Effect of TNBS-induced morphological changes on pharmacological contractility of the rat ileum

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Abstract

Intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol is a classical model of colitis in the rat. Little is known about the time-related effect of TNBS on the contractility and morphology of the rat ileum. After 36 h, TNBS induced acute ileitis. Spontaneous activity of longitudinal muscle strips was decreased, as were receptor- and nonreceptor-mediated contractions and contractions induced by electrical stimulation. After 1 week, mucosal integrity was restored, although the thickness of both mucosal and muscle layers was increased. Spontaneous activity, receptor- and nonreceptor-mediated contractions and electrically induced contractions of longitudinal muscle strips were increased due to hypertrophy and hyperplasia of smooth muscle cells. This was confirmed in the contractility study of individual muscle cells. Functional alterations after 1 week were restricted to a decreased response to substance P. TNBS-ileitis in the rat lacks a chronic phase and is accompanied by functional hypocontractility of longitudinal smooth muscle cells during the acute inflammation, whereas the contractility of the longitudinal muscle layer is increased in the postinflammation phase due to structural alterations. There is a selective inhibition of the response to substance P in the postinflammation phase.

Introduction

Gastrointestinal inflammation is accompanied by structural and functional changes of the gut, leading to gastrointestinal motility disturbances (Collins, 1996). These motility disturbances contribute to the generation of symptoms, such as nausea, dyspepsia, abdominal cramps and diarrhea.

Intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol is a classical model of intestinal inflammation, mimicking some aspects of Crohn's disease (Elson et al., 1995). Initially, TNBS was administered in an enema to develop a rat model of colitis (Morris et al., 1989). Nowadays, the model of TNBS-induced inflammation has been further adapted to the mouse colon (Neurath et al., 1995), the guinea pig small intestine (Miller et al., 1993), the rabbit small intestine (Sjogren et al., 1994) and also the rat small intestine (Goldhill et al., 1999).

The model of TNBS-induced colitis is used in several studies to investigate motility disturbances occurring in the inflamed rat colon. Dysmotility was shown in vivo as an initial increase in colonic transit and a decrease in colonic myoelectrical activity within the first few hours after TNBS instillation, followed by a progressive decrease of the transit and an increase in the colonic myoelectrical activity Pons et al., 1992, Morteau et al., 1993. In vitro studies evidenced functional changes at the colonic neuromuscular level. The contractility of the circular smooth muscle layer is increased 4 h after TNBS instillation, whereas after 1 week, the contractility of both the circular and the longitudinal muscle layer is decreased, irrespective of the type of the contractile stimulus Grossi et al., 1993, Hosseini et al., 1999. Also, the expression and the release of specific neurotransmitters from the myenteric plexus are disturbed Jacobson et al., 1997, Miampamba and Sharkey, 1998. Finally, TNBS-induced inflammation of the rat colon leads to morphological changes of the neuromuscular apparatus. Important structural changes of the rat colonic wall have been shown to appear from 1 week onward after TNBS administration. The thickness of the muscularis externa is significantly increased, which is partially due to smooth muscle cell hypertrophy Morris et al., 1989, Miampamba and Sharkey, 1998, Hosseini et al., 1999. This increase in muscle mass may contribute to the inflammation-induced dysmotility.

Although little information is as yet available on the effect of TNBS on the rat small intestine, it appears that the inflammatory response to TNBS in the small intestine is different from the well-characterized response in the rat colon. The cellular components of the inflammatory infiltrate are comparable in the colon and the small intestine Morris et al., 1989, Grossi et al., 1993, Morteau et al., 1993, Coméra et al., 1999, Hosseini et al., 1999. However, the time course of the inflammatory response is different. Whereas TNBS-induced colitis persists for several weeks Morris et al., 1989, Pons et al., 1992, preliminary reports of different groups show that the transmural inflammation of the rat small intestine recovers 1 week after TNBS Demedts et al., 1999a, Demedts et al., 1999b, Moreels et al., 1999. Furthermore, it was shown that the myoelectrical activity of the small intestine was disrupted with a loss of phase I quiescence and decreased phase III activity 1 week after TNBS (Demedts et al., 1998). Contractility studies of the acutely inflamed rat ileum show that the response of circular muscle strips to substance P was selectively increased (Goldhill et al., 1999). However, in the acutely inflamed colon, circular muscle contractility is increased irrespective of the contractile stimulus used, with the exception of the selectively decreased response to neurokinin A (Hosseini et al., 1999). At this moment, no data are available on the TNBS-induced modulation of ileal longitudinal muscle contractility in the rat.

Therefore, the present study was undertaken to investigate the effect of TNBS-induced ileitis on the structural and contractile properties of the longitudinal muscle layer of the rat ileum during the course of inflammation. To study receptor-specific alterations, different contractile receptor agonists were used. To determine the portion of the contractile response that was accounted for by mere structural changes of the muscle layer, longitudinal muscle strip contractility was always presented in g contraction and in g contraction normalized to the cross-sectional area of the longitudinal muscle layer. Using the two methods to express the contractile data enables to differentiate between structural and functional alterations of the neuromuscular apparatus during TNBS-induced ileitis. In addition, we compared the contractility of longitudinal muscle strips with the contractility of individual longitudinal smooth muscle cells. The inflammatory response was assessed both histologically and biochemically.

Section snippets

Animals

Before the ileal treatment with TNBS or saline, male Wistar rats (Iffa Credo, St.-Germain sur l'Abresle, France) weighing 300–400 g were fasted for 48 h with free access to drinking water. This prolonged fasting period was necessary for the ileum to empty its content completely into the caecum. Preliminary experiments showed that TNBS treatment of chyme-containing ileum failed to induce a pronounced inflammatory response. Before tissues were harvested for histology, myeloperoxidase activity

Induction of ileitis

After TNBS treatment, rats developed black loose stools for 2 to 3 days. Thereafter, the fecal pellets regained normal colour and consistency. This short period of diarrhea was followed by a significantly slower increase in body weight as compared to rats treated with saline (Fig. 1). Although by the time of 1 week and after a fasting period of 12 h before experimentation, this difference in body weight between controls and TNBS-treated rats was no longer significant.

Histology

No inflammatory infiltrates

Discussion

An enema of TNBS in ethanol is a well-characterized model of acute and chronic colitis in the rat (Morris et al., 1989). Recently, this model was adjusted to the rat small intestine (Goldhill et al., 1999). However, it appears that the inflammatory response of the rat ileum to TNBS differs from the response in the rat colon. In the present study, we elaborated the knowledge on TNBS-induced ileitis in the rat by studying the structural and functional alterations of the longitudinal muscle layer.

Acknowledgements

The work was supported by the Fund for Scientific Research Flanders (Grant no. G.0220.96) and by the Interuniversitary Poles of Attraction Program (Grant no. P4/16, Services of the Belgian Prime Minister, Federal Agency for Scientific, Technical and Cultural Affairs).

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