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.