Bleomycin-Induced Lung Injury Assessment

At days 7, 14, and 21 days posttreatment the first bleomycin challenge, animals were anesthetized with isoflurane and sacrificed by bleeding from the abdominal aorta. Bronchoalveolar lavage fluid (BALF) was collected by gently washing the lungs with 0.6 mL sterile solution [Hank’s balanced salt solution × 10; ethylenediaminetetraacetic acid 100 mM; 4-(2-hydroxy-ethyl)-1-piperazineethansulphonic acid 1 mM; and distilled water] for three times in the bronchial tree and collected for subsequent analysis (14 (link)). After centrifugation at 400 g for 10 min, the BAL supernatants were frozen at −80°C for simultaneous quantitation of multiple cytokines/chemokines using a Bio-Plex™ Cytokine Assay Kit (Bio-Rad Laboratories, Segrate, Milano, Italy). The cell pellet was resuspended in 0.2 ml of PBS. Cell number was counted with an automated cell counter (Dasit XT 1800J). The concentration of MMP proteins in BAL fluid was determined by enzyme-linked immunosorbent assay (ELISA). MMP-2, MMP-9, and TIMP-1 were measured with specific ELISA kit (R&D System), and MMP-1 was measured with Mouse Matrix Metalloproteinase 1 ELISA Kit (MyBioSource) according to the manufacturer’s instructions.