Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death

Abstract

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H⁺]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Keywords: DCD; donation after circulatory death; hypothermic machine perfusion; liver; machine perfusion; mitochondria; osmium-maceration; pH; transplantation.

Figure 1

(a) Perfusate pH during HMP at 7–10 °C: Although the pH of UW-MPS^® was adjusted to 7.6, 7.8, and 8.0 before starting the HMP, it gradually dropped with perfusion. Therefore, the pH was adjusted by adding NaOH during the HMP. In contrast, the pH decreased in the MP Group; (b) Portal vein resistance during reperfusion at 37 °C The graft was perfused at a constant pressure (8 cmH₂O in the CT group and 12 cmH₂O in the other groups) on an IPRL. PVR maintained the lowest value in the CT group compared to the other groups throughout reperfusion. The PVR was 8.0 to 9.0 (cmH₂O/mL × min × g liver) in the CS group and 9.0 to 10.0 (cmH₂O/mL × min × g liver) in MP and MP-pH groups with no significant differences.

Figure 2

(a–c) Liver enzyme leakage in perfusate at 90 min after reperfusion. AST, ALT, and LDH in the MP and MP-pH groups were significantly lower than those in the CS group. Only AST in the MP-pH 7.8 group appeared to be significantly lower than that of the MP group. (d) OCR at 90 min after reperfusion. OCR was highest in the CT group. There were no significant differences between the CS and MP-pH groups. (e) Bile production during reperfusion: Bile production was highest in the CT group and significantly reduced in the CS group. Bile production in the MP-pH 7.6, 7.8, and 8.0 groups was significantly higher than that in the CS group. (f) Apoptotic index. TUNEL-positive cells were undetectable in the CT group. The TUNEL-positive cell ratio was increased in the CS and MP groups, whereas it was decreased in the MP-pH 7.8 group. Data are expressed as means ± SD (n = 6). *,†: A p-value less than 0.05 was considered significant: *: CS versus MP and MP-pH groups, †: MP versus MP-pH groups.

Figure 3

Liver histopathological examination at 90 min after reperfusion: (a) HE staining showed an almost normal appearance in the CT group. Vacuolization and condensed or swollen nuclei with heterogeneous staining were observed in the CS and MP groups, whereas these findings were suppressed in the MP-pH groups, especially at pH 7.8. (b) TUNEL staining showed the highest positive cell ratio in the CS group and the least in the CT group. The number of positive cells tended to decrease in MP-pH groups.

Figure 4

Liver scanning electron microscopy (SEM) findings at 90 min after reperfusion: The blue areas are nuclei, and the green areas are mitochondria. The mitochondria in the MP-pH 7.6 and pH 8.0 groups had pendulous, flat structures, few rod-like structures, and low cristae density (arrow). In contrast, rod-like structures and dense cristae were preserved in the pH 7.8 group. Single hepatocyte in MP-pH 7.6, 7.8, and 8.0 groups were presented, respectively (A,C,E), and subfigures (higher magnification; B,D,F). The photos in the right column are the enlarged version of the middle column. The scale bar in the figure represents 1 µm.

Figure 5

Autophagy and inflammatory signals by western blotting. The degree of phosphorylation was represented by the ratio of signal intensity derived from against phosphorylated and pan antibodies (p/pan). (a) AMPK-α (p/pan), (b) SQSTM-1 (p/pan), (c) ATG5, (d) PARKIN, (e) PINK-1, (f) cytosolic JNK (p/pan), and (g) mitochondrial JNK (p/pan). The band intensity was normalized by the total protein in (c–e). The representative photos were shown in the lower right. There were no differences among all groups for all signals.

Figure 6

Vitamin B2 in the effluent. (a) FMN showed an increasing trend over time in all MP and MP-pH groups. MP-pH 7.8 showed a significantly lower value at the end of 180 min of HMP as compared to that of the MP group. (b,c) FAD and RF did not show any significant changes among the groups. Data are expressed as means ± SD (n = 6). *: A p-value less than 0.05 was considered significant: *: MP versus MP-pH groups.