Volume 44, Issue 4, Pages 694-701 (April 2006) Mouse livers with macrosteatosis are more susceptible to normothermic ischemic injury than those with microsteatosis  Nazia Selzner, Markus Selzner, Wolfram Jochum, Beatrice Amann-Vesti, Rolf Graf, Pierre-Alain Clavien  Journal of Hepatology  Volume 44, Issue 4, Pages 694-701 (April 2006) DOI: 10.1016/j.jhep.2005.07.032 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Pattern of fat distribution in lean and fatty livers. (a) Total amount of intrahepatic lipid content in lean and fatty mice. Both ob/ob and choline deficient mice contained comparable amounts of intrahepatic lipid (56±23.4 and 57.5±10.4μg/mg of protein, respectively), which was five times higher than lean control mice (9.6±4μg/mg of protein) (n=5, in each group, *P<0.05, Student's t-test). (b) Macro- vs. microvesicular steatosis in ob/ob and choline deficient mice. Ob/ob mice had a significantly larger preponderance of macrovesicular (58±6%) vs. microvesicular (27.5±4.4%) steatosis. In contrast, the liver from mice with a choline deficient diet contained a higher percentage of micro (66.7±5%) than macrovesicular (16.7±4.5%) inclusion (n=5 in each group, *P<0.05, Student's t-test). (c) Oil-red O stained section from the lean and fatty livers. Ob/ob mice had a higher predominance of macro- vs. microvesicular fat in the liver. In contrast, the liver of the choline deficient mice contained a higher percentage of micro than macrovesicular steatosis. Of note, no fat staining was observed in the lean animals. Magnification of the histologic sections ×100 (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article.). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 AST levels after reperfusion. Ob/ob ▪ mice had a significantly higher AST release at 30min (7433±713U/l) and 4h (19940±4471U/l) after reperfusion compared to choline deficient mice ▴ (4933±309 and 8025±998IU/ml) and to the lean controls ♦ (4033±601 and 6873±873U/l). In contrast to the lean mice, AST levels increased in the choline deficient mice at later time of reperfusion, with values comparable to the ob/ob mice (14–500±3395 vs. 16,566±5731U/l) at 24h of reperfusion (n=5 in each group, *P<0.05 compared to the choline deficient and lean animals, **P<0.05 compared to the lean animals, Student's t-test). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Necrosis in liver tissue after reperfusion. (a) Four hours after reperfusion □, Ob/ob mice had a significantly higher percentage of necrosis (70±8.5%) compared to choline deficient (26±5%) and to the lean controls (14±3%). *P<0.05. (b) At 24h of reperfusion ▪, only a small percentage of necrosis was noted in lean animals (25±12%), while both macro- (80±21%) and microvesicular (65±24%) steatosis were associated with large area of necrosis (n=5 in each group,**P<0.05, Student's t-test). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Intrahepatic energy content in liver tissue. Both types of fatty livers (ob/ob ▪ and choline deficient ▴) had lower ATP levels at baseline (sham) as well as at different times of reperfusion in comparison to the lean mice (♦). However, no difference was observed regarding ATP contents between the two types of fatty livers. Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Portal vein and sinusoidal flow after reperfusion. (a) Ob/ob mice (▪) had a significantly higher drop on portal vein flow after 45min of ischemia (18±2.5%). *P<0.05 compared to choline deficient (▴) (60±5%) and lean (♦) mice (55±7%) (n=5 in each group). (b) Similarly, Ob/ob (▪) mice had also a significant higher drop of sinusoidal flow after 45min of ischemia (31±2.5%). *P<0.05, compared to choline deficient (▴) (72±5%) and lean (♦) mice (86±7%) (n=5 in each group). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 AST levels and necrosis in liver tissue after ischemic pre-conditioning. (a) Ischemic pre-conditioning improved AST level in both macro- (13,620±4692U/l) and microvesicular (3975±869U/l) steatosis in comparison with their respective control group without pre-conditioning (19940±4471 and 8025±998U/l) (n=5 in each group, *P<0.05, Student's t-test). (b) Necrosis in liver tissue was significantly improved in the pre-conditioning groups in both macro- (24±3%) and microvesicular (6±0.2%) steatosis compared to their respective control group without pre-conditioning (70±8.5%) and (26±5%) (n=5 in each group, *P<0.05, Student's t-test). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Portal vein and sinusoidal flow after ischemic pre-conditioning. Portal vein blood flow and sinusoidal flow were investigated immediately after 45min of ischemia and 4h of reperfusion. (a and b) Pre-conditioning improved portal vein flow in both types of fatty livers (Ob/ob, choline deficient) and lean animals at the end of ischemia (a) and after 4h of reperfusion (b). The protection conferred by ischemic pre-conditioning was more prominent in microvesicular steatosis with a complete recovery of portal vein flow to baseline values (100%) after 4h of reperfusion compared to their control group without pre-conditioning (*P<0.05). In contrast, despite ischemic pre-conditioning the portal vein flow in macrovesicular fatty livers remained impaired with 69% of the baseline flow (**P<0.05) (n=5 in each group). (c and d) Pre-conditioning improved sinusoidal flow in both types of fatty livers (Ob/ob, choline deficient) and also in lean mice at the end of ischemia (c) and after 4h of reperfusion (d) compared to their respective control group without pre-conditioning (Ob/ob, choline deficient and lean) (*P<0.05, n=5 in each group). Journal of Hepatology 2006 44, 694-701DOI: (10.1016/j.jhep.2005.07.032) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions