Otilonium Bromide Prevents Cholinergic Changes in the Distal Colon Induced by Chronic Water Avoidance Stress, a Rat Model of Irritable Bowel Syndrome

Abstract

Irritable Bowel syndrome (IBS) is a highly widespread gastrointestinal disorder whose symptomatology mainly affect the large intestine. Among the risk factors, psychosocial stress is the most acknowledged. The repeated water avoidance stress (rWAS) is considered an animal model of psychosocial stress that is capable of mimicking IBS. Otilonium bromide (OB), which is orally administered, concentrates in the large bowel and controls most of the IBS symptoms in humans. Several reports have shown that OB has multiple mechanisms of action and cellular targets. We investigated whether the application of rWAS to rats induced morphological and functional alterations of the cholinergic neurotransmission in the distal colon and whether OB prevented them. The results demonstrated that rWAS affects cholinergic neurotransmission by causing an increase in acid mucin secretion, in the amplitude of electrically evoked contractile responses, abolished by atropine, and in the number of myenteric neurons expressing choline acetyltransferase. OB counteracted these changes and also showed an intrinsic antimuscarinic effect on the post-synaptic muscular receptors. We assume that the rWAS consequences on the cholinergic system are linked to corticotrophin-releasing factor-1 (CRF1) receptor activation by the CRF hypothalamic hormone. OB, by interfering with the CFR/CRFr activation, interrupted the cascade events responsible for the changes affecting the rWAS rat colon.

Keywords: atropine; choline acetyl transferase; contractile responses; corticotropin-releasing factor; methacholine; mucus secretion; muscarinic receptors; protein gene product 9.5; tetrodotoxin.

Figure 1

Mucus secretion. Periodic Acid and Schiff’s reagent (PAS) (A–C) and Toluidine Blue (TB) staining (D–F). The two stainings were located in the glandular funds and in the goblet cells. Quantitation of PAS staining showed no difference in the total quantity of mucus production among the groups of rats ((G), right columns). Conversely, the quantitation of the acidic component, measured as TB metachromasia, demonstrated a significant increase in the rWAS group compared with the other three ((G), left columns). ((A–F) bar = 50 µm). One-way ANOVA followed by Newman–Keuls post-test, ** p < 0.005. (n = 7), Bar = 100 μm.

Figure 2

EFS in preparations from the different animal groups and effects of TTX and atropine. (A): Typical tracings showing the contractile response to EFS in preparations from Ctrl rats. The response elicited by EFS (4 Hz) is abolished following the addition to the bath medium of either TTX (left-hand panel) or atropine (right-hand panel). (B): Typical tracings showing the contractile response to EFS in preparations from: Ctrl, rWAS, OB, and rWAS+OB rats. The amplitude of the neurally induced contractile response to EFS is higher in preparations from rWAS in respect with that recorded from the Ctrl ones. In preparations from both OB and rWAS+OB rats, the amplitude of the contractile response to EFS appears greatly reduced in comparison with that of the rWAS or even Ctrl ones. (C): Bar chart showing the mean amplitude of the EFS-induced contractile responses in preparations from the different groups. No statistical differences were observed in the amplitude of the contractile responses to EFS between preparations from Ctrl and Sham rats, so all these data were put together and analyzed (Ctrl/Sham). The mean amplitude of the contractile responses to EFS in preparations from rWAS animals is increased in comparison with the Ctrl/Sham rats. At variance, that of both OB and rWAS+OB rats is reduced, without statistical differences between these two groups, in comparison with Ctrl/Sham and rWAS ones. All values are means ± S.E.M. of 8–12 preparations. * p < 0.05 vs. Ctrl/Sham; # p < 0.05 vs. Ctrl/Sham or rWAS; § p < 0.05 vs. Ctrl/Sham or rWAS, and p > 0.05 vs. OB (ANOVA and Newman–Keuls post-test).

Figure 3

Direct muscular responses elicited by methacholine in strips from the different animal groups. (A): Typical tracing showing the muscular contraction elicited by the addition of methacholine to the bath medium in preparations from Ctrl, rWAS, OB, and rWAS+OB rats. Note the similar amplitude of the response in preparations from Ctrl and rWAS animals and its reduction in both OB and rWAS+OB rats. (B): Bar chart showing the mean amplitude of the direct contraction induced by methacholine in preparations from Ctrl/Sham, rWAS, OB, and rWAS+OB rats. The amplitude of the response to methacholine was not statistically different between Ctrl and Sham rats, so all the data were put together and analyzed (Ctrl/Sham). No statistical differences are present in the mean amplitude of the response to methacholine between preparations from Ctrl/Sham and rWAS rats. At variance, the mean amplitude of the direct response to methacholine appears reduced in preparations from both OB and rWAS+OB rats, without statistical differences between these two groups, in comparison with the Ctrl/Sham and rWAS ones. All values are means ± S.E.M. of 6–8 preparations. * p < 0.05 vs. Ctrl/Sham or rWAS; # p < 0.05 vs. Ctrl/Sham or rWAS, and p > 0.05 vs. OB (ANOVA and Newman–Keuls post-test).

Figure 4

Protein Gene Product 9.5 (PGP9.5) and Choline Acetyl-Transferasi (ChAT)-immunoreactive (IR) myenteric neurons. PGP9.5 (A–D) and ChAT (E–H) labeling (green) in the myenteric neurons of Sham (A,E), rWAS (B,F), OB (C,G), and rWAS+OB (D,H) rat distal colon. Hoechst 33342 labeled the nuclei (blue). The arrows indicate the neurons that express both PGP9.5 and ChAT. Bar = 50 μm. Quantitative analysis of the percentage (%) of ChAT-IR neurons respect to the total PGP9.5-IR neurons and of the total number of ChAT-IR neurons. (I). The columns express the %. The number of ChAT-IR neurons is reported inside the columns. Both parameters showed a significant increase in the rWAS compared with all the other groups. Only the labelled neurons containing the nucleus were included in the statistical analysis. One-way ANOVA followed by Newman–Keuls post-test, ** p < 0.005. (n = 5).