An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis.

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (alpha-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl(4) and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl(4) mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and alpha-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl(4) and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.