| Objective:To expand the liver donor pool,marginal livers including Donation after Circulatory Death(DCD)has been considered.However,the risk of postoperative complications is increased in DCD liver transplantation,which limits their utilization.Regional perfusion assisted by Extracorporeal Membrane Oxygenation(ECMO)is an important technique to maintain DCD liver function.This study aimed to establish a rat model of DCD with ECMO regional perfusion and investigate the optimal perfusion temperature and potential mechanisms during ECMO regional perfusion.Methods:First,the breath-holding technique was used to induce asphyxia in rats,causing cessation of blood circulation throughout the body,to establish a DCD rat model.Then,after catheterization of the right jugular vein and right femoral artery,the thoracic aorta was clamped and blood was drained from the jugular vein and perfused through the femoral artery into the abdominal organs to establish an ECMO regional perfusion rat model.At the same time,an allogeneic liver transplantation model was established.Based on the successful establishment of the rat models,different warm ischemia time groups were set up,followed by the establishment of a simple static cold preservation group and an ECMO regional perfusion group.Then,rats were randomly divided into different perfusion temperature groups using a heat exchanger.Blood serum aspartate transaminase(AST)and alanine transaminase(ALT)levels were measured,and liver function and pathology were evaluated by HE staining of the liver tissues in the different groups.The postoperative survival rate of each liver transplantation group was calculated to determine the optimal warm ischemia time of the model.The reliability of the DCD regional perfusion rat model was also validated.Finally,the optimal perfusion temperature was determined,and the room temperature perfusion group and subnormal temperature perfusion group were selected for transcriptomic analysis to explore the possible mechanisms.By detecting inflammatory factors in blood and liver tissues of different temperature groups and using immunohistochemical staining,it was determined whether immune regulation is a possible mechanism for the effects of different temperature perfusion on liver function.Results:(1)Successfully established a rat DCD-ECMO regional perfusion model;(2)Comparing different groups with varying warm ischemic times,it was found that after 30 minutes of warm ischemia,liver enzyme levels in the blood increased rapidly in rats.In addition,severe congestion of the liver and massive death of liver cells and infiltration of inflammatory cells were observed through HE staining.(3)Compared with traditional static cold storage,DCD-ECMO regional perfusion in rats can improve liver function based on both ALT,AST levels and HE pathological scores.(4)Compared with traditional static cold storage,DCD-ECMO regional perfusion can improve the survival rate of rats after liver transplantation.(5)Among the different temperature perfusion groups,the ALT and AST levels in the room temperature group were lower than those in other temperature groups.HE staining showed that the liver injury pathological score was the lightest in the room temperature perfusion group,indicating that room temperature perfusion may be the most suitable regional perfusion temperature.(6)Transcriptomic analysis results showed that there were 491 differentially expressed genes between the normal temperature perfusion group and the low temperature perfusion group,with 342 up-regulated and 149 down-regulated genes in the low temperature perfusion group compared with the normal temperature perfusion group.Enrichment analysis of differentially expressed genes showed that immune regulation-related signaling pathways such as "TNF signaling pathway","cytokine-cytokine receptor interaction" and "Toll-like receptor signaling pathway"were significantly enriched,indicating that liver damage in the low temperature perfusion group is closely related to rat immune regulation.(7)Using immunohistochemical staining,neutrophils and macrophages were labeled by MPO and CD68 respectively.It was found that the infiltration of neutrophils and macrophages in the room temperature perfusion group of ECMO was significantly reduced compared with other temperature groups.(8)ELISA detection of the levels of inflammatory factors in the serum and liver tissue of different perfusion temperature groups showed that compared with other temperature groups,IL-6,IL-1β,TNF-α,and HMGB-1 were significantly decreased in the serum and liver tissue of the ECMO room temperature perfusion group.Conclusions:We have successfully established a rat model of DCD followed by ECMO regional perfusion,and found that room temperature perfusion is the optimal temperature for liver protection,possibly by inhibiting the inflammatory response.This study provides a mature and reliable rat model for subsequent research on DCD liver transplantatio,and identifies room temperature perfusion as the optimal temperature during regional perfusion. |
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