Euthanasia- and Lavage-mediated Effects on Bronchoalveolar Measures of Lung Injury and Inflammation

Accurate and reproducible assessments of experimental lung injury and inflammation are critical for basic and translational research. In particular, investigators use various methods for BAL and euthanasia; however, the impact of these methods on assessments of injury and inflammation is unknown. To define potential effects, we compared methods of lavage and euthanasia in uninjured mice and after a mild lung injury model (ozone). C57BL/6J male mice (8-10 weeks old) underwent BAL after euthanasia with ketamine/xylazine, carbon dioxide (CO₂), or isoflurane. BAL methods included 800 μl of isotonic solution instilled and withdrawn three times, and one or three passive fills and drainage to 20 cm H₂O. Parallel experiments were performed 24 hours after 3 hours of ozone (O₃) exposure at 2 ppm. BAL total cell counts/differentials and total protein/albumin were determined. Lung histology was evaluated for lung inflammation or injury. BAL cells were cultured and stimulated with PBS, PMA, or LPS for 4 hours and supernatants were evaluated for cytokine content. In uninjured mice, we observed differences due to the lavage and euthanasia methods used. The lavage method increased total cells and total protein/albumin in uninjured and O₃-exposed mice, with the 800-μl instillation having the highest values. Isoflurane increased total BAL cells, whereas CO₂ euthanasia increased the total protein/albumin levels in uninjured mice. These effects limited our ability to detect differences in BAL injury measures after O₃ exposure. In conclusion, the method used for lavage and euthanasia affects measures of lung inflammation/injury and should be considered a variable in model assessments.