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Int J Clin Exp Pathol 2013;6(12):2683-2696

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

Hiroko Itagaki, Kazuhiko Shimizu, Shunichi Morikawa, Kenji Ogawa, Taichi Ezaki

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan;
Department of Surgery, Tokyo Women’s Medical University Medical Center East, Tokyo, Japan

Received August 26, 2013; Accepted September 23, 2013; Epub November 15, 2013; Published December 1, 2013

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)

Address correspondence to: Dr. Taichi Ezaki, Department of Anatomy and Developmental Biology, Graduate School of Medicine, Tokyo
Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: 81-3-5269-7407; Fax: 81-3-5269-7407; E-mail: