Histological lung assessment

Immediately after pulmonary function tests, deeply anesthetized animals were euthanized, and lungs were removed en bloc via thoracotomy. The right lung was snap frozen for molecular analysis, and the left lung was pressure fixed for 24 h at 25 cmH2O in 4% paraformaldehyde (PFA). After fixation, 6 sections from the superior and inferior lobe were embedded and cut as described before³³. Alveolar morphology was measured on digitally scanned 4 µm hematoxylin and eosin-stained slides using a semi-automated, validated Fiji-plugin (ImageJ; http://fiji.sc/Fiji)³⁴. For each lung, 3 slides from each of the 6 segments were analyzed (18 slides per lung). Calculations of the mean linear intercept (Lm), alveolar air space (Lma), and alveolar wall thickness (Lmw) were made as previously described³⁵. ASMC and vascular morphology were evaluated using immunohistochemistry. A primary α-smooth muscle actin (α-SMA) antibody (mouse anti-human, M0851; DakoCytomation) was used in combination with a secondary goat anti-mouse antibody (115-035-044; Jackson ImmunoResearch). Aminoethyl carbazole was used as a chromogen.

ASMC was measured in 10 randomly selected airways per lung. Airways with a diameter of 150–250 µm if cut in cross section to their long axis were included. Airways with a long axis-to-short axis ratio of > 2:1 were excluded. ASMC was analyzed in QuPath 0.2.0³⁶ by manually selecting the fraction of stained muscle tissue and the full perimeter of the airway. Vascular morphometry was performed examining a minimum of 10 intra-acinar pulmonary arteries per lung with an external diameter of 40–200 µm, measuring both internal and external diameter of the media to calculate the vascular medial thickness^32,37.