A fast and accurate mouse model for inducing non-alcoholic steatohepatitis

Abstract

Aim: This study aimed to perform a head-to-head comparison of changes during NASH progression throughout 6-11 weeks of an experiment to supply a faster nutritional model in mimicking NASH to decrease the duration and cost of in vivo studies.

Background: New therapies are urgently needed because of the growing prevalence of non-alcoholic steatohepatitis (NASH) and the lack of an effective treatment approach. Currently, dietary interventions are the most efficient options.

Methods: This study compared features of NASH in a murine model using protocol that combined special nutritional regimes based on the combination of 21.1% fat, 41% sucrose, and 1.25% cholesterol with weekly intraperitoneal injections of carbon tetrachloride (CCl4). Male C57BL/6J mice received either special compositions + CCl4 (NASH group) or standard chow diet (healthy control group) for 11 weeks. Liver histopathology based on hematoxylin and eosin (H&E) and Masson's Trichrome (TC) staining and biochemical analyses were used to assess disease progression.

Results: In C57BL/6J mice administered a high fat, high cholesterol, high sucrose diet and CCl4 for 8 weeks, steatohepatitis with pronounced hepatocyte ballooning, inflammation, steatosis, and fibrosis was observed. According to the NAFLD activity scoring system, the maximum NAS score was manifested after 8-9 weeks (NAS score: 6.75). Following this protocol also led to a significant increase in AST and ALT, total cholesterol, and total triglyceride serum levels in the NASH group.

Conclusion: Following the special nutritional regime based on high fat, cholesterol, and sucrose in combination with CCL4 injections resulted in a NASH model using C57BL/6J mice in a shorter time compared to similar studies. The obtained histopathological NASH features can be advantageous for preclinical drug testing.

Keywords: Animal model; Carbon tetrachloride; Liver diseases; Nonalcoholic; Steatohepatitis.

Figure 1

Protocol for the control and NASH experimental groups. Mice were categorized into 2 main groups: the healthy control group and the NASH group. NASH mice were separated into 3 subgroups. In each group, mice were sacrificed one week after the final injection. (ND: normal diet, PBS: phosphate-buffered saline, WD: western diet, CCl4: Carbon tetrachloride)

Figure 2

Histopathological analysis of liver section at 6-11 weeks post-induction in comparison with the control group. A) Macroscopic view of the liver tissue from control and NASH mice. H&E stains of liver samples (magnification: X100 and X400) represent steatosis (black arrows), lobular inflammation (blue arrows), ballooning (gray arrows), and TC stains (magnification X100) represent fibrotic septa (red arrows) (scale bar: 100µm). B) Histological scoring for steatosis, hepatocyte ballooning, lobular inflammation. Data is expressed as mean ± SEM and compared by one-way ANOVA (*p <0.05, **p <0.01, ****p <0.0001)

Figure 3

(A) total triglyceride, (B) total cholesterol, (C) serum AST, and (D) ALT were measured in the 8-9 weeks treatment group in comparison with controls. Data is expressed as mean ± SEM and compared by t-test (*p<0.05, **p<0.01, ***p<0.001; ALT, alanine aminotransferase; AST, aspartate aminotransferase)