DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome

Abstract

Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

“…Future testing of whether resin can be delivered via glass capillary would therefore be of interest. DUCT was further adapted to inject other tubular systems such as the airways and pulmonary artery vasculature of the lungs 9 . The double resin injection could also be modified to be used with other commercially available resins, or this protocol could be used for injections with carbon ink.…”

“…DUCT can only be used for resin casting of tubular structures above a diameter of 5 µm. In this data set, the resin could penetrate tubes with the smallest diameter of 5 µm 9 . This size limitation precludes the analysis of fine ductules and small capillaries.…”

“…In Hankeova et al 9 , DUCT was compared to two other commonly used techniques, double carbon ink injections followed by tissue clearing and standard photography, and iDISCO+ with staining of the blood vessels with alpha-smooth muscle cell actin and bile ducts with cytokeratin 7, followed by 3D imaging 9 . DUCT outperformed the other two methods in terms of dual analysis (which was challenging for iDISCO+ due to high liver autofluorescence), 3D imaging, and quantification (not possible with carbon ink injection), and lumenization (DUCT provides data for the internal lumen architecture and system perfusion).…”

“…DUCT can be applied to study any two tubular networks in 3D. As proof of principle, DUCT was used to visualize the liver biliary and portal vein systems and the pulmonary artery vasculature and airways in lung 9 . The intrahepatic bile ducts develop adjacent to the portal vein, and the portal vein provides a structural template and signaling center that regulates the growth and differentiation of the biliary tree 12 .…”

“…DUCT is based on the extensive existing knowledge of single-system liver resin casting 3 , 4 , 5 , 6 , 7 , 8 and extends to simultaneous 3D visualization and analysis of two systems 9 . DUCT advanced single resin casting to double resin casting by mixing two radiopaque resins of different contrast and injecting these resins into two different networks, specifically the common bile duct and portal vein.…”