Expression and function of inducible nitric oxide synthase during rat colon anastomotic healing.
Nitric oxide plays a significant but incompletely understood role in fibroblast function and cutaneous wound collagen synthesis; however, the participation of inducible nitric oxide synthase (iNOS) in gastrointestinal anastomotic healing has not been studied. Male Sprague-Dawley rats underwent single-layer left colonic anastomosis. Animals were killed at 24-hour intervals postoperatively and the anastomosis was excised. Parallel uninjured colon tissue samples were also analyzed. Reverse transcriptase-polymerase chain reaction confirmed the absence of iNOS messenger RNA in control colon and expression of the gene in anastomotic tissue on all study days. Northern hybridization demonstrated maximal iNOS messenger RNA transcription on day 1 with decreased levels on days 3 and 5. iNOS enzyme activity, measured biochemically by the conversion of [(3) H-arginine to [(3) H]-citrulline ex vivo, was also maximal on day 1 (7.35 +/- 1.34 pmol/mg protein/min [+/- standard error of the mean], n = 10) and decreased on days 3 (4.37 +/- 2.32 pmol/mg protein/min; n = 6) and 5 (2.80 +/- 0.92 pmol/mg protein/min; n = 6). Immunohistochemical staining demonstrated that (1) iNOS expression is confined to a discrete cell population in the region of the anastomosis containing inflammatory cells; (2) those cells assume a highly conserved position on the luminal edge of the proliferating scar; and (3) the iNOS-expressing cells are present throughout the fibroplastic phase of healing. To functionally assess the role of iNOS in colonic healing, rats were treated with a continuous intravenous infusion of S-methylisothiourea (a selective inhibitor of iNOS) at a dosage of 200 mg/kg/day for 5 days after anastomosis. There was a significantly reduced anastomotic bursting pressure in rats treated with the inhibitor as compared to rats treated with intravenous normal saline solution (108.4 +/- 13.2 mm Hg vs. 148.4 +/- 10.3 mm Hg; P <0.05). These results suggest that iNOS gene expression is induced during colonic anastomotic healing, that it is present through all phases of healing but is maximal through the inflammatory phase, and that iNOS activity is required for optimal anastomotic healing.