Methacholine chloride

Mice were sensitized to allergic responses by intranasal administration of papain, an allergenic protease or HDM extract (Der p 1, Greer Laboratories)^{28 (link)}. In brief, mice were anaesthetized with isoflurane (Isothesia, Henry Schein Animal Health) and 10 μg of papain (5125, Calbiochem) in 30 μl of PBS was administered for 2 consecutive days (day 0 and 1). For HDM, mice were sensitized by 25 μg (protein content) in 25 μl of PBS for 3 days (days 0, 1 and 2), and challenged after two weeks with 5 μg (protein content) in 25 μl of PBS for 3 days (days 14, 15 and 16)^{28 (link)}. The mechanical properties of the respiratory system were measured using a rodent lung function testing system (FlexiVent, SCIREQ). In brief, the mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (100 mg kg⁻¹, Esconarkon, Streuli Pharma), then tracheotomized and mechanically ventilated followed by intraperitoneal administration of pancuronium (0.08 mg kg⁻¹, Hospita). Airway constriction was induced by administering increasing aerosolized doses (0.3125 kg⁻¹, 12.5 kg⁻¹ and 50 mg kg⁻¹) of methacholine chloride (Sigma-Aldrich). Airway resistance was calculated from forced-oscillation measurements of changes in pressure, volume and flow.

Mice were mechanically ventilated and challenged with increasing concentrations of methacholine as described previously (60 (link)). Briefly, mice were anesthetized with diazepam (17.5 mg/kg) and ketamine (450 mg/kg), intubated, connected to a ventilator (flexiVent; SCIREQ, Montreal, PQ, Canada) and ventilated at a rate of 160 breaths per minute at a tidal volume of 0.2 ml with a positive end-expiratory pressure of 3 cm H₂O. Total respiratory system resistance (R) and elastance (E) were recorded continuously as previously described (ibid.). Baseline lung volume was set via deep inhalation. Increasing concentrations of methacholine chloride (0–50 mg/ml, Sigma-Aldrich, St Louis, MS) were administered via aerosolization within an administration time of 10 sec. Airway responsiveness was recorded every 15 seconds for 3 minutes after each aerosol challenge. Broadband perturbation was used and impedance was analyzed via constant phase model.