Hypothermic machine perfusion with metformin-University of Wisconsin solution for ex vivo preservation of standard and marginal liver grafts in a rat model

Figure 1 Analysis of biochemical indicators in the perfused liquid. Aspartate aminotransferase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) levels (mean ± SD) in the perfused liquid of the ex vivo livers of the three groups: CTRL (control groups A and D), UWP (UW solution perfusion groups B and E), and MUWP (metformin perfusion groups C and F). Data are presented as mean ± SD (n = 6) and compared by two-way analysis of variance and the Sidak's multiple comparisons test.

Figure 2 Analysis of inflammatory factors in the perfused liquid. The IL-18 and TNF-α levels in the perfused liquid (mean ± SD) of the ex vivo livers of the three groups: CTRL (control groups A and D), UWP (UW solution perfusion groups B and E), and MUWP (metformin perfusion groups C and F). Data are presented as mean ± SD (n = 6) and compared by two-way analysis of variance and the Tukey's and Sidak's multiple comparisons tests. ^aP < 0.05 vs group B; ^bP < 0.05 vs group E; ^cP < 0.05 vs group B; ^dP < 0.05 vs group E.

Figure 3 Histograms of relative gray values of the expression levels of p-eNOS, t-eNOS, p-AMPK, and t-AMPK in the liver sinusoidal endothelial cells of young rats. AMPK/eNOS pathway was activated ex vivo by metformin. A: Phosphorylation of AMPK and eNOS was increased in the MUWP group at 12 h after HMP; B-E: Quantitative analysis of total AMPK, total NOS, phosphorylated AMPK, and eNOS expression in CTRL (control groups A and D), UWP (UW solution perfusion groups B and E), and MUWP (metformin perfusion groups C and F) groups. Data are presented as mean ± SD (n = 3) and compared by ordinary one-way analysis of variance and the Dunnett's multiple comparisons test. ^aP < 0.05 vs CTRL group; ^bP < 0.05 vs CTRL group; ^cP < 0.05 vs UWP group.

Figure 4 Staining of the slices of ex vivo rat liver tissue. A and D: The young control group A and the aged control group D, respectively; B and E: The young group B and aged group E of UWP, respectively; C and F: The young group C and the aged group F of MUWP, respectively; G: Histopathologic scoring of structural injury in each group. Data are presented as mean ± SD (n = 6) and compared by Kruskal-Wallis one-way analysis of variance on ranks and Mann-Whitney U test. ^aP < 0.05 vs group B of UWP; ^bP < 0.05 vs group E of UWP. Original magnification (A-F): × 400. UWP: University of Wisconsin solution perfusion; MUWP: Metformin perfusion solution.

Figure 5 Ultrastructural changes of hepatocytes. A and D: Hepatocytes of the young control group A and the aged control group D, respectively; B and E: Hepatocytes of the young group B and aged group E of UWP without metformin, respectively; C and F: Hepatocytes of the young group C and aged group F of MUWP, respectively. The black arrows indicate the nuclei, the white arrows indicate the intracellular mitochondria of the young groups, and the white triangles indicate the intracellular mitochondria of the aged groups. UWP: University of Wisconsin solution perfusion; MUWP: Metformin perfusion solution. Original magnification (A-F): ×10000.

Figure 6 Ultrastructural changes of liver sinusoidal endothelial cells. A and D: Sinusoidal endothelial cells of the young control group A and the aged control group D, respectively; B and E: Sinusoidal endothelial cells of the young group B and aged group E of UWP, respectively; C and F: Sinusoidal endothelial cells of the young group C and aged group F of MUWP, respectively. UWP: University of Wisconsin solution perfusion; MUWP: Metformin perfusion solution. Original magnification (A–F): ×10000.