however , graft dysfunction remains a problem affecting up to one - third of the recipients , despite reports of good to excellent long - term outcome .
the organ dysfunction is considered multifactorial , but ischemia and reperfusion ( i / r ) injury is probably the most important contributing factor , although the detailed steps of pathogenesis are controversially debated .
i / r injury is a major cause of liver graft dysfunction resulting in adenosine triphosphate ( atp ) decrease , evidence of intracellular acidosis , and cell swelling of the hepatocytes .
this aspect is also accompanied by or linked to bile duct loss or ductopenia , cholestasis , and biliary ductular proliferations in the posttransplant liver biopsy . however , biliary marker levels increase usually only 57 days after transplantation stimulating several fields of research in the last couple of decades .
cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation , and the steps reaching this state are not completely understood .
we reviewed the canine liver transplantation model as i / r injury model to delineate in detail the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes , which seems to be a post - transplant event occurring in the liver at an earlier stage than frank cholestasis . here , we speculate that i / r liver injury through a phenotypical switch of the liver cells may contribute to the poor outcome of the liver graft .
liver transplantation is widely known as the most effective therapy for both acute and chronic liver failure . in 1963 , thomas starzl , an american surgeon , northwestern university medical school graduate with degrees in anatomy , neurophysiology , and medicine , was the first to perform the liver transplantation on a child suffering from biliary atresia . despite the significant success of liver transplantation , infection , poor graft function , and rejection are major problems that may still contribute to death of the transplanted patient or to deterioration of the graft .
reperfusion following long ischemia of liver during liver transplantation causes severe injury , which is now universally indicated as i / r injury .
i / r injury of liver is a major cause of morbidity and mortality in patients undergoing liver transplantation .
several mechanisms and distinct pathways may involve i / r leading to both initial poor function and primary nonfunction of the liver allograft .
three cytoskeletal proteins form a network that provide the cellular structure and fundamental integrity of the eukaryotic cells : microfilaments , microtubules , and intermediate filaments .
the intermediate filament proteins have an important role in liver protection against mechanical and nonmechanical injury , which have been demonstrated in animal models .
overexpression of proteins and mutations of the corresponding keratin genes have been reported to contribute to several human oncological and nononcological diseases .
eukaryotic cells have a unique cytoplasmic structure labeled as cytoskeleton , which consists of three distinct kinds of cytoskeletal filaments , including microfilaments , microtubules , and intermediate filaments . besides giving the rigidity of the cell and maintaining cell shape and borders or cell  scaffolding , 
cytoskeleton plays important and probably crucial roles in intracellular transport , cell division , gene regulation , and signal transduction of the genetic information [ 6 , 7 ] .
actin , which is widely accepted to be a highly conserved structure among different species , is the major protein that constitutes the microfilaments
. there are three known classes of human actin gene that have been identified as - , - , and -actin .
cell - cell interaction , signal transduction , cell shape maintenance , and cell motility are the central functions of actin .
microtubules , which have a diameter of around 23  nm , are protofilaments that are important for the intracellular transport , movement of cilia and flagella , mitotic spindle , and cell wall synthesis .
both microfilaments and microtubules have several groups of binding proteins that modulate their stability and biological functions , such as signal transduction .
intermediate filaments with around 10  nm of diameter are more stable than actin filaments and function in the maintenance of cell shape by carrying tension .
intermediate filaments , which include , for example , vimentin , glial fibrillary acidic protein , neurofilament proteins , keratins , and nuclear lamins , organize the internal 3d cell structure anchoring cyto - organelles and serving as structural scaffolds of the nucleus and cytoplasm .
cytokeratins ( ks ) are the main protein family that constitutes the intermediate filaments with more than fifty members that have been identified .
polypeptides of the ks are variants and have been divided into type i and type ii , or acidic and basic , respectively .
studies have shown that each epithelial cell has a distinct content of cytokeratins ( i.e. , expression of keratins is described as tissue - specific manner ) playing a major role in the tissue identification of metastasis of unknown primary carcinomas .
ks usually present in cells as heteropolymers pairs composed of type i ( k9k20 ) and type ii ( k1k8 ) .
k8 ( mw 52kd ) and k18 ( mw 45kd ) are considered as the only ks that are normally expressed in human hepatocytes . moreover ,
both k8 and k18 have been found to be expressed in early development stage of mouse embryos .
immunohistochemical studies have revealed that cholangiocytes ( bile duct cells ) express k7 ( 54 kd ) and k19 ( 40 kd ) in addition to k8 and k18 , and those ks have been used as valid markers for both studies on development of the intrahepatic biliary system and for assessment of bile duct damage .
it has also been well established that cholangiocytes are derived from hepatoblasts found around portal vein .
these cells show a particularly intense expression of k8 and k18 . as indicated above , k7 and k19
have been linked to development of the intrahepatic bile duct system in both humans and animal models .
the expression of k19 is useful to identify primitive biliary cells , while the expression of k7 appears after 20 weeks of gestation in humans .
both k7 and k19 are consistently expressed in the development of the intrahepatic bile duct with elevated levels until one month of postnatal age .
thus , in order to phenotypically switch hepatoblasts into cholangiocytes , these cells should firstly express k19 followed by expression of k7 .
however , it has been shown that hepatocytes might express k7 in response to different conditions , such as ductopenia and cholestasis , and , interestingly , it has been found that hepatocellular carcinoma cells expressing k19 are significantly associated with reoccurrence of neoplasm after transplantation .
a recent study has demonstrated that hepatocytes are frequently expressing k7 in case of chronic liver allograft rejection .
have demonstrated that hepatocytes intensively expressed k7 and k19 early following cold ischemia in a canine isolated perfused liver transplantation model .
furthermore , they demonstrated that bile duct cytokeratins are very useful markers to diagnose an early sign of cholestasis .
it has become clear that cholestasis is significantly associated with high morbidity and mortality in patients undergoing liver transplantation .
therefore , investigations that lead to discover the mechanisms of the cholestasis are dramatically required .
i / r injury is a phenomenon that occurs when blood flow and oxygen delivery return to reperfused tissue .
it is considered one of the major causes of morbidity and mortality among patients undergoing liver transplantation .
i / r injury has been demonstrated in several diseases such as cerebrovascular diseases , peripheral vascular diseases , sepsis , myocardial infarction , and organ transplantation .
it involves several mechanisms , which lead to organ failure , circulatory dysfunction , and , finally , death of the transplanted patient .
cellular mechanisms of i / r injury include a few essential cell cascades that suggest the role of activation of endothelial cells , kupffer cells , reactive oxygen species ( ros ) , and polymorphonuclear leukocytes ( pmn ) or neutrophils in the pathogenesis of i / r injury .
there may also be mechanisms involving t lymphocytes that seem to have a key role in short- and long - term damage during i / r injury .
t lymphocytes act as mediator in the subacute inflammatory phase of neutrophilic recruitment following i / r injury .
another important constituent of tissue injury following i / r injury that needs to be emphasized is the production of oxygen free radicals ( ofrs ) .
these ofrs can arise from different sources , such as kupffer cells , pmn , and xanthine oxidase ( xo ) that is the most significant source .
several etiological factors are involved in i / r injury , including atp reduction , activation of proteases , and alteration in the intracellular concentration of cytokines and chemokines , and cell swelling .
investigations of posttransplantation surgery have indicated that reperfusion plays an essential role in primary graft nonfunction , which is one of the most serious complications of liver transplantation .
i / r injury also causes early organ failure up to 10% as well as increases the acute and chronic rejection .
several studies demonstrated that during i / r injury , morphological changes of liver tissues occur , and these changes can have a prognostic significance .
the intermediate filament ( if ) cytoskeletal protein is one of the three major cytoskeletal proteins whose result is important in maintaining both cellular structure and integrity of eukaryotic cells .
the other two filament proteins are microfilaments ( mf ) and microtubules ( mt ) . beside their cellular functions like cell motility , division , and stress responses
, they also have an essential role in human diseases due to mutations in filament proteins .
if proteins consist of five types of keratins according to structure of genome and composition of amino acid , if proteins types i  iv are cytoplasmic , and type v if contain nuclear lamins .
the keratins are obligating noncovalently heteropolymers because they consist of one of each type i and type ii keratins as pairs .
neutral to basic  keratins are the largest group of if proteins   classified as type i ( k9k20 ) and type ii ( k1k8 ) . another type of if proteins is type iii if that includes vimentin , peripherin , glial fibrillary acidic protein , and desmin of mesenchymal cells , peripheral neurons , glia cells and astrocytes , and muscle cells , respectively .
type iv if proteins include neurofilament proteins ( nf - l , nf - m , and nf - h ) , synemin , nestin , syncoilin , and -internexin ; lamins a 
as indicated above , the adult hepatocytes express k8 and k18 only compared to other epithelial cells that express 2 or more type i or type ii keratins   such as bile duct , which expresses additional keratins k7 and k19 .
the first disease to be discovered related to keratin mutation was epidermolysis bullosa simplex ( ebs ) with mutations in k5 and k14 .
additionally , mutations in k8 and/or k18 lead to acute or chronic liver diseases . normally in hepatocytes ,
further studies indicated that reperfusion alone causes alteration of theses microfilaments ( f - actin ) leading to contraction of canaliculi , relocalization of enzymes and transporters of canaliculi , and increasing permeability of cellular tight junction .
cholestatic liver disease occurs when there is a decrease in bile flow , which is an abnormal physiologic state , and retention of toxic bile acids .
clear cytoplasmic hepatocytes ( cholate stasis ) presented in cirrhotic nodules and showed a decrease in keratin if network in the cytoplasm .
studies on mice reported that keratin was overexpressed in epithelia of bile duct due to ligation of bile duct and in hepatocytes . in cholestatic liver disease , bile duct epithelial - type keratin ( k7 and k19 )
intermediate hepatocytes express k7 , while reactive bile ductules express k7 and k19 . in post - transplant period ,
keratins of biliary type were detected in the hepatocytes in the early period of i / r injury . moreover
, bile canaliculus progressively dilate after ischemia , and a change of the microvillous integrity is demonstrable .
the liver is one of the organs with an incredible capacity for in vivo tissue engineering which allow restoration of the liver architecture and reestablishment of certain vital functions .
the study of liver regeneration in humans arise ethical issues , and it is difficult to carry out because of a plethora of heterogeneous liver lesions . accordingly , using experimental animal models is more useful and helpful for studying liver regeneration . in vitro studies need to be followed by in vivo ones to simulate the interaction between liver cell populations . in our opinion ,
the use of large animals such as dog , pig , or sheep is more advantageous than small animals like rat or mouse ; large animals are similar to human beings in their physiology and anatomy , and techniques of microsurgery are not necessary to carry out determinate experiments .
only few studies have been carried out on i / r animal models in liver .
the canine liver model following i / r injury has been used as a liver transplantation model to study excretion of bile and intrahepatic intermediate filaments expression involved in morphological changes of the biliary system . to the best of our knowledge ,
in a canine isolated and perfused liver model , there was the first clear - cut evidence of cholestatic changes starting early following cold ischemia despite prompt recovery of the bile flow .
according to some other authors , the use of the canine liver transplantation model seems to allow a better vascular perfusion under ex vivo conditions in contrast to the rat model .
's study revealed an ischemia - dependent impairment of an experimental biliary dye excretion during early stage of reperfusion and a progressive cytokeratin expression of biliary type in the hepatocytes despite a prompt recovery of the bile flow after i / r .
the recovery of the bile output after i / r represents a key event in lt .
the nonstimulated bile flow rate was unaffected by cold ischemia up to 10  h in our canine isolated perfused liver model , but the decrease of the biliary dye excretion during early stage of reperfusion correlated with the peak output rate of the dye across the canalicular membrane .
remarkably , the dye uptake at the sinusoidal membrane was not affected by cold ischemia , leading to a possible accumulation of toxic compounds in the hepatocytes .
hepatic elimination of the dye following i / r has been analyzed mainly by plasma clearance .
very importantly was the study of the hepatic elimination of the dye after a bolus injection by compartmental spectrophotometric analysis both in the perfusate by transhepatic sampling and in the bile .
differential mechanisms can be suggested for the impaired biliary excretion of organic anions following i / r .
previously , a positive correlation between biliary dye excretion , viability of the graft , and hepatic atp content has been found in cholestatic liver disease , and reduced biliary dye excretion has been attributed to the lack of atp .
atp content does not seem to be the main limiting factor for the hepatocellular transport following i / r .
an impairment of the intracellular transport or a decreased transport rate across the canalicular membrane might play a major role as contributing factor .
the step across the canalicular membrane generally represents the rate - limiting one in the hepatocellular transport . in the canine liver transplantation model ,
the flow in the portal vein was continuous , because roller pump , oxygenator , and heat exchanger in the perfusion line were connected in series with the roller pump upstream .
the oxygen content of the saline reperfusion solution was 2% in volume ( 10% of arterial hepatic blood and about 15% of the portal venous blood ) .
thus , one can expect to have reactive - oxygen - species- ( ros- ) linked damage at the beginning of the reperfusion after 8 or 10  h of ischemia .
however , the excellent reperfusion results of the control group and data of the 2  h ischemia group indicate that this is not a general problem , but may be an ischemia - time - linked problem .
high o2 partial pressure in the liver stimulates ros formation after extensive long ischemia times , and this may be one of the most important limiting factors of the ischemia tolerance .
electron microscopy studies in hepatocytes revealed microfilaments , which are distributed along the plasma membrane and in the region of the bile canaliculus . as indicated above ,
cytoskeleton filaments are also essential for the maintenance of cell shape , bile canalicular architecture , and integrity of the cellular tight junctions .
the microfilaments network surrounding the canalicular pole and extending into the canalicular microvilli is damaged during cholestasis .
reperfusion , but not ischemia , has been considered faulty , inducing an alteration of f - actin microfilaments , suggesting an impairment of the canalicular contraction , an increase of the tight junction permeability , and a probable relocalization of canalicular enzymes and transporters .
bile canalicular size change as detected in our study may be coincident with the reorganization of the pericanalicular filaments and the colocalization of actin and myosin .
in fact , contraction in this study has been associated with shortening and/or twisting of bile canaliculi .
however , graft dysfunction remains a problem affecting up to one - third of the recipients , despite good to excellent long - term outcome .
the organ dysfunction is considered multifactorial , but i / r injury is probably the most important contributing factor .
the detailed steps of the pathogenesis of i / r injury continue to be a topic of vivid debate .
i / r injury is a major cause of liver graft dysfunction resulting in atp decrease , intracellular acidosis , and cell swelling of the hepatocytes .
this feature is also linked to bile duct loss or ductopenia , cholestasis , and biliary ductular proliferations in the liver biopsy . however , the plasmatic levels of biliary markers increase usually only 57 days after transplantation prompting on - going research to address new markers of early liver damage
. this could also be a stimulus to support more grant approvals for liver posttransplantation studies .
cholestasis is associated with high morbidity and mortality in patients undergoing liver transplantation , but the steps reaching this state are still deemed elusive .
we reviewed the canine lt model as an i / r injury model and reviewed the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes .
in fact , this phenotypic switch of the liver cells , which is also sometimes called pseudo- or neoacinar transformation in diseases with cholestasis , seems to occur at a stage earlier than frank cholestasis .
we emphasized that i / r injury through this peculiar phenotypic switch of the hepatocytes may be a milestone to investigate the pathways contributing to the poor outcome of the liver graft .
reperfusion injury following ischemia affects graft function in liver transplantation by induction of phenotypical changes in hepatocellular keratins .
keratin phenotype of hepatocytes leads to hepatocellular damage that is intimately connected to several intracellular processes , which are under intense investigation in our laboratory , and the appropriate choice of an animal model is crucial in forwarding research in very complex areas requiring not only human , but also comparative ( veterinary ) pathology knowledge .
 liver transplantation has been a successful therapy for liver failure . 
 however , a significant number of recipients suffer from graft dysfunction . 
 considerably , ischemia and reperfusion ( i / r ) injury is the most important factor leading to organ dysfunction , although the pathogenesis has not been fully described . 
 i / r injury have several established features that are accompanied by and/or linked to bile duct loss or ductopenia , cholestasis , and biliary ductular proliferations in the posttransplant liver biopsy . however , biliary marker levels increase usually only 57 days after transplantation . 
 intermediate filaments are one of the three cytoskeletal proteins that have a major role in liver protection and maintaining both cellular structure and integrity of eukaryotic cells . 
 we reviewed the canine liver transplantation model as i / r injury model to delineate the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes . 
 remarkably , this interesting feature seems to occur earlier than frank cholestasis . 
 we speculate that i / r liver injury through a phenotypical switch of the hepatocytes may contribute to the poor outcome of the liver graft . 