Carrageenan-Induced Pleurisy

Rats were anesthetized with 4% enflurane mixed with 0.5 L/min O₂ and 0.5 L/min N₂O, and a skin incision at the level of the left sixth intercostal space was performed. The underlying muscle was dissected, and 0.2 ml of λ-carrageenan type IV (1% w/v; Sigma-Aldrich, Milan, Italy) was injected into the pleural cavity. The CD73 inhibitor, APCP (400 μg/rat; Tocris Bioscience, Bristol, UK), or an equal volume of the vehicle (distilled water), was injected into the pleural cavity immediately before carrageenan injection. The skin was then sutured, and animals were returned to their cages and allowed to have food and water ad libitum; 4 and 72 h following pleurisy induction, rats were sacrificed by CO₂ inhalation. The chest was carefully opened, and the pleural cavity was washed with 2 ml of sterile saline containing 10 U/ml heparin (Sigma-Aldrich, Milan Italy). Any lavage fluid with blood contamination was rejected. The volume of pleural lavage fluid collected from each animal was measured; then, fluids were centrifuged at 180 × g for 10 min and the pellet suspended in phosphate-buffered saline (PBS). Cells were counted with TC20^™ Automated Cell Counter (Bio-Rad, Italy). Samples of supernatants and cell pellets were then frozen at −80°C to be successively analyzed for cytokine content and AMPase activity, whereas differential cell count was performed in smears by May-Grunwald-Giemsa staining (Carlo Erba, Italy). Lung samples were harvested from each rat and immediately frozen at −80°C or fixed in formaldehyde solution (4% v/v, in distilled water) for 1 week at room temperature and successively utilized for further analyses.