Acetyl β methyl choline methacholine

Mice were anaesthetized by i.p. injection of 50 μl Ketavet/Rompun (76 mg/ml and 4.8 mg/ml respectively, Pfizer/Bayer). Muscle relaxation was induced by administration of 50 μl of Esmeron (10 mg/ml, Organon). Airway hyperresponsiveness (AHR) was measured in anesthetized mice that were mechanically ventilated using a FlexiVent (SciReq) system as described [24 (link)]. Aerosolized Acetyl-β-Methyl-Choline (methacholine) (0, 2.5, 5, 10, 25, and 50 mg/ml; Sigma-Aldrich) was generated by an ultrasonic nebulizer and delivered in-line through the inhalation port for 10 seconds. Airway resistance was measured two minutes later.

Toluene-2,4-diisocyanate (TDI) (98%; Fluka, CAS 584-84-9), acetyl-β-methylcholine (methacholine) and acetone were obtained from Sigma-Aldrich (Bornem, Belgium). Pentobarbital (Nembutal®) was obtained from Sanofi Santé Animale (CEVA, Brussels, Belgium). Anti-mouse IL-13 antibody (262A-5-1) and mouse IgG1 anti-human gp120 were kindly provided by Genentech (San Francisco, California, USA). The vehicle (acetone/olive oil, AOO), used to dissolve TDI consisted of a mixture of 2 volumes of acetone and 3 volumes of olive oil (Selección de Almazara, Carbonell, Madrid, Spain) for both the dermal sensitization and the intranasal challenge. Concentrations of TDI are given as percent (v/v) in AOO.