龍莉莉,肖波,李國(guó)良,李蜀渝,易芳,陳鍶,畢方方

中南大學(xué)湘雅醫(yī)院神經(jīng)內(nèi)科,長(zhǎng)沙 410008

龍莉莉,肖波#,李國(guó)良,李蜀渝,易芳,陳鍶,畢方方

中南大學(xué)湘雅醫(yī)院神經(jīng)內(nèi)科,長(zhǎng)沙 410008

目的:探討氯化鋰(LiCl)-匹羅卡品(PILO)致大鼠的行為學(xué)特征及海馬病理改變。方法:建立改良的LiCl-PILO致大鼠模型,觀察大鼠行為,尼氏染色觀察不同時(shí)間點(diǎn)大鼠海馬病理改變,FJB染色觀察大鼠海馬神經(jīng)元及其軸突、樹突變性。結(jié)果:大鼠腹腔注射LiCl-PILO后,癲持續(xù)狀態(tài)(SE)誘發(fā)成功率為92.5%,死亡率21.25%;尼氏染色結(jié)果顯示,實(shí)驗(yàn)組大鼠海馬神經(jīng)元于SE后7 d和60 d缺失最明顯,與對(duì)照組相比,齒狀回顆粒細(xì)胞減少(P＜0.05),CA 1、CA 3區(qū)錐體細(xì)胞和門區(qū)神經(jīng)元均顯著減少(P＜0.01);FJB染色結(jié)果顯示,實(shí)驗(yàn)組大鼠海馬神經(jīng)元于SE后7 d和60 d變性最明顯,主要集中在CA 1、CA 3區(qū)錐體細(xì)胞層和門區(qū),SE后7 d海馬腔隙-分子層亦可見變性的神經(jīng)元軸突和樹突。結(jié)論:改良后的LiCl-PILO致模型SE誘發(fā)成功率高,死亡率低,可基本復(fù)制人類顳葉癲的發(fā)作特點(diǎn)及病理改變;顳葉癲海馬神經(jīng)元的缺失可能是由于神經(jīng)元的變性死亡。

顳葉癲;氯化鋰-匹羅卡品;大鼠;海馬

1 材料與方法

1.1 材料 ①動(dòng)物:6～8周齡健康雄性SD大鼠120只,體質(zhì)量(250±20)g,由中南大學(xué)湘雅醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物學(xué)部提供,在室溫18～25℃、相對(duì)濕度50%～60%、人工12 h晝/夜循環(huán)照明環(huán)境中用全價(jià)營(yíng)養(yǎng)飼料分籠飼養(yǎng),能自由攝食及飲水,每日定時(shí)清洗籠舍。②主要試劑與材料:LiCl、0.1%焦油紫(購于美國(guó) Sigma公司);PILO(購于美國(guó)Boehringer M annheim公司);FJB(購于美國(guó)Chemicon公司);冰凍切片機(jī)(購于德國(guó)SLEE Tecknik公司);LSM 510型激光共聚焦顯微鏡(購于德國(guó)ZEISS公司);HPIAS-1000高清晰彩色病理圖象分析系統(tǒng)(購于武漢大學(xué)影像工程公司)。

1.2 方法

1.2.2 標(biāo)本取材與制備 在各時(shí)間點(diǎn)以4%多聚甲醛對(duì)大鼠進(jìn)行灌注取材,取腦置4℃4%多聚甲醛固定過夜后,蔗糖過夜沉底。冰凍切片機(jī)行20μm腦冠狀連續(xù)切片(隔3取1,每只大鼠取4套),收集有海馬的腦片置于0.01mo l/L磷酸鹽緩沖液中。將腦片貼片于用多聚賴氨酸包被的載玻片上,切片充分晾干后,-80℃冰箱保存?zhèn)溆谩?/p>

1.2.3 尼氏染色 切片自-80℃冰箱取出后晾干,蒸餾水漂洗,0.1%焦油紫室溫下染色10min,鏡下觀察,如染色過深,將切片浸入95%乙醇中分色,鏡下監(jiān)視至滿意效果;自來水沖洗,脫水(70%乙醇3m in、95%乙醇3min、100%乙醇1m in×3次),二甲苯透明(5min×2次),中性樹脂封片;鏡下觀察海馬區(qū)域的病理改變。

1.2.4 FJB染色 50℃烤箱內(nèi)使切片充分干燥(≥0.5 h),浸入蒸餾水2m in,浸入含1%NaOH的80%乙醇(20m L 5%NaOH加入80m L無水乙醇中)內(nèi)5m in;浸入70%乙醇2min,浸入蒸餾水2min,浸入0.06%高錳酸鉀溶液10min(搖床上輕微振動(dòng)以使背景均勻),蒸餾水漂洗 2 m in后放入0.0004%FJB溶液(4m L 0.01%FJB固定液加入96m L 0.1%醋酸)染色20 min(該步驟及以下步驟均在暗室中進(jìn)行),蒸餾水漂洗1min×3次后,傾掉剩余水,50℃烤箱內(nèi)充分烤干(約5～10min),二甲苯透明≥1min,中性樹脂封片,激光共聚焦顯微鏡下觀察。

1.2.5 細(xì)胞計(jì)數(shù) 用HPIAS-1000高清晰彩色病理圖像分析系統(tǒng)對(duì)切片進(jìn)行圖像分析,由2名不熟悉實(shí)驗(yàn)的技術(shù)員進(jìn)行尼氏染色的神經(jīng)元計(jì)數(shù)。每個(gè)時(shí)間點(diǎn)每只大鼠隨機(jī)選3～4張腦切片,每組24張切片,用于細(xì)胞計(jì)數(shù)及統(tǒng)計(jì)學(xué)分析。2組大鼠選取斷面水平相似的組織切片,所有切片在相同強(qiáng)度及相同放大倍數(shù)下進(jìn)行觀察計(jì)數(shù)。在40倍物鏡下,對(duì)CA 1、CA 3區(qū)錐體細(xì)胞層、齒狀回顆粒細(xì)胞層和門區(qū)隨機(jī)選取5個(gè)不重疊視野,計(jì)算每個(gè)視野神經(jīng)元數(shù)目,取均數(shù)作為各時(shí)間點(diǎn)各區(qū)域尼氏染色的細(xì)胞數(shù)目。

1.3 統(tǒng)計(jì)學(xué)處理 用SPSS 12.0統(tǒng)計(jì)軟件處理數(shù)據(jù),結(jié)果以(x±s)表示,成組設(shè)計(jì)多樣本均數(shù)用單因素方差分析,2組之間比較用t檢驗(yàn),P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 行為學(xué)特征 LiCl腹腔注射后,實(shí)驗(yàn)組大鼠的行為活動(dòng)無變化;PILO注射5～30min后,大鼠開始呈現(xiàn)外周膽堿能反應(yīng):立毛、流涎、顫抖及流血淚,同時(shí)或先后出現(xiàn)刻板行為:凝視不動(dòng)、咀嚼、吸鼻或探索行為、濕狗樣震顫、反復(fù)頭頸上仰,隨后出現(xiàn)眨眼、面肌痙攣、點(diǎn)頭,最后出現(xiàn)反復(fù)雙側(cè)前肢陣攣,伴直立、跌倒或翻轉(zhuǎn),部分動(dòng)物出現(xiàn)四肢強(qiáng)直-陣攣發(fā)作。開始發(fā)作不頻繁,隨時(shí)間延長(zhǎng)發(fā)作頻率增高。實(shí)驗(yàn)組中74只達(dá)到RacineⅢ～Ⅴ級(jí)并進(jìn)入SE;進(jìn)入SE后,13只抽搐致死,其余大鼠在24～72 h后進(jìn)入靜止期;靜止期大部分行為正常,4只抽搐嚴(yán)重的大鼠進(jìn)入靜止期后活動(dòng)遲鈍,不能主動(dòng)進(jìn)食水,數(shù)天后衰竭死亡;存活的大鼠在15～45 d后均出現(xiàn)自發(fā)性強(qiáng)直-陣攣性發(fā)作,持續(xù)時(shí)間短,約0.5～1min,發(fā)作次數(shù)從1 d數(shù)次至數(shù)天1次,慢性期內(nèi)無死亡。SE誘發(fā)成功率為92.5%,總死亡率為21.25%,模型成功率為71.25%。

2.2 尼氏染色結(jié)果 對(duì)照組大鼠CA1、CA 3區(qū)及齒狀回有大量致密的錐體細(xì)胞及顆粒細(xì)胞,門區(qū)有中等量神經(jīng)元,排列整齊,形態(tài)完整,胞漿內(nèi)尼氏小體豐富。PILO致SE后,海馬齒狀回、門區(qū)和CA 1、CA 3區(qū)均有不同程度神經(jīng)元減少;CA 1和CA3區(qū)錐體細(xì)胞層神經(jīng)元排列紊亂,可見部分形態(tài)不完整,細(xì)胞腫脹或皺縮、輪廓模糊、界限不清,細(xì)胞間距加大,胞漿尼氏小體減少,齒狀回顆粒細(xì)胞邊界不整齊,排列松散、增寬,呈現(xiàn)發(fā)散改變;SE后7 d和60 d,CA1區(qū)膠質(zhì)細(xì)胞增生明顯。急性期和靜止期以SE后7 d神經(jīng)元減少最明顯,與對(duì)照組相比,齒狀回顆粒細(xì)胞減少(P＜0.05),CA1、CA 3區(qū)及門區(qū)神經(jīng)元均顯著減少(P＜0.01);慢性自發(fā)發(fā)作期以SE后60 d神經(jīng)元減少最明顯,與對(duì)照組相比,CA 1區(qū)錐體細(xì)胞缺失最嚴(yán)重(P＜0.01),CA3區(qū)錐體細(xì)胞和門區(qū)神經(jīng)元顯著減少(P＜0.01),齒狀回顆粒細(xì)胞也出現(xiàn)丟失(P＜0.05),見表 1,圖 1A-1L 。

2.3 FJB染色結(jié)果 FJB染色檢測(cè)實(shí)驗(yàn)組大鼠海馬變性神經(jīng)元結(jié)果顯示,各時(shí)間點(diǎn)中,SE后7 d和60 d神經(jīng)元變性最明顯。SE后7d,CA 1區(qū)和門區(qū)均可見FJB染色陽性的變性神經(jīng)元,在CA1區(qū)變性主要集中于錐體細(xì)胞層;CA 3區(qū)未見明顯FJB標(biāo)記的神經(jīng)元;海馬腔隙-分子層可見變性的神經(jīng)元軸突及樹突。SE后60 d,CA 1區(qū)、CA3區(qū)和門區(qū)均可見明顯的變性神經(jīng)元及其軸、樹突,變性在CA 1和CA 3區(qū)主要集中于錐體細(xì)胞層,CA 1區(qū)始層和輻狀層亦有少許,見圖2A-2F。

表1 2組大鼠SE后 7 d及60 d海馬不同部位尼氏染色神經(jīng)元計(jì)數(shù)(x-±s,個(gè))

圖 1A-L 對(duì)照組海馬(A)、CA 1區(qū)(D)、門區(qū)(G)、CA 3區(qū)(J),實(shí)驗(yàn)組大鼠SE后7 d海馬(B)、CA 1區(qū)(E)、門區(qū)(H)、CA 3區(qū)(K),SE后60 d海馬(C)、CA 1區(qū)(F)、門區(qū)(I)、CA 3區(qū)(L)尼氏染色結(jié)果 A-C:比例尺為 200μm,D-I:比例尺為100μm,J-L:比例尺為50μm

3 討論

圖2 A-F 實(shí)驗(yàn)組大鼠SE后7 d CA 1區(qū)(A)、門區(qū)(B)、海馬腔隙-分子層(C),SE后60 d CA 1區(qū)(D)、門區(qū)(E)、CA 3區(qū)(F)FJB染色結(jié)果 A,B,D,E,F:比例尺為50μm,C:比例尺為20μm

以海馬神經(jīng)元變性缺失和神經(jīng)膠質(zhì)增生為主要表現(xiàn)的海馬硬化是顳葉癲的標(biāo)志性病理改變[13],約60%～70%的顳葉癲患者伴有海馬硬化[14]。海馬硬化最早由Falcomer等[15]提出,又稱為顳葉內(nèi)側(cè)硬化(medial tem poral sclerosis)或Ammon角硬化(Ammon's sclerosis),其主要病理改變?yōu)?CA 1區(qū)和CA 3區(qū)錐體細(xì)胞嚴(yán)重缺失,門區(qū)神經(jīng)元減少,齒狀回顆粒細(xì)胞減少。神經(jīng)元丟失具有一定區(qū)域選擇性,一般以CA1起始部最明顯,其次為齒狀回門區(qū)和CA 3區(qū),齒狀回顆粒細(xì)胞層受累相對(duì)輕微,下托(subicu lum)不受累或輕度受累,另外可能見到杏仁核神經(jīng)元減少伴膠質(zhì)增生[15]。本研究尼氏染色結(jié)果顯示,PILO致后CA 1、CA3區(qū)錐體細(xì)胞和門區(qū)神經(jīng)元大量減少,齒狀回顆粒細(xì)胞部分減少,可見膠質(zhì)細(xì)胞增生;FJB染色顯示CA 1、CA 3區(qū)和門區(qū)有部分變性神經(jīng)元;以上改變均類似于人類顳葉癲海馬硬化的病理特征。尼氏染色所示的神經(jīng)元缺失與FJB染色所示的變性神經(jīng)元增多相對(duì)應(yīng),提示顳葉癲海馬神經(jīng)元的缺失可能是由于神經(jīng)元的變性死亡。

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A Rat Temporal Lobe Epilepsy M odel Induced by LiCl-pilocarpine

LONG Li-li▲,X IAO Bo,LI Guo-liang,LIShu-yu,YI Fang,CHEN Si,B I Fang-fang.▲Department of Neurology,X iangya Hospital of Central South University,Changsha 410008,China

Ob jective:To investigate the behavioral characteristics and neuro-histological progression of hippocam pus in lithium-pilocarpine induced tem poral lobe epilepsy m odel in rats.Methods:M odified LiCl-pilocarpine induced animalm odel w as estab lished.Nissl stainning was used to observe the pathological changes of hippocam pus;FJB stainning was used to detect the degeneration of hippocam pal neurons and their neurophils.Results:A fter lithium-chloride and pilocarpine administration,92.5%rats generated SE successfu lly,and the Mortality rate was 21.25%.Nissl stain revealed that loss of hippocam palneurons wasmostevident on 7 d and 60 d after SE.Significant lossof hilar neurons and pyramidal neurons was present in CA 1 area(P＜0.01),while loss of the granu lar cells in the dentate gy rusw as relatively slight(P＜0.05).FJB stain showed that degeneration of the hippocampalneurons wasmost evidenton day 7 and day 60 after SE,main ly restricted in the pyramidal layer and hilar area.Degenerated neurophils cou ld be seen in the lacunosum-moleculare(lm)layer on 7d after SE.Conclusion:M odified LiCl-pilocarpine induced temporal lobeepilepsy model could generate SE in rats successfully with low mortality.It canm imic the clinical features and pathologic changes of hum an temporalepilepsy.Loss of neurons in the hippocampusof temporal lobeepilepsy may be due to the cellular degeneration and death.

temporal lobe epilepsy;LiCl-pilocarpine;rat;hippocam pus

R741;R742.1

A

1001-117X(2010)02-0083-06

10.3870/sjsscj.2010.02.002

衛(wèi)生部部屬醫(yī)院臨床學(xué)科重點(diǎn)項(xiàng)目(2007-353);湖南省科技廳科技計(jì)劃項(xiàng)目(2007TP4005)

2009-12-30

#【通訊作者】肖波,Tel:86-731-84327236,E-mail:xiaobo62_xy@yahoo.com.cn。