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.
