Acetyl β methacholine chloride

All in vivo studies were conducted 48 hours after HRV-1B inoculation, which coincided with 24 hours after the last HDM exposure (the “day-of-study”). This timing was based on preliminary kinetics studies which showed that the peak of HRV-1B induced pulmonary inflammation occurs 48 hours post infection in adult female BALB/c mice (Figure S1). Mice were surgically prepared, and lung function/responsiveness to methacholine (MCh; acetyl β-methacholine chloride, Sigma-Aldrich, MO, USA) was assessed as previously described [38] (link). Briefly, we used a modification of the low frequency forced oscillation technique (FOT) to measure respiratory system impedance (Z_rs) [31] (link). We fit the single compartment and constant phase models to Z_rs to generate respiratory system resistance (R_rs) and to partition the impedance into airway resistance (R_aw), tissue damping (G) and tissue elastance (H). Tissue hysteresivity (η) was calculated as the ratio of G/H [39] (link). Sensitivity to MCh was calculated as the dose required for respiratory system resistance, airway resistance, tissue damping and tissue elastance to increase by 150% (R_rsEC₁₅₀, R_awEC₁₅₀, GEC₁₅₀ and HEC₁₅₀).

After measurement of TGV and lung mechanics, a randomized selection of half the mice from each group (n = 12) were transferred to a small animal ventilator (Legacy flexiVent; SCIREQ, Montreal, QC, Canada) for assessment of responsiveness to methacholine (MCh; acetyl β-methacholine chloride; Sigma-Aldrich, St. Louis, MO, USA) as previously described [110 (link)]. Briefly, 5x forced oscillation technique (FOT) measurements were taken at FRC/baseline (1 per minute), then after a 10 s saline aerosol and again after increasing doses of MCh from 0.1 to 30 mg/mL. Peak responses to MCh at each dose were used to construct dose–response curves.