Hypothermia Inhibits Fas-Mediated Apoptosis of Primary Mouse Hepatocytes in Culture

Abstract

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37°C to 32°C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32°C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.

The extrinsic and intrinsic apoptotic pathways are differentially affected by temperature upstream of mitochondrial damage

“…Our observations with anti-Fas antibody contrast with results obtained in other cell types, such as freshly isolated hepatocytes and mouse fibroblasts, in which mild hypothermia blocked Fas-induced apoptosis without affecting activation of caspase 8 and cleavage of Bid [45,46]. This discrepancy could be cell type-specific or due to differences in the incubation temperature, 32 • C versus RT.…”

“…This protective effect has been associated with elevated Akt activity [44]. In vitro, decreasing the culture temperature to 32 • C protects mammalian cells from apoptosis induced by various stimuli, including antiFas antibody [45,46]. Moreover, ex vivo handling of blood cells for conservation or manipulation and subsequent in vivo transplantation may expose cells to various thermal conditions that might compromise cell viability [47].…”

Effect of mild hypothermia preconditioning against low temperature (4°C) induced rat liver cell injury in vitro

“…In 2004, Fu et al first evaluated the hepatocyte apoptosis with the treatment of mild hypothermia, and their research indicated that mild hypothermia (26℃) could suppress Fas-mediated apoptotic signaling pathways in liver cells. This function mainly depended on the inhibition of some signaling events, such as cytochrome c release, effector caspase activation, and so on [10]. In recent years, the protective effect of mild hypothermia was also evaluated in some other kinds of liver cell injury models to clarify more detailed mechanisms.…”

Keeping cool in acute liver failure: Rationale for the use of mild hypothermia

“…Studies suggest that hypothermia reduces liver injury [164], [165] and [166], but it may impair liver regeneration [167]. Further research will be needed to clarify these issues.…”