Pneumologie 2015; 69 - A15
DOI: 10.1055/s-0035-1556607

Development of Confocal Reflection Microscopy for the Study of Airway Surface Liquid Dysregulation in Cystic Fibrosis

A Seyhan Agircan 1, 2, M Lampe 1, 2, 3, J Duerr 1, 2, R Pepperkok 2, 3, MA Mall 1, 2
  • 1Department of Translational Pulmonology, University of Heidelberg
  • 2Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL)
  • 3European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

In cystic fibrosis(CF) patients and βENaC-overexpressing mice, impaired airway epithelial ion transport produces airway surface liquid(ASL) depletion, leading to mucociliary dysfunction, mucus obstruction, chronic airway inflammation and infection. ASL homeostasis can be studied by measuring ASL height in primary epithelial cultures from murine and human airways. The height can be determined using confocal fluorescence microscopy; however the current protocol requires fluorescently labelled liquid addition to the apical side of the epithelium resulting in nonphysiological 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 is detecting the inherent differences in refractive indices between the permeable membrane, cytosol of cells and liquid layer. Primary airway epithelial cells were prepared from βENaC-overexpressing mice as a CF model and their wild-type littermate controls, and were cultured under physiological conditions at air-liquid interface. ASL homeostasis was investigated by confocal fluorescent and reflection microscopy.

In airway cultures from wild-type mice, following addition of fluorescent dye to the apical compartment, fluorescence microscopy detected an initial ASL height as 30.8 ± 1 µm. At 2h after the volume challenge, the ASL height was reduced to 4.9 ± 0.1 µm and at 24h ASL height was recovered to normal levels (6.3 ± 0.2 µm, n = 10). In cultures from mutant mice, ASL height was similar at early time points however it remained reduced at 24h (4.5 ± 0.2 µm, n = 9 – 10). Initial comparative studies support that reflection measurements are sensitive to detect these changes without the requirement of apical dye/volume addition.

Reflection confocal microscopy has the potential to conduct ASL height measurements under more physiological conditions and avoid addition of fluorescently labelled dye. This approach can be used to study the basis of abnormal regulation and strategies to restore ASL on airway surfaces from mouse models and patients with CF disease.