Determination of bronchial hyperresponsiveness

For invasive measurement of dynamic resistance, mice were anesthetized by intraperitoneal injection of solution containing ketamine (100 mg/kg, Merial, Duluth, Ga) and xylazine (10 mg/kg, Bayer, Leverkusen, Germany), paralyzed by using D-tubocuranine (0.125%, Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per minute with a tidal volume of 0.2 mL and a positive end-expiratory pressure of 2 mL of H₂O. Increasing concentrations of aerosolized methacholine (9.375, 18.75, 37.5, 75, and 150 mg/mL) were administered. Resistance was recorded by using an invasive plethysmograph (Buxco, London, United Kingdom). Baseline resistance was restored before administration of the subsequent doses of methacholine.^E7