Published online Jun 14, 2016. doi: 10.3748/wjg.v22.i22.5154
Peer-review started: February 9, 2016
First decision: March 7, 2016
Revised: March 23, 2016
Accepted: April 7, 2016
Article in press: April 7, 2016
Published online: June 14, 2016
Processing time: 120 Days and 19.7 Hours
AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn’s disease (CD).
METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry.
RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time.
CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.
Core tip: Intestinal strictures in Crohn’s disease (CD) cause hardly treatable complications in patients. The aim of this study was to find the correlation between the intestinal stricture formation, the damaged innervation of smooth muscle cells (SMCs) and the changed expression of TGF-beta 2, 3 and MMP9/TIMP1 in rats with CD by using different light- and electron microscopic and molecular biological methods. Our findings indicate that disintegration of SMCs due to the up-regulation of TGF-beta 2 and off-balance in MMP9/TIMP1 expression rather than neuronal cell death play the primary role in the formation of intestinal strictures in CD.