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) by exposing them to 2% isoflurane along with 1 L/min of oxygen during TNBS administration. Check for reflexes or pinch toes to confirm anesthetization.</li>
	<li>Prepare fresh TNBS solution according to body weights. 
	&#8203;NOTE: TNBS - 65 mg/kg of body weight in 250 &#181;L of 40% ethanol/saline&#160;was used.</li>
	<li>Put rats in a supine position on the anesthesia table. To induce colitis, insert through the anus a medical-grade open-end polyurethane catheter for ~7-8 cm from the anal verge and gently instill TNBS (prepared in step 1.3) into the colon19. Administer the sham control rats with 250 &#181;L of saline only.</li>
	<li>After instilling TNBS or saline, keep rats in supine and slightly head-down position (~30&#176;), with the anus closed for 2 min to help TNBS distribution and avoid spills.</li>
	<li>Provide rats with food and water ad libitum for 7 days and observe the rats daily for body weight, food uptakes, feces, and general health condition.</li>
</ol>
2. Tissue preparations
<ol>
	<li>On the day of euthanasia, euthanize the rats using CO2 inhalation and confirm euthanasia with cervical dislocation.</li>
	<li>Open the rat abdomen using surgical-grade scissors and forceps.</li>
	<li>Carefully remove the entire colon (above the anal canal) and transfer the colon immediately to ice-cold 1x HBSS buffer.</li>
	<li>Straighten the colon in the buffer and measure the colon length using a ruler. Take nylon thread and circle around the colon to measure the external circumference of the colon segments in control and TNBS-treated rats. Take full-thickness tissues for histology.</li>
	<li>Cut open the colon along the mesenteric board and clean the colon well with HBSS buffer. Assess the colon for macroscopic inflammation score based on the criteria as previously described19 with minimal modifications. 
	NOTE: 0 = normal mucosa; 1 = localized hyperemia but no erosions or ulcers; 2 = ulcer and stenosis (affected area &#60; 5 mm); 3 = severe ulcer, scar, and stenosis (affected area &#62; 5 mm).</li>
	<li>Collect colonic tissue samples from site P (portion 2-3 cm before the oral margin of inflammation site), site I (inflammation site, typically 4-6 cm from the end of the colon, where TNBS is instilled to), and site D (portion 1-2 cm distal to the aboral margin of inflammation site), respectively from TNBS-treated rats. 
	NOTE: Colon tissue of ~1-2 cm-long was taken from each segment. In addition, the colon tissues of 2 cm long (~4-6 cm from the end of the colon) of the saline-treated rats were taken as sham control (S) (Figure 1).</li>
	<li>Take tissue samples from each site for full-thickness preparation, and if desired, mucosa/submucosa and muscularis externa layers, respectively, as well21,22.</li>
	<li>Freeze tissue samples in liquid nitrogen first before storing them at -80 &#176;C for storage up to one year and for future purposes (i.e., RNA preparations).</li>
</ol>
3. Histopathologic assessment of gut inflammation and fibrosis
<ol>
	<li>Fix the full-thickness colon tissues in 10% formalin for 48 h, then transfer to 70% ethanol for 24-48 h.</li>
	<li>Use a microtome to cut paraffin sections of 5 &#181;m thickness for Hematoxylin and eosin (H&#38;E) and Masson&#39;s Trichrome stains6,19,23 (see Table of Materials), respectively.</li>
	<li>Acquire and view images with an upright microscope equipped with a high-resolution camera with compatible software (see Table of Materials).</li>
	<li>Grade inflammation and fibrosis indexes by two independent investigators, including a gastrointestinal surgical pathologist according to criteria described previously6,23 with modifications. See Supplementary File 1 for the scores.</li>
	<li>Measure the thickness and cell numbers of the circular and longitudinal muscle layers per cross-section in four views of each H&#38;E stained specimen and take the mean of the four measurements for each specimen.</li>
</ol>
4. RNA extraction and quantitative RT-PCR
<ol>
	<li>Homogenize excised colon tissues obtained from the sham control and three sites (P, I, D) of TNBS colitis rats in the extraction reagent of an RNA extraction kit (see Table of Materials).</li>
	<li>Isolate RNA from each sample utilizing the kit. Elute the RNA pellet in 30 &#181;L of RNase-free water.</li>
	<li>Quantify RNA concentration and check for purity using a microvolume UV-Vis spectrophotometer (see Table of Materials).</li>
	<li>Use 1 &#181;g of total RNA to synthesize cDNA21,22 using the RNA synthesis kit (see Table of Materials).</li>
	<li>Analyze and quantify gene expression levels by performing real-time PCR with 50 ng of cDNA as a template, probes of IL-6, and CTGF using a commercial PCR kit for real-time PCR system (see Table of Materials).</li>
	<li>Use control gene 18S rRNA to normalize the samples and quantify relative gene expression utilizing the Cq values obtained.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Utilize statistical analysis software (see Table of Materials) to compare sham control and TNBS colitis rats.</li>
	<li>Consider p value &#60; 0.05 to be statistically significant15,19.</li>
	<li>To test the differences between two groups, use Student&#39;s t-test analysis and perform an ANOVA test if comparisons are more than two groups15,19.</li>
</ol>

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The authors report no conflict of interest and have nothing to disclose.

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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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.

<table><tbody><tr><td>ACT-1 Control Software Ver2.63</td><td>Nikon</td><td>DXM1200F</td><td></td></tr><tr><td>C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module</td><td>BIO-RAD</td><td>1851196</td><td></td></tr><tr><td>CFX96 Optical Reaction Module for Real-Time PCR Systems</td><td>BIO-RAD</td><td>1845097</td><td></td></tr><tr><td>Dako Agilent Artisan Link Pro Special stainer</td><td>Dako</td><td>AR310</td><td></td></tr><tr><td>Dako-Agilent Masson&#039;s Trichrome Kit ref# AR173</td><td>Dako</td><td>AR173</td><td></td></tr><tr><td>DXM1200 Digital Color HR Camera</td><td>Nikon</td><td>DXM1200</td><td></td></tr><tr><td>Eukaryotic 18S rRNA Endogenous Control</td><td>ThermoFisher Scientific</td><td>4352930E</td><td></td></tr><tr><td>E-Z Anesthesia</td><td>E-Z Systems Inc.</td><td>EZ-155</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad</td><td>9.0.2 (161)</td><td></td></tr><tr><td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear</td><td>BIO-RAD</td><td>HSP9601</td><td></td></tr><tr><td>HBSS (Corning Hank&#039;s Balanced Salt Solution, 1x without calcium and magnesium)</td><td>CORNING</td><td>21-021-CV</td><td></td></tr><tr><td>HM 325 Microtome</td><td>Thermo Scientific</td><td>23-900-667</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>NDC 66794-017-10</td><td></td></tr><tr><td>LI-COR Odyssey Digital Imaging System</td><td>LI-COR</td><td>9120</td><td></td></tr><tr><td>Mastercycler epGradient Thermal Cycler with Control Panel 5340 Thermal Cycler</td><td>Eppendorf</td><td>5341</td><td></td></tr><tr><td>Medical grade open end polyurethane catheter</td><td>Covidien</td><td>8890703013</td><td></td></tr><tr><td>NanoDrop 2000/2000c Spectrophotometers</td><td>Thermo Fisher Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>Nikon Eclipse E800 Upright Microscope</td><td>Nikon</td><td>E800</td><td></td></tr><tr><td>Nitrocellulose/Filter Paper Sandwiches Pkg of 50, 0.45 &amp;mu;m, 7 x 8.5 cm</td><td>BIO-RAD</td><td>1620215</td><td></td></tr><tr><td>Polyethylene Glycol 3350, Osmotic Laxative</td><td>Miralax</td><td>C8175</td><td>Dose: 17g in 226 mL of water</td></tr><tr><td>RNeasy Mini Kit (250)&lt;br/&gt; 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><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>ThermoFisher Scientific</td><td>18080051</td><td></td></tr><tr><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></tr><tr><td>Tissue-Tek Prisma H&amp;amp;E Stain Kit #1</td><td>Sakura</td><td>6190</td><td></td></tr><tr><td>Tissue-Tek Prisma &lt;em&gt;Plus &lt;/em&gt;Automated Slide Stainer</td><td>Sakura</td><td>6171</td><td></td></tr><tr><td>TNBS (Picrylsulfonic acid solution)</td><td>SIGMA-ALDRICH</td><td>92822</td><td></td></tr></tbody></table>

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 ...

a-tnbs-induced-rodent-model-to-study-pathogenic-role-mechanical

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

3.8K Views.  the University of Texas Medical Branch. 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.

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

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

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

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.

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 ...

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

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 ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Author Spotlight: Developing a Rat Model for Pouchitis Research and Treatment

This protocol describes the method for establishing a rat model of pouchitis. The ileal pouch model was created by performing ileal pouch-anal anastomosis (IPAA) surgery using microsurgical techniques. After the surgery, the rat was treated with 4% dextran sulfate sodium (DSS) for 4 days.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing ...

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

Summary

Explore More Videos

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

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

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

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

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

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4⁺ T Cells to Immunodeficient Mice

A Mouse Model of Intestinal Partial Obstruction

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium