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Keywords:

  • inflammation;
  • leucocytes;
  • liver transplantation;
  • microvasculature;
  • NADPH oxidase;
  • nitric oxide;
  • pro-inflammatory cytokines;
  • reactive oxygen species

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES
  • 1
    Hepatic resection with concomitant periods of ischaemia and reperfusion (I/R) is required to perform reduced-size liver (RSL) transplantation procedures, such as living donor or split liver transplantation. Although a great deal of progress has been made using these types of surgical procedures, a significant number of patients develop tissue injury from these procedures, ultimately resulting in graft failure.
  • 2
    Because of this, there is a real need to understand the different mechanisms responsible for the tissue injury induced by I/R of RSL transplantation (RSL + I/R), with the ultimate goal to develop new and improved therapeutic agents that may limit the tissue damage incurred during RSL transplantation.
  • 3
    The present paper reviews the recent studies that have been performed examining the role of reactive metabolites of oxygen and nitrogen in a mouse model of RSL + I/R. In addition, we present data demonstrating how the pathophysiological mechanisms identified in this model compare with those observed in a model of RSL transplantation in rats.

INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

Liver transplantation has become the primary treatment for patients with fulminant hepatic failure and end-stage chronic liver disease, as well as certain metabolic liver diseases.1 Thus, the demand for transplantation now greatly exceeds the availability of donor organs. It has been estimated that more than 18 000 patients are currently registered with the United Network for Organ Sharing (UNOS) and that an additional 9000 patients are added to the liver transplant waiting list each year, yet less than 5000 cadaveric donors are available for transplantation.2,3 Not surprisingly, the paucity of donor livers has resulted in increases in both the mean waiting time to undergo liver transplantation and mortality while on the UNOS waiting list.4 This problem is especially acute for the paediatric population, where mortality of infants or small children waiting for appropriately sized-matched grafts may exceed 25%.5 In an attempt to expand the size of the donor pool, a variety of surgical techniques has been developed over the past 15 years, including reduced-size liver (RSL) transplantation, split liver transplantation (SLT) and living donor liver transplantation (LDLT).6

Bismuth and Houssin were the first surgeons to perform RSL transplantation in which they transplanted the left lateral segment of the left liver lobe from a cadaveric donor into a small child and discarded the remainder of the donor liver.7 Despite the fact that these types of surgeries significantly reduced the mortality of children waiting for liver transplantation and solved some of the problems associated with size-matched grafts for infants and small children, it became apparent that discarding the un-used portion of the donor liver was wasteful and significantly reduced the number of donor grafts available for adults awaiting transplant. In an attempt to overcome this inefficient use of donor grafts, new surgical techniques were developed to preserve both parts of the liver for subsequent transplantation. The improved techniques resulted in the development of SLT in which one cadaver liver is split into the larger right lobe for use in adult transplantation and the left lobe or left lateral segment, which is used for small adults or children.8 The development and use of SLT has proven to be effective in lowering the mortality of paediatric patients waiting for liver transplants; however, it has also become clear that decreased graft survival, as well as an increase in the number of hepatobiliary complications, compared with whole liver transplantation represent significant limitations with this type of surgery. Many of the complications are thought to be associated with graft size and the duration of cold (and warm) ischaemia produced by the complicated surgical procedures necessary for the ex vivo or in situ dissection of the liver and preservation.9

Living donor liver transplantation was first reported more than 15 years ago with an adult-to-child transplantation of the mother's left lateral segment.10 This procedure has become very effective in treating end-stage liver disease over the past several years and represents the standard of care at several different paediatric liver transplant centres across North America and the world. Data obtained for adult-to-adult LDLT are more limited than for other transplantation surgeries; however, its popularity is rapidly growing within the US and Japan. One of the major disadvantages of LDLT is the risk to both the recipient and donor. Although the risk of death to the donor has not been clearly defined, a few deaths have been recorded for living donors, as well as a 20–30% morbidity rate in living donors.4,11 As with SLT, graft viability for LDLT appears to be critically dependent upon the size of the graft as well as the duration of cold preservation.

Table 1. List of abbreviations:
ASTAspartate amino transferaseRSLReduced-size liver
I/RIschaemia and reperfusionSLTSplit liver transplantation
NONitric oxideSODSuperoxide dismutase
inline imageSuperoxideTNF-αTumour necrosis factor-α
ROSReactive oxygen speciesUWUniversity of Wisconsin

The pathophysiological mechanisms responsible for graft injury and failure remain largely undefined at the present time. It is known, for example, that hepatic resection with concomitant periods of ischaemia and reperfusion (I/R) is commonly used in the different RSL surgeries.12,13 It is not uncommon for patients undergoing RSL transplantation to withstand anywhere from 30–40 min to several hours of hepatic ischaemia, depending upon the specific type of RSL surgery. However, many surgeons have experienced poor outcome in patients subjected to these relatively long periods of liver ischaemia in association with liver resection and transplant. Because patients typically develop delayed liver failure within 3–7 days following surgery, it is thought that the I/R results in hepatocellular injury that may impair liver regeneration and may lead to primary graft dysfunction and failure.13,14 In fact, recent work by Selzner et al. has shown that I/R impairs the regenerative capacity of the liver.14

Numerous studies using the experimental model of in situ (warm) liver I/R in the absence of resection indicate that reactive oxygen species (ROS), pro-inflammatory cytokines (e.g. tumour necrosis factor (TNF)-α) and neutrophils contribute to hepatocellular injury in the postischaemic tissue.15–19 Indeed, previous studies have demonstrated protective effects using a variety of anti-oxidants and/or free radical scavengers in I/R-induced liver injury;16,20–24 however, the identity of the specific reactive metabolites of oxygen, the sources of these oxidants and free radicals and the mechanisms by which these ROS promote hepatocellular injury have not been defined clearly. In fact, the use of low molecular weight ROS scavengers and/or enzymatic anti-oxidants has proven as much problematic as informative because many of the compounds used were either non-specific in nature and/or possessed short circulating half-lives in vivo.25,26 It has been proposed that ROS may be generated during the early and late phases of liver I/R by xanthine oxidase (XO), mitochondrial respiration and/or Kupffer cell and polymorphonuclear leucocyte (PMN)-associated NADPH oxidase.15,27 Although different investigators have implicated XO or mitochondrial metabolism as potential sources for ROS generation in the postischaemic liver, there has been little investigation of the role of NADPH oxidase. This multimeric inline image-generating complex is composed of several protein subunits, including the membrane-spanning gp91 and p22 subunits, the cytosolic p47 and p67 subunits and the gp91-associated rac-1 protein.28 Experimental evidence suggests that this enzyme complex is activated in Kupffer cells following a variety of inflammatory stimuli and may contribute to hepatocellular injury.17,29,30 In addition, NADPH oxidase has been found in vascular tissue, most likely associated with the endothelium.31 Production of even small quantities of ROS at this location following I/R may be important for the initiation of events leading to liver injury, as well as cellular adhesion and PMN infiltration.

REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

Despite the wealth of information that has been generated using full-size liver I/R to mimic the events that occur during liver transplantation, this type of model may or may not accurately mimic the pathophysiological mechanisms that occur with surgeries involving liver resection combined with I/R. In fact, few, if any, studies have examined how liver resection may affect the responses of the liver to warm I/R.

In an attempt to more closely mimic RSL transplantation, a mouse model of RSL + I/R was developed.32 In this model, livers of anaesthetized male and female mice were rendered ischaemic by cross-clamping the hepatic arterial and portal venous blood supply for 45 min, followed by resection of the perfused bypass lobes, which represented removal of approximately 30% of the total liver mass. Blood supply was restored to the remaining 70% of the liver immediately following the resection, thereby producing a model of RSL combined with liver I/R. Initial studies using this model demonstrated that 100% of female mice survived indefinitely following surgery, whereas all male mice died within 5 days following RSL + I/R.32 The protective effect afforded to females correlated with decreased liver injury compared with their male counterparts, as assessed by lower serum alanine aminotransferase (ALT) levels and decreased histopathological scores in females at 20 h following surgery. In addition, significant periportal infiltration of mononuclear leucocytes (monocytes, lymphocytes) but not neutrophils was observed in the livers of both male and female mice.32

To address the mechanisms by which female mice were protected from the injurious effects of RSL + I/R, female mice were ovariectomized and subjected to RSL + I/R. These mice showed a significant reduction in the post-surgery 7 day survival rates compared with sham-operated female mice (14 vs 100%), which correlated well with increased liver injury.32 Similarly, administration of the selective oestrogen receptor antagonist ICI 182,780 to female mice prior to RSL + I/R resulted in increased liver injury and mortality of these animals compared with their vehicle-treated counterparts. Conversely, treatment of male mice with 17β-oestradiol (oestrogen; E2) significantly improved their survival compared with vehicle-treated controls, from 0% survival to 87% at 7 days post-surgery, suggesting that oestrogen-induced, endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) may be protective to the liver. Histopathological analysis of the livers revealed that E2 administration attenuated hepatocellular injury in male mice.32

ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

Numerous investigations have suggested that ROS may be important mediators of reperfusion injury in standard full-size liver I/R preparations.16,20,21,23,24,33 However, many of these studies reported varying degrees of protection against liver I/R using relatively large concentrations of various specific (and non-specific) anti-oxidants and/or free radical scavengers (for a review, see Jaeschke16). In order to assess the role that superoxide (inline image) plays in RSL + I/R-induced liver hepatocellular injury, we tested the effectiveness of a genetically engineered, polycationic form of human Mn-superoxide dismutase (SOD). This enzyme was engineered to possess the catalytic activity of human Mn-SOD (SOD2) and the binding characteristics of human extracellular Cu,Zn-SOD (SOD3). This SOD2/3 fusion protein has been shown to bind to the heparin sulphate binding regions on the surface of endothelial cells and the extracellular matrix (ECM), thereby markedly increasing the half-life to 30 h in vivo.26,34 These characteristics are particularly important physiologically because localization of this cationic Mn-SOD to the surface of sinusoidal endothelial cells (and ECM) increases the effective concentration of the anti-oxidant (as well as its lifetime) in the vasculature and extracellular space. It is well known that both these compartments are remarkably deficient in anti-oxidants. We found that Mn-SOD2/3, but not native human Mn-SOD, decreased the mortality of male mice from 90 to 10%, as well as attenuated liver injury by greater than 60%.35 In addition, Mn-SOD2/3 administration reduced the expression of TNF-α. Taken together, these data suggest that inline image plays an important role in the pathophysiology of RSL + I/R-induced liver injury, possibly by virtue of its ability to enhance expression of TNF-α.35

Having established that inline image plays an important role in postischaemic liver injury, we wished to examine the role that the NADPH oxidase complex played as a major source of this free radical in RSL + I/R-induced injury. This multicomponent enzyme complex has been characterized extensively in phagocytic leucocytes and Kupffer cells.17,36–39 More recent data suggest that a similar inline image-generating complex is found in endothelial cells and may be important for regulating vascular tone and blood flow.31 In order to assess the importance of NADPH oxidase in the mouse model of RSL + I/R, we used male gp91phox-deficient mice (gp91−/–), which have a defective NADPH oxidase and lack the ability to generate inline image.40 We found that gp91phox deficiency resulted in decreased mortality and reduced liver injury by 60–80% compared with wild-type male mice.35 Because our previous studies demonstrated that postischaemic liver injury occurs in the absence of significant neutrophil infiltration, the most likely cellular sources of NADPH oxidase in this model are Kupffer cells and possibly the sinusoidal and/or microvascular endothelial cells.31,41 Indeed, we showed that inactivation of Kupffer cell function by pretreatment of mice with gadolinium chloride 24 h prior to the induction of ischaemia, resection and reperfusion increased the 7-day survival from 0 to 60%.35 Exactly how NADPH oxidase-derived inline image directly (or indirectly) promotes liver injury is not known with certainty. It may be that generation of inline image early on during reperfusion may damage mitochondrial membrane lipids and proteins, leading to the loss of inner membrane potential and the ATP-generating capacity of these organelles.42 Intracellular inline image generation may also interact with and modify a number of cellular enzymes. For example, inline image has been shown to interact with aconitase, a non-heme iron enzyme that is part of the citric acid cycle responsible for the conversion of citrate to isocitrate. Inactivation of this enzyme can result in significant reductions in cellular ATP production. If inactivation of this and other cellular enzymes occurs to a significant level, as it may during I/R, then hepatocellular dysfunction, injury and death may occur.43 Furthermore, inline image may mediate hepatocellular damage indirectly via the upregulation of certain injurious cytokines, such as TNF-α.21,33,44 This is supported by our observations that the protective effect of Mn-SOD2/3 and gp91−/– was associated with reductions in serum TNF-α.35 In order to test the hypothesis that the NADPH oxidase-derived inline image is involved in TNF-α expression, we subjected wild-type or gp91−/– mice to the surgery and quantified serum levels of TNF-α. We found that gp91−/– mice expressed significantly lower serum levels of TNF-α compared with wild-type mice, indicating that NADPH oxidase-derived inline image may be responsible for the upregulation of TNF-α expression.

Taken together, our data support the hypothesis that NADPH oxidase-derived inline image directly or indirectly promotes the expression of TNF-α, which, in turn, mediates the RSL + I/R-induced liver damage. Therapies directed at scavenging inline image, inhibiting NADPH oxidase and/or immunoneutralizing TNF-α may prove useful in attenuating liver damage induced by RSL + I/R.

NITRIC OXIDE AND RSL + I/R INJURY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

A plethora of investigations, including some from our laboratory, has demonstrated that oxidants and free radicals damage cellular components leading to cell death. Although inline image or H2O2 are not particularly potent oxidizing agents, they will interact with certain transition metals, such as iron (Fe) or copper (Cu), to produce short lived oxidants, such as the hydroxyl radical (OH.):

  • image
  • Fe2+ (Cu+) + H2O2 [RIGHTWARDS ARROW] Fe3+ (or Cu2+) + OH+ OH

In addition, ROS may interact certain haemoproteins (HP-Fe; haemoglobin, myoglobin, cytochromes) to generate the potent oxidizing agent, the porphyrin cationic radical (+.HP-Fe4+):

  • HP-Fe3+ + H2O2[RIGHTWARDS ARROW]+.HP-Fe4+ = O + H2O

Historically, direct oxidation of cellular constituents by these free radicals has been thought to be the primary mechanism for postischaemic liver injury; however, more recent experimental data suggest that ROS may mediate postischaemic liver injury by a multitude of other mechanisms. Jaeschke et al. have examined the oxidation of membrane lipids as a marker of direct postischaemic cellular oxidation. They found that although lipid peroxidation occurred in the postischaemic liver, it did not occur at a time or in sufficient quantities to induce liver injury.27

Although membrane lipid peroxidation may not be a principal mechanism of ROS-induced liver injury, inline image may mediate cell and tissue injury by rapidly interacting with other intracellular and extracellular radical species, including NO. This interaction is known to produce the potent cytotoxic oxidizing and nitrating specie peroxynitrite (ONOO) and its conjugate peroxynitrous acid (ONOO).

  • image

We reasoned that if inline image mediates its apparent damaging effects through the production of ONOO, then one would expect protection if NO production was reduced or eliminated by NOS inhibition or genetic deletion. In fact, we found that eNOS-deficient (eNOS−/–) or NG-nitro-l-arginine methyl ester (l-NAME)-treated female mice were much more sensitive to the damaging effects of RSL + I/R compared with wild-type females, as assessed by increased serum ALT levels.45 In fact, all eNOS−/– and l-NAME-treated female mice died within 2 days following RSL + I/R.45 These data agree well with other studies using full-size liver I/R injury in which eNOS−/– or l-NAME-treated male mice suffered substantially more liver injury in response to 45 min ischaemia followed by 3 or 6 h reperfusion.46 The question then became, how does eNOS-derived NO protect the liver from the damaging effects of I/R? We have evidence to suggest that the superoxide-mediated interaction with and degradation of NO within the postischaemic liver removes an important hepatoprotective molecule (e.g. NO). For example, reductions in the availability of NO in the liver are known to increase the microvascular pressure through the liver via decreases in vessel diameter. This occurs because, under normal circumstances, NO counteracts other vasoconstrictor substances, including endothelin and phenylephrine. Inhibition or genetic disruption of the production of NO by eNOS results in an imbalance in the vasorelaxer/vasoconstrictor ratio, leading to contraction of the extrasinusoidal hepatic stellate (or Ito) cells. These cells, in addition to being important storage cells for certain vitamins and fat and sources of certain growth factors and collagen synthesis, are capable of contracting, thereby reducing the diameter of the sinusoid. Studies using endothelin receptor antagonists and endothelin receptor agonists prior to liver I/R have further demonstrated the importance of sinusoidal perfusion in the postischaemic liver. Therefore, the action of NO on stellate cells may represent an important perfusion-related function of NO and interaction of this molecule with inline image would effectively remove it from the system.

REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

Liver injury and recipient survival following RSL transplantation

Reduced-size liver transplantation is known to increase the risk for primary graft failure. Data obtained from several different studies suggest that this increased risk is due to increased microvascular dysfunction and injury, more severe postischaemic hepatocellular damge and/or a more accelerated immune-mediated rejection of the graft.47–53 Indeed, many investigators have used rodent models of warm (in situ) liver I/R to mimic some of the pathophysiological events that occur during liver transplantation. Although a great deal of useful information has been generated from these studies, the overriding question remains, are the mechanisms responsible for transplant-mediated liver injury and dysfunction the same as those that have been reported for warm liver I/R injury? The answer is yes and no; that is, some of the mechanisms are similar, but many are not. For example, graft size appears to represent an important risk factor for both the donor and recipient. In adult-to-adult LDLT, harvesting larger grafts presents the donor with a higher risk; however, harvesting smaller grafts may not be adequate for normal function for the recipient.4,11 In addition to the size of the graft, the duration of cold preservation (i.e. ischaemia) is thought to represent an important pathophysiological aspect of partial liver transplantation that is in need of investigation. Indeed, few studies have systematically evaluated how graft size and duration of cold storage affect liver function and survival in an animal model of partial liver transplantation. We have found, using a syngeneic RSL transplantation model in rats, that graft size and the duration of cold ischaemia are important determinants of graft viability and host survival.54 In addition, data obtained from our studies have revealed some rather surprising differences in terms of pathophysiological mechanisms of graft injury compared with results obtained from studies of warm RSL + I/R injury.

In order to study only those events associated with surgery, cold ischaemia and warm reperfusion of the full-size or partial liver grafts and to avoid the confounding mechanisms associated with acute (and/or chronic) graft rejection, we have used a syngeneic rat liver transplant model for these studies. Not surprisingly, we found that graft size and duration of cold preservation are critical determinants of graft viability and host survival. Implantation of liver grafts that approximated 30% of the normal mass of a rat liver that had been stored in cold University of Wisconsin (UW) solution for 4 or 6 h resulted in 50 or 75% mortality of the recipients within 2–3 days, following surgery, respectively (Fig. 1). Liver injury and dysfunction, as measured by increases in serum ALT and total bilirubin at 24 h following surgery, were enhanced by approximately 5- to 10-fold in the 4 and 6 h groups compared with animals receiving full-size (100%) grafts that were stored for the same amount of time in the UW solution (Figs 2,3). However, when the duration of cold preservation was reduced to 2 h, all rats survived indefinitely (> 30 days) regardless of graft size and yet their serum ALT and total bilirubin values were enhanced to the same extent as those subjected to 4 or 6 h of cold storage. These data demonstrated a clear time dependence between cold storage and graft viability and agree well with other studies in humans and experimental animals where the duration of cold storage was found to be critical for maintaining graft viability and recipient survival.1,8,55–58 The mechanisms responsible for this apparent time-dependent decrease in graft viability in cold UW solution are not clear at the present time, but may be due to a time-dependent increase in microvascular/endothelial cell injury, as described by other investigators.55,59–61 This cold-induced microvascular injury is thought to initiate an acute inflammatory response resulting in leucocyte and/or platelet adhesion and infiltration resulting in tissue damage.55,59,60 Histopathological analyses revealed hepatocellular necrosis and a mild monuclear leucocyte infiltrate in the 30% group subjected to 4 or 6 h of cold preservation,54 but not at 2 h. Together with the AST and bilirubin data, these data suggest that the inflammatory response may not account for the increased tissue injury and decreased survival observed in rats receiving a 30% graft that has been stored for 4 or 6 h in cold UW solution.

image

Figure 1. Effects of different durations of cold storage on the survival of recipients receiving 30% liver grafts. When 30% grafts were maintained for 4 and 6 h in cold University of Wisconsin (UW) solution, survival rates decreased to 54 and 27%, respectively, whereas 2 h of preservation resulted in 100% survival (P < 0.05 for 30% graft with 4 and 6 h storage groups vs all other groups; generalized Wilcoxon's test). Rats receiving 40, 50 and 100% isografts all survived indefinitely, regardless of the duration of preservation (data not shown). Data from Urakami and Grisham.54

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image

Figure 2. Serum alanine aminotransferase (ALT) levels 24 h following liver transplantation. Serum ALT levels were significantly increased in the smaller graft groups. *P < 0.01 compared with others; P < 0.01 compared with 100% graft; P < 0.05 compared with 50% graft; §P < 0.05 compared with 100% graft. Data from Urakami and Grisham.54

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image

Figure 3. Serum total bilirubin 24 h following partial or whole liver transplantation and for different durations of cold preservation *P < 0.01 compared with 100% graft; P < 0.05 compared with 50% graft; P < 0.05 compared with 100% graft. Data from Urakami and Grisham.54

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These results are rather surprising given the extensive literature on leucocyte (i.e. neutrophil) infiltration in the postischaemic liver in rats and mice.62–66 The reasons for these differences are not clear; however, several possibilities exist. The one obvious difference between our model and the in situ liver I/R models in rats and mice is the use of warm ischaemia for varying lengths of time in the I/R models. It is known that, in rats, warm ischaemia activates the complement system and promotes the expression of a variety of different pro-inflammatory cytokines, chemokines and lipid mediators, some of which are derived from the Kupffer cells.67–69 These inflammatory mediators are thought to activate the microvascular endothelial cells to induce the expression of different endothelial cell adhesion molecules, thereby promoting leucocyte and platelet adhesion, extravasation and tissue injury. In our model of RSL transplantation, we subject the small-for-size livers to extended periods of cold ischaemia (2–6 h) followed by implantation and warm reperfusion. Surprisingly, a small but significant mononuclear, but not neutrophil, inflammatory infiltrate was observed histologically only in animals receiving 30% grafts that had been stored for 4 or 6 h in cold UW solution. These data imply that, in the absence of tissue rejection, implantation of grafts equal to or greater than 40% will not result in significant inflammation (or liver failure) even when storage times are extended to 6 h. Furthermore, these data suggest that most, if not all, of the liver injury and dysfunction occur by leucocyte-independent pathways. Obviously, when transplantations are performed using major histocompatibility complex (MHC) mismatched livers, acute and chronic liver rejection will ensue and a major portion of graft damage will occur by invading leucocytes (e.g. lymphocytes).

Another possible mechanism for enhanced graft failure and mortality in the 30% graft + 6 h storage group may be increased expression of potentially damaging cytokines, such as TNF-α. Indeed, we observed large increases in circulating levels of this pro-inflammatory mediator only in the 30% graft + 6 h group, in accordance with another study.70 A more likely mechanism may be that extending the duration of cold preservation decreases the regenerative capacity of the liver, resulting ultimately in liver failure and death. Selzner et al. reported that a short period (30 min) of cold preservation was associated with enhanced liver regeneration, whereas a prolonged time of cold preservation (10 and 16 h) was associated with a markedly decreased liver regeneration at 2 days after surgery in their rat 30% liver transplant model.1 In another study,54 we observed that when the preservation period was extended, liver regeneration was reduced at 7 days after RSL transplantation. In addition, the expression of interleukin (IL)-6, a cytokine known to be important in liver regeneration,71 was actually enhanced in the 30% graft + 4 h and 30% graft + 6 h groups ,which were both destined to fail.54 Obviously, these data do not exclude the possibility that liver regeneration in those animals with 30% grafts and 4 or 6 h of cold storage is compromised at later times following surgery.

In addition to duration of preservation in UW solution, we found that graft size was important for normal liver function and host survival. We observed 100% survival of all recipient rats that were implanted with 40, 50 or 100% (whole) livers, regardless of the duration of preservation (Fig. 1). Although we observed increased graft injury and dysfunction (as measured by increases in serum ALT and total bilirubin) in the animals receiving 40 and 50% grafts and extended periods of cold storage, the values for ALT and total bilirubin were substantially less than those recorded for rats receiving 30% grafts at all storage times tested (Figs 2,3). These data suggest that graft sizes of 40% or greater are sufficient to meet the metabolic demands of the recipients and that cold storage up to 6 h does not appear affect graft viability or survival of recipients in the absence of tissue rejection. These data agree with those of other investigators, who have demonstrated that most, if not all, rats receiving 40% isografts survive.53,72 Taken together, our data describe the relationship between graft size, duration of preservation, graft viability and host survival.

Role of reactive oxygen-derived free radicals in the pathophysiology of RSL transplantation

As mentioned previously, a number of different studies has demonstrated that ROS are important mediators of postischaemic liver injury in rodents.16,20,21,23,24,33 However, many of these studies used non-specific ‘anti-oxidants’ or ‘free radical scavengers’, which makes interpretation of their results problematic. In order to assess the role that superoxide plays in partial liver transplantation, we tested the effectiveness of Mn-SOD2/3 in our RSL transplantation model in rats.34,54 Briefly, 30% grafts were stored in cold UW solution containing 1 U/mL Mn-SOD2/3 for 6 h and recipients were injected 1000 U/kg, i.v., Mn-SOD2/3 immediately before harvesting the recipient liver. Surprisingly, we found that all recipients treated with Mn-SOD2/3 died within 3 days following transplantation, indicating that the SOD2/3 fusion protein did not prolong host survival.54 In addition to SOD2/3, we tested the efficacy of α-lipoic acid (LA), a naturally occurring dithiol compound that is known to be an essential anti-oxidant as well as a cofactor for mitochondrial bioenergetic enzymes and is known to attenuate I/R-induced liver injury in rats.73,74 We treated rats with LA (100 mg/kg, i.p.) 24 h prior to surgery and grafts were stored in cold UW solution containing 0.1 mg/mL LA for 6 h before implantation. In addition, recipients received LA (100 mg/kg, i.p.) daily for 3 days following transplantation. Again, we found that this treatment protocol provided no protection to the rats transplanted with a 30% graft compared with their vehicle controls.54 In a similar set of experiments, we assessed the role of the NO donor S-nitrosoglutathione (GSNO; 200 µmol/L) added to the UW solution for the 6 h storage period. We found that GSNO did not prevent the mortality incurred by rats implanted with 30% grafts compared with their GSH controls.54 Taken together, these data suggest that neither superoxide nor NO modulate RSL transplantation-induced liver injury and recipient mortality.

CONCLUSIONS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES

In conclusion, our data demonstrate that, in the absence of acute and/or chronic tissue rejection, transplantation of small-for-size livers (< 40%) that have been stored for periods longer than 2 h results in significant liver injury and dysfunction, as well as increased mortality of the recipients. In contrast with studies performed with warm RSL + I/R, tissue injury and recipient mortality do not appear to correlate with leucocyte infiltration, nor do they appear to be modulated by superoxide or NO. Taken together, our data have important implications for the different types of RSL transplantations currently being performed in several different institutions. First, graft size would appear to be a more critical consideration for SLT than for LDLT because grafts for SLT generally require several hours of cold ischaemia/storage time (8–12 h) because of the transportation times needed for cadaveric or brain-dead grafts.75–77 Because the preservation times for living donor transplants are generally much shorter (1–2 h) owing to the proximity of donor and recipient in the operating room, the findings suggest that smaller grafts may be adequate in adult-to-adult or adult-to-child LDLT surgeries provided the metabolic demands of the recipient are met by the implanted liver.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. REDUCED-SIZE LIVER COMBINED WITH I/R AS A MODEL FOR PARTIAL LIVER TRANSPLANTATION
  5. ROLE OF SUPEROXIDE IN RSL + I/R-INDUCED LIVER INJURY
  6. NITRIC OXIDE AND RSL + I/R INJURY
  7. REDUCED-SIZE LIVER TRANSPLANTATION: CAN WE APPLY THE SAME RULES?
  8. CONCLUSIONS
  9. ACKNOWLEDGEMENT
  10. REFERENCES
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