Nitric oxide (NO) is an important regulator of gut function but its role in gut injury and inflammation is uncertain. Under acute conditions NO protects the gut from a variety of vascular and luminal insults. However under more chronic conditions inhibition of NO formation prevents gut injury. The reasons for this disparity appears to lie in the enzyme source for NO production. Nitric oxide-mediated gut injury is the result of inducible nitric oxide synthase (iNOS), whereas the protective effects of NO reflect the constitutive form (cNOS). Unlike cNOS, iNOS is transcriptionally regulated and releases large amounts of NO (1000x more than cNOS) for the life of the enzyme. Thus, iNOS is a marker and a mediator of gut injury. Necrotizing enterocolitis (NEC) is a devastating neonatal disease of unknown etiology. Gut necrosis with NEC probably represents the end result of diverse events. Infectious agents, limited mucosal maturation and gut motility as a result of the prematurity, and aggressive feeding patterns are thought to contribute, probably in combination. The working hypothesis of this proposal is the iNOS is induced in NEC and the iNOS gene expression will be detectable in both intestinal tissue and circulating leukocytes by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, NOS inhibitors selective for the inducible form may protect neonatal gut from damage. These hypotheses will be tested in several adult and neonatal models of NEC, with studies on NOS gene expression supported by biochemical tests quantifying NO production. Rat and guinea pig models of NEC gut inflammation include trinitrobenzene sulfonic acid, luminal acetic and + casein, and luminal fast fermenting bacteria (anerobes or aerobes) with infant formula. We have published extensively with all models. Gut injury will be assessed by a variety of in vivo functional tests, morphology (including immunohistochemistry) and mediator release. The advantages of this approach to the diagnosis of NEC is that iNOS is normally quiescent with gene expression induced by bacterial products (endotoxin) and/or cytokine responses. Furthermore we have demonstrated that iNOS mRNA is detectable in circulating leukocytes following in vivo exposure to LPS, suggesting that a direct application to premature infants is readily accomplished. A lack of cNOS may also contribute to the pathophysiology of NEC, possible due to developmental deficiencies. While a cNOS deficiency will also be testable in our animal models, this hypothesis is not easily evaluated in premature infants due to problems in obtaining endothelial cells or gut biopsies. This proposal focuses on a novel and sensitive approach to the diagnosis of NEC, one which provides insight into the pathophysiology and the possibility of new therapeutic approaches.
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