Histological Analysis of Lung Architecture

The left lung was fixed in Milloning buffer solution (pH 7.4) with 4% paraformaldehyde to preserve the pulmonary architecture. Lung sections were stained using Sirius Red (pH 10.2) or Masson’s trichrome for the analysis of peribronchiolar leukocyte infiltration and extracellular matrix deposits, respectively, as described previously^{19 (link),20 (link)}. The slides stained using Masson’s trichrome were scanned with 3DHISTECH–Pannoramic MIDI whole slide scanner, and the images were captured using a 20× objective lens. The analysis was performed in 10 bronchioles per animal in a double-blind experiment evaluating the use of Image-Pro-Plus^® software version 6.2 (Media Cybernetics Inc,). The quantification of eosinophils, neutrophils and mononuclear cells number on the peribronchiolar region was performed under an optical microscope (BX40; Olympus) through an integrating eyepiece. The morphometric reticulum had a known area of 10⁴ μm² at the final magnification of 1,000× and was randomly positioned over the peribronchiolar regions. Three different fields were analyzed for bronchioles in 7 bronchioles per animal^{19 (link),20 (link)}.