Effects of Nebulized Diethylenetetraamine-NONOate in a Mouse Model of Acute Pseudomonas aeruginosa Pneumonia: DETA-NO Preparation and Delivery
Mice in the pneumonia and sham groups were randomized to receive nebulized DETA-NO, iNO, or room air (RA). Two doses were studied for both DETA-NO (12.5 or 125 |j,mol; 2.0 or 20 mg, respectively) and iNO (10 or 40 ppm). Thus, the following 10 groups were studied: pneumonia/RA; pneumonia/iNO (10 and 40 ppm); pneumonia/DETA-NO (12.5 and 125 |j,mol); sham/RA; sham/iNO (10 and 40 ppm); and sham/DETA-NO (12.5 and 125 |j,mol).
DETA-NO was chosen because it has the longest half-life of commercially available NONOates (56 h at 22°C in a 0.1 M phosphate buffer at pH 7.4) flovent inhaler flovent inhaler. Solutions of DETA-NO in PBS were prepared fresh immediately before nebulization. For exposure to DETA-NO, mice were housed in a 5-L plastic (Plexiglas; Rohm and Haas; Philadelphia, PA) chamber into which 2.5 mL DETA-NO/PBS were completely nebulized over 10 min (LC plus jet-nebulizer; PARI Respiratory Equipment, Inc; Mississauga, ON, Canada; and Medi-mist compressor, model 1802; Mountain Medical Equipment; Littleton, CO). The estimated average chamber DETA-NO concentration during nebulization would be approximately 1.0 |j,mo]/L and approximately 10 |j,mo]/L for the 12.5 and 125 |j,mol doses, respectively. Given that two molecules of NO are released by each molecule of DETA-NO, these DETA-NO chamber concentrations are equivalent to iNO levels of approximately 40 ppm and approximately 400 ppm, respectively.
In pilot studies, the intrapulmonary release of NO was assessed following a single exposure to 125 |j,mol nebulized DETA-NO (4 h after surgery) in animals in both the pneumonia and sham groups. Separate groups of animals were sacrificed before and at several time points (ie, 0.5, 1.5, 2.5, and 12 h) after DETA-NO exposure for the measurement of plasma and BAL levels of nitrites/nitrates (NOx_). At each time point, mice were killed, blood was aspirated via cardiac puncture, and a tracheostomy was performed with a 24-gauge catheter. BAL was carried out by instilling and withdrawing a single 1-mL aliquot of PBS three times.