The dual effect of cannabinoid receptor-1 deficiency on the murine postoperative ileus.

INTRODUCTION: Intestinal inflammatory responses play a critical role in the pathogenesis of postoperative ileus (POI). As cannabinoid receptor-1 (CB1) is involved in inhibiting gastrointestinal (GI) motility and anti-inflammation, we aimed to explore its contribution to POI. METHODS: Experimental POI was induced in adult female CB1-deficient (CB1-/-) mice and wild-type littermates (C57BL/6N) by standardized small bowel manipulation. Twenty-four hours after surgery, GI transit was assessed by charcoal transport. FITC avidin, F4/80, and myeloperoxidase immunohistochemistry techniques were used to evaluate the inflammatory response in the muscularis of ileum and colon. Expressions of p38MAPK and its phosphorylated form (pp38) in the intestine were determined. Plasma levels of proinflammatory cytokines and chemokines were measured by ELISA as well. RESULTS: POI was characterized by decreased GI transit (p<0.01) and accompanied by a marked intestinal and systematic inflammatory response in wild-type and CB1-/- mice. Increased numbers of inflammatory cells, including macrophages, neutrophils, and mast cells were observed in the muscularis of ileum and colon (p<0.01, or p<0.05). Plasma levels of interleukin-6 (IL-6), cytokine-induced neutrophil chemoattractant-1 (CINC-1/KC), and monocyte chemoattractant protein-1 (MCP-1) were elevated (p<0.01, or p<0.05). Expression of p38 and pp38 increased in the intestine (p<0.01, or p<0.05). CB1-/- mice showed an increased inflammatory response during POI, especially the systemic inflammatory markers, such as IL-6, KC, CINC1, and pp38 expression were increased as compared to those in WT mice (p<0.05). CONCLUSIONS: Intestinal motility was inhibited during POI. In this condition, inhibition of motility did not seem to be altered by the absence of CB1 receptors, however, an increased inflammatory response was observed in CB1-/- mice. Hence, CB1 receptor activation rather than inhibition may reduce the inflammatory response in POI, which has a remote potential to relate into reduced inhibition of intestinal motility during POI.

Inhibition of p38/Mk2 signaling pathway improves the anti-inflammatory effect of WIN55 on mouse experimental colitis.

P38/Mk2 (mitogen-activated protein kinase (MAPK)-activated protein kinase-2, also known as MAKAP kinase-2) is a member of the mitogen-activated protein kinases (MAPKs) family, and participates in inflammatory responses directly or indirectly. WIN55, 212-2 (WIN55) is a synthetic non-selective agonist of cannabinoid (CB) receptors with remarkable anti-inflammatory properties. This study was to explore the roles of WIN55 and p38/Mk2 signaling pathway in dextran sodium sulfate (DSS)-induced mouse colitis and ascertain their anti-inflammatory mechanisms. Colitis was induced in C57BL Mk2 gene homozygous deletion (Mk2-/-) and wild-type mice by replacing the drinking water with 4% DSS solution for 7 days. DSS-treated mice developed bloody stool, weight loss, and eye-visible multiple bleeding ulcers on colon mucosa. The mRNA expressions levels of TNF-α and IL-6, as well as the protein levels of p38 and its phosphorylated form (p-p38), were upregulated in the colon. The plasma levels of TNF-α, IL-6, cytokine-induced neutrophil chemoattractant-1 (CINC-1), monocyte chemoattractant protein-1 (MCP-1), and lung myeloperoxidase (MPO) activities were raised; however, all these changes were less severe in Mk2-/- mice. After WIN55 intervention, the Mk2-/- mice recovered faster and better from the induced colitis than their wild-type counterparts. The results indicate that the Mk2 homozygous deletion in mice impedes the induction of experimental colitis by DSS, confirming the notion that p38/Mk2 is involved in this inflammatory response. WIN55 protects mice against DSS-induced colitis, in particular when the p38/Mk2 pathway is obstructed, implying that the activation of CB system, together with blocking of p38/Mk2 pathway, serves as a potential drug target for colitis treatment.

Vagal innervation and early postoperative ileus in mice.

INTRODUCTION: Postoperative ileus is characterized by infiltrates of leukocytes in the gut wall 24 h after surgery, which is subject to vagal modulation. We hypothesized that vagal modulation is irrelevant during earlier hours of postoperative ileus and aimed to determine whether afferent neuronal feedback to the central nervous system is altered by vagal innervation during this early period. METHODS: C57BL6 mice were laparotomized and received standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments while control animals were sham-operated. Three or 9 h later a 2-cm jejunal segment was harvested for multi-unit mesenteric afferent nerve recordings in vitro. Intestinal motility was monitored continuously and intestinal muscularis was stained for myeloperoxidase to determine infiltration of leukocytes. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different in all subgroups. Afferent discharge to 5-HT (500 µM) was virtually absent following vagotomy at 3 and 9 h of postoperative ileus (POI) compared to controls (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was not different in all subgroups while luminal distension from 10 to 30 mmHg was lower at 3 h of POI following vagotomy compared to controls (p < 0.05). The number of myeloperoxidase positive cells was similar at 3 h of POI in both subgroups; however, at 9 h of POI, ileus counts were increased to 713 ± 99 cells following vagotomy compared to 47 ± 6 cells per square millimeter in control animals. CONCLUSIONS: Vagal afferents mediate sensitivity to low-threshold distension and 5-HT during postoperative ileus but not to high-threshold distension and bradykinin. Vagal inhibition of the intestinal immune response is present at 9 h but not detectable earlier, i.e., at 3 h of postoperative ileus when spinal reflex inhibition may prevail.

Role of the vagus nerve on the development of postoperative ileus.

INTRODUCTION: Postoperative ileus involves reflex inhibition of intestinal motility within hours after surgery and a subsequent intestinal inflammatory response that is characterized by efferent vagal modulation via acetylcholine receptors on intestinal macrophages. We aimed to characterize the role of vagal modulation of intestinal motility during the early hours after surgery. METHODS: C57BL6 mice underwent laparotomy and standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments or received pharmacological inhibition of the acetylcholine alpha7 subunit with the inhibitor alpha-bungarotoxin, while control animals were sham operated and remained otherwise untreated. Three hours later, a 2-cm jejunal segment was harvested with the mesentery attached. Mesenteric afferent nerve recordings were established in an organ bath generating a multiunit signal with subsequent computerized analysis. Intraluminal pressure was continuously recorded to assess intestinal motility. Afferent nerve responses were quantified at baseline and to chemical stimulation with bradykinin (0.5 microM) or serotonin (5-HT; 500 microM) and following mechanical stimulation by continuous ramp distension to 60 mmHg. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different following chronic vagotomy, alpha-bungarotoxin or sham operation. Maximum afferent discharge to 5-HT following alpha-bungarotoxin was comparable to sham controls, while the response was reduced in chronically vagotomized animals (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was similar in the different subgroups. At luminal distension from 10 to 30 mmHg, afferent discharge was lower in vagotomized animals compared to sham controls (p < 0.05) but unchanged after alpha-bungarotoxin. CONCLUSIONS: Sensitivity to low-threshold distension and 5-HT is mediated via vagal afferents during postoperative ileus, while sensitivity to high-threshold distension and bradykinin is independent of vagal afferent innervation. Early inhibition of intestinal motility at 3 h after onset of postoperative ileus does not appear to depend on vagal innervation.