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

Summary

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.

Abstract

Inflammatory bowel diseases (IBD) such as Crohn'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'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.

Introduction

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn'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 formation^2,3. 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 process^3,4. 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 treatment^2,6. However, post-surgery recurrences are almost 100%, given sufficient time^2,7. As an inflammatory response, fibrosis and SMC hyperplasia may also develop in non-inflammatory conditions (i.e., bowel obstruction) in the gut^8,9; it is believed that both inflammation-dependent and independent mechanisms are involved in stricture formation^3,4. 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 stenosis^10,11,12,13 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 site^10,14. 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-β) 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 stress^17,18. 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'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's colitis.

Protocol

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

1. Fast rats for 24 h and treat them with laxative (bowel cleanser, see Table of Materials) overnight.
2. 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.
3. Prepare fresh TNBS solution according to body weights.
   ​NOTE: TNBS - 65 mg/kg of body weight in 250 µL of 40% ethanol/saline was used.
4. 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 µL of saline only.
5. After instilling TNBS or saline, keep rats in supine and slightly head-down position (~30°), with the anus closed for 2 min to help TNBS distribution and avoid spills.
6. 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.

2. Tissue preparations

1. On the day of euthanasia, euthanize the rats using CO₂ inhalation and confirm euthanasia with cervical dislocation.
2. Open the rat abdomen using surgical-grade scissors and forceps.
3. Carefully remove the entire colon (above the anal canal) and transfer the colon immediately to ice-cold 1x HBSS buffer.
4. 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.
5. 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 < 5 mm); 3 = severe ulcer, scar, and stenosis (affected area > 5 mm).
6. 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).
7. Take tissue samples from each site for full-thickness preparation, and if desired, mucosa/submucosa and muscularis externa layers, respectively, as well^21,22.
8. Freeze tissue samples in liquid nitrogen first before storing them at -80 °C for storage up to one year and for future purposes (i.e., RNA preparations).

3. Histopathologic assessment of gut inflammation and fibrosis

1. Fix the full-thickness colon tissues in 10% formalin for 48 h, then transfer to 70% ethanol for 24-48 h.
2. Use a microtome to cut paraffin sections of 5 µm thickness for Hematoxylin and eosin (H&E) and Masson's Trichrome stains^6,19,23 (see Table of Materials), respectively.
3. Acquire and view images with an upright microscope equipped with a high-resolution camera with compatible software (see Table of Materials).
4. Grade inflammation and fibrosis indexes by two independent investigators, including a gastrointestinal surgical pathologist according to criteria described previously^6,23 with modifications. See Supplementary File 1 for the scores.
5. Measure the thickness and cell numbers of the circular and longitudinal muscle layers per cross-section in four views of each H&E stained specimen and take the mean of the four measurements for each specimen.

4. RNA extraction and quantitative RT-PCR

1. 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).
2. Isolate RNA from each sample utilizing the kit. Elute the RNA pellet in 30 µL of RNase-free water.
3. Quantify RNA concentration and check for purity using a microvolume UV-Vis spectrophotometer (see Table of Materials).
4. Use 1 µg of total RNA to synthesize cDNA^21,22 using the RNA synthesis kit (see Table of Materials).
5. 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).
6. Use control gene 18S rRNA to normalize the samples and quantify relative gene expression utilizing the Cq values obtained.

5. Statistical analysis

1. Utilize statistical analysis software (see Table of Materials) to compare sham control and TNBS colitis rats.
2. Consider p value < 0.05 to be statistically significant^15,19.
3. To test the differences between two groups, use Student's t-test analysis and perform an ANOVA test if comparisons are more than two groups^15,19.

Results

Macroscopic view of Crohn'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, ...

Discussion

TNBS-induced colitis was introduced in 1989 and has been used as an experimental model of Crohn's disease since then^19,20,23. Significant features of this model in rodents include the development of a transmural inflammation that closely resembles the histopathological lesions developed in human Crohn's disease^19,20. Previous studies on the model have focuse...

Materials

Name	Company	Catalog Number	Comments
ACT-1 Control Software Ver2.63	Nikon	DXM1200F
C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module	BIO-RAD	1851196
CFX96 Optical Reaction Module for Real-Time PCR Systems	BIO-RAD	1845097
Dako Agilent Artisan Link Pro Special stainer	Dako	AR310
Dako-Agilent Masson's Trichrome Kit ref# AR173	Dako	AR173
DXM1200 Digital Color HR Camera	Nikon	DXM1200
Eukaryotic 18S rRNA Endogenous Control	ThermoFisher Scientific	4352930E
E-Z Anesthesia	E-Z Systems Inc.	EZ-155
GraphPad Prism 9	GraphPad	9.0.2 (161)
Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear	BIO-RAD	HSP9601
HBSS (Corning Hank's Balanced Salt Solution, 1x without calcium and magnesium)	CORNING	21-021-CV
HM 325 Microtome	Thermo Scientific	23-900-667
Isoflurane	Piramal	NDC 66794-017-10
LI-COR Odyssey Digital Imaging System	LI-COR	9120
Mastercycler epGradient Thermal Cycler with Control Panel 5340 Thermal Cycler	Eppendorf	5341
Medical grade open end polyurethane catheter	Covidien	8890703013
NanoDrop 2000/2000c Spectrophotometers	Thermo Fisher Scientific	ND2000CLAPTOP
Nikon Eclipse E800 Upright Microscope	Nikon	E800
Nitrocellulose/Filter Paper Sandwiches Pkg of 50, 0.45 μm, 7 x 8.5 cm	BIO-RAD	1620215
Polyethylene Glycol 3350, Osmotic Laxative	Miralax	C8175	Dose: 17g in 226 mL of water
RNeasy Mini Kit (250) 250 RNeasy Mini Spin Columns, Collection Tubes (1.5 mL and 2 mL), RNase-free Reagents and Buffers	QIAGEN	74106
SuperScript III First-Strand Synthesis System	ThermoFisher Scientific	18080051
TaqMan Gene Expression Assays Rn00573960_g1 CTGF Probe	ThermoFisher Scientific	4331182
TaqMan Gene Expression Assays Rn99999011_m1 IL6 Probe	ThermoFisher Scientific	4331182
TaqMan Fast Advanced Master Mix	ThermoFisher Scientific	4444557
Tissue-Tek Prisma H&E Stain Kit #1	Sakura	6190
Tissue-Tek Prisma Plus Automated Slide Stainer	Sakura	6171
TNBS (Picrylsulfonic acid solution)	SIGMA-ALDRICH	92822