Name: a tnbs-induced rodent model to study the pathogenic role of mechanical stress in crohn's disease
Uploaded: 2022-03-01T14:30:00
Description: Watch this Scientific Journal Video about A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease at JoVE.com

This work is supported in part by grants from NIH (R01 DK124611 to XZS) and the US Department of Defense (W81XWH-20-1-0681 to XZS). The&#160;histology work was done with the help of the UTMB Surgical Pathology Lab.

All animal experiments were conducted according to the institutional animal care and use committee of the University of Texas Medical Branch (#0907051C). Male or female Sprague-Dawley rats, ~8-9 weeks old, were used for the study.
1. Animal preparation
<ol>
	<li>Fast rats for 24 h and treat them with laxative (bowel cleanser, see Table of Materials) overnight.</li>
	<li>The next day, anesthetize rats using an anesthesia system (see Table of Materials...

<ol>
	<li>Kappelman, M. 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=The+prevalence+and+geographic+distribution+of+Crohn's+disease+and+ulcerative+colitis+in+the+United+States.">The prevalence and geographic distribution of Crohn's disease and ulcerative colitis in the United States.</a> Clinical Gastroenterology and Hepatology. 5 (12), 1424-1429 (2007).</li><li>Hwang, J. M., Varma, 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=Surgery+for+inflammatory+bowel+disease.">Surgery for inflammatory bowel disease.</a> World Journal of Gastroenterology. 14 (17), 2678-2690 (2008).</li><li>Latella, G., Rieder, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+fibrosis:+Ready+to+be+reversed.">Intestinal fibrosis: Ready to be reversed.</a> Current Opinion in Gastroenterology. 33 (4), 239-245 (2017).</li><li>Rieder, F., Fiocchi, C., Rogler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms,+management,+and+treatment+of+fibrosis+in+patients+with+inflammatory+bowel+diseases.">Mechanisms, management, and treatment of fibrosis in patients with inflammatory bowel diseases.</a> Gastroenterology. 152 (2), 340-350 (2017).</li><li>Bettenworth, 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=Assessment+of+Crohn's+disease-associated+small+bowel+strictures+and+fibrosis+on+cross-sectional+imaging:+A+systematic+review.">Assessment of Crohn's disease-associated small bowel strictures and fibrosis on cross-sectional imaging: A systematic review.</a> Gut. 68 (6), 1115-1126 (2019).</li><li>Chen, W., Lu, C., Hirota, C., Iacucci, M., Ghosh, S., Gui, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+hyperplasia/hypertrophy+is+the+most+prominent+histological+change+in+Crohn's+fibrostenosing+bowel+strictures:+A+semiquantitative+analysis+by+using+a+novel+histological+grading+scheme.">Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: A semiquantitative analysis by using a novel histological grading scheme.</a> Journal of Crohn's and Colitis. 11 (1), 92-104 (2017).</li><li>Olaison, G., Smedh, K., Sj&#246;dahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+course+of+Crohn's+disease+after+ileocolic+resection:+Endoscopically+visualised+ileal+ulcers+preceding+symptoms.">Natural course of Crohn's disease after ileocolic resection: Endoscopically visualised ileal ulcers preceding symptoms.</a> Gut. 33 (3), 331-335 (1992).</li><li>Lin, Y. M., Li, F., Shi, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stress+is+a+pro-inflammatory+stimulus+in+the+gut:+In+vitro,+in+vivo+and+ex+vivo+evidence.">Mechanical stress is a pro-inflammatory stimulus in the gut: In vitro, in vivo and ex vivo evidence.</a> PLoS One. 9, 106242 (2014).</li><li>Gabella, G., Yamey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+collagen+by+smooth+muscle+in+the+hyertrophic+intestine.">Synthesis of collagen by smooth muscle in the hyertrophic intestine.</a> Experimental Physiology. 62 (3), 257-264 (1977).</li><li>Katsanos, K. H., Tsianos, V. E., Maliouki, M., Adamidi, M., Vagias, I., Tsianos, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obstruction+and+pseudo-obstruction+in+inflammatory+bowel+disease.">Obstruction and pseudo-obstruction in inflammatory bowel disease.</a> Annals of Gastroenterology. 23 (4), 243-256 (2010).</li><li>Johnson, L. A., 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=Matrix+stiffness+corresponding+to+strictured+bowel+induces+a+fibrogenic+response+in+human+colonic+fibroblasts.">Matrix stiffness corresponding to strictured bowel induces a fibrogenic response in human colonic fibroblasts.</a> Inflammatory Bowel Disease. 19 (5), 891-903 (2013).</li><li>Gayer, C. P., Basson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+forces+on+intestinal+physiology+and+pathology.">The effects of mechanical forces on intestinal physiology and pathology.</a> Cell Signalling. 21 (8), 1237-1244 (2009).</li><li>Cox, C. 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=Hypertonic+saline+modulation+of+intestinal+tissue+stress+and+fluid+balance.">Hypertonic saline modulation of intestinal tissue stress and fluid balance.</a> Shock. 29 (5), 598-602 (2008).</li><li>Shi, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+gene+expression+in+gut+smooth+muscle+cells.">Mechanical regulation of gene expression in gut smooth muscle cells.</a> Frontiers in Physiology. 8, 1000 (2017).</li><li>Shi, X. Z., Lin, Y. M., Powell, D. W., Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+motility+dysfunction+in+bowel+obstruction:+Role+of+stretch-induced+COX-2.">Pathophysiology of motility dysfunction in bowel obstruction: Role of stretch-induced COX-2.</a> American Journal of Physiology-Gastrointestinal and Liver. 300 (1), 99-108 (2011).</li><li>Gutierrez, J. A., Perr, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+modulates+TGF-beta1+and+alpha1(I)+collagen+expression+in+fetal+human+intestinal+smooth+muscle+cells.">Mechanical stretch modulates TGF-beta1 and alpha1(I) collagen expression in fetal human intestinal smooth muscle cells.</a> American Journal of Physiology. 277 (5), 1074-1080 (1999).</li><li>Lipson, K. E., Wong, C., Teng, Y., Spong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTGF+is+a+central+mediator+of+tissue+remodeling+and+fibrosis+and+its+inhibition+can+reverse+the+process+of+fibrosis.">CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis.</a> Fibrogenesis Tissue Repair. 5, 24 (2012).</li><li>Chaqour, B., Goppelt-Struebe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+the+Cyr61/CCN1+and+CTGF/CCN2+proteins.">Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins.</a> The FEBS Journal. 273 (16), 3639-3649 (2006).</li><li>Shi, X. Z., Winston, J. H., Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+immune+and+genetic+responses+in+rat+models+of+Crohn's+colitis+and+ulcerative+colitis.">Differential immune and genetic responses in rat models of Crohn's colitis and ulcerative colitis.</a> American Journal of Physiology-Gastrointestinal and Liver. 300 (1), 41-51 (2011).</li><li>Antoniou, 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=The+TNBS-induced+colitis+animal+model:+An+overview.">The TNBS-induced colitis animal model: An overview.</a> Annals of Medicine and Surgery (London). 11, 9-15 (2016).</li><li>Shi, X. Z., Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+of+Cav1.2+channel+with+VIP+and+VIP+receptor+agonists+and+antagonists:+A+novel+approach+to+designing+promotility+and+antimotility+agents.">Gene therapy of Cav1.2 channel with VIP and VIP receptor agonists and antagonists: A novel approach to designing promotility and antimotility agents.</a> American Journal of Physiology-Gastrointestinal and Liver. 295 (1), 187-196 (2008).</li><li>Lin, Y. M., Sarna, S. K., Shi, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophylactic+and+therapeutic+benefits+of+COX-2+inhibitor+on+motility+dysfunction+in+bowel+obstruction:+Roles+of+PGE2+and+EP+receptors.">Prophylactic and therapeutic benefits of COX-2 inhibitor on motility dysfunction in bowel obstruction: Roles of PGE2 and EP receptors.</a> American Journal of Physiology-Gastrointestinal and Liver. 302 (2), 267-275 (2012).</li><li>Morris, G. P., Beck, P. L., Herridge, M. S., Depew, W. T., Szewczuk, M. R., Wallace, J. L. <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+chronic+inflammation+and+ulceration+in+the+rat+colon.">Hapten-induced model of chronic inflammation and ulceration in the rat colon.</a> Gastroenterology. 96 (3), 795-803 (1989).</li><li>Mudter, J., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Il-6+signaling+in+inflammatory+bowel+disease:+Pathophysiological+role+and+clinical+relevance.">Il-6 signaling in inflammatory bowel disease: Pathophysiological role and clinical relevance.</a> Inflammatory Bowel Disease. 13 (8), 1016-1023 (2007).</li><li>Geesala, R., Lin, Y. M., Zhang, K., Shi, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+mechano-transcription+process+as+therapeutic+intervention+in+gastrointestinal+disorders.">Targeting mechano-transcription process as therapeutic intervention in gastrointestinal disorders.</a> Frontiers in Pharmacology. 12, 809350 (2021).</li></ol>

TNBS-induced colitis was introduced in 1989&#160;and has been used as an experimental model of Crohn&#39;s disease since then19,20,23. Significant features of this model in rodents include the development of a transmural inflammation that closely resembles the histopathological lesions developed in human Crohn&#39;s disease19,20. Previous studies on the model have focuse...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63499">A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn&#39;s Disease</a>. The Introduction and Protocol were updated.
The first sentence of the Introduction was updated from:
Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,00,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis.
to:
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.
Step 1.3 in the Protocol was updated from:
Prepare fresh TNBS solution according to body weights. 
NOTE: TNBS - 65 mg/kg of body weight in 250 &#181;L of 40% ethanol was used.
to:
Prepare fresh TNBS solution according to body weights. 
NOTE: TNBS - 65 mg/kg of body weight in 250 &#181;L of 40% ethanol/saline was used.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63499">A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn&#39;s Disease</a>. The Introduction and Protocol were updated.

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

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn&#39;s disease (CD), is characterized by chronic inflammation in the gastrointestinal (GI) tract. It affects ~1-2 million Americans1. The estimated annual costs for IBD care in the US are $11.8 billion. Unlike UC, the CD is characterized by transmural inflammation and stricture formation2,3. Stricture formation (stenosis) occurs in up to 70% of CD patients3 and may be caused by transmural inflammation (inflammatory stenosis) or intestinal fibrosis (fibrotic stenosis)4,5. Intestinal fibrosis is characterized by excessive collagen deposition and other extracellular matrices (ECM) with smooth muscle cells (SMC) as one of the main mesenchymal cell types involved in the process3,4. Smooth muscle hyperplasia associated with hypertrophy is another significant histological change in fibrotic stenosis in CD6. Although stricture formation in CD is associated with chronic inflammation, no anti-inflammatory treatment is effective, except surgical treatment2,6. However, post-surgery recurrences are almost 100%, given sufficient time2,7. As an inflammatory response, fibrosis and SMC hyperplasia may also develop in non-inflammatory conditions (i.e., bowel obstruction) in the gut8,9; it is believed that both inflammation-dependent and independent mechanisms are involved in stricture formation3,4. Given that extensive research into the inflammation-dependent mechanisms has not translated into any effective therapy for stricture formation, studies into the possible role of inflammation-independent mechanisms in intestinal fibrosis are needed.
As a non-inflammatory factor, mechanical stress (MS) associated with edema, inflammatory cell infiltration, tissue deformation, fibrosis, and stenosis10,11,12,13 is commonly encountered in IBD, especially CD, which is characterized by transmural inflammation. Mechanical stress is most remarkable in stenotic CD, where stenosis (inflammatory or fibrotic) in the inflammation site presents mechanical stress in the local tissue and leads to lumen distention in the segment proximal to the obstruction site10,14. Previous in vitro studies have demonstrated that mechanical stress alters gene expression of specific inflammatory mediators (i.e., COX-2, IL-6)8,14,15 and growth factors (i.e., TGF-&#946;) in the gastrointestinal tissues, especially gut smooth muscle cells (SMC)16. Recent studies also found that the expression of specific pro-fibrotic mediators such as connective tissue growth factor (CTGF) is highly sensitive to mechanical stress17,18. It was hypothesized that mechanical stress might play an independent pathogenic role in CD-associated inflammation, fibrosis, and tissue remodeling. However, the pathogenic significance of mechanical stress in gut inflammation, fibrosis, and smooth muscle hyperplasia in CD remains largely unexplored. This may be partly because inflammation is a more visible and better-studied process than mechanical stress. More importantly, there has been no well-defined animal model of IBD to distinguish the effect of mechanical stress from that of inflammation.
The current work describes a rodent model of Crohn&#39;s-like colitis induced by intracolonic injection of hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS)19,20, which may serve the purpose to study the role of mechanical stress in CD. It was found that TNBS instillation induced a localized (~2 cm in length) transmural inflammation with lumen narrowing (stenosis) in the distal colon. The stenosis leads to marked bowel distention (mechanical stress)14,15 but not visible inflammation in the colonic segment proximal to the instillation site. On the contrary, the colon segment distal to the stenosis site presents neither inflammation nor mechanical stress. Significant site-specific changes in gene expression, inflammation, fibrosis, and SMC hyperplasia were observed in the three different sites. The results suggest that mechanical stress, particularly mechanical stress-induced gene expression, may play a critical role in developing fibrosis and hyperplasia in Crohn&#39;s colitis.

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			<td>ACT-1 Control Software Ver2.63</td>
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			<td>C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module</td>
			<td>BIO-RAD</td>
			<td>1851196</td>
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			<td>CFX96 Optical Reaction Module for Real-Time PCR Systems</td>
			<td>BIO-RAD</td>
			<td>1845097</td>
			<td></td>
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			<td>Dako Agilent Artisan Link Pro Special stainer</td>
			<td>Dako</td>
			<td>AR310</td>
			<td></td>
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			<td>Dako-Agilent Masson&#39;s Trichrome Kit ref# AR173</td>
			<td>Dako</td>
			<td>AR173</td>
			<td></td>
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			<td>DXM1200 Digital Color HR Camera</td>
			<td>Nikon</td>
			<td>DXM1200</td>
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			<td>Eukaryotic 18S rRNA Endogenous Control</td>
			<td>ThermoFisher Scientific</td>
			<td>4352930E</td>
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			<td>E-Z Anesthesia</td>
			<td>E-Z Systems Inc.</td>
			<td>EZ-155</td>
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			<td>GraphPad Prism 9</td>
			<td>GraphPad</td>
			<td>9.0.2 (161)</td>
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			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear</td>
			<td>BIO-RAD</td>
			<td>HSP9601</td>
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			<td>HBSS (Corning Hank&#39;s Balanced Salt Solution, 1x without calcium and magnesium)</td>
			<td>CORNING</td>
			<td>21-021-CV</td>
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			<td>HM 325 Microtome</td>
			<td>Thermo Scientific</td>
			<td>23-900-667</td>
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			<td>Isoflurane</td>
			<td>Piramal</td>
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			<td>LI-COR Odyssey Digital Imaging System</td>
			<td>LI-COR</td>
			<td>9120</td>
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			<td>Mastercycler epGradient Thermal Cycler with Control Panel 5340 Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>5341</td>
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			<td>Medical grade open end polyurethane catheter</td>
			<td>Covidien</td>
			<td>8890703013</td>
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			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND2000CLAPTOP</td>
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			<td>Nikon Eclipse E800 Upright Microscope</td>
			<td>Nikon</td>
			<td>E800</td>
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			<td>Nitrocellulose/Filter Paper Sandwiches Pkg of 50, 0.45 &#956;m, 7 x 8.5 cm</td>
			<td>BIO-RAD</td>
			<td>1620215</td>
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			<td>Polyethylene Glycol 3350, Osmotic Laxative</td>
			<td>Miralax</td>
			<td>C8175</td>
			<td>Dose: 17g in 226 mL of water</td>
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			<td>RNeasy Mini Kit (250) 
			250 RNeasy Mini Spin Columns, Collection Tubes (1.5 mL and 2 mL), RNase-free Reagents and Buffers</td>
			<td>QIAGEN</td>
			<td>74106</td>
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			<td>SuperScript III First-Strand Synthesis System</td>
			<td>ThermoFisher Scientific</td>
			<td>18080051</td>
			<td></td>
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			<td>TaqMan Gene Expression Assays Rn00573960_g1 CTGF Probe</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assays Rn99999011_m1 IL6 Probe</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>4444557</td>
			<td></td>
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			<td>Tissue-Tek Prisma H&#38;E Stain Kit #1</td>
			<td>Sakura</td>
			<td>6190</td>
			<td></td>
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			<td>Tissue-Tek Prisma Plus Automated Slide Stainer</td>
			<td>Sakura</td>
			<td>6171</td>
			<td></td>
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			<td>TNBS (Picrylsulfonic acid solution)</td>
			<td>SIGMA-ALDRICH</td>
			<td>92822</td>
			<td></td>
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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.

Macroscopic view of Crohn&#39;s-like colitis induced by intra-colonic instillation of TNBS 
As shown in Figure 1, intracolonic instillation of TNBS in rats induced a localized transmural inflammation (~2 cm in length) with thickened bowel wall and narrowed lumen (stenosis) in the site of instillation in the distal colon (Figure 1A). The site of TNBS instillation is referred to as site I. As a result of transmural inflammation and stenosis, ...

Watch this Scientific Journal Video about A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease at JoVE.com

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

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.

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

So in our protocol, intra-colonic instillation of TNBS leads to transmural inflammation at the instillation site and the mechanical distention in the segment proximal to inflammation. So this mimics stenotic Crohn's disease. As the proximal segment is mechanically distended but does not show a visible inflammation, a comparative study of the proximal segment and the inflammation site helps to determine if mechanical stress plays a role in Crohn's colitis.Previous studies focus on the inflammation site only. Our protocol working on different sites in the model highlights a critical role of mechanical stress in Crohn's disease, particularly in stenotic Crohn's disease. To begin, place the rats on the anesthesia table in a supine position.To induce colitis, insert a medical-grade, open-end, polyurethane catheter through the anus for approximately seven to eight centimeters from the anal verge, and gently instill TNBS into the colon. Administer the sham control rats with 250 microliters of saline. To help the distribution of TNBS or saline and avoid spills, keep the rats in a supine position with their head facing downwards at approximately a 30-degree angle, with the anus closed for two minutes.Open the rat abdomen using surgical-grade scissors and forceps. Carefully remove the entire colon above the anal canal, and transfer the colon immediately to ice-cold 1x HBSS buffer. Straighten the colon in the buffer, and measure the colon length using a ruler.Take a nylon thread, and circle it around the colon to measure the external circumference of the colon segments in control and TNBS-treated rats. Take full-thickness tissues for histology. Cut open the colon along the mesenteric board, and clean the colon thoroughly with HBSS buffer.Assess the colon for macroscopic inflammation score based on the predefined criteria with minimal modifications. Collect colonic tissue samples from TNBS-treated rats at sites P, I, and D.Site P is the portion two to three centimeters before the oral margin of the inflammation site. Site I is the inflammation site where TNBS is instilled, typically four to six centimeters from the end of the colon.Finally, site D is the portion one to two centimeters distal to the aboral margin of the inflammation site. Intra-colonic instillation of TNBS in rats induced a localized transmural inflammation approximately two centimeters in length with thickened bowel wall and narrowed lumen in the site of instillation in the distal colon. The site of TNBS instillation was referred to as site I.As a result of transmural inflammation and stenosis, both inflammation and mechanical stress were present in site I.The stenosis in site I led to marked lumen distension in the segment proximal to the site of TNBS instillation, referred to as site P.The colon circumference was significantly increased in sites P and I compared to sham control colon.While mechanically distended, site P did not show visible inflammation. On the contrary, the colonic segment distal to the TNBS instillation referred to as site D presented neither inflammation nor mechanical distension. The macroscopic score of inflammation in site I in the TNBS-treated rats was dramatically increased compared to sham controls in sites P and D of colitis rats.The inflammation scores in sites P and D were not significantly increased compared to sham. So it is important to study not just site I but sites P and D in the model, as this helps to recognize the pathogenic significance of mechanical stress in Crohn's disease. To further determine if mechanical stress is a pathogenic factor in gut inflammation, one may also try to release mechanical distention by feeding the animals exclusively with liquid diet and see if this helps with inflammation and bowel function.

Research

JoVE Journal

Immunology and Infection

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