Impact of Different Clinical Perfusates During Normothermic Ex Situ Liver Perfusion on Pig Liver Transplant Outcomes in a DCD Model.

BACKGROUND: Human albumin/dextran (HA-D), bovine-gelatin (BG), and packed red blood cells plus plasma have been used in European and North-American clinical trials of normothermic ex situ liver perfusion (NEsLP). We compared the effects of these perfusates in a porcine model during NEsLP and after transplantation. METHODS: Porcine livers were retrieved 30 minutes after circulatory death. After 5 hours of NEsLP, grafts were transplanted. Three groups (n = 6) were assessed (HA-D vs BG vs whole blood [WB]). One group of static cold storage (SCS) was evaluated for comparison with the perfusion groups. Hemodynamic variables, liver and endothelial injury, and function were assessed during NEsLP and posttransplantation. RESULTS: Hepatic artery flow was higher since the beginning of NEsLP in the HA-D group (HA-D, 238 ± 90 mL/min vs BG, 97 ± 33 mL/min vs WB, 148 ± 49 mL/min; P = 0.01). Hyaluronic acid was lower in the HA-D at the end of perfusion (HA-D, 16.28 ± 7.59 ng/µL vs BG, 76.05 ± 15.30 ng/µL vs WB, 114 ± 46 ng/µL; P < 0.001). After transplant, aspartate aminotransferase was decreased in the HA-D group when compared with the rest of the groups (HA-D, 444 ± 226 IU/L vs BG, 1033 ± 694 IU/L vs WB, 616 ± 444 IU/L vs SCS, 2235 ± 1878 IU/L). At 5 hours after transplant, lactate was lower in the HA-D group (HA-D, 3.88 ± 1.49 mmol/L vs BG, 7.79 ± 2.68 mmol/L vs WB, 8.16 ± 3.86 mmol/L vs SCS, 9.06 ± 3.54 mmol/L; P = 0.04). International Normalized Ratio was improved in HA-D group compared to the rest of the groups (HA-D, 1.23 ± 0.30 vs BG, 1.63 ± 0.20 vs WB, 1.50 ± 0.31 vs SCS, 1.97 ± 1.55; P = 0.03) after transplantation. In contrast, BG displayed lower aspartate aminotransferase levels during NEsLP (HA-D, 183 ± 53 IU/L vs BG, 142 ± 52 IU/L vs WB, 285 ± 74 IU/L; P = 0.01) and less cleaved-caspase-3 staining (HA-D, 2.05 ± 0.73% vs BG, 0.95 ± 1.14% vs WB, 1.74 ± 0.54% vs SCS, 7.95 ± 2.38%) compared with the other groups. On the other hand, the bile from the WB showed higher pH (HA-D, 7.54 ± 0.11 vs BG, 7.34 ± 0.37 vs WB, 7.59 ± 0.18) and lower glucose levels (HA-D, 0.38 ± 0.75 mmol/L vs BG, 1.42 ± 1.75 mmol/L vs WB, 0 ± 0 mmol/L) by the end of perfusion. CONCLUSIONS: Overall HA-D displayed more physiologic conditions during NEsLP that were reflected in less graft injury and improved liver function and survival after transplantation. Optimization of the perfusates based on the beneficial effects found with these different solutions would potentially improve further the outcomes through the use of NEsLP in marginal grafts.

PPAR-gamma activation is associated with reduced liver ischemia-reperfusion injury and altered tissue-resident macrophages polarization in a mouse model.

BACKGROUND: PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI. MATERIALS AND METHODS: Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+). RESULTS: Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs. CONCLUSION: PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.

Comparison of BQ123, Epoprostenol, and Verapamil as Vasodilators During Normothermic Ex Vivo Liver Machine Perfusion.

BACKGROUND: The optimal vasodilator to avoid hepatic artery vasospasm during normothermic ex vivo liver perfusion (NEVLP) is yet to be determined. We compared safety and efficacy of BQ123 (endothelin1 antagonist), epoprostenol (prostacyclin analogue), and verapamil (calcium channel antagonist). METHODS: Livers from porcine heart beating donors were perfused for 3 hours and transplanted into recipient pigs. Four groups were compared: group 1, livers perfused with a dose of 1.25 mg of BQ123 at baseline and at 2 hours of perfusion; group 2, epoprostenol at a continuous infusion of 4 mg/h; group 3, verapamil 2.5 mg at baseline and at 2 hours of perfusion; group 4, no vasodilator used during ex vivo perfusion. Liver injury and function were assessed during perfusion, and daily posttransplantation until postoperative day (POD) 3. All groups were compared with a cold storage group for postoperative graft function. RESULTS: Hepatic artery flow during NEVLP was significantly higher in BQ123 compared with verapamil, epoprostenol, and no vasodilator-treated livers. Aspartate aminotransferase levels were significantly lower with BQ123 and verapamil compared with epoprostenol and control group during perfusion. Peak aspartate aminotransferase levels were lower in pigs receiving BQ123 and verapamil perfused grafts compared with epoprostenol and control group. International Normalized Ratio, alkaline phosphatase, and total bilirubin levels were lower in the BQ123 and verapamil groups compared to epoprostenol group. Cold storage group had increased markers of ischemia reperfusion injury and slower graft function recovery compared to machine perfused grafts. CONCLUSION: The use of BQ123, epoprostenol, and verapamil during NEVLP is safe. Livers perfused with BQ123 and verapamil have higher hepatic artery flow and reduced hepatocyte injury during perfusion compared with epoprostenol. Hepatic artery flow is significantly reduced in the absence of vasodilators during NEVLP.