In a subset of animals, exhaled NO (eNO) levels were serially measured noninvasively before and 0.5, 2, 6, 12, and 24 h after the single DETA-NO nebulization, as previously described. In brief, spontaneously breathing mice were individually placed in a small plastic chamber that was flushed with NO-free medical-grade air at 60 mL/min. The chamber effluent, containing exhaled gas, was sampled in triplicate, and the concentration of gas-phase NO measured by chemiluminescence. Samples were referenced to a calibration curve of signal mV vs standard concentrations of gas-phase NO (0 to 527 parts per billion; R2 > 0.999). Data are reported as the concentration of eNO in parts per billion. asthma medications inhalers
In order to assess the effects of DETA-NO on pulmonary bacterial burden and inflammation, mice in both the sham and pneumonia groups were twice exposed (at 4 and 12 h) to DETA-NO during the 24-h experimental protocol.
During the 24-h protocol, mice were housed in a sealed plastic chamber that was continuously flushed with NO gas in medical-grade air at a final chamber NO concentration of either 10 ± 1 or 40 ± 2 ppm, as continuously measured by chemiluminescence (Pac III NO analyzer; Drager; Mississauga, ON, Canada). Chamber NO2 concentrations were intermittently monitored and were consistently < 2 ppm. Mice exposed to RA were housed in an identical plastic chamber continuously flushed with medical-grade air (final chamber NO concentration, < 0.5 ppm) for 24 h.
Twenty-four hours following surgery, animals were killed with 0.5 mg/g body weight of intraperitoneal pentobarbital (65 mg/mL). Blood was collected in a heparinized syringe via cardiac puncture. Heparinized blood samples were centrifuged at 10,000g for 10 min at 4°C, and the plasma supernatant was collected and stored under nitrogen at — 20°C until assayed for NOx— levels (vide infra).