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Keywords:

  • colitis;
  • electromyography;
  • ileum;
  • rat;
  • trinitrobenzene sulphonic acid

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Changes in gastric emptying and orocaecal transit time in patients with ulcerative colitis suggest that disturbances in gut motility may not be restricted to inflamed sites. This study sought to characterize changes in the motility of noninflamed ileum in a rat colitis model and to explore the mechanism(s) potentially involved. The myoelectrical activity of the ileum was recorded in rats with trinitrobenzene sulphonic acid (TNBS)-induced colitis. The degree of ileal and colonic inflammation was assessed by quantification of macroscopic damage and myeloperoxidase activity (MPO). The effect on ileal motility of pretreatment with atropine, indomethacin and NG-nitro- L-arginine-methyl ester (L-NAME) was investigated. TNBS-induced inflammation was restricted to the distal colon, as evidenced by morphological scores and MPO. Colitis was associated with increased frequency of ileal migrating motor complexes, characterized mainly by a decrease in the duration of phases I and III. The occurrence of ileal giant migrating complexes remained unchanged. The myoelectrical changes observed in the ileum persisted after treatment with atropine, indomethacin and L-NAME. Distal colitis is associated with abnormal myoelectrical activity in the noninflamed ileum of rats. Neither acetylcholine nor prostaglandins and nitric oxide seem to be involved.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

In inflammatory bowel diseases, the inflammatory process not only affects intestinal mucosa but also the deeper layers of the intestinal wall, including nerves and muscles.1, 2 Studies in humans and animals indicate that inflammatory bowel diseases are associated with significant changes in intestinal motor activity of the gut segment affected by inflammation.3[4]–5 However, changes in gastric emptying and orocaecal and small intestinal transit times have also been observed in patients with ulcerative colitis, suggesting that motor dysfunction can affect segments beyond the inflammation site.6, 7 As the enteric nervous system plays a major role in the regulation of motor intestinal motility, it is likely that the morphological changes and alterations in myenteric nerve functions observed in the noninflamed intestine induce motor disorders.1, 8 Moreover, changes in smooth muscle function may also contribute to motor disorders reported in noninflamed ileum.1 Previous studies have shown that prostaglandin mediators as well as nitrergic or cholinergic pathways may be involved in the motility changes observed in the inflamed intestine.9[10][11]–12 The involvement of these mediators has not been detected in the noninflamed ileum. The present study used electromyography to characterize the ileal motor disorders associated with distal colitis in rats treated by trinitrobenzene sulphonic acid (TNBS) and to investigate the potential role of certain mediators such as nitric oxide (NO), prostaglandins (PG) and acetylcholine (ACh) in these disorders.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Electromyography

Three groups of six male Wistar rats weighing 350–400 g were used. All experimental procedures were approved by the local INRA Animal Care and Use Committee. After an overnight fast, surgery was performed in animals anaesthetized by intraperitoneal administration of pentobarbital sodium (20 mg kg−1; Sanofi Santé Animale, France). Three groups of three Nichrome wire electrodes (Microfil Industries, Renens, Switzerland), 80 μm in diameter and 60 cm in length, were implanted through the serosal and muscular layers, using a needle as a trocar, and anchored with a tie close to the distal ileal wall. The three groups of electrodes were positioned at 2-cm intervals, the first being 4 cm from the ileocaecal valve. The free ends of the electrodes were brought subcutaneously to the back of the neck. Each rat was then allowed to recover from the operation for 5 days. Electrical activity of the ileum was recorded with an electroencephalograph (Schwarzer ED 24, Picker, Germany) at a sensitivity of 10 μV cm−1, with a low filter of 16 Hz, a high filter of 50 Hz and a paper speed of 0.8 mm sec−1.

2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis

TNBS colitis was induced during the same surgical time as electrode implantation using a protocol adapted from Morris et al.13 Briefly, TNBS was dissolved in 40% ethanol to a concentration of 120 mg mL−1. After implantation of the electrodes, a catheter was inserted through the anus so that its tip was located approximately at the level of the splenic flexure (8 cm proximal to the anal verge). The colon was infused with 1 mL of TNBS plus an ethanol solution at a dose of 25 mg mL−1. Control rats were similarly intubated but infused with 1 mL of 0.9% NaCl solution.

Assessments

Inflammation

Eight days after intrarectal administration of saline or TNBS, rats were killed by cervical dislocation, and the distal ileum and colon were excised and opened longitudinally. Samples prepared from full-thickness ileal and colonic wall (50–200 mg) were removed, snap-frozen in liquid nitrogen and stored at − 70 °C before determination of the extent of colitis, as previously described.14 The degree of ileal and colonic inflammation was assessed by measuring macroscopic damage (Table 1) and myeloperoxidase activity (MPO). MPO, a specific granule-associated marker enzyme primarily found in polymorphonuclear neutrophils, is commonly used as an index of both neutrophil infiltration into intestinal tissue and inflammation.15 The activity of MPO was expressed in units per gram of wet tissue, one unit being the quantity of MPO required to convert 1 μmol of hydrogen peroxide to water in 1 min at room temperature.

Table 1.  Criteria for macroscopic scoring of colonic ulceration and inflammation. Thumbnail image of
Motor activity 1. Myoelectrical activity in control and TNBS rats.

Recordings started 6 days after operations were performed for 17 days. The experiments were conducted daily with fasted rats. Briefly, 8 h before each experiment, the animals were fasted but free access to water was allowed. Then, intestinal electrical activity was recorded from 16:00 to 19:00 h. After each experiment, the animals were placed again in their cage and fed ad libitum from 20:00 to 08:00 h.

Phase III of migrating motor complex (MMC) was identified as a period of clear intense spiking activity with an amplitude of at least twice that of the preceding baseline, propagating aborally through the whole recording segment and followed by a period of quiescence, phase I of MMC. Phase II was characterized by irregular spiking preceding the activity front. Giant migrating complexes (GMCs) were identified as brief spiking activity with an amplitude at least twice that of the preceding baseline. This type of electromyographical activity has been shown to the associated with GMCs recorded by strain gauge.16

2. Acute effect of synthesis inhibitors and antagonist.

The preventive effect of pharmacological agents on ileal myoelectrical activity was tested 8 days after TNBS or saline administration in fasted rats. Control recordings were done for at least 1 h before infusion of test substances. The test drugs were given to the same animal at 48-h intervals. PG synthesis inhibitor (indomethacin, 1 mg kg−1, i.p.), NO synthesis inhibitor (NG-nitro- L-arginine methyl ester: L-NAME, 10 mg kg−1, i.p.) or atropine (5 mg kg−1, s.c.) was then administered during phase I of the MMC. Motility recording was continued for at least 2 h after drug administration.

Preparation of test substances

Indomethacin, L-NAME and atropine sulphate were purchased from Sigma Chemical Co. (St Louis, MO, USA). Indomethacin was dissolved in 0.3 mL of 5% sodium bicarbonate, whereas L-NAME and atropine were dissolved in sterile saline.

Data processing and statistical methods

The following characteristics of ileal MMC were measured: (i) frequency of MMC; (ii) duration of phase I, II, III; and (iii) propagation velocity of phase III. The number per hour of GMC was also determined. Results are expressed as the means ± SEM, and n indicates the number of rats in each group. Statistical studies of the results were performed by analysis of variance ( ANOVA). A P-value < 0.05 was considered as significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Morphological scores and MPO assays

Eight days after intrarectal administration of saline, no macroscopically detectable damage was noted in the colon and ileum of control rats ( Fig. 1A). Conversely, a significant increase in colonic morphological scores was noted in TNBS-treated rats as compared to controls rats ( Fig. 1A), whereas no macroscopically detectable damage was found in the ileum of TNBS-treated rats ( Fig. 1A). MPO activity in the colon was significantly higher in TNBS-treated than in control rats ( Fig. 1B). However, no significant difference was noted between ileal MPO activity in TNBS-treated rats and controls ( Fig. 1B).

image

Figure 1.  Macroscopic damage score (A) and myeloperoxidase (MPO) activity (B) in full-thickness preparations from rat distal ileum and colon 8 days after intrarectal administration of saline or trinitrobenzene sulphonic acid (TNBS). Data are mean ± SEM (n = 4 rats for each group). *< 0.05, TNBS vs saline.

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Myoelectrical ileal activity

Control rats

During the interdigestive state, the myoelectrical activity of the ileum was organized into MMC which occurred at a rate of 2.1 ± 0.1 cycles per hour on day 6 after saline administration ( Figs 2 and 3A). Each MMC consisted of irregular spiking activity (phase II, 11.5 ± 1.1 min, Fig. 4B) followed by regular spiking activity (phase III, 6.1 ± 0.3 min, Fig. 4C). These phases of activity were separated by a period of quiescence (phase I, 12.4 ± 1.1 min, Fig. 4A). Moreover, MMC frequency and the duration of phases I, II and III, respectively, remained unchanged during the 17 days of recording ( Figs 2 and 4).

image

Figure 2.  Time-related changes in the number of migrating motor complexes (MMC) during the 17 days after intracolonic administration of saline or trinitrobenzene sulphonic acid (TNBS). Bars are means ± SEM (n = 5 for control rats and n = 6 for TNBS-treated rats). *< 0.05 TNBS vs saline.

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image

Figure 3.  Examples of myoelectric tracings obtained with three electrodes implanted in distal ileum (E1 = 8 cm, E2 = 6 cm; E3 = 4 cm) to ileocaecal valve). A: Fasting motor pattern in control rat. B: Increase in migrating motor complex frequency in TNBS-treated rat.

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image

Figure 4.  Time-related changes in phase I (A), II (B) and III (C) during the 17 days after intracolonic administration of saline or trinitrobenzene sulphonic acid (TNBS). Bars are means ± SEM (n = 5 for control rats and n = 6 for TNBS-treated rats). *< 0.05 TNBS vs saline.

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TNBS-treated rats

Ileal myoelectrical activity remained well organized in TNBS-treated rats, with well-identified MMC as in control rats. However, the number of MMC was significantly higher than in control rats ( Figs 2 and 3B), owing to a reduction in the duration of phases I and III ( Fig. 4A,C), whereas phase II remained unchanged ( Fig. 4B). This motor pattern was consistently observed during the period from day 6 to 15. Then, animals returned to the control pattern on days 16 and 17 following TNBS administration ( Fig. 2). During the 17 days of the experiment, the velocity of phase III propagation in TNBS-treated rats was not significantly different from that of controls (1.18 ± 0.16 cm min−1vs 0.98 ± 0.23 cm min−1 on day 6).

The GMC frequency remained unchanged during the 17 days of recording (0.41 ± 0.08 vs 0.51 ± 0.02 GMCs per hour on day 6; Fig. 3A,B).

Effects of drugs on myoelectrical activity in TNBS-treated rats

Although L-NAME stimulated ileal myoelectrical activity significantly in TNBS-treated rats and control rats, a significant difference between the two groups was maintained ( Figs 5A and 6A). Administration of indomethacin increased MMC frequency in TNBS-treated rats but not in control rats ( Figs 5B and 6B), so that the difference in MMC frequency persisted. Treatment of TNBS-treated rats and controls with atropine was associated with a reduction in MMC frequency, but the difference between the two groups was maintained ( Figs 5C and 6C). Therefore, none of the three drugs administered at day 8 following TNBS (i.e. atropine, indomethacin or L-NAME) was able to suppress the difference in the motility pattern observed in TNBS-treated as compared to control rats ( Figs 5 and 6A–C).

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Figure 5.  Effects of treatment with L-NAME (10 mg kg−1, i.p.; A), indomethacin (1 mg kg−1, i.p.; B) or atropine (5 mg kg−1, s.c.; C) on migrating motor complex (MMC) frequency of rat ileum 8 days after intrarectal administration of vehicle or trinitrobenzene sulphonic acid (TNBS). Bars are means ± SEM (n = 6 for control rats and n = 6 for TNBS-treated rats). *< 0.05 pretreatment vs vehicle alone.

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image

Figure 6.  Effects of different drugs on electrical activity of the distal ileum recorded using three electrodes (same symbols as in Fig. 3). A: L-NAME (10 mg kg−1, i.p.); B: Indomethacin (1 mg kg−1, i.p.); C: Atropine (5 mg kg−1, s.c.). Note the lack of difference in response to these drugs between control and trinitrobenzene sulphonique acid (TNBS)-treated rats.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Our results show that the myoelectrical activity of a noninflamed segment (the ileum) was altered in TNBS-induced colitis. These changes were mainly characterized by an increase in the number of MMC associated with a decrease in the duration of phases I and III. In contrast, MMC propagation velocity and GMC frequency were not modified.

The fact that the disturbances of intestinal motor activity observed in our work were not restricted to the site of intestinal inflammation corroborates previous studies showing changes in gastric emptying and orocaecal transit time in patients with ulcerative colitis.6, 7 It has also been recently reported that a colonic antigen challenge of sensitized rats resulted in jejunal motor disturbances.17

Motor alterations at intestinal inflamed sites persisted at least 2 weeks after an intestinal infection.18 This event does not seem restricted to the inflamed site since, in our experiment, the myoelectrical ileal disturbances occurring at a site remote from colon TNBS challenge lasted at least 15 days after TNBS administration. The literature provides support for disruption of ileal MMC periodicity during ileitis.18, 19 For example, myoelectrical patterns from the inflamed ileum of infected rats were characterized by a decrease in phase III frequency.18 In contrast, there was an increase in ileal MMC frequency in our study during colitis. Taken together, these results suggest that the mechanism(s) potentially involved in MMC regulation may be different in noninflamed and inflamed ileum. However, it is possible that the decrease in phase III frequency may be more relevant to nematode infection (mast cell mediated) than to IBD.

GMCs are powerful lumen-occluding contractions occurring in the small intestine that rapidly propel intestinal secretions and undigestible food into the colon, thereby increasing its osmotic load. A recent study has shown that one of the major changes in ileal motility during ileitis is the increase of GMC frequency.19 This event seems to be restricted to the inflamed site since in our study GMC frequency was not different in rats with noninflamed colitis and control rats. However, our conclusion should be tempered by noting methological differences (electromyography vs strain gauge).

It is quite unlikely that alterations of ileal motor activity in our experiment reflect a transmural extension of the inflammatory process in the distal colon. Macroscopic scores and ileal levels of MPO activity were normal, and there was a long intervening segment of normal mucosa. It cannot be excluded that abnormal ileal motility was related to a systemic neurotoxic effect of the TNBS used to induce inflammation. However, this seems unlikely since changes in myenteric nerve function at noninflamed sites have been observed in two quite different models of rat inflammation (TNBS and Trichinella spiralis).20

Increased levels of prostaglandins (PGE2) have been observed in mucosa from patients with ulcerative colitis and in experimental animal colitis.21, 22 Moreover, it has been reported that enhanced PGE2 production may affect muscle contractility and thus contribute to the smooth muscle dysfunction associated with intestinal inflammation.9 In our study, a mechanism of this type does not seem to have been responsible for the increase of MMC frequency observed at the noninflamed site since it was not abolished by pretreatment with indomethacin. However, it is noteworthy that indomethacin significantly increased MMC frequency in TNBS-treated animals but had no effect in control rats, as noted previously for normal rats.17, 23 These findings suggest that PGE2s, though not directly responsible for the increase in MMC periodicity observed in noninflamed ileum, probably cause significant modulation in the ileum during inflammation.

NO, which is considered to be the final mediator regulating the MMC cycle in rats, contributes to TNBS-induced changes in the smooth-muscle adrenoceptor population in guinea-pig inflamed intestine.10, 24 Under inflammatory conditions, the resulting excess NO produced by myenteric neurones may facilitate the altered motility observed during colitis.25 In our study, administration of a NO synthase inhibitor failed to affect the ileal disturbances associated with TNBS-induced colitis. Therefore, NO was not involved in the intestinal motor alterations observed in noninflamed sites. Administration of L-NAME significantly increased MMC frequency in control rats, which corroborates earlier findings indicating that inhibitors of NO synthase increase intestinal intraluminal pressure and shorten the duration of small intestinal MMC in rats.24, 26

In a recent study, Shi et al.12 reported that a decrease in contractile response to muscarinic receptor activation may be one of the factors involved in the disruption of the phasic contractions observed during ileal inflammation in the dog. Moreover, the contractility of the noninflamed colon to ACh increased in a rat model of colitis.27 This led us to speculate that ACh may be the neuromediator responsible for the increase in MMC periodicity described in noninflamed ileum. In fact, in our investigations, the significant difference observed between MMC frequency in noninflamed ileum of TNBS-treated rats and ileum of control rats persisted after atropine administration. This finding suggests that the increase in MMC frequency recorded in noninflamed ileum of TNBS-treated rats did not involve a cholinergic pathway. Pretreatment with atropine significantly reduced MMC frequency in both groups of rats. These results are consistent with earlier works reporting that the cholinergic pathway plays an important physiological role in the regulation of MMC cycling.28

In conclusion, our results suggest that motor disturbances are not restricted to the inflammation site since an increase in MMC frequency was observed in noninflamed ileum in rats with TNBS-induced colitis. Neither PG nor NO appears to be involved in this abnormal motor activity, which is also independent of a muscarinic pathway. Further studies are required to determine the mechanism(s) involved in this motor change.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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