Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.
OBJECTIVE
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.


CONCLUSIONS
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.
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.
Petra
P
Talapka
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.
Anikó
A
Berkó
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
LI
Nagy
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.
Lalitha
L
Chandrakumar
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.
Mária
M
Bagyánszki
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.
László Géza
LG
Puskás
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.
Éva
É
Fekete
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.
Nikolett
N
Bódi
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D003092:Colitis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D003106:Colon / Q000378:metabolism / Q000648:ultrastructure*; D003251:Constriction, Pathologic; D003424:Crohn Disease / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D007415:Intestinal Obstruction / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000235:genetics / Q000378:metabolism; D014302:Trinitrobenzenesulfonic Acid
D012333:RNA, Messenger; C576839:TIMP1 protein, rat; C509043:Tgfb2 protein, rat; C509046:Tgfb3 protein, rat; D019715:Tissue Inhibitor of Metalloproteinase-1; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D014302:Trinitrobenzenesulfonic Acid; D020780:Matrix Metalloproteinase 9; C572337:Mmp9 protein, rat
Crohn’s disease; Intestinal strictures; MMP9; Rat model; TGF-beta; TIMP1