Animals, experimental design, and mouse models of NASH

In the present study, we used liver tissue samples from male C57BL/6J mice subjected to two different models of NASH induced by feeding: a high-fat diet [Stelic animal model (STAM)] and a CFD diet (CFD model). STAM is the first mouse model of NASH that depicts the sequential development of clinical and pathomorphologic features of NASH in diabetic patients (23). In brief, 2-d-old male C57BL/6J mice were injected with streptozotocin (200 μg/mouse). Starting from 4 wk of age, mice were continuously fed a high-fat diet (HFD-32; Clea, Tokyo, Japan; Supplemental Table 1) throughout the study. Control mice were maintained on standard animal chow for the duration of the study. Liver samples from male STAM mice in the steatotic (6 wk), NASH fibrotic (12 wk), and full-fledged (20 wk) HCC stages of liver carcinogenesis and from age-matched C57BL/6J mice were purchased from SMC Laboratories (Tokyo, Japan). These experimental procedures were performed according to the Japanese Pharmacological Society Guidelines and the experimental protocols were approved by the Research Animal Care and Use Committee of SMC Laboratories, Inc.

The CFD model of NASH is characterized by uniform morphologic features of human NASH (24) and is an ideal model for studying the subgroup of NASH patients with histologically advanced NASH (25). The in-life portion of the CFD study, mouse treatment, tissue sample collection, and liver histopathology are detailed in Pogribny et al. (12). In brief, mice in the CFD experimental group were maintained on a diet low in methionine (0.17% w/w) and lacking in choline and folic acid (diet no. 519541, CFD, iron-supplemented, and l-amino acid-defined diet; Dyets, Bethlehem, PA, USA; Supplemental Table 1) for 12 wk. Mice in the control group were fed the same diet supplemented with 0.4% methionine, 0.3% choline bitartrate, and 2 mg/kg folic acid. Mice from both groups were euthanized by exsanguination following deep isoflurane anesthesia 12 wk after diet initiation. These experimental procedures were reviewed and approved by the National Center for Toxicological Research Animal Care and Use Committee.

In addition to liver tissue samples from mice subjected to NASH-related liver carcinogenesis, HCC tissue samples were obtained from male C57BL/6J mice subjected to fibrosis- and inflammation-associated hepatocarcinogenesis induced by N,N-diethylnitrosamine (DEN) and carbon tetrachloride (CCl₄), as detailed in Uehara et al. (26). These animal experiments were approved by the University of North Carolina at Chapel Hill Animal Care and Use Committee.