Pulmonary dysfunction was measured as we previously described (Campa et al., 2018; Russo et al., 2018). For invasive in vivo assessment, mice were anesthetized and tracheostomized, then were placed in a whole-body plethysmograph to maintain spontaneous breathing connected to a computer-controlled ventilator (Forced Pulmonary Maneuver System®, Buxco Research Systems©, Wilmington, North Carolina, United States). Under mechanical respiration the Dynamic Compliance (Cdyn) and Lung Resistance (Rl) were determined by Resistance and Chord Compliance RC test. To measure the Inspiratory Capacity (IC) a Pressure-Volume maneuver was performed, which inflates the lungs to a standard pressure of +30 cm H2O and then slowly exhales until a negative pressure of −30 cm H2O is reached. To evaluate airway hyperresponsiveness (AHR), the same mice used in previous maneuvers (basal condition) received Methacholine, 1 mg Kg–1 (Acetyl-β-methylcholine chloride, A-2251, Sigma-Aldrich St. Louis, MO, United States) i.v. and after 10 s, a new set of maneuvers were conducted to assess Rl changes. Suboptimal maneuvers were rejected and for each test at least three acceptable maneuvers were conducted in every single mouse to obtain a reliable mean for all numeric parameters.

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