NF-kappaB activation precedes increases in mRNA encoding neurokinin-1 receptor, proinflammatory cytokines, and adhesion molecules in dextran sulfate sodium-induced colitis in rats.

Nuclear factor kappa B (NF-kappa B) plays a key role in initiating inflammation associated with colitis. A systematic study was conducted in the rat DSS colitis model to determine the temporal relationship between NF-kappa B activation and expression of substance P (SP), neurokinin-1 receptor (NK-1R), proinflammatory cytokines, and adhesion molecules. Rats were given 5% DSS in their water and sacrificed daily for 6 days. Colon tissue was collected for assessment of histological changes, NF-kappa B activation, myeloperoxidase (MPO) activity, and expression of NK-1R, SP, TNFalpha, IL-1beta, VCAM-1, ICAM-1, E-selectin, CINC-1, MIP-1alpha, and iNOS. NF-kappa B activation increased, biphasically, on Day 1 and again on Days 4-6. The mRNA levels for ICAM-1, CINC-1, IL-1beta, TNFalpha, VCAM-1, and NK-1R rose significantly (P < 0.05) by 2-4 days. Increased iNOS mRNA levels, MPO activity, and mucosal damage occurred on Day 6. These data demonstrate that NF-kappa B activation substantially precedes the onset of physical disease signs and active inflammation.

A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.

Fibrous adhesions remain a major sequela of abdominal surgery. The proinflammatory peptide substance P (SP), known to participate in inflammatory events, may play a key role in adhesion formation. This hypothesis was tested by using an antagonist, CJ-12,255 (Pfizer), that blocks the binding of SP to the neurokinin 1 receptor (NK-1R). Adhesion formation was surgically induced in the peritoneum of rats receiving daily doses of the NK-1R antagonist (NK-1RA; 5.0 or 10.0 mg/kg per day) or saline. On postoperative day 7, both the low and high doses of NK-1RA significantly (P < 0.05) reduced adhesion formation by 45% and 53%, respectively, compared with controls. Subsequently, the effect of NK-1RA administration on peritoneal fibrinolytic activity was investigated to determine a potential mechanism for SP action in the peritoneum. Samples were collected from nonoperated controls and from animals 24 h postsurgery that were administered either NK-1RA or saline. Fibrinolytic activity in peritoneal fluid was assayed by zymography, and expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1, both regulators of fibrinolytic activity, was assessed in peritoneal tissue and fluid by RT-PCR and bioassay, respectively. NK-1RA administration led to a marked (P < 0.05) increase in tPA mRNA levels in peritoneal tissue compared with nonoperated and saline-administered animals. Likewise, NK-1RA administration significantly (P < 0.05) increased tPA in the peritoneal fluid. These data suggest that activation of the NK-1R promotes peritoneal adhesion formation by limiting fibrinolytic activity in the postoperative peritoneum, thus enabling fibrinous adhesions to persist.

A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus.

Ileal pouch-anal anastomosis (IPAA) is an excellent surgical option for patients with chronic ulcerative colitis (CUC) requiring colectomy; however, persistent episodes of ileal pouch inflammation, or pouchitis, may result in debilitating postoperative complications. Because considerable evidence implicates substance P (SP) as an inflammatory mediator of CUC, we investigated whether SP participates in the pathophysiology of pouchitis. With the use of a rat model of IPAA that we developed, we showed that ileal pouch MPO levels and neurokinin 1 receptor (NK-1R) protein expression by Western blot analysis were significantly elevated 28 days after IPAA surgery. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was localized to the lamina propria and epithelia of pouch ileum. The intraperitoneal administration of the NK-1R antagonist (NK-1RA) CJ-12,255 for 4 days, starting on day 28, was effective in reducing MPO levels. Starting on day 28, animals with IPAA were given 5% dextran sulfate sodium (DSS) in their drinking water for 4 days, which caused histological and physical signs of clinical pouchitis concomitant with significant increases in ileal pouch MPO concentrations as well as NK-1R protein expression by Western blot analysis. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was especially evident in crypt epithelia of pouch ileum. When the NK-1RA was administered 1 day before starting DSS and continued for the duration of DSS administration, the physical signs of clinical pouchitis and the rise in MPO were prevented. These data implicate SP in the pathophysiology of pouchitis and suggest that NK-1RA may be of therapeutic value in the management of clinical pouchitis.

Colonic metaplasia in the ileal pouch is associated with inflammation and is not the result of long-term adaptation.

Ileal pouch-anal anastomosis (IPAA) is the preferred surgical therapy for chronic ulcerative colitis (CUC) and familial adenomatous polyposis (FAP). Previous studies have demonstrated morphologic changes in pouch mucosa such as villous atrophy and crypt hyperplasia. These changes have been labeled "colonic metaplasia." The aims of this study were to determine whether these changes represent "normal" long-term adaptation of the nondiseased pouch or instead are present only in the setting of inflammation. Twenty-four patients were identified, greater than 5 years status post-IPAA for CUC, who underwent pouchoscopy for surveillance and had no history of pouchitis. Thirty-one patients were identified greater than 5 years status post-IPAA for CUC, who had a history of pouchitis and had undergone pouchoscopy at least 5 years status post-IPAA. Eight patients status post-IPAA for FAP were also identified. Biopsy specimens were reevaluated by a single, blinded pathologist for degree of inflammation, the presence of villous atrophy and crypt hyperplasia, and evidence of dysplasia. Among the patients with CUC, the inflammation score was greater in the pouchitis group, 13.2 +/- 1.2, compared to the nonpouchitis group, 4.0 +/- 0.5 (P < 0.0001). Median colonic metaplasia score was greater in the pouchitis group (4 [range 2 to 6]) vs. 2 (9 [range 0 to 6]; P < 0.0001). The colonic metaplasia score correlated with the inflammation score (Spearman coefficient r = 0.83; P < 0.0001). In the eight patients with FAP, the inflammation score was 5.1 +/- 0.9 and the median colonic metaplasia score was 1 (range 0 to 4). There was no evidence of dysplasia in any of the biopsy specimens. Patients without a history of pouchitis or symptoms of pouchitis have only a minimal degree of villous atrophy and crypt hyperplasia. These morphologic changes in the ileal pouch are found primarily in the setting of inflammation, and likely represent a reparative response.

Neurokinin-1 receptor and substance P messenger RNA levels increase during intraabdominal adhesion formation.

BACKGROUND: Intraabdominal peritoneal adhesions are a significant cause of postoperative morbidity and remain one of the major long-term complications associated with abdominal surgery. Adhesion formation at the molecular level involves a complex interaction of cytokines, growth factors, cell adhesion molecules, and neuropeptides, as well as many other factors secreted by cells proximate to the traumatized area. Limited studies exist which investigate the molecular processes involved in adhesion formation. Therefore, the aim of the present study was to determine the pattern of gene expression for substance P, the neurokinin-1 receptor, and downstream mediators of substance P action during the early stages of adhesion formation in the rat. METHODS: Four ischemic buttons were created on one side of the peritoneum in male Wistar rats. Animals were sacrificed at 3, 6, and 12 h and 1, 3, 5, and 7 days following surgery. Peritoneal tissue from ischemic buttons and from the opposite sidewall was harvested for total RNA isolation. Semiquantitative RT-PCR was used to measure changes at the transcript level for the neurokinin-1 receptor, substance P, TGFbeta-1, and the cell adhesion molecules ICAM-1 and VCAM-1. RESULTS: Messenger RNA levels for substance P, neurokinin-1 receptor, TGFbeta-1, ICAM-1, and VCAM-1 were significantly increased in peritoneal tissue taken from ischemic button sites (P < 0.05) when compared with controls. In the peritoneal tissues taken from the opposite sidewall, there was a significant (P < 0.05) early increase in substance P mRNA levels. TGF-beta1, neurokinin-1 receptor, and ICAM-1 mRNA levels were also significantly (P < 0.05) increased when compared to controls, while the mRNA levels for VCAM-1 did not change. CONCLUSIONS: The increased levels of mRNA for substance P, the neurokinin-1 receptor, and the downstream mediators of substance P action, TGF-beta1, ICAM-1, and VCAM-1, in peritoneal tissue associated with intraabdominal adhesions support a role for substance P in adhesion formation.