Lotowska Joanna M, Sobaniec-Lotowska Maria E, Bockowska Sylwia B, Lebensztejn Dariusz M
Joanna M Lotowska, Department of General Pathomorphology, Medical University of Bialystok, 15269 Bialystok, Poland.
World J Gastroenterol. 2014 Apr 21;20(15):4335-40. doi: 10.3748/wjg.v20.i15.4335.
To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH).
Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm(3) volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope.
Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles.
Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.
研究小儿非酒精性脂肪性肝炎（NASH）活检组织中异常肝细胞线粒体的超微结构，包括其在细胞内和肝小叶带的分布。
对10例年龄在2至14岁、先前经临床病理诊断为NASH的儿童（6例男孩和4例女孩）的肝活检标本进行超微结构研究。如果肝活检显示脂肪变性、炎症、气球样变肝细胞、马洛里小体或局灶性坏死，以及在无感染性肝病、自身免疫性肝炎、代谢性肝病或乳糜泻的临床、血清学或组织学表现的情况下出现不同程度的纤维化，则诊断为该疾病。为进行超微结构分析，将新鲜的小肝块（体积为1立方毫米）固定于含有2%多聚甲醛和2.5%戊二醛的0.1摩尔/升二甲胂酸钠缓冲液中。标本用四氧化锇后固定，随后依次通过一系列梯度乙醇和环氧丙烷脱水，并包埋于Epon 812中。材料在Reichert超薄切片机上切片以获得半薄切片，用硼酸钠中的亚甲蓝染色。超薄切片用醋酸铀和柠檬酸铅进行对比，并使用Opton EM 900透射电子显微镜检查。
对非酒精性脂肪性肝炎患儿活检组织的超微结构分析显示，肝细胞内存在特征性的重复性线粒体异常；主要有线粒体多态性，如巨型线粒体、线粒体嵴缺失，以及线粒体基质内存在电子密度增加的线性晶体包涵体。晶体包涵体在巨型线粒体（MMC）内尤为明显，其在肝实质细胞和肝小叶带内似乎随机分布，数量上无特殊差异。包涵体纵向观察呈束状，横断面观察呈均匀间隔的基质，且常导致线粒体变形。这些线性结构的平均直径为10纳米，它们之间的平均间距为20纳米。有时在其附近可见线粒体颗粒增大。发现肝细胞的细胞质呈泡沫状，这是由于滑面内质网增生和糖原蓄积所致。狄氏间隙的血管周围间隙常扩张，含有过渡性肝星状细胞以及成熟和/或新形成的胶原纤维束。
在肝细胞线粒体中观察到的明显超微结构异常，尤其是以巨型线粒体形式存在的多态性和线粒体嵴缺失，伴有泡沫状细胞质，清楚地表明这些细胞器在小儿NASH的形态发生中起主要作用。我们的研究结果似乎证明了电子显微镜在该疾病诊断中的高效性。
