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Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice

Abstract

BACKGROUND Studies using inhibitors of nitric oxide synthase (NOS) to date are inconclusive regarding the role of inducible NOS (iNOS) in intestinal inflammation.

AIMS (1) To examine the role of iNOS in the development of chronic intestinal inflammation; (2) to identify the cellular source(s) of iNOS.

METHODS Colitis was induced by an intrarectal instillation of trinitrobenzene sulphonic acid (TNBS, 60 mg/ml, 30% ethanol), in wild type (control) or iNOS deficient mice. Mice were studied over 14 days; the colons were scored for injury and granulocyte infiltration was quantified. Blood to lumen leakage of51Cr-EDTA was measured as a quantitative index of mucosal damage.

RESULTS At 24 and 72 hours, iNOS deficient mice had significantly increased macroscopic inflammation compared with wild type mice. Granulocyte infiltration increased significantly at 24 hours and remained elevated in iNOS deficient mice at 72 hours, but significantly decreased in controls. However, by seven days post-TNBS macroscopic damage, microscopic histology, granulocyte infiltration, and mucosal permeability did not differ between wild type and iNOS deficient mice. A four- to fivefold increase in iNOS mRNA was observed in wild type mice at 72 hours and seven days post-TNBS and was absent in iNOS deficient mice. Immunohistochemistry techniques showed that iNOS expression was predominantly localised in neutrophils, with some staining also in macrophages.

CONCLUSIONS These results suggest that leucocyte derived iNOS ameliorates the early phase, but does not impact on the chronic phase of TNBS induced colitis despite the presence of iNOS.

  • nitric oxide synthase
  • neutrophils
  • epithelial permeability
  • inflammatory bowel disease
  • Abbreviations used in this paper

    bNOS
    brain nitric oxide synthase
    cpmpr
    counts per minute per ml perfusate
    cpmpl
    counts per minute per ml plasma
    ecNOS
    endothelial constitutive nitric oxide synthase
    FITC
    fluorescein isothiocyanate
    GAPDH
    glyceraldehyde-3-phosphate dehydrogenase
    iNOS
    inducible nitric oxide synthase
    MPO
    myeloperoxidase
    NO
    nitric oxide
    NOS
    nitric oxide synthase
    PBS
    phosphate buffered saline
    PBS-T
    PBS-Triton X-100
    RT-PCR
    reverse transcriptase polymerase chain reaction
    TNBS
    trinitrobenzene sulphonic acid
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