necrosis following cerebral ischemia and reperfusion injury.Key Words：Cerebral ischemia and reperfusion injury Mild hypothermia Apoptosis在腦缺血損傷機(jī)制中,神經(jīng)元發(fā)生壞死及凋亡是共存的

6、,二者對神經(jīng)元的功能損害均具有重要作用,由于細(xì)胞凋亡出現(xiàn)早且持續(xù)時(shí)間長,如何阻止其發(fā)生或發(fā)展的研究已越來越受到國內(nèi)外學(xué)者關(guān)注1，2。目前,輕度低溫(即體溫3134)被視為最理想的腦保護(hù)措施,它可以縮小梗死面積,并能明顯促進(jìn)神經(jīng)功能恢復(fù)3,能否影響細(xì)胞凋亡罕有報(bào)道?，F(xiàn)將我們的動物實(shí)驗(yàn)結(jié)果報(bào)道如下:1材料和方法1.1 動物分組及腦缺血動物模型：雄性沙鼠40只,購自上海畜物醫(yī)學(xué)技術(shù)服務(wù)站,體重50100克,隨機(jī)分5組各8只。采用常規(guī)前腦缺血再灌注模型,1%戊巴比妥鈉30mg.kg-1 麻醉,頸正中切口,分離雙側(cè)頸總動脈。A組:假手術(shù)組,不夾閉血管;B組:缺血組,血管夾夾雙側(cè)頸總動脈20min時(shí)恢復(fù)血

7、流,再灌注72h斷頭處死,以上兩組術(shù)中用反饋熱墊保持顳肌溫度36.537(半導(dǎo)體測溫儀測定);C組:輕度低溫2h組,自夾閉雙側(cè)頸總動脈始,頭枕部浸入冰水,至10min使顳肌溫度降至3233,維持2小時(shí)后自然復(fù)溫;D、E組:即輕度低溫6h組、12h組,分別維持低溫6h、12h,余步驟同B組;將海馬組織置于-70電冰箱中。取右側(cè)半球頂葉冠狀切面用多聚甲醛固定。1.2實(shí)驗(yàn)儀器及試劑 ：SACSAN440型流式細(xì)胞儀(FCM),BectonDickinson公司生產(chǎn);碘化丙啶(PI)美國Sigma公司生產(chǎn)。1.3FCM檢測凋亡細(xì)胞百分率：將海馬組織研磨,用PBS制成單細(xì)胞懸液,離心10min,PBS洗

8、2次,70%酒精固定4過液，4離心去上清,加200L RNaseA37水浴30min,加800L PI染色液,4避光30min。用FCM測定并記錄激發(fā)波長488nm處紅色熒光,經(jīng)計(jì)算機(jī)分析,繪制細(xì)胞數(shù)和凋亡細(xì)胞分布。1.4HE染色：將固定的組織常規(guī)制成蠟塊,切成5m厚切片,常規(guī)脫蠟,常規(guī)HE染色,脫水透明封片,光鏡下觀察并記錄結(jié)果。1.5統(tǒng)計(jì)學(xué)處理：凋亡細(xì)胞及損傷細(xì)胞百分?jǐn)?shù)用均數(shù)±標(biāo)準(zhǔn)差,凋亡細(xì)胞組間比較用直接計(jì)算概率法,損傷細(xì)胞用二項(xiàng)分布兩樣本率比較檢驗(yàn)。2結(jié)果2.1FCM檢測凋亡細(xì)胞百分率結(jié)果見1。 1FCM檢測凋亡細(xì)胞百分率(注：A組：B組，B組：E組，均為P<

9、0.001;A組：C組，B組：D組，均為P<0.01；A組：D組，B組：C組，均為P<0.05；A組：E組，P>0.05。從中可以看出,A組8海馬組織極小有凋亡細(xì)胞,B組凋亡細(xì)胞數(shù)明顯增多。而C組、D組及E組凋亡細(xì)胞較B組明顯減少,E組凋亡細(xì)胞減少最顯著,提示輕度低溫能抑制缺血再灌注損傷所致細(xì)胞凋亡作用。2.2HE染色結(jié)果見表1,2,3。表1HE染色海馬CA1區(qū)損傷神經(jīng)元比較 組別  例數(shù) 損傷神經(jīng)元(%) 假手術(shù)組  8 3.2±2.6 缺血組  8 26.6±8.6* 輕度低溫2h 8  14.8

10、77;4.5* 輕度低溫6h 8  8.4±4.2* 輕度低溫12h 8 4.4±3.6* 注：各組與缺血組比較*P<0.0012HE染色缺血組海馬CA1區(qū)損傷神經(jīng)元(400) 3HE染色輕度低溫組海馬CA1損傷神經(jīng)元(400)B組及C組海馬區(qū)CA1、紋狀體可見有散在凋亡細(xì)胞呈核固縮,著色深,邊聚或裂解的凋亡小體;壞死細(xì)胞胞體腫脹,胞內(nèi)結(jié)構(gòu)破壞,胞漿淡染,而A組、D組及E組偶有上述變化。B組、C組海馬CA1區(qū)凋亡細(xì)胞各占損傷細(xì)胞的70%、50%,提示遲發(fā)性神經(jīng)元損傷以凋亡細(xì)胞為主。隨著低溫時(shí)間延長損傷細(xì)胞數(shù)進(jìn)一步減少。3討論近年來,一些研究表明,腦

11、缺血后細(xì)胞凋亡是缺血神經(jīng)元死亡的另一種形式,它參與梗死灶的形成。Chanrant1證實(shí)缺血1h,再灌注6h后,凋亡細(xì)胞占死亡細(xì)胞總數(shù)的比例,在皮質(zhì)約65%、紋狀體約51%、海馬區(qū)約85%。而全腦12min缺血模型中2,缺血第23天海馬區(qū)出現(xiàn)以單體200bp和雙體400bp為主的DNA條帶,無明顯DNA降解所致DNA涂片狀帶。提示輕中度缺血易導(dǎo)致以細(xì)胞凋亡為主的遲發(fā)性神經(jīng)元損害。我們通過兩種檢測手段綜合確定凋亡細(xì)胞存在。FCM繪制的DNA上表現(xiàn)出特征性亞二倍體峰,依據(jù)該峰所占比例進(jìn)行凋亡定量分析,五組中以缺血組該峰值最高,輕度低溫2h治療凋亡細(xì)胞明顯減少,隨著輕度低溫時(shí)間延長,凋亡細(xì)胞也相應(yīng)進(jìn)一

12、步減少。假手術(shù)組也測到極小量凋亡細(xì)胞,可能是腦組織的正常生理性死亡。HE染色在缺血組見凋亡細(xì)胞主要分布在海馬CA1區(qū)及紋狀體區(qū),以神經(jīng)元細(xì)胞為主,其形態(tài)特征是胞漿濃縮、胞核固縮、邊聚,有的核裂變成凋亡小體。近年來的實(shí)驗(yàn)表明,輕度低溫能明顯減輕腦缺血后腦組織損害,其作用機(jī)理主要包括以下幾個(gè)方面:(1)降低氧耗,減少乳酸堆積4。(2)保護(hù)血腦屏障,減輕腦水腫5。(3)抑制內(nèi)源性有毒產(chǎn)物(如谷氨酸、自由基)的產(chǎn)生6。(4)抑制Ca2+內(nèi)流,阻斷Ca2+超載引發(fā)一系列有害反應(yīng)7。沙鼠短暫腦缺血后實(shí)施低溫3233持續(xù)212h,壞死細(xì)胞及凋亡細(xì)胞與缺血組相比明顯減少,表明輕度低溫不但能抑制腦缺血后神經(jīng)元壞

13、死,而且能抑制細(xì)胞凋亡,這可能是輕度低溫對神經(jīng)元另一保護(hù)途徑。目前腦缺血后細(xì)胞凋亡的發(fā)生機(jī)制不十分清楚。許多實(shí)驗(yàn)顯示腦缺血能誘導(dǎo)腦組織c-fos、c-jun、p53、bc1-2等基因表達(dá),提高易損區(qū)p53基因表達(dá)能誘導(dǎo)細(xì)胞凋亡，抑制p53基因表達(dá)能減少細(xì)胞凋亡。Martinou采用轉(zhuǎn)基因鼠過量表達(dá)bc1-2能保護(hù)缺血性神經(jīng)元損傷。提示缺血過程中細(xì)胞凋亡的發(fā)生受基因調(diào)控710。此外,腦缺血時(shí)細(xì)胞內(nèi)Ca2+濃度增高被認(rèn)為是細(xì)胞凋亡的始動因素,Ca2+濃度增高可將核酸內(nèi)切酶激活,染色質(zhì)DNA被核酸內(nèi)切酶在核小體單位之間降解,產(chǎn)生300kbpDNA片段1。Greenland11發(fā)現(xiàn)超氧化物岐化酶能抑制

14、神經(jīng)元去除神經(jīng)營養(yǎng)因子出現(xiàn)的細(xì)胞凋亡。Banfoco12發(fā)現(xiàn)皮質(zhì)神經(jīng)元暴露短時(shí)間或低濃度過氧亞硝酸鹽環(huán)境能誘導(dǎo)以凋亡為特征遲發(fā)性神經(jīng)元損害,相反暴露長時(shí)間或高濃度過氧亞硝酸鹽環(huán)境能誘導(dǎo)壞死性細(xì)胞損傷?？梢娾}超載、自由基、NO在腦缺血所致的細(xì)胞凋亡發(fā)生過程中亦起重要作用。輕度低溫抑制凋亡發(fā)生可能是通過減少自由基生成,抑制大量Ca2+內(nèi)流,從而抑制核酸內(nèi)切酶活性而起作用。關(guān)于輕度低溫抑制細(xì)胞凋亡機(jī)理,還有待于進(jìn)一步研究。作者單位：劉雪平（山東省立醫(yī)院神經(jīng)內(nèi)科250021）王鳳焰（山東省立醫(yī)院神經(jīng)內(nèi)科250021）冷珍璞（山東省立醫(yī)院神經(jīng)內(nèi)科250021）高聆（山東省立醫(yī)院神經(jīng)內(nèi)科250021）薛瑞

15、賢（山東省立醫(yī)院神經(jīng)內(nèi)科250021）參考文獻(xiàn)：1Marlangue CC,Margain I,Represa A.et al,Apoptosis and necrosis after reversible focal ischemia an in situ DNA fragmentation analysis,J Cereb Blood Metab,1996，16:1862Kihara SI,Shiraishi T,Nakagawa S,et al.Visualization of DNA double strand breaks in the gerbil hippocampal CA1

16、following transient ischemia.Neurosci Lett,1994，175:1333Hoffman WE,Werner C,Baughman VL,et al.Postischemic treatment with hypothermia improves out come from incomplete cerebral ischemia in rats,J neurosurg Anesthesiol,1991，3:344Chopp M,Knight R,Tidwell CD,et al.The metabolic effect of mild hypotherm

17、ia on global cerebral ischemia and recirculation in the rat.J cereb blood Flow Metab,1989,9:1415Dietrich WD,Busto R,Halley M,et al.The important of brain temperature in alterations of the blood-brain-barrier following cerebral ischemia,J Neuropath Exp Neurol，1990，49:4866Globus MY,Alonso ON,Dietrich

18、WD,et al.Glutamate release and free radical production following brain injury:effect of posttraumatic hypothermia.J Neurochem.1995，65:17047Mitani A Kadoya F,Kataoke,Temperature dependence of hypoxia-iduced calcium accumulation in gerbil hippocampal slices. Brain Res,1991，68(4):10038Estus S,Zaks W,Freeman RS,et al. Altered gene expression in neurous during programmed cell death:Identification of c-jun as necessary for neuronal apoptosis.J Cell Brain,1994，127:17179Crumrina RC,Thomas AL,Moegan PF.Attenuation of p53 expression protects against focal ischemic damage in transgen