Apoptosis is a dynamic process of programmed cell death and is 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. 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) providing 0.7 μm resolution (1,000-fold magnification). Different fluorescence staining protocols were used for cellular staining, vascular and cellular barrier function imaging, and caspase activation visualization. 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. 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.
"Apoptotic indices in individual ducks were counted in haematoxylin and eosin stained sections (data not shown) and were found to be in the range of 0.005–0.2%, which might contribute a maximum daily cell death rate of about 2% (Goetz et al., 2011). However, cell death would decrease levels of cccDNA and decrease the relative contribution of loss of cccDNA at mitosis. "
[Show abstract][Hide abstract] ABSTRACT: Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis.
[Show abstract][Hide abstract] ABSTRACT: Confocal laser endomicroscopy (CLE) is a novel imaging modality providing in vivo histology and allowing capture of very high-resolution images of human and animal liver. The manuscript from Goetz and colleagues in this issue of American Journal of Physiology GI and Liver, used CLE to continuously follow distinct morphological, functional and molecular features of apoptosis in intact liver in vivo and at high resolution. This new technology allowed the authors to make several important observations whilst highlighting the enormous promise of this technology for future in vivo studies of hepatic apoptosis and its pharmacological manipulation in animals and humans.
[Show abstract][Hide abstract] ABSTRACT: Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.
The Journal of Cell Biology 06/2013; 201(7):969-979. DOI:10.1083/jcb.201212130 · 9.83 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.