Distinctive protein expression in elderly livers in a Sprague-Dawley rat model of normothermic ex vivo liver machine perfusion.

BACKGROUND: Liver grafts are frequently declined due to high donor age or age mismatch with the recipient. To improve the outcome of marginal grafts, we aimed to characterize the performance of elderly vs. young liver grafts in a standardized rat model of normothermic ex vivo liver machine perfusion (NMP). METHODS: Livers from Sprague-Dawley rats aged 3 or 12 months were procured and perfused for 6 h using a rat NMP system or collected as a reference group (n = 6/group). Tissue, bile, and perfusate samples were used for biochemical, and proteomic analyses. RESULTS: All livers cleared lactate during perfusion and continued to produce bile after 6 h of perfusion (614 mg/h). Peak urea levels in 12-month-old animals were higher than in younger animals. Arterial and portal venous pressure, bile production and pH did not differ between groups. Proteomic analysis identified a total of 1477 proteins with oxidoreductase and catalytic activity dominating the gene ontology analysis. Proteins such as aldehyde dehydrogenase 1A1 and 2-Hydroxyacid oxidase 2 were significantly more present in livers of older age. CONCLUSIONS: Young and elderly liver grafts exhibited similar viability during NMP, though proteomic analyses indicated that older grafts are less resilient to oxidative stress. Our study is limited by the elderly animal age, which corresponds to mature but not elderly human age typically seen in marginal human livers. Nevertheless, reducing oxidative stress could be a promising therapeutic target in the future.

Quality Assessment by Bile Composition in Normothermic Machine Perfusion of Rat Livers.

Background: The persistent challenge of organ scarcity in liver transplantation leads to an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) is used for improved preservation. Due to the mimicked in vivo conditions during normothermic machine perfusion, the liver is metabolically active, which allows quality assessment during perfusion. Bile seems to be of rising interest in clinical studies, as it is easily collectible for analysis. As there are currently no data on biliary bile acids during NMP, the primary objective of this study was to use our experimental rodent NMP model to assess changes in bile composition through organ damage during perfusion to inform clinical evaluation of donor organs during NMP. Methods: Thirty livers from male Sprague-Dawley rats in five groups underwent 6 h of NMP using either erythrocyte-supplemented DMEM or Steen solution, with or without 30 min of warm ischemia time (WIT). We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at 3-hour intervals. Bile samples were analyzed for biliary pH, LDH, and gamma glutamyltransferase, as well as biliary bile acids via mass spectrometry and UHPLC. Results: Compared with regular livers, liver injury parameters were significantly higher in our donation after circulatory death (DCD) model. Bile production was significantly reduced in livers exposed to WIT, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers experiencing WIT. However, regular livers produced a higher total amount of biliary bile acids during perfusion. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced during perfusion due to the missing enterohepatic circulation. Conclusions: WIT-induced liver injury affects bile composition within our small-animal NMP model. We hypothesize this phenomenon to be attributed to the energy-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile. Our results may inform clinical studies, in which biliary bile acids might have a potential as a quantifiable viability marker in human NMP liver transplantation studies.

Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome.

BACKGROUND: Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess potential metabolic changes in response to differing enrichment levels. METHODS: We conducted a six-month study involving 24 male Sprague Dawley rats, randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an activity focused rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed. RESULTS: Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p = 0.007). We observed 1871 distinct proteins in liver tissue, with 77% being common across groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p = 0.024 and p = 0.009). Rats in the intervention groups with weekly playtime displayed the least amount of reported distress during inspection or upon room entry and were less prone to accepting treats. Removing animals from their enclosure was most effortless for those in the large cage group. Over time, there was a decrease in conflicts among rats that interacted only twice weekly during playpen time. DISCUSSION: In summary, refining husbandry practices for aging rats is both simple and budget-friendly, with no apparent adverse effects on stress levels, animal development, or relevant metabolic changes in the liver.

Validation of risk scores for allograft failure after liver transplantation in Germany: a retrospective cohort analysis.

A growing number of clinical risk scores have been proposed to predict allograft failure after liver transplantation. However, validation of currently available scores in the Eurotransplant region is still lacking. We aimed to analyze all clinically relevant donor and recipient risk scores on a large German liver transplantation data set and performed a retrospective cohort analysis of liver transplantations performed at the Charité-Universitätsmedizin Berlin from January 2007 until December 2021 with organs from donation after brain death. We analyzed 9 previously published scores in 906 liver transplantations [Eurotransplant donor risk index (ET-DRI/DRI), donor age and model for end-stage liver disease (D-MELD), balance of risk (BAR), early allograft dysfunction (EAD), model for early allograft function (MEAF), liver graft assessment following transplantation (L-GrAFT7), early allograft failure simplified estimation (EASE), and a score by Rhu and colleagues). The EASE score had the best predictive value for 3-month, 6-month, and 12-month graft survival with a c-statistic of 0.8, 0.77, and 0.78, respectively. In subgroup analyses, the EASE score was suited best for male recipients with a high-MELD (>25) and an EAD organ. Scores only based on pretransplant data performed worse compared to scores including postoperative data (eg, ET-DRI vs. EAD, p<0.001 at 3-month graft survival). Out of these, the BAR score performed best with a c-statistic of 0.6. This a comprehensive comparison of the clinical utility of risk scores after liver transplantation. The EASE score sufficiently predicted 12-month graft and patient survival. Despite a relatively complex calculation, the EASE score provides significant prognostic value for patients and health care professionals in the Eurotransplant region.