005 mg/kg iv), and ventilated with a constant flow ventilator (Samay VR15; Universidad de la Republica, Montevideo, Uruguay) with the following parameters: frequency of 100 breaths/min,

BMS754807 tidal volume (VT) of 0.2 ml, and fraction of inspired oxygen of 0.21. The anterior chest wall was surgically removed and a positive end-expiratory pressure (PEEP) of 2 cm H2O was applied. After a 10-min ventilation period, lung mechanics were computed. At the end of the experiments (approximately 30 min), lungs were prepared for histology. Airflow and tracheal pressure (Ptr) were measured ( Burburan et al., 2007). Lung mechanics was analyzed using the end-inflation occlusion method ( Bates et al., 1988). In an open chest preparation, Ptr reflects transpulmonary pressure (PL). Static lung elastance (Est) was determined by dividing the elastic recoil pressure of the lung by VT. Lung mechanics measurements were performed 10 times in each animal. All data were analyzed using the ANADAT data analysis software (RHT-InfoData, Inc., Montreal, Quebec, Canada). A laparotomy was done immediately

after determination of lung mechanics, and heparin (1000 IU) was intravenously injected in the vena CB-839 mw cava. The trachea was clamped at end-expiration (PEEP = 2 cm H2O), and the abdominal aorta and vena cava were sectioned, yielding a massive haemorrhage that quickly killed the animals. The right lung, liver and kidney were then removed, fixed in 3% buffered formaldehyde and paraffin-embedded. Four-micrometer-thick slices were cut and stained with hematoxylin-eosin. Lung morphometry analysis was performed with an integrating eyepiece with a coherent system consisting of a grid with

100 points and 50 lines (known length) coupled to a conventional light microscope (Olympus MycoClean Mycoplasma Removal Kit BX51, Olympus Latin America-Inc., Brazil). Fraction areas of collapsed and normal lung areas were determined by the point-counting technique (Hsia et al., 2010 and Weibel, 1990) across 10 random, non-coincident microscopic fields (Menezes et al., 2005, Santos et al., 2006 and Chao et al., 2010). Polymorphonuclear (PMN) and mononuclear (MN) cells and lung tissue were evaluated at x 1000 magnification. Points falling on PMN and MN cells were counted, and divided by the total number of points falling on tissue area in each microscopic field. Collagen fibres (picrosirius-polarization method) were quantified in alveolar septa at 400× magnification (Rocco et al., 2001 and Chao et al., 2010). Three 2 mm × 2 mm × 2 mm slices were cut from three different segments of the left lung and fixed [2.5% glutaraldehyde and phosphate buffer 0.1 M (pH = 7.4)] for electron microscopy (JEOL 1010 Transmission Electron Microscope, Tokyo, Japan) analysis.