Non-heart-beating Donor Livers Research Articles

Induction of apoptosis in non-heart-beating donor livers by peroxynitrite

Objective To investigate the role of peroxynitrite in induction of apoptosis in non-heart-beating donor (NHBD) liver grafts. Methods Rat livers were harvested and perfused via the portal vein with 60 ml of cold 4℃ HTK solution from heart beating donors (HB) or 60 min after car-diac arrest from NHBD. A third group consisted of NHBD livers which were perfused with HTK con-taining 7500 IU of superoxide dismutase (SOD) (n=6, resp.). After cold storage for 24 h, the func-tional integrity of the liver was evaluated by isolated reperfusion with Krebs-Henseleit Buffer in a re-circulating system at 37℃ for 45 min. Results Compared to the NHBD livers, HB and SOD groups were characterized by a significantly reduced hepatic enzyme leakage(P<0. 05) as well as decreased portal venous pressure(P<0.01). Bile production upon reperfusion was increased significantly (P< 0.01). Prominent decreasing nitrite oxide(NO) (P<0.05), nitrotyrosine(P<0.01, P<0.05) and apoptosis(P<0.05) in effluent perfusate was encountered. Slight nitrotyrosine staining was concen-trated on the endothelial cells. Conclusion Peroxynitrite may play a fundamental role in the apoptosis caused by enhanced oxygen free radical production in NHBD livers. Key words: Apoptosis; Non-heart-beating donor; Peroxynitrite

Exogenous Superoxide Dismutase Prevents Peroxynitrite-Induced Apoptosis in Non-Heart-Beating Donor Livers

Objective: To investigate the role of oxygen free radicals in the induction of apoptosis in non-heart-beating donor (NHBD) livers, and if superoxide dismutase (SOD) ameliorates these alterations. Methods: Rat livers were perfused via the portal vein with histidine/tryptophan/α-ketoglutarate solution from heart-beating donors (HBD) or 60-min warm ischemia from NHBD, with or without the addition of SOD. After 24 h, cold storage livers were evaluated by isolated reperfusion. Results: NHBD showed significantly higher enzyme leakage and elevated portal venous pressure (PVP) versus HBD. Bile and total adenine nucleotides (TAN) were significantly decreased. Apoptosis was prominent in sinusoidal lining cells, coupled with strong nitrotyrosine staining (NTR). The concentrations of nitric oxide and lipoperoxides were largely increased. SOD medication reduced hepatic enzyme release by 30% and lipoperoxides by nearly 50%. Apoptosis and NTR were significantly decreased, and PVP was strikingly reduced to normal values. A 3-fold enhancement in bile production and 1.5-fold increase in TAN of the liver tissue were also observed. Conclusion: NHBD livers are prone to severe reoxygenation injury promoted by oxygen free radicals, massive nitrite oxide production and peroxynitrite-induced apoptosis within the sinusoids. Antioxidant medication with SOD should be considered as a useful means of preserving NHBD livers.

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

Preservation of non-heart-beating donor livers in extracorporeal liver perfusion and histidine-trytophan-ketoglutarate solution

To compare the preservation of non-heart-beating donor (NHBD) livers in cold histidine-trytophan-ketoglutarate (HTK) solution and extracorporeal liver perfusion (ECLP). Livers harvested from healthy pigs were stored for 10 h in cold HTK solution (group A, n = 4) or perfused with oxygenated autologous blood at body temperature (group B, n = 4). Both groups were then tested on the circuit for 4 h. Bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of extracorporeal livers were tested in each group. Liver tissues from each group were examined at the end of reperfusion. At 1, 2, 3 and 4 h after reperfusion, bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of livers in group A were statistically different from those in group B (P < 0.05 or P < 0.01). ECLP is better than HTK solution to preserve NHBD livers. ECLP can assess the graft viability before liver transplantation.

Novel short-term hypothermic oxygenated perfusion (HOPE) system prevents injury in rat liver graft from non-heart beating donor.

To assess a machine perfusion system in rescuing liver grafts from non-heart-beating donors (NHBD). The introduction of extracorporeal liver perfusion systems in the clinical routine depends on feasibility. Conceivably, perfusion could be performed during recipient preparation. We investigated whether a novel rat liver machine perfusion applied after in situ ischemia and cold storage can rescue NHBD liver grafts. We induced cardiac arrest in male Brown Norway rats by phrenotomy and ligation of the subcardial aorta. We studied 2 experimental groups: 45 minutes of warm in situ ischemia + 5 hours cold storage versus 45 minutes of warm in situ ischemia + 5 hours cold storage followed by 1 hour hypothermic oxygenated extracorporeal perfusion (HOPE). In both groups, livers were reperfused in a closed sanguineous isolated liver perfusion device for 3 hours at 37 degrees C. To test the benefit of HOPE on survival, we performed orthotopic liver transplantation in both experimental groups. After cold storage and reperfusion, NHBD livers showed necrosis of hepatocytes, increased release of AST, and decreased bile flow. HOPE improved NHBD livers significantly with a reduction of necrosis, less AST release, and increased bile flow. ATP was severely depleted in cold-stored NHBD livers but restored in livers treated by HOPE. After orthotopic liver transplantation, grafts treated by HOPE demonstrated a significant extension on animal survival. We demonstrate a beneficial effect of HOPE by preventing reperfusion injury in a clinically relevant NHBD model.

Predisposing Factors for Biliary Complications Following Liver Transplantation

Introduction Biliary complications remain a major cause of morbidity and mortality in patients following liver transplantation. We sought to identify possible risk factors predisposing to biliary complications after OLT using duct-to-duct biliary reconstruction. Materials and Methods We retrospectively reviewed 5 years of prospectively collected donor and recipient data between April 1999 and April 2004. We evaluated the presence of biliary complications, donor and recipient age, cold ischemia time, hepatic artery thrombosis, non-heart-beating donor (NHBD), and graft steatosis (>30%). The results were compared with a control group of OLT patients without biliary complications. Results Among 173 OLT recipients, biliary complications occurred in 28 patients (16.2%), of whom 12 were leaks, 15 strictures, and 1 a nonanastomotic intrahepatic stricture. The mortality following biliary complications was 11%, compared to 6% in the control group. Conclusion Biliary complications remain a persistent problem in OLT. Analysis of risk factors identified hepatic artery thrombosis and steatosis as predisposing factors. With greater experience, NHBD livers may also prove to be at greater risk of biliary complications.

Wash‐out of the non‐heart‐beating donor liver: a matter of flush solution and temperature?

Ischemically damaged donor livers are prone to graft non-function. This can in part be explained by a suboptimal wash-out during procurement. An enriched machine perfusion (MP) preservation solution for livers, named Polysol, was developed. The aim of this study was to investigate the type of flush solution, temperature and anticoagulant content on the wash-out of the non-heart-beating donor (NHBD) rat liver. Rat livers were flushed after 30 min warm ischemia. After excision, livers were reperfused at 37 degrees C, with analysis of damage and function, concerning (1) solutions (University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK) and Polysol); (2) temperature (4 degrees C, 18 degrees C and 37 degrees C); (3) addition of heparin and (4) wash-out followed by 24 h MP. (1) Reperfusion results were inferior in the UW group; (2) less damage and improved function were seen after wash-out using Polysol at 37 degrees C; (3) No effects were seen of the addition of heparin to Polysol; (4) MP after wash-out using HTK resulted in more liver damage and decreased liver function as compared with wash-out using Polysol. Polysol is applicable as a flush solution for the NHBD liver, resulting in equal to better wash-out as compared with UW and HTK. The best temperature for this NHBD wash-out is 37 degrees C.

Reduced Liver Apoptosis After Venous Systemic Oxygen Persufflation in Non-Heart-Beating Donors

Graft injury caused by warm ischemia in livers from non-heart-beating donors (NHBDs) strongly affects posttransplantation outcome and is associated with liver apoptosis, which is mediated by death receptors, such as Fas, a surface receptor of the tumor necrosis factor (TNF)-α family. The aim of this study was to test the ability of venous systemic oxygen persufflation (VSOP) to reduce apoptotic changes and Fas activation in the liver after warm ischemic insult in vivo. Livers of male Wistar rats were harvested 30 min after cardiac arrest from non-heart-beating donors (NHBD) with (NHBD + O2) or without (NHBD) application of gaseous oxygen during the cold storage period via the suprahepatic caval vein. After 24 h of storage in University of Wisconsin solution at 4°C, viability of the livers was assessed upon isolated reperfusion in vitro. Conventional signs of tissue damage like enzyme release and bile production showed a significantly elevated nonspecific cell injury in the NHBD group. TUNEL staining revealed increased DNA fragmentation of sinusoidal endothelial cells in the NHBD group and more apoptotic hepatocytes than in the control group. All these alterations could be almost abrogated by the use of VSOP in the NHBD + O2 group. The immunohistochemical staining of Fas antigen expression showed a significantly elevated Fas receptor expression in the NHBD and NHBD + O2 groups, in accord with an eightfold increase of Fas receptor mRNA detected by real-time reverse-transcription polymerase chain reaction (RT-PCR). These results demonstrate that the postischemic apoptotic rate of sinusoidal endothelial cells in NHBD livers can be reduced by the use of VSOP. A significant improvement in liver integrity and viability was obtained with this technique, without influencing the expression of Fas expression.

Isolation of hepatocytes from livers from non-heart-beating donors for cell transplantation

One of the limitations to hepatocyte transplantation is the restricted availability of donor liver tissue. The aim of this study was to evaluate livers from non-heart-beating donors (NHBDs) as a source of hepatocytes for cell transplantation. A total of 20 livers/segments obtained from NHBD were perfused under good manufacturing practices using a standard collagenase digestion method. The donor liver median warm ischemia time was 15 minutes (range, 11-40 minutes), and cold ischemia time was 13 hours (range, 6-30 hours) prior to cell isolation. The cell viability of the hepatocytes obtained was 52% (1-81%), with a yield of 2.2 x 10(6)(0.2-29.7 x 10(6)) cells per gram of tissue. There was a significant negative correlation between hepatocyte viability and length of both warm ischemia (r = -0.544, P = 0.013) and cold ischemia (r = -0.510, P = 0.022). Preliminary experiments were performed on the viability testing of NHBD livers based on digestion of needle biopsies with collagenase and assessment of the hepatocytes produced. Two of the NHBD cell preparations, which had been cryopreserved, were used as part of a series of cell infusions for hepatocyte transplantation. A 3.5-yr-old girl with Crigler-Najjar syndrome type I received 9.7 x 10(8) NHBD hepatocytes (viability on thawing, 65%), and a 4-month-old boy with inherited clotting factor VII deficiency received 5.0 x 10(8) hepatocytes (viability, 57%). In conclusion, hepatocytes suitable for cell transplantation can be obtained from NHBD livers. Higher viability values may be obtained if both warm and cold ischemia times of donor liver can be reduced prior to processing.

The Effect of Normothermic Recirculation is Mediated by Ischemic Preconditioning in NHBD Liver Transplantation

We have evaluated the involvement of hepatic preconditioning mediators (adenosine, adenosine A1 and A2 receptors) during normothermic recirculation (NR) in a model of liver transplantation from non-heart-beating donor (NHBD) pigs. Application of NR after 20 min of warm ischemia (WI) reversed the lethal injury associated with transplantation of NHBD livers (achieving 5-day survival and diminishing glutathione S-transferase (GST), aspartate aminotransferase (AST) and hyaluronic acid (HA)). Adenosine administration prior to WI simulated the effect of NR. Measuring adenosine, we found that during NR, hepatic adenosine levels increased and xanthine levels decreased. Then when we blocked A2 receptors the effect of NR was abolished, whereas the blocking of A1 receptors further protected the liver. Furthermore, A2 blocking improved hepatic perfusion during NR whereas A1 blocking reduced it. The study suggests that NR has a preconditioning effect by maintaining adequate adenosine and xanthine levels. During NR, adenosine protects the liver through A2 activation and damages it through A1 activation although simultaneous stimulation of both receptors exerts a clear beneficial effect. The possible relation of NR mechanism with other preconditioning mediators such as cAMP and nitric oxide synthesis are discussed.

Wash-out of the non–heart-beating donor liver: A comparison between ringer lactate, HTK, and polysol

The solution of choice for wash-out of non–heart-beating donor (NHBD) livers ishistidine tryptophan ketoglutarate (HTK). This solution has a lower viscosity, due to absenceof a colloid, and is less expensive as compared to the University of Wisconsin (UW) solution.A new preservation solution for machine perfusion was developed, named Polysol. In orderto apply Polysol clinically in NHBD organ retrieval, the efficacy as a wash-out solution wasinvestigated. Methods After a warm ischemic time of 30 minutes, the rat liver was washed out via theportal vein with 50 mL of either ringer lactate (RL), HTK or Polysol. After wash-out andharvesting, the liver was reperfused with Krebs-Henseleit buffer. Samples were taken toassess hepatocellular injury and liver function. Results Liver damage parameters were elevated in the RL group as compared to the HTKand Polysol groups. Liver/rat weight ratios were significantly lower after wash-out withPolysol. Overall, no differences were seen in ammonia clearance and bile production. Inconclusion, wash-out of the NHBD liver with Polysol results in equal to improvedreperfusion results as compared to HTK. Polysol is feasible as a wash-out solution incombination with machine perfusion using Polysol.

Machine perfusion preservation of the non–heart-beating donor rat livers using polysol, a new preservation solution

Aims The increasing shortage of donor organs has led to a focus on extended criteria donors, including the non–heart-beating donor (NHBD). An optimal preservation method is required to facilitate successful transplantation of these ischemically damaged organs. The recent literature has shown clear advantages of hypothermic machine perfusion (MP) over cold storage (CS). For MP, modified University of Wisconsin perfusion solution (UW-G) is often used, which, however, is known to cause microcirculatory obstruction, is difficult to obtain, and is expensive. Therefore, Polysol was developed as a MP preservation solution that contains specific nutrients for the liver, such as amino acids, energy substrates, and vitamins. The aim of this study was to compare Polysol with UW-G in a NHBD rat liver model. Methods After 24 hours hypothermic MP of NHBD rat livers using UW-G or Polysol, liver damage and function parameters were assessed during 60 minutes of reperfusion with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours CS in UW. Results Liver enzyme release was significantly higher among the CS-UW group compared to MP using UW-G or Polysol. Flow during reperfusion was significantly higher when using Polysol compared to UW-G. Bile production and ammonia clearance were highest when using Polysol compared to UW-G. There was less cellular edema after preservation with Polysol compared to UW-G. Conclusions MP of NHBD rat livers for 24 hours using UW-G or Polysol resulted in less hepatocellular damage than CS in UW. MP of NHBD livers for 24 hours using Polysol is superior to MP using UW-G.

Liver fatty acid-binding protein: An early and sensitive plasma marker of hepatocellular damage and a reliable predictor of graft viability after liver transplantation from non-heart-beating donors

Objective Liver fatty acid-binding protein (L-FABP) is a small protein (15 kD) involved in the intracellular transport of long-chain fatty acids in the liver. The L-FABP is regarded as a sensitive marker for liver cell damage. In a pig model for liver transplantation (LTx) from non-heart-beating donors (NHBD), we evaluated plasma changes of L-FABP early after reperfusion of grafts exposed to increasing periods of warm ischemia (WI). Methods Porcine livers were procured after 0, 15, 30, 45, and 60 minutes' WI. After 4 hours' cold ischemia (CI), LTx was performed. Primary graft nonfunction (PNF) and day 4 survival were recorded. Plasma samples were collected prior to and 15, 60, and 180 minutes after graft reperfusion for determination of L-FABP and aspartate transaminase (AST). Results Early after reperfusion, levels of L-FABP correlated well with the duration of WI. The PNF developed in 100% of animals after 60 minutes of WI, 50% after 30, and 45 minutes' WI, and was absent after no WI and 15 minutes of WI. Day 4 survival was 100% in 0 minutes' WI, 83% in 15 minutes' WI, 50% in 30 and 45 minutes' WI, and 0% in 60 minutes of WI. Conclusions Plasma levels of L-FABP correlated well with WI and concomitant hepatocellular damage in LTx from NHBD. Monitoring of posttransplant L-FABP plasma levels is a valuable new tool to quantify early the extent of parenchymal cell damage of NHBD livers and to predict their viability and function.

WASH-OUT OF THE NON-HEART-BEATING DONOR LIVER: A COMPARISON BETWEEN RINGERS LACTATE, HTK AND POLYSOL

P39 Aims: Complete wash-out of the non-heart-beating donor (NHBD) liver during harvest, is of major importance for optimal preservation. The most commonly used wash-out solution when retrieving NHBD livers is histidine tryptophan ketoglutarate (HTK). This solution has a lower viscosity and is less expensive compared to the University of Wisconsin (UW) solution. Machine perfusion (MP) has proven to be beneficial for the preservation of NHBD livers as compared to static, cold storage. A new preservation solution for MP was developed, named Polysol. In previous studies we have shown the superiority of Polysol over UW-Gluconate in MP of rat livers. In order to apply Polysol clinically in NHBD liver retrieval, it will also need to be effective as wash-out solution. The aim of this study is to assess Polysol as a wash-out solution for the NHBD liver and to compare it to HTK and Ringers Lactate (RL). Methods: Male Wistar rats (250-300 g) were used as liver donor. After midline laparotomy and heparinization, phrenotomy was performed to obtain circulatory arrest. After a warm ischemic time (=NHBD time) of 30 minutes the bile duct, portal vein and caval vein were canulated. Thereafter the liver was flushed with 50 cc of either RL, HTK or Polysol through the portal vein, at a pressure of 40 cm H2O. After wash-out, the liver was harvested, weighed and connected to a reperfusion system, using the isolated perfused rat liver (IPRL) system. In the IPRL, the liver was reperfused with normothermic, oxygenated Krebs Henseleit buffer during 60 minutes. During this period, samples were taken to assess hepatocellular injury (AST, ALT and LDH) and liver function (bile production, ammonia clearance, oxygen consumption). The perfusate flow was recorded to calculate intravascular resistance. Liver weight after wash-out was determined as a parameter of fluid extravasation, wet-dry weight ratios were used to determine tissue edema after reperfusion. Results: Hepatocellular damage was significantly increased in the RL group, but there were no differences between the HTK and Polysol groups. Bile production was highest after wash-out with Polysol (395 μl/hour± 34.9) as compared to HTK (311 μl/hour± 11.11) and RL (290 μl/hour± 42.86). Liver weight after flush was less after flush with Polysol (13.5 g± 0.16) as compared to HTK (16.67 g± 0.40) or RL (17.33 g± 0.59) (p<0.005). Overall, no differences were seen in ammonia clearance, oxygen consumption and wet-dry weight ratios after reperfusion. Conclusions: Wash-out of the non-heart-beating donor liver with Polysol results in equal to even better reperfusion results as compared to HTK. Therefore Polysol can be applied as a wash-out solution, as well as a preservation solution for machine perfusion.

Preservation of porcine non-heart-beating donor livers by sequential cold storage and warm perfusion.

Normothermic perfusion has been shown to resuscitate and maintain viability of non-heart-beating donor (NHBD) livers that have undergone significant warm ischemic injury. However, the logistics of clinical organ retrieval are complex, and a period of cold storage before warm preservation would simplify the process. We have investigated the effects of short duration of cold preservation before normothermic preservation on the function of porcine NHBD livers. Porcine livers were subjected to 60 minutes of warm ischemia and then assigned to the following groups: group W (n=5), normothermic preservation for 24 hours; and group C (n=4), cold preservation in University of Wisconsin solution for 4 hours followed by normothermic preservation for 20 hours (total preservation time 24 hours). Outcome parameters that were measured included bile production, serum transaminases and hyaluronic acid levels (cellular damage), and base deficit and glucose use (metabolic function). Group W livers had superior bile production, metabolic activity (base deficit and greater glucose use), and less evidence of hepatocellular damage (alanine aminotransferase, aspartate aminotransferase), and sinusoidal endothelial cell dysfunction (hyaluronic acid). Group C livers showed greater necrosis and destruction of architecture on histology. Normothermic perfusion failed to resuscitate porcine livers after 60 minutes of warm ischemia and 4 hours of cold preservation. Even a short period of cold ischemia is significantly deleterious to the function of ischemically damaged (NHBD) livers.

Non-heart-beating donor porcine livers: The adverse effect of cooling

Normothermic preservation has been shown to be advantageous in an experimental model of preservation of non-heart-beating donor (NHBD) livers, which have undergone significant warm ischemic injury. The logistics of clinical organ retrieval might dictate a period of cold preservation prior to warm perfusion. We have investigated the effects of a brief period of cold preservation on NHBD livers prior to normothermic preservation. Porcine livers were subjected to 60 minutes of warm ischaemia and then assigned to following groups: Group W (n = 5), normothermic preservation for 24 hours; and Group C (n = 6), cold preservation in University of Wisconsin solution for 1 hour followed by normothermic preservation for 23 hours (total preservation time, 24 hours). Synthetic function (bile production and factor V production) and cellular damage were compared on the ex vivo circuit during preservation. There was no significant difference in the synthetic function of the livers (bile production and factor V production). Markers of hepatocellular damage (alanine aminotransferase and aspartate aminotransferase release), sinusoidal endothelial cell dysfunction (hyaluronic acid), and Kupffer cell injury (beta-galactosidase) were significantly higher in Group C. The histology of the livers at the end of perfusion was similar. In conclusion, a brief-period cold preservation prior to normothermic perfusion maintains the synthetic function and metabolic activity but results in significant hepatocellular damage, sinusoidal endothelial cell dysfunction, and Kupffer cell injury. Transplant studies are required to establish whether livers treated in this way are viable for transplantation.

Liver transplantation from non-heart-beating donors: current status and future prospects.

Liver transplantation is the treatment of choice for many patients with acute and chronic liver failure, but its application is limited by a shortage of donor organs. Donor organ shortage is the principal cause of increasing waiting lists, and a number of patients die while awaiting transplantation. Non-heart-beating donor (NHBD) livers are a potential means of expanding the donor pool. This is not a new concept. Prior to the recognition of brainstem death, organs were retrieved from deceased donors only after cardiac arrest. Given the preservation techniques available at that time, this restricted the use of extrarenal organs for transplantation. In conclusion, after establishment of brain death criteria, deceased donor organs were almost exclusively from heart-beating donors (HBDs). To increase organ availability, there is now a resurgence of interest in NHBD liver transplantation. This review explores the basis for this and considers some of the published results.

Survival transplantation of preserved non–heart-beating donor rat livers: preservation by hypothermic machine perfusion1

Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Fas Receptor Is Upregulated in Livers from Non-Heart-Beating Donors

Cell injury in livers from non-heart-beating donors (NHBDs) is not only caused by necrosis, but it is increasingly being recognized that apoptotic transformation contributes to postischemic cell death. It remains unclear which pathways lead to apoptosis. We suggested that apoptosis in NHBD livers might be mediated by the Fas receptor. Therefore the aim of our study was the detection and quantification of the Fas receptor, Fas mRNA and apoptosis in NHBD livers. Livers of male Wistar rats were harvested either from heart-beating donors (control, n = 15) or 30 min after cardiac arrest from NHBDs (n = 15). All livers were stored in University of Wisconsin solution for 24 h at 4°C, and reperfused using a recirculating model with oxygenated Krebs-Henseleit buffer for 120 min at 37°C. Conventional signs of cell damage, such as enzyme release and bile production, showed an elevated nonspecific cell injury in NHBD livers. The immunohistochemical staining of Fas antigen expression revealed a 7% rate of Fas receptor-expressing hepatocytes in the control group, but a considerable increase up to 38% in the NHBD group. No Fas antigen expression was detected on sinusoidal endothelial cells. Real-time RT-PCR detected an approximately eightfold elevated concentration of Fas mRNA transcripts in NHBD livers. We conclude that upregulation of the Fas receptor on hepatocytes in NHBDs will prime these cells to eventually undergo apoptosis upon reperfusion in vivo. Therefore, therapeutic modulation of the Fas pathway must be considered as a new strategy in order to additionally protect NHBD livers which are otherwise prone to parenchymal apoptosis.

Adenosine A2 receptor stimulation protects the predamaged liver from cold preservation through activation of cyclic adenosine monophosphate-protein kinase a pathway

The shortage of organ donors has led to reconsideration for the use of non-heart-beating donors (NHBDs). However, graft injury caused by warm ischemia in livers from NHBDs strongly affects posttransplantation outcome. The aim of the present study is to investigate the role of adenosine A2 receptor with regard to hepatic viability after cold preservation of NHBD livers. Cardiac arrest was induced in Wistar rats by phrenotomy of the anesthetized nonheparinized animal. After 60 minutes, the livers were excised and flushed with 60 mL of histidine-tryptophan-ketoglutarate (HTK) and stored submerged in HTK at 4 degrees C for 24 hours. Reperfusion was performed in vitro after all livers were incubated at 22 degrees C in saline solution to account for the period of slow rewarming during surgical implantation in vivo. Addition of the selective A2-receptor agonist (CGS 21680; 30microg/100 mL) to the preservation solution resulted in a significant reduction to one quarter of the parenchymal enzyme release of alanine aminotransferase or lactate dehydrogenase on reperfusion and promoted a 2-fold increase in hepatic bile production. This salutory effect was accompanied by a significant increase (40%) in the activity ratio of protein kinase A (PKA) in the liver tissue and could be abrogated in large part by the PKA inhibitor, Rp-cAMPs. Stimulation of the adenosine A2 receptor during harvest and storage of the graft improves maintenance of tissue integrity in liver grafts. A major part of this effect, which may represent a promising approach for the use of NHBD grafts, seems to be mediated through activation of PKA.