, Montreal, Quebec, Canada). The duration of lung mechanics data collection was 20 min per animal. A laparotomy was performed immediately after determination of lung mechanics, and heparin (1000 IU) injected directly into the vena cava. The trachea was clamped at end-expiration (PEEP = 2 cm H2O) and the abdominal aorta and vena cava were severed, producing massive hemorrhage and rapid death by exsanguination. The right lung was then removed, fixed in 3% buffered formalin and embedded in paraffin. Slices (4 μm thick)

were cut and stained with hematoxylin and eosin. Lung morphometry analysis was performed with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines of known selleck compound length coupled to a conventional light microscope (Olympus BX51, Olympus Latin America Inc.,

Brazil). The volume fractions of the lung occupied by collapsed alveoli or normal tissue were determined by the point-counting technique (Weibel, 1990) across find more 10 random, non-coincident fields of view (Santos et al., 2012). The number of neutrophils and macrophages in lung tissue was evaluated at 1000× magnification. Points falling on neutrophils and macrophages were counted and divided by the total number of points falling on tissue in each field of view. Apoptotic cells in lung, kidney, liver, and small intestine specimens were quantified using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Staining was performed in a blinded fashion by two pathologists to assay cellular apoptosis (Oliveira et al., Nutlin-3 cost 2009). Ten fields per section from regions with cell apoptosis were examined under 1000× magnification. A 5-point, semi-quantitative, severity-based scoring system was used to assess the degree of apoptosis, graded as: 0 = normal parenchyma; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100% of examined tissue. Quantitative real-time reverse transcription polymerase

chain reaction (RT-PCR) was performed to measure the relative expression of the inducible nitric oxide synthase (iNOS), nuclear factor E2-related factor 2 (Nrf2), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) genes (Santos et al., 2012). Central slices of right lung were cut, collected in cryotubes, flash-frozen by immersion in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from frozen tissues using the SV total RNA Isolation System (Promega Corporation, Fitchburg, WI, USA) in accordance with manufacturer recommendations. RNA levels were measured by spectrophotometry in a Nanodrop ND-1000 system. First-strand cDNA was synthesized from total RNA using the GoTaq® 2-STEP RT qPCR System (Promega Corporation, Fitchburg, WI, USA).

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