Pipe-3D: A Pipeline Based on Immunofluorescence, 3D Confocal Imaging, Reconstructions, and Morphometry for Biliary Network Analysis in Cholestasis.
Pipe-3D: A Pipeline Based on Immunofluorescence, 3D Confocal Imaging, Reconstructions, and Morphometry for Biliary Network Analysis in Cholestasis.
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DOI：
10.1007/978-1-4939-9420-5_3
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发表时间：
2019
影响因子：
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通讯作者：
A. Damle-Vartak;Brigitte Begher-Tibbe;G. Gunther;F. Geisler;N. Vartak;J. Hengstler
中科院分区：
文献类型：
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作者：
A. Damle-Vartak;Brigitte Begher-Tibbe;G. Gunther;F. Geisler;N. Vartak;J. Hengstler
胆汁淤积，即肝外胆汁流量的损害，是许多病理性肝脏疾病的常见并发症，如胆管病、原发性胆道硬化和原发性胆汁性肝硬化。除了胆汁酸在肝脏和血液中的积累外，它还导致胆道树的增殖反应，称为导管反应。胆管反应的特点是形成胆管上皮的胆管细胞增殖增强。据报道，胆道树的这种强烈反应可以产生祖细胞来源，这些祖细胞可以在再生过程中分化为肝细胞或胆管细胞。另一方面，它可引起门周纤维化，最终发展为肝硬化和死亡。在二维组织学上，这导致每个组织区域的管腔数量增加。然而，胆道树是一个三维结构，在薄片上的管腔的出现可能是由于新的导管的出现，或者是由于现有导管在三维上的分支或卷积。在许多这样的方面，传统的二维薄切片组织学限制了我们对胆道树反应的理解。全面了解胆汁淤积中胆道网络的结构重塑取决于强大的3D样品制备和分析方法。为此，我们描述了管道3D，这是一种基于免疫荧光、共聚焦成像、表面重建和自动形态测量的胆道网络三维亚细胞分辨率的管道可视化处理和分析。该管道已被用于发现胆汁淤积时小叶间胆管的广泛重塑，其中延伸，分支和环在门静脉分支周围形成密集的导管网。由共聚焦数据生成的Pipe-3D表面重建也显示，通过上皮层的波纹，管腔管表面增强了约5倍，这可能会增加胆汁重吸收并减轻胆汁淤积。小叶间管对胆汁淤积的反应与胚胎发育过程中新生胆管形成的大胆管形成鲜明对比。它也不同于其他肝损伤模型中的导管反应，如胆碱缺乏、蛋氨酸补充饮食，其中观察到实质组织受到导管及其分支的侵犯。Pipe-3D适用于任何肝损伤模型，并可选择集成组织清除技术，用于厚（>500 μm）组织切片的3D分析。
Cholestasis, the impairment of bile flux out of the liver, is a common complication of many pathological liver disorders, such as cholangiopathies, primary biliary sclerosis, and primary biliary cirrhosis. Besides accumulation of bile acids in the liver and blood, it leads to a proliferative response of the biliary tree termed as a ductular reaction. The ductular reaction is characterized by enhanced proliferation of cholangiocytes, which form the epithelial lining of bile ducts. This strong reaction of the biliary tree has been reported to generate a source of progenitor cells that can differentiate to hepatocytes or cholangiocytes during regeneration. On the other hand, it can cause periportal fibrosis eventually progressing to cirrhosis and death. In 2D histology, this leads to the appearance of an increased number of duct lumina per area of tissue. Yet, the biliary tree is a 3D vstructure and the appearance of lumina in thin slices may be explained by the appearance of novel ducts or by ramification or convolution of existing ducts in 3D. In many such aspects, traditional 2D histology on thin slices limits our understanding of the response of the biliary tree. A comprehensive understanding of architecture remodeling of the biliary network in cholestasis depends on robust 3D sample preparation and analysis methods. To that end, we describe pipe-3D, a processing and analysis pipeline visualization based on immunofluorescence, confocal imaging, surface reconstructions, and automated morphometry of the biliary network in 3D at subcellular resolution. This pipeline has been used to discover extensive remodeling of interlobular bile ducts in cholestasis, wherein elongation, branching, and looping create a dense ductular mesh around the portal vein branch. Surface reconstructions generated by Pipe-3D from confocal data also show an approximately fivefold enhancement of the luminal duct surface through corrugation of the epithelial lamina, which may increase bile reabsorption and alleviate cholestasis. The response of interlobular ducts in cholestasis was shown to be in sharp contrast to that of large bile ducts, de novo duct formation during embryogenesis. It is also distinct from ductular response in other models of hepatic injury such as choline-deficient, ethionine-supplemented diet, where parenchymal tissue invasion by ducts and their branches is observed. Pipe-3D is applicable to any model of liver injury, and optionally integrates tissue clearing techniques for 3D analysis of thick (>500 μm) tissue sections.