Chronic ileal inflammation leads to transmural changes in the iNOS - NO signaling pathway

Abstract

Alterations in mucosal production of NO and expression of iNOS are known to occur in lBD . Changes in the iNOS-NO pathway during lBD were further studied using a rabbit model of chronic ileitis. Chronic inflammation in pathogen free rabbits was produced by intragastric inoculation with Eimeria magna oocytes . Experiments were done on ileal specimens from 14 rabbits day 14 post-inoculation; means are average values from 3-4 rabbits. NO concentration was measured by chemiluminescence. The production of NO in the mucosa of inflamed animals increased from 22.23±.04 ~M to 84.76=: .51~M (p=O.OOOI). The activity of mucosal NOS increased from 2.75=:.29 to 7.34:!:.45 nmoles/mg DNA/hr (p=0.0222). Inflammation increased blood NO from 12.39:!:.40 to 19.36:!:.34 p.M (p=0.0153. No clear change in NO production occured in smooth muscle-myenteric plexus tissues (l2.95:!:.22p.M vs 15.35:!:.35p.M, p> .05). Labeling studies with a monoclonal anti-iNOS IgGI antibody were used to analyze the cellular distribution of iNOS immunoreactivity (IR) . Laser confocal imaging showed that inflammation did not increase the number of iNOS-positive myenteric or submucous neurons (inflamed,6.39:!:.95 myenteric neurons / ganglion, n=2,738 iNOS cells counted vs normal,6.77 :!: 0.92 myenteric neurons / ganglion , n= 2,734 iNOS cells counted, p> .05). Inflammation caused a 21% increase in iNOS intensity/expression in myenteric neurons (intensity increased from 163.81:!:.70 to 197.87:!:.11 units. n=514 neurons , p< .OOOOI). Inflammation caused a tiny 7:!:.02% increase in iNOS expression in submucous neurons (n=327 cells, p= .OO8). Inducible NOS was expressed in neurons with diverse morphologies. Intense focal expression of iNOS was observed in inflamed villus and crypt epithelia near abscesses, but was marginally detectable in non-inflamed villus epithelium. Diffuse staining occurred in the cytoplasm of epithelia. MCID Imaging Analysis revealed a 4-5 fold increase in iNOS IR / mm? proportional area. Intense iNOS staining occured at the apical and basolateral borders of the inflamed villus epithe lia, apical borders of some inflamed crypts , as well as in mononuclear leukocytes of the inflamed lamina propria. The results are consistent with mucosal alterations in the iNOS-NO pathway known to occur in IBD, suggesting that the rabbit enteritis model is a useful model to further study the pathway. Chronic enteritis increases the expression of iNOS-NO production in epithelia, mononuclear leukocytes and myenteric neurons (seed funds & NIH NCRR ISlO RRI1434).