Effects of Nebulized Diethylenetetraamine-NONOate in a Mouse Model of Acute Pseudomonas aeruginosa Pneumonia: Plasma NO Metabolites

Analysis of Plasma NO Metabolites (Nitrites and Nitrates)
Plasma levels of nitrites and nitrates were collectively measured as NOx— by chemiluminescence, as previously described. Briefly, plasma was refluxed in saturated vanadium chloride in hydrochloric acid, resulting in the reduction of NOx— to NO, which is detected by chemiluminescence (model 270B NO analyzer; Sievers Instruments; Boulder, CO). The analyzer was calibrated daily, and samples were referenced to a standard curve generated from NO3— standards (0.05 to 500 |j,M; R2 > 0.999).
Histology
An experienced pulmonary pathologist blindly evaluated the morphologic changes in various experimental groups. In a subset of 26 animals (sham group mice, 3; pneumonia mice that were untreated, 3; and pneumonia mice that were treated, 20 [7 with DETA-NO and 13 with iNO]), one lung was harvested and fixed for 24 h in buffered formalin under constant vacuum-induced inflation at 20 cm H2O. Representative lung sections were examined for a variety of histologic parameters including atelectasis, vascular congestion, hemorrhage, alveolar edema, accumulation of inflammatory cells, and the presence of bacteria. The distribution of the above changes was assessed as either diffuse (uniform) or patchy (nonuniform). Using a semi-quantitative scoring system, the degree of bronchopneumonia was graded as follows: 0, normal; 1, mild pneumonia; 2, moderate pneumonia; and 3, severe pneumonia. flovent inhaler

Effects of DETA-NO Exposure on P aeruginosa Growth In Vitro
P aeruginosa cultures (initial concentrations of 107 and 108 cfu/mL) were incubated at 37°C for 24 h (5% CO2 in medical air) in the presence and absence of DETA-NO (0.5, 5, and 50 mM in PBS). Bacteria incubated with DETA-NO also were exposed inadvertently to two other species as follows: (1) DETA itself, following the release of NO by DETA-NO; and (2) NO2~ (in pilot studies 59 ± 2% of the NO released from DETA-NO was oxidized in vitro to nitrite).

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