Abstract

Introduction and aims: The respiratory tract is lined by a thin layer of fluid, the airway surface liquid (ASL), which plays an important role in the mucociliary clearance (MCC). ASL is composed by a mucus layer that contains secreted mucins and the periciliar fluid layer that keeps the mucus separated from the epithelial surface. ASL volume is critically regulated by sodium absorption and chloride secretion in order to maintain the MCC function. Measurement of ASL height on primary airway cell cultures by confocal microscopy is a powerful tool which has allowed the study ASL physiology and pharmacology, nevertheless, the use of mouse cells requires an elevated number of animals per culture. We have successfully adapted the use of a fluorescent probe to determine ASL height in the acute isolated mouse trachea. We asked whether changes in fluid absorption relate to ASL height using the Kca3.1-/- mice that showed a significant reduction of ENaC mediated sodium absorption. Methods: To measure the ASL thickness, freshly isolated mouse tracheas were placed in a humidified chamber bathed in physiological buffer (37° C) under constant flow and ASL was labelled with 8 µl PBS containing 2 mg ml-1 Texas red--dextran (10 kD). Tracheas were then placed in a sealed chamber and images were obtained in XZ-scanning mode using a Olympus FV1000 confocal microscope with a water immersion lens (20×/1.0 numerical aperture ) and exited at 540/440 nm. We imaged the ASL height at 10-20 min after dye application and up to 150 min. Images were processed using the ImageJ software and Texas Red-dependent fluorescence intensity along the z-axis was calculated. After derivation of the intensity signal, curves with Gaussian functions were fitted to identify the points with higher rate of fluorescence increase or decrease to determine the distance between these two points. The distance obtained corresponds to the thickness of the ASL. Finally, the mean of the values obtained from the entire scanned area, corresponds to the ASL height. Results: The ASL height of wild type mice and Kca3.1-/- was similar at the starting time (40 µm). However, 60 minutes after addition of the dye the mean ASL height was higher in Kca3.1-/- trachea (18.43 ± 1.003 µm; n=4) than wild type (11.41 ± 0.4730 µm; n=9) and the difference was maintained up to 150 min. Conclusions: We successfully determined ASL height in acute isolated mouse tracheas of mice. Our results demonstrate that the reduced sodium absorption by KCa3.1 inhibition causes an increase in the ASL height which could explain the enhanced MCC previously observed. Preliminary results using tracheas from the Beta-ENaC transgenic animal indicate a reduction in ASL height as predicted for the augmented absorption of sodium. We are now adapting the technique to test the effect of agonists and inhibitors of ion channels of interests for the maintenance of ASL and MCC.