Repair of naphthalene-injured microdissected airways in vitro

Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary toxicants and the progenitor cell for repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. Previously, we described a model system whereby differentiated Clara cells can be maintained in culture using explants of microdissected distal airways. The purpose of this study is to establish whether distal airway explants can be used to study bronchiolar epithelial repair in vitro. Lungs from adult mice treated with naphthalene, a metabolically activated Clara cell cytotoxicant, or vehicle were inflated with agarose and distal airways were microdissected. Distal airway explants were cultured for up to 7 days in serum-free medium. Clara cells in explants from naphthalene-treated mice exhibited the characteristic cytotoxic responses previously reported in vivo when maintained in vitro: cell swelling, formation of cytoplasmic vacuoles, and exfoliation of injured cells into the airway lumen 1 to 2 days after injury (DAI). Epithelial cells squamated to cover the injured area 2 to 4 DAI. At 7 DAI, the epithelium generally consisted of cuboidal cells. Proliferating cells and marker proteins for differentiated Clara cells (Clara cell 10 kD secretory protein, or CC10, and cytochrome P450 monooxygenase isozyme 2B, or CYP2B) were detected immunochemically and their pattern of distribution during the injury and repair response in vitro paralleled the pattern of cell regeneration seen previously in vivo. We conclude that Clara cells in explants from defined regions of murine tracheobronchial airways can be used to study the early phases of the repair response to naphthalene injury, including differentiation and proliferation, in vitro.