Mucociliary clearance measurement in the upper airways.

To determine the rate of MCC in the anterior nasopharynx (ANP) of the nasal cavity, the mice were anesthetized (2.5% isoflurane) and then euthanized by severing the abdominal aorta. The lower jaw was removed and a small incision made in the lateral wall of the ANP (hard palate) at the level of the nasopalatine duct. A 35-gauge silica cannula (WPI, Sarasota, FL) was dipped to a depth of ∼5 mm into an aliquot of dry green fluorescent 7-μm beads (Thermo Scientific, Fremont, CA). The dry beads adhered to the outside of the cannula by static electricity. The cannula with the adherent beads was then introduced into the ANP via the incision. Enough beads were deposited on the surface of the nasal tissue by contact with the cannula to allow easy quantification of MCC by tracking the beads in the ANP. The preparation was placed under a dissecting microscope with a fluorescent lamp outfitted with a video camera (MTI, Michigan City, IN). The camera was interfaced to a DVD recorder for image acquisition. The beads could be clearly seen moving caudally inside the nasal cavity toward the posterior nasopharynx. The rate of MCC was measured downstream of the site of bead introduction. Before the MCC was recorded, a slide micrometer was placed on the stage of the dissecting scope to calibrate distance measurements. Once the video was recorded, MCC was determined by playing back the video and determining the time it took fluorescent particles to traverse a calibrated distance on the screen monitor (corresponding to an in vivo distance of 0.5–1 mm). Usually 10–30 particles were tracked per mouse over a 10-min period. There was no systematic change in the rate of MCC over the 10-min time period in the majority of the mice. The rate of MCC was calculated as millimeters per minute.