Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 14, 2016; 22(22): 5154-5164
Published online Jun 14, 2016. doi: 10.3748/wjg.v22.i22.5154
Structural and molecular features of intestinal strictures in rats with Crohn's-like disease
Petra Talapka, Anikó Berkó, Lajos István Nagy, Lalitha Chandrakumar, Mária Bagyánszki, László Géza Puskás, Éva Fekete, Nikolett Bódi
Petra Talapka, Anikó Berkó, Lalitha Chandrakumar, Mária Bagyánszki, Éva Fekete, Nikolett Bódi, Department of Physiology, Anatomy and Neuroscience, Faculty of Sciences and Informatics, University of Szeged, H-6726 Szeged, Hungary
Lajos István Nagy, László Géza Puskás, Avidin Ltd., H-6726 Szeged, Hungary
Author contributions: Talapka P and Berkó A contributed equally to this work; Talapka P performed the majority of experiments and analyzed the data; Berkó A and Nagy LI performed the molecular investigations; Chandrakumar L and Bódi N participated equally in treatment of animals; Bagyánszki M, Puskás LG, Fekete E and Bódi N designed and coordinated the research; Talapka P, Fekete E and Bódi N wrote the paper; Talapka P and Berkó A contributed equally to this work.
Supported by Hungarian Scientific Research Fund, No. OTKA PD 108309 to Bódi N; and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences to Bagyánszki M.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of the Department of Anatomy, Physiology and Neuroscience, Faculty of Sciences, University of Szeged.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and use committee of the University of Szeged (protocol number: EIK-645/2014.02.05).
Conflict-of-interest statement: We certify that there is no actual or potential conflict of interest in relation to this article.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author, Mária Bagyánszki at bmarcsi@bio.u-szeged.hu.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Mária Bagyánszki, PhD, Associate Professor, Department of Physiology, Anatomy and Neuroscience, Faculty of Sciences and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary. bmarcsi@bio.u-szeged.hu
Telephone: +36-62-544123 Fax: +36-62-544291
Received: February 2, 2016
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
Abstract

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.

Keywords: Crohn’s disease; Rat model; TGF-beta; Intestinal strictures; MMP9; TIMP1

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.