This work was supported by operating grants to KJ from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Crohn's and Colitis Foundation of Canada (CCFC). VM is supported by a CFRI Fellowship and GB a graduate studentship from the Canadian Institutes for Health Research (CIHR). BAV is a Children with Intestinal and Liver Disorders Foundation (CHILD) Chair of Pediatric IBD Research and the Canada Research Chair in Pediatric Gastroenterology and KJ is a Senior Clinician Scientist supported by CHILD and the Child and Family Research Institute (CFRI) Clinician Scientists Award Program, University of British Columbia.

Procedures involving animal subjects follow animal care guidelines outlined by the Institutional Animal Care and Use Committee (IACUC) at the University of British Columbia.
Procedures mentioned in this protocol will be described for both rats and mice, while the representative results will be presented from Sprague-Dawley rats.
1. Preparation of DNBS and Intrarectal Administration
<ol>
	<li>For rectal instillation of DNBS in rats, freshly prepare 15-30 mg of DNBS/250 &#181;l of 50% ethanol. For mice, prepare 2-6 mg of DNBS/100 &#181;l of 50% ethanol. Note: Optimize the exact DNBS concentration to be administered at each facility. Male Sprague-Dawley rats, and C57BL/6 mice are preferred for induction of DNBS colitis, however other mouse and rat strains can be used. Mice and rats treated only with 50% ethanol can be used as control groups however given the effects of ethanol are mild or absent, one can omit the inclusion of this group for a study12.</li>
	<li>Attach a 1 ml&#160;syringe to a 19 G needle that has a polyethylene catheter (PE-90 - rats or PE-50 - mice) fastened to its end.</li>
	<li>Lightly anesthetize the rat or mouse resting on a heating pad (e.g. by administering 1.5% isoflurane via nose cone, animals should still have blink and swallowing reflexes and display regular respiration). Apply tear gel to its eyes to avoid the eyes drying out.</li>
	<li>Gently apply pressure with fingertips to the posterior end of the animal to remove any stool that may be present in the distal colon.</li>
	<li>Gently insert the catheter intrarectally, with the catheter reaching approximately 8 cm proximal to the anus for rats, or 3-4 cm for mice. Inject a small amount of the solution while inserting the catheter to lubricate, allowing easier insertion. Note: If any resistance is felt while inserting the catheter, do not continue to insert as this may lead to perforation of the intestinal wall. Remove the catheter and attempt to gently reinsert, again lubricating the colon as proceeding.</li>
	<li>Slowly inject 250 (rats) or 100 (mice) &#181;l&#160;of DNBS, or an equal volume or 50% ethanol for controls. After injecting the DNBS, position the animal head-down for 90 sec to avoid loss of the DNBS.</li>
	<li>After DNBS administration, feed the animals 8% sucrose water in 0.2% saline to prevent dehydration during the first week.</li>
	<li>Carefully observe the animals, with daily monitoring for weight loss and signs of dehydration or distress for the duration of the experiment. Note: Weight loss may rapidly approach the CCAC recommendations of maximum permissible weight loss. Since this mostly derived from dehydration, weight loss should be used as an indicator for administering lactated Ringer&#39;s solution to limit dehydration. Please be sure to consult with the facility veterinarian on health issues as appropriate.</li>
</ol>
2. Collecting Tissues and Assessing Histological Damage in DNBS Challenged Mice
<ol>
	<li>At the desired time point after challenge with DNBS, euthanize animals by overdosing with isoflurane in an anesthetic chamber supplemented with sufficient oxygen. Observe the animal until no rising or falling of the chest is seen and there is no palpable heartbeat. Confirm the animal has been correctly euthanized by absence of a response to toe pinch or touching of the cornea, followed by cervical dislocation.</li>
	<li>Open the abdominal cavity and remove the entire large intestine. Open the intestine longitudinally; macroscopic mucosal ulcers should be observed approximately 5-6 cm proximal from the distal end of the colon in rats (2-3 cm in mice). Note: After removing the large intestine, assess macroscopic damage scores if desired. Collect segments of colon from corresponding anatomical location from control animals. Note: The individuals scoring the tissue for macroscopic damage scores should be blinded to the identity of the treatment groups. Score tissue sections using the following or similar criteria: severity and extent of ulceration (0-10), summed with scores for absence or presence of diarrhea (0 or 1), adhesions (0, 1, or 2) and maximal colonic wall thickness in millimeters11.</li>
	<li>Collect 0.5 cm sections proximal to the site of maximal macroscopic damage. Snap freeze one 0.5 cm section in liquid nitrogen to later assess myeloperoxidase (MPO) activity (a measure of neutrophil infiltration), and place the other section in 10% neutral buffered formalin for histological analysis. Note: Snap frozen tissues for myeloperoxidase assay can be stored at -80 &#176;C for up to 1 month.</li>
	<li>Allow the tissues collected for histological analysis in step 2.3 to fix in formalin overnight, and then wash with 70% ethanol. Embed tissues in paraffin and cut to desired thickness (3-5 &#181;m), stain with hemotoxylin and&#160;eosin (HE) for histological scoring.</li>
	<li>Have two observers score at least three tissue sections from each animal under a light microscope to determine the histological damage scores for each group. Note: The individuals scoring the tissue for histological damage scores should be blinded to the identity of the treatment groups. Score tissue sections using the following or similar criteria: extent of inflammatory cell infiltrate (0 = absent, 3 = transmural), loss of mucosal architecture (0 = absent, 3 = severe), presence or absence of crypt abscesses (0-1), and goblet cell depletion (0-3). Determine histological damage score as the sum of these scores11.</li>
</ol>
3. Performing Myeloperoxidase (MPO) Assay on DNBS Challenged Intestinal Tissue
<ol>
	<li>Assay MPO activity in the 0.5 cm colonic segments obtained in step 2.3. Homogenize tissues in MPO homogenization buffer (0.5% hexadecyltrimethyl-ammonium bromide in 50 mM potassium phosphate buffer (pH 6.0)) to give a 50 mg colon segment/ml&#160;of homogenization buffer suspension.</li>
	<li>Aliquot the homogenized solution into 1 ml&#160;portions and centrifuge at 4 &#176;C for 2 min at 10,000 x g.</li>
	<li>Mix 100 &#181;l&#160;of each aliquot with 2.9 ml&#160;of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml&#160;of o-dianisidinedihydrochloride and 0.0005% hydrogen peroxide in a cuvette.</li>
	<li>Measure the rate of absorbance using a spectrophotometer at 460 nm. Note: MPO activity is defined as the quantity of enzyme degrading 1 &#181;mol/min of H2O2 to H2O&#160;at room temperature and is expressed in units per mg of tissue.</li>
</ol>

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	<li>Per&#353;e, M., Cerar, A., Biotechnol, J. .. . B. i. o. m. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dextran sodium sulphate colitis mouse model: traps and tricks. , (2012).</li><li>Ghia, J. E., Blennerhassett, P., Deng, Y., Verdu, E., Khan, W., Collins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+inflammatory+bowel+disease+in+a+mouse+model+of+depression.">Reactivation of inflammatory bowel disease in a mouse model of depression.</a> Gastroenterology. 136 (7), 2280-2288 (2009).</li><li>Reber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+animal+models+of+inflammatory+bowel+disease-an+update+on+the+role+of+the+hypothalamo-pituitary-adrenal+axis.">Stress and animal models of inflammatory bowel disease-an update on the role of the hypothalamo-pituitary-adrenal axis.</a> Psychoneuroendocrinology. 37 (1), 1-19 (2012).</li><li>Qiu, B., Vallance, B., Blennerhassett, P., Collins, S. <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+CD4++lymphocytes+in+the+susceptibility+of+mice+to+stress-induced+reactivation+of+experimental+colitis.">The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis.</a> Nat. Med. 5 (10), 1178-1182 (1999).</li><li>Boyer, L., 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=Myenteric+plexus+injury+and+apoptosis+in+experimental+colitis.">Myenteric plexus injury and apoptosis in experimental colitis.</a> Auto. Neurosci. Basic Clin. 117 (1), 41-53 (2005).</li><li>Saunders, P., 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=Noradrenergic+and+cholinergic+neural+pathways+mediate+stress-induced+reactivation+of+colitis+in+the+rat.">Noradrenergic and cholinergic neural pathways mediate stress-induced reactivation of colitis in the rat.</a> Auto. Neurosci. Basic Clin. 124 (1-2), 56-68 (2006).</li><li>Cuzzocrea, 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=Calpain+inhibitor+I+reduces+colon+injury+caused+by+dinitrobenzene+sulphonic+acid+in+the+rat.">Calpain inhibitor I reduces colon injury caused by dinitrobenzene sulphonic acid in the rat.</a> Gut. 48 (4), 478-488 (2001).</li><li>Cuzzocrea, 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=Melatonin+reduces+dinitrobenzene+sulfonic+acid-induced+colitis.">Melatonin reduces dinitrobenzene sulfonic acid-induced colitis.</a> J. Pineal Res. 30 (1), 1-12 (2001).</li><li>Innis, S., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+lipids+in+early+development+and+intestinal+inflammatory+disease.">Dietary lipids in early development and intestinal inflammatory disease.</a> Nutr. Rev. 65, S188-S193 (2007).</li><li>Innis, S. M., de La Presa Owens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+fatty+acid+composition+in+pregnancy+alters+neurite+membrane+fatty+acids+and+dopamine+in+newborn+rat+brain.">Dietary fatty acid composition in pregnancy alters neurite membrane fatty acids and dopamine in newborn rat brain.</a> J. Nutr. 131 (1), 118-122 (2001).</li><li>Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mundra+H.,Innis+S.M.+Intestinal+responsiveness+to+experimental+colitis+in+young+rats+is+altered+by+maternal+diet.">Mundra H.,Innis S.M. Intestinal responsiveness to experimental colitis in young rats is altered by maternal diet.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 289, 13-20 (2005).</li><li>Sanovic, S., Lamb, D. P., Blennerhassett, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+the+enteric+nervous+system+in+experimentalcolitis.">Damage to the enteric nervous system in experimentalcolitis.</a> Am. J. Pathol. 155 (4), 1051-107 (1999).</li><li>Vilaseca, J., Salas, A., Guarner, F., Rodriguez, R., Martinez, M., Malagelada, 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=Dietary+fish+oil+reduces+progression+of+chronic+inflammatory+lesions+in+a+rat+model+of+granulomatous+colitis.">Dietary fish oil reduces progression of chronic inflammatory lesions in a rat model of granulomatous colitis.</a> Gut. 31, 539-544 (1990).</li><li>Andoh, A., Tsujikawa, T., Ishizuka, I., Araki, Y., Sasaki, M., Koyama, S., Fujiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-3+fatty+acid-rich+diet+prevents+early+response+of+interleukin-6+elevation+in+trinitrobenzene+sulfonic+acid-induced+enteritis.">N-3 fatty acid-rich diet prevents early response of interleukin-6 elevation in trinitrobenzene sulfonic acid-induced enteritis.</a> Int. J. Mol. Med. 12, 721-725 (2003).</li><li>Barros, K. V., Xavier, R. A., Abreu, G. G., Martinez, C. A., Ribeiro, M. L., Gambero, A., Carvalho, P. O., Nascimento, C. M., Silveira, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soybean+and+fish+oil+mixture+increases+IL-10,+protects+against+DNA+damage+and+decreases+colonic+inflammation+in+rats+with+dextran+sulfate+sodium+(DSS)+colitis.">Soybean and fish oil mixture increases IL-10, protects against DNA damage and decreases colonic inflammation in rats with dextran sulfate sodium (DSS) colitis.</a> Lipids Health Dis. 9, 68 (2010).</li><li>Hekmatdoost, A., Wu, X., Morampudi, V., Innis, S. M., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+oils+modify+the+host+immune+response+and+colonic+tissue+damage+following+Citrobacter+rodentium+infection+in+mice.">Dietary oils modify the host immune response and colonic tissue damage following Citrobacter rodentium infection in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 304 (10), 917-928 (2013).</li><li>Innis, S. M., Dai, C., Wu, X., Buchan, A. M., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+lipid+nutrition+alters+early+intestinal+development+and+programs+the+response+to+experimental+colitis+in+young+adult+rats.">Perinatal lipid nutrition alters early intestinal development and programs the response to experimental colitis in young adult rats.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 299 (6), 1376-1385 (1152).</li></ol>

The authors declare that they have no competing financial interests.

The DNBS model described in this protocol is a valuable, inexpensive, and reproducible colitis model that can be utilized to study various aspects of IBD. When administered intrarectally to rats or mice, DNBS induces a substantial degree of inflammation and tissue injury in the colon, resembling human Crohn&#39;s disease in terms of its various histological features including transmural infiltration of polymorphonuclear cells and predominant NF-&#954;B-dependent Th1 activation.
This model has ...

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation in the gastro-intestinal (GI) tract, resulting in symptoms of diarrhea, weight loss, and abdominal pain. Ulcerative colitis (UC) and Crohn&#39;s disease (CD) are the two major forms of IBD, and can be distinguished by the location of the inflammation within the GI tract. In UC patients, the inflammation typically involves the rectum and extends contiguously up the colon for a variable extent affecting only the superficial mucosa. In contrast, CD can affect any part of the GI tract, although it predominantly affects the ileum and cecum. CD frequently manifests as transmural inflammation often associated with granulomas and leading to stricturing (fibrostenotic) and/or penetrating (fistulizing) disease. Although the etiology of IBD remains elusive, it is well accepted that IBD is multifactorial, involving interactions among the hosts immune system, genetic susceptibility and responses to environmental and microbial factors.
To date, different models of IBD have been proposed that display various clinical, histological and immune responses characteristic of UC and CD. The most commonly used models include genetically modified mice (IL-2, IL-10, SAMP/Yit), infection induced models (Citrobacter rodentium, Salmonella typhimurium), adoptive transfer models (CD45+ RB High, CD62L+ cell transfer into SCID mice) and chemically induced colitis models (Dextran Sodium Sulfate (DSS), Trinitrobenzene sulfonic acid (TNBS), and Dinitrobenzene sulfonic acid (DNBS)). Owing to their low cost and rapid onset of disease, chemical models are considered invaluable to the study of various aspects of IBD. Each of the chemical colitis models listed has advantages as well as limitations in some aspects of their clinical, immunological and histopathological relevance to IBD. DSS is one of the most common chemical methods employed to induce colitis in rodents1. Administration of 3-10% DSS (MW: 42 kDa) for 7-10 days in the drinking water of mice can induce symptoms and signs of colitis including weight loss, diarrhea with blood, colonic shortening, mucosal ulceration and neutrophil infiltration. This model is particularly useful for drug screening studies, as well as exploring the mechanisms of epithelial regeneration, the impact of innate immunity on mucosal homeostasis, and the role of inflammation in promoting intestinal dysplasia and adenocarcinoma development. There are however some drawbacks to the DSS model, including variation in the concentration of DSS needed to induce colitis in different animal facilities, as well as inconsistent water uptake by mice and thus inconsistent exposure to DSS, resulting in variation in the degree, extent and distribution of mucosal injury and ulceration in the colon. All these features lead to heterogeneity of results and limit the ability to compare results across studies from different research groups.
An alternative to the DSS model is the hapten-induced DNBS or TNBS models of colitis. This model employs rectal instillation of the mucosal sensitizing agents DNBS or TNBS, diluted in varying concentrations of ethanol. The administration of ethanol is a prerequisite to break the colonic mucosal barrier to allow penetration of DNBS or TNBS into the lamina propria. DNBS/TNBS will then haptenize the localized colonic and gut microbial proteins to become immunogenic, thereby triggering the host innate and adaptive immune responses. In general, this model is associated with severe and sometimes bloody diarrhea, weight loss and intestinal wall thickening however symptoms vary depending upon the type of rodent used, as well as the timing, dose and degree of exposure to the DNBS or TNBS used in the study. Important distinctions between rats and mice should be noted, with the benefits of lower cost for purchase and board, as well as lower body mass for decreasing per-animal costs of treatment in vivo. This should be set against the more rapid and severe course of colitis in mice, where the more fragile and responsive animals may quickly reach a humane endpoint.
The intestinal inflammation initially results from ethanol induced damage to intestinal epithelial cells, leading to increased epithelial permeability, microbial penetration into the mucosa, haptenization of host proteins, all of this resulting in infiltration of neutrophils, macrophages and Th1 T lymphocytes into the damaged mucosa. In comparison to DNBS, TNBS is considered as a hazardous chemical due to its highly oxidative properties that can pose a risk of explosion upon contact with bases such as sodium and potassium hydroxide. Therefore DNBS is currently regarded as a preferred choice of chemical over TNBS to induce colitis. In rodents, DNBS colitis is considered as one of the most convenient methods to study the following IBD associated modifiers of disease:
<ol>
	<li>Depression and reactivation of colitis: It has been shown that stress, anxiety and depression in IBD patients are frequently associated with disease relapse. DNBS colitis is a suitable model to study the role of depression and its consequences on reactivation of colitis in mice. The method typically involves initial induction of colitis by DNBS, followed by the resolution of the colitis by leaving the mice for 6-8 weeks. The mice are then administered with depression causing agents such as reserpine or by olfactory bulbectomy to induce depression followed by testing them for reactivation of colitis through challenge with a subcolitic dose of DNBS2.</li>
	<li>Stress and reactivation of colitis: Stress is another common environmental factor that has been linked to IBD pathogenesis. There is a growing body of evidence that suggests a strong association between chronic stress and the onset of symptoms of UC and CD in rodents as well as in humans and nonhuman primates3. DNBS colitis is a good model to study stress-associated reactivation of colitis in both mice and rats. The method is usually employed in a similar fashion as mentioned above for depression except in place of depression, the animals are exposed to stressors such as sonic and restraint stress4.</li>
	<li>Neurogenic inflammation: A number of studies have described either transient or permanent alterations in the enteric nervous system (ENS) structure and function, as seen in tissue samples from animal models and from patients with IBD. DNBS is a good model to explore the effects of inflammation on the ENS5 and to study both noradrenergic and cholinergic neural pathways6.</li>
	<li>Injury-repair mechanisms: During IBD pathogenesis, host-derived injury mediators such as reactive oxygen species (ROS), nitric oxide (NO), intercellular adhesion molecule 3 (ICAM-3), and P-selectin have all been shown to play a role in intestinal epithelial disruption. DNBS is an excellent model to study injuries caused by the up-regulation of these mediators as well as repair mechanisms that are regulated by using selective drugs or inhibitors7,8.</li>
	<li>Transmural inflammation: In addition to the above mentioned applications of DNBS, the model can also be applied to study transmural inflammation of the gut, a classical feature found in patients with CD. Both DNBS and TNBS-induced colitis are associated with significant infiltration of lymphocytes, into the colonic mucosa making these models particularly useful to study T-cell dependent immune mechanisms.</li>
</ol>
In comparison with the DSS model, the advantages of DNBS and TNBS-induced colitis include low cost, rapid development of colitis (usually requires 1-3 days to show reproducible ulceration and inflammation) and consistent localized damage to the distal colon. However the drawbacks are a requirement for a greater level of technical expertise, optimization of DNBS/ TNBS dose, and the need for anesthesia for rectal administration.
In this methodological paper we studied the effects of varying concentrations of n-6 and n-3 polyunsaturated fatty acids on altering the colonic mucosal response to DNBS-induced colitis in rats using the vegetable oils safflower oil (SO) and canola oil (CO), and fish oil (FO). It has been shown that n-6 and n-3 fatty acids are important mediators of intestinal inflammatory disease through their role as acyl moieties of cell membrane phosopholipids9. In contrast to the proinflammatory potential of n-6 fatty acids, n-3 fatty acids at sufficiently high intake are potentially potent anti-inflammatory agents. The anti-inflammatory actions of n-3 fatty acids are mediated directly through replacement of arachidonic acid as an eicosanoid substrate, inhibiting arachidonic acid metabolism and indirectly through alteration of proinflammatory gene expression and cell signaling. The n-3 fatty acids also give rise to resolvins, a family of anti-inflammatory mediators. High intake of n-3 fatty acids is associated with a decrease in production of proinflammatory eicosanoids, cytokines, chemokines, reactive oxygen species and expression of adhesion molecules. In this study, rats were fed ad libitum diets identical in all nutrients except fatty acids, with as a percent energy from fat, 20% SO, 20% CO, or 18% fish oil plus 2% safflower oil (FO)10-11. As a percentage&#160;of the daily energy, the SO diet provided 15% linoleic acid (LA), with &#60;0.06% &#945;-linolenic acid (ALA) and no eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the CO diet had 4.2% LA and 1.9% ALA with no EPA or DHA, and the FO diet provided 1.4% EPA, 4.9% DHA, 0.32% LA, and 0.12% ALA. Three weeks after initiation of the lipid diets, the mice were administered intrarectal DNBS or 50% ethanol and sacrificed 5 days later. The inflammatory response was evaluated by assessment of weight loss, histological damage scores and tissue myeloperoxidase activity.

<table><tbody><tr><td>1 ml Syringe</td><td>BD Biosciences</td><td>309659</td><td>&amp;nbsp;</td></tr><tr><td>19 G Needle</td><td>BD Biosciences</td><td>305187</td><td>&amp;nbsp;</td></tr><tr><td>Polyethylene tubing PE-50</td><td>BD Biosciences</td><td>427517</td><td>for intrarectal administration in mice</td></tr><tr><td>Polyethylene tubing PE-90</td><td>BD Biosciences</td><td>427519</td><td>for intrarectal administration in rat</td></tr><tr><td>DNBS</td><td>MP Bio</td><td>150959</td><td>&amp;nbsp;</td></tr><tr><td>Tear gel</td><td>Novartis</td><td>63601662596</td><td>&amp;nbsp;</td></tr><tr><td>10% Formalin</td><td>Fisher</td><td>5F93-4</td><td>&amp;nbsp;</td></tr><tr><td>Warming pad</td><td>Kent Scientific</td><td>TPZ-0510</td><td>&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>A-962.4</td><td>&amp;nbsp;</td></tr><tr><td>Hexadecyltrimethyl-ammonium bromide</td><td>Sigma-Aldrich</td><td>H5882</td><td>&amp;nbsp;</td></tr><tr><td>Potassium phosphate buffer solution</td><td>Sigma-Aldrich</td><td>79628-</td><td>&amp;nbsp;</td></tr><tr><td>o-Dianisidine dihydrochloride</td><td>Sigma-Aldrich</td><td>D3252</td><td>&amp;nbsp;</td></tr><tr><td>30% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>31642</td><td>hydrogen peroxide</td></tr><tr><td>Spectrophotometer</td><td>BioRad</td><td>Benchmarker Plus</td><td>&amp;nbsp;</td></tr><tr><td>Light Microscope</td><td>Zeiss</td><td>Axio Image.Z1</td><td>&amp;nbsp;</td></tr><tr><td>Data analysis software program&amp;nbsp;</td><td>GraphPad Software</td><td>GraphPad Prism Software Version 4.00</td><td>&lt;a href=&quot;http://www.graphpad.com/&quot;&gt;www.graphpad.com&lt;/a&gt;</td></tr></tbody></table>

dnbs/tnbs colitis models: providing insights into inflammatory bowel disease and effects of dietary fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

Challenge of rats (Sprague-Dawley) or mice (C57BL/6) with DNBS typically results in transient weight loss and diarrhea, sometimes with blood present in the stool. However, factors such as genetics, diet and the intestinal microbiota can modify susceptibility to DNBS induced colitis. Therefore, animals included in the experiments should be from the same institution, either derived locally or ordered in from one animal supplier. All animals should be carefully monitored for the degree of weight loss and symptoms of distres...

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DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

dnbstnbs-colitis-models-providing-insights-into-inflammatory-bowel

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47.1K Views.  BC Children's Hospital. DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Video: DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Biochemistry

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Biology

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Immunology and Infection

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

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.

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 ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.