Machine perfusion of fatty livers at 20° reduces sinusoidal cell and hepatocyte apoptosis respect to conventional cold storage

The demand for liver transplantation is in excess of available deceased donor livers, stimulating the research to improve the function of marginal donor livers, in particular fatty livers (FL); these are extremely vulnerable to conventional cold-storage (CS) and show more cell death after ischemia/reperfusion. Our group has developed a machine perfusion system with recirculation of an oxygenated medium at 20°C (MP20) that appears a promising strategy to protect normal and fatty liver (1). To further support the assumption that MP20 is a valid alternative for the preservation of FL, we investigated the response of FL to MP20, respect to CS, in terms of apoptotic cell death, using Zucker rats (ZR) as a model of hepatic steatosis. Methods: DNA fragmentation was detected with the TUNEL assay (ApopTag) and early apoptosis by the immunodetection of activated caspase-3 and of M30, a neo-epitope of CK18 degraded by activated caspasi-3 (present only in hepatocytes). Results: For obese Zucker rat statistically significant decrease of apoptotic hepatocytes and sinusoidal lining cells (SLC) after MP20 was observed respect to CS. The same was found for lean animals but only for SLC; TUNEL-stained cells were located mainly in periportal area (PP) and in the midzone (MZ). Whereas cholangioles were in general TUNEL and caspase-3 negative, occasionally positive bile ducts were seen after both treatments, suggesting that the oxygenation of these duct was compromised. Statistically significant decrease of early apoptotic hepatocytes after MP20 respect to CS both for lean and obese Zucker rat was observed; positive cells were seen mainly in the PP area. These results were confirmed by caspase-3 immunodetection. Conclusions: Apoptosis is crucial in cases of mild or moderate steatosis since apoptotic cells can exacerbate microcirculatory disturbances of FL and can be thrombogenic. It has been shown that pharmacological strategies to inhibit caspase-3 activity and ischemic preconditioning confer protection. We demonstrated that MP20 can reduce both SLC and hepatocyte apoptosis without need of further protective strategies. These new findings support the possibility of increasing the donor pool for transplantation by preserving FL with MP20. (COFIN 2006 funds). 1- Vairetti et al. 2008. Liver Transpl. In press.