Matrix metalloproteinase-9 contributes to parenchymal hemorrhage and necrosis in the remnant liver after extended hepatectomy in mice

Figure 1 Histological and hematological differences between groups I and II at 6 h after 80%-partial hepatectomy. A: Histological findings in group I mice; B: Representative images of remnant liver histology demonstrating significant multiple foci of hemorrhage and necrosis in group II mice; C: Quantitation of the size (area percentage) of hemorrhage and necrosis: Significantly larger areas and more hemorrhagic and necrotic foci were observed in group II mice than in group I mice (^aP < 0.05); D: Quantitation of the number of foci of hemorrhage and necrosis per mm²: Significantly larger areas and more hemorrhagic and necrotic foci were observed in group II mice than in group I mice (^bP < 0.01); E: Aspartate aminotransferase (AST) levels were significantly higher in group II mice than those in group I mice (^aP < 0.05); F: Alanine aminotransaminase (ALT) levels were significantly higher in group II mice than those in group I mice (^aP < 0.05); G: Total bilirubin levels were significantly higher in group II mice than those in group I mice (^bP < 0.01).

Figure 2 Western blotting analysis for matrix metalloproteinase-9 in remnant livers with (group II) and without (group I) foci of hemorrhage and necrosis after 80%-partial hepatectomy. Representative immunoblot (A) and histogram (B) show enhanced expression of matrix metalloproteinase (MMP)-9 protein in group II mice compared with that in group I mice and sham controls (^bP < 0.01).

Figure 3 Immunohistochemical staining for matrix metalloproteinase-9 in the remnant liver. A: Representative images of matrix metalloproteinase (MMP)-9 staining in a non-necrotic area in group II; B: Representative images of MMP-9 staining in a necrotic area in group II; C: Histogram of MMP-9 expression as described in the material and methods section: Enhanced MMP-9 expression was observed in areas close to the focus of necrosis (^aP < 0.05).

Figure 4 Co-localization analysis by dual immunofluorescence in the remnant liver after 80%-partial hepatectomy. Co-localization analysis by dual immunofluorescence in the remnant liver for (A) matrix metalloproteinase (MMP)-9 labeled in red (Alexa Fluor 568), (B) CD68 labeled in green (Alexa Fluor 488), and (C) both MMP-9 and CD68; for (D) MMP-9 labeled in red (Alexa Fluor 568), (E) desmin labeled in green (Alexa Fluor 488), and (F) both MMP-9 and desmin; and for (G) MMP-9 labeled in red (Alexa Fluor 568), (H) CD11b labeled in green (Alexa Fluor 488), and (I) both MMP-9 and CD11b. These co-localization results demonstrate that MMP-9 protein expression is mainly localized in CD11b-positive cells, and to a lesser extent in desmin-positive cells.

Figure 5 Histological analysis in each strain. Histological analysis between wild-type (WT, n = 15) (A), matrix metalloproteinase (MMP)-9(-/-) (n = 15) (B), and tissue inhibitors of metalloproteinases (TIMP)-1 (-/-) mice (n = 14) (C) at 6 h after 80%-partial hepatectomy (PH). Representative images of remnant liver histology at 6 h after 80%-PH in WT, MMP-9(-/-), and TIMP-1(-/-) mice. Foci of hemorrhage and necrosis are denoted by white arrows. The percentage of the necrotic area (D) and the number of necrotic foci per mm² (E) in remnant livers of study mice are shown. Significantly smaller and fewer necrotic foci in the remnant liver were observed in MMP-9(-/-) mice compared with WT and TIMP-1(-/-) mice (^aP < 0.05, ^bP < 0.01).

Figure 6 Expression and activity of matrix metalloproteinase-9. Expression and activity of matrix metalloproteinase (MMP)-9 were determined using (A) western blotting analysis for MMP-9 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control, (B) polyacrylamide gel electrophoresis gelatin zymography with human MMP-9 and -2 standards (S), and in situ fluorescence gelatin zymography in the remnant livers of wild-type (WT), MMP-9(-/-), and tissue inhibitors of metalloproteinases (TIMP)-1(-/-) mice at 6 h after 80%-partial hepatectomy. We observed MMP-9 protein expression and activity in WT mice (C), but they were absent in MMP-9(-/-) mice (D). Enhancement of MMP-9 protein expression and activity in TIMP-1(-/-) mice was observed (E).

Figure 7 Immunohistochemical analysis of accumulated neutrophils with myeloperoxidase staining in remnant livers of wild-type, matrix metalloproteinase-9(-/-), and tissue inhibitors of metalloproteinases-1(-/-) mice at 6 h after 80%-partial hepatectomy. Representative images of myeloperoxidase staining, labeled in green (Alexa Fluor 488), of cytoplasmic azurophilic granules in neutrophils (arrows) in wild-type (WT) mice in remnant liver sections in (A) WT, (B) matrix metalloproteinase (MMP)-9(-/-), and (C) tissue inhibitors of metalloproteinases (TIMP)-1(-/-) mice. A histogram shows the number of accumulated neutrophils in the remnant liver (D). Significantly fewer neutrophils were observed in the remnant liver in MMP-9(-/-) mice than in WT and TIMP-1(-/-) mice (^aP < 0.05, ^bP < 0.01).

Figure 8 Effect of matrix metalloproteinase-9 monoclonal antibody on 80%-partial hepatectomy. A, B: Treatment with matrix metalloproteinase-9 monoclonal antibody (MMP-9 mAb) in mice with 80%-partial hepatectomy (PH) resulted in a significant reduction in the size (percent area) and number of hemorrhagic and necrotic foci 6 h after 80%-PH compared with treatment using control IgG (^aP < 0.05); C, D: Suppression of MMP-9 activity as determined by in situ fluorescence gelatin zymography compared with control IgG-treated mice; E, F: There was significantly less myeloperoxidase staining (arrows) in remnant liver sections in mice treated with MMP-9 mAb compared with that in control IgG-treated mice; G: A histogram shows the number of accumulated neutrophils in the remnant liver 6 h after 80%-PH. Significantly fewer neutrophils were observed in MMP-9 mAb-treated mice than in control IgG-treated animals (^aP < 0.05).