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 chronically injured due to long-term viral infection, alcoholic or non-alcoholic fatty liver disease, the inflammatory microenvironment triggers the activation of fibrosis-driving hepatic stellate cells and the proliferation of liver progenitor cells (LPCs) with the potential to differentiate into either cholangiocytes or hepatocytes^2,3,4,5. The precise origin, differentiation fate of LPCs, their contribution to liver regeneration, and hepatocarcinogenesis have been topics of intense debate and most likely depend on the injury severity and context². The order of early regeneration-associated events is also controversially discussed, with some investigators stating that hepatic stellate cell activation and matrix remodeling is essential for generation of a LPC-favoring niche⁶, while others report that LPC expansion and the so-called Ductular Reaction are required to trigger fibrogenesis⁷. There are numerous animal models to study specific aspects of injury and regeneration, in an attempt to understand all the underlying factors that regulate disease progression and to ultimately develop new treatment strategies for patients⁸.

The choline-deficient and ethionine-supplemented (CDE) dietary model was originally developed for use in rats and later modified for chronic liver injury induction in mice^9,10. Dietary deficiency of choline results in impaired assembly and secretion of very low-density lipoproteins. Combined with the hepatocarcinogen DL-ethionine, this regimen leads to excessive hepatic fat loading, continuous inflammation, periportal fibrosis, LPC response and long-term to hepatocellular carcinoma development^11,12. However, importantly, different mouse strains exhibit distinctive patterns of inflammatory, fibrogenic and LPC response dynamics¹³. This protocol describes chronic liver injury induction in C57BL/6J mice, the most commonly used inbred mouse strain.

In chronic liver disease research, typical analyses include histological assessments by hematoxylin and eosin as well as Sirius Red staining to visualize collagen depositions, immunohistochemical, or immunofluorescent detection of hepatic cell populations, and transcriptomic analyses of the liver microenvironment that orchestrates the induced cellular changes through complex growth factor and cytokine networks^{14,15,16,17,18}.

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, which has been depleted of visible grains and stalks and keep mice on 12-hour day/night cycles in individually ventilated cages. Change bedding on days three and seven, then weekly thereafter.
   3. Provide mice with ad libitum access to the choline-deficient diet and drinking water supplemented with 0.15% of DL-ethionine. Keep the DL-ethionine-supplemented water at 4°C and replace drinking water every second day to ensure freshness and encourage drinking. Top up the choline-deficient chow every second day with a complete change of chow once a week.
   4. Observe mice at rest and during handling. Monitor standard signs of animal health, including overall appearance, posture, social interaction, grooming, coat condition, and body weight.
   5. Weigh mice daily during the first two weeks of the experiment to ensure any animals exhibiting more than 20% body weight loss are euthanized to limit undue suffering. This is typically the case in less than 5% of animals. After two weeks, the frequency of weighing can be reduced to three times weekly (e.g., Monday, Wednesday, Friday).
3. Liver perfusion and isolation
   1. Anesthetize mice at indicated time points (in this study one, two, and six weeks on the CDE diet) with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Test the pedal withdrawal reflex to ensure adequate anesthesia.
   2. Wet the fur with 70% ethanol and make a vertical midline incision in the abdominal wall up to the diaphragm using Mayo scissors. The rib cage can be removed for easier access to the heart.
   3. Collect blood by slow cardiac puncture using a 25 G x 1/2" regular wall needle to avoid collapsing of the heart. Allow the blood to clot in a microcentrifuge tube at room temperature to obtain serum samples after centrifugation at 2,000 x g for 10 min. Later, measure serum alanine transaminase levels by standard procedures¹².
   4. Move the stomach and small intestine to the side and expose the portal vein. Cut the heart using Mayo scissors to allow fluids to exit and perfuse the liver with sterile phosphate buffered saline (pH=7.4) by cannulating the portal vein using a 27 G x 1/2" regular wall needle. An evenly blanched liver indicates successful perfusion of all liver lobes.
   5. Carefully detach the liver and place it in a petri dish using forceps and Mayo scissors. Remove excess, non-liver tissue and the gallbladder.
4. Liver preservation
   1. After the total liver weight has been recorded, cut one small lobe into several small pieces of a few square millimeters, transfer them to sterile tubes, and snap-freeze in liquid nitrogen for later RNA and protein extraction, as per standard protocols. Snap-freeze these tissue pieces as soon as possible after organ collection to avoid any tissue degradation.
   2. Collect one lobe in 10% neutral buffered formalin for later processing and paraffin embedding, as per standard protocols.
   3. Place one lobe into a mold filled with optimum cutting temperature cryomatrix embedding resin and snap-freeze in liquid nitrogen.

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