Abstract
Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.
Highlights
Et al successfully showed perturbation of the ciliary beating direction in an Odf[2] gene-mutated mouse[5]
Since mouse tracheae are sufficiently thin so as to observe ciliary movement through transmitted light, live images of wheat germ agglutinin (WGA)-stained cilia can be compared with those obtained using transmitted light
Tracheal epithelial cilia were successfully visualized with fluorescein isothiocyanate-WGA (FITC-WGA) in 5 out of 5 mice (Fig. 2A, Movie 1)
Summary
Et al successfully showed perturbation of the ciliary beating direction in an Odf[2] gene-mutated mouse[5]. Labeling cilia with fluorescent materials may be necessary to observe the movement of human airway cilia in situ. The surface of the airway cilia is covered with a mesh-like network of membrane-tethered mucin and cell surface proteoglycans, which are considered to be important feature for maintaining appropriate physiological properties for mucociliary clearance. This implies the possibility that agglutinins could be used for labeling cilia[12,13]. Okada et al have shown that WGA can be attached to the cilia of cultured airway epithelial cells. We report a method for live imaging of cilia, and show aberrant ciliary movement in 2 tracheal samples with abnormal cilia, obtained from patients who underwent radiation therapy and tracheotomy
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