Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Warm Ischemia Induces Spatiotemporal Changes in Lysophosphatidylinositol That Affect Post-Reperfusion Injury in Normal and Steatotic Rat Livers.

Warm ischemia-reperfusion injury is a prognostic factor for hepatectomy and liver transplantation. However, its underlying molecular mechanisms are unknown. This study aimed to elucidate these mechanisms and identify the predictive markers of post-reperfusion injury. Rats with normal livers were subjected to 70% hepatic warm ischemia for 15, 30, or 90 min, while those with steatotic livers were subjected to 70% hepatic warm ischemia for only 30 min. The liver and blood were sampled at the end of ischemia and 1, 6, and 24 h after reperfusion. The serum alanine aminotransferase (ALT) activity, Suzuki injury scores, and lipid peroxidation (LPO) products were evaluated. The ALT activity and Suzuki scores increased with ischemic duration and peaked at 1 and 6 h after reperfusion, respectively. Steatotic livers subjected to 30 min ischemia and normal livers subjected to 90 min ischemia showed comparable injury. A similar trend was observed for LPO products. Imaging mass spectrometry of normal livers revealed an increase in lysophosphatidylinositol (LPI (18:0)) and a concomitant decrease in phosphatidylinositol (PI (18:0/20:4)) in Zone 1 (central venous region) with increasing ischemic duration; they returned to their basal values after reperfusion. Similar changes were observed in steatotic livers. Hepatic warm ischemia time-dependent acceleration of PI (18:0/20:4) to LPI (18:0) conversion occurs initially in Zone 1 and is more pronounced in fatty livers. Thus, the LPI (18:0)/PI (18:0/20:4) ratio is a potential predictor of post-reperfusion injury.

Rapid and Reliable Steatosis Rat Model Shrsp5-Dmcr for Cold Storage Experiment.

Interventions for liver grafts with moderate macrovesicular steatosis have been important in enlarging donor pools. Here, we tested a high-fat and cholesterol (HFC) diet to create a steatosis model for cold hepatic preservation and reperfusion experiments. The aim of the present study was to assess the steatosis model's reliability and to show the resulting graft's quality for cold preservation and reperfusion experiment. Male SHRSP5-Dmcr rats were raised with an HFC diet for up to 2 weeks. The fat content was evaluated using magnetic resonance imaging (MRI) proton density fat fraction (PDFF). The nonalcoholic fatty liver disease activity score (NAS) was evaluated after excision. Steatosis created by 2 weeks of HFC diet was subjected to 24-hour cold storage in the University of Wisconsin and the original test solution (new sol.). Grafts were applied to isolated perfused rat livers for simulating reperfusion. The NAS were 2.2 (HFC 5 days), 3.3 (HFC 1 week), and 5.0 (HFC 2 weeks). Ballooning and fibrosis were not observed in any group. An MRI-PDFF showed 0.2 (HFC 0 days), 12.0 (HFC 1 week), and 18.9 (HFC 2 weeks). The NAS and MRI-PDFF values correlated. Many indices in the isolated perfused rat liver experiment tended to improve in the new sol. group but were insufficient. Although the new sol. failed to be effective, it acted at multiple sites under difficult conditions. In conclusion, the HFC diet for 2 weeks in SHRSP5-Dmcr rats, together with MRI-PDFF evaluation, is a reliable method for creating simple steatosis and provides good-quality cold preservation and reperfusion experiments.

Hypothermic Machine Perfusion with Hydrogen Gas Reduces Focal Injury in Rat Livers but Fails to Restore Organ Function.

BACKGROUND: We have previously reported the efficacy of post-reperfusion H2 gas treatment in cold storage (CS) and subsequent reperfusion of the rat liver. The present study aimed to evaluate the effect of H2 gas treatment during hypothermic machine perfusion (HMP) in rat livers retrieved from donation after circulatory death (DCD) and elucidate the mechanism of action of H2 gas. METHODS: Liver grafts were procured from rats after 30 min of cardiopulmonary arrest. The graft was subjected to HMP for 3 hours at 7°C using Belzer MPS with or without dissolved H2 gas. The graft was reperfused using an isolated perfused rat liver apparatus at 37°C for 90 minutes. Perfusion kinetics, liver damage, function, apoptosis, and ultrastructure were evaluated. RESULTS: Portal venous resistance, bile production, and oxygen consumption rates were identical in the CS, MP, and MP-H2 groups. Liver enzyme leakage was suppressed by MP (vs control), whereas H2 treatment did not show a combination effect. Histopathology revealed poorly stained areas with a structural deformity just below the liver surface in the CS and MP groups, whereas these findings disappeared in the MP-H2 group. The apoptotic index in the CS and MP groups was high but decreased in the MP-H2 group. Mitochondrial cristae were damaged in the CS group but preserved in the MP and MP-H2 groups. CONCLUSIONS: In conclusion, HMP and H2 gas treatment are partly effective in DCD rat livers but insufficient. Hypothermic machine perfusion can improve focal microcirculation and preserve mitochondrial ultrastructure.

No impact of donor sex on the recurrence of hepatocellular carcinoma after liver transplantation.

BACKGROUND/PURPOSE: We aimed to verify a recent theory that female donors reduced the risk of hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT). METHODS: A total of 1118 recipients registered in the Japanese Liver Transplantation Society database were evaluated for HCC, of whom 446 received a graft from female donors (F-D group) and 672 from male donors (M-D group). RESULTS: Between the groups, donor age, recipient age and sex, positivity of hepatitis viruses, and graft type were different, whereas tumor-related factors were all comparable. The 5-year overall recurrence rates were 14% and 16% in the F-D and M-D groups, respectively (P = 0.59). The 5-year graft recurrence rate was also comparable between the groups (4% and 6%, respectively, P = 0.17). Neither univariate nor multivariate analysis identified donor sex as a significant risk factor for recurrence. Propensity score matching showed similar 5-year overall recurrence rates (15% in the F-D group and 14% in the M-D group, P = 0.63) and graft recurrence rates (5% and 5%, respectively, P = 0.94) between the groups. CONCLUSION: Donor sex did not affect post-LT recurrence of HCC in the Japanese cohort and should not be considered in the process of donor selection or organ allocation.

Metformin Preconditioning Improves Hepatobiliary Function and Reduces Injury in a Rat Model of Normothermic Machine Perfusion and Orthotopic Transplantation.

BACKGROUND: Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. METHODS: Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. RESULTS: Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. CONCLUSIONS: Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation.

Heavy Water (D2O) Containing Preservation Solution Reduces Hepatic Cold Preservation and Reperfusion Injury in an Isolated Perfused Rat Liver (IPRL) Model.

BACKGROUND: Heavy water (D2O) has many biological effects due to the isotope effect of deuterium. We previously reported the efficacy of D2O containing solution (Dsol) in the cold preservation of rat hearts. Here, we evaluated whether Dsol reduced hepatic cold preservation and reperfusion injury. METHODS: Rat livers were subjected to 48-hour cold storage in University of Wisconsin (UW) solution or Dsol, and subsequently reperfused on an isolated perfused rat liver. Graft function, injury, perfusion kinetics, oxidative stress, and cytoskeletal integrity were assessed. RESULTS: In the UW group, severe ischemia and reperfusion injury (IRI) was shown by histopathology, higher liver enzymes leakage, portal resistance, and apoptotic index, oxygen consumption, less bile production, energy charge, and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio (versus control). The Dsol group showed that these injuries were significantly ameliorated (versus the UW group). Furthermore, cytoskeletal derangement was progressed in the UW group, as shown by less degradation of α-Fodrin and by the inactivation of the actin depolymerization pathway, whereas these changes were significantly suppressed in the Dsol group. CONCLUSION: Dsol reduced hepatic IRI after extended cold preservation and subsequent reperfusion. The protection was primarily due to the maintenance of mitochondrial function, cytoskeletal integrity, leading to limiting oxidative stress, apoptosis, and necrosis pathways.

Transplantation of High-risk Donor Livers After Ex Situ Resuscitation and Assessment Using Combined Hypo- and Normothermic Machine Perfusion: A Prospective Clinical Trial.

OBJECTIVE: The aim of this study was to evaluate sequential hypothermic and normothermic machine perfusion (NMP) as a tool to resuscitate and assess viability of initially declined donor livers to enable safe transplantation. SUMMARY BACKGROUND DATA: Machine perfusion is increasingly used to resuscitate and test the function of donor livers. Although (dual) hypothermic oxygenated machine perfusion ([D]HOPE) resuscitates livers after cold storage, NMP enables assessment of hepatobiliary function. METHODS: In a prospective clinical trial, nationwide declined livers were subjected to ex situ NMP (viability assessment phase), preceded by 1-hour DHOPE (resuscitation phase) and 1 hour of controlled oxygenated rewarming (COR), using a perfusion fluid containing an hemoglobin-based oxygen carrier. During the first 2.5 hours of NMP, hepatobiliary viability was assessed, using predefined criteria: perfusate lactate <1.7 mmol/L, pH 7.35 to 7.45, bile production >10 mL, and bile pH >7.45. Livers meeting all criteria were accepted for transplantation. Primary endpoint was 3-month graft survival. RESULTS: Sixteen livers underwent DHOPE-COR-NMP. All livers were from donors after circulatory death, with median age of 63 (range 42-82) years and median Eurotransplant donor risk index of 2.82. During NMP, all livers cleared lactate and produced sufficient bile volume, but in 5 livers bile pH remained <7.45. The 11 (69%) livers that met all viability criteria were successfully transplanted, with 100% patient and graft survival at 3 and 6 months. Introduction of DHOPE-COR-NMP increased the number of deceased donor liver transplants by 20%. CONCLUSIONS: Sequential DHOPE-COR-NMP enabled resuscitation and safe selection of initially declined high-risk donor livers, thereby increasing the number of transplantable livers by 20%. TRIAL REGISTRATION: www.trialregister.nl; NTR5972.

Transplantation of high-risk donor livers after resuscitation and viability assessment using a combined protocol of oxygenated hypothermic, rewarming and normothermic machine perfusion: study protocol for a prospective, single-arm study (DHOPE-COR-NMP trial).

INTRODUCTION: Extended criteria donor (ECD) livers are increasingly accepted for transplantation in an attempt to reduce the gap between the number of patients on the waiting list and the available number of donor livers. ECD livers; however, carry an increased risk of developing primary non-function (PNF), early allograft dysfunction (EAD) or post-transplant cholangiopathy. Ischaemia-reperfusion injury (IRI) plays an important role in the development of these complications. Machine perfusion reduces IRI and allows for reconditioning and subsequent evaluation of liver grafts. Single or dual hypothermic oxygenated machine perfusion (DHOPE) (4°C-12°C) decreases IRI by resuscitation of mitochondria. Controlled oxygenated rewarming (COR) may further reduce IRI by preventing sudden temperature shifts. Subsequent normothermic machine perfusion (NMP) (37°C) allows for ex situ viability assessment to facilitate the selection of ECD livers with a low risk of PNF, EAD or post-transplant cholangiopathy. METHODS AND ANALYSIS: This prospective, single-arm study is designed to resuscitate and evaluate initially nationwide declined ECD livers. End-ischaemic DHOPE will be performed for the initial mitochondrial and graft resuscitation, followed by COR of the donor liver to a normothermic temperature. Subsequently, NMP will be continued to assess viability of the liver. Transplantation into eligible recipients will proceed if all predetermined viability criteria are met within the first 150 min of NMP. To facilitate machine perfusion at different temperatures, a perfusion solution containing a haemoglobin-based oxygen carrier will be used. With this protocol, we aim to transplant extra livers. The primary endpoint is graft survival at 3 months after transplantation. ETHICS AND DISSEMINATION: This protocol was approved by the medical ethical committee of Groningen, METc2016.281 in August 2016 and registered in the Dutch Trial registration number TRIAL REGISTRATION NUMBER: NTR5972, NCT02584283.

Ex Situ Machine Perfusion of Human Donor Livers via the Surgically Reopened Umbilical Vein: A Proof of Concept.

BACKGROUND: Machine perfusion of donor livers is typically performed via the portal vein main stem. Instead, cannulation of a reopened umbilical vein could allow machine perfusion during organ procurement and subsequent implantation in the recipient without interruption of the portal venous circulation. We aimed to assess the feasibility of portal venous machine perfusion via the umbilical vein. METHODS: During back table inspection of 5 human livers declined for transplantation, the umbilical vein was surgically reopened, dilated, and cannulated. Hypothermic and normothermic oxygenated machine perfusion (NMP) were performed using the umbilical vein for portal inflow. Three livers were perfused with hypothermic machine perfusion, 1 full liver graft underwent NMP for 4 hours, and 1 left lateral split procedure was performed under continuous NMP with portal perfusion via the umbilical vein. RESULTS: In all livers, access to the portal venous system via the umbilical vein was successfully achieved with good portal flows and macroscopically homogeneous perfusion. The full liver graft that underwent NMP via the umbilical vein for 4 hours showed good lactate clearance, normalized pH, and achieved good bile production with pH >7.55. During the split procedure under continuous NMP via the umbilical vein, the left lateral segment and extended right lobe remained equally perfused, as demonstrated by Doppler ultrasound. CONCLUSIONS: Machine perfusion with portal perfusion via the umbilical vein is feasible. Portal venous flows were similar to those obtained after cannulation of the portal vein main stem. This technique enables continuous oxygenated perfusion of liver grafts during procurement, splitting, and implantation.

First report of successful transplantation of a pediatric donor liver graft after hypothermic machine perfusion.

One of the main limiting factors in pediatric liver transplantation is donor availability. For adults, DCD liver grafts are increasingly used to expand the donor pool. To improve outcome after DCD liver transplantation, ex situ machine perfusion is used as an alternative organ preservation strategy, with the supplemental value of providing oxygen to the graft during preservation. We here report the first successful transplantation of a pediatric DCD liver graft after hypothermic oxygenated machine perfusion. The full-size liver graft was derived from a 13-year-old, female DCD donor and was end-ischemic pretreated with dual hypothermic oxygenated machine perfusion. Arterial and portal pressures were set at 18 and 4 mm Hg, slightly lower than protocolized settings for adult livers. During 2 hours of machine perfusion, portal and arterial flows increased from 100 to 210 mL/min and 30 to 63 mL/min, respectively. The pretreated liver graft was implanted in a 16-year-old girl with progressive familial intrahepatic cholestasis type 2. Postoperative AST, ALT, and prothrombin time normalized within a week. The recipient quickly recovered and was discharged from the hospital after 18 days. One year after transplantation, she is in excellent condition with a completely normal liver function and histology. This case is the first report of successful transplantation of a pediatric DCD liver graft after hypothermic oxygenated machine perfusion and illustrates the potential role of ex situ machine perfusion in expanding the donor pool and improving outcome after pediatric liver transplantation.

Opposite acute potassium and sodium shifts during transplantation of hypothermic machine perfused donor livers.

Liver transplantation is frequently associated with hyperkalemia, especially after graft reperfusion. Dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia/reperfusion injury and improves graft function, compared to conventional static cold storage (SCS). We examined the effect of DHOPE on ex situ and in vivo shifts of potassium and sodium. Potassium and sodium shifts were derived from balance measurements in a preclinical study of livers that underwent DHOPE (n = 6) or SCS alone (n = 9), followed by ex situ normothermic reperfusion. Similar measurements were performed in a clinical study of DHOPE-preserved livers (n = 10) and control livers that were transplanted after SCS only (n = 9). During DHOPE, preclinical and clinical livers released a mean of 17 ± 2 and 34 ± 6 mmol potassium and took up 25 ± 9 and 24 ± 14 mmol sodium, respectively. After subsequent normothermic reperfusion, DHOPE-preserved livers took up a mean of 19 ± 3 mmol potassium, while controls released 8 ± 5 mmol potassium. During liver transplantation, blood potassium levels decreased upon reperfusion of DHOPE-preserved livers while levels increased after reperfusion of SCS-preserved liver, delta potassium levels were -0.77 ± 0.20 vs. +0.64 ± 0.37 mmol/L, respectively (P = .002). While hyperkalemia is generally anticipated during transplantation of SCS-preserved livers, reperfusion of hypothermic machine perfused livers can lead to decreased blood potassium or even hypokalemia in the recipient.

Biliary Bicarbonate, pH, and Glucose Are Suitable Biomarkers of Biliary Viability During Ex Situ Normothermic Machine Perfusion of Human Donor Livers.

BACKGROUND: Ex situ normothermic machine perfusion (NMP) can be used to assess viability of suboptimal donor livers before implantation. Our aim was to assess the diagnostic accuracy of bile biochemistry for the assessment of bile duct injury (BDI). METHODS: In a preclinical study, 23 human donor livers underwent 6 hours of end-ischemic NMP to determine biomarkers of BDI. Livers were divided into groups with low or high BDI, based on a clinically relevant histological grading system. During NMP, bile was analyzed biochemically and potential biomarkers were correlated with the degree of BDI. Receiver operating characteristics curves were generated to determine optimal cutoff values. For clinical validation, identified biomarkers were subsequently included as viability criteria in a clinical trial (n = 6) to identify transplantable liver grafts with low BDI. RESULTS: Biliary bicarbonate and pH were significantly higher and biliary glucose was significantly lower in livers with low BDI, compared with high BDI. The following cutoff values were associated with low BDI: biliary bicarbonate greater than 18 mmol/L (P = 0.002), biliary pH greater than 7.48 (P = 0.019), biliary glucose less than 16 mmol/L (P = 0.013), and bile/perfusate glucose ratio less than 0.67 (P = 0.013). In the clinical trial, 4 of 6 livers met these criteria and were transplanted, and none developed clinical evidence of posttransplant cholangiopathy. CONCLUSIONS: Biliary bicarbonate, pH, and glucose during ex situ NMP of liver grafts are accurate biomarkers of BDI and can be easily determined point of care, making them suitable for the pretransplant assessment of bile duct viability. This may improve graft selection and decrease the risk of posttransplant cholangiopathy.

Expanded living-donor liver transplantation criteria for patients with hepatocellular carcinoma based on the Japanese nationwide survey: the 5-5-500 rule - a retrospective study.

Expansion of the liver transplantation indication criteria for patients with hepatocellular carcinoma (HCC) has long been debated. Here we propose new, expanded living-donor liver transplantation (LDLT) criteria for HCC patients based on a retrospective data analysis of the Japanese nationwide survey. A total of 965 HCC patients undergoing LDLT were included, 301 (31%) of whom were beyond the Milan criteria. Here, we applied the Greenwood formula to investigate new criteria enabling the maximal enrollment of candidates while securing a 5-year recurrence rate (95% upper confidence limit) below 10% by examining various combinations of tumor numbers and serum alpha-fetoprotein values, and maintaining the maximal nodule diameter at 5 cm. Finally, new expanded criteria for LDLT candidates with HCC, the 5-5-500 rule (nodule size ≤5 cm in diameter, nodule number ≤5, and alfa-fetoprotein value ≤500 ng/ml), were established as a new regulation with a 95% confidence interval of a 5-year recurrence rate of 7.3% (5.2-9.3) and a 19% increase in the number of eligible patients. In addition, the 5-5-500 rule could identify patients at high risk of recurrence, among those within and beyond the Milan criteria. In conclusion, the new criteria - the 5-5-500 rule - might provide rational expansion for LDLT candidates with HCC.

Pretransplant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin-based oxygen carrier perfusion solution.

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin-based oxygen carrier (HBOC)-based perfusion fluid (DHOPE-COR-NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE-COR-NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3-month graft survival, was a 100%. In conclusion, sequential DHOPE-COR-NMP using an HBOC-based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.

Post-reperfusion hydrogen gas treatment ameliorates ischemia reperfusion injury in rat livers from donors after cardiac death: a preliminary study.

BACKGROUND AND PURPOSE: We reported previously that hydrogen gas (H2) reduced hepatic ischemia and reperfusion injury (IRI) after prolonged cold storage (CS) of livers retrieved from heart-beating donors. The present study was designed to assess whether H2 reduced hepatic IRI during donation of a cardiac death (DCD) graft with subsequent CS. METHODS: Rat livers were harvested after 30-min cardiac arrest and stored for 4 h in University of Wisconsin solution. The graft was reperfused with oxygenated buffer, with or without H2 (H2 or NT groups, respectively), at 37° for 90 min on isolated perfused rat liver apparatus. RESULTS: In the NT group, liver enzyme leakage, apoptosis, necrosis, energy depletion, redox status, impaired microcirculation, and bile production were indicative of severe IRI, whereas in the H2 group these impairments were significantly suppressed. The phosphorylation of cytoplasmic MKK4 and JNK were enhanced in the NT group and suppressed in the H2 group. NFkB-p65 and c-Fos in the nucleus were unexpectedly unchanged by IRI regardless of H2 treatment, indicating the absence of inflammation in this model. CONCLUSION: H2 was observed to ameliorate IRI in the DCD liver by maintaining microcirculation, mitochondrial functions, and redox status, as well as suppressing the cytoplasmic MKK4-JNK-mediated cellular death pathway.