Gauri Khandekar1, Jessica Llewellyn1, Alyssa Kriegermeier2, Orith Waisbourd-Zinman3, Nicolette Johnson4, Yu Du1, Roquibat Giwa1, Xiao Liu5, Tatiana Kisseleva5, Pierre A Russo6, Neil D Theise7, Rebecca G Wells8. 1. Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States. 2. Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States. 3. Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States; Schneider Children's Hospital, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 4. Cell and Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States. 5. Department of Surgery University of California, San Diego, La Jolla, CA, United States. 6. Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, United States. 7. Department of Pathology, New York University School of Medicine, New York, NY, United States. 8. Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Department of Bioengineering, School of Engineering and Applied Sciences, The University of Pennsylvania, Philadelphia, PA, United States; Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Center for Engineering MechanoBiology, The University of Pennsylvania, Philadelphia, PA, United States. Electronic address: rgwells@pennmedicine.upenn.edu.
Abstract
BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.
BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.
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