The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Summary

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Abstract

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

Introduction

The liver is the largest glandular metabolic organ of the body and has many complex functions. Key roles for the liver include digestion, metabolism, detoxification, storage of essential nutrients, production of blood plasma protein components, and immunity mediated through resident macrophages or Kupffer cells. The liver has a great ability to regenerate even if up to 70-90% of its total mass is lost. In the event of acute liver injury, such as seen following a partial hepatectomy or acetaminophen poisoning, the remaining healthy hepatocytes proliferate to repair the damage in a highly coordinated process¹. However, when the hepatocytes are ch....

Protocol

1. Animal Experimentation

All animal studies described in this investigation were approved by the Curtin University Animal Ethics Committee (Approval number: AEC_2014_28) prior to commencement of the experiments and performed in accordance with the Australian code for the care and use of animals.

1. Animals
   1. Use six-week old male C57BL/6J mice for the experiments.
2. Experimental design
   1. Following arrival at the Animal Facility, allow mice to acclimatize for four days prior to commencement of any experiments.
   2. House mice on wheaten chaff bedding,....

Results

Throughout the six-week time course of CDE-induced chronic liver injury, parameters were assessed on days 7 (induction phase), 14 and 21 (establishment phase) and 42 (maintenance phase). Compared to control mice, CDE-treated mice lost up to 20% of their initial body weight in an initial adaptation phase and tend to regain weight in the establishment and maintenance phases (Figure 1). The body weight was inversely correlated with serum alanine.......

Discussion

Chronic liver disease is often a silent disease with most patients being asymptomatic and it is one of the major contributors to morbidity and mortality worldwide. Chronic alcoholism and hepatitis C virus infection are the leading causes. Chronic liver injury is characterized by hepatic inflammation, fibrosis and in severe cases cirrhosis, carcinoma and ultimately liver failure. There is currently no cure available and although major advances have been made to understand the mechanisms of liver disease, new therapeutic a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Six-week-old male C57BL/6J mice	Animal Resource Centre of Western Australia, Murdoch, WA,  Australia	N/A
10 Kg Steam Cut Wheaten Chaff	Specialty Feeds, Glen Forrest, WA, Australia	N/A
Water for irrigation 1000ml bottle (Baxter)	Surgical House, Perth, WA, Australia	AHF7114A
Choline- deficient diet, modified (pellets)	MP Biomedicals Australasia Pty Limited, WA, Australia 02960210
DL-Ethionine	Sigma-Aldrich, Castle Hill, NSW,  Australia	E5139-25G
Ketamil injection	Troy Laboratories Pty Limited, Glendenning, NSW, Australia	N/A
Ilum Xylazil-20 injection	Troy Laboratories Pty Limited, Glendenning, NSW, Australia	N/A
27G x 1/2", Regular Wall Needle	Terumo Australia Pty Limited, NSW, Australia	NN-2713R
Syringes Terumo 1ml and 10ml	Terumo Australia Pty Limited, Macquarie Park, NSW, Australia	1018242, 1018037
Tissue-Tek OCT compound	VWR International Pty Limited, Tingalpa, QLD, Australia	25608-930
Neutral buffered formalin	Amber Scientific, Midvale, WA, Australia	NBF-2.5L
Ethanol absolute anaLaR normalpur	VWR International Pty Limited, Tingalpa, QLD, Australia	20821.33
Superfrost Plus slides	Grale Scientific Pty Limited, Ringwood, VIC, Australia	SF41296SP
Dako Protein Block, serum-free	Dako Australia Pty Limited, North Sydney, NSW, Australia	X090930-2
Dako antibody diluent	Dako Australia Pty Limited, North Sydney, NSW, Australia	s0809
rat anti-CD45	eBioscience, San Diego, California, USA	m0701	1/200 dilution
rabbit anti-panCK	Dako Australia Pty Limited, North Sydney, NSW, Australia	Z0622	1/300 dilution
Goat anti-rabbit (Alexa Fluor 488)	Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia	A-11008	1/500 dilution
Goat anti-rat IgG (Alexa Fluor 594)	Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia	A-11007	1/500 dilution
ProLong Gold Antifade Reagent with DAPI	Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia	P36935
Picrosirius Red Stain Kit	Polysciences Inc., Warrington, PA, USA	ab150681