Effects of Nebulized Diethylenetetraamine-NONOate in a Mouse Model of Acute Pseudomonas aeruginosa Pneumonia: Materials and Methods
Thus, we hypothesized that nebulized diethyl-enetetraamine-NONOate (DETA-NO), through in-trapulmonary NO release, would have beneficial antibacterial and anti-inflammatory effects in a mouse model of acute P aeruginosa pneumonia.
Murine Model of P aeruginosa Pneumonia
The experimental protocol was approved by our institutional Animal Care and Use Committee, in accord with the guidelines of the Canadian Council on Animal Care, and was supervised by a veterinarian. Male C57Bl/6 mice (weight range, 20 to 25 g; age, 6 to 7 weeks; Charles River Laboratories; St. Constant, PQ, Canada) were allowed at least a 48-h period of environmental acclimatization before experimentation and were provided access to food and water ad libitum.
An established rat model of P aeruginosa pneumonia, previously described by ourselves, was adapted to the mouse. Under general anesthesia (halothane in oxygen) and aseptic conditions, an anterior tracheotomy was performed with a 24-gauge needle. Mice were randomized to either a pneumonia group (n = 99) or a sham group (n = 49). Pneumonia was induced by intratracheal instillation of a 50-^L aliquot of a suspension of P aeruginosa (approximately 6 X 108 cfu/mL; strain No. 27853; American Type Culture Collection; Manassas, VA) followed by a 200-^L bolus of air. Animals in the sham group underwent anesthesia and tracheostomy but did not undergo intratracheal instillation more natural asthma treatment.
Mice were allowed to recover in cages with access to fluid and water ad libitum and were monitored for 24 h until they were killed. During this time, the mice received a total of 6 mL potassium phosphate (10 mM)-buffered saline solution (PBS; 0.9%; pH 7.4) subcutaneously in divided doses at 4-h intervals (0.01 mL/g/h). The mice also received buprenorphine (0.1 ^g/g body weight) subcutaneously for analgesia at 0 and 12 h.