Twelve-hour normothermic liver perfusion in a rat model: characterization of the changes in the ex-situ bio-molecular phenotype and metabolism

Abstract

The partial understanding of the biological events that occur during normothermic machine perfusion (NMP) and particularly during prolonged perfusion might hinder its deployment in clinical transplantation. The aim of our study was to implement a rat model of prolonged NMP to characterize the bio-molecular phenotype and metabolism of the perfused organs. Livers (n = 5/group) were procured and underwent 4 h (NMP4h) or 12 h (NMP12h) NMP, respectively, using a perfusion fluid supplemented with an acellular oxygen carrier. Organs that were not exposed to any procedure served as controls (Native). All perfused organs met clinically derived viability criteria at the end of NMP. Factors related to stress-response and survival were increased after prolonged perfusion. No signs of oxidative damage were detected in both NMP groups. Evaluation of metabolite profiles showed preserved mitochondrial function, activation of Cori cycle, induction of lipolysis, acetogenesis and ketogenesis in livers exposed to 12 h-NMP. Increased concentrations of metabolites involved in glycogen synthesis, glucuronidation, bile acid conjugation, and antioxidant response were likewise observed. In conclusion, our NMP12h model was able to sustain liver viability and function, thereby deeply changing cell homeostasis to maintain a newly developed equilibrium. Our findings provide valuable information for the implementation of optimized protocols for prolonged NMP.

Figure 1

Oxygen consumption and release of markers of liver viability during the prolonged NMP procedure. (A) Oxygen delivery (DO₂) was stable throughout the perfusion procedure (p = 0.873), while oxygen consumption (VO₂), after an intial decrease from 0 to 2 h of perfusion, remained then stable; Two-way repeated measures ANOVA, Tukey’s post hoc test. (B) Lactate concentration decreased over time in both study groups (p < 0.001 vs time; p = 0.683 vs 4 h), despite 7.99 mmol/h lactate were continuously infused; Two-way repeated measures ANOVA, Tukey’s post hoc test. (C) and (D) Release of biomarkers of liver cell viability during NMP. Caspase cleaved cytokeratin 18 (CK18) and adenylate kinase (AK) were assessed in perfusate samples. One-way repeated measures ANOVA, Tukey’s post hoc test; p value vs Wash-out (W) ***p < 0.001, *p < 0.05; p value vs 1 h ^§§§p < 0.001, ^§§p < 0.01; p value vs 4 h ^###p < 0.001; p value vs 8 h ^°°°p < 0.001.

Figure 2

Histopathology analyses of the liver tissue at the end of prolonged NMP. (A) Representative of Hematoxylin and eosin (H&E—magnification 500x), Ki67 antigen (magnification 200x), PAS (magnification 50x) and TUNEL (magnification 50×) stain. (B) Graphical representation of Suzuki’s histological score and (C) Ki67 positive cells. Data are expressed as mean ± SEM.

Figure 3

Markers of oxidative stress during prolonged NMP. (A) An increased concentration of 8-hydroxydeoxyguanosine (8-OHdG) was observed in wash-out (W) samples, reflecting the oxidative stress suffered during liver procurement and cold storage; One-way repeated measures ANOVA, Tukey’s post hoc test; p value vs Wash-out (W): **p < 0.01. (B) Malondialdehyde (MDA) was measured to assess oxidative stress-dependent lipid peroxidation in snap-frozen liver biopsies; One-way ANOVA. (C) NAD+/NADH content in liver biopsies; One-way ANOVA, Tukey’s post hoc test; p value vs native: *p < 0.05; ***p < 0.001.

Figure 4

Cell metabolism and energy charge in livers subjected to prolonged NMP. (A) ATP content in liver tissue biopsies. Bars denote mean ± SEM; One-way ANOVA, Tukey’s post hoc test; p value vs native: *p < 0.05 NMR spectroscopy-based metabolomic analysis was performed to assess the concentration of specific metabolites in liver homogenates. (B) Top 18 significant metabolite heatmap illustrating individual sample metabolite concentration variation; Ward clustering algorithm, auto scaled concentration values between -2 and 2 (red—high, blue—low). (C) Energy metabolism-related metabolites lactate and glucose showed a reduced concentration in livers exposed to prolonged NMP. (D) UPD-glucuronate and UDP-glucose content was lower in perfused organs compared to native livers. (E) Acetate, citrate, and 3-hydroxybutyrate were induced by both short-term and prolonged NMP. (F) Oxalacetic acid, 1-methylnicotinamide, and taurine displayed increased concentrations in the NMP12h group. Bar plots illustrate mean ± SD; One-way ANOVA, Fisher’s post hoc test. White bar, white circles, native group; light grey bar, grey squares, NMP4h; black bar, grey squares, NMP12h. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 5

Schematic representation of the normothermic machine perfusion circuit. 1, thermal probe; 2, Bile duct tube; 3, portal vein cannula (portal flow 30 mL/min); 4, cava vein for perfusate drainage; (A) reservoir; (B) suction line used to remove perfusate; (C) inflow line to add fresh perfusate to the circuit (20 mL/h); (D) pump; (E) membrane oxygenator; (F) gas flow (50% O₂–5% CO₂ at a flow of 200 mL/min); G, bubble trap and heating coil with a stopcock for perfusate sampling.