Effects of Nebulized Diethylenetetraamine-NONOate in a Mouse Model of Acute Pseudomonas aeruginosa Pneumonia: MPO
The heart and lungs were removed en bloc, and the pulmonary vasculature was perfused with 10 mL saline solution through a right ventriculotomy. Lung tissue was homogenized in 1 mL 10 mM bicarbonate-free N-2-hydroxyeth-ylpiperazine-N’-2-ethanesulfonic acid buffer (containing 0.1 mM ethylenediaminetetraacetic acid, 1 mg/mL phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.32 M sucrose [pH 7.4]) at 4°C for analysis of myeloperoxidase (MPO) content and pulmonary bacterial load.
Pulmonary leukocyte infiltration was assessed by the measurement of MPO activity, as previously described. Briefly, solubilized MPO activity was assessed in quadruplicate in a sonicated lung homogenate by assessing the H2O2-dependent oxidation of tetramethylbenzidine for > 15 min at 37°C. One unit of MPO activity was defined as a change of 1.0 optical density units at 655 nm/min and was expressed as total pulmonary MPO in milliunits.
The pulmonary bacterial load was determined as previously described. Briefly, a lung homogenate was serially diluted in a sterile 0.9% saline solution, and aliquots were incubated in duplicate on sheep’s blood agar plates at 37°C for 24 h. The total pulmonary bacterial load was calculated and was expressed as a multiple of the instilled number of bacteria. The consistency of the bacterial load in the mice with pneumonia on each experimental day was ensured by twice quantitating the concentration of the P aeruginosa suspension (approximately 6 X 108 cfu/mL) that had been used for inducing pneumonia, both before the first mouse and after the last mouse had undergone intratracheal instillation. The coefficient of variation for within-day variability of the bacterial suspension concentration was < 2%. ventolin inhaler