Hydrogen Rich Solution Attenuates Cold Ischemia-Reperfusion Injury in Rat Liver Transplantation

Introduction: Although liver transplantation is the only curative and es lished treatment for end-stage liver disease, patients that could have ben ted from transplantation are restricted by the severe donor organ short The transplantable whole-liver engineering using decellularization techn is one of the approaches to resolve this problem, however, coagulatio the decellularized liver stopped the blood flow and there was no study to ach clinical relevant recellularized liver grafts. Inappropriate cell distribution and c ulation with blood perfusion were tasks to be solved for practical use. The cu study proposed a method substituted for transplantation as clinical applicatio recellularized liver graft, reproducing appropriate cell distribution. Methods: The Lewis male rat livers were used for generating the wh organ decellularized scaffolds and cell isolation. Isolated primary hepatoc and liver sinusoidal endothelial cells (LSECs) were recellularized via bile and portal vein, respectively, and perfusion culture was performed. Histolo analysis was evaluated for cell distribution and function in recellularized After perfusion culture, we put our engineered liver graft into the extraco real perfusion system using an alive Lewis rat. Results: Histological analysis revealed appropriate distribution of hep cytes into the parenchymal space without intra-portal thrombosis, resu in LSECs attachment along the portal vein, maintaining characteristic phology and phenotype. Platelet deposition evaluated by measuring fluo cent intensity of integrin αIIb+ was significantly decreased in hepatocytes LSECs co-seeded group than in hepatocyte alone seeded group (p < 0.0 Blood clotting did not stop the blood flow during 3 h extracorporeal perfus Conclusion: Biliary duct seeding of parenchymal cells could allow endo lial cell attachment along the vasculature and did not obstruct blood flow vessels. LSECs could maintain their morphology and phenotype, and sess anti-coagulation ability in engineered liver grafts. Extracorporeal p sion might be one of the solutions for clinical use of recellularized liver g Further study is needed to investigate our fabricated liver graft could sup liver function in the extracorporeal perfusion system.


Methods:
The Lewis male rat livers were used for generating the wholeorgan decellularized scaffolds and cell isolation. Isolated primary hepatocytes and liver sinusoidal endothelial cells (LSECs) were recellularized via bile duct and portal vein, respectively, and perfusion culture was performed. Histological analysis was evaluated for cell distribution and function in recellularized liver. After perfusion culture, we put our engineered liver graft into the extracorporeal perfusion system using an alive Lewis rat.
Results: Histological analysis revealed appropriate distribution of hepatocytes into the parenchymal space without intra-portal thrombosis, resulting in LSECs attachment along the portal vein, maintaining characteristic morphology and phenotype. Platelet deposition evaluated by measuring fluorescent intensity of integrin αIIb+ was significantly decreased in hepatocytes and LSECs co-seeded group than in hepatocyte alone seeded group (p < 0.001). Blood clotting did not stop the blood flow during 3 h extracorporeal perfusion.
Conclusion: Biliary duct seeding of parenchymal cells could allow endothelial cell attachment along the vasculature and did not obstruct blood flow into vessels. LSECs could maintain their morphology and phenotype, and possess anti-coagulation ability in engineered liver grafts. Extracorporeal perfusion might be one of the solutions for clinical use of recellularized liver graft. Further study is needed to investigate our fabricated liver graft could support liver function in the extracorporeal perfusion system.
3. Stefanovich P. Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology. Introduction: Liver transplantation (LT) is considered as the standard treatment for end stage liver disease. However, there is a problem of donor shortage, and the need of grafts from marginal donors has increased. Attenuation of ischemia and reperfusion injury (IRI) in such marginal donors is crucial for less possibility of primary non-function and the graft loss. There have been some reports that hydrogen (H2) shows the antioxidant and anti-inflammatory effects, and eventually prevents IRI, in some non-hepatic transplant models [1] [2] . Therefore, we investigated whether the H2 attenuates IRI in LT model using rats.

Methods:
We made and used the H2 rich water bath (HRWB), in which the H2 ion was dissolved in the UW solution. Isogenic LT model of Lewis rats was used. Without arterial reconstruction, orthotopic LT was performed according to Kamada's cuff method. The animals were divided into four groups; sham operation (Sham), not preserved (NP), preserved 12 hours in UW solution (UW), preserved 12 hours in H2 rich UW solution (UW+H2). H2 ion solution in the graft liver was measured every hour after preservation in the preliminary study. Blood and tissue samples were corrected 6 hours after the reperfusion. Hepatic enzymes in serum were measured. Pathological findings including the expressions of cytokines and heme oxygenase-1 (HO-1) in liver tissues were evaluated.
Result: H2 concentration of graft tissue increased depending on the storing time in the HRWB, and it became plateau after 1 hour. AST, ALT, and LDH levels of serum showed significantly lower in UW+H2 groups. In the UW group, liver histology showed focal hemorrhage, cell ballooning, and infiltration of neutrophils and macrophages, and those findings were much attenuated in the UW+H2 group. UW+H2 group also showed less oxidative damage and hepatocyte apoptosis. UW+H2 groups tended to have lower proinflammatory cytokines and higher HO-1 levels in mRNA expressions, and protein levels of HO-1 increased significantly.
Conclusion: By using the HRWB, sufficient H2 distribution in the liver graft was obtained. Storage of the liver grafts in H2 rich UW solution presented superior functional and morphologic protection for IRI. Up-regulation of HO-1 was suggested as one mechanism of this effect. Result of our present study demonstrated that H2 rich solution decrease oxidative stress and inflammatory changes by IRI in rat LT model.