Development of a Representative Mouse Model with Nonalcoholic Steatohepatitis

Jef Verbeek1, Ans Jacobs2, Pieter Spincemaille3, David Cassiman4

1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Maastricht University Medical Center, Maastricht, The Netherlands, 2 Department of Hepatology, University Hospitals KU Leuven, Leuven, Belgium, 3 Department of Laboratory Medicine, University Hospitals KU Leuven, Leuven, Belgium, 4 Metabolic Center, University Hospitals KU Leuven, Leuven, Belgium
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.1
Online Posting Date:  June, 2016
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Abstract

Non‐alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the Western world. It represents a disease spectrum ranging from isolated steatosis to non‐alcoholic steatohepatitis (NASH). In particular, NASH can evolve to fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure. The development of novel treatment strategies is hampered by the lack of representative NASH mouse models. Here, we describe a NASH mouse model, which is based on feeding non–genetically manipulated C57BL6/J mice a ‘Western style’ high‐fat/high‐sucrose diet (HF‐HSD). HF‐HSD leads to early obesity, insulin resistance, and hypercholesterolemia. After 12 weeks of HF‐HSD, all mice exhibit the complete spectrum of features of NASH, including steatosis, hepatocyte ballooning, and lobular inflammation, together with fibrosis in the majority of mice. Hence, this model closely mimics the human disease. Implementation of this mouse model will lead to a standardized setup for the evaluation of (i) underlying mechanisms that contribute to the progression of NAFLD to NASH, and (ii) therapeutic interventions for NASH. © 2016 by John Wiley & Sons, Inc.

Keywords: fibrosis; fructose; mouse model; NAFLD; NASH; sucrose

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of NASH In C57BL/6J Mice and In Vivo Phenotyping
  • Basic Protocol 2: Sacrifice, Sample Collection, and Analyses of Collected Tissues
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of NASH In C57BL/6J Mice and In Vivo Phenotyping

  Materials
  • 6‐week‐old male C57BL/6J mice (The Jackson Laboratory)
  • HF‐HSD with 44.6% of kcal derived from fat (of which 61% are saturated fatty acids) and 40.6% of kcal derived from carbohydrates (primarily sucrose 340 g/kg diet) (45% kcal Fat Diet; Harlan Laboratories Inc., cat. no. TD.08811; store up to 6 months at 4°C)
  • A regular NCD (ssniff R/M‐H; ssniff Spezialdiäten GmbH, cat. no. V1534‐000 ) as control
  • Glucose dissolved in normal saline 0.9 % NaCl
  • Standard housing cages
  • Balance
  • Scalpel
  • Bayer Contour glucometer and glucose test strips
  • Gastric gavage needles
  • 1‐ml syringes
  • PIXImus densitometer (Lunar Corp.) or EchoMRI (EchoMRI LLC)
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, )

Basic Protocol 2: Sacrifice, Sample Collection, and Analyses of Collected Tissues

  Materials
  • Mice from NASH‐induction experiment ( protocol 1)
  • Nembutal (Ceva) diluted to 5 mg/ml in normal saline (0.9% NaCl in distilled water)
  • Liquid nitrogen
  • Tissue‐Tek (Sakura Finetek, Inc.)
  • 2‐methylbutane (Sigma‐Aldrich)
  • Mouse Insulin ELISA Kit (Crystal Chem. Inc.)
  • Bayer Contour glucometer and glucose test strips
  • Dissecting board
  • Balance
  • Scissors
  • Tweezers
  • Marked microcentrifuge tubes
  • Heparinized 1 ml syringe prepared by flushing with Heparin LEO (LEO Pharma A/S)
  • Tissue‐Tek disposable vinyl specimen cryomolds (Sakura Finetek, Inc.)
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, ), blood collection by cardiac puncture (Donovan and Brown, ), and fixation and paraffin embedding of tissues (Zeller, ), H&E staining (Mangiavini et al., ), and Oil Red O and picrosirius red staining (Mark et al., )
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Figures

Videos

Literature Cited

Literature Cited
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