Localization of lipid peroxidation and protein nitrosylation in the diaphragm during endotoxin mediated sepsis

In previous studies we demonstrated that free radicals play an important role in mediating sepsis-induced diaphragmatic dysfunction. In this past work, however, we failed to identify which specific portions of the diaphragm were modified by free radicals. The purpose of the present study was to localize sites of lipid peroxidation and protein nitrosylation within the septic diaphragm. Rats were sacrificed 18 hr after injection with saline (n=5) or endotoxin (8 mg/kg, n=5). In vitro force generation was then assessed on a portion of the diaphragm, and the remaining diaphragm was examined immunohistochemically. Lipid peroxidation was determined using anti-4-hydroxynonenal (4HN) antibodies, protein nitrosylation was detected using anti-nitrotyrosine (NT) antibodies, and neutrophils were localized with a Wright's stain. We found that both 4HN and NT were increased for endotoxin-group diaphragms when compared to controls. Moreover, both 4HN and NT staining for the endotoxin group was especially prominent along the outer rim of muscle fibers and in areas adjacent to vascular structures (e.g. muscle perimeter NT staining for control and endotoxin groups averaged 17.2 ±0.1 and 24.0 ±0.1 AU, respectively, p<.05). Extravascular neutrophils were increased 31-fold for the endotoxin group, and these cells were located in areas of perivascular NT/4HN staining. Force generation by septic muscles was reduced when compared to controls (p<.01). We speculate that perivascular lipid/protein modification of the diaphragm in sepsis results from the action of neutrophil-derived free radicals, while myocyte-derived radicals cause NT/4HN formation along the muscle perimeter.