In vivo real time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis Running head: In vivo imaging of hepatocyte apoptosis

Background: Apoptosis is a dynamic process of programmed cell death and involved in multiple diseases. However, its mechanisms and sequence of events are still incompletely understood, partly because of the inability to visualize single cells continuously in vivo. The aim of the present study was to monitor hepatocyte apoptosis with confocal endomicroscopy in living rodents. Methods: In 73 anaesthetized mice, apoptotic liver injury was induced by injection of the CD95-agonistic antibody Jo2. Individual hepatocytes were followed for up to 240 min with a handheld confocal probe (FIVE1, Optiscan, Australia) providing 0.7 μm resolution (1000fold magnification). Different fluorescence staining protocols were used for cellular staining, vascular and cellular barrier function imaging, and caspase activation visualization. Results: The time course of apoptosis could be visualized in vivo while liver perfusion and tissue integrity were maintained. In contrast to most ex vivo studies, initial cell swelling was observed that coincided with early defects in barrier function of sinusoids and hepatocytes. Cytoplasmic vesicle formation, nuclear condensation, cellular disintegration and macrophage infiltration were captured sequentially. Labeling of caspases allowed molecular imaging. Conclusions: Our study allowed for the first time to continuously follow distinct morphological, functional and molecular features of apoptosis in a solid organ in vivo and at high resolution. Intravital confocal microscopy may be a valuable tool to study the effects of therapeutic intervention on apoptosis in animal models and humans.