All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for

the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA. Thirty-two male BALB/c mice (25 ± 5 g) received intraperitoneal injections of saline (100 μL, 0.9% NaCl, N = 16) or ovalbumin (OVA, 10 μg in 100 μL, 0.9% NaCl, N = 16) on each of seven alternate days (days 1, 3, 5, 7, 9, 11 and 13). Forty days after the first instillation, PD0325901 ic50 the mice were challenged three times with intratracheal instillations of ovalbumin (20 μg, 20 μL, 0.9% NaCl) or saline (20 μL, days 41, 44 and 47). Immediately after the last

challenge they were divided into four groups (N = 8, each) and intranasally instilled with 10 μL of saline (SAL-SAL and OVA-SAL, respectively) or 10 μL of ROFA (20 μg/mL, SAL-ROFA and OVA-ROFA). For the instillation, the mice were anesthetized with sevoflurane and solutions (saline or ROFA) were gently instilled into their snouts with the aid of a precision pipette. The animals recovered rapidly after instillation. Our ROFA was extracted from an incinerator located at the University Hospital, University of São Paulo, Brazil. selleck screening library The distribution of particle sizes was determined by laser diffraction (Long Bench Mastersizer S, Malvern Instruments Ltd, Malvern, Worcestershire, United Kingdom). The particulate matter was visualized by

scanning electron microscopy (JEOL 5310, Tokyo, Japan). Twenty-four hours after the intranasal instillation of ROFA, the animals were sedated (diazepam, 1 mg i.p.), anesthetized (pentobarbital sodium, 20 mg/kg BW i.p.), tracheotomized, and a snugly fitting cannula (0.8 mm i.d.) was introduced into the trachea. The animals were then paralyzed (pancuronium bromide, 0.1 mg/kg) and the anterior chest wall was surgically removed; thus, the pressure measured in the airway represents transpulmonary pressure (PL). A constant-flow ventilator (Samay VR15, Universidad de la Republica, Montevideo, Uruguay) provided artificial ventilation with a frequency of 100 breaths/min, a tidal volume of 0.2 mL, flow of 1 mL/s, and positive end-expiratory pressure amounting to 2 cmH2O. For the determination of pulmonary mechanics a 5-s end-inspiratory pause could Fludarabine solubility dmso be generated by the ventilator. A pneumotachograph with 1.5 mm i.d., length of 4.2 cm and distance between side ports of 2.1 cm was connected to the tracheal cannula for the measurements of airflow (V′). Lung tidal volume (VT) was determined by V′ signal integration. The pressure gradient across the pneumotachograph was determined by means of a differential pressure transducer (Validyne MP45-2, Engineering Corp., Northridge, CA, USA). The equipment resistance (Req) including the tracheal cannula was previously measured using different flow rates (Req = 0.