**4. Steatosis in hepatic ischemia-reperfusion**

Several hypotheses have been suggested to explain the decreased tolerance of steatotic liver to I/R injury compared with non-steatotic livers. The impairment of the microcirculation is considered a major event of reperfusion injury in steatotic livers (Ijaz et al., 2003). A reduction in hepatic microcirculation has been observed in human fatty donor livers and in experimental models of hepatic steatosis (Ijaz et al., 2003; Seifalian et al., 1999). An imbalance between vasoconstrictors (e.g., ET1) and vasodilators (e.g., NO) negatively affect the hepatic microcirculation (Massip-Salcedo et al., 2007; Peralta et al., 2000a). In addition, fatty accumulation in the cytoplasm of hepatocytes is associated with an increase in cell volume that reduces the size of the hepatic sinusoid space by 50% compared with a normal liver and may result in partial or complete obstruction of the hepatic sinusoid space (Ijaz et al., 2003; Seifalian et al., 1999). Using Doppler flowmetry, Seifalian et al., 1999 demonstrated reduced sinusoidal perfusion in fatty human liver donors compared with healthy livers.

(necrotic cell death) or programmed cellular resorption (apoptosis), depending on factors

Fig. 2. Scheme of possible cell death pathway in hepatic I/R. (Alfany et al., 2009; Ben

Mosbah et al., 2010; Casillas et al., 2006; Fernández et al., 2004; Ghobrial et al., 2005; Jaeschke &Lemasters, 2003; Malhi et al., 2006; Massip-Salcedo et al., 2007; Selzner et al., 2000; Yin,

Several hypotheses have been suggested to explain the decreased tolerance of steatotic liver to I/R injury compared with non-steatotic livers. The impairment of the microcirculation is considered a major event of reperfusion injury in steatotic livers (Ijaz et al., 2003). A reduction in hepatic microcirculation has been observed in human fatty donor livers and in experimental models of hepatic steatosis (Ijaz et al., 2003; Seifalian et al., 1999). An imbalance between vasoconstrictors (e.g., ET1) and vasodilators (e.g., NO) negatively affect the hepatic microcirculation (Massip-Salcedo et al., 2007; Peralta et al., 2000a). In addition, fatty accumulation in the cytoplasm of hepatocytes is associated with an increase in cell volume that reduces the size of the hepatic sinusoid space by 50% compared with a normal liver and may result in partial or complete obstruction of the hepatic sinusoid space (Ijaz et al., 2003; Seifalian et al., 1999). Using Doppler flowmetry, Seifalian et al., 1999 demonstrated reduced sinusoidal perfusion in fatty human liver donors compared with healthy livers.

such as the decline of cellular ATP levels (see Fig. 2).

**4. Steatosis in hepatic ischemia-reperfusion** 

2000)

Analogous studies in rabbits with diet-induced steatosis confirmed that this reduction in perfusion correlated with the severity of fat accumulation in hepatocytes. The reductions in sinusoidal perfusion appear to arise initially from the effects of enlarged hepatic parenchymal cells, swollen with accumulated lipid, which widen the parenchymal cell plates and narrow and distort the lumens of sinusoids. Other investigators have shown that as a result of the structural alterations around them, the sinusoids become inefficient conduits of blood with resulting impairment of tissue perfusion, evidenced by the significant reductions in the numbers of perfused sinusoids per microscopic field (Teoh et al., 2010).

Hepatocyte damage appears remarkably higher in steatotic livers than in non-steatotic livers (Casillas et al., 2006; Selzner et al., 2000). Several evidences indicate that an increased sensitivity of fatty hepatocytes to the injurious effects of ROS could explain the poor tolerance of steatotic livers to I/R (Koneru et al., 2005; Soltys et al., 2001). It has been postulated that steatotic livers are more susceptible than nonsteatotic livers to lipid peroxidation because of either their lower antioxidant defenses or their greater production of ROS or both (Fernández et al., 2004). Mitochondrial ROS generation dramatically increases during reperfusion and mitochondrial structures are exposed to the attack of the ROS generated both outside and inside these organelles leading eventually to the dysfunction of important mitochondrial processes including those responsible for the ATP synthesis. In ROS generation systems, the inhibition of XOD with allopurinol effectively protected against the greater liver and lung damage in transplantation of steatotic livers (Fernández et al., 2004). Higher levels of IL-1β and lower IL-10 levels were observed in steatotic livers compared with non-steatotic livers after I/R. This imbalance between proand anti-inflammatory ILs was responsible for the vulnerability of steatotic livers to I/R (Serafin et al., 2004). Previous studies form our group indicated less glutathione (GSH) and SOD levels in steatotic livers than in non-steatotic livers as consequence of hepatic I/R (Fernández et al., 2004; Serafin et al., 2002).

It is well-known that steatotic livers synthesise less ATP than non-steatotic livers during post-ischemic reperfusion (Caraceni et al., 2005). Fatty degeneration induces a series of ultra-structural and biochemical alterations in both human and animal mitochondria. The lower ATP and adenine nucleotide content observed in steatotic livers preserved in UW solution could be caused by mitochondrial damage (Ben Mosbah et al., 2006; Caraceni et al., 2005; Massip-Salcedo et al., 2007). Caraceni et al., 2004 reported that alterations in oxidative phosphorylation during preservation is greatly enhanced by fatty infiltration resulting from damage to respiratory chain complex I and F0F1–ATP synthase. Others studies have discovered that in steatotic livers under conditions of either warm ischemia or transplantation, the content of mitochondrial uncoupling protein-2 (UCP-2) is four to five times higher than in non-steatotic livers (Chavin et al., 2004; Wan et al., 2008). This finding was associated with reduced ability to synthesize ATP upon reperfusion (Chavin et al., 2004). If cold storage time exceeds 10-12 h, complications in biliary structures occur in more than 25% of liver transplant recipients (Kukan & Haddad, 2001). Several factors, including poor recovery after ATP depletion appear to contribute to bile duct cell damage after liver transplantation. Furthermore, isolated rat bile duct epithelial cells are noticeably sensitive to oxidative stress, possibly because their cellular stores of reduced glutathione are seven times lower than those of hepatocytes (Noack et al., 1993). Taking these observations into account, bile production failure in steatotic livers could be explained, at least partially, by the lower ATP and increased oxidative stress presented by this type of liver compared with nonsteatotic liver.

Toll-like receptor 4 (TLR4) has been implicated as a mediator of steatotic liver damage after I/R (Ellett et al., 2009). The loss of TLR4 in steatotic livers from TLR4-knockout HFD animals reduces pro-inflammatory cytokines and liver injury and improves survival (Ellett et al., 2009). Although TLR4 signaling is relevant in hepatic I/R injury, there is some controversy over which of the pathways [(myeloid differentiation factor 88 (My-D88) dependent) or Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF/IRF-3 signalling pathway)] is activated in hepatic I/R (Kang et al., 2011). Neutrophils have been involved in the increased vulnerability of steatotic livers to I/R injury, especially in alcoholic steatotic livers. However, neutrophils do not account for the differentially greater injury in the non-alcoholic steatotic liver during the early or late hours of reperfusion. Similarly, the role of TNF-α in the vulnerability of steatotic livers to I/R injury may be dependent on the type of steatosis (Serafin et al., 2002). These observations could be of clinical interest because pharmacological strategies that could be effective in alcoholic fatty livers by reducing the neutrophil infiltration and or TNF-α action may not be sufficient to reduce the hepatic I/R injury in non-alcoholic fatty livers.

Cell death can occur by either necrosis or apoptosis and intracellular ATP level appear to play a role as a putative apoptosis/necrosis switch: when ATP depletion is severe, necrosis ensues before the activation of the energy-requiring apoptotic pathway (Casillas et al., 2006; Massip-Salcedo et al., 2007) (See Fig. 2). In steatotic liver graft undergoing 6 h of cold ischemia, necrosis was the predominant cell death whereas no apoptosis signs were found (Alfany et al., 2009; Fernández et al., 2004). Since apoptosis is an energy-requiring process, the impaired maintenance of ATP levels observed after reperfusion in steatotic livers submitted to long periods of cold ischemia may be linked with a failure to induce apoptosis. Thus, it is not surprising that data reported previously indicate that necrosis rather than apoptosis is the predominant process by which steatotic livers undergo cell death (Alfany et al., 2009; Fernández et al., 2004; Selzner et al., 2000).

Previous studies from our group have indicated that steatotic livers differed from nonsteatotic livers in their response to UPR and ER stress. Steatotic livers showed a reduced ability to respond to ER stress as the activation of two UPR arms, IRE1 and PERK, was weaker in the presence of steatosis. (Ben Mosbah et al., 2010). Different hypotheses, including decreased ATP production and dysfunction of regulators of apoptosis, such as Bcl-2, Bcl-xL and Bax have been proposed to explain the failure of apoptosis in steatotic livers. The results on ER stress in steatotic livers undergoing I/R may throw some light on this question. Reduced proapoptotic factors related to ER stress such as caspase 12, C/EDPhomologos protein (CHOP) and Jun N-terminal kinase (JNK) were observed in steatotic livers under conditions of I/R compared with non-steatotic livers. This may be related to the reduced activation of the two UPR arms, inositol-requiring enzyme-1 (IRE1) and PERK, which are responsible for caspase 9 and 12 activation, JNK activation and CHOP induction (Ben Mosbah et al., 2010) (see Fig. 2). We believe that the damaged ER and mitochondria are intimately linked and that mitochondrial cell death and ER-induced cell death cannot be separated in hepatic I/R. Thus, caspase activation and Cyt *c* release from mitochondria consequently to hepatic I/R (Ben Mosbah et al., 2010) can be attributed to ischemic disturbance or damage to the ER. Given these results in steatotic livers under warm ischemia conditions, it is therefore tempting to speculate that increased ER stress may be involved in the vulnerability of steatotic liver grafts to I/R injury associated with transplantation and in the sensitivity of other marginal grafts to I/R injury, such as liver grafts from aging donors. Indeed, aging donors have an increased incidence of steatosis, which may favor cold preservation injury (Busuttil & Tanaka, 2003; Massip-Salcedo et al., 2007). Alterations in the activation of inflammatory transcription factors and expression of cytoprotective proteins, increased intracellular oxidants and decreased mitochondrial function and protein misfolding accumulation, and aggregation also characterize many agerelated diseases (Massip-Salcedo et al., 2007; Pallet et al., 2009).
