Name: the murine choline-deficient, ethionine-supplemented (cde) diet model of chronic liver injury
Uploaded: 2017-10-21T14:00:00
Description: Watch this Scientific Journal Video about The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury at JoVE.com

This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (APP1042370, APP1061332, APP1087125). The authors would like to thank the Curtin Health Innovation Research Institute staff for technical assistance.

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.
<ol>
	<li>Animals
	<ol>
		<li>Use six-week old male C57BL/6J mice for the experiments.</li>
	</ol>
	</li>
	<li>Experimental design
	<ol>
		<li>Following arrival at ...

<ol>
	<li>Taub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+from+myth+to+mechanism.">Liver regeneration: from myth to mechanism.</a> Nat Rev Mol Cell Biol. 5, 836-847 (2004).</li><li>Kohn-Gaone, J., Gogoi-Tiwari, J., Ramm, G. A., Olynyk, J. K., Tirnitz-Parker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+liver+progenitor+cells+during+liver+regeneration,+fibrogenesis,+and+carcinogenesis.">The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis.</a> Am J Physiol Gastrointest Liver Physiol. 310, G143-G154 (2016).</li><li>Lu, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+progenitor+cells+of+biliary+origin+with+liver+repopulation+capacity.">Hepatic progenitor cells of biliary origin with liver repopulation capacity.</a> Nat Cell Biol. 17, 971-983 (2015).</li><li>Prakoso, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+intrahepatic+ductular+reaction+and+progenitor+cell+responses+in+hepatitis+C+virus+recurrence+after+liver+transplantation.">Analysis of the intrahepatic ductular reaction and progenitor cell responses in hepatitis C virus recurrence after liver transplantation.</a> Liver Transpl. 20, 1508-1519 (2014).</li><li>Tirnitz-Parker, J. E., Tonkin, J. N., Knight, B., Olynyk, J. K., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+immortalisation+of+hepatic+oval+cells+from+adult+mice+fed+a+choline-deficient,+ethionine-supplemented+diet.">Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.</a> Int J Biochem Cell Biol. 39, 2226-2239 (2007).</li><li>Van Hul, N. K., Abarca-Quinones, J., Sempoux, C., Horsmans, Y., Leclercq, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+liver+progenitor+cell+expansion+and+extracellular+matrix+deposition+in+a+CDE-induced+murine+model+of+chronic+liver+injury.">Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.</a> Hepatology. 49, 1625-1635 (2009).</li><li>Clouston, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+correlates+with+a+ductular+reaction+in+hepatitis+C:+roles+of+impaired+replication,+progenitor+cells+and+steatosis.">Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis.</a> Hepatology. 41, 809-818 (2005).</li><li>Forbes, S. J., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration+-+mechanisms+and+models+to+clinical+application.">Liver regeneration - mechanisms and models to clinical application.</a> Nat Rev Gastroenterol Hepatol. 13, 473-485 (2016).</li><li>Akhurst, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+choline-deficient,+ethionine-supplemented+diet+protocol+effectively+induces+oval+cells+in+mouse+liver.">A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver.</a> Hepatology. 34, 519-522 (2001).</li><li>Shinozuka, H., Lombardi, B., Sell, S., Iammarino, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+histological+and+functional+alterations+of+ethionine+liver+carcinogenesis+in+rats+fed+a+choline-deficient+diet.">Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.</a> Cancer Res. 38, 1092-1098 (1978).</li><li>Knight, B., Tirnitz-Parker, J. E., Olynyk, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-kit+inhibition+by+imatinib+mesylate+attenuates+progenitor+cell+expansion+and+inhibits+liver+tumor+formation+in+mice.">C-kit inhibition by imatinib mesylate attenuates progenitor cell expansion and inhibits liver tumor formation in mice.</a> Gastroenterology. 135, 969-979 (2008).</li><li>Kohn-Gaone, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Inflammatory,+Fibrogenic,+and+Liver+Progenitor+Cell+Dynamics+in+Two+Common+Mouse+Models+of+Chronic+Liver+Injury.">Divergent Inflammatory, Fibrogenic, and Liver Progenitor Cell Dynamics in Two Common Mouse Models of Chronic Liver Injury.</a> Am J Pathol. 186, 1762-1774 (2016).</li><li>Knight, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+liver+progenitor+(oval)+cell+and+fibrogenic+responses+to+the+choline+deficient,+ethionine+supplemented+diet+in+the+BALB/c+inbred+strain+of+mice.">Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice.</a> J Hepatol. 46, 134-141 (2007).</li><li>Dwyer, B. J., Olynyk, J. K., Ramm, G. A., Tirnitz-Parker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TWEAK+and+LTbeta+Signaling+during+Chronic+Liver+Disease.">TWEAK and LTbeta Signaling during Chronic Liver Disease.</a> Front Immunol. 5, 39 (2014).</li><li>Karin, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+inflammation+takes+charge+of+tissue+regeneration.">Reparative inflammation takes charge of tissue regeneration.</a> Nature. 529, 307-315 (2016).</li><li>Ruddell, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin-beta+receptor+signaling+regulates+hepatic+stellate+cell+function+and+wound+healing+in+a+murine+model+of+chronic+liver+injury.">Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.</a> Hepatology. 49, 227-239 (2009).</li><li>Tirnitz-Parker, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-like+weak+inducer+of+apoptosis+is+a+mitogen+for+liver+progenitor+cells.">Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells.</a> Hepatology. 52, 291-302 (2010).</li><li>Viebahn, C. S., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+fires+prometheus?+The+link+between+inflammation+and+regeneration+following+chronic+liver+injury.">What fires prometheus? The link between inflammation and regeneration following chronic liver injury.</a> Int J Biochem Cell Biol. 40, 855-873 (2008).</li><li>Hayner, N. T., Braun, L., Yaswen, P., Brooks, M., Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isozyme+profiles+of+oval+cells,+parenchymal+cells,+and+biliary+cells+isolated+by+centrifugal+elutriation+from+normal+and+preneoplastic+livers.">Isozyme profiles of oval cells, parenchymal cells, and biliary cells isolated by centrifugal elutriation from normal and preneoplastic livers.</a> Cancer Res. 44, 332-338 (1984).</li><li>Tee, L. B., Smith, P. G., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+alpha,+mu+and+pi+class+glutathione+S-transferases+in+oval+and+ductal+cells+in+liver+of+rats+placed+on+a+choline-deficient,+ethionine-supplemented+diet.">Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet.</a> Carcinogenesis. 13, 1879-1885 (1992).</li><li>Chayanupatkul, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+in+the+absence+of+cirrhosis+in+patients+with+chronic+hepatitis+B+virus+infection.">Hepatocellular carcinoma in the absence of cirrhosis in patients with chronic hepatitis B virus infection.</a> J Hepatol. 66, 355-362 (2017).</li><li>Mittal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+Carcinoma+in+the+Absence+of+Cirrhosis+in+United+States+Veterans+is+Associated+With+Nonalcoholic+Fatty+Liver+Disease.">Hepatocellular Carcinoma in the Absence of Cirrhosis in United States Veterans is Associated With Nonalcoholic Fatty Liver Disease.</a> Clin Gastroenterol Hepatol. 14, 124-131 (2016).</li></ol>

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...

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 hepatocytes2,3,4,5. 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 mice9,10. 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&#160;to hepatocellular carcinoma development11,12. 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 networks14,15,16,17,18.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Six-week-old male C57BL/6J mice&#160;</td>
			<td style="width:367px;">Animal Resource Centre of Western Australia, Murdoch, WA,&#160; Australia</td>
			<td style="width:121px;">N/A</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 Kg Steam Cut Wheaten Chaff&#160;</td>
			<td>Specialty Feeds, Glen Forrest, WA, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water for irrigation 1000ml bottle (Baxter)&#160;</td>
			<td>Surgical House, Perth, WA, Australia&#160;</td>
			<td>AHF7114A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Choline- deficient diet, modified (pellets)&#160;</td>
			<td colspan="2">MP Biomedicals Australasia Pty Limited, WA, Australia 02960210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DL-Ethionine&#160;</td>
			<td>Sigma-Aldrich, Castle Hill, NSW,&#160; Australia&#160;</td>
			<td>E5139-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamil injection&#160;</td>
			<td>Troy Laboratories Pty Limited, Glendenning, NSW, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ilum Xylazil-20 injection&#160;</td>
			<td>Troy Laboratories Pty Limited, Glendenning, NSW, Australia&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">27G x 1/2&#34;, Regular Wall Needle&#160;</td>
			<td>Terumo Australia Pty Limited, NSW, Australia&#160;</td>
			<td>NN-2713R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringes Terumo 1ml and 10ml&#160;</td>
			<td>Terumo Australia Pty Limited, Macquarie Park, NSW, Australia&#160;</td>
			<td>1018242, 1018037</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek OCT compound&#160;</td>
			<td>VWR International Pty Limited, Tingalpa, QLD, Australia&#160;</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neutral buffered formalin&#160;</td>
			<td>Amber Scientific, Midvale, WA, Australia&#160;</td>
			<td>NBF-2.5L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol absolute anaLaR normalpur&#160;</td>
			<td>VWR International Pty Limited, Tingalpa, QLD, Australia&#160;</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superfrost Plus slides&#160;</td>
			<td>Grale Scientific Pty Limited, Ringwood, VIC, Australia&#160;</td>
			<td>SF41296SP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dako Protein Block, serum-free&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;</td>
			<td>&#160;X090930-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dako antibody diluent&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;&#160;</td>
			<td>s0809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rat anti-CD45&#160;</td>
			<td>eBioscience, San Diego, California, USA&#160;</td>
			<td>m0701&#160;</td>
			<td>1/200 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit anti-panCK&#160;</td>
			<td>Dako Australia Pty Limited, North Sydney, NSW, Australia&#160;&#160;</td>
			<td>Z0622</td>
			<td>1/300 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit (Alexa Fluor 488)</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia</td>
			<td>A-11008</td>
			<td>1/500 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rat IgG (Alexa Fluor 594)&#160;</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia</td>
			<td>&#160;A-11007</td>
			<td>1/500 dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies Australia Pty Limited, Mulgrave, VIC, Australia&#160;</td>
			<td>P36935&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Picrosirius Red Stain Kit</td>
			<td>Polysciences Inc., Warrington, PA, USA&#160;</td>
			<td>ab150681&#8195;</td>
			<td></td>
		</tr>
	</tbody>
</table>

the murine choline-deficient, ethionine-supplemented (cde) diet model of chronic liver injury

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.

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...

Watch this Scientific Journal Video about The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury at JoVE.com

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

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.

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

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

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