李曉青，種 莉，劉 鵬，劉 玥，唐 鵬，陳 麗，張 欣，侯 辰，李 銳，郭民俠

?

肝性腦病大鼠腦超微結(jié)構(gòu)的改變

李曉青*，種 莉，劉 鵬，劉 玥，唐 鵬，陳 麗，張 欣，侯 辰，李 銳，郭民俠

（陜西省人民醫(yī)院神經(jīng)內(nèi)三科，西安 710068）

通過觀察肝性腦病大鼠腦部分區(qū)域及氨對體外培養(yǎng)大鼠神經(jīng)元超微結(jié)構(gòu)的改變，討論其病理發(fā)生機(jī)制。選用健康雄性SD大鼠12只，隨機(jī)分為肝性腦病模型組和正常對照組兩組，每組6只。用電子透射顯微鏡觀察硫代乙酰胺誘導(dǎo)的肝性腦病大鼠和體外氨中毒大鼠皮質(zhì)神經(jīng)元的超微結(jié)構(gòu)。肝性腦病大鼠神經(jīng)元細(xì)胞數(shù)量減少；神經(jīng)元線粒體腫脹，尼氏體數(shù)量明顯減少；可見凋亡各期表現(xiàn)。神經(jīng)膠質(zhì)細(xì)胞細(xì)胞器減少，黑質(zhì)的超微結(jié)構(gòu)改變程度較基底核略重。體外培養(yǎng)氨中毒神經(jīng)元變化：神經(jīng)元細(xì)胞數(shù)量明顯減少；細(xì)胞明顯水腫，線粒體明顯腫脹，尼氏體顯著減少；可見不同時(shí)期的凋亡表現(xiàn)。肝性腦病大鼠腦超微結(jié)構(gòu)改變明顯，其主要機(jī)制可能與氨中毒引起的神經(jīng)元凋亡有關(guān)。

肝性腦??；氨；細(xì)胞培養(yǎng)；超微結(jié)構(gòu)

肝性腦病（hepatic encephalopathy）是一種由于急、慢性肝功能嚴(yán)重障礙或各種門靜脈-體循環(huán)分流異常所致的、以代謝紊亂為基礎(chǔ)的、輕重程度不同的神經(jīng)精神異常綜合征[1]。其發(fā)病機(jī)制與病理生理較復(fù)雜，迄今仍未完全闡明。目前認(rèn)為游離氨濃度升高是導(dǎo)致肝性腦病的最主要的因素[2]，黑質(zhì)-紋狀體通路形態(tài)學(xué)及功能改變也起一定作用[3,4]。其神經(jīng)組織超微結(jié)構(gòu)的改變鮮見報(bào)道。本研究分別對肝性腦病大鼠基底節(jié)區(qū)、黑質(zhì)以及體外培養(yǎng)的氨中毒神經(jīng)元超微結(jié)構(gòu)進(jìn)行觀察，以證明肝性腦病神經(jīng)超微結(jié)構(gòu)改變的主要機(jī)制與氨中毒引起的神經(jīng)元凋亡有關(guān)，進(jìn)一步闡明黑質(zhì)?紋狀體系統(tǒng)損傷在肝性腦病發(fā)病機(jī)制中的作用。

1 材料與方法

1.1 動物模型

健康雄性SD大鼠12只，西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動物中心，體質(zhì)量（230±17）g，隨機(jī)分為兩組，每組6只：（1）肝性腦病模型組，硫代乙酰胺350mg/kg腹腔注射，連續(xù)3d，急性肝性腦病造模[5]，血氨濃度（592±70）μmol/L，顯示造模成功；（2）正常對照組，同等劑量生理鹽水腹腔注射，連續(xù)3d，血氨濃度（34±12）μmol/L（＜0.01）。

1.2 肝性腦病大鼠各腦區(qū)超微結(jié)構(gòu)改變研究

大鼠麻醉后，用4%多聚甲醛/0.1mol/L磷酸緩沖液，含0.25% 戊二醛灌注固定。斷頭取出大腦，取基底核和黑質(zhì)部位標(biāo)本，切成約1mm3的小塊，迅速放入2.5%戊二醛固定液內(nèi)固定＞2h，0.1mol/L磷酸緩沖液浸洗，1%四氧化鋨后固定2h。再用磷酸緩沖液浸洗，乙醇梯度脫水，Epon 812包埋、聚合，半超薄切片機(jī)切片約1～2μm，光學(xué)顯微鏡下定位后，瑞典LKB超薄切片機(jī)切片，厚度為50～70nm，醋酸鈾-檸檬酸鉛染色，日本日立H-600型透射電子顯微鏡下觀察、拍照。

1.3 神經(jīng)元培養(yǎng)

新生SD大鼠，西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動物中心，無菌取腦，剝除腦膜。取雙側(cè)大腦皮質(zhì)剪成1～2mm3，0.25%胰蛋白酶消化。終止消化后吹打分散成單細(xì)胞懸液，離心，棄上清。加入等體積D-Hanks，吹打重新懸浮細(xì)胞，離心，棄上清。加入20%血清的DMEM，輕柔吹打成單細(xì)胞懸液。過篩，錐蟲藍(lán)（臺盼藍(lán)）排斥法檢測細(xì)胞活力，計(jì)數(shù)后稀釋至1×106個(gè)/ml接種入預(yù)先經(jīng)過多聚賴氨酸包被的、放置有處理好的蓋玻片的培養(yǎng)皿中。37℃、5%CO2培養(yǎng)箱培養(yǎng)。24h后更換為10%血清DMEM培養(yǎng)液培養(yǎng)，同時(shí)加入阿糖胞苷10μmol/L以抑制非神經(jīng)元細(xì)胞生長。以后隔天半量換液。

1.4 氨中毒神經(jīng)元模型

取培養(yǎng)7d成熟的皮質(zhì)神經(jīng)元細(xì)胞隨機(jī)分為兩組，氨中毒組和對照組。無血清DMEM洗3次后，氨中毒組加入含0.5mmol/LNH4Cl的無血清DMEM，對照組加入無血清DMEM，培養(yǎng)24h。

1.5 氨中毒神經(jīng)元超微結(jié)構(gòu)改變研究

終止培養(yǎng)后，取出爬有神經(jīng)元細(xì)胞的蓋玻片，用0.1mol/L 磷酸緩沖液洗2min×3次。2.5%戊二醛固定2h，0.1mol/L磷酸緩沖液浸洗，1%四氧化鋨后固定2h。再用磷酸緩沖液浸洗，乙醇梯度脫水，Epon 812倒扣包埋、聚合，瑞典LKB超薄切片機(jī)切成50～70nm的切片，醋酸鈾?檸檬酸鉛染色，日本日立H-600型透射電子顯微鏡下觀察、拍照。

1.6 倫理

本文所涉及實(shí)驗(yàn)均得到西安交通大學(xué)動物倫理委員會批準(zhǔn)。

1.7 統(tǒng)計(jì)學(xué)處理

2 結(jié) 果

2.1 肝性腦病大鼠腦基底核及黑質(zhì)超微結(jié)構(gòu)改變

2.1.1 基底核 神經(jīng)元細(xì)胞數(shù)量減少；神經(jīng)元細(xì)胞腫脹，胞漿內(nèi)可見空泡形成，細(xì)胞器減少，尼氏體（Nissl body）數(shù)量明顯減少（圖1A，圖1B）；部分細(xì)胞體積變小，細(xì)胞核電子密度增加，細(xì)胞核染色質(zhì)輕度凝集，核周間隙加大（圖1C，圖1D）。神經(jīng)膠質(zhì)細(xì)胞核固縮，染色質(zhì)凝集，邊集（圖1E，圖1F）。微血管管腔擴(kuò)張，血管周圍局部溶解，內(nèi)皮細(xì)胞局部溶解消失，連續(xù)性中斷（圖1G，圖1H）。

2.1.2 黑質(zhì) 神經(jīng)元細(xì)胞數(shù)量明顯減少；神經(jīng)黑色素顆粒數(shù)量不多，線粒體腫脹明顯，嵴斷裂溶解，尼氏體數(shù)量明顯減少，高爾基體扁平囊擴(kuò)張，核膜連續(xù)性中斷（圖2A，圖2B）；部分細(xì)胞體積變小，細(xì)胞質(zhì)濃縮，電子密度增加，細(xì)胞核內(nèi)染色質(zhì)高度凝聚，沿核周分布（或邊集），核仁肥大邊移（圖2C，圖2D）?？梢姷蛲鲂◇w（圖2E）。神經(jīng)膠質(zhì)細(xì)胞細(xì)胞器減少，核形不規(guī)則，核內(nèi)染色質(zhì)凝集，邊集（圖2F，圖2G）。微血管管腔擴(kuò)張，血管周圍溶解，內(nèi)皮細(xì)胞腫脹，表面皺襞增多，局部內(nèi)皮細(xì)胞膜向管腔膨出，基底膜電子密度降低，局部溶解消失，連續(xù)性中斷。其程度較基底核略重（圖2H，圖2I）。

2.2 氨中毒神經(jīng)元細(xì)胞超微結(jié)構(gòu)改變

神經(jīng)元細(xì)胞數(shù)量明顯減少；細(xì)胞腫脹明顯，細(xì)胞質(zhì)內(nèi)空泡增多，線粒體明顯腫脹，尼氏體顯著減少；部分細(xì)胞體積變小，細(xì)胞質(zhì)濃縮，核染色質(zhì)高度盤結(jié)出現(xiàn)空泡結(jié)構(gòu)；也有細(xì)胞核染色質(zhì)高度凝聚，沿核膜內(nèi)側(cè)排列、邊集而形成半月形；細(xì)胞核固縮，電子密度增加，核畸形，核膜迂曲；細(xì)胞核核碎裂，在細(xì)胞內(nèi)可見多個(gè)電子密度增強(qiáng)的胞核碎塊（圖3A，圖3B）。

圖1 肝性腦病大鼠及正常大鼠腦基底核電鏡表現(xiàn)

Figure 1 Ultrastructure of basal nuclei in HE and healthy rats (TEM)

HE: hepatic encephalopathy; A: neuron of basal nuclei in HE rats (×15000); B: neuron of basal nuclei in healthy rats (×20000); C: neuron nucleus of basal nuclei in HE rats (×4000); D: neuron nucleus of basal nuclei in healthy rats (×4000); E: neurogliocyte of basal nuclei in HE rats (×10000); F: neurogliocyte of basal nuclei in healthy rats (×10000); G: capillaries of basal nuclei in HE rats (×5000); H: capillaries of basal nuclei in healthy rats (×5000)

圖2 肝性腦病大鼠及正常大鼠黑質(zhì)電鏡表現(xiàn)

Figure 2 Ultrastructure of substantia nigra in HE and healthy rats (TEM)

HE: hepatic encephalopathy; A: neuron of substantia nigra in HE rats(×20000); B: neuron of substantia nigra in healthy rats (×20000); C:neuron nucleus of substantia nigra in HE rats (×4000); D: neuron nucleus of substantia nigra in healthy rats (×4000); E: apoptosis body in neuron nucleus of substantia nigra in HE rats (×4000); F: neurogliocyte of substantia nigra in HE rats (×10000); G: neurogliocyte of substantia nigra in healthy rats (×10000); H: capillaries of substantia nigra in HE rats (×4000); I: capillaries of substantia nigra in healthy rats (×5000)

圖3 氨中毒神經(jīng)元及正常神經(jīng)元電鏡超微結(jié)構(gòu)改變

Figure 3 Ultrastructure ofcultured rat neurons induced by ammonia and healthy neurons (TEM ×4000)

A:cultured rat neurons induced by ammonia; B: healthy neurons

3 討 論

肝性腦病腦組織病理改變主要表現(xiàn)為水腫、神經(jīng)元退行性變、尼氏小體溶解、軟化灶、神經(jīng)膠質(zhì)細(xì)胞增生[6]。本研究發(fā)現(xiàn)肝性腦病大鼠基底核與黑質(zhì)的超微結(jié)構(gòu)均有顯著改變。主要表現(xiàn)為神經(jīng)元細(xì)胞數(shù)量減少；神經(jīng)元線粒體腫脹，尼氏體數(shù)量明顯減少；可見凋亡各期表現(xiàn)。神經(jīng)膠質(zhì)細(xì)胞細(xì)胞器減少，可見凋亡Ⅰ期表現(xiàn)。微血管擴(kuò)張，血管周圍溶解，基底膜破壞。黑質(zhì)的超微結(jié)構(gòu)改變程度較基底核略重，可見細(xì)胞凋亡的晚期表現(xiàn)凋亡小體。

有學(xué)者研究發(fā)現(xiàn)，肝硬變大鼠紋狀體中神經(jīng)元數(shù)量減少，染色變淺、尼氏小體減少或消失[7]，與本研究結(jié)果相符。肝性腦病時(shí)神經(jīng)元數(shù)目減少的原因可能為：內(nèi)毒素、低氧血癥使腦內(nèi)毛細(xì)血管內(nèi)皮腫脹、通透性增加，有害物質(zhì)易進(jìn)入腦內(nèi)，干擾能量代謝，神經(jīng)細(xì)胞受損；肝功不全，血中升高的膽酸亦可損傷內(nèi)皮細(xì)胞。另外，一氧化氮也可能參與了神經(jīng)元的損傷。尼氏體是由顆粒內(nèi)質(zhì)網(wǎng)及游離的多核糖體組成的易染色質(zhì)，對缺血、缺氧、中毒極為敏感，其數(shù)量減少可能與肝性腦病時(shí)的缺氧狀態(tài)有關(guān)[8]。

研究表明，肝性腦病的患者腦部可見到明顯的大腦皮質(zhì)萎縮，病理學(xué)檢查可見大腦或小腦神經(jīng)元及神經(jīng)纖維的消失[9]。本研究發(fā)現(xiàn)，肝性腦病大鼠基底核和黑質(zhì)神經(jīng)細(xì)胞均有不同程度的凋亡表現(xiàn)，因此認(rèn)為神經(jīng)元及神經(jīng)纖維的消失與神經(jīng)細(xì)胞凋亡有關(guān)。肝性腦病時(shí)神經(jīng)細(xì)胞凋亡的原因可能是由于高血氨引起神經(jīng)細(xì)胞產(chǎn)生過量的細(xì)胞凋亡。

本研究發(fā)現(xiàn)肝性腦病大鼠微血管管腔擴(kuò)張，基底膜破壞。提示肝性腦病時(shí)血腦屏障損傷，通透性增加，是肝性腦病血管源性腦水腫的發(fā)生原因。有研究發(fā)現(xiàn)肝性腦病患者大腦皮質(zhì)毛細(xì)血管內(nèi)皮細(xì)胞明顯腫脹，囊泡增多，基底膜受損[10]；肝性腦病大鼠微血管明顯擴(kuò)張，內(nèi)皮皺褶增多，內(nèi)皮下基底膜電子密度降低，甚至局部溶解消失；微血管周圍星形膠質(zhì)細(xì)胞突起及皮質(zhì)內(nèi)膠質(zhì)細(xì)胞明顯腫脹，細(xì)胞器成分減少，基質(zhì)溶解，膠質(zhì)細(xì)胞出現(xiàn)水腫[11]，與本研究結(jié)果相符。但早期有學(xué)者在半乳糖胺誘導(dǎo)的暴發(fā)性肝衰竭兔腦超微結(jié)構(gòu)研究中發(fā)現(xiàn)，毛細(xì)血管內(nèi)皮細(xì)胞是正常的[12]，其差異可能與不同的建模機(jī)制有關(guān)。

本研究發(fā)現(xiàn)，氨中毒神經(jīng)元超微結(jié)構(gòu)改變主要為：神經(jīng)元細(xì)胞數(shù)量明顯減少；細(xì)胞明顯水腫，細(xì)胞質(zhì)內(nèi)空泡增多，線粒體明顯腫脹，尼氏體顯著減少；可見不同時(shí)期的凋亡表現(xiàn)。因此可以認(rèn)為，氨主要導(dǎo)致體外培養(yǎng)的神經(jīng)元細(xì)胞水腫，同時(shí)可誘導(dǎo)體外培養(yǎng)神經(jīng)細(xì)胞產(chǎn)生過量的細(xì)胞凋亡。有學(xué)者通過觀察體外培養(yǎng)的大鼠神經(jīng)細(xì)胞在氨作用下的形態(tài)學(xué)、超微結(jié)構(gòu)的變化，發(fā)現(xiàn)一定濃度的氨可以誘導(dǎo)神經(jīng)細(xì)胞產(chǎn)生核染色質(zhì)濃縮邊集及由膜包裹形成的“凋亡小體”的典型細(xì)胞凋亡，與本研究結(jié)果基本一致[13]。結(jié)合項(xiàng)目組前期的細(xì)胞存活率研究結(jié)果[14]，可以認(rèn)為氨作用下神經(jīng)元細(xì)胞的過量凋亡可能也是細(xì)胞存活率降低的原因之一。因此，凋亡在肝性腦病的發(fā)生中也起重要作用，進(jìn)一步證實(shí)了氨中毒引起的神經(jīng)元凋亡可能為肝性腦病大鼠腦超微結(jié)構(gòu)改變的重要機(jī)制之一。

γ-氨基丁酸（gamma-aminobutyric acid，GABA）是黑質(zhì)紋狀體通路中最重要的抑制性遞質(zhì)[15]。項(xiàng)目組前期通過對肝性腦病大鼠GABA-A受體亞單位及γ-氨基丁酸轉(zhuǎn)運(yùn)體的研究發(fā)現(xiàn)GABA能使神經(jīng)張力增高也在肝性腦病的發(fā)病中起重要作用[16,17]。其改變均是以黑質(zhì)網(wǎng)狀部改變?yōu)樽睿坠?jié)區(qū)次之。本研究電子顯微鏡觀察發(fā)現(xiàn)肝性腦病大鼠基底核與黑質(zhì)的超微結(jié)構(gòu)均有顯著改變，而黑質(zhì)的改變程度較基底核更重。這些微觀結(jié)構(gòu)的改變，進(jìn)一步表明黑質(zhì)紋狀體通路在肝性腦病發(fā)生中發(fā)揮作用。

[1] Chinese Society of Gastroenterology and Chinese Society of Hepatology of Chinese Medical Association. Consensus on the diagnosis and treatment of hepatic encephalopathy in China(Chongqing, 2013)[J]. Chin J Hepatol, 2013, 21(9): 641?651. [中華醫(yī)學(xué)會消化病學(xué)分會, 中華醫(yī)學(xué)會肝病學(xué)分會. 中國肝性腦病診治共識意見(2013年, 重慶)[J]. 中華肝臟病雜志, 2013, 21(9): 641?651.]

[2] Qureshi MO, Khokhar N, Shafqat F. Ammonia levels and the severity of hepatic encephalopathy[J]. J Coll Physicians Surg Pak, 2014, 24(3): 160?163.

[3] Cauli O, Rodrigo R, Llansola M,. Glutamatergic and gabaergic neurotransmission and neuronal circuits in hepatic encephalopathy[J]. Metab Brain Dis, 2009, 24(1): 69?80.

[4] Choi JM, Kim YH, Roh SY. Acute hepatic encephalopathy presenting as cortical laminar necrosis: case report[J]. Korean J Radiol, 2013, 14(2): 324?328.

[5] Chu CJ, Lee FY, Wang SS,. Establishment of an animal model of hepatic encephalopathy[J]. Zhonghua Yi Xue Za Zhi (Taipei) [Chin Med J (Taipei)], 2000, 63(4): 263?269.

[6] Mustafa HN, El Awdan SA, Hegazy GA. Protective role of antioxidants on thioacetamide-induced acute hepatic encephalopathy: biochemical and ultrastructural study[J]. Tissue Cell, 2013, 45(5): 350?362.

[7] Rivera-Mancía S, Montes S, Méndez-Armenta M,. Morphological changes of rat astrocytes induced by liver damage but not by manganese chloride exposure[J]. Metab Brain Dis, 2009, 24(2): 243?255.

[8] Méndez M, Méndez-López M, Lopez L,. Mammillary body alterations and spatial memory impairment in Wistar rats with thioacetamide-induced cirrhosis[J]. Brain Res, 2008, 1233: 185?195.

[9] Zhang LJ, Qi R, Zhong J,. The effect of hepatic encephalopathy, hepatic failure, and portosystemic shunt on brain volume of cirrhotic patients: a voxel-based morphometry study[J]. PLoS One, 2012, 7(8): e42824.

[10] Kato M, Hughes RD, Keays RT,. Electron microscopic study of brain capillaries in cerebral edema from fulminant hepatic failure[J]. Hepatology, 1992, 15(6): 1060?1066.

[11] Jayakumar AR, Tong XY, Ospel J,. Role of cerebral endothelial cells in the astrocyte swelling and brain edema associated with acute hepatic encephalopathy[J]. Neuroscience, 2012, 218: 305?316.

[12] Traber PG, Dal Canto M, Ganger DR,. Electron microscopic evaluation of brain edema in rabbits with galactosamine-induced fulminant hepatic failure: ultrastructure and integrity of the blood-brain barrier[J]. Hepatology, 1987, 7(6): 1272?1277.

[13] Back A, Tupper KY, Bai T,Ammonia-induced brain swelling and neurotoxicity in an organotypic slice model[J]. Neurol Res, 2011, 33(10): 1100?1108.

[14] Li XQ,Chong L.The study of toxicity by ammonia in rats neurons[J]. Shaanxi Med J, 2009, 38(8): 956?959. [李曉青, 種 莉. 氨對體外培養(yǎng)大鼠神經(jīng)細(xì)胞毒性的研究[J]. 陜西醫(yī)學(xué)雜志, 2009, 38(8): 956?959.]

[15] Salgado M, Cortes Y. Hepatic encephalopathy: etiology, pathogenesis, and clinical signs[J]. Compend Contin Educ Vet, 2013, 35(6): e1?e8.

[16] Li XQ, Dong L, Liu ZH,. Expression of gamma-aminobutyric acid A receptor subunits alpha1, beta1, gamma2 mRNA in rats with hepatic encephalopathy[J]. World J Gastroenterol, 2005, 11(21): 3319?3322.

[17] Li XQ, Dong L. Expression of γ-aminobutyric acid transporter mRNA in rats with hepatic encephalopathy[J]. Chin J Neuromed, 2009, 8(7): 658?660. [李曉青, 董 蕾.肝性腦病大鼠γ-氨基丁酸轉(zhuǎn)運(yùn)體mRNA的表達(dá)[J]. 中華神經(jīng)醫(yī)學(xué)雜志, 2009, 8(7): 658?660.]

（編輯: 周宇紅）

Ultrastructure change of brain in rats with hepatic encephalopathy

LI Xiao-Qing*, CHONG Li, LIU Peng, LIU Yue, TANG Peng, CHEN Li, ZHANG Xin, HOU Chen, LI Rui, GUO Min-Xia

(The Third Department of Neurology, Shaanxi Provincial People’s Hospital, Xi’an 710068, China)

To observe the ultrastructure change of the brain tissues in the rats with hepatic encephalopathy and determine theeffect of ammonia on the ultrastructure of rat neurons in order to investigate the pathological mechanism of these changes.Twelve healthy male SD rats were randomly divided into two groups: hepatic encephalopathy group and normal control group (with six in each group). Transmission electron microscopy was employed to observe the ultrastructure of the brain tissues in rats with hepatic encephalopathy induced by thioacetamide and ofcultured rat neurons induced by ammonia.In rats with hepatic encephalopathy, the neurons were reduced in number, with swollen mitochondria, significantly decreased Nissl body quantity, and arrested in each stage of apoptosis. While, the cell organelles in glial were also decreased, and the ultrastructure changes were more serious in the nigra than in the basal nuclei.Forinjured rat neurons induced by ammonia, the neurons were decreased in number, with edema, swollen mitochondria, and significantly reduced Nissl body quantity, and displayed apoptosis at each stage.Obvious changes are seen in the ultrastructure of nervous cells in rats with hepatic encephalopathy andinjured neurons, which might be associated with neuron apoptosis caused by ammonia.

hepatic encephalopathy; ammonia; cell culture; ultrastructure

(2012KCT-17).

R747.9

A

10.3724/SP.J.1264.2014.000142

2014?06?03;

2014?07?12

陜西省重點(diǎn)領(lǐng)域科技創(chuàng)新團(tuán)隊(duì)項(xiàng)目（2012KCT-17）

李曉青, E-mail: goodlxq@sina.com