% of animals
Pellet Diarrhea Loose stool
Control
1 wk 4 wks
*
Fig. 7. Stool consistency before and after 1 week and 4weeks of TNBS treatment. Score 0: normal well formed pellets, score 1: loose stool/easy to smear, score 2:
diarrhea/watery stool 3. Results are expressed as cumulative percentage of total scored stool, *, p < 0.05, n=7.
DISCUSSION
The TNBS model (Morris et al., 1989) is one of the most frequently used models, since a single intrarectal administration of this hapten induces colitis. TNBS is a hapten, and when coupled to colonic tissue proteins, it has been shown to elicit an immune response (Fidler et al., 1985, Little et al., 1966). The TNBS model of colitis was designed on the principle that the ethanol vehicle would damage the colonic epithelium, thereby permitting entry into the lamina propria where it would bind to tissue and act as an antigen (Morris et al., 1989). It has been presumed that an increase of mucosal permeability allows a luminal antigen to enter the lamina propria that is not cleared by the mucosal immune system of IBD patient (Shorter et al., 1972 and Ward et al., 1977). TNBS-ethanol-induced colitis models have been developed in several laboratory animals such as rats, rabbits and guinea pigs (Morris et al., 1989, Depoortere et al, 2002, Lomax et al., 2007). Although various doses of TNBS have been used to induce colitis in rats and guinea pigs from 5-50mg per animal (Boughton-Smith et al., 1988, Linden et al, 2003, Morris et al., 1989), but in this study I used intrarectal administration of 10 mg per animal induced severe colonic injury, which was accompanied by a lack of body weight gain and a significant increase in colonic size, when compared to control hamsters (Fig 4, table 3). The data presented by Menozzi et al, (2006) and Hirota et al, (2005), support the present finding in the hamster colitis.
Thus, the dose of 10 mg TNBS on the ethanol was selected in this study to induce colitis.
It has been reported that the administration of ethanol alone induced acute damage, which spontaneously reverted in a few days (Poli et al., 2001, Rachmilewitz et
ethanol alone was fatal for these hamsters (Hirota et al., 2005). Therefore I considered it was inappropriate to use such a high concentration of ethanol as the vehicle of TNBS.
In the present study macroscopic alterations of the colonic mucosa that take place in the acute and chronic phases of the inflammation, following three different concentrations of ethanol (15, 30 and 40%) with TNBS. The study revealed marked quantitative differences between the three concentrations of ethanol for the severity of acute inflammation and the rate of recovery from colonic damage. In particular, the lower concentration 15 and 30% ethanol plus TNBS did not induce macroscopically visible colonic injury and changes of mucosal architecture (Fig. 4). Thus, these concentrations seem to be suitable for neither short-term nor long-term studies. By contrast, the higher concentration of ethanol 40% with TNBS induced maximal colonic damage during the first week after treatment; epithelial regeneration was slower and areas of necrosis were still present in some animals after 4 weeks from colitis induction. Thus present result shows species differences of ethanol sensitivity in rat and hamster. Therefore, 40%
ethanol is a concentration large enough to break the mucosal barrier, which allows TNBS to penetrate into the mucosa and induction of colitis in 100% of treated animals.
When a colon segment to be examined for its motility in vitro is isolated from the acute inflammatory phase, we can easily judge the severity and extend of colonic damage. To examine the restoration of motility disorder TNBS-injected animals would have to be kept until the expected colitis had recovered. However, there is no reliable marker to judge the severity and extent of prior colonic damage in the healed colon. As mentioned previously TNBS-induced colitis was highly reproducible with relatively small individual difference in the hamster, accordingly, I selected hamsters to induce
of rats and guinea pigs reached maximal severity at about 1 week and returned to normal in terms of the presence of hyperemia, ulceration or bowel wall thickening within 4 weeks after TNBS injection (Lomax et al, 2007, Kiyosue et al, 2006poli et al, 2001). Consistent with the time course observed in rats and guinea-pigs, enlarged size of the colon at the acute inflammatory phase (1 week after TNBS treatment) in the hamster model was restored at 4 weeks after TNBS treatment as judged by macroscopic and microscopic observation (Fig. 5, E and F, Fig. 6, E and F). In addition histological scoring shows significantly low score at 4 weeks after colitis in contrast to acute inflammation phase (Table. 4). Thus, in the present study, colonic segments were isolated at 1 week and 4 weeks after TNBS treatment as representative samples for acute inflammatory phase and post-inflammatory phase, respectively.
Since the size or weight of the colon segment is recognized to be a reliable and sensitive indicator of the severity of colonic damage (Poli et al, 2001), in the present study macroscopic observation showed that colonic inflammation is apparently resolved within 4 weeks after TNBS treatment in hamsters (Fig. 5). Measurement of the body weight and stool consistency has been used previously for indicative marker to determine the colitis (Poli et al, 2001, Rijnierse et al 2006). Presence of diarrhea and significant reduction in the body weight shows the acute colitic condition during 1 week after TNBS instillation had recovered in 4 weeks of time point with well formed pellet and gain in the body weight (Fig. 4 and Fig. 7). Present result differs from that obtained in rat (Menozzi et al, 2006) which shows maximal damage of colitis remain until 2 weeks and complete regeneration of mucosal architecture and histology were obtained in 2 months whereas, in hamster almost complete regeneration was possible within 1
This data indicates that appropriate concentration of ethanol is necessary for induction of colitis. It also shows that recovery from colitis in hamsters is more rapid as compared to other lab animals. Diarrhea in response to TNBS colitis shows that this model is suitable for the study of colonic motility disorder, similar to that found in human inflammatory bowel disease.
CHAPTER 2
Progressive alteration of spontaneous contractile activity of longitudinal smooth muscles in the TNBS-inflamed hamster distal colon
INTRODUCTION
Smooth muscle preparations from the gastrointestinal tract usually develop spontaneous contractions of varying frequency and amplitude. The nature of this activity is not fully understood although involvement of interstitial cells of Cajal (ICCs) and enteric nerves has been implied (Keef et al., 1997; Horowitz et al., 1999). This unpropagated smooth muscle contractions and relaxation causes mixing of the gastric content and electrolyte transport. Clinical studies have suggested that the contractile response is decreased when colon inflammation is present. The lack of contractility is believed to decrease movements of the colonic contents, and accentuates diarrhea in patients with inflammatory bowel disease (Snape et al., 1980, Rao et al., 1987).
Both excitatory and inhibitory neurotransmitters from myenteric neurons are important in the regulation of colonic smooth muscle contraction. Alteration of the myenteric nerve system might lead to spontaneous dysmotility. Previous studies have suggested a role for inhibitory neurotransmitters in controlling smooth muscle contraction (Hosseini et al., 1999, Koch et al., 1988). NO has been identified as an important inhibitory neurotransmitter or neuromodulator in the ENS (Boekxstaens et al., 1993). Morphological studies of canine colon suggest that NO is also a critical intermediary in the communication between ICC, enteric inhibitory nerves, and smooth
that impaired nitric oxide synthase (NOS) activity in nerves was implicated in the reduced ability of smooth muscle to relax in colitis induced by dextran sulfate sodium in rats (Mizuta et al., 2000).
Myogenic contractions induced by high K+ and carbachol were decreased in TNBS-induced colitis (Kiyosue et al 2006), indicating substantial damage of smooth muscle during the inflammatory process. In addition, the network between ICC and the myenteric plexus is also disrupted by inflammation (Der et al., 2000, Lu et al., 1997, Wang et al., 2002). Collectively, impairments of the ENS, ICC-myenteric network and smooth muscle contractility may cause disorder of intestinal motility.
In addition to the acute structural and functional changes, persistent alterations in gastrointestinal function are commonly observed after the resolution of intestinal inflammation (Barbara et al., 1997). These include altered motility patterns, abnormal secretion and changes in visceral sensation (Linden et al., 2003, Rao et al., 1987, Reddy et al., 1991). Recently it has been shown in the guinea pig colon that sustained alterations in enteric neural signaling, such as increased amplitude of fast excitatory postsynaptic potential in submucosal S neurons and shortened action potential durations and decreased afterhyperpolarization in AH neurons, were promoted following transient intestinal inflammation (Lomax et al., 2007). Thus, the apparent recovery from inflammation is not necessarily accompanied by complete restoration of neural components.
Previous studies have shown that inflammation alters circular smooth muscle contractile properties and responses to neurotransmitters or nerve stimulation in the colon (Kiyosue et al, 2006, Bossone et al, 2001). In the present study, it was aimed to
of a hamster colon preparation, with the expectation of better understanding of the effects of inflammation on colonic spontaneous motility.
RESULTS
1) Effect of TNBS treatment on spontaneous motility in the isolated distal colon The segment of the distal colon isolated from the control hamster exhibited spontaneous contractile motility. There was a variation in the motility pattern between preparations. To show these variations, two typical examples were presented in Fig. 8.
Some preparations (6 of 16 preparations) showed a relatively well-synchronized rhythmic motility (left panel in Fig. 8), whereas the others (10 of 16 preparations) showed an irregular pattern (right panel in Fig. 8). At 1 week after TNBS treatment, the spontaneous activity was strongly suppressed. The suppressed spontaneous motility reappeared at 4 weeks after the initiation of colitis, with similar probability of variations to controls.
2) Effect of nitrergic blockade on spontaneous contraction isolated segment of normal and TNBS treated distal colon
Application of a nitric oxide synthase (NOS) inhibitor; NG-nitro-L-arginine methyl ester (L-NAME, 200 µM), increased the amplitude of spontaneous contractions Fig. 9. The blocker had little effect on the suppressed motility in the colon at 1 week after TNBS treatment. L-NAME application enhanced amplitude of spontaneous contractions in the colonic segment at 4 weeks after TNBS treatment.
3) Response to cholinergic blockade on spontaneous contraction isolated segment of normal and TNBS treated distal colon
Application of a muscarinic blocker, atropine (1 µM), significantly reduced the
in the colon at 1 week after TNBS treatment, but the drug suppressed spontaneous activity of the colon at 4 weeks after the treatment in a manner similar to that in the control.
4) Exogenous application of muscarinic agonist on contractility of isolated segments of control and TNBS treated distal colon
The relationship between time after TNBS enema and contractile response to ACh is presented in Fig 11. Application of ACh to the organ bath at a final concentration of 10 µM evoked contractions in the control distal colon. The longitudinal smooth muscle contractility after application of ACh remained constant in the colon at 1 week or 4 weeks after TNBS treatment. The tensions generatedby the tissues from normal, colitic and recovered animals were 4.5 ± 0.3, 4.1 ± 0.8 and 4.5 ± 0.2 g, respectively (n=4-6).