Lung Histology and Lung Injury Scoring

Mice were killed by a pentobarbital overdose followed by exsanguination. The lungs were fixed in a 4% paraformaldehyde solution overnight and subsequently embedded in paraffin, sectioned to 5 μm-thick sections, and stained with hematoxylin and eosin (H&E) as described previously (26) for histopathological scoring or anti-myeloperoxidase (MPO) antibodies for immunohistochemistry (IHC) analysis.

Two random H&E-stained tissue sections were examined by a pathologist who was blinded to the genetic background and treatment of the mice. ALI was scored as described previously (27), in accordance with the following criteria: 1) alveolar congestion; 2) hemorrhage; 3) infiltration or aggregation of neutrophils in the airspace or vessel wall; and 4) thickness of the alveolar wall/hyaline membrane formation. For each subject, a five-point scale was applied: 0, minimal (little) damage; 1+, mild damage; 2+, moderate damage; 3+, severe damage; and 4+, maximal damage. Points were totaled and expressed as the median ± range.