Paraffin-embedded 4 µm tissue sections were stained with haematoxylin and eosin (H&E) and analyzed for inflammation and tissue damage as described [22] (link)–[25] (link). Briefly, all slides were coded and scored by a pathologist blinded for the experimental groups. Lung tissues were scored for the following parameters: interstitial inflammation, necrosis, endothelialitis, bronchitis, edema, pleuritis, presence of thrombi and percentage of lung surface with pneumonia. All parameters were rated separately from 0 (condition absent) to 4 (most severe condition). The total histopathological score was expressed as the sum of the scores of the individual parameters, with a maximum of 24. Granulocyte stainings, using fluorescein isothiocyanate-labeled rat-anti-mouse Ly-6G mAb (BD Pharmingen, San Diego, CA) were done as described previously [23] (link)–[25] (link). Slides were counterstained with methylgreen (Sigma-Aldrich, St. Louis, MO). The total tissue area of the Ly-6G-stained slides was scanned with a slide scanner (Olympus dotSlide, Tokyo, Japan) and the obtained scans were exported in TIFF format for digital image analysis. The digital images were analyzed with ImageJ (version 2006.02.01, National Institutes of Health, Bethesda, MD) and the immunopositive (Ly6G+) area was expressed as the percentage of the total lung surface area.
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