Value of α glutathione S‐transferase for in vitro evaluation of preservation injury in normal and steatotic livers

Abstract Liver steatosis is frequently encountered at organ harvest and, although functionally in‐apparent in the donor, may seriously affect the functional recovery of the graft after ischemic preservation. The present study was aimed to investigate the diagnostic value of alpha‐glutathione S‐transferase (GST) in non‐schemic and ischemic livers with or without compensated steatosis. A histologically documented mild to moderate steatosis was induced in livers of male Wistar rats by fasting for 2 days and subsequent feeding of a fat‐free diet enriched in carbohydrates. Fatty livers (FL) were retrieved and perfused in vitro for 45 min either immediately or after ischemic preservation at 4°C in HTK solution. Effluate was collected during isolated perfusion and later analysed for liver specific enzymes, including GST. Normal livers (NL) were excised from healthy rats and underwent the same protocol. Non‐ischemic livers showed similar enzyme release (FL versus NL) for ALT or GLDH but significant differences in GST. After ischemic preservation of NL, enzyme release increased mildly with respect to the non‐ischemic reference values for ALT, remained unchanged for GLDH and rose substantially for GST. In FL, there was a more than 10‐fold increase in all parameters, being most pronounced for GLDH as a marker of mitochondrial damage. It is concluded that GST may discriminate between healthy and suboptimal steatotic livers prior to ischemia and that the release of GST upon postischemic reperfusion of normal livers proves to be the most sensitive indicator for hepatocellular injury. However, GST turned out to be less useful for the detection of postischemic reperfusion injury in steatotic grafts.     

Safe use of highly steatotic livers by utilizing a donor/recipient clinical algorithm

The aim of this study was to assess the long‐term safety and clinical outcomes associated with the utilization of highly steatotic donor livers utilizing a specific donor/recipient matching algorithm. This was a prospective, observational, single‐center, 10‐yr follow‐up study. Highly steatotic livers were utilized according to a donor/recipient algorithm that guided the surgeon to use highly steatotic donor organs judiciously in low‐risk recipients. This study initially compared fat assessment based on frozen‐section Ehrlich's hematoxylin and eosin (H&E) to reperfusion biopsy fat assessment and demonstrated that H&E is an insensitive analysis to determine degree of steatosis. Patients were divided into three groups based on donor steatosis (group 1: <30% steatosis, group 2: 30–60% steatosis, group 3: >60% steatosis), and clinical outcomes were assessed. One hundred and sixteen patients were included in the analysis. Patients that received severely steatotic livers (>60% fat) showed increased reperfusion liver injury and delayed return of liver function in the early postoperative period, demonstrated by biochemical markers. However, there were no differences in primary non‐function, postoperative complications, length of stay, and patient and graft survival. Using rigorous donor/recipient matching through a detailed algorithm, these data demonstrate that normal liver allograft outcomes are not superior to those in highly steatotic grafts.     

The effects of vascular bed expansion in steatotic rat liver graft viability

Disturbed microcirculation caused by fat accumulation in hepatocytes has been implicated in poor graft preservation and reperfusion. The aim of this study was to investigate the effect of vascular bed expansion (VBE) during cold preservation in graft survival Moderate liver steatosis in male Wistar rats (240–280 g) was induced by choline-deficient diet. Normal, steatotic or VBE-pretreated steatotic grafts were transplanted after 1 h or 9 h of cold preservation. Graft viability was determined by 7-day survival, serum liver enzymes, plasma tumour necrosis factor (TNF)-, interleukin (IL)-6, and malondialdehyde (MDA) levels. Post-reperfusion bile flow and liver histology were also examined. After 9 h of preservation, VBE-pretreated steatotic liver grafts were associated with significantly reduced serum liver enzyme, plasma TNF-, IL-6, and MDA levels, as well as increased bile flow and higher survival rates compared with untreated ones. The present study shows that VBE protects fatty liver grafts from subsequent long-term cold preservation and reperfusion injury in a rat liver transplantation model.     

Warm ischemia and reperfusion injury in diet-induced canine fatty livers

BACKGROUND: Fatty liver is associated with primary nonfunction after liver transplantation, contributing a shortage of suitable liver grafts. Because extensive investigation of mechanisms underlying such nonfunction has been limited largely to rodents, we made a new fatty liver model in dogs and studied primary nonfunction after warm ischemia. METHODS: We developed a diet rich in fat but deficient in choline to induce fatty change in canine liver and investigated effects of 60 min of warm ischemia and reperfusion in dogs with such fatty livers. RESULTS: Microscopically evident steatosis increased with duration of dietary manipulation (up to 12 weeks), as did hepatic total lipid and triglyceride levels. No dog with >30% of steatotic hepatocytes, >445 mg/g hepatic total lipid or >145 mg/g hepatic triglyceride survived after 60 min of warm ischemia. Arterial ketone body ratios decreased and blood endotoxin increased after reperfusion in nonsurvivors. The main histologic finding in livers of nonsurvivors was marked sinusoidal congestion. CONCLUSIONS: Damage to hepatocytes and nonparenchymal cells after warm ischemia and reperfusion was thought to be closely related to sinusoidal microcirculatory disturbances in fatty livers. The canine fatty liver model reported here may be useful in studying the pathology of primary nonfunction and in establishing criteria for allowable degrees of fatty change in potential liver grafts.     

Micro- and macrovesicular steatotic liver model for transplantation induced by ethanol and protein-deficient diet

Steatotic liver grafts are associated with a high incidence of primary nonfunction and initial poor function. Due to the increasing number of liver transplant candidates, centers are inclined to accept marginal donors more frequently. For a lack of a reliable fatty liver model, preservation concepts for fatty livers have hardly been evaluated. Moreover, there is an ongoing debate on the relevance and impact of micro- versus macrovesicular steatotic organs. We therefore intended to establish a steatotic liver model in pigs comprising both micro- and macrovesicular steatotic livers. Five groups of pigs received daily 1 to 6 g ethanol/kg body weight and/or a protein-deficient diet for up to 72 days. Liver biopsy was carried out at days 24, 48, and 72. With an increasing amount and duration of ethanol intake, higher levels of microvesicular steatosis were induced. Ethanol and protein deficient diet resulted in more than 60% microvesicular steatosis after 72 days. Exclusively protein-deficient diet without ethanol induced macrovesicular steatosis of more than 70% after 72 days. For the first time, we established a porcine model of hepatic steatosis that comprises both histologic types of fatty liver: micro- and macrovesicular steatosis induced by ethanol and a protein-deficient diet. We would like to conclude that our model is particularly qualified to study new concepts of preservation for steatotic livers to improve on the posttransplant outcome.     

Elimination of Kupffer cells and nafamostat mesilate rinse prevent reperfusion injury in liver grafts from agonal non-heart-beating donors.

BACKGROUND: We hypothesized that microcirculatory disturbance was an obstacle to liver transplantation (LTx) from non-heart-beating donors (NHBDs) and that it was attributed mainly to a deterioration of sinusoidal endothelial cells (SECs) and sinusoidal narrowing. This study was designed to examine porcine orthotopic LTx using livers obtained from pretreated agonal NHBDs, and to determine whether the maintenance of the liver microcirculation would result in successful LTx from agonal NHBDs. METHODS: Pigs were allocated to five groups: (i) control group; (ii) NM group, in which grafts were rinsed with nafamostat mesilate (NM) rinse; (iii) LD group, in which Kupffer cells in grafts were eliminated by liposome-encapsulated dichloromethylene diphosphonate (L-DMDP); (iv) LDNM group, in which grafts pretreated with L-DMDP were rinsed with NM rinse; (v) heart-beating donor (HBD) group. In all groups, but the HBD group, the livers were pretreated with FK506 and prostaglandin I2 analogue, and were preserved in University of Wisconsin solution after cardiac arrest. Thereafter orthotopic LTx was performed. RESULTS: After reperfusion, it was histologically demonstrated that elimination of Kupffer cells prevented SECs deterioration and NM rinse prevented sinusoidal narrowing. The hepatic energy charge recovered in all groups except the control group. In the LDNM group, three of four recipients survived more than 7 days. CONCLUSIONS: For a successful LTx from agonal NHBDs, it is important to prevent microcirculatory disturbance caused by SEC deterioration and sinusoidal narrowing after reperfusion. Combination therapy consisting in the elimination of Kupffer cells and NM rinse prevented primary graft non-function in liver grafts from agonal NHBDs.     

Protection of sinusoidal endothelial cells against storage/reperfusion injury by prostaglandin E2 derived from Kupffer cells.

BACKGROUND: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats. METHODS: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections. RESULTS: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants. CONCLUSION: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.     

Synergistic effects of brain death and liver steatosis on the hepatic microcirculation

BACKGROUND: The routine transplantation of steatotic livers could potentially mitigate the donor shortage, but so far is associated with a high rate of graft dysfunction. Steatosis and brain death have been perceived as independent risk factors, but they may synergistically target the hepatic microcirculation. This study compares the effects of brain death on the microcirculation of steatotic and normal livers. METHODS: Brain death was induced in obese and lean Zucker rats. Lean and obese sham-operated animals served as controls. Liver microcirculation was investigated using intravital fluorescence microscopy. Serum liver enzyme and reduced glutathione, expression of P-selectin, ICAM-1 and VCAM-1 mRNA in the liver were determined. The ultrastructural alterations were compared by electron microscopy. RESULTS: In nonbrain-dead animals, liver steatosis was associated with smaller sinusoidal diameters, but did not impair sinusoidal perfusion. During brain death, sinusoidal diameter and perfusion were reduced in normal and, to a greater extent, in steatotic livers. Also, more leukocytes were recruited to the microvasculature of steatotic livers than to normal livers in brain-dead state. The highest liver enzyme activities and the lowest hepatic GSH concentrations were measured in brain-dead animals with steatotic livers; only in these organs was endothelial cell swelling regularly observed. In brain-dead state, only the P-selectin mRNA expression was increased in steatotic livers as compared to normal livers. CONCLUSIONS: Brain death amplifies the adverse effects of steatosis on the hepatic microcirculation. Our results underline the need for therapeutic intervention in brain-dead state when steatotic livers are to be used for transplantation.     

Relevance of epidermal growth factor to improve steatotic liver preservation in IGL-1 solution

Aim

Static preservation solution is critical for liver graft outcomes, especially when steatosis is present. Institut Georges Lopez (IGL)-1 solution protects fatty livers effectively against cold ischemia reperfusion injury. Its benefits are mediated by nitric oxide and prevention of oxidative stress. The supplementation of IGL-1 with epidermal growth factor (EGF) enhances steatotic graft preservation by increasing adenosine triphosphate content, thereby mitigating oxidative stress and mitochondrial damage.

Methods

After steatotic livers were preserved for 24 hours in IGL-1 solution with or without EGF supplements, they were perfused ex vivo for 2 hours at 37°C. The benefits of EGF were assessed by evidences of hepatic damage and function—transaminases, bile production, and flow rate—as well as by other factors presumably associated with the poor tolerance of fatty livers toward cold ischemia-reperfusion injury (IRI)—energy metabolism, mitochondrial damage, oxidative stress, eNOS activity and proinflammatory interleukin (IL) beta content.

Results

Steatotic livers preserved in IGL-1 solutions supplemented with EGF (10 μg/L) showed lower transaminase levels, greater bile production, and ameliorated flow rates when compared to IGL-1 alone. In addition, energy metabolism deterioration, mitochondrial damage, oxidative stress, and cytokine IL-1 beta release were prevented.

Conclusion

EGF addition to IGL-1 increased fatty liver graft preservation, thereby reducing steatotic liver damage against cold IRI.     

Correlation between von Willebrand factor levels and early graft function in clinical liver transplantation

Cold preservation/reperfusion leads to sinusoidal endothelial cell (SEC) activation and damage in nearly every liver transplantation; the extent of these changes influences early graft function. Upon reperfusion, activated SEC show increased expression of adhesion molecules, including von Willebrand factor (vWF) which is released into the circulation. This study was designed to evaluate the levels of vWF measured in the caval effluent and correlate these findings with known markers of SEC damage and early graft function. Data were obtained from 35 patients undergoing orthotopic liver transplantation (LTx). Two samples were taken from each patient for measurement of vWF: a) from the portal vein immediately prior to reperfusion; and b) from the first 50 ml of the caval effluent. Commercial assays were used to measure vWF, as well as hyaluronic acid (HA), thrombomodulin (TM), IL-1β, IL-6, IL-8 and TNF-α. Patients were divided into two groups based on early graft function. Poor early graft function (PEGF) was defined as a peak aspartate transaminase (AST) or alanine transaminase (ALT) level> 2500 U/L during the first three postoperative days (POD) and a prothrombin time (PT)> 16 s on POD 2 (n=8). The remaining 27 patients had good early graft function (GEGF). In patients with GEGF, vWF levels dropped significantly between the two time points. This change was not observed in those with PEGF. A positive linear correlation was observed in the PEGF group between vWF and HA and IL-6. The different pattern of change in vWF between the two groups, as well as the positive correlation between HA, IL-6 and vWF in PEGF, suggest that vWF may be a useful marker of early graft function.     

Mild steatosis impairs functional recovery after liver resection in an experimental model

BACKGROUND: Mild steatosis has been thought not to affect outcome after liver resection. However, recent studies have reported impaired postoperative recovery of patients with mild steatosis. This study evaluated the recovery of hepatic functional reserve during regeneration in a rat model of mild steatosis and liver resection. METHODS: Male Wistar rats had a standard methione- and choline-deficient diet to induce mild steatosis before 70 per cent liver resection. Evaluation of hepatobiliary function was by (99m)Tc-labelled mebrofenin scintigraphy. Mebrofenin uptake rate, the time for maximum uptake (T peak) and the time required for peak activity to decrease by 50 per cent (T(1/2) peak) were assessed 1, 2, 3 and 7 days after liver resection, along with regeneration of the remnant liver, hepatocellular and sinusoidal damage, and hepatic adenosine 5'-triphosphate (ATP) levels. RESULTS: Liver regeneration and proliferative response in mild steatotic rats were no different from those in controls. However, the mebrofenin uptake rate was lower (P < 0.050) and the recovery of hepatic ATP impaired (P < 0.050) in animals with mild steatosis. Hepatocellular damage was increased (P < 0.050) but sinusoidal endothelial cell function was not affected after liver resection in mildly steatotic rats. CONCLUSION: Mild steatosis impaired functional recovery and increased hepatocellular damage after liver resection.     

Impact of stable PGI₂ analog iloprost on early graft viability after liver transplantation: a pilot study

Bärthel E, Rauchfuß F, Hoyer H, Habrecht O, Jandt K, Götz M, Voigt R, Heise M, Marx G, Settmacher U. Impact of stable PGI₂ analog iloprost on early graft viability after liver transplantation: a pilot study.
Clin Transplant 2012: 26: E38–E47.
© 2011 John Wiley & Sons A/S. Abstract: Background: Ischemia/reperfusion injury after liver transplantation (LT) may be associated with primary graft dysfunction (PDF) or non‐function. Prostaglandins were demonstrated to be beneficial in reducing ischemic injury by improving microcirculation and protecting endothelial cells. The aim of this study was to analyze the effect of the continuously administered prostaglandin I₂ analog iloprost on allograft function after LT. Methods: Eighty patients were prospectively randomized and assigned to two groups. Patients in the treatment group received iloprost for seven d after transplantation, and those in the control group did not. The primary end point was graft dysfunction. Results: The incidence of PDF was 20% (n = 8) in the control group and 5% (n = 2) in the treatment group, respectively (p = 0.087). Four patients in the control group underwent re‐transplantation for initial non‐function (INF). There was no evidence for INF in the treatment group. Iloprost was associated with improved allograft function. Clinical course and outcome were comparable. Conclusions: We suggest iloprost to be beneficial for early post‐transplant liver function. If the rate of PDF can be significantly reduced with this treatment concept, it should be analyzed in a larger number of patients (ISRCTN95672167).     

Assignment of steatotic livers by the Mayo model for end-stage liver disease

Prognosis after liver transplantation depends on a combination of recipient and donor variables. The purpose of this study is to define an allocation system of steatotic donor livers relative to recipient model for end-stage liver disease (MELD) score. We reviewed 500 consecutive OLT, computing the MELD score for each recipient. Fatty infiltration in grafts was categorized in no steatosis, 10-30%, 30-60% and > or = 60% steatosis. MELD score did not affect preservation injury and graft dysfunction, which were increased with fat content. Recipient and graft survivals lowered when increasing MELD score. Outcome in low-risk recipients (MELD < or = 9) was not altered with steatosis, except those with > or = 60%. Survival functions in moderate-risk recipients (MELD 10-19) were moderately affected with 10-30% steatosis and severely with those with >30. Exactly 30-60% steatotic grafts work poorly in high-risk recipients (MELD > or = 20), and very poorly with > or = 60% steatosis. Prognosis of candidates is optimally influenced when divergence of recipient-donor risks is presented.     

Ischemia–reperfusion injury in patients with fatty liver and the clinical impact of steatotic liver on hepatic surgery

Hepatic steatosis is one of the most common hepatic disorders in developed countries. The epidemic of obesity in developed countries has increased with its attendant complications, including metabolic syndrome and non-alcoholic fatty liver disease. Steatotic livers are particularly vulnerable to ischemia/reperfusion injury, resulting in an increased risk of postoperative morbidity and mortality after liver surgery, including liver transplantation. There is growing understanding of the molecular and cellular mechanisms and therapeutic approaches for treating ischemia/reperfusion injury in patients with steatotic livers. This review discusses the mechanisms underlying the susceptibility of steatotic livers to ischemia/reperfusion injuries, such as mitochondrial dysfunction and signal transduction alterations, and summarizes the clinical impact of steatotic livers in the setting of hepatic resection and liver transplantation. This review also describes potential therapeutic approaches, such as ischemic and pharmacological preconditioning, to prevent ischemia/reperfusion injury in patients with steatotic livers. Other approaches, including machine perfusion, are also under clinical investigation; however, many pharmacological approaches developed through basic research are not yet suitable for clinical application.     

Sinusoidal microcirculatory changes after small‐for‐size liver transplantation in rats

Small‐for‐size graft injury is characterized by portal venous hypertension and loss of intracellular homeostasis early after transplant. The long‐term alteration of sinusoidal microcirculatory hemodynamic state remains unknown. A syngeneic rat orthotopic liver transplantation model was developed using small‐for‐size grafts (35% of recipient liver weight) or whole grafts (100% of recipient liver weight). Graft survival, portal pressure, liver function, hepatocellular apoptosis as well as morphological changes (by light microscopy and electron microscopy) were assessed. Sinusoidal microcirculatory hemodynamics was examined by intravital fluorescence microscopy. Although portal hypertension lasted only for 1 h after performance of small‐for‐size liver transplantation, a sustained microcirculatory disturbance was accompanied by dramatic reduction of sinusoidal perfusion rate, elevation of sinusoidal diameter as well as increase in the number of apoptotic hepatocytes during the first 7 days. These resulted in lower survival rate (50% vs. 100%, P = 0.012), higher level of liver function, and more severe morphological changes, which could induce small‐for‐size syndrome. In conclusion, persistent microcirculatory hemodynamic derangement during the first 7 days after reperfusion as well as transient portal hypertension is significant manifestation after small‐for‐size liver transplantation. Long‐term microcirculation disturbance displayed as decrease of sinusoidal reperfusion area and increase of spread in functional liver mass seems to be the key factor for graft injuries.     

Preservation of steatotic livers: a comparison between cold storage and machine perfusion preservation.

Liver grafts are frequently discarded due to steatosis. Steatotic livers can be classified as suboptimal and deteriorate rapidly during hypothermic static preservation, often resulting in graft nonfunction. Hypothermic machine perfusion (MP) has been introduced for preservation of donor livers instead of cold storage (CS), resulting in superior preservation outcomes. The aim of this study was to compare CS and MP for preservation of the steatotic donor rat liver. Liver steatosis was induced in male Wistar rats by a choline-methionine-deficient diet. After 24 hours hypothermic CS using the University of Wisconsin solution (UW) or MP using UW-Gluconate (UW-G), liver damage (liver enzymes, perfusate flow, and hyaluronic acid clearance) and liver function (bile production, ammonia clearance, urea production, oxygen consumption, adenosine triphosphate [ATP] levels) were assessed in an isolated perfused rat liver model. Furthermore, liver biopsies were visualized by hematoxylin and eosin staining. Animals developed 30 to 60% steatosis. Livers preserved by CS sustained significantly more damage as compared to MP. Bile production, ammonia clearance, urea production, oxygen consumption, and ATP levels were significantly higher after MP as compared to CS. These results were confirmed by histology. In conclusion, MP improves preservation results of the steatotic rat liver, as compared to CS.     

Quantification of Degree of Steatosis in Extended Criteria Donor Grafts With Standardized Histologic Techniques: Implications for Graft Survival

The gap between the availability of livers from organ donors and the increased demand has led many centers to apply strategies to reduce this deficit. Splitting of cadaveric organs for use in 2 recipients; domino transplantation; and organs from living donors, non-heart-beating donors, and extended-criteria donors (ECDs) are all currently used in orthotopic liver transplantation (OLT). Fatty changes in the donor liver are a risk factor for poor function after OLT; however, the presence of steatosis, frequently present in livers from ECDs, does not exclude the use of these organs. Since January 2000 at our institution, we observed 39 steatotic grafts that were stratified istologically as follows: low steatosis, 5% to 15%; mild steatosis, 16% to 30%; moderate steatosis, 31% to 60%; and severe steatosis (>60%). Histologic techniques can enable identification of the type of fatty change as macrovesicular and microvesicular. These alterations have different effects on primary nonfunction and primary dysfunction. Fifteen grafts, all with severe or moderate, macrovesicular changes were discarded. Twenty-four fatty grafts with low to moderate steatosis were utilized for transplant. Sections from 2 liver biopsies (1 wedge in the left lobe and 1 needle in the right lobe) were stained with hematoxylin-eosin, Masson trichrome, Gomori reticulin, and oil red O. The OLT was performed only in patients with a MELD (Model for End-Stage Liver Disease) score lower than 27. The rate of primary dysfunction was 12.5%, and of primary nonfunction 8.4%. The 6-month graft survival for all fatty livers was 80%. We encourage the careful use of grafts with low to moderate steatosis in recipients without additional risks.     

Effect of ischemia-reperfusion injury on the microcirculation of the steatotic liver of the Zucker rat.

BACKGROUND: Much discussion has been focused on the use of steatotic livers for transplantation due to the prevalence of steatosis in the potential donor liver pool (1). The aim of this study was to investigate the possibility that the microcirculation of steatotic liver is more sensitive to the ischemia-reperfusion (IR) injury than normal liver. METHODS: The left liver lobe of obese (n=9) and lean Zucker rats (n=9) were subjected to 40 min of warm ischemia followed by 60 min of reperfusion. Fluorescent probes rhodamine 123 (Rh123), bisbenzimide (Bis), and rhodamine 6G (Rh6G) were administered for the identification by intravital fluorescence microscopy (IVFM) of mitochondrial membrane potential, hepatocyte nuclei and leukocytes, respectively before hepatic ischemia and at 15, 30, 45, and 60 min after reperfusion. Blood samples were obtained before and after 60 min of reperfusion. Liver tissue was taken at the end of experiment for histological analysis. RESULTS: The liver of the obese rats showed prominent macro- and microvesicular fatty changes (MAFC and MIFC) and hepatocyte swelling. Under IVFM, the obese animals had significantly wider hepatic cords (23.1+/-0.8 microm) than the lean ones (15.9+/-0.5 microm) (P<0.01), whereas no significant difference in sinusoidal diameters was noted. The number of functional sinusoids significantly decreased after 30 min of reperfusion in both groups but no significant change was noted in the nucleus count throughout the experiment. Rh123 fluorescence intensity dropped significantly in the obese group after 60 min of reperfusion but not in the lean rats. Leukocyte adherence showed a significant rise after reperfusion in both groups. Plasma AST and ALT levels were 40- and 24-fold higher respectively for the obese animals after IR compared with their preischemic values, whereas the corresponding increase were 4.2- and 3.4-fold for the lean animals, respectively. CONCLUSIONS: Our results indicate that the liver of the obese Zucker rat is steatotic and presents with an abnormal microcirculation manifested by a reduced sinusoidal density. IR led to significantly greater hepatic injury in the steatotic than in the normal liver. This injury was accompanied by a significant reduction in the functional sinusoidal density and mitochondrial membrane potential as assessed by Rh123-associated fluorescence in the steatotic liver. In conclusion, the increased sensitivity of the steatotic liver to IR injury would appear to involve both alterations in blood flow in the microcirculation and to cellular changes.     

Tauroursodeoxycholic Acid Affects PPARγ and TLR4 in Steatotic Liver Transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia‐reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator‐activated receptor‐γ (PPARγ) and damage. When PPARγ was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPARγ, and the PPARγ antagonist treatment up‐regulated toll‐like receptor 4 (TLR4), specifically the TIR domain‐containing adaptor inducing IFNβ (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPARγ antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPARγ and this in turn up‐regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating‐based strategies could reduce the inherent risk of steatotic liver failure after transplantation.     

Use of severely steatotic grafts in liver transplantation: a matched case-control study

BACKGROUND: Although there is a worldwide need to expand the pool of available liver grafts, cadaveric livers with severe steatosis (>60%) are discarded for orthotopic liver transplantation (OLT) by most centers. METHODS: We analyzed patients receiving liver grafts with severe steatosis between January 2002 and September 2006. These patients were matched 1:2 with control patients without severe steatosis according to status the waiting list, recipient age, recipient body mass index (BMI), and model for end-stage liver disease (MELD) score. Primary end points were the incidence of primary graft nonfunction (PNF), and graft and patient survival. Secondary end points included primary graft dysfunction (PDF), the incidence of postoperative complications, and histologic assessment of steatosis in follow-up biopsies. We also conducted a survey on the use of grafts with severe steatosis among leading European liver transplant centers. RESULTS: During the study period, 62 patients dropped out of the waiting list and 45 of them died due to progression of disease. Of 118 patients who received transplants 20 (17%) received a graft with severe steatosis during this period. The median degree of total liver steatosis was 90% (R = 65%-100%) for the steatotic group. The steatotic (n = 20) and matched control group (n = 40) were comparable in terms of recipient age, BMI, MELD score, and cold ischemia time. The steatotic group had a significantly higher rate of PDF and/or renal failure. Although the median intensive care unit (ICU) and hospital stay were not significantly different between both groups, the proportion of patients with long-term ICU (> or =21 days) and hospital (> or =40 days) stay was significantly higher for patients with a severely steatotic graft. Sixty-day mortality (5% vs. 5%) and 3-year patient survival rate (83% vs. 84%) were comparable between the control and severe steatosis group. Postoperative histologic assessment demonstrated that the median total amount of liver steatosis decreased significantly (median: 90% to 15%, P < 0.001). Our survey showed that all but one of the European centers currently reject liver grafts with severe steatosis for any recipient. CONCLUSION: Due to the urgent need of liver grafts, severely steatotic grafts should be no longer discarded for OLT. Maximal effort must be spent when dealing with these high-risk organs but the use of severely steatotic grafts may save the lives of many patients who would die on the waiting list.