2.7 | Pathological examinations
At the end of the experiments, the animals were euthanized under deep anesthesia with a high dose of 15% potassium chloride (3 mL). Within 5 min after death, the ventilator was stopped, and a positive airway pressure equivalent to PEEP of 5 cm H2O was applied. The chest wall was then opened, and the trachea was clamped. The airway and lung were incised and fixed in 10% formaldehyde solution for histological preparations and examinations. Routine techniques were used to prepare the lung tissues for paraffin embedding. Thin sections were stained with hematoxylin and eosin stain. The sections were examined under light microscopy (Olympus AX-80, Yuanyu Industry CO., Ltd, Taipei, Taiwan), and histology was scored using a quantitative system by an investigator blinded to the identity of the specimens. Injury scores were based on the following pathohistological characteristics: alveolar inflammation, interstitial inflammation, alveolar hemorrhage, interstitial hemorrhage, atelectasis, necrosis, and overdistension. Injury score was graded 0, 1, 2, 3, and 4 for abnormalities noted in 0%, 25%, 50%, 75%, and diffusely covering the lung field, respectively. Multiple (> 10) fields of the lung section for each pathohistological characteristic were examined, and the scores were averaged to obtain a mean value.