Machine perfusion preservation with hemoglobin based oxygen vesicles alleviate ultrastructural damages in porcine liver donated after cardiac death.

Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.

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.

Combined Use of Subnormothermic Extracorporeal Support and Hypothermic Oxygenated Machine Perfusion for Liver Graft After Cardiac Death in Pigs.

BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. This study aims to determine the benefits of extracorporeal membrane oxygenation (ECMO) and hypothermic oxygenated machine perfusion (HOPE) in a large animal model of DCD liver. METHODS: After cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 45 minutes of cardiac death. Then, the livers were perfused for 4 hours by cold storage (CS) or HOPE. In group 1, non-in situ ECMO and grafts were preserved by HOPE. In group 2, in situ ECMO and grafts were preserved by HOPE. In group 3, in situ ECMO and grafts were preserved by CS. After preservation, all grafts were evaluated using an isolated reperfusion model (IRM) with autologous blood for 2 hours. RESULTS: During HOPE, aspartate aminotransferase (AST) levels and hepatic arterial pressure in group 2 tended to be lower than in group 1. Hematoxylin-eosin staining findings after HOPE showed more massive sinusoidal congestion and hepatocyte cytoplasmic vacuolization in group 1 than in group 2. The AST and LDH levels in group 2 at the start-up of IRM tended to be lower than in group 1. CONCLUSIONS: The combined use of in situ subnormothermic ECMO and HOPE is essential for the functional recovery of DCD liver grafts.

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.