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To resolve the critical donor shortage worldwide, enlarging the potential donor pool to include expanded criteria donors is necessary. Despite numerous attempts to establish new preservation solutions, no dramatic innovation has occurred since University of Wisconsin (UW) solution displaced Euro Collins' solution; UW solution remains the global gold standard. We previously developed a heavy water (D 2 O)-containing organ storage solution, Dsol, which is effective for livers subjected to extended cold storage (CS), and reported its effectiveness. Dsol is a modified UW solution; however, the substances or conditions that exhibit a synergistic or additive effect with D 2 O are unclear. Here we made UWD solution by removing hydroxyethyl starch (HES) from and adding 30%-D 2 O to UW solution, and compared the effects of these solutions. After 48 hours of CS, the livers were reperfused at 37 °C on an isolated perfused rat liver apparatus, and their perfusion kinetics, functions, and injuries were compared. In the UW group, portal vein resistance significantly increased and the oxygen consumption rate and bile production decreased; in contrast, these changes were suppressed in the UWD group. Organ expansion and liver damage progressed in both groups. These results confirmed that the removal of HES from and addition of D 2 O to the UW solution reduced CS-induced cellular function impairments and microcirculatory disorders. However, to reduce injury during reperfusion after CS, it is necessary to provide conditions that inhibit injury progression after reperfusion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Moto Fukai reports financial support was provided by Japan Society for the Promotion of Science. Tsuyoshi Shimamura reports financial support was provided by Japan Society for the Promotion of Science. Moto Fukai reports a relationship with Japan Society for the Promotion of Science that includes: funding grants. Tsuyoshi Shimamura reports a relationship with Japan Society for the Promotion of Science that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 (D 2 O)-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 D 2 O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D 2 O or DFX (mDsol-D 2 O[-], 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-D 2 O(-) 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: We have previously reported the efficacy of post-reperfusion H 2 gas treatment in cold storage (CS) and subsequent reperfusion of the rat liver. The present study aimed to evaluate the effect of H 2 gas treatment during hypothermic machine perfusion (HMP) in rat livers retrieved from donation after circulatory death (DCD) and elucidate the mechanism of action of H 2 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 H 2 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-H 2 groups. Liver enzyme leakage was suppressed by MP (vs control), whereas H 2 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-H 2 group. The apoptotic index in the CS and MP groups was high but decreased in the MP-H 2 group. Mitochondrial cristae were damaged in the CS group but preserved in the MP and MP-H 2 groups., Conclusions: In conclusion, HMP and H 2 gas treatment are partly effective in DCD rat livers but insufficient. Hypothermic machine perfusion can improve focal microcirculation and preserve mitochondrial ultrastructure., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 H 2 treatment group (UW-H 2 ), and the CS with Dsol and post-reperfusion H 2 group (Dsol-H 2 ). We first compared the Dsol-H 2 , UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H 2 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-H 2 , and Dsol-H 2 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Because of the serious organ shortage in Japan, the use of extended criteria (EC) donors is inevitable to increase the number of deceased donors. However, the influence of this practice on recipient outcomes has not been clarified yet. We analyzed donor and recipient factors to determine whether those factors, especially from EC donors impacted early recipient outcomes. From February 1999 to January 2011, 100 deceased liver transplantations were performed in Japan, including 85 consecutive adult cases (age≥18 years) who were studied to evaluate whether 6 recipient and 16 donor factors affected 3-month (90-day) recipient survival. Upon univariate analysis, Model for End-stage Liver Disease (MELD) score≥25 (P=.018), donor age≥55 years (P=.040), and cold ischemia time (CIT)≥10 hours (P=.00013) significantly reduced 3-month survival. Multivariate analysis confirmed the independent contributions of, three adverse factors including MELD score≥25 (P=.0133, odds ratio [OR]=12.3, 95% confidence interval [CI]=1.7-90.3), donor age≥55 years (P=.013, OR=14.0, 95% CI=1.6-119.5), and CIT≥10 hours (P=.0024, OR=67.6, 95% CI=4.5-1024.9). Three-month recipient survivals with 0, 1, 2, and 3 positive factors were 100% (n=34), 94.4% (n=36), 53.8% (n=13), and 0% (n=2), respectively (P<.0001). In conclusion, to improve recipient short-term survivals, minimizing CIT is the first priority. In the long-term, we must promote deceased donation to reduce recipient MELD scores by shortening the waiting time, and revise the allocation system to minimize CIT by giving priority to the local area., (Copyright © 2012 Elsevier Inc. All rights reserved.)