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Hepatic ischemia and reperfusion injury: Effects on the liver sinusoidal milieu

  • Carmen Peralta
    Affiliations
    Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
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  • Mónica B. Jiménez-Castro
    Affiliations
    Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
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  • Jordi Gracia-Sancho
    Correspondence
    Corresponding author. Address: Barcelona Hepatic Hemodynamic Laboratory, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/Rosselló 153, 4th floor, room 4.5, 08036 Barcelona, Spain. Tel.: +34 932275400x4306; fax: +34 932279856.
    Affiliations
    Barcelona Hepatic Hemodynamic Laboratory, IDIBAPS, CIBEREHD, Barcelona, Spain
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Open AccessPublished:June 26, 2013DOI:https://doi.org/10.1016/j.jhep.2013.06.017

      Summary

      Ischemia-reperfusion injury is an important cause of liver damage occurring during surgical procedures including hepatic resection and liver transplantation, and represents the main underlying cause of graft dysfunction post-transplantation. Cellular and biochemical processes occurring during hepatic ischemia-reperfusion are diverse and complex, and include the deregulation of the healthy phenotype of all liver cellular components. Nevertheless, a significant part of these processes are still unknown or unclear. The present review aims at summarizing the current knowledge in liver ischemia-reperfusion, but specifically focusing on liver cell phenotype and paracrine interaction deregulations. Moreover, the most updated therapeutic strategies including pharmacological, genetic and surgical interventions, as well as some of the scientific controversies in the field will be described. Finally, the importance of considering the subclinical situation of liver grafts when translating basic knowledge to the bedside is discussed.

      Abbreviations:

      I/R (ischemia-reperfusion), LSEC (liver sinusoidal endothelial cells), KC (Kupffer cells), HSC (hepatic stellate cells), NAD (nicotinamide adenine dinucleotide), ROS (reactive oxygen species), NO (nitric oxide), ET (endothelin), TXA2 (thromboxane A2), KLF2 (Kruppel-like factor 2), eNOS (endothelial nitric oxide synthase), TNF (tumour necrosis factor), IL (interleukin), INF-γ (interferon-gamma), ICAM-1 (intracellular adhesion molecule-1), MIP-2 (macrophage inflammatory protein-2), ENA-78 (epithelial neutrophil activating protein-78), CINC (cytokine-induced neutrophil chemoattractant-1), GM-CSF (granulocyte-macrophage colony-stimulating factor), GdCl3 (gadolinium chloride), NFκB (nuclear factor kappa B), HO-1 (heme oxygenase-1), PPAR (peroxisome proliferator-activated receptor)

      Keywords

      Deregulation of hepatic cell phenotype due to ischemia-reperfusion injury

      Ischemia-reperfusion (I/R) injury is an important cause of liver damage during surgical procedures such as hepatic resection and liver transplantation. I/R injury is a biphasic phenomenon whereby cellular damage due to hypoxia and lack of biomechanical stimulus is accentuated upon restoration of oxygen delivery and shear stress. The signaling events contributing to local hepatocellular damage are diverse and complex, and involve the interaction between hepatocytes, liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), hepatic stellate cells (HSC), as well as infiltrating neutrophils, macrophages, and platelets [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Montalvo-Jave E.E.
      • Escalante-Tattersfield T.
      • Ortega-Salgado J.A.
      • Piña E.
      • Geller D.A.
      Factors in the pathophysiology of the liver ischemia-reperfusion injury.
      ,
      • Jaeschke H.
      Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning.
      ,
      • Gracia-Sancho J.
      • Villarreal Jr., G.
      • Zhang Y.
      • Yu J.X.
      • Liu Y.
      • Tullius S.G.
      • et al.
      Flow cessation triggers endothelial dysfunction during organ cold storage conditions: strategies for pharmacologic intervention.
      ]. It is important to note that I/R injury represents the main reason of liver graft dysfunction post-transplantation, independently of liver basal characteristics, being even more relevant when using organs from extended-criteria donors.
      Hepatocytes are very much negatively affected by I/R, especially when ischemia is normothermic. Most early changes in the anoxic hepatocytes occur in the mitochondria. The lack of O2, as a terminal electron carrier for the mitochondrial respiratory chain, immediately interrupts the electron flow causing the respiratory chain to become reduced. Since mitochondria are no longer accepting electrons from substrates, a reduction in pyridine nucleotides occurs, resulting in an increase in the intracellular NADH/NAD+ ratio. The abruption of oxidative phosphorylation rapidly leads to cellular ATP depletion, acceleration of glycolysis, increased formation of lactate, and alterations on H+, Na+, and Ca2+ homeostasis, altogether inducing serious deleterious effects on the hepatocyte. Ischemia also leads to a considerable increase in cAMP, which is an important factor in glucose metabolism. cAMP, through the action of cAMP-dependent protein kinase, leads to the phosphorylation/deregulation of key enzymes involved in the control of carbohydrate metabolism [
      • Gasbarrini A.
      • Borle A.B.
      • Farghali H.
      • Bender C.
      • Francavilla A.
      • Van Thiel D.
      Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.
      ,
      • Peralta C.
      • Bartrons R.
      • Riera L.
      • Manzano A.
      • Xaus C.
      • Gelpí E.
      • et al.
      Hepatic preconditioning preserves energy metabolism during sustained ischemia.
      ]. Reperfusion injury mainly derives from toxic reactive oxygen species (ROS) generated upon reintroduction of O2 to ischemic tissues. ROS are produced from both intracellular and extracellular sources, being the mitochondria their major source in liver cells [
      • Caraceni P.
      • Domenicali M.
      • Vendemiale G.
      • Grattagliano I.
      • Pertosa A.
      • Nardo B.
      • et al.
      The reduced tolerance of rat fatty liver to ischemia reperfusion is associated with mitochondrial oxidative injury.
      ] (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Hepatocyte phenotype deregulations due to ischemia and reperfusion injury. A summary of the molecular mechanisms affecting the hepatocyte is given, the specific modifications due to I/R are indicated in red. AC, adenine cyclase; AMPK, 5′ adenosine monophosphate-activated kinase; ADP, adenine diphosphate; ATP, adenine triphosphate; A2aR, A2A adenosine receptor; Ang, angiotensin; cAMP, 3′-5′-cyclic adenosine monophosphate; ER, endoplasmic reticulum; FBPase-1, fructose 1,6-bisphosphatase; GK, glucokinase; GSH, gluthatione; HO-1, heme oxygenase-1; HSPs, heat shock proteins; MAPK, mitogen-activated protein kinase; NO, nitric oxide; Nrf2, nuclear factor (erythroid-derived 2)-like 2; O2, superoxide; OAA, oxaloacetic acid; ONOO, peroxynitrite; PEP, phosphoenolpyruvate; PFK, phosphofructokinase; PK, protein kinase; RBP4, retinol-binding protein 4; SOD, superoxide dismutase; UPR, unfolded protein response; X/XOD, xanthine/xanthine oxidase.
      Liver sinusoidal endothelial cells (LSEC) form the vascular wall of the hepatic sinusoid, lack an organized basal membrane, and the cytoplasm of these flattened cells is penetrated by open fenestrations that form clusters called sieve plates, making the hepatic microvascular endothelium discontinuous [
      • Wisse E.
      An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells.
      ]. LSEC play important protective roles controlling vascular homeostasis, inflammation, vascular tone, and toxicants clearance. Thus, maintenance of a healthy LSEC phenotype is indispensable to minimize any type of liver injury.
      LSEC are particularly vulnerable to I/R injury and develop serious alterations during cold storage (Fig. 2). In fact, pioneering studies by Caldwell-Kenkel and colleagues described the deregulation of LSEC viability due to I/R injury as plasma membranes discontinuation, nuclear membranes vacuolization, and cell shape rounding [
      • Caldwell-Kenkel J.C.
      • Thurman R.G.
      • Lemasters J.J.
      Selective loss of nonparenchymal cell viability after cold ischemic storage of rat livers.
      ]. These very initial observations, which were usually reported at the Kupffer Cell Foundation meetings (currently the International Society for Hepatic Sinusoidal Research), have been extended during the last decades. Nowadays, it is accepted that hepatic endothelium damage occurring during cold preservation represents the initial factor leading to hepatic I/R injury, determining poor graft microcirculation, platelet activation, persistent vasoconstriction, upregulation of adhesion molecules, oxidative stress, Kupffer cell activation, neutrophil infiltration, and hepatocyte death.
      Figure thumbnail gr2
      Fig. 2Liver endothelial deregulations and sinusoidal cellular interactions during ischemia and reperfusion injury. A summary of the molecular mechanisms affecting the liver sinusoidal cells is shown, the specific alterations due to I/R are indicated in red. CAM, cell adhesion molecule; cGMP, cyclic guanilate monophosphate; eNOS, endothelial nitric oxide synthase; ESAM, endothelial selective adhesion molecule; ET-1, endothelin 1; H2O2, hydrogen peroxide; HO-1, heme oxygenase-1; HSC, hepatic stellate cell; IL-1, interleukin 1; ICAM, intracellular cell adhesion molecule; KLF2, Kruppel-like factor 2; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; MMPs, metalloproteinases; NO, nitric oxide; O2, superoxide; OH, hydroxyl; ONOO, peroxynitrite; PMN, polymorphonuclear neutrophil; ROS, reactive oxygen species; ROCK, rho kinase; TM, thrombomodulin; vWF, von Willebrand factor.
      During the ischemic period, the lack of energetic substrate interferes with active transmembrane transport, producing edema in KC and LSEC [
      • Vollmar B.
      • Glasz J.
      • Leiderer R.
      • Post S.
      • Menger M.D.
      Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion.
      ]. This fact, together with the imbalance between low nitric oxide (NO) bioavailability and exacerbated endothelin (ET) and thromboxane A2 (TXA2) production, contributes to narrowing the sinusoidal lumen, and thus to microcirculatory dysfunction. Diminished NO levels within the liver during I/R are derived from both decreased production and increased scavenging by elevated levels of ROS, and ultimately modulate the intensity of the I/R injury by regulating neutrophil adhesion, platelet aggregation, and HSC contraction [
      • Serracino-Inglott F.
      • Habib N.A.
      • Mathie R.T.
      Hepatic ischemia-reperfusion injury.
      ,
      • Cywes R.
      • Packham M.A.
      • Tietze L.
      • Sanabria J.R.
      • Harvey P.R.
      • Phillips M.J.
      • et al.
      Role of platelets in hepatic allograft preservation injury in the rat.
      ,
      • Clemens M.G.
      Nitric oxide in liver injury.
      ]. In addition, recent studies have demonstrated that lack of biomechanical stimuli occurring during cold preservation for transplantation markedly deteriorates LSEC protective phenotype by downregulating the expression of the transcription factor Kruppel-like Factor 2 (KLF2), which orchestrates the transcription of a variety of protective genes including the endothelial synthase of NO (eNOS), the anti-thrombotic molecule thrombomodulin, or the antioxidant transcription factor Nrf2 [
      • Gracia-Sancho J.
      • Villarreal Jr., G.
      • Zhang Y.
      • Yu J.X.
      • Liu Y.
      • Tullius S.G.
      • et al.
      Flow cessation triggers endothelial dysfunction during organ cold storage conditions: strategies for pharmacologic intervention.
      ,
      • Russo L.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Marrone G.
      • García-Pagán J.C.
      • García-Cardeña G.
      • et al.
      Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.
      ].
      Concomitantly to LSEC deregulation, KC suffer from a profound activation process that is promoted by neighbour hepatic cells-released damage-associated molecular patterns (DAMPs) and, under conditions of sepsis or endotoxemia, also by pathogen-associated molecular patterns (PAMPs) [
      • Huang H.
      • Evankovich J.
      • Yan W.
      • Nace G.
      • Zhang L.
      • Ross M.
      • et al.
      Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice.
      ,
      • Van Bossuyt H.
      • Wisse E.
      Structural changes produced in kupffer cells in the rat liver by injection of lipopolysaccharide.
      ]. Activated KC significantly increase their release of ROS and pro-inflammatory cytokines, including tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), interferon-γ (INF-γ) and interleukin-12 (IL-12) (Fig. 2) [
      • Bilzer M.
      • Gerbes A.
      Preservation injury of the liver: mechanisms and novel therapeutic strategies.
      ,
      • Lentsch A.
      • Kato A.
      • Yoshidome H.
      • McMasters K.
      • Edwards M.
      Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury.
      ]. Both TNF-α and IL-1 upregulate Mac-1 (CD11b/CD18) adhesion proteins on neutrophils and induce IL-8 synthesis, further promoting neutrophil chemotaxis within the parenchyma [
      • Witthaut R.
      • Farhood A.
      • Smith C.W.
      • Jaeschke H.
      Complement and tumor necrosis factor-alpha contribute to Mac-1 (CD11b/CD18) up-regulation and systemic neutrophil activation during endotoxemia in vivo.
      ]. Moreover, IL-1 has the potential to stimulate the release of ROS by neutrophils, which will further increase TNF-α synthesis by KC [
      • Perry B.C.
      • Soltys D.
      • Toledo A.H.
      • Toledo-Pereyra L.H.
      Tumor necrosis factor-α in liver ischemia/reperfusion injury.
      ]. TNF-α stimulates the expression of the intracellular adhesion molecule-1 (ICAM-1) on the intraluminal side of LSEC, contributing to neutrophil rolling, binding, and parenchymal extravasation [
      • Perry B.C.
      • Soltys D.
      • Toledo A.H.
      • Toledo-Pereyra L.H.
      Tumor necrosis factor-α in liver ischemia/reperfusion injury.
      ]. TNF-α also induces P-selectin expression in LSEC, being essential for the recruitment of neutrophils [
      • Peralta C.
      • Fernández L.
      • Panés J.
      • Prats N.
      • Sans M.
      • Piqué J.M.
      • et al.
      Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat.
      ]. TNF-α has been shown to increase the release of other molecules, including interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), epithelial neutrophil activating protein-78 (ENA-78), cytokine-induced neutrophil chemoattractant-1 (CINC), and a number of CXC motif chemokines (including CXL-1, -2, and -3). In addition, IL-1 and TNF-α recruit and activate CD4+ T-lymphocytes, which produce granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (INF-γ) and tumour necrosis factor beta (TNF-β). These cytokines amplify KC activation and promote neutrophil recruitment and adherence into the liver sinusoids [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Teoh N.C.
      • Farrell G.C.
      Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection.
      ,
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ].
      Platelets also play an important role in hepatic I/R injury. In fact, in response to I/R, platelets synthesize and release several factors that intervene in liver transplant and hepatic regeneration [
      • Lesurtel M.
      • Graf R.
      • Aleil B.
      • Walther D.J.
      • Tian Y.
      • Jochum W.
      • et al.
      Platelet-derived serotonin mediates liver regeneration.
      ]. These include platelet activating factor, cytokines, growth factors including NO, TGF-β, serotonin, and calpain. Platelet activating factor, which can also be produced by KC and LSEC, primes neutrophils for ROS generation and leukotriene B4 release, further contributing to the amplification of the neutrophil response [
      • Jaeschke H.
      Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning.
      ]. Platelets also adhere to the hepatic sinusoids and induce programmed LSEC death through the production of NO, which ultimately will result in the potent toxicant peroxynitrite (Fig. 2) [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Sindram D.
      • Porte R.J.
      • Hoffman M.R.
      • Bentley R.C.
      • Clavien P.A.
      Platelets induce sinusoidal endothelial cell apoptosis upon reperfusion of the cold ischemic rat liver.
      ].

      Controversial mechanisms of liver cells deregulation in hepatic I/R injury

      Although most of published data converge on the currently known molecular mechanisms underlying liver cells deregulation during I/R injury, other findings are not completely understood or validated up to now. The following data summarizes some of these controversies, and may provide a better understanding of why hepatic I/R injury still remains an unresolved problem in clinical practice.

      Sinusoidal endothelial cells

      Although LSEC injury due to I/R was reported in the late 1980s [
      • Caldwell-Kenkel J.C.
      • Thurman R.G.
      • Lemasters J.J.
      Selective loss of nonparenchymal cell viability after cold ischemic storage of rat livers.
      ], this cell type has somehow been relegated to a secondary place and only few research groups have devoted their efforts to better understand the pathophysiology of LSEC during I/R. Rauen and colleagues did demonstrate that LSEC under cold ischemia and warm reperfusion conditions exhibit markedly increased levels of ROS that ultimately lead to decrease in LSEC viability [
      • Rauen U.
      • Elling B.
      • Gizewski E.R.
      • Korth H.G.
      • Sustmann R.
      • de Groot H.
      Involvement of reactive oxygen species in the preservation injury to cultured liver endothelial cells.
      ]. Other studies demonstrated upregulation of adhesion molecules [

      Nishimura Y, Takei Y, Kawano S, Goto M, Nagai H, Ohmae A, et al. Expression of ICAM-1 is involved in the mechanism of liver injury after orthotopic liver transplantation. In: Wisse E, Knook DL, Wake K, editors. Cells of the hepatic sinusoid, vol. 5; 1994. p. 231–233.

      ], actin disassembly and cell deregulation due to an increment in calcium-derived calpain activity [
      • Upadhya G.A.
      • Topp S.A.
      • Hotchkiss R.S.
      • Anagli J.
      • Strasberg S.M.
      Effect of cold preservation on intracellular calcium concentration and calpain activity in rat sinusoidal endothelial cells.
      ], and LSEC death due to elastase-mediated paracrine interactions with leucocytes [

      Lasnier E, Blanc MC, Roch-Arveiller M, Housset C. Interaction between polymorphonuclear leukocytes and hepatic sinusoidal endothelial cells. Role of elastase. In: Wisse E, Knook DL, de Zanger R, Fraser R, editors. Cells of the hepatic sinusoid, vol. 7; 1998, p. 125–126.

      ]. Nevertheless, scarce reports investigated the physiological consequences of SEC deregulation on hepatic I/R injury, and more importantly, very little is currently known about the relative importance of LSEC de-regulation due to I/R on the global reduction in organ viability and function post-transplantation. In fact, very recent data obtained in experimental models of liver preservation for transplantation demonstrated that cold storage per se negatively affects the LSEC phenotype, becoming pro-inflammatory, pro-thrombotic, and vasoconstrictor, which is translated into acute endothelial dysfunction development [
      • Russo L.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Marrone G.
      • García-Pagán J.C.
      • García-Cardeña G.
      • et al.
      Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.
      ]. Importantly, the alterations in LSEC phenotype occur due to the abolition of the blood-derived biomechanical stimuli, which have been demonstrated to be protective, and suggest that part of the beneficial effects of the normothermic machine perfusion may derive from the continuous vascular stimulation obtained with this novel and promising strategy [
      • Yuan X.
      • Theruvath A.J.
      • Ge X.
      • Floerchinger B.
      • Jurisch A.
      • García-Cardeña G.
      • et al.
      Machine perfusion or cold storage in organ transplantation: indication, mechanisms, and future perspectives.
      ,
      • Fondevila C.
      • Hessheimer A.J.
      • Maathuis M.H.
      • Muñoz J.
      • Taurá P.
      • Calatayud D.
      • et al.
      Superior preservation of DCD livers with continuous normothermic perfusion.
      ,
      • García-Valdecasas J.C.
      • Fondevila C.
      In-vivo normothermic recirculation: an update.
      ].

      Kupffer cells

      It has been shown that KC, isolated after in vivo I/R, spontaneously release increased amounts of TNF-α compared to controls, thus proposing them as main generators of this pro-inflammatory cytokine during liver I/R [
      • Wanner G.A.
      • Ertel W.
      • Müller P.
      • Höfer Y.
      • Leiderer R.
      • Menger M.D.
      • et al.
      Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation.
      ]. However, other studies have contradicted this hypothesis demonstrating that inactivation of KC with gadolinium chloride before rat liver transplantation does not reduce expression of TNF-α, then suggesting that KC are not the only source of TNF-α in the liver [
      • Bradham C.A.
      • Schemmer P.
      • Stachlewitz R.F.
      • Thurman R.G.
      • Brenner D.A.
      Activation of nuclear factor-kappaB during orthotopic liver transplantation in rats is protective and does not require Kupffer cells.
      ]. Of note, the stimulatory state of KC after I/R depends on the duration of ischemia, and may also differ between ischemia at 4 °C and that at 37 °C, and between liver grafts types, which altogether probably leads to different mechanisms of liver damage [
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ].

      Neutrophil accumulation

      As described above, activation of neutrophils has been implicated in the hepatic microvascular dysfunction and parenchymal damage associated with I/R [
      • Cutrin J.C.
      • Perrelli M.G.
      • Cavalieri B.
      • Peralta C.
      • Rosello-Catafau J.
      • Poli G.
      Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning.
      ]. Still, a controversial topic is the question of how neutrophils actually accumulate within the liver. The classical theory argues that the increased expression of adhesion molecules, such as ICAM-1 and P-selectin, plays a key role in neutrophil collection and the subsequent liver damage associated with I/R [
      • Cutrin J.C.
      • Perrelli M.G.
      • Cavalieri B.
      • Peralta C.
      • Rosello-Catafau J.
      • Poli G.
      Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning.
      ]. By contrast, it has also been reported that neutrophil accumulation in the liver after I/R occurs independently of the upregulation of either ICAM-1 or P-selectin [
      • Peralta C.
      • Fernandez L.
      • Panes J.
      • Prats N.
      • Sans M.
      • Pique J.M.
      • et al.
      Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat.
      ]. This alternative theory proposed that mechanical factors such as active vasoconstriction, vascular cell swelling and injury, and reduced membrane flexibility after activation of neutrophils are involved in trapping these leukocytes into the sinusoids [
      • Jaeschke H.
      Preservation injury: mechanisms, prevention and consequences.
      ]. Indeed, the extensive vascular injury happening during reperfusion partly eliminates the LSEC barrier, thus the neutrophils have direct access to hepatocytes [
      • Jaeschke H.
      Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning.
      ]. According to this alternative hypothesis, anti-ICAM-1 therapies failed to protect liver grafts against I/R injury [
      • Farhood A.
      • McGuire G.M.
      • Manning A.M.
      • Miyasaka M.
      • Smith C.W.
      • Jaeschke H.
      Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver.
      ]. Regarding the possible role of P-selectin in neutrophil recruitment, although LSEC neither contain Weibel Palade bodies nor transcriptionally upregulate relevant levels of P-selectin after liver injury [
      • Essani N.A.
      • Fisher M.A.
      • Simmons C.A.
      • Hoover J.L.
      • Farhood A.
      • Jaeschke H.
      Increased P-selectin gene expression in the liver vasculature and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock.
      ], a variety of interventions directed against selectins in the experimental scenario of liver I/R injury reduced hepatic neutrophil accumulation and hepatocellular injury [
      • Amersi F.
      • Dulkanchainun T.
      • Nelson S.K.
      • Farmer D.G.
      • Kato H.
      • Zaky J.
      • et al.
      A novel iron chelator in combination with a P-selectin antagonist prevents ischemia/reperfusion injury in a rat liver model.
      ]. Since these findings cannot be explained by the prevention of P-selectin-dependent rolling over the hepatic sinusoids, it has been suggested that most models of liver I/R may include some degree of intestinal ischemia, which would lead to neutrophil accumulation in remote organs including the liver [
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ,
      • Kubes P.
      • Payne D.
      • Woodman R.C.
      Molecular mechanisms of leukocyte recruitment in postischemic liver microcirculation.
      ]. Thus, the lower amount of neutrophils in the liver when selectins are blocked may be a secondary effect derived from the protection of anti-selectin therapy against intestinal reperfusion injury [
      • Kubes P.
      • Payne D.
      • Woodman R.C.
      Molecular mechanisms of leukocyte recruitment in postischemic liver microcirculation.
      ].

      Time-dependent sensitivity of hepatocyte and LSEC to ischemia

      Two of the liver cell types mostly affected during I/R injury are hepatocytes and sinusoidal endothelial cells, however, they show differential sensitivity to different types of ischemia: hepatocytes are more sensitive to warm ischemia, and LSEC to cold ischemia [
      • Bilzer M.
      • Gerbes A.
      Preservation injury of the liver: mechanisms and novel therapeutic strategies.
      ,
      • Ikeda T.
      • Yanaga K.
      • Kishikawa K.
      • Kakizoe S.
      • Shimada M.
      • Sugimachi K.
      Ischemic injury in liver transplantation: difference in injury sites between warm and cold ischemia in rats.
      ]. In fact, although most hepatocytes remain viable for 48 h after cold preservation and warm reperfusion, LSEC rapidly suffer from severe damage (up to 50% of them end up as non-viable cells) [
      • Caldwell-Kenkel J.C.
      • Currin R.T.
      • Tanaka Y.
      • Thurman R.G.
      • Lemasters J.J.
      Reperfusion injury to endothelial cells following cold ischemic storage of rat livers.
      ].
      The severity of hepatocyte damage depends on the length of warm ischemia. Short periods of ischemia (60 min) result in reversible cell injury in which liver oxygen consumption returns to normal levels when oxygen is resupplied during reperfusion. Nevertheless, reperfusion after more prolonged periods of warm ischemia (120–180 min) results in irreversible cell damage, being 90 min of warm ischemia the estimated time limit for hepatocytes survival [
      • Gonzalez-Flecha B.
      • Cutrin J.C.
      • Boveris A.
      Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion.
      ]. In cold ischemia, in vitro studies demonstrated maintenance of hepatocyte function and viability up to 72 h of preservation [
      • Pazo J.A.
      • Rodriguez M.E.
      • Vega F.
      • De la Cruz L.
      • Guilbert E.E.
      • Mediavilla M.G.
      • et al.
      Primary culture of rat hepatocytes after cold storage in the university of wisconsin solution: a tool to study the effects of hypothermic preservation.
      ], however, experimental models of liver transplantation indicate that 24 h, or even shorter periods, of cold ischemia may be accompanied by the development of graft primary non-function after transplantation [
      • Fernández L.
      • Heredia N.
      • Grande L.
      • Gómez G.
      • Rimola A.
      • Marco A.
      • et al.
      Preconditioning protects liver and lung damage in rat liver transplantation: role of xanthine/xanthine oxidase.
      ]. The divergence between in vitro and in vivo studies is attributed to the significant sinusoidal damage occurring during cold storage, which will result in microcirculatory abnormalities and hepatocyte injury. In fact, in vitro experiments demonstrated that LSEC function and viability are significantly compromised after 8–16 h of cold storage, and completely blunted afterwards [
      • Reinders M.E.
      • van Wagensveld B.A.
      • van Gulik T.M.
      • Frederiks W.M.
      • Chamuleau R.A.
      • Endert E.
      • et al.
      Hyaluronic acid uptake in the assessment of sinusoidal endothelial cell damage after cold storage and normothermic reperfusion of rat livers.
      ,
      • Rauen U.
      • Hanssen M.
      • Lauchart W.
      • Becker H.D.
      • de Groot H.
      Energy-dependent injury to cultured sinusoidal endothelial cells of the rat liver in UW solution.
      ]. Moreover, studies using experimental models of liver preservation for transplantation showed acute endothelial dysfunction development after 16 h of cold storage, associated with significant hepatocellular injury and death [
      • Russo L.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Marrone G.
      • García-Pagán J.C.
      • García-Cardeña G.
      • et al.
      Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.
      ]. Considering the intimate cellular cross-talk between LSEC and hepatocytes, it is very possible that LSEC injury due to cold storage may negatively affect hepatocyte viability, and vice versa. In fact, hepatocytes from cold stored livers exhibit perturbations in key hepatocellular functions like solute transport or drug metabolism, together with low levels of adenine nucleotides that may trigger proteolytic events, altogether contributing to liver graft dysfunction [
      • Kukan M.
      • Haddad P.
      Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts.
      ]. These observations indicate that aside from reducing LSEC damage, I/R injury therapy may also benefit from strategies aimed at maintaining appropriate hepatocyte functions [
      • Kukan M.
      • Haddad P.
      Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts.
      ,
      • Vajdova K.
      • Graf R.
      • Clavien P.
      ATP-supplies in the cold-preserved liver: a long-neglected factor of organ viability.
      ].
      Another factor affecting the time-dependent sensitivity of liver cells to I/R is the percentage of ischemia applied. The extent of hepatic injury, as well as part of the underlying mechanisms, depends on whether a total or partial hepatic ischemia is conducted [
      • Hasselgren P.
      • Jennische E.
      • Fornander J.
      • Hellman A.
      No beneficial affect of ATP-MgCl2 on impaired transmembrane potential and protein synthesis in liver ischemia.
      ,
      • Clemens M.G.
      • Mcdonagh P.F.
      • Chaudry I.H.
      • Baue A.E.
      Hepatic microcirculatory failure after ischemia and reperfusion: improvement with ATP-MgCl2 treatment.
      ]. Different authors have suggested that this fact could be explained by the “stealing phenomenon”: In contrast to what happens in the 100% hepatic ischemia, during ischemia of the left and median lobes (70% of ischemia), the hepatic flow is totally shunted through the right lobes, and even after the release of the occlusion, a significant amount of flow is still deflected (“steeled”) through the right lobes, until vascular resistance of the post-ischemic lobes decreases. This is due to the physical fact that blood will flow through the vascular path with less resistance. Thus, the recovery of blood flow and hepatic perfusion in the post-ischemic lobes occurs much later in the case of partial than total hepatic ischemia [
      • Anderson M.D.
      • Garrison R.N.
      • Ratcliffe D.J.
      • Fry D.E.
      Hepatic “no reflow” following transient ischemia.
      ]. In line with these observations, the benefits of some drugs such as ATP–MgCl2 depend on the extent of the hepatic ischemia used.

      Type of cell death occurring in hepatocytes and LSEC subjected to ischemia

      The exact mechanism of cell death in hepatic I/R injury remains unclear. Apoptosis of hepatic cells, including hepatocytes and LSEC, due to I/R has been described by different reports [
      • Gao W.
      • Bentley R.
      • Madden J.
      • Clavien P.
      Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation.
      ,
      • Kohli V.
      • Selzner M.
      • Madden J.
      • Bentley R.
      • Clavien P.
      Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver.
      ], and partly occurs through the c-Jun N-terminal kinase 2-mediated mitochondrial permeability transition pathway that ultimately leads to cytochrome C release and caspase activation [
      • Theruvath T.P.
      • Czerny C.
      • Ramshesh V.K.
      • Zhong Z.
      • Chavin K.D.
      • Lemasters J.J.
      C-Jun N-terminal kinase 2 promotes graft injury via the mitochondrial permeability transition after mouse liver transplantation.
      ,
      • Rauen U.
      • Kerkweg U.
      • Weisheit D.
      • Petrat F.
      • Sustmann R.
      • de Groot H.
      Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.
      ]. On the other hand, other groups oppose the view that the majority of cells undergo apoptosis in response to either warm or cold I/R injury, believing that necrosis is the main form of cell death [
      • Massip-Salcedo M.
      • Roselló-Catafau J.
      • Prieto J.
      • Avila M.
      • Peralta C.
      The response of the hepatocyte to ischemia.
      ]. This last hypothesis derives from the idea that the proportion of cells undergoing apoptosis described in other studies is not of significant magnitude, and that the degree of caspase activation does not correlate with the number of LSEC and hepatocytes supposedly undergoing apoptosis. Thus, a controversy has emerged over the past years as to whether necrotic or apoptotic cell death accounts for the severe parenchymal injury observed during hepatic reperfusion. Remarkably, and although it has been assumed that necrosis and apoptosis are different processes, a new term “necrapoptosis” has been coined to describe a process that begins with a common death signal and culminates in either cell lysis (necrotic cell death) or programmed cellular resorption (apoptosis), depending on factors such as the decline of cellular ATP levels [
      • Lemasters J.J.
      V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis.
      ].

      Relevance of the type of liver undergoing I/R

      A variety of subclinical factors including starvation, donor age, and graft steatosis contribute to enhance liver susceptibility to I/R injury, thus increasing patient risks. It is well-known that the shortage of organs has led care centres to expand their criteria for the acceptance of graft donors. Some of these criteria include the use of organs from elderly donors and steatotic liver grafts [
      • Grazi G.L.
      • Cescon M.
      • Ravaioli M.
      • Ercolani G.
      • Pierangeli F.
      • D’Errico A.
      • et al.
      A revised consideration on the use of very aged donors for liver transplantation.
      ,
      • Fernandez L.
      • Carrasco-Chaumel E.
      • Serafin A.
      • Xaus C.
      • Grande L.
      • Rimola A.
      • et al.
      Is ischemic preconditioning a useful strategy in steatotic liver transplantation?.
      ,
      • Zamboni F.
      • Franchello A.
      • David E.
      • Rocca G.
      • Ricchiuti A.
      • Lavezzo B.
      • et al.
      Effect of macrovescicular steatosis and other donor and recipient characteristics on the outcome of liver transplantation.
      ]. However, donor age higher than 70 years, as well as use of grafts with moderate steatosis, is associated with lower patient and graft survival [
      • Busuttil R.W.
      • Tanaka K.
      The utility of marginal donors in liver transplantation.
      ,
      • Busquets J.
      • Xiol X.
      • Figueras J.
      • Jaurrieta E.
      • Torras J.
      • Ramos E.
      • et al.
      The impact of donor age on liver transplantation: influence of donor age on early liver function and on subsequent patient and graft survival.
      ]. Moreover, hepatic steatosis is the current major cause of graft rejection for liver transplantation, exacerbating organ shortage problem [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ,
      • Massip-Salcedo M.
      • Roselló-Catafau J.
      • Prieto J.
      • Avila M.
      • Peralta C.
      The response of the hepatocyte to ischemia.
      ]. Therefore, minimizing the adverse effects of I/R injury especially in grafts from extended-criteria donors would increase the number of both suitable grafts and patients who successfully recover from a liver transplant. The first step towards achieving this objective is a full understanding of the mechanisms involved in I/R injury in these suboptimal organs.

      Starvation

      In clinical liver transplant, starvation of the donor due to prolonged intensive care unit hospitalization or to the lack of adequate nutritional support increases the incidence of hepatocellular injury and primary non-function [
      • Stadler M.
      • Nuyens V.
      • Seidel L.
      • Albert A.
      • Boogaerts J.G.
      Effect of nutritional status on oxidative stress in an ex vivo perfused rat liver.
      ]. The pre-existent nutritional status is a major determinant of hepatocyte injury associated with I/R. Based on the nutritional status, several experimental and clinical studies support the hypothesis that the availability of glycolytic substrates is important for maintenance of hepatic ATP levels during ischemia and for a functional recovery during reperfusion [
      • Jaeschke H.
      Preservation injury: mechanisms, prevention and consequences.
      ,
      • Cywes R.
      • Greig P.D.
      • Sanabria J.R.
      • Clavien P.A.
      • Levy G.A.
      • Harvey P.R.
      • et al.
      Effect of intraportal glucose infusion on hepatic glycogen content and degradation, and outcome of liver transplantation.
      ]. Fasting exacerbates I/R injury because the low pool of glycogen results in more rapid ATP depletion during ischemia [
      • Cywes R.
      • Greig P.D.
      • Sanabria J.R.
      • Clavien P.A.
      • Levy G.A.
      • Harvey P.R.
      • et al.
      Effect of intraportal glucose infusion on hepatic glycogen content and degradation, and outcome of liver transplantation.
      ]. In addition, fasting causes alterations in tissue antioxidant defences, accelerates the conversion of xanthine dehydrogenase to xanthine oxidase during hypoxia, and induces mitochondrial alterations [
      • Stadler M.
      • Nuyens V.
      • Seidel L.
      • Albert A.
      • Boogaerts J.G.
      Effect of nutritional status on oxidative stress in an ex vivo perfused rat liver.
      ]. Considering these observations, an artificial nutritional support may represent a new approach for the prevention of reperfusion injury in fasted patients [
      • Domenicali M.
      • Vendemiale G.
      • Serviddio G.
      • Grattagliano I.
      • Pertosa A.M.
      • Nardo B.
      Oxidative injury in rat fatty liver exposed to ischemia-reperfusion is modulated by nutritional status.
      ]. However, fasting has been reported to improve organ viability and survival when long periods of ischemia are applied, as it reduces KC phagocytosis and the generation of TNF-α [
      • Sankary H.N.
      • Chong A.
      • Foster P.
      • Brown E.
      • Shen J.
      • Kimura R.
      • et al.
      Inactivation of Kupffer cells after prolonged donor fasting improves viability of transplanted hepatic allografts.
      ]. To understand these apparently contradictory results, it is important to consider the different experimental conditions of those investigations. A beneficial effect of high glycogen content is mainly expected under conditions of short ischemia, when high metabolic reserves within the liver may attenuate ischemic cell injury and preserve defence functions against cytotoxic mediators of KC. Conversely, long ischemic periods may preferentially demand lower metabolic reserves since KC become de-activated and will not contribute to early graft injury.

      Age

      Under warm hepatic ischemia, mature adult mice show markedly increased neutrophil activity and intracellular ROS, and decreased mitochondrial function compared with young adult mice. Mature adult mice have much lower hepatic expression of the cytoprotective heat shock protein 70 (HSP70) than young adult mice. In contrast, serum HSP70 levels, which have been linked to subsequent tissue injury, are higher in mature than in young adult mice [
      • Okaya T.
      • Blanchard J.
      • Schuster R.
      • Kuboki S.
      • Husted T.
      • Caldwell C.C.
      • et al.
      Age-dependent responses to hepatic ischemia/reperfusion injury.
      ]. These alterations may contribute to the exacerbated liver injury after I/R observed in elderly mice compared with young animals. On the other hand, using the experimental model of the isolated perfused liver, previous studies demonstrated lower production of oxyradicals in old animals compared to livers from young rats. This fact was explained by lower KC activity, reduction in liver blood flow, and the impaired functions and structural alterations observed in the livers of old rats [
      • Gasbarrini A.
      • Pasini P.
      • Nardo B.
      • De Notariis S.
      • Simoncini M.
      • Cavallari A.
      • et al.
      Chemiluminescent real time imaging of post-ischemic oxygen free radicals formation in livers isolated from young and old rats.
      ]. In hepatocytes from mature adult mice, delayed activation of nuclear factor kappa B (NFκB) in response to TNF-α and virtually no production of macrophage inflammatory protein 2 have been detected [
      • Okaya T.
      • Blanchard J.
      • Schuster R.
      • Kuboki S.
      • Husted T.
      • Caldwell C.C.
      • et al.
      Age-dependent responses to hepatic ischemia/reperfusion injury.
      ]. Evidently, further investigations are required to better understand the pathophysiological status of liver grafts from old donors.

      Steatosis

      The first step to minimize the adverse effects of I/R in steatotic livers is a full understanding of the mechanisms involved in I/R injury in these marginal organs. Microcirculatory dysfunction probably represents the main underlying mechanism of steatotic liver I/R injury [
      • Domenicali M.
      • Vendemiale G.
      • Serviddio G.
      • Grattagliano I.
      • Pertosa A.M.
      • Nardo B.
      Oxidative injury in rat fatty liver exposed to ischemia-reperfusion is modulated by nutritional status.
      ,
      • Gracia-Sancho J.
      • García-Calderó H.
      • Hide D.
      • Marrone G.
      • Guixé-Muntet S.
      • Peralta C.
      • et al.
      Simvastatin maintains function and viability of steatotic rat livers procured for transplantation.
      ,
      • Ijaz S.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Impairment of hepatic microcirculation in fatty liver.
      ], nevertheless, other hypotheses have also been suggested. Hepatocyte damage is remarkably higher in steatotic livers than in non-steatotic livers [
      • Selzner M.
      • Rudiger H.A.
      • Sindram D.
      • Madden J.
      • Clavien P.A.
      Mechanisms of ischemic injury are different in the steatotic and normal rat liver.
      ], and may contribute to their poor tolerance to I/R. Hepatocyte de-regulation has several causes, it may be partially explained due to an increased sensitivity to ROS that will affect mitochondrial processes including those responsible for ATP synthesis. In fact, steatotic livers synthesise less ATP than non-steatotic livers during post-ischemic reperfusion [
      • Caraceni P.
      • Domenicali M.
      • Vendemiale G.
      • Grattagliano I.
      • Pertosa A.
      • Nardo B.
      • et al.
      The reduced tolerance of rat fatty liver to ischemia reperfusion is associated with mitochondrial oxidative injury.
      ,
      • Nardo B.
      • Caraceni P.
      • Pasini P.
      • Domenicali M.
      • Catena F.
      • Cavallari G.
      • et al.
      Increased generation of reactive oxygen species in isolated rat fatty liver during postischemic reoxygenation.
      ], being the upregulation of the mitochondrial uncoupling protein 2 a key underlying mechanism [
      • Evans Z.P.
      • Palanisamy A.P.
      • Sutter A.G.
      • Ellett J.D.
      • Ramshesh V.K.
      • Attaway H.
      • et al.
      Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation.
      ]. In addition, it has been described that hepatocytes with fatty infiltration develop massive necrosis after I/R injury, instead of apoptosis observed in non-steatotic livers [
      • Fernandez L.
      • Carrasco-Chaumel E.
      • Serafin A.
      • Xaus C.
      • Grande L.
      • Rimola A.
      • et al.
      Is ischemic preconditioning a useful strategy in steatotic liver transplantation?.
      ]. This fact may be due to low ATP production and dysfunction of regulators of apoptosis (Bcl-2, Bcl-xL, and Bax), and may explain why caspase inhibition, a highly protective strategy in non-steatotic livers, had no effects on hepatocyte injury in steatotic livers [
      • Selzner M.
      • Rudiger H.A.
      • Sindram D.
      • Madden J.
      • Clavien P.A.
      Mechanisms of ischemic injury are different in the steatotic and normal rat liver.
      ]. In experimental models of liver transplantation, exogenous NO administration protected non-steatotic grafts but was ineffective, or even noxious, in livers with steatosis. The injurious effects of exogenous NO donors was explained by exaggerated peroxynitrite generation caused by ROS overproduction [
      • Carrasco-Chaumel E.
      • Rosello-Catafau J.
      • Bartrons R.
      • Franco-Gou R.
      • Xaus C.
      • Casillas A.
      • et al.
      Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation.
      ]. Heme oxygenase-1 (HO-1) activators such as cobalt (III) protoporphyrin IX might protect livers against warm I/R injury. However, a much lower dose of the HO-1 activator is required to protect steatotic livers, as they show higher HO-1 basal levels than non-steatotic livers [
      • Massip-Salcedo M.
      • Casillas-Ramirez A.
      • Franco-Gou R.
      • Bartrons R.
      • Ben Mosbah I.
      • Serafin A.
      • et al.
      Heat shock proteins and mitogen-activated protein kinases in steatotic livers undergoing ischemia-reperfusion: some answers.
      ]. Steatotic livers also differed from non-steatotic grafts in their response to the unfolded protein response and endoplasmic reticulum stress, indeed the expression of inositol-requiring enzyme 1 and PKR-like endoplasmic reticulum kinase is lower in the presence of steatosis [
      • Ben Mosbah I.
      • Alfany-Fernández I.
      • Martel C.
      • Zaouali M.
      • Bintanel-Morcillo M.
      • Rimola A.
      • et al.
      Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia-reperfusion.
      ]. Differences have also been observed analyzing the role of the renin-angiotensin system, non-steatotic grafts exhibited higher angiotensin (Ang)-II and lower Ang-(1–7) levels than steatotic grafts [
      • Alfany-Fernández I.
      • Casillas-Ramírez A.
      • Bintanel-Morcillo M.
      • Brosnihan K.
      • Ferrario C.
      • Serafin A.
      • et al.
      Therapeutic targets in liver transplantation: angiotensin II in nonsteatotic grafts and angiotensin-(1–7) in steatotic grafts.
      ]. Moreover, reduced retinol binding protein 4 and increased peroxisome proliferator-activated receptor gamma (PPAR-γ) levels were observed in steatotic livers compared to non-steatotic livers [
      • Casillas-Ramírez A.
      • Alfany-Fernández I.
      • Massip-Salcedo M.
      • Juan M.E.
      • Planas J.M.
      • Serafín A.
      • et al.
      Retinol-binding protein 4 and peroxisome proliferator-activated receptor-γ in steatotic liver transplantation.
      ]. The increased vulnerability of steatotic livers subjected to I/R has been also associated with increased adiponectin, oxidative stress and IL-1 levels, and reduced capability to generate IL-10, PPAR-α and Retinol-Binding Protein 4 [
      • Casillas-Ramírez A.
      • Alfany-Fernández I.
      • Massip-Salcedo M.
      • Juan M.E.
      • Planas J.M.
      • Serafín A.
      • et al.
      Retinol-binding protein 4 and peroxisome proliferator-activated receptor-γ in steatotic liver transplantation.
      ,
      • Massip-Salcedo M.
      • Zaouali M.
      • Padrissa-Altés S.
      • Casillas-Ramírez A.
      • Rodés J.
      • Roselló-Catafau J.
      • et al.
      Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.
      ,
      • Serafin A.
      • Rosello-Catafau J.
      • Prats N.
      • Gelpi E.
      • Rodes J.
      • Peralta C.
      Ischemic preconditioning affects interleukin release in fatty livers of rats undergoing ischemia/reperfusion.
      ].
      Very importantly, it should be considered that the mechanisms involved in hepatic I/R injury may differ depending on the method used to induce experimental steatosis. In contrast with other models of steatosis, both dietary and alcohol exposure induces the production of superoxide dismutase/catalase-insensitive ROS, which may partly justify steatotic liver failure after transplantation [
      • Gao W.
      • Connor H.D.
      • Lemasters J.J.
      • Mason R.P.
      • Thurman R.G.
      Primary nonfunction of fatty livers produced by alcohol is associated with a new, antioxidant-insensitive free radical species.
      ]. Neutrophils, which have been involved in the increased vulnerability of alcohol-induced steatotic livers to I/R injury, would not account in I/R damage in non-alcoholic steatotic livers. Similarly, the role of TNF-α in the vulnerability of steatotic livers to I/R injury may depend on the type of steatosis [
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ,
      • Yamada S.
      • Iida T.
      • Tabata T.
      • Nomoto M.
      • Kishikawa H.
      • Kohno K.
      • et al.
      Alcoholic fatty liver differentially induces a neutrophil-chemokine and hepatic necrosis after ischemia-reperfusion in rat.
      ]. All the aforementioned observations indicate that therapies that are effective in non-steatotic livers may either be useless in the presence of steatosis, or the effective drug dose may differ between types of grafts [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Carrasco-Chaumel E.
      • Rosello-Catafau J.
      • Bartrons R.
      • Franco-Gou R.
      • Xaus C.
      • Casillas A.
      • et al.
      Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation.
      ,
      • Massip-Salcedo M.
      • Casillas-Ramirez A.
      • Franco-Gou R.
      • Bartrons R.
      • Ben Mosbah I.
      • Serafin A.
      • et al.
      Heat shock proteins and mitogen-activated protein kinases in steatotic livers undergoing ischemia-reperfusion: some answers.
      ]. On the other hand, some pharmacological compounds would only be effective in steatotic livers [
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ,
      • Massip-Salcedo M.
      • Roselló-Catafau J.
      • Prieto J.
      • Avila M.
      • Peralta C.
      The response of the hepatocyte to ischemia.
      ]. In summary, the different mechanisms involved in hepatic I/R injury depending on the type of steatosis could explain the difficulties to translate our current knowledge to the bedside.

      Strategies to prevent hepatic I/R injury

      As a consequence of the incipient amount of uncovered molecular mechanisms responsible for hepatic I/R injury, a variety of new therapeutic strategies have been developed (Table 1, Table 2, Table 3, Table 4). Due to limitation in review length, only those strategies reported during the last 5 years have been included in the present review. For less recent studies please refer to previous articles [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.
      Protective strategies against ischemic injury of the liver.
      ,
      • Casillas-Ramírez A.
      • Ben Mosbah I.
      • Ramalho F.
      • Rosello-Catafau J.
      • Peralta C.
      Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation.
      ,
      • Massip-Salcedo M.
      • Roselló-Catafau J.
      • Prieto J.
      • Avila M.
      • Peralta C.
      The response of the hepatocyte to ischemia.
      ,
      • Jaeschke H.
      • Woolbright B.L.
      Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species.
      ,
      • Iñiguez M.
      • Dotor J.
      • Feijoo E.
      • Goñi S.
      • Prieto J.
      • Berasain C.
      • et al.
      Novel pharmacologic strategies to protect the liver from ischemia-reperfusion injury.
      ].
      Table 1Pharmacological strategies to protect livers against ischemia/reperfusion injury
      • Gracia-Sancho J.
      • García-Calderó H.
      • Hide D.
      • Marrone G.
      • Guixé-Muntet S.
      • Peralta C.
      • et al.
      Simvastatin maintains function and viability of steatotic rat livers procured for transplantation.
      ,
      • Ben Mosbah I.
      • Alfany-Fernández I.
      • Martel C.
      • Zaouali M.
      • Bintanel-Morcillo M.
      • Rimola A.
      • et al.
      Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia-reperfusion.
      ,
      • Alfany-Fernández I.
      • Casillas-Ramírez A.
      • Bintanel-Morcillo M.
      • Brosnihan K.
      • Ferrario C.
      • Serafin A.
      • et al.
      Therapeutic targets in liver transplantation: angiotensin II in nonsteatotic grafts and angiotensin-(1–7) in steatotic grafts.
      ,
      • Casillas-Ramírez A.
      • Alfany-Fernández I.
      • Massip-Salcedo M.
      • Juan M.E.
      • Planas J.M.
      • Serafín A.
      • et al.
      Retinol-binding protein 4 and peroxisome proliferator-activated receptor-γ in steatotic liver transplantation.
      ,
      • Massip-Salcedo M.
      • Zaouali M.
      • Padrissa-Altés S.
      • Casillas-Ramírez A.
      • Rodés J.
      • Roselló-Catafau J.
      • et al.
      Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.
      ,
      • Shi Y.
      • Rehman H.
      • Ramshesh V.K.
      • Schwartz J.
      • Liu Q.
      • Krishnasamy Y.
      • et al.
      Sphingosine kinase-2 inhibition improves mitochondrial function and survival after hepatic ischemia-reperfusion.
      ,
      • Liu P.G.
      • He S.Q.
      • Zhang Y.H.
      • Wu J.
      Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice.
      ,
      • Ramalho F.S.
      • Alfany-Fernandez I.
      • Casillas-Ramirez A.
      • Massip-Salcedo M.
      • Serafín A.
      • Rimola A.
      • et al.
      Are angiotensin II receptor antagonists useful strategies in steatotic and nonsteatotic livers in conditions of partial hepatectomy under ischemia-reperfusion?.
      ,
      • Ichiki A.
      • Miyazaki T.
      • Nodera M.
      • Suzuki H.
      • Yanagisawa H.
      Ascorbate inhibits apoptosis of Kupffer cells during warm ischemia/reperfusion injury.
      ,
      • Llacuna L.
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      ,
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      Cardiotrophin-1 reduces ischemia/reperfusion injury during liver transplant.
      ,
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      • Helfrich W.
      Carbon monoxide-releasing molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats.
      ,
      • Casillas-Ramírez A.
      • Zaouali A.
      • Padrissa-Altés S.
      • Ben Mosbah I.
      • Pertosa A.
      • Alfany-Fernández I.
      • et al.
      Insulin-like growth factor and epidermal growth factor treatment: new approaches to protecting steatotic livers against ischemia-reperfusion injury.
      ,
      • Hochhauser E.
      • Pappo O.
      • Ribakovsky E.
      • Ravid A.
      • Kurtzwald E.
      • Cheporko Y.
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      Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model.
      ,
      • Yang X.
      • Qin L.
      • Liu J.
      • Tian L.
      • Qian H.
      17β-Estradiol protects the liver against cold ischemia/reperfusion injury through the Akt kinase pathway.
      ,
      • Kuroda S.
      • Tashiro H.
      • Igarashi Y.
      • Tanimoto Y.
      • Nambu J.
      • Oshita A.
      • et al.
      Rho inhibitor prevents ischemia-reperfusion injury in rat steatotic liver.
      ,
      • Jiang W.
      • Kong L.
      • Wu X.
      • Pu L.
      • Wang X.
      Allograft inflammatory factor-1 is up-regulated in warm and cold ischemia-reperfusion injury in rat liver and may be inhibited by FK506.
      ,
      • Lehne K.
      • Nobiling R.
      Metabolic preconditioning with fructose prior to organ recovery attenuates ischemia-reperfusion injury in the isolated perfused rat liver.
      ,
      • Xue F.
      • Zhang J.J.
      • Xu L.M.
      • Zhang C.
      • Xia Q.
      Protective effects of HGF-MSP chimer (metron factor-1) on liver ischemia-reperfusion injury in rat model.
      ,
      • Jiménez-Castro M.B.
      • Elias-Miro M.
      • Mendes-Braz M.
      • Lemoine A.
      • Rimola A.
      • Rodés J.
      • et al.
      Tauroursodeoxycholic acid affects PPARã and TLR4 in steatotic liver transplantation.
      ,
      • Zúñiga J.
      • Cancino M.
      • Medina F.
      • Varela P.
      • Vargas R.
      • Tapia G.
      • et al.
      N-3 PUFA supplementation triggers PPAR-α activation and PPAR-α/NF-κB interaction: anti-inflammatory implications in liver ischemia-reperfusion injury.
      ,
      • Ocuin L.M.
      • Zeng S.
      • Cavnar M.J.
      • Sorenson E.C.
      • Bamboat Z.M.
      • Greer J.B.
      • et al.
      Nilotinib protects the murine liver from ischemia/reperfusion injury.
      ,
      • Li F.
      • Chen Z.
      • Pan Q.
      • Fu S.
      • Lin F.
      • Ren H.
      • et al.
      The protective effect of PNU-282987, a selective α7 nicotinic acetylcholine receptor agonist, on the hepatic ischemia-reperfusion injury is associated with the inhibition of high-mobility group box 1 protein expression and nuclear factor κB activation in mice.
      ,
      • Elias-Miró M.
      • Massip-Salcedo M.
      • Raila J.
      • Schweigert F.
      • Mendes-Braz M.
      • Ramalho F.
      • et al.
      Retinol binding protein 4 and retinol in steatotic and nonsteatotic rat livers in the setting of partial hepatectomy under ischemia/reperfusion.
      ,
      • Schmeding M.
      • Rademacher S.
      • Boas-Knoop S.
      • Roecken C.
      • Lendeckel U.
      • Neuhaus P.
      • et al.
      RHuEPo reduces ischemia-reperfusion injury and improves survival after transplantation of fatty livers in rats.
      ,
      • Zhang Y.
      • Ji H.
      • Shen X.
      • Cai J.
      • Gao F.
      • Koenig K.M.
      • et al.
      Targeting TIM-1 on CD4 T cells depresses macrophage activation and overcomes ischemia-reperfusion injury in mouse orthotopic liver transplantation.
      ,
      • Hide D.
      • Guixé-Muntet S.
      • Mancici A.
      • Bosch J.
      • Gracia-Sancho J.
      A novel recombinant form of the manganese superoxide dismutase protects rat liver grafts procured for transplantation.
      ,
      • Busuttil R.W.
      • Lipshutz G.S.
      • Kupiec-Weglinski J.W.
      • Ponthieux S.
      • Gjertson D.W.
      • Cheadle C.
      • et al.
      RPSGL-Ig for improvement of early liver allograft function: a double-blind, placebo-controlled, single-center phase II study.
      ,
      • Beck-Schimmer B.
      • Breitenstein S.
      • Bonvini J.M.
      • Lesurtel M.
      • Ganter M.
      • Weber A.
      • et al.
      Protection of pharmacological postconditioning in liver surgery: results of a prospective randomized controlled trial.
      ,
      • Liu Y.X.
      • Jin L.M.
      • Zhou L.
      • Xie H.Y.
      • Jiang G.P.
      • Chen H.
      • et al.
      Sirolimus attenuates reduced-size liver ischemia-reperfusion injury but impairs liver regeneration in rats.
      ,
      • Jiménez-Castro M.B.
      • Casillas-Ramirez A.
      • Massip-Salcedo M.
      • Elias-Miro M.
      • Serafin A.
      • Rimola A.
      • et al.
      Cyclic adenosine 3′,5′-monophosphate in rat steatotic liver transplantation.
      ,
      • López-Neblina F.
      • Toledo-Pereyra L.H.
      Anti-ischemic effect of selectin blocker through modulation of tumor necrosis factor-alpha and interleukin-10.
      ,
      • Hassan-Khabbar S.
      • Cottart C.H.
      • Wendum D.
      • Vibert F.
      • Clot J.P.
      • Savouret J.F.
      • et al.
      Postischemic treatment by trans-resveratrol in rat liver ischemia-reperfusion: a possible strategy in liver surgery.
      ,
      • Cheng F.
      • Li Y.
      • Feng L.
      • Li S.
      Effects of tetrandrine on ischemia/reperfusion injury in mouse liver.
      ,
      • Xu S.Q.
      • Li Y.H.
      • Hu S.H.
      • Chen K.
      • Dong L.Y.
      Effects of Wy-14,643 on hepatic ischemia reperfusion injury in rats.
      ,
      • Teoh N.C.
      • Williams J.
      • Hartley J.
      • Yu J.
      • McCuskey R.S.
      • Farrell G.C.
      Short-term therapy with peroxisome proliferation-activator receptor-alpha agonist Wy-14,643 protects murine fatty liver against ischemia-reperfusion injury.
      AIF-1, allograft inflammatory factor 1; GSH, glutathione; HMGB1, high mobility group box 1; HSC, hepatic stellate cells; IL, interleukin; JNK, jun N-terminal kinase; MCP-1, monocyte chemoattractant protein-1; MDA, malondialdehyde; MIP-2, macrophage inflammatory protein-2; MPO, myeloperosidase; NADPH, nicotinamide adenine dinucleotide phosphate; NF-kB, nuclear factor kappa B, NO, nitric oxide; PPAR, peroxisome proliferator-activated receptor; PTEN, phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; LSEC, liver sinusoidal endothelial cells; SOD, superoxide dismutase; TLR4, toll-like receptor 4; TNF, tumor necrosis factor; XOD, xanthine oxidase.
      Table 2Additives for preservation solutions to protect livers against ischemia/reperfusion injury
      • Russo L.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Marrone G.
      • García-Pagán J.C.
      • García-Cardeña G.
      • et al.
      Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.
      ,
      • Hide D.
      • Guixé-Muntet S.
      • Mancici A.
      • Bosch J.
      • Gracia-Sancho J.
      A novel recombinant form of the manganese superoxide dismutase protects rat liver grafts procured for transplantation.
      ,
      • Kuriyama N.
      • Isaji S.
      • Hamada T.
      • Kishiwada M.
      • Ohsawa I.
      • Usui M.
      • et al.
      The cytoprotective effects of addition of activated protein C into preservation solution on small-for-size grafts in rats.
      ,
      • Ben Mosbah I.
      • Roselló-Catafau J.
      • Alfany-Fernandez I.
      • Rimola A.
      • Parellada P.P.
      • Mitjavila M.T.
      • et al.
      Addition of carvedilol to University Wisconsin solution improves rat steatotic and nonsteatotic liver preservation.
      ,
      • Zaouali M.A.
      • Padrissa-Altés S.
      • Ben Mosbah I.
      • Alfany-Fernandez I.
      • Massip-Salcedo M.
      • Casillas-Ramirez A.
      • et al.
      Improved rat steatotic and nonsteatotic liver preservation by the addition of epidermal growth factor and insulin-like growth factor-I to University of Wisconsin solution.
      ,
      • Bezinover D.
      • Ramamoorhy S.
      • Uemura T.
      • Kadry Z.
      • McQuillan P.M.
      • Mets B.
      • et al.
      Use of a third-generation perfluorocarbon for preservation of rat DCD liver grafts.
      ,
      • Abbas R.
      • Kombu R.S.
      • Dignam D.
      • Gunning W.
      • Stulberg J.J.
      • Brunengraber H.
      • et al.
      Polyethylene glycol modified-albumin enhances the cold preservation properties of University of Wisconsin solution in rat liver and a hepatocyte cell line.
      ,
      • Defamie V.
      • Laurens M.
      • Patrono D.
      • Devel L.
      • Brault A.
      • Saint-Paul M.C.
      • et al.
      Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.
      ,
      • Yue L.H.
      • Zhao Y.L.
      • Chen J.
      • Lu D.R.
      Effect of fusion protein TAT and heme oxygenase-1 on liver sinusoidal endothelial cells apoptosis during preservation injury.
      ,
      • Anderson C.D.
      • Upadhya G.
      • Conzen K.D.
      • Jia J.
      • Brunt E.M.
      • Tiriveedhi V.
      • et al.
      Endoplasmic reticulum stress is a mediator of posttransplant injury in severly steatotic liver allografts.
      APC, activated protein C; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; EGF, epidermal growth factor; HO-1, heme oxygenase-1; IGF-1, insulin-like growth factor 1; IL-6, interleukin-6; MMP, metalloproteinase; NO, nitric oxide; PAF, platelet activating factor; LSEC, liver sinusoidal endothelial cells; TNFα, tumor necrosis factor α.
      Table 3Gene therapy strategies aimed at protecting liver grafts against ischemia/reperfusion injury
      • Massip-Salcedo M.
      • Zaouali M.
      • Padrissa-Altés S.
      • Casillas-Ramírez A.
      • Rodés J.
      • Roselló-Catafau J.
      • et al.
      Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.
      ,
      • Saito Y.
      • Shimada M.
      • Utsunomiya T.
      • Ikemoto T.
      • Yamada S.
      • Morine Y.
      • et al.
      The protective effect of adipose-derived stem cells against liver injury by trophic molecules.
      ,
      • Sun C.K.
      • Chang C.L.
      • Lin Y.C.
      • Kao Y.H.
      • Chang L.T.
      • Yen C.H.
      • et al.
      Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia-reperfusion injury in rats.
      ,
      • Selzner N.
      • Liu H.
      • Boehnert M.U.
      • Adeyi O.A.
      • Shalev I.
      • Bartczak A.M.
      • et al.
      FGL2/fibroleukin mediates hepatic reperfusion injury by induction of sinusoidal endothelial cell and hepatocyte apoptosis in mice.
      ,
      • Watanabe G.
      • Uchinami H.
      • Yoshioka M.
      • Abe Y.
      • Kikuchi I.
      • Iwasaki W.
      • et al.
      Transfection of naked nuclear factor-κB decoy oligodeoxynucleotides into liver by rapid portal vein infusion in rats: its effect on ischemia-reperfusion injury of liver.
      ,
      • Schneider M.
      • Van Geyte K.
      • Fraisl P.
      • Kiss J.
      • Aragonés J.
      • Mazzone M.
      • et al.
      Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.
      ,
      • Hui W.
      • Jinxiang Z.
      • Heshui W.
      • Zhuoya L.
      • Qichang Z.
      Bone marrow and non-bone marrow TLR4 regulates hepatic ischemia/reperfusion injury.
      ,
      • Ellett J.D.
      • Evans Z.P.
      • Atkinson C.
      • Schmidt M.G.
      • Schnellmann R.G.
      • Chavin K.D.
      Toll-like receptor 4 is a key mediator of murine steatotic liver warm ischemia/reperfusion injury.
      FGL2, fibrinogen-like protein 2; IL-6, interleukin-6; NF-kB, nuclear factor kappa B; PHD1, prolyl hydroxylase 1; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor α; LSEC, liver sinusoidal endothelial cells; VEGF, vascular endothelial growth factor.
      Table 4Surgical strategies to protect livers against ischemia/reperfusion injury
      • Casillas-Ramírez A.
      • Alfany-Fernández I.
      • Massip-Salcedo M.
      • Juan M.E.
      • Planas J.M.
      • Serafín A.
      • et al.
      Retinol-binding protein 4 and peroxisome proliferator-activated receptor-γ in steatotic liver transplantation.
      ,
      • Fondevila C.
      • Hessheimer A.J.
      • Maathuis M.H.
      • Muñoz J.
      • Taurá P.
      • Calatayud D.
      • et al.
      Superior preservation of DCD livers with continuous normothermic perfusion.
      ,
      • García-Valdecasas J.C.
      • Fondevila C.
      In-vivo normothermic recirculation: an update.
      ,
      • Hong F.
      • Yang S.
      Ischemic preconditioning decreased leukotriene C4 formation by depressing leukotriene C4 synthase expression and activity during hepatic I/R injury in rats.
      ,
      • Jang J.H.
      • Kang K.J.
      • Kang Y.
      • Lee I.S.
      • Graf R.
      • Clavien P.A.
      Ischemic preconditioning and intermittent clamping confer protection against ischemic injury in the cirrhotic mouse liver.
      ,
      • Shimoda M.
      • Iwasaki Y.
      • Sawada T.
      • Kubota K.
      Protective effect of ischemic preconditioning against liver injury after major hepatectomy using the intermittent Pringle maneuver in swine.
      ,
      • Esposti D.D.
      • Domart M.C.
      • Sebagh M.
      • Harper F.
      • Pierron G.
      • Brenner C.
      • et al.
      Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis.
      ,
      • Heizmann O.
      • Loehe F.
      • Volk A.
      • Schauer R.J.
      Ischemic preconditioning improves postoperative outcome after liver resections: a randomized controlled study.
      ,
      • Schiesser M.
      • Wittert A.
      • Nieuwenhuijs V.B.
      • Morphett A.
      • Padbury R.T.
      • Barritt G.J.
      Intermittent ischemia but not ischemic preconditioning is effective in restoring bile flow after ischemia reperfusion injury in the livers of aged rats.
      ,
      • Wang F.
      • Birch S.E.
      • He R.
      • Tawadros P.
      • Szaszi K.
      • Kapus A.
      • et al.
      Remote ischemic preconditioning by hindlimb occlusion prevents liver ischemic/reperfusion injury: the role of High Mobility Group-Box 1.
      ,
      • Schlegel A.
      • Rougemont O.
      • Graf R.
      • Clavien P.A.
      • Dutkowski P.
      Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts.
      ,
      • Vairetti M.
      • Ferrigno A.
      • Carlucci F.
      • Tabucchi A.
      • Rizzo V.
      • Boncompagni E.
      • et al.
      Subnormothermic machine perfusion protects steatotic livers against preservation injury: a potential for donor pool increase?.
      ,
      • Hessheimer A.J.
      • Fondevila C.
      • Taurá P.
      • Muñoz J.
      • Sánchez O.
      • Fuster J.
      • et al.
      Decompression of the portal bed and twice-baseline portal inflow are necessary for the functional recovery of a “small-for-size” graft.
      IC, intermittent clamping; IP, ischemic preconditioning; LTC4, leukotriene C4; MP, Machine perfusion; RP, remote preconditioning; LSEC, liver sinusoidal endothelial cells.

      Concluding remarks and future directions

      Over the past years, our knowledge about the mechanisms involved in the development of liver injury due to I/R has significantly improved, and it has consequently been accompanied by a long list of potential therapeutic alternatives. However, ischemia-reperfusion injury still represents a serious problem in the clinical practice probably because very few basic (or translational) studies have successfully been applied at the bedside. This important drawback in hepatic I/R experimental research may have several origins.
      Firstly, and as stated along this review, it should be considered that the mechanisms involved in hepatic I/R very much depend on the experimental conditions used: which type of research is done (in vitro, ex vivo, in vivo), type of ischemia applied (warm or cold), period of ischemia (ranging from minutes to days), extension of hepatic ischemia (partial or total), graft subclinical situation (healthy, steatotic, aged…), etc. Thus, new therapeutic strategies from experimental studies should be considered specific to the concrete experimental/surgical conditions used, and most probably they cannot automatically be validated for a different I/R situation. Secondly, although we certainly know part of the underlying molecular mechanisms of I/R injury in hepatocytes and Kupffer cells, much fewer knowledge has been gained for other key hepatic cells such as liver sinusoidal endothelial cells or hepatic stellate cells. Thus further investigations are still required. Nevertheless, liver cells phenotype modifications due to I/R injury should be investigated in experimental conditions that maximally mimic the hepatic sinusoid environment, including the possibility of paracrine interactions and under physiological biomechanical stimuli. Third, it should be taken into consideration that the possible applicability of basic research knowledge is even more difficult in clinical situations with extended-criteria donors. The relative homogeneity of liver steatosis obtained in genetically modified animals or animals under dietary alterations cannot be compared to humans, where surgeons are still looking for a reliable method for hepatic lipid content quantification, and similarly happens with elderly donors, non-beating heart donors, etc. Overall foster complicating the applicability of basic research.
      Figure thumbnail fx5

      Financial support

      Spanish Ministry of Economy (Instituto de Salud Carlos III – FIS PI11/0235 to JG-S) and (SAF2012-31238 to CP). Co-funded by FEDER (Fondos para el Desarrollo Económico Regional, “una manera de hacer Europa”) from the European Union. JG-S has a Ramón y Cajal contract from the Spanish Ministry of Economy, MBJ-C has a fellowship from the Catalan Society of Transplantation. CIBEREHD is funded by the Instituto de Salud Carlos III.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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