In vivo imaging of liver injury and regeneration by functional two-photon microscopy

Intravital two-photon based imaging allows real-time observation of biological processes. Videos can be recorded at sub-cellular resolution up to 200nm. Although the mechanisms of acetaminophen (APAP) induced liver injury are intensively studied, this technique gave us surprising insights both during the destruction and the regeneration processes. Videos recorded directly after administration of a toxic dose of APAP (300 mg/kg) identified two different scenarios of cell death. Hepatocytes which are close the central veins undergo necroptosis as already described in literatures. Surprisingly, the outer layer of the pericentral hepatocytes are killed by in dependent mechanism which is mediated by bile salt overload. First, dilatation of the bile canaliculi was observed within 30 min after APAP administration. Subsequently, the widened bile canaliculi form protrusions into the adjacent hepatocytes. These protrusions continue to increase in size and finally burst leading to bile regurgitation into hepatocytes and cell death. Interestingly, similar scenario was observed in cholestasis induced by bile duct ligation. The regeneration process after a single APAP challenge is characterized by a transient activation of hepatic stellate cells (HSCs) which reach to the peak on day two and disappear between days four and six after APAP injection. This was associated with macrophages infiltration into the dead cell area. In contrast to the situation during fibrosis resolution, the activated HSCs did not undergo apoptosis following regeneration from an acute challenge. Although work for a formal proof is still in progress, it seems as if the infiltrating macrophages interact with and finally phagocytose the activated HSCs in the dead cell area. This stimulated us to deplete macrophages by repeated administration of clodronate. Removal of macrophages lead to prolonged activation of HSCs. Interestingly, shortly after macrophages depletion backup infiltration of CD45+ immune cells was observed followed by apoptosis of HSCs. In conclusion, the direct observation of cellular and sub-cellular events in the living liver allows insights into the sequence of pathophysiological events which are difficult to obtain by conventional methods.