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.

<table border="0" cellpadding="0" cellspacing="0" width="982">
	<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 ...

The overall goal of this protocol is to demonstrate a reliable method to induce chronic liver injury in mice and analyze liver injury parameters during the induction, establishment and maintenance phases over a course of six weeks. The CDE diet of chronic liver injury reliably induces steatosis, or fatty liver, inflammation, liver progenitor cell proliferation and hepatic fibrosis. Collection and analysis of sera and tissues at indicated time points allow us to follow the molecular and cellular processes that underlie liver regeneration and chronic liver disease progression.The techniques are demonstrated by Jully Gogoi-Tiwari, at post doc in my lab. New and chronic liver injury is induced through feeding of a choline deficient diet and supplementation of DL-ethionine, the drinking water. At the selected time points, mice are anesthetized, a cardio puncture is performed to collect sera for the analysis of blood based markers of liver injury.The liver is perfused through the portal vein and is then excised for further processing and analysis. Mice on the CDE diet are observed daily in the first two weeks and three weeks, a week thereafter. Once water is supplemented with ethionine, it is kept at four degrees.Bottles are refreshed every two to three days and the mice have free access to pellets of choline deficient chow which is topped up as needed. For bedding, wheaten chaff is used after each has been depleted of visible grains and stalks. Standard science of animal health are monitored and include overall appearance, posture, social interaction, grooming and coat condition, as well as body weight.CDE treated mice will lose up to 20%of their initial body weight in the induction phase while they adapt to the diet but will recover thereafter. Mice on the CDE diet sometimes develop a greasy coat. This is not an indicator of poor grooming but a consequence of the high fat content of the diet.After the mouse has been weighed and anesthetized, the feral withdraw reflex is tested to ensure the anesthesia is adequate. If anesthesia is just deep enough to commence surgery, the fur is wetted with 70%ethanol and the vertical midline incision is made in the abdominal wall up to the diaphragm. For easier access to the heart, the ribcage can be removed to expose the thoracic cavity.A cardiac puncture is then performed where the blood is slowly collected from the ventricle to avoid collapsing of the heart. The blood is then transferred to a sterile tube and allowed to clot at room temperature to later obtain sera samples after low speed centrifugations. The intestine and stomach are gently swept to the side to expose the portal vein.The heart is cut to allow fluids to exit and the portal vein is perfused with sterile phosphate saline solution until the liver blanches and indicates full perfusion of all liver lobes. The liver is then carefully detached and placed in a petri dish to remove potential non liver tissue and the gallbladder. After the liver weight has been recorded the liver is separated into individual lobes.One liver lobe is further cut into small cubes of a few millimeters in diameter. One cube is collected in a separate tube for later RNA extraction, while the other cubes are collected in a second tube for future DNA and protein analysis as well potential extra RNA extractions. These tubes are snap frozen in liquid nitrogen as soon as possible after organ collection.Once the larger lobes is used for formal infixation, and later paraffin embedding for sectioning on a microtome and histology and immunohistochemistry evaluations. Two other lobes are embedded in a cryo matrix while making sure that the tissue piece is completely covered by the matrix. These liver lobes are then snap frozen in liquid nitrogen for sectioning on a cryo stet and immunofluorescent or confocal microscopy experiments.The enzyme alanine transaminase can be measured in the sera as a biomarker for hepatocellular injury. While healthy animals display low sera ALT levels, at all major time points, CDE treated mice release ALT into the bloodstream. With a major peak in the induction phase and slow normalization in the establishment and maintenance phase.Formalin fixed and paraffin embedded liver tissue can be stained with hematoxylin and eosin to evaluate the liver histology. CDE treated livers show vastly disrupted liver architecture and the parenchymal infiltration of many basophilic cells in the induction and establishment phase with normalization in the maintenance phase. To assess fibrogenesis, formalin fixed and paraffin embedded liver tissue can also be stained with the polyazo dye sirius red, which visualizes hepatic collagen deposition.In the induction phase, collagen accumulates in the parenchyma and normalizes in establishment and maintenance phase. Cryo matrix embedded and snap frozen liver tissue can be cut into thin sections of about seven millimeter on a cryo stet. Immunofluorescent staining for biliary and liver progenitor cell marker panCK and the inflammatory cell marker CD45 can then be performed.An early increase in the transcription levels of cytokines and growth factors can be observed. These include tumor necrosis factor, tumor necrosis factor like weak inducer of apoptosis, or TWEAK, lymphotoxin beta, hepatocide growth factor, transforming growth factor beta and interleukin six, for example. After watching this video, you should have a good understanding of the CDE model of chronic liver injury.You should have gained some knowledge of the processes involved in perfusing and isolating livers for further analysis and the histological cellular and molecular changes induced in livers of mice fed a choline deficient, ethionine supplemented diet over a time course of six weeks.

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JoVE Journal

Biology

11.6K 次观看.  Curtin University. The overall goal of this protocol is to demonstrate a reliable method to induce chronic liver injury in mice and analyze liver injury parameters during the induction, establishment and maintenance phases over a course of six weeks. The CDE diet of chronic liver injury reliably induces steatosis, or fatty liver, inflammation, liver progenitor cell proliferation and hepatic fibrosis. Collection and analysis of sera and tissues at indicated time points allow us to follow the molecular and cellul...