The quantification of fibrosis in cleared human and mouse liver tissues by light-sheet fluorescence microscope using a slide-free approach

Background & Aim: Liver fibrosis is a common end point of any type of liver injury. Liver biopsy is the only reliable modality for determining the extent of fibrosis and subsequently providing patients with appropriate treatment. However, liver biopsy is not without limitations. Sampling error, observer variability, and semi-quantitative assessment are the significant limitations of the procedure, which may limit the accurate assessment of hepatic fibrosis. Besides, the utility of such small samples increases the staging inaccuracy. Problems in assessment of hepatic fibrosis limit the development of effective biomarkers and therapeutics particularly in NASH clinical trials. A slide-free examination of optically cleared liver biopsy samples may assist in accurate quantification of hepatic fibrosis. To this end, we developed a platform for quantifying hepatic fibrosis in human and mouse liver tissues utilizing a modified-CLARITY and custom-made light sheet fluorescence microscope. Material and Method: A hundred and sixty-eight liver specimens from human subjects were included in this study. The cohort included fifty-seven NAFLD, thirty-eight viral hepatitis patients, and thirteen healthy subjects who underwent hepatic resection due to benign liver diseases. Sirius-red stained mouse and human liver sections were scored by an expert pathologist and Collagen proportionate area (CPA) analysed for 2D quantification of liver fibrosis. Liver specimens were cleared using the modified-CLARITY method and visualized with a custom-made light sheet microscope. To test the applicability of the method in intermediate stages of fibrosis, five animal models of liver disease were employed in C57Bl/6 mice. Collagen type I and Elastin were quantified in cleared human liver tissues using a slide-free approach via custom-made light sheet microscope. Multiple optical sections of whole liver samples were used to calculate collagen proportionate volume (CPV) and elastin proportionate volume (EPV). Results: In this project, we successfully obtained transparent liver tissues for volumetric quantification of hepatic fibrosis. The modified-CLARITY method efficiently cleared human and mouse liver tissues. The major ECM proteins, Collagen type I and Elastin, were quantified in the human NAFLD and viral hepatitis cohort. Cut-off values were determined for CPV and EPV values for each fibrotic stages of human NASH. Further analysis demonstrated substantial sectional variability between optical slices of volumetric imaging data. Coefficient of variation analysis determined the extent of variation and found 84.6% intermediate and high level variation for CPV and EPV sections of F3-4 groups of NAFLD. 3D fibrosis staging of optical sections with established cut-off values for CPV and EPV demonstrated that only 44% and 47% of optical sections would be staged the same for F3 and F4, respectively. Conclusion: We developed a slide-free method for assessing the 3D pathology of hepatic fibrosis using a custom-made light sheet microscope. Volumetric image analysis of whole liver biopsies revealed substantial heterogeneity within the biopsy. Comprehensive profiling of extracellular matrix proteins in hepatic fibrosis and quantification with a slide-free non-destructive platform may improve the staging accuracy. Thus, utilizing this method may advance the treatments for NASH clinical studies.