6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease

Abstract

Background: Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model.

Methods: Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K⁺ Krebs-induced contractility and spontaneous contractions.

Conclusions: This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

Figure 1

Tissue dissection and preparation and 6-OHDA-induced dopamine (DA) lesion. The distal colon, proximal colon, and distal ileum tissues were excised by first cutting three, each 4–5 cm long tissue pieces, from ≈3 cm from the anus, and 3 cm from each side of the aboral and oral end of the caecum, respectively (shaded sections in a). Segments were then prepared from these 4–5 cm long tissue pieces by further cutting ≈10–15 mm long tubes of each tissue type (scissor markings), followed by attaching a silk thread to each end closing the luminal space (b). In a subset of the ≈10–15 mm long tubes, inverted segments were created by turning the tissue inside out, exposing the luminal space but closing the mucosal space (c). The longitudinal and circular strips were also prepared from the 4 to 5 cm long tissue pieces. The longitudinal strip was prepared by cutting a ≈10–15 mm long segment, opening it up along the mesenteric line, splitting in half with a width of ≈5 mm, and attaching a thread at each end (d). The circular strips were prepared by cutting a ≈5 mm long segment, rotating it 90°, opening it up along the mesenteric line, and attaching a thread at each end (e). The saline injection (Sham) did not affect the DA fibers (f), whereas the 6-OHDA injections (6-OHDA) in the medial forebrain bundle induced an almost complete DA fiber degeneration in the striatum (g).

Figure 2

Distal colon contractile responses to electrical field stimulation (EFS) and methacholine. Following EFS, frequency-dependent responses were observed in all tissues. The segments showed significant differences in the 6-OHDA group in both EFS- (e) and methacholine-induced contractions (f) compared with Sham (n = 16 and n = 17, respectively). However, no differences in EFS- and methacholine-mediated contraction were observed between the 6-OHDA and Sham animals in circular (a and b; n = 10 and n = 9, respectively) and longitudinal strips (c and d; n = 9 and n = 8, respectively). ∗ = Significant difference from Sham group; two-way ANOVAs (e) group-concentration interaction (5, 155) = 3.88, p = 0.0024; (f) group-concentration interaction (5, 155) = 4.38, p = 0.0009.

Figure 3

Contractions of distal colon segments and inverted segments following electrical field stimulation (EFS) and methacholine in the absence and presence of the nitric oxide synthase inhibitor L-NAME. Significant and similar reduced contractile responses in EFS by L-NAME were observed in the segments in both Sham (n = 6) and 6-OHDA animals (n = 5; a). However, L-NAME reduced the methacholine-mediated contractility in the 6-OHDA animals (n = 6), but not in the Sham group (n = 5; b). The inverted segments showed significant increased responses to EFS (c) and methacholine (d) in the 6-OHDA (n = 4) as compared with Sham group (n = 6) in the absence of L-NAME. In the presence of L-NAME the EFS response showed no difference within respective groups, but a trend to decrease could be seen in the 6-OHDA animals. The methacholine response on the other side was significantly increased in the Sham group after L-NAME. The increase was however, not significant in the 6-OHDA animals. ∗Significant difference from Sham group; # = significant difference between presence and absence of L-NAME in respective group. Two-way ANOVAs (a) ∗ = group F(1, 9) = 15.31, p = 0.00036; and # = group F(1, 5) = 14.29, p = 0.0129; F(1, 4) = 8.44, p = 0.0438 for Sham and 6-OHDA, respectively; (b) ∗ = group F(1, 9) = 6.36, p = 0.0327 and # = group F(1, 4) = 7.84, p = 0.0488 for 6-OHDA; (c): ∗ = group F(1, 8) = 6.80, p = 0.0312; (d) ∗ = group F(1, 8) = 38.32, p = 0.0003; # = F(1, 5) = 18.72, p = 0.0075 for Sham. The data here are representing a subset of the data included in Figures 2(e) and 2(f).

Figure 4

Proximal colon contractile responses to electrical field stimulation (EFS) and methacholine. EFS in the circular strips showed a trend for increased contractile responses in the 6-OHDA (n = 7) as compared with Sham animals (n = 6; a), whereas the proximal colon segments displayed a non-significant decreased response in the 6-OHDA animals (n = 9) compared with Sham animals (n = 8; e). The methacholine-induced contraction in the circular strips (b: 6-OHDA, n = 10; Sham, n = 7) and the segments (f: 6-OHDA, n = 11; Sham, n = 11) was not significantly changed between the groups. No changes were observed in the longitudinal strips either by EFS (c; 6-OHDA, n = 7 and Sham, n = 6) or methacholine-induced contraction (d; n = 10 in each group); two-way ANOVAs a: group F(1, 11) = 3.30, p = 0.0966; (e) group F(1, 15) = 3.41, p = 0.0848.

Figure 5

Electrical field stimulation (EFS) and methacholine-induced contractions in distal ileum segments. No significant difference in the smooth muscle contractility (mN mg⁻¹) was observed after either EFS (a: Sham n = 10; 6-OHDA, n = 9) or methacholine (b: Sham n = 11; 6-OHDA, n = 11). In addition, the frequency of the spontaneous contraction was unaltered by 6-OHDA-induced dopamine lesions (c: Sham n = 11; 6-OHDA, n = 11).

Figure 6

Spontaneous contractions in distal and proximal colon. No changes were observed between the groups in distal colon in either circular strips (a: Sham n = 10; 6-OHDA, n = 11), longitudinal strips (b: Sham n = 10; 6-OHDA, n = 11) or segments (c: Sham n = 17 6-OHDA, n = 16). In the proximal colon segments (f) an increased spontaneous contractility after the high K⁺ Krebs activation were observed in the 6-OHDA (n = 11) as compared with Sham animals (n = 11). This was however, not observed in either circular (d: Sham n = 7, 6-OHDA, n = 10), or longitudinal strips (e: Sham n = 10; 6-OHDA, n = 10) of the proximal colon. ∗ = Significant difference from Sham group; Mann–Whitney U test (f) p = 0.0133.