Morphometry and Biomarkers assessment
The animals underwent endotracheal intubate soon after euthanasia. For further orphologic and morphometric study, one side of the lungs was prepared through the intratracheal instillation of a 4% paraformaldehyde solution with a hydrostatic pressure of 20 cm that was maintained for at least 20 minutes, before being dissected and sent for additional sample processing procedures, e.g., fixation in 4% paraformaldehyde, paraffin embedding, sagittal slice preparation and staining with hematoxylin-eosin (HE), etc.. Morphometric studies were conducted in accordance with methods previously reported (Knudsen, Weibel, Gundersen, Weinstein & Ochs, 2010), briefly, twenty non-overlapping fields in each slice were randomly selected under an Olympus DP72 camera system (×40 magnification power). The degree of airspace enlargement or emphysema was assessed by mean linear intercept (Lm, μm) using STEPanizer software (Bern, Switzerland) based on the number of intersections of a given line across the alveolar septa. The morphometry was conducted in a blind manner and the scores separately calculated by two experts in pulmonary anatomy, before being averaged and used as the final scores.
The other side of the lungs underwent intratracheal instillation of 0.5 ml of ice-cold phosphate-buffered saline (PBS), three times, while the contralateral main bronchus was ligated. The bronchoalveolar lavage fluid (BALF) was recovered by gentle aspiration and subjected to filtration and centrifugation at 750g for 10 min at 4 ºC. The supernatant was stored at -80 ºC for the subsequent measurements. The lungs of the mice not subjected to BAL or paraformaldehyde fixation were excised and stored at -80 ºC for HDAC-2 assessment. The expression of IL-8 and TNF-α in the BALF were measured using a commercial ELISA kit (Neobioscience, Wuhan, China) according to the manufacturer’s instructions.
For assessment of reactive oxygen species (ROS), the frozen sections (6 μm) of the lung tissue were stained with DHE (2 µmol/L) in a humidified and light-protected chamber at 37 ºC for 30 min as described (Faller et al., 2013). Fluorescent images were photographed under a confocal laser scanning microscope (Olympus) and the intensity of fluorescence was measured using Image-Pro Plus 6.0® (Media Cybermetics, Bethesda, MD) software and expressed as arbitrary units of fluorescence.
The nuclear components in the homogenized lung tissue were extracted using a nuclear extraction kit (Aspen, Wuhan, China). HDAC-2 activity in the nuclear extracts was quantified using an HDAC-2 assay kit (Epigentek Group, Brooklyn, NY), in accordance with the manufacturer’s instructions (Sugiura et al., 2012).