Measurement of methacholine airway hyperresponsiveness

Twenty-four hours after the last challenge, airway hyperresponsiveness (AHR) was assessed in conscious, unrestrained mice using noninvasive whole-body plethysmography (Allmedicus, Seoul, Republic of Korea), as described previously [27]. Briefly, mice were placed in the plethysmography chamber and exposed to increasing concentrations of aerosolized methacholine at 0, 12.5, 25, and 50 mg/mL for 10 min. Enhanced pause (Penh) was calculated automatically based on the mean pressure generated in the plethysmography chamber during inspiration and expiration combined with the time of each phase. Then the Penh values calculated during each 3 min interval were averaged.