Alveolar-capillary permeability was assessed with Evans blue albumin (EBA) as previous described5455. Evans blue (0.5% EB, Sigma-Aldrich, St Louis, MO, USA) was dissolved in Ca2+/Mg2+-free phosphate-buffered saline (PBS; Sigma-Aldrich), and conjugated to albumin (4% EBA) that was prepared by adding bovine serum albumin (Sigma-Aldrich). After thoroughly dissolving by gently stirring with a magnetic bar, the EBA solution was filtered through a 0.22 μm syringe filter and aliquots were stored at −80 °C until use. Each aliquot was used only once for each experiment. To evaluate alveolar-capillary barrier function, EBA (25 mg/kg body weight) was injected into the internal jugular vein 1 h before euthanasia and lung harvesting. Blood samples were obtained from the right heart, and the pulmonary vasculature was subsequently infused with 1 mL PBS. The right lung was ligated at the level of the right mainstem bronchus, excised, blotted dry, weighed and stored in liquid nitrogen until these samples were used for EBA analysis. After freeze/thaw, the lung tissue was homogenized in 2 mL PBS and incubated with an additional 2 mL of formamide (Sigma-Aldrich) (18 h; 60 °C). Formamide extracts were centrifuged (15,000 g × 30 min; 4 °C), and the centrifuged supernatants were collected to quantify lung EBA content using a dual-wavelength (620 nm and 740 nm) spectrophotometric method. Pulmonary EBA absorbance at 620 nm was corrected by a correction factor with EBA absorbance at 740 nm. The EBA permeability index was calculated by dividing pulmonary EBA absorbance at 620 nm/g of lung tissue by plasma EBA absorbance at 620 nm.