Morphological and functional characterization of non-alcoholic fatty liver disease induced by a methionine-choline-deficient diet in C57BL/6 mice

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. Its histopathology and the effects of nutrition on liver function have not been fully determined.

Aim: To elucidate the cellular mechanisms of NAFLD induced by a methionine-choline-deficient (MCD) diet in mice. Particular focus was placed on the role of phagocytic cells.

Methods: Male C57BL/6 mice were fed an MCD diet for 30 weeks. A recovery model was also established wherein a normal control diet was provided for 2 weeks after a period of 8, 16, or 30 weeks.

Results: Mice fed the MCD diet for ≥ 2 weeks exhibited severe steatohepatitis with elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Steatohepatitis was accompanied by the infiltration of CD68-positive macrophages (Kupffer cells). The severity of steatohepatitis increased in the first 16 weeks but was seen to lessen by week 30. Fibrosis began to develop at 10 weeks and continued thereafter. Steatohepatitis and elevated serum hepatic enzyme concentrations returned to normal levels after switching the diet back to the control within the first 16 weeks, but fibrosis and CD68-positive macrophages remained.

Conclusions: The histopathological changes and irreversible fibrosis seen in this model were caused by prolonged feeding of an MCD diet. These results were accompanied by changes in the activity of CD68-positive cells with temporary elevation of CCL-2, MMP-13, and MMP-9 levels, all of which may trigger early steatohepatitis and late fibrosis through phagocytosis-associated MMP induction.

Keywords: CD68 antigen; Kupffer cells; liver fibrosis; matrix metalloproteinase (MMP); mouse; non-alcoholic fatty liver disease (NAFLD).

Figure 1

Changes in body weight and liver weight. A: Direct comparison of changes in body weight (circles) and liver weight (triangles) between the control diet (open marks) and MCD diet (closed marks) group mice. Effects of diet reverse at 8, 16, and 30 weeks on body weight (B) and liver weight (C). Control diet (control group: open columns), MCD diet (group M: black column), and reversed diet (group R: gray columns). **P<0.01, ND (not determined).

Figure 2

Serum levels of liver-derived enzymes. Serum levels of AST (A) and ALT (B) were measured. Note that the levels of both enzymes in group M (black columns) increased from 2 to 16 weeks, but markedly decreased at 30 weeks. In group R (gray columns) the levels of both enzymes decreased to almost normal. *P<0.05, ND (not determined).

Figure 3

H&E staining of liver sections in groups M and R. A: Normal control liver section. B-F: Liver sections of the MCD diet group (group M) fed for 2 weeks (B), 4 weeks (C), 8 weeks (D), 16 weeks (E), and 30 weeks (F). G-I: Liver sections of the reversed diet group (group R) at 8 weeks (G), 16 weeks (H), and 30 weeks (I). Bar=5 μm in all panels.

Figure 4

Development of hepatic steatosis by the MCD diet and effect of diet reverse. Oil Red O staining of the liver in controls (A), group M (B: 2 weeks, C: 4 weeks, D: 8 weeks, E: 16 weeks, and F: 30 weeks), and group R (G: 8 weeks, H: 16 weeks, and I: 30 weeks). Note that fat deposits developed from 2 to 16 weeks, but deposit levels were reduced at 30 weeks in group M. In group R (G-I), the fat deposition areas decreased remarkably. Bar=10 μm in all panels. J: Quantification of fat deposition in the liver. Areas of lipid deposition in liver sections were identified by Oil Red O staining and expressed as multiple numbers; the control level was set to 1, as described in the Materials and Methods. Note that the Oil Red O-stained areas increased significantly from 2 weeks in group M, but decreased almost completely in group R. **P<0.01, ND (not determined).

Figure 5

Quantification of PCNA-positive cells in the liver. The average number of PCNA-positive cells was counted in group M (black columns) and group R (gray columns) liver sections. The number of PCNA-positive cells increased significantly from 2 weeks in group M. In group R, they decreased significantly after changing the diet back to control. **P<0.01, ND (not determined).

Figure 6

Histological evaluation of hepatic fibrosis by Azan staining. A: Control liver section. Sections of MCD-diet-fed livers from group M after 2 weeks (B), 4 weeks (C), 8 weeks (D), 16 weeks (E), and 30 weeks (F). Liver sections of the reversed diet group R at 8 weeks (G), 16 weeks (H), and 30 weeks (I). Note that fibrosis typically progressed to stage 3 in portal areas and bridging areas after 16 weeks in groups M and R. Bar=10 μm in all panels.

Figure 7

Evaluation of NAS and hepatic fibrosis. A: NAFLD Activity Score (NAS). B: Ten-week fibrosis scores in groups M and R. NAFLD estimation by NAS and fibrosis scores is shown in Table 1. ND (not determined).

Figure 8

Immunohistochemical identification of CD68-positive cells in liver sections. A: Normal control liver section. Liver sections in group M at 2 weeks (B), 4 weeks (C), 8 weeks (D), 16 weeks (E), and 30 weeks (F). Liver sections in group R at 8 weeks (G), 16 weeks (H), and 30 weeks (I). CD68-positive cells became swollen at 16 weeks in groups M and R. Bar=10 μm in all panels. J: Quantification of CD68-positive cells in the liver. The number of CD68-positive cells per view field in liver sections was compared between groups M (black columns) and R (gray columns). The number of CD68-positive cells increased significantly in group M from 2 weeks and continued thereafter. **P<0.01, ND (not determined).

Figure 9

Ultrastructures at the semi-thin section level. A: Normal control liver section. Liver sections in group M at 2 weeks (B), 8 weeks (C), 16 weeks (D), and 30 weeks (E). Swollen cells appeared among hepatocytes from 16 weeks in group M (D and E). F: Immunohistochemical identification of swollen cells with a CD68 marker in semi-thin sections. A liver section in group M at 16 weeks. A typical swollen CD68-positive cell is seen. Bar=1 μm applies to all panels.

Figure 10

mRNA levels of CCL-2, MMP-13, and MMP-9 by real-time PCR. The mRNA levels of CCL-2 (A), MMP-13 (B), and MMP-9 (C) were determined and evaluated as arbitrary units, whereas those of normal control livers were set to 1. MCD-diet-fed group M (black columns). Reversed-diet-fed group R (gray columns). **P<0.01.

Figure 11

Summary of the histopathological changes in MCD-diet-induced NAFLD.