Ischemia-Reperfusion Injury and Ischemic-Type Biliary Lesions following Liver Transplantation

Ischemia-reperfusion (I-R) injury after liver transplantation (LT) induces intra- and/or extrahepatic nonanastomotic ischemic-type biliary lesions (ITBLs). Subsequent bile duct stricture is a significant cause of morbidity and even mortality in patients who underwent LT. Although the pathogenesis of ITBLs is multifactorial, there are three main interconnected mechanisms responsible for their formation: cold and warm I-R injury, injury induced by cytotoxic bile salts, and immunological-mediated injury. Cold and warm ischemic insult can induce direct injury to the cholangiocytes and/or damage to the arterioles of the peribiliary vascular plexus, which in turn leads to apoptosis and necrosis of the cholangiocytes. Liver grafts from suboptimal or extended-criteria donors are more susceptible to cold and warm I-R injury and develop more easily ITBLs than normal livers. This paper, focusing on liver I-R injury, reviews the risk factors and mechanisms leading to ITBLs following LT.


Introduction
After liver transplantation (LT), the incidence of biliary complications, which include a wide spectrum of functional and anatomical abnormalities varies from 10 to 30% [1][2][3]. These biliary complications lead to an increase of graft dysfunction and patient morbidity and in some cases even to graft loss [4] and retransplantation [5]. They are associated with an increased mortality rate (8 to 15%) [6].
Liver ischemia-reperfusion (I-R) injury during transplantation occurs at different periodes [7]. The first, after liver explantation from the donor and storage on ice at 0 • to 4 • C, is a variable but generally long period of cold ischemia. The time of vascular anastomosis, when the liver is removed from ice until its implantation in the recipient, represents the second, relatively shorter period of warm I-R injury. In this period of ischemia, the liver warms slowly up to a temperature of 12.5 • C during the realization of suprahepatic cava and portal vein anastomoses, and to a temperature of 34 • C, once hepatic artery anastomosis is performed [8]. Now the liver is fully revascularized and graft temperature stabilizes. Normothermic reperfusion of the implanted liver with the recipient's blood at 37 • C delineates the third period.
The ischemic injury itself, a localized process of cellular metabolic disturbances, results from glycogen consumption, lack of oxygen supply and adenosine triphosphate (ATP) depletion [14]. Reperfusion, which consists of initial phase injury (within 2 h after reperfusion) and late phase injury (6-48 hours after reperfusion), aggravates the cellular injuries caused by the ischemic period [9,[15][16][17].
Although all types of ischemia share common mechanisms cold ischemia of the liver is characterized mainly by injury to sinusoidal lining cells and disruption of the microcirculation, whereas warm ischemia results primarily in

Anatomy and Blood Supply of the Biliary System
The human biliary system is divided into extrahepatic and intrahepatic bile ducts and is lined by biliary epithelial cells (or cholangiocytes). The classical extrahepatic biliary anatomy consists of a right and left hepatic duct draining the right and left liver lobes, respectively [21][22][23]. The fusion of the right and left hepatic ducts gives rise to the common hepatic duct (choledochus) [21][22][23]. The intrahepatic bile ducts are further sub-divided into large and small bile ducts [24][25][26]. They represent that part of the biliary tree proximal to the confluence of the hepatic ducts [27] extending from the canals of Hering to the large extrahepatic ducts [24][25][26]. Small ductules that are lined by 4-5 cholangiocytes have a basement membrane, tight junctions between cells, and microvilli projecting into the bile duct lumen [25]. In larger bile ducts cholangiocytes too are progressively larger and more columnar in shape. Ten to twelve cholangiocytes line a larger bile duct [28,29]. The vascular plexus of the biliary system is composed of branches arising directly from the right and left hepatic arteries (and accessory hepatic arteries when present) and their segmental branches and indirectly from the gastroduodenal artery via the arteries supplying the common bile duct [21][22][23]. This peribiliary vascular plexus is arranged around the extra-and intrahepatic biliary tree in normal liver [25]. The peribiliary vascular plexus delivers blood to the sinusoids both through lobular branches and through peribiliary branches into the portal vein [25]. In very small portal spaces a small capillary, representing the terminal branches of the hepatic artery, can continue the course of the arteriole and eventually run into the sinusoids [25]. In large portal spaces, the peribiliary vascular plexus is an anastomotic network between short collateral arterioles arising from the same arterial branches. Since the blood flows in the opposite direction to the bile (from the large towards the small vessels), the peribiliary vascular plexus presents a countercurrent stream [25,30]. The veins draining the surface of the bile ducts follow closely the arterial plexus and drain into the marginal veins. The marginal veins end in subcapsular capillaries related to the hilum of the liver [31].

Classification of Biliary Complications Following LT
Bile duct strictures following LT have been classified as anastomotic strictures (ASs) and nonanastomotic biliary strictures (NAS) [32]. ASs are isolated strictures at the site of the bile duct anastomosis (choledochocholedochostomy or choledochojejunostomy reconstruction), while NASs concern strictures located in both, the extrahepatic and intrahepatic biliary system of the liver graft [32]. NASs occur after hepatic artery thrombosis (HAT), but also with an open hepatic artery [32]. NASs with an open hepatic artery represent a separate entity and are generally referred to as ischemic-type biliary lesions (ITBLs) [32,33]. ITBLs were also termed as "ischemic-type biliary complications or ITBC" [3], "ischemic cholangitis" [34] and "ischemic cholangiopathy" [35]. NAS were subclassified according to their etiology: (a) NAS secondary to miaroangiopathic injury (hepatic arterial thrombosis or stenosis), (b) ITBLs secondary to microangiopathic injury (preservation injury, prolonged cold and warm ischemia times, donation after cardiac death, and prolonged use of dopamine in the donor), and (c) ITBLs secondary to immunogenetic injury (ABO incompatibility, rejection, autoimmune hepatic disease, CMV infection, and chemokines polymorphisms) [36].

Incidence and Risk Factors of ITBLs Following LT
The incidence of ITBLs following LT is 5-15% [33]. This great variability may be partially due to the different definitions used for ITBLs. Although most ITBLs secondary to ischemic lesions occur within 1 year after the transplantation, their prevalence continues to increase with time after liver transplantation [5]. ITBLs appearing more than 1 year after transplantation are mainly related to immunological causes [5]. Risk factors involved in the development of ITBLs are old donor age [36,37], prolonged cold and warm ischemia times [4,[10][11][12], non-heart-beating donors (NHBD) [38,39], graft steatosis [40,41], some graft perfusion methods [42], high viscosity of cold storage solutions [43,44], prolonged use of dopamine in the donor [45], and posttransplant liver cytolysis and cholestasis due to I-R injury [46,47]. In liver transplantation, the increasing gap between the number of patients awaiting an organ and the number of available organs has led to the use of extended-criteria donor (ECD) organs, including organs, which present risk factors mentioned above [48]. As livers from suboptimal donors or ECD are more susceptible to I-R and preservation injury, primary nonfunction (PNF), initial poor graft function (IPGF), delayed graft function (DFG), and also ITBLs are more frequent in these organs [4,12,37,38,49,50].

Cold and Warm I-R Injury and ITBLs Following LT (Figures 1 and 2)
Cold ischemic storage of the liver graft and its reperfusion produces injury to the biliary epithelium [2,13] and is strongly associated with the development of biliary strictures including ITBLs [77]. As Kupffer cells are situated within the lumen of the sinusoid, they are in direct contact with the endothelial surface. From this position when activated by I-R, they release ROS, proinflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and oxidants into the circulation as well as directing them to the endothelial layer and the underlying hepatocytes [78]. Although ROSs are essential to cell life at physiological levels, when overproduced they may be responsible of IPGF after LT [79]. Increased production of ROS is associated to reduced basal levels of intracellular glutathione, a principal nonprotein thiol responsible for maintaining intracellular redox status and protecting cells against oxidative injury [80]. Glutathione has an important role in the prevention of cellular ischemia-related oxidative injury during liver preservation by reducing biliary tract cell ROS production [10]. Glutathione present in bile may prevent cholangiocyte injury by counteracting the cytotoxic effects of ROS within the biliary tract [2,10]. Glutathione is also one of the major determinants of bile acid-independent bile flow [81]. In animals, impaired biliary excretion of glutathione may contribute to the decreased bile flow after cold ischemia [82]. Decreased biliary glutathione excretion is due to impaired transport across the canalicular membrane [82], but also to increased intrabiliary degradation by solubilized γ-glutamyltranspeptidase (GGT) [83]. The resulting lower biliary glutathione concentrations diminish the resistence of the cholangiocytes to oxidative stress provoked by I-R [83,84] and induce cholangiocyte apoptosis [84] through the loss of the antiapoptotic protein B-cell CLL/lymphoma 2 protein (Bcl-2) [85]. Thus, glutathione depletion might explain the intense injury of bile ducts seen in LT [10,11,84,86].
The late or subacute phase of I-R injury is a polymorphonuclear (PMN) leukocyte-dependent process in which the above described ROS generation is associated to cytokine and chemokine expression [78,87,88].
The epithelial-lining cells of the biliary system are not only exposed to proinflammatory mediators deriving from intrahepatic sources, but also to those deriving from extrahepatic sources via arterial circulation [89]. These inflammatory mediators promote the invasion of PMNs into the interstitium via the upregulation of adhesion molecules and formation of chemotactic agents [87,90]. PMNs can penetrate the ductal basal membrane and thus contribute to bile duct injury [91]. Ductal cells are desquamated to the lumen of the bile duct and, together with PMNs, they appear in bile during the first few days after LT [92][93][94]. There is a clear relationship between postreperfusion hepatic biopsy findings (the degree of PMN infiltration and hepatocellular necrosis of the graft) and biliary complications after liver transplantation, including ITBLs [77].
PMNs and platelets synergistically exacerbate sinusoidal endothelial cell injury by induction of apoptosis during reperfusion. During cell anoxia, cholangiocytes are significantly more resistant to cell death than hepatocytes [10]. This is inverted after reoxygenation of the anoxic cells (which mimics tissue reperfusion), when hepatocytes are more resistant to cell death than cholangiocytes. The rate of ROS formation by cholangiocytes during reoxygenation is greater than in hepatocytes at this moment with concomitant low basal levels of the antioxidant glutathione in cholangiocytes [10]. These findings suggest that bile duct injury after LT is mainly caused during the reperfusion period [10]. Liver reoxygenation upregulates other apoptotic receptor expression than Fas and enhances apoptosis in human biliary epithelial cells [20]. Tumor necrosis factor-related apoptosisinducing ligand (TRAIL) which binds TRAIL recep-tor1/death receptor 4 (DR4) and TRAIL receptor2/death receptor 5 (DR5) membrane death receptors can activate apoptosis [95]. Reoxygenation up-regulates DR4 and DR5 expression and enhances TRAIL-mediated apoptosis in human intrahepatic biliary epithelial cells [20].
Human bile epithelial cells [20] do not normally express DR5 [96], but during reoxygenation an even increased DR5 expression of cholangiocytes can be observed [20]. Reoxygenation increases also the activity of caspase-8 and caspase-3 in a TRAIL-dependent manner [20]. Some studies demonstrated an association of a longer warm ischemia time and a marked cholangiocyte apoptosis [20,97]. Cholangiocyte apoptosis after cold and warm liver I-R is at least partly involved in the pathogenesis of ITBLs after LT [86].

Intrahepatic Cholestasis.
Bile formation requires the coordinated function of hepatocytes and intrahepatic cholangiocytes, which represent 2 to 5% of liver cells [98,99]. Hepatocytes produce primary or hepatic bile, which percolates through the intrahepatic bile ducts. During this journey, cholangiocytes modify the bile via secretory and absorptive processes that provide additional bile water and alkalinity [100][101][102]. Cholestasis is an impairment of bile secretion, which results either from a functional defect at the level of hepatocytes (hepatocellular cholestasis) or from an impairment in bile secretion and flow at the level of bile ductules or ducts (ductular/ductal cholestasis) [103]. Intrahepatic cholestasis following liver transplantation is common and generally subclinical [104][105][106][107]. However, when severe, cholestasis may be associated with irreversible liver damage, requiring retransplantation [104,108]. One of the main causes of intrahepatic cholestasis after LT is cold and warm I-R injury [104,109]. Under normal conditions, bile production requires an active vectorial secretion of biliary constituents from portal blood plasma into bile canaliculi [110]. An intact cytoskeleton is required for bile canalicular contraction, which is based on a pericanalicular web of contractile proteins, actin microfilaments, and cytokeratin intermediate filaments [111] acting as a pump to facilitate bile flow into the intrahepatic canalicular system [112,113]. The bile canaliculus is one of the liver structures that is early damaged by I-R [105]. This oxidative stress-dependent structural damage contributes to perturbate the bile acid transport during ischemia. The resulting loss of microvilli and the canalicular atony, decrease the bile flow and lead to cholestasis [105][106][107]. The impairment of bile canaliculi structure following I-R, and postreperfusion biliary complications observed in patients undergoing LT, may be due to an altered reassociation of Ras GTPase-activating-like protein IQGAP1, a regulator molecule of bile canaliculi structure, with the endocytic machinery, particularly with the endocytic multimeric (AP-2) and monomeric (clathrin) adaptors (proteins that mediate the interactions between "address tickets" on cargo proteins and clathrin, as clathrin cannot bind directly to cargo or membranes) [114]. The maintenance of the hepatocyte bile secretion properties would then depend on their ability to rapidly rereform integral adherent junctions and maintain bile canaliculi structure upon reperfusion [114]. Although during 120 min of ischemia or ATP depletion, cell viability and integrity of tight junctions supported by adherent junctions in cholangiocytes were maintained, striking alterations in the secondary structure of their plasma Increased susceptibility of Na(+), K(+)-ATPase membrane, with decrease of the basolateral interdigitations and apical microvilli have been observed [115]. This reorganization of cholangiocyte membrane domains represents an early event in rat liver ischemia and contributes to impaired vectorial bile duct secretion and postischemic cholestasis [115]. During the ischemic phase failure of the sodium pump or Na(+), K(+)-ATPase [116] leads to intracellular accumulation of Na(+), edema, and swelling of Kupffer cells, sinusoidal endothelial cells, and hepatocytes [117]. Hepatocellular Na(+), K(+)-ATPase is an important driving force for bile secretion and has been localized in the basolateral plasma membrane domain [118,119]. Bile acid uptake by the hepatocyte is a secondary active transport that is energized by the Na(+) gradient maintained by the Na(+), K(+)-ATPase. Thus, Na(+), K(+)-ATPase appears important in coupling the energy from ATP to transport activity, resulting in so-called bile acid-dependent bile flow [120]. Decreased Na(+), K(+)-ATPase activity following cold and warm I-R results in apoptosis, necrosis, and shedding of biliary tract epithelial cells [121]. Reasons for alterations of Na(+), K(+)-ATPase activity after hypoxia and reoxygenation in the perfused rat liver [122] and after cold and warm I-R in human LT [123], may be direct alteration of the enzyme catalytic subunit and modification of its environment; ROS released from activated Kupffer cells, changes in ATP levels and in membrane lipid fluidity and ionic distribution may also contribute to Na(+), K(+)-ATPase activity disturbances [122]. Moreover, a marked delay of functional recovery in cultured biliary epithelial cells, which was provoked by ATP depletion, induced intrahepatic bile duct injury following I-R [124].

Pathological Effects of Bile Salts.
Cholestasis induced by I-R injury is characterized by dilatation of bile canaliculi and loss of microvilli [105] and exposes hepatocytes and cholangiocytes to an elevated concentration of toxic bile acids [125].
Bile formation is an energy consuming process, which is regulated by specific transport proteins situated in the membrane of hepatocytes and cholangiocytes [126]. I-R can induce selective and/or temporary modification of the expression and function of some biliary transporters, leading to abnormal bile composition and to toxic injury to the cholangiocytes [110,127], as well as to the hepatocytes [98].
The toxic bile composition early after LT, characterized by a low biliary phospholipid/bile salt ratio, is associated with histological signs of injury of the small bile ducts in the liver [51,54,56]. The most important apoptotic initiator in cholangiocytes is the Fas receptor/Fas ligand pathway [128]. Human cholangiocytes express Fas receptor [129]. Activated Fas receptor complexes on the plasma membrane cause caspase-8 activation and trigger apoptosis [128]. By liver I-R activated Kupffer cells can potentiate cholestatic injury through the synthesis of the proapoptotic Fas-independent receptor TRAIL [130]. Then, as in a vicious circle, during cholestasis bile acids themselves may initiate or aggravate hepatocellular damage [131]. Toxic hydrophobic bile acids retained in the hepatocytes during cholestasis initiate the generation of ROS metabolites from mitochondria, leading to lipid peroxidation and loss of cell viability [132][133][134]. The mitochondrial oxidative stress triggers the mitochondrial permeability transition (MPT), resulting in exaggerated mitochondrial cytochrome c release and apoptosis [135].
Biliary secretion of HCO 3 (−) prevents the uncontrolled membrane permeation of cytotoxic hydrophobic bile salts by maintaining an alkaline pH near the apical surface of hepatocytes and cholangiocytes [13,136].
The cholangiocyte "protector" HCO 3 (−) secretion may be disturbed after LT, as I-R results in altered expression of the anion exchanger 2 (AE2) and of the cystic fibrosis transmembrane conductance regulator (CFTR) proteins, which regulate the biliary secretion [137].
Prolonged cold ischemia time during LT is associated to a downregulation of membrane-associated Mucine 1 (Muc1), 3A (Muc3A), and 5B (Muc5B) expression [138,139]. Mucines are expressed on the apical membrane of the biliary epithelial cells and lubricate and protect these cells from diverse injuries, including injury by cytotoxic bile salts [140]. Decreased expression of Muc1 and Muc3A after LT may favour the development of ITBLs [138].

Immunologically Mediated ITBLs
Following LT 8.1. ABO Incompatibility. In the past, liver transplantation across ABO blood group barriers has been discouraged because of multiple complications, particularly acute rejection and biliary complications [58]. However, organ shortage and new developed immunosuppressive agents decreasing humoral rejection have led to an increased use of ABOincompatible liver for transplantation with acceptable results concerning patient and graft survival rate [65,141,142]. Although in children, there is no obvious difference in the outcome of ABO-compatible LT and ABO-incompatible LT, in adults graft survival rate after ABO-incompatible LT is not so satisfactory [65]. Moreover, the incidence of ITBLs after ABO-incompatible LT in adults is much higher than in ABOcompatible LT [60,65,67]. ABO blood group antigens are expressed on both, bile duct epithelium and vascular endothelial cells [143,144]. Donor ABH antigen expression up to 150 days after LT is associated with a high incidence of late, severe biliary strictures (82%), hepatic artery complications (24%), decreased graft survival (44%), and acute cellular rejection [60]. Persisting ABH antigen expression after ABO-incompatible LT is often the consequence of the vascular occlusion. Subsequent ischemic injuries caused by endothelial lesions increase the susceptibility to immunologic injury of biliary cells leading to ITBLs [62,67,145]. Preexisting primary sclerosing cholangitis and autoimmune hepatitis are also associated with a higher incidence of ITBLs [75,76,146].

Acute and Chronic Rejection.
In liver allograft rejection, most tissue damage occurs as a consequence of direct cellular immunologic injury to the bile duct epithelium [147].
Acute cellular rejection, occurring generally within 90 days of LT, concerns 50 to 75% of liver allograft recipients [148]. The targets of activated lymphocytes are donorderived bile duct epithelial cells and vascular endothelium [147]. Acute rejection is associated with lymphocytic cholangitis, a cytotoxic T-cell-mediated nonsuppurative destructive cholangitis of the small intrahepatic bile ducts that can induce cholestasis [147,149]. Activated Kuppfer cells migrate into the rejecting liver and release cytokines, resulting in the loss of the Na(+), K(+)-ATPase activity, which plays an important role in bile secretion [119]. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, can protect from cholestatic and hepatocellular injury by enhancing the secretory capacity of the cholestatic liver cells and by its cytoprotective action against hydrophobic bile salts [150][151][152]. During allograft rejection, the loss of Na(+), K(+)-ATPase activity, a cotransporter for hepatocyte taurocholate uptake, leads to impaired secretion of TUDCA and results in subsequent cholestatic injury [119]. In patients presenting ITBLs following LT, the response of lymphocyte T helper 1 (Th1) was decreased, while the response of Th2 was increased [153]. Whether these immunological changes were induced by the damage of the bile ducts or occur as an additional damaging factor or are found as an epiphenomen in patients with liver transplant dysfunction remains unclear.
Although some studies did not show that chronic graft rejection was a risk factor for development of ITBLs following LT [154,155], others demonstrated the association of chronic allograft rejection and development of ITBLs [33,46,60,[66][67][68]. Chronic allograft rejection after LT also termed "ductopenic rejection", is characterized by ischemic injury and paucity of bile ducts [148]. It occurs within the first year after LT with an incidence rate of 2 to 5% [156]. The zone of the central venous drainage or zone 3 of liver parenchyma is poorest in oxygen concentration and more sensitive to ischemia. During cellular chronic rejection, progressive intimal and subintimal infiltration of second-and thirdorder branches of the hepatic artery with foam macrophages accompanied by foam cells or obliterative arteritis can result in arterial stenoses and ultimately in ischemic injury to interlobular bile ducts and hepatocytes of zone 3 [104,157]. However, in the early stages of chronic rejection, the necroinflammatory lesions of interlobular bile ducts and hepatocytes, frequently associated with typical portal inflammatory infiltrates of acute cellular rejection, seem more likely to be the results of direct immune-mediated mechanisms [158].

Gender.
Gender-mismatched liver transplant recipients had a higher likelihood of graft failure when compared with gender-matched liver transplant recipients [74]. In male recipients receiving female donor organs there is an increased risk of graft failure compared with a female recipient receiving a liver from a male donor [74]. Moreover a female to male donor/recipient match is associated with late occurrence of ITBLs [76].

Cytomegalovirus (CMV) Infection.
The overall rate of CMV infection in liver transplant recipients varies from 30 to 50% [159,160]. In CMV-infected rat liver allografts undergoing acute rejection there was a significant increase in portal inflammation and more severe bile duct injury compared with CMV-negative liver allografts [161]. In transplanted patients developping ITBLs during CMV infection, histological examination of specimens from bile duct strictures showed CMV inclusions [71]. CMV infection induces injury of endothelial cells of the peribiliary capillary plexus, with subsequent microthrombi formation and insufficient oxygenation of the biliary epithelium, ultimately leading to ischemic injury of bile duct cells and development of ITBLs [13,72]. CMV infection can damage the bile duct cells in a direct manner by infecting directly biliary epithelial cells and in an indirect manner by immune attack evoked against infected biliary epithelial cells [13].

Chemokine Polymorphism CCR5 Delta 32.
Loss-offunction mutation in the CC chemokine receptor 5 (CCR5delta32) has been associated with development of ITBLs following LT [61]. Functional changes in the immune system resulting from CCR-5 delta 32 mutation, which include impaired chemotaxis of regulatory T cells to the site of injury may be responsible [162]. Indeed, a greater risk of developing ITBLs after LT was observed in liver transplant recipients carrying CCR5-delta 32 polymorphism compared with CCR5 wild-type transplant recipients [13,61,73].

Metalloproteinase-2 (MMP-2) Polymorphism in Liver
Graft Donor and Recipient. A large family of proteolytic enzymes involved in the degradation of extracellular matrix called matrix metalloproteinases (MMPs), secreted by Kupffer and stellate cells [163], are involved in the mechanisms of neutrophil infiltration and in the alteration of liver microcirculation due to the loss of the normal sinusoidal extracellular matrix following cold [164] and warm [165,166] I-R injury.
The activation of one of these MMPs, the metalloproteinase-2 (MMP-2), also called Gelatinase-2, takes place at the cell surface, which confers to this unique MMP a pivotal role in cellular migration during processes requiring the remodelling of basement membranes, the thin extracellular matrices underlying endothelial and epithelial cells [167,168].
MMPs are subject to complex regulation at multiple levels: gene transcription, proenzyme activation, and inhibition of activity by tissue inhibitors of matrix metalloproteinases (TIMPs) [167,168]. At gene level, it has been demonstrated that several single nucleotide polymorphisms (SP) in the gene promoter regions of MMPs have an impact on the transcription rate into the cells [169,170]. The SP C/T transition at position-1306 in the promoter of MMP-2, abolishes the single polymorphism 1 binding site and leads to decreased mRNA transcription and protein expression of MMP-2 [169,170]. After LT, in association with the-1306 CT genotype of donor and recipient, the serum levels of MMP-2 were decreased in patients that developed ITBLs [75].
MMP-2 CT genotype in both, donor and recipient is strongly and independently related to the development of ITBLs within 4 years after LT [75]. The presence of the MMP-2 CT genotype in donor and/or recipient was found to increase the incidence of ITBLs incidence stepwise from 9% when absent, increasing to 16% when present in either donor or recipient, further increasing to 29% when present in both donor and recipient [75]. These findings indicate that a genetically determined reduced MMP-2 tissue remodelling contributes to the development of ITBLs after LT.

Extended Donor Criteria and ITBLs
9.1. Donor Age and ITBLs. As old livers are more susceptible to warm and cold I-R injury than young livers , donor age seems to be a relevant risk factor in the development of ITBLs after LT [4,12,36,37,76,173].
The activation of peroxisome proliferators-activated receptor gamma (PPARγ), which belongs to the hormone nuclear receptor superfamily [174], is significantly reduced in old mice compared to young mice [175]. During liver ischemia, its activation is suppressed [176]. In old mice, PPARγ activation significantly improves liver I-R injury [176] by modulating inflammatory response and apoptosis [177].
Different from young livers, the initiation of apoptosis by nonparenchymal cells in older livers is increased and is driven by the enhanced release of TNFα [171]. TNFα is a cytokine mainly released by activated Kupffer cells following liver I-R [19,178,179]. Aging may directly affect Kupffer cells resulting in TNFα release [180,181] and apoptosis [171]. Apoptosis is a highly regulated ATP-requiring form of cell death [182]. Despite decreased ATP levels and reduced hepatic mitochondrial function in older livers [117], apoptosis seems to be a predominant feature of liver cell death following ischemic injury in these old livers [171]. Lower ATP content in older liver does not directly affect the apoptotic cascade but facilitates the activation of apoptotic mediators and inhibits survival mechanisms [171].

Cold Ischemia Time and ITBLs.
Prolonged cold ischemia time is an independent risk factor for liver preservation injury, even more so than donor age [183]. Cold graft preservation for more than 14 h has been associated with a two-fold increase in preservation injury, resulting in biliary stricures and decreased graft survival [183][184][185]. Accordingly, the risk of graft loss increases by 1% for each additional hour of cold ischemia [186]. Although several studies failed to show a correlation between the incidence of ITBLs and cold ischemia time [44,68,155,173,187,188], in other studies this incidence was increased after a prolonged cold ischemia time [2,12]. After a cold ischemia time less than 13 hours the percentage of ITBLs was 7%, whereas the percentage increased to 52% when the cold ischemia time was longer than 13 hours, and to 69% if it was longer than 15 hours [2,12]. In a recent large retrospective study with an overall incidence of post liver transplantation ITBLs of 3.9% [12], 10 hours of cold ischemia time turned out to be the threshold that should not be excessed in order to avoid ITBLs, [12].

NHBDs and ITBLs.
In liver transplantation, the use of NHBDs has been introduced in order to expand the organ donor pool [189]. However, the addition of donor warm ischemia time to the subsequent cold preservation time and warm reperfusion injury negatively impacts graft function following LT [190].
Compared with donation after brain death (DBD), livers from NHBD inevitably sustain a period of warm ischemia from circulatory arrest until start of preservation, resulting in ischemic injury with higher risk of biliary complications including ITBLs [39]. Also the incidence of IPGF, PNF, acute and chronic rejection, and retransplantation is higher with NHBD [190][191][192][193][194].
In NHBD, low blood flow during the period of hypotension after tracheal extubation and no blood flow during the period between cardiac arrest and organ recovery result in formation of microthrombi that obstruct the capillaries and limit liver perfusion [196]. Inadequate flush of these capillaries leads to suboptimal cold preservation and subsequently to exacerbated ischemic injury [196]. In a pig NHBD model of liver transplantation, prolonged warm ischemia time resulted in a high biliary salt-to-phospholipid ratio, which contributes to the development of ITBLs [51].

Graft Steatosis and ITBLs.
Steatosis of the liver is considered pathologic when the hepatic fat content, consisting mainly of triglycerides, exceeds 5% of the actual wet weight of the liver [197]. Hepatic steatosis is present in approximately 20% of liver donors, and 5-6% of cadaveric livers are discarded due to steatosis [198]. Liver steatosis is histologically classed as "macrovesicular" when the hepatocytes are distended by a single large fat vacuole that displaces the nucleus to one side of the cell and as "microvesicular" when multiple small droplets finely are dispersed in the cytoplasm without nuclear displacement [197]. More than 30% of macrovesicular steatosis on donor liver biopsy is an independent risk factor for allograft loss at one year along with other elements of the donor risk index [199]. Early biliary complications seems to be associated with moderate macrovesicular steatosis [200,201]. In a recent study, the time interval between portal and arterial reperfusion and macrovesicular steatosis of the graft of more than 25% revealed to be significant predictors of biliary complications [40,41]. At Univariate analysis macrosteatosis of more of 25% of the graft is the only independent risk factor predicting biliary complications after liver transplantation [40,41]. The increased susceptibility of the steatotic liver to I-R injury is due to the perturbation of both, blood flow microcirculation and changes in the cells [202]. Brain death of the liver donor may amplify the adverse effects of preexisting steatosis by inducing hypotension, and reducing portal venous and hepatic microcirculation [203,204].

Graft Perfusion and ITBLs.
Although approximately 75% of the total liver blood flow is provided by the portal vein, the hepatic artery supplies approximately 50% of the oxygen consumed by the liver in physiologic conditions [205]. There are two main methods for revascularization of the liver graft: sequential and simultaneous revascularization [42]. In the first method, sequential revascularization, the graft is first reperfused via either the portal vein or the hepatic artery (anterograde reperfusion), or via the inferior vena cava (IVC) (retrograde reperfusion) with subsequent reconstruction of the remaining vessels. In the second method, simultaneous revascularization, the graft is reperfused simultaneously via the portal vein and the hepatic artery. The sequence of graft reperfusion may be relevant for the development of ITBLs, particularly in grafts from ECD [42]. Liver transplantation standard technique involves initial blood perfusion by the portal vein to shorten the anhepatic period and graft rewarming in situ. In this period, the graft is exclusively perfused through the portal vein for at least 10 min until the realization of the hepatic arterial anastomosis [40]. The delay of rearterialization in sequential revascularization is associated with more pronounced microvascular disturbances and subsequent graft dysfunction [206]. Indeed rearterialization of the graft during liver transplantation causes an increased volumetric blood flow within the sinusoids called "reactive hyperemia" [207]. A long interval between portal and arterial reperfusion of the liver, in case of sequential revascularization, is associated to a higher incidence of biliary complications following DBD LT [40]. Simultaneous revascularization elicits a remarkable improvement in oxygen tension and maintenance of tissue ATP, compared to sequential revascularization [208]. The disadvantage of simultaneous revascularization is the prolongation of warm ischemia time and the anhepatic phase, Journal of Transplantation 9 which can be detrimental to postoperative graft function and survival [184,209]. Whether simultaneous revascularization is better than sequential revascularization remains unclear [42,207,210,211].
In some retrospective studies, the incidence of ITBLs in patients who underwent simultaneous revascularization of the graft [45,212] was lower compared to patients who had sequential revascularization [42,211]. Particularly in a recent study, simultaneous revascularization resulted in a minor incidence of ITBls compared to sequential revascularization (none versus 26%, resp.) [213], suggesting that simultanous revascularization may be more suitable to protect the integrity of the intrahepatic biliary tree [213].
Retrograde perfusion of the liver graft via the vena cava, followed by anterograde sequential reperfusion of the portal vein and the hepatic artery, decreases liver I-R injury and IPGF [210]. However, on the biliary epithelium or other cells of the biliary tract retrograde reperfusion has detrimental effects with an increased risk of ITBLs [210]. Improvement in flushing the microscopic biliary vasculature and possibly preventing microvascular thrombosis in the biliary tree may be obtained by adding the high-pressure aortal perfusion technique to the main graft perfusion methods, [66] and additional arterial back-table pressure perfusion [36]. These graft perfusion methods seem to reduce the rate of ITBLs following LT [36,66].

ITBLs: Importance of Portal Venous Blood Flow.
The blood supply to the biliary tree is almost solely arterial, with no significant contribution from the portal vein in physiological conditions [31,214,215]. However, some support the hypothesis that the peribiliary vascular plexus is not only sustained by blood from the hepatic artery as traditionally reported, but also by blood from the portal vein [216]. The hepatic artery is in essence an end artery for the donor biliary tree, as collaterals from the lower extrahepatic biliary tree are interrupted in the process of liver procurement and transplantation. In case of hepatic artery thrombosis, new collateral vessels can form and limit additional biliary stricture formation [155]. As ITBLs occur in the absence of hepatic artery thrombosis, it has been suggested that the portal venous blood flow has an important impact on the pathogenesis of ITBLs after liver transplantation [216]. In a recent study, patients with partial portal vein thrombosis and intact hepatic arterial blood supply developed ITBLs in the hepatic segments affected by portal vein thrombosis [216]. In many cases of hepatic artery thrombosis it seems that the portal perfusion maintains hepatocytes [216]. Thus, the contribution of the portal blood flow to the biliary microcirculation is not negligible and a compromised portal venous blood supply can predispose to the development of ITBLs [216].

Static Cold Storage Solutions of the Graft and ITBLs.
Liver preservation techniques do influence the graft quality [9]. Static and dynamic preservation are the two current methods of liver preservation in LT [217]. Static preservation means simple cold storage while dynamic preservation comprises hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation [217]. Until today, only static cold storage preservation is clinically approved for liver transplantation in humans [217].
Cold preservation injuries to the biliary tract of the donor liver were decreased by efficient flushing of the biliary tract in animals [218,219] and in humans [220,221]. In a recent study, an effective biliary flush reduced the effects of bile salt toxicity to the epithelium, reduced cell edema, prevented cell acidification, and provided adequate ATP precursor substances, resulting in reduction of biliary cold preservation injuries [222]. Generally, static cold storage UW solution is used for organ preservation [223], however the histidinetryptophan-ketoglutarate (HTK) static preservation solution has started to compete with UW [224] and is now mainly used in deceased donor liver transplantation (DDLT) in Europe and North America and in living-related liver transplantation (LRLT) in Japan and Hongkong [43,221,[225][226][227][228][229][230][231][232][233]. UW cold storage solution has more hepatocytoprotective effects than HTK cold storage solution [234][235][236], but its viscous nature may hinder an efficient flushing of the small bile duct capillaries, so residual bile can crystallize and obstruct capillary ducts, thus aggravating the cold ischemic insult to the epithelial biliary cells [237,238]. HTK cold storage solution has the same viscosity as water and its average velocity is three times greater than UW solution under the same perfusion pressure [221]. The time of liver cooling with HTK cold storage solution is shorter and improves the perfusion of the biliary vascular plexus resulting in reduced biliary tract preservation injury [36,220,221,239,240]. Also the lack of macroaggregate formation of adenosine crystals and the absence of plastic byproducts in HTK solution, responsible for occlusion of small capillaries, which exacerbates small bile duct ischemia following reperfusion, contribute to the beneficial effects of HTK cold storage solution [237,238]. HTK cold storage solution may be used particularly in livers with existing I-R injury, with high risk of I-R injury, or with biliary injury such as ECD organs [221,223,241]. A combined use of both cold storage solutions, HTK with its low viscosity and UW with its hepatocytoprotective effects, may have additional benefits for the biliary system [219,242]. Thrombolytic agents as urokinase [243], which may help flushing the microscopic biliary vasculature, were employed to prevent microvascular thrombosis in the biliary tree [244].
As there are no standardized guidelines regarding the methods of liver graft perfusion in terms of solution type, amount of solution, route of perfusion, perfusion pressure, and the time of perfusion, adequately powered randomized clinical trials with long follow-up periods are needed to evaluate the long-term impact on warm and cold I-R injury and induction of ITBLs after LT.

Conclusion
The main pathomechanisms leading to ITBLs following LT are cold and warm I-R injury, stagnation of cytotoxic bile salts and changes in bile composition, and immunological mechanisms. These mechanisms are mutually connected, one inducing or reinforcing the other, that it may be difficult sometimes to settle the "culprit". Besides, knowledge of these mechanisms remains superficial and in the beginnings. Naturally, that goes too for the possibilities of ITBL prevention and treatment. Until sustained progressions are not made in the field of ITBL research, the only way to keep the incidence of ITBLs after LT as low as possible is to reduce as much as possible their risk factors.