Name: investigating intestinal inflammation in dss-induced model of ibd
Uploaded: 2012-02-01T13:00:00
Description: Watch this Scientific Journal Video about Investigating Intestinal Inflammation in DSS-induced Model of IBD at JoVE.com

This work is supported by grants from Canadian Institutes of Health Research (CIHR) and by Crohn's and Colitis Foundation of Canada (CCFC).

1. Murine model of DSS-induced acute colitis
<ol>
	<li>Add dextran sulphate sodium (DSS) to autoclaved drinking water to the desired final concentration (1-5%) (wt/vol) (i.e. To make a 5% DSS solution, add 25 g of DSS powder to 500 mL of autoclaved water). Stock solution can be left at room temperature for up to one week or at 4&deg;C until use.</li>
	<li>In a biosafety hood, pour stock DSS solution into 50 mL Falcon tubes (one needed per cage). Keep the stock solution to refill tubes when needed.</li>
	<li>Replace dri...

<ol>
	<li>Sands, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+symptom+to+diagnosis:+clinical+distinctions+among+various+forms+of+intestinal+inflammation.">From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation.</a> Gastroenterology. 126, 1518-1532 (2004).</li><li>Danese, S., Fiocchi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiopathogenesis+of+inflammatory+bowel+diseases.">Etiopathogenesis of inflammatory bowel diseases.</a> World J. Gastroenterol. 12, 4807-4812 (2006).</li><li>Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+acute+murine+dextran+sodium+sulphate+colitis:+cytokine+profile+and+dose+dependency.">Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency.</a> Digestion. 62, 240-248 (2000).</li><li>Krawisz, J. E., Sharon, P., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+for+acute+intestinal+inflammation+based+on+myeloperoxidase+activity.+Assessment+of+inflammation+in+rat+and+hamster+models.">Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models.</a> Gastroenterology. 87, 1344-1350 (1984).</li><li>Cooper, H. S., Murthy, S. N., Shah, R. S., Sedergran, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathologic+study+of+dextran+sulfate+sodium+experimental+murine+colitis.">Clinicopathologic study of dextran sulfate sodium experimental murine colitis.</a> Lab. Invest. 69, 238-249 (1993).</li><li>Smith, J. W., Castro, G. 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+of+peroxidase+activity+in+gut+mucosa+to+inflammation.">Relation of peroxidase activity in gut mucosa to inflammation.</a> Am. J. Physiol. 234, R72-R79 (1978).</li><li>Ghia, J. E., Blennerhassett, P., Kumar-Ondiveeran, H., Verdu, E. F., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vagus+nerve:+a+tonic+inhibitory+influence+associated+with+inflammatory+bowel+disease+in+a+murine+model.">The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model.</a> Gastroenterology. 131, 1122-1130 (2006).</li><li>Okayasu, I., Hatakeyama, S., Yamada, M., Ohkusa, T., Inagaki, Y., Nakaya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98, 694-702 (1990).</li><li>Solomon, L., Mansor, S., Mallon, P., Donnelly, E., Hoper, M., Loughrey, M., Kirk, S., Gardiner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dextran+sulphate+sodium+(DSS)+model+of+colitis:+an+overview.">The dextran sulphate sodium (DSS) model of colitis: an overview.</a> Comparative Clinical Pathology. 19, 235-239 (2010).</li><li>Mähler, M., Bristol, I. J., Leiter, E. H., Workman, A. E., Birkenmeier, E. H., Elson, C. O., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am. J. Physiol. 274, G544-G551 (1998).</li><li>Hans, W., Scholmerich, J., Gross, V., Falk, W. <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+the+resident+intestinal+flora+in+acute+and+chronic+dextran+sulfate+sodium-induced+colitis+in+mice.">The role of the resident intestinal flora in acute and chronic dextran sulfate sodium-induced colitis in mice.</a> Eur. J. Gastroenterol. Hepatol. 12, 267-273 (2000).</li><li>Rath, H. C., Schultz, M., Freitag, R., Dieleman, L. A., Li, F., Linde, H., Schölmerich, J., Sartor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+subsets+of+enteric+bacteria+induce+and+perpetuate+experimental+colitis+in+rats+and+mice.">Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice.</a> Infect. Immun. 69, 2277-2285 (2001).</li><li>Ghia, J. E., Li, N., Wang, H., Collins, M., Deng, Y., El-Sharkawy, R. T., Côté, F., Mallet, J., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+has+a+key+role+in+pathogenesis+of+experimental+colitis.">Serotonin has a key role in pathogenesis of experimental colitis.</a> Gastroenterology. 137, 1649-1660 (2009).</li><li>Elson, C. O., Beagley, K. W., Sharmanov, A. T., Fujihashi, K., Kiyono, H., Tennyson, G. S., Cong, Y., Black, C. A., Ridwan, B. W., McGhee, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hapten-induced+model+of+murine+inflammatory+bowel+disease:+mucosa+immune+responses+and+protection+by+tolerance.">Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance.</a> J. Immunol. 157, 2174-2185 (1996).</li><li>Dieleman, L. A., Ridwan, B. U., Tennyson, G. S., Beagley, K. W., Bucy, R. P., Elson, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+sulfate+sodium-induced+colitis+occurs+in+severe+combined+immunodeficient+mice.">Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice.</a> Gastroenterology. 107, 1643-1652 (1994).</li></ol>

DSS colitis is a widely used chemically induced model of intestinal inflammation. In this model, mice are given drinking water supplemented with DSS, which is thought to be toxic to gut epithelial cells and disrupt the integrity of the mucosal barrier.10 Administration of DSS induces an acute colitis that is characterized by loose stool, fecal bleeding, and infiltration with granulocytes.10 During DSS administration, colitis is usually associated with significant weight loss and presence of blood in...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent/equipment:</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Eppendorf Safe-Lock Microcentrifuge Tube (2mL)</td>
 <td>eppendorf</td>
 <td>0030 120.094</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biotek EL808 Absorbance plate reader </td>
 <td>Biotek </td>
 <td>EL808 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran sulphate sodium salt reagent grade (molecular weight 36,000-50,000 Da)</td>
 <td>MP Biomedicals</td>
 <td>160110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gen5 (software)</td>
 <td>Biotek</td>
 <td>Version 1.10.8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hexadecyltrimethylammonium bromide (HTAB)</td>
 <td>Sigma-Aldrich</td>
 <td>H5882-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide, 30 wt.% solution in water</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 <td>Store at 2-8&deg;C</td>
 </tr>
 <tr>
 <td>Microtest plate 96-well flat bottom</td>
 <td>Sarstedt</td>
 <td>82.1581</td>
 <td>For single use only</td>
 </tr>
 <tr>
 <td>o-Dianisidine</td>
 <td>Sigma-Aldrich</td>
 <td>D-3252</td>
 <td>Light sensitive. Store at 2-8&deg;C</td>
 </tr>
 <tr>
 <td>Potassium phosphate, dibasic</td>
 <td>Caledon</td>
 <td>6620-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate, monobasic</td>
 <td>EMD Chemicals</td>
 <td>PX1565-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail</td>
 <td>Sigma-Aldrich </td>
 <td>P8340</td>
 <td>Store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T8787</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tungsten Carbide beads for Tissue Lyser II</td>
 <td>QUIAGEN</td>
 <td>69997</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

investigating intestinal inflammation in dss-induced model of ibd

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Watch this Scientific Journal Video about Investigating Intestinal Inflammation in DSS-induced Model of IBD at JoVE.com

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

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