title: Reflection confocal microscopy for the study of airway surface liquid dysregulation in cystic fibrosis
Creators: name: Seyhan Agircan, Ayca
Publisher: name: Heidelberg University Library
Year: 2019

description: Measurements of airway surface liquid (ASL) on primary airway epithelial cultures (AECs) grown at air-liquid interface (ALI) identified ASL depletion as a characteristic abnormality in cystic fibrosis (CF) and may be used as an endpoint for preclinical testing of strategies to improve airway surface hydration in patients with CF. Traditionally, ASL height has been determined by confocal fluorescence microscopy after addition of fluorescently labelled liquid to the apical side of the epithelium, however, several hours are required to restore steady state ASL depth after this volume challenge. Moreover, the current protocol requires fluorescently labelled liquid addition to the apical side of the epithelium resulting in non-physiological conditions.
The aim of this project is to determine the suitability of confocal reflection microscopy as a novel approach to study ASL regulation without requirement of fluorescent labelling/volume. Reflection microscopy detects the inherent differences in refractive indices between the permeable membrane, cytosol of cells and liquid layer. Primary airway epithelial cells were prepared from wild-type mice and βENaC-overexpressing mice as a model of CF lung disease, and also from non-CF and CF patients, and were cultured under physiological conditions at air-liquid interface. ASL homeostasis was investigated by confocal fluorescence and reflection microscopy.
Using the fluorescence-based volume challenge protocol, directly after the addition of the fluorescent dye in a volume of 20 µl, ASL height on AECs from wild-type mice determined by confocal reflection microscopy did not differ from values obtained by fluorescence microscopy. Two hours after volume challenge, ASL height was reduced and at 24 hour time point ASL height recovered to normal levels that did not differ between the two techniques. In AECs from βENaC-overexpressing mice, ASL height was similar at early time points, but remained reduced at 24 hour time point. Similar to studies in wild-type AECs, ASL height data did not differ between reflection and fluorescence based measurements. Moreover, continuous measurements of unperturbed steady state measurements using reflection confocal microscopy revealed a stable ASL height measured in both wild-type and ENaC-overexpressing AECs for 5 hours. Similar to comparison studies wild-type mice derived AECs showed higher ASL height than ENaC-overexpressing cells. In addition to murine primary AECs, measurements were performed also with human primary AECs from non-CF and CF patients. Steady state measurements of non-CF patient derived AECs showed a higher ASL height than CF patient derived AECs.With the basolateral addition of benzamil, due to blocked Na+ and liquid absorption ASL height of non-CF AECs was increased, showing that reflection confocal microscopy is capable of measuring even the small changes in ASL height. However, due to lack of functional CFTR channels, ASL height did not change in CF AECs.
In conclusion, these results support that reflection confocal microscopy can be used for accurate measurements of ASL height (dys)regulation without the need of addition of fluorescently labelled dye. This approach may facilitate preclinical evaluation of novel drugs designed to improve airway surface hydration in patients with CF under more physiological steady state conditions.