Visualization of Hepatocellular Regeneration in Mice After Partial Hepatectomy

Background: Although hepatocellular regeneration is the cornerstone of liver homeostasis, current techniques for assessing such regeneration are limited. A method for visualizing the regeneration process would provide a means for advanced studies. Therefore, we examined the possibility of using fluorescence ubiquination-based cell cycle indicator (Fucci) mice for direct visualization of hepatocellular regeneration. Materials and methods: We performed a two-thirds partial hepatectomy in conventional and Fucci mice. Fucci animals have orange Cdt1 expressed in the G₁ phase and green Geminin expressed in S/G₂/M phases. Regenerating livers were procured daily for 7 d. Immunohistochemical staining was performed for proliferative Ki67 and mitotic pHH3 serine 10 (pHH3) markers on formalin-fixed, paraffin-embedded tissue sections from conventional mice. The orange Cdt1 and green Geminin fluorescence indicative of the G₁ and S/G₂/M phases, respectively, were assessed in liver tissues, in vivo and ex vivo, with two-photon laser scanning microscopy. Results: Immunostaining with Ki67 and pHH3 revealed a typical profile of hepatocellular regeneration after hepatectomy in conventional mice, although immunostaining required more than a week to process. In contrast, hepatocellular regeneration could be visualized with two-photon microscopy within a few hours in regenerating livers of the Fucci mice. Only orange G₁ hepatocytes were seen in the baseline liver specimens; however, multiple bright green and yellow hepatocytes were seen 48 h after hepatectomy, indicating active hepatocytes in the S/G₂/M phases of the cell cycle. Conclusions: Hepatocellular regeneration is readily visualized in regenerating livers of Fucci mice. The Fucci model is an exciting tool for advanced studies of hepatocellular and liver regeneration.