An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

The liver lymphatic system is essential for maintaining tissue fluid balance and immune function. The detailed structure of lymphatic vessels (LVs) in the liver remains to be fully demonstrated. The aim of this study is to reveal LV structures in normal and diseased livers by developing a tissue-clearing and co-immunolabeling protocol optimized for the tissue size and the processing time for 3D visualization and quantification of LVs in the liver. We showed that our optimized protocol enables in-depth exploration of lymphatic networks in the liver, consisting of LVs along the portal tract (deep lymphatic system) and within the collagenous Glisson's capsule (superficial lymphatic system) in different species. With this protocol, we have shown 3D LVs configurations in relation to blood vessels and bile ducts in cholestatic mouse livers, in which LVs were highly dilated and predominantly found around highly proliferating bile ducts and peribiliary vascular plexuses in the portal tract. We also established a quantification method using a 3D volume rendering approach. We observed a 1.6-fold (p<0.05) increase in average diameter of LVs and a 2.4-fold increase (p<0.05) in the average branch number of LVs in cholestatic/fibrotic livers compared to control livers. Furthermore, cholestatic/fibrotic livers showed a 4.3-fold increase (p<0.05) in total volume of LVs compared to control livers. Our optimized protocol and quantification method demonstrate an efficient and simple liver tissue-clearing procedures that allow the comprehensive analysis of liver lymphatic system.