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 ...

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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Research

JoVE Journal

Biology

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which negatively influences clinically relevant parameters, such as strength and retraction of the muscle-tendon/myotendinous unit, ultimately causing loss of shoulder function and negatively affecting RC repair outcomes. Animal models that mimic aspects of human RC anatomy and pathophysiology are crucial for advancing the conceptual understanding of injury progression and developing effective tissue engineering and regenerative medicine-based therapeutics.
Within this context, a rabbit subscapularis (SSC) model is suitable due to (i) its anatomical similarity to the human supraspinatus (SSP) bone-tendon-muscle unit, which is the most frequently injured RC site; (ii) its pathophysiological similarity to humans in terms of fibrosis and muscle fatty degeneration (FD); and (iii) its amenability to surgical procedures. Therefore, the goal of this study is to describe the surgical techniques for inducing SSC RC injury. Briefly, the procedure involves the isolation of the SSC by identifying the coracobrachialis muscle followed by a full-thickness transection at the muscle-tendon junction and wrapping the free end of the muscle-tendon junction with a silicone-based penrose tubing to prevent spontaneous reattachment. Histologic evaluations are performed to monitor the progression of muscle FD at 4 weeks post-surgery using hematoxylin and eosin (H&#38;E) as well as Masson&#39;s trichrome staining.
Loss of muscle and FD were evident 4 weeks after transection of the SSC muscle-tendon junction, similar to human RC pathophysiological conditions. This protocol demonstrates the steps for successfully establishing a chronic-like rabbit SSC RC injury model, which can serve as a powerful tool to study skeletal muscle changes associated with RC pathophysiology and aid the development of novel therapeutic strategies for chronic-like RC tears.

This study details procedures for establishing a chronic-like rabbit rotator cuff (RC) injury. Specifically, the injury is created in the subscapularis (SSC) muscle-tendon/myotendinous unit to mimic human RC anatomy and pathophysiology, including severe muscle fatty degeneration (FD). This protocol can be applied to study RC injuries and assess regenerative therapies.

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which ...

A Chronic High-Intensity Interval Training and Diet-Induced Obesity Model to Maximize Exercise Effort and Induce Physiologic Changes in Rats

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that results in similar physiologic benefits. This paper presents a HIIT protocol that can be used to assess various health markers in a Sprague-Dawley rat model of diet-induced obesity. Female Sprague Dawley rats aged 21 days old were randomly assigned to the following groups: control (CON, n = 10), exercise-trained (TRN, n = 10), high-fat diet (HFD, n = 10), and high-fat diet/exercise training (HFD/TRN, n = 10). The control diets consisted of commercial laboratory chow with 10% kilocalories (kcal) from fat (3.82 kcal/g), and the high-fat diets (HFD) consisted of 45% kcal from fat (4.7 kcal/g). The animals had ad libitum access to their assigned diet throughout the study. After an 8 week diet induction period, the exercise cohorts completed four HIIT sessions per week for 8 weeks. Each HIIT session consisted of 10 intervals of 1 min sprints/2 min rest using a rodent treadmill with a motor-driven belt. After the 8 weeks of training, the animals were sacrificed for tissue collection. The results revealed no differences in the distance run between the TRN and HFD/TRN groups, and the training speed steadily increased over the duration of the study, with a final running speed of 115 cm/s and 111 cm/s for the TRN and HFD/TRN groups, respectively. The weekly caloric intake was decreased (p &#60; 0.05) in the TRN group relative to the CON group but increased (p &#60; 0.05) in the HFD/TRN group relative to the HFD group. Lastly, the animals on the HFD had greater (p &#60; 0.05) adiposity, and the trained animals had reduced (p &#60; 0.05) adiposity relative to controls. This protocol demonstrates an efficient method to evaluate the effects of HIIT on various physiologic outcomes in a diet-induced obesity model.

This paper presents the morphometric responses and training performance outcomes of a high-intensity interval training (HIIT) protocol in a Sprague-Dawley rat model of diet-induced obesity. The purpose of this protocol was to maximize exercise intensity and determine the physiologic responses to HIIT in lean and obese rats.

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that ...