The histological analyses were performed by observers who were not aware of the groups of mice from which the samples originated. Images were captured with a digital camera. At least 10 bronchioles with 150–200 μm inner diameter were selected and counted in each slide. For the thickness of tracheal basement membrane, three measures were taken, Compound Library and the average basement membrane thickness was calculated. The area of airway wall (WAt) and area of smooth muscle (WAm) were determined

by morphometric analysis (image-pro plus 6.0; MediaCybernetics Co., Bethesda, MD, USA) on transverse sections after haematoxylin & eosin staining. Basement membrane perimeter (Pbm) was measured for normalization of WAt and WAm. Then we used the ratios of WAt to Pbm (WAt/Pbm) and WAm to

Pbm (WAm/Pbm) to evaluate airway remodelling. Mucus production was determined on transverse sections from the upper left lobe of the lung. The mucus index was calculated as follows: the percentage of the area of mucus on the epithelial surface stained with PAS was determined by image-pro plus 6.0. The area of the respiratory epithelium was outlined, and the image analyser quantified the area of PAS-stained mucus within this reference area. At least 10 bronchioles were counted in each slide. Results were expressed as the percentage of PAS-positive cells/bronchiole, which is calculated from the area of PAS-positive epithelial cells per bronchus divided by the total number of epithelial cells of each bronchiole. Staining with MT was used to determine collagen deposition in the lung. The image-pro plus 6.0 allowed for manual outlining of the trichrome-stained collagen Roxadustat Methisazone layer and computed the area within

the outlined ring of tissue. Briefly, two to four specimens of the MT-stained histological preparations of the lung lobe, in which the total length of the epithelial basement membrane of the bronchioles was 1·0–2·5 mm, were selected and the fibrotic area (stained in blue) beneath the basement membrane in 20 μm depth was measured. The mean score of the fibrotic area divided by the basement membrane perimeter in two to four preparations of one mouse were calculated, then the mean values of subepithelial fibrosis were calculated in 10 mice.21–23 Total RNA was isolated from the right lung tissue using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. One millilitre of trizol reagent was added to frozen airway samples and the resulting preparation was ground using a mortar and pestle for 5 min. Chloroform (200 μl) was added and the solution was centrifuged (6750 g, 4°) for 20 min. The aqueous layer was removed by aspiration with a pipette, and an equal volume of isopropanol was added to the aqueous layer. After centrifugation for 17 min as above, the supernatant was discarded and the remaining pellet was washed in 75% ethanol and suspended in 20 μl DNase-free and RNase-free water.