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Functional Live Cell Imaging of the Pulmonary Neuroepithelial Body Microenvironment

¹ Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium; ² Institute for Risk Assessment Sciences, University of Utrecht and Department of Surgery, UMC Utrecht, The Netherlands; and ³ Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom

Correspondence and requests for reprints should be addressed to Dirk Adriaensen, Ph.D., Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. E-mail: dirk.adriaensen{at}ua.ac.be

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction–linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca²⁺]_i) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K⁺]_o) evoked a fast and reproducible [Ca²⁺]_i increase in NEB cells, while Clara-like cells displayed a delayed (± 4 s) [Ca²⁺]_i increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.

Key Words: mouse • lung • vibratome slices • NEBs • calcium imaging

CLINICAL RELEVANCE A unique functional live cell imaging model is developed for studying the airway neuroepithelial body "microenvironment," which is believed to represent an important lung stem cell niche involved in airway epithelial regeneration and lung carcinogenesis.