Imaging of Wound Closure of Small Epithelial Lesions in the Mouse Trachea

Integrity of the airway epithelium is essential for normal lung function. However, studies analyzing the repair process of small epithelial lesions in pseudostratified airway epithelium are missing. To follow airway-epithelial wound closure over time, we lesioned small areas of the mouse tracheal epithelium (1 to 12 cells) using a femtosecond laser and followed wound closure up to 6 hours by autofluorescence multiphoton microscopy. Selected lesions were also examined by scanning and transmission electron microscopy and by staining of filamentous actin. Most lesions with a size up to six cells closed by elongation of the surrounding epithelial cells within 6 hours, and all damaged cells were extruded from the epithelium. Electron microscopy confirmed that the surrounding epithelial cells directly closed lesions up to six cells. Most lesions larger than six cells did not close in the observation period of 6 hours, but we observed that basal cells flattened to cover the basement membrane. Delayed wound closure was, in part, attributable to damage of the basement membrane. Cells facing the lesion exhibited increased filamentous actin staining, indicating active cell movement. Not all cells initially facing the lesion participated directly in wound closure, indicating that closure is driven by movement of individual cells rather than a transepithelial coordinated process. Small wounds in the pseudostratified airway epithelium close within hours to preserve epithelial integrity.