In the ovalbumin group (OVA), mice were immunized using an adjuvant-free protocol with intraperitoneal injection of ovalbumin (10 μg in 0.1 mL sterile saline) on each of seven alternate days. Forty days after the beginning of sensitization, 20 μg of OVA in 20 μL

sterile saline were intratracheally instilled. This procedure was performed three times at 3-day intervals. The control group (C) received saline using the same protocol. Eighty-four animals were used for analysis of lung mechanics and histology, and a second group of 84 animals was used for analysis of airway responsiveness and bronchoalveolar lavage fluid (BALF). The BCG Moreau vaccine was donated by the Ataulpho de Paiva Foundation, Brazil. Twenty-four hours after the last challenge, mice were sedated (diazepam 1 mg i.p.), anesthetized (thiopental sodium 20 mg/kg i.p.), tracheotomized, paralyzed (vecuronium bromide, 0.005 mg/kg i.v.), and mechanically PF-02341066 in vitro ventilated with the following settings: respiratory frequency 100 breaths/min, tidal volume (VT) 0.2 mL, and fraction of inspired oxygen (FiO2) 0.21. The anterior chest wall was surgically removed and a positive end-expiratory pressure (PEEP) of 2 cmH2O was applied, and the lung mechanics were computed. At the end of the experiment, the lungs were prepared for histology Vorinostat ic50 and molecular biology.

Airflow, volume and tracheal pressure (Ptr) were measured ( Hsia et al., 2010). In an open chest preparation, Ptr reflects transpulmonary pressure (PL). Lung static elastance and airway resistance were computed by the end-inflation ifenprodil occlusion method ( Bates et al., 1985) using the ANADAT data analysis software (RHT-InfoData, Inc., Montreal, Quebec, Canada). Twenty-four hours after the last challenge, airway responsiveness was measured. Increasing doses of methacholine (Sigma Chemical Co., Saint Louis, MI, USA) (100, 300, 1000, 3000, and 10,000 μg/kg) were administered via a silastic catheter placed in the jugular vein. Data were stored at 30 s, 1, 3, and 5 min after agonist injection. Shortly after each intravenous infusion of methacholine, the maximal increase in Ptr was reached, and the respective airflow

was measured at this moment (Antunes et al., 2009). Respiratory system resistance (R) was obtained using the equation of motion of the respiratory system: Ptr(t) = E·V(t) + R·V′(t), where (t) is time. The right lung was removed, fixed in 4% buffered formaldehyde, paraffin-embedded, and cut into 4 μm-thick slices, which were stained with hematoxylin and eosin (Vetec Química Fina, Rio de Janeiro, Brazil). Fraction area of collapsed and normal lung areas were determined by the point-counting technique at a magnification of 200× across 10 random, non-coincident microscopic fields (Hsia et al., 2010). Points falling on collapsed or normal pulmonary areas were counted and divided by the total number of points in each microscopic field.