Visualization of Hepatocellular Regeneration in Mice After Partial Hepatectomy

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.

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.

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.

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.