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Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, allowing to study fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow-cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artefacts or contamination with other cells. The described isolation procedure takes approximately four hours to complete.

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Tumor necrosis factor (TNF)-␣ signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the "ceramide/ S1P rheostat") may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-␣-induced apoptosis of primary hepatocytes. Endogenous C 16 -ceramide was elevated during TNF-␣-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-␣-induced apoptosis and C 16 -ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-␣-induced increase of C 16 -ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with D-galactosamine plus TNF-␣. This protective effect was abrogated by the sphingosine kinase inhibitor N,Ndemethylsphingosine, suggesting that the survival effect of NCDase is due to not only C 16 -ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C 16 -ceramide contributed to TNF-␣-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C 16 -ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-␣.