Changes in 3-Dimensional Intrahepatic Biliary Structures in Patients With Hepatobiliary Diseases Visualized Using Tissue Clearing Methods

Biliary lesions, including ductular reactions and biliary obstruction, are involved in the pathophysiology of human hepatobiliary diseases. However, little is known about the 3-dimensional (3D) biliary structures associated with these diseases. We examined pathological changes in 3D structures in human chronic liver diseases using tissue clearing methods.

Thirty autopsied liver tissues were obtained from 2 patients with advanced primary biliary cholangitis, 6 with cirrhosis (viral hepatitis, 3; alcoholic liver disease, 2; and metabolic dysfunction-associated steatotic liver disease, 1), 4 with mild fibrosis (viral, 1; alcoholic liver disease, 1; and metabolic dysfunction-associated steatotic liver disease, 2), and 18 with nonspecific reactive (NSR) changes. Liver tissues were fixed in formalin and cleared using the clear, unobstructed brain/body imaging cocktails methods or rapid and nondestructive tissue clearing system. Three-dimensional imaging of biliary structures was performed using immunostaining with Alexa Fluor 488-labeled anti-cytokeratin (CK)19 and light sheet fluorescence microscopy. The number of CK19-positive biliary components per portal tract and the degrees of biliary networks in the portal tract, anastomoses, bridging formation, meandering, and caliber variation in 3D images were evaluated semiquantitatively.

Three-dimensional biliary structures were clearly visualized by tissue clearing methods and light sheet fluorescence microscopy. The number of biliary components was significantly higher with cirrhosis than with NSR changes (P < .05). The degrees of anastomoses and bridging formation were significantly increased in cirrhosis compared with other groups (P < .01). The degrees of biliary networks and meandering were significantly higher with cirrhosis than with mild fibrosis and NSR changes (P < .01). The number of biliary components and other factors was not elevated in the 2 cases of advanced primary biliary cholangitis examined. The number of biliary components and degrees of 3D biliary features correlated with the results of various liver function tests (P < .01).

Three-dimensional imaging using the tissue clearing methods may be a promising strategy to examine pathological changes in human intrahepatic biliary structures in patients with various human hepatobiliary diseases.