Department of Gastroenterology and Hepatology, University Hospital of Cologne, Cologne, Germany.
Histol Histopathol. 2013 Aug;28(8):1013-20. doi: 10.14670/HH-28.1013. Epub 2013 Mar 4.
Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes.
Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively.
Upon histomorphological analysis of cryo- and paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size.
Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE.
当前用于可视化血管系统、胆管树和分离重要胆管细胞的方法存在许多技术难题，尤其是在小鼠中。在本项目中，我们提出了一种新颖、可靠且简单的替代技术，用于组织学显示血液和胆管系统，并分离重要的胆管细胞。
对二十只野生型小鼠进行逆行胆管内活体灌注。通过正中剖腹术暴露肝脏和胆囊。使用静脉导管对胆囊进行插管，切除肝脏边缘的几毫米，以允许灌流液自由流出，并逆行注入羧基荧光素二乙酸琥珀酰亚胺酯（CFDA-SE）溶液。此后，通过同一导管注入甲醛溶液，或立即将肝脏解离成单细胞悬液进行 FACS 分析。通过直接向腹主动脉内注射 CFDA-SE 对十只 Lewis 大鼠进行血管系统的活体灌注。使用印度墨水或细胞角蛋白 19 免疫组织化学分别控制 CFDA-SE 标记的特异性和敏感性。
对冷冻和石蜡切片进行组织形态学分析后，在整个肝脏的大、小胆管以及 CFDA-SE 溶液输注后的血管系统中均观察到强烈的荧光。在初步的 FACS 实验中，我们成功地根据 CFDA-SE 染色和细胞大小分离出胆管细胞。
使用逆行灌注和 CFDA-SE 对小鼠胆管上皮和血管内皮的快速、经济有效的活体荧光标记方法，可实现肝组织结构的可视化和胆管细胞的分离。
