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.

Limited sampling strategies have been developed to predict full AUCs. The goal of this study was to develop a limited sampling strategy to estimate the AUC of tacrolimus in adult renal transplant patients and to evaluate its predictive performance in an independent patient population. A total of 27 tacrolimus pharmacokinetic profiles were studied. Blood samples were collected before the dose (0) and at 0.5, 1, 2, 4, 6, 8, and 12 hours postdose. The study was divided into 2 phases. In phase 1, the goal was to obtain a sampling strategy from 14 pharmacokinetic profiles. In phase 2, the bias and precision of the model were evaluated in another 13 pharmacokinetic profiles. The best correlation was achieved at 4 hours after dose (r(2) = 0.790). Stepwise multiple regression analysis determined that the abbreviated AUC at 0, 1, and 4 hours could accurately predict total AUC (r(2) = 0.965). The following formula was developed: AUC = 8.90 + 4.0C0h+ 1.77C1h + 5.47C4h. No significant differences were found between calculated and estimated AUC (165.6 +/- 41.1 and 166.7 +/- 43.2 ng.h/mL, respectively). The mean prediction error (MPE), the relative prediction error (PE), and the mean squared error (MSE) were 0.48 ng.h/mL, 0.16%, and 40.0 ng.h/mL, respectively. The limited sampling with use of the 3 levels at 0, 1, and 4 hours postdose provides accurate, reliable determination of tacrolimus AUC in renal transplant patients.

Background. This study ascertained the effect of S-adenosyl-L-methionine (SAMe) administration on the ischemia-reperfusion injury associated with pig liver transplantation from non-heart-beating donors (NHBDs) after prolonged warm ischemia.Method. Twenty-five animals underwent transplantation with an allograft from an NHBD. After donor cardiac arrest, cardiopulmonary bypass and normothermic recirculation (NR) were performed for 30 min. Ten animals were given SAMe during NR. Donors were cooled to 15degreesC, and liver procurement was performed.Results. SAMe reduced histologic liver damage 5 days after transplantation. The necrotic area affected 15.9+/-14.5% of the liver biopsies in controls and 7.4%+/-9% in SAMe livers. Six of eight controls and only one of eight survivors in the SAMe group developed ischemic cholangitis. SAMe reduced apoptosis of hepatocytes 5 days after transplantation and apoptosis of sinusoidal endothelial cells at reperfusion and at 5 days. SAMe increased energy charge at the end of NR and favored the balance between adenosine and xanthine. It was also associated with higher portal blood flow (740+/-59.2 vs. 475.2+/-65.0 mL/min(-1)/m(-2)), hepatic hyaluronic acid extraction (132+/-72.2 vs. -205.8+/-64.6 mug/L), and lower levels of alpha-glutathione-S-transferase after reperfusion (2,601% +/-581% with respect to baseline vs. 6,488%+/-5,612%).Conclusion. SAMe administration during liver procurement from NHBDs prevents liver endothelial, parenchymal, and biliary tract damage. The protective role of SAMe may be partially mediated by the effect of adenosine during liver procurement.

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0–4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.The re-beating time was significantly shorter in hearts stored in the new solution (10.3 ± 2.6 min vs. HTK 14.2 ± 4.1 min; p < 0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3 ± 0.4 vs. HTK 1.6 ± 0.5; p < 0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8 ± 0.2 vs. HTK 2.2 ± 0.7), less interstitial fibrosis (4.8 ± 1.9% vs. HTK 8.5 ± 3.8%, p < 0.05), less vasculopathy (14.7 ± 6.9% vs. 22.0 ± 23.2%; p = 0.06) and lower TGF-β1-expression (6.6 ± 1.4% vs. HTK 12.0 ± 4.6%).The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-l-histidine, glycine, alanine, arginine and sucrose.

Background The aim of the present study was to evaluate hepatic content of adenine nucleotides and their degradation products in non-heart-beating donor (NHBD) pigs and its relationship with recipient survival,Methods. Thirty animals were transplanted with an allograft, from NHBDs. After warm ischemia (WI) time (20, 30, or 40 min), cardiopulmonary bypass and normothermic recirculation (NR) were run for 30 min. Afterward, the animals were cooled to 15 degreesC and liver procurement was performed.Results, Survival rate was 100% in the 20WI, 70% in the 30WI, and 50% in the 40WI, Livers from non-surviving animals had higher levels of xanthine after NR than Livers from surviving animals. Logistic regression analysis revealed that xanthine at the end of NR was the only variable able to predict survival with a calculated sensitivity of 80% and a specificity of 60%. Prolongation of warm ischemic period leaded to a greater xanthine accumulation as well as increased plasma cu-glutathione S-transferase levels at reperfusion. Xanthine at NR and alpha -glutathione S-transferase at reperfusion significantly correlated, indicating that donor xanthine contributes to some extent to the severity of the lesion by ischemia-reperfusion.Conclusions, It is suggested that xanthine content in the donor is able to predict survival after transplantation. Xanthine is significantly involved in the hepatic lesion elicited by warm ischemia and subsequent ischemia-reperfusion associated to liver transplantation from a NHBD.

Net, Marc; Almenara, Raul; Valero, Ricard; Boado, Miguel Angel Lopez; Blasi, Anabel; Capdevila, LLuis; Taura, Pilar; Deulofeu, Ramon; Valdecasas, Juan Carlos Garcia Author Information

Background. To evaluate whether L-arginine reduces liver and biliary tract damage after transplantation from non heart-beating donor pigs.Methods. Twenty-five animals received an allograft hom non-heart-beating donors, After 40 min of cardiac arrest, normothermic recirculation was run for 30 min. The animals were randomly treated with L-arginine (400 mg kg(-1) during normothermic recirculation) or saline (control group). Then, the animals were cooled and their livers were transplanted after 6 hr of cold ischemia. The animals were killed on the 5th day, liver damage was assessed on wedged liver biopsies by a semiquantitative analysis and by morphometric analysis of the necrotic areas, and biliary tract damage by histological examination of the explanted liver.Results. Seventeen animals survived the study period. The histological parameters assessed (sinusoidal congestion and dilatation, sinusoidal infiltration by polymorphonuclear cells and lymphocytes, endothelitis, dissociation of liver cell plates, and centrilobular necrosis) were significantly worse in the control group. The necrotic area affected 15.9+/-14.5% of the liver biopsies in the control group and 3.7+/-3.1% in the L-arginine group (P<0.05), Six of eight animal in the control group and only one of eight survivors in the L-arginine group developed ischemic cholangitis (P<0.01), L-Arginine administration was associated with higher portal blood flow (676.9+/-149.46 vs. 475.2+/-205.6 ml.min.m(-2); P<0.05), higher hepatic hialuronic acid extraction at normothermic recirculation (38.8+/-53.7% vs. -4.2+/-18.2%; P<0.05) and after reperfusion (28.6+/-55.5% vs. -10.9+/-15.5%; P<0.05) and lower levels of alpha-glutation-S-transferase at reperfusion (1325+/-1098% respect to baseline vs, 6488+/-5612%; P<0.02).Conclusions. L-Arginine administration during liver procurement from non heart beating donors prevents liver and biliary tract damage.

Our aim was to investigate the potential of the preservation solution Celsior to protect rat cremaster muscle microcirculation during ischemia and reperfusion, and to compare its effects with those of HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution). Because of its anti-oxidant contents, we expected Celsior to be more protective than HTK.Capillary perfusion and leukocyte—endothelium interactions were examined in rat cremaster muscle using intravital microscopy. After perfusion with Celsior or HTK (4°C), the cremaster was subjected to 4 or 6 h of warm (33–34°C) ischemia and 2 h of reperfusion. Measurements were performed prior to perfusion and/or ischemia, and 0, 1, and 2 h after restoration of flow.Without Celsior or HTK, capillary perfusion transiently decreased to 50% of baseline after 4 h of ischemia; it remained low (45%) after 6 h of ischemia. Whereas HTK had no significant influence, Celsior deteriorated capillary perfusion: it remained low after 4 h of ischemia (39–48%) and decreased even further after 6 h of ischemia (18–8%). Both preservation solutions similarly reduced the increase in leukocyte—endothelium interactions after ischemia.Preischemic tissue perfusion with Celsior had an adverse effect on capillary perfusion in rat cremaster muscle after 4 and 6 h of ischemia, whereas HTK did not significantly influence this parameter. Both preservation solutions similarly prevented the increase in leukocyte—endothelium interactions after ischemia. These data suggest that HTK is more suited as a preservation solution for muscular tissue than Celsior, especially when the known protective effects of HTK on muscle function are taken into account.

201 INTRODUCTION: We have previously shown in an experimental model of liver transplantation from non heart beating donors that livers procured after up to 40 minutes of warm ischemia were able to sustain life. However long term survival was seriously compromised due biliary tract necrosis. Along this line, strategies of conditioning by adding certain substances during liver procurement would be useful in order to ameliorate the ischemia reperfusion injury. AIM: The aim of this study was to asses the effect of three substances that have been described to work through different cellular mechanisms: L-arginine (NO precursor), S-adenosyl L-methionine (SAMe, glutathione precursor) and Glycine (membrane structure stabilizer and glutathione precursor), on liver graft quality. MATERIAL AND METHODS: Forty five out-bread weanling pigs (±30 Kg) were transplanted (LTx) from a NHBD with the following characteristics: After a period of 40′ of cardiac arrest (KCl), normothermic recirculation (NR) by using cardiopulmonary bypass (CPB) was performed during 30 minutes. Afterwards liver procurement was done and liver transplantation performed after six hours of cold ischemia (UW), in an standard manner. Four groups were designed: L-Arginine group (AG, n=10) in which a bolus of L-Arginine (400 mg/Kg) was added during NR, SAMe group (SG, n=10) treated with the addition of SAMe (200 mg/Kg) during NR, Glycine group (GG, n=10) treated with continous infusion of D-Gly (0.6mg/Kg/min) during NR, and Control group (CG, n=15) where no substances were added during NR. Blood samples to determine hepatocellular (AST, α-GST), and endothelial damage (hyaluronic acid, HA) were taken (in the donor) at the beginning (BL), after cardiac arrest (CA), after 30′ of NR; (in the recipient): one hour after reperfusion (RP); and five (5D) days after Ltx. Values were expressed as percentage of baseline. HA clearance (marker of endothelial cell function) was calculated as follows: (systemic HA- suprahepatic vein HA)*100/systemic HA. Biliary tract necrosis (BTN) was evaluated by light microscopy in liver specimens taken on the fifth postoperativeday. RESULTS:(Table)TableCONCLUSION: The use of L-arginine, SAMe and Gly during liver procurement from NHBD ameliorates the ischemia reperfusion injury in terms of the hepatocellular and endothelial damage produced after 40′ of CA. While in the CG the presence of biliary tract necrosis burdens long term survival, the use of these substances during liver procurement are capable to maintain biliary tract viability.

The aim of this study was to assess liver viability after different periods of cardiac arrest and the predictive value of two markers of ischemia-reperfusion injury. Methods: A pig liver transplantation model of non-heart-beating donors was studied. Four donor groups were designed; three groups were submitted to different periods of cardiac arrest (20, 30 and 40 min), and the fourth group served as the control group (without cardiac arrest). In the non-heart-beating donor groups, normothermic recirculation was established 30 min prior to total body cooling. Aminotransferase, α-glutathione-S-transferase, and hyaluronic acid determinations as well as liver biopsies, were serially performed. Results: Although hepatocellular function could be preserved after 40 min of cardiac arrest, histological lesions at 5 days were considered irreversible due to the presence of a necrotic biliary tract. An overall significant relationship was found between the time period of cardiac arrest (20, 30 or 40 min) and the levels of hyaluronic acid (p = 0.004) or α-glutathione-S-transferase (p = 0.01) obtained during liver procurement and transplantation. Conclusions: The period of cardiac arrest is the determinant factor of liver viability after liver transplantation from non-heart-beating donors. As early markers of endothelial or hepatocellular damage, hyaluronic acid or α-glutathione-S-transferase levels may help to evaluate the ischemic injury of a potential donor.

181 We have previously shown in an experimental model of liver transplantation (LTx) from non heart beating donors (NHBD), the beneficial effect of the use of normothermic recirculation (NR) during liver procurement. However, the mechanism through which NR is useful is unknown. It has also been stablished that brief periods of ischemia-reperfusion previous to long ischemia ameliorates the ischemia reperfusion injury and that this preconditioning effect is mediated by an increase in extracellular adenosine levels. AIM: The aim of this study was to investigate whether NR has a certain effect of conditioning and therefore its action would be mediated by an increase in extracellular adenosine (ADE) levels. MM: Fifteen out-bread weanling pigs (±30 Kg) were transplanted (LTx) from a NHBD with the following characteristics: Control group (CG, n=5): After a period of 20′ of cardiac arrest (KCl) liver was procured and transplantation performed after six hours of cold ischemia in an standard manner. Adenosine group (AG, n=5): as CG but adenosine was administered as a continous infusion (350μg/Kg/min) before cardiac arrest. Normothermic Recirculation Group (NRG, n=5): as CG but normothermic recirculation (NR) by using cardiopulmonary bypass (CPB) was performed during 30 minutes after cardiac arrest. Blood samples to determine hepatocellular (AST, α-GST), and endothelial damage (hyaluronic acid, HA) were taken (in the donor) at the beginning (BL), after cardiac arrest (CA), after 30′ of NR; (in the recipient): at the end of total ischemia (TI) one hour after reperfusion (RP); and five (5D) days after Ltx. Values were expressed as percentage of baseline. HA clearance (marker of endothelial cell function) was calculated as follows: (systemic HA- suprahepatic vein HA)*100/ systemic HA. Liver biopsies were obtained at the same time as blood to determine tissue adenine nucleotides and adenosine levels by High Performance Liquid Chromatography (HPLC). RESULTS: Survivals were 1/5(CG), 4/5 (AG) and 5/5 (NRG). At the end of organ procurement ADE levels were significantly higher in AG and NRG than in CG (126.22±31.7; 193.3±154.6 vs. 48.4±16, p<0.05). Throughout the study ASAT increase was higher in CG than in AG and NRG, reaching statistical significance at 2D post LTx between CG and AG (6732±3365 vs. 1390±889, p=0.03). α-GST increase was also higher throughout the study in CG; being the values in AG and NRG similar in all phases studied. HA clearance at RP was negative in CG, slightly negative or near to zero in NRG, and positive values were only obtained in AG animals, indicating a better endothelial cells function in this group. CONCLUSION: Preliminary results suggests that adenosine administration seems to obtain the same beneficial effect as normothermic recirculation during liver procurement in terms of survival, parenchymal and endothelial damage. Normothermic recirculation produces adenosine levels at the end of organ procurement similar to those obtained by adenosine administration. Therefore, donor conditioning may be the mechanism through which normothermic recirculation works.