Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

Abstract

Objectives: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.

Design: Using Ca²⁺ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.

Results: EFS reduced the ATP-induced Ca²⁺ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.

Conclusion: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.

Trial registration number: NCT02425774.

Keywords: Macrophages; anti-inflammatory; enteric neuron; ileus; prucalopride.

Figure 1

Cholinergic enteric neurons modulate resident muscularis macrophages. (A) Muscularis externa whole mount from the jejunal tissue of wild-type (WT) mice was stained for choline acetyltransferase (ChAT)-positive enteric neurons and F4/80-positive muscularis macrophages (mMφ) (left panel). Muscularis externa whole mount from human small intestinal tissue was stained for CD68-positive mMφ and ChAT-positive enteric fibres (right panel). Scale bars are 50 µm. (B) Upper panel shows representative images and tracing of enteric neurons in the jejunal muscularis of Wnt.1^GCAMP3 mouse before stimulation (no stimulation (NS)) and during electrical field stimulation with 1, 5 or 20 Hz (EFS; pulse width: 1 ms, current: 20 mA). Scale bar is 10 µm. (C) Representative images from a Fluo-4 loaded mMφ in the jejunal muscularis of CX3CR1^GFP/WT mouse in response to 100 µM of ATP before and after EFS (20 Hz; pulse width: 1 ms, current: 20 mA). Scale bar is 20 µm. (D–E) Effect of Krebs (CTRL), EFS, EFS+tetradotoxin (TTX; 10 µM), EFS+ω-conotoxin (0.1 µM) or 150 µM of prucalopride (PRUC) on ATP-induced mMφ activation, expressed as mean decrease in Ca²⁺-induced amplitude (D) and as percentage of mMφ maximally inhibited (E) for n=74–100 mMφ. Data are from 4 to 7 animals/condition. *P<0.05, **p<0.01. One-way analysis of variance with Bonferonni post hoc test (D) and two-tailed χ² test (E). (F) 5-HT4R expression in sorted mMφ, isolated ganglia and whole mount muscularis tissue. (G) Maximum intensity projection of confocal stacks showing muscularis externa whole mount from the jejunum of WT mice was stained for 5-HT4R, ChAT and F4/80.

Figure 2

Pharmacological activation of enteric neurons reduces intestinal inflammation and improves postoperative ileus. (A) Schematic representation of experimental protocol. Mice subjected to intestinal manipulation (IM) were treated with vagus nerve stimulation (VNS) or prucalopride (PRUC; oral gavage 1.5 hour prior to or 22.5 hours after IM) and compared to sham/placebo (SHAM/PLAC). Fluorescently labelled dextran was gavaged 22.5 hours after IM and mice were sacrificed 1.5 hours later. Geometrical centre (GC) of dextran distribution was used to quantify the GI transit. (B) Dextran distribution through the GI tract (left panel) and GC (right panel) for each group of mice. St, stomach; Sb, small bowel; C, colon. (C) Representative image (left panel) and number of myeloperoxidase (MPO)-positive cells/0.5 mm² (right panel) in jejunal muscularis. Scale bars are 50 µm. (D–E) Relative mRNA levels for Il6 and Il1α normalised to the housekeeping gene rpl32 from jejunal muscularis tissue. (B–E) Data are expressed as mean±SEM from n=6–10 mice/group. *P<0.05, **p<0.01. One-way ANOVA with Bonferonni post hoc test for PREOP and unpaired t-test for POSTOP. (F–G) Vagotomised (VGX) mice treated with PLAC or PRUC 1.5 hours prior to IM. (F) Bar graph represents the mean GC 24 hours after IM. (G) Mean of relative mRNA levels in the jejunal muscularis for Il1α and Il6 normalised to the housekeeping gene rpl32. *P<0.05, **p<0.01. Unpaired t-test for VGX-PLAC vs VGX-PRUC. n=6–8 mice/group.

Figure 3

Cholinergic modulation of muscularis macrophages (mMφ) is mediated by alpha7 nicotinic acetylcholine receptor (α7nAChR). (A) Maximum intensity projection of confocal stacks showing small intestinal muscularis whole mount tissue stained for α7nAChR (red) on F4/80-positive muscularis macrophages (mMφ; green) in a wild-type (WT) mouse (right panel) and on CD68-positive mMφ (green) in human (left panel). Scale bars are 20 µm. (B) Heatmap of nAChR subunit expression in sorted lamina propria macrophages (LpMφ) and mMφ. (C) Heatmap of nAChR subunit expression in naïve bone marrow-derived macrophages (BMDM) incubated with conditioned medium (CM) collected from myenteric ganglia cultures or control medium (CTRL). (D) Bar graph of anti-inflammatory gene expression of naïve BMDM incubated with CM collected from myenteric ganglia cultures or CTRL. Data are expressed as mean log10 ± SEM. *P<0.05. Unpaired t-test. n=3-5/experiment. (E) Heatmap of nAChR subunit expression in BMDM (M0) and polarised M2 BMDM. (F) Western blot analysis of sorted mMφ and sorted muscularis immune cells (CD45⁺CD11c^-IA/IE^-CD64^-CD11b^-) purified on α-bungarotoxin affinity column (α7nAChR). Brain extracts are from WT and α7nAChR knock-out (KO) mice. (G–J) α7nAChR KO mice were treated with either placebo (PLAC) or prucalopride (PRUC) 1.5 hours prior to intestinal manipulation (IM). (G) Bar graph represents the GC values for each group 24 hours after IM. (H) Bar graph represents the number of MPO-positive cells/0.5 mm² 24 hours after IM. (G–H) Data are expressed as mean±SEM. **P<0.01. Unpaired t-test. n=6–8 mice/group. (I–J) Relative mRNA levels in jejunal muscularis for Il1α and Il6 normalised to the housekeeping gene rpl32. Data are expressed as median with 5–95 percentile whiskers. **P<0.01. Mann-Whitney U test. n=6–8 mice/group.

Figure 4

(A) Patient flow chart. (B) Schematic overview of study protocol. PLAC, placebo; PRUC, prucalopride; VNS, vagus nerve stimulation.

Figure 5

Preoperative administration of prucalopride reduces intestinal inflammation and improves postoperative ileus in human. (A) Relative mRNA levels in the muscularis externa for Il6, Il8 and Ccl2 normalised to the housekeeping gene c10fr43. Duodenal tissue was taken at the beginning of the procedure (0 hour) and 2 hours into the surgery (2 hours) for patients treated with sham/placebo (SHAM/PLAC), abdominal vagus nerve stimulation (VNS) or prucalopride (PRUC). Data are expressed as mean±SEM *P<0.05. Repeated two-way analysis of variance (ANOVA) with Bonferroni correction for multiple testing; n=9–10 patients/group. (B) Time until removal of nasogastric tube (NGT) (upper panel) and the NGT output on postoperative day 3 (lower panel). (C) Other clinical (secondary) end points (ie, days until tolerance to first solids, first defecation, combination of time to tolerance to solids and first defecation and days until discharge) for SHAM/PLAC-treated, abdominal VNS-treated or PRUC-treated groups without site-specific complications (SSC). (C) NGT output data on postoperative day 3 is expressed as median±interquartile range for SHAM/PLAC-treated, abdominal VNS-treated or PRUC-treated groups without SSC. *P<0.05. Kruskal-Wallis test with Dunn’s test for multiple comparison test; n=7–9 patients without SSC/group. (B–C) The other dot plots are expressed as mean±SEM *P<0.05, **p<0.01. One-way ANOVA with Bonferroni correction for multiple testing; n=7–9 patients without SSC/group.