Functional Live Cell Imaging of the Pulmonary Neuroepithelial Body Microenvironment

Abstract

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.

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells

“…In perinatal and neonatal airways, NEB function is believed to be related to hypoxia-chemosensing and important for respiratory adaptation during transition to air-breathing (Cutz and Jackson, 1999), a time window in which carotid body function -a wellaccepted arterial hypoxia sensor in control of breathing -is still immature (Bollé et al, 2000;Cutz and Jackson, 1999 (De Proost et al, 2008). In this way, we were able to demonstrate that ATP released from activated NEB cells typically gives rise to a delayed activation of Clara-like cells (De Proost et al, 2009;Lembrechts et al, 2012).…”

“…Preparation of lung slices and staining of neuroepithelial bodies Tissue slices were prepared from individual mouse lung lobes (WT C57-Bl6; PD14; n515) and stained for NEBs using 4-Di-2-ASP as previously published (De Proost et al, 2008;Pintelon et al, 2005). In short, lung tissue was stabilized by instillation of a 2% agarose solution (low-melt agarose A4018).…”

“…In many species, the PNECs in NEBs are largely shielded from the airway lumen and the surrounding epithelial cells by a tightjunction-sealed specialized cell type, the Clara-like cells, which together with NEB cells and nerve terminals are referred to as the 'NEB microenvironment' (De Proost et al, 2008). In a previous report, we were able to show the delayed activation of Clara-like cells through P2Y 2 ATP receptors upon NEB stimulation and subsequent ATP release (De Proost et al, 2009).…”

“…Because of the invariably observed close interaction between NEB cells, complex nerve terminals and Clara-like cells, in vitro models for functional studies should preferentially include all components of the NEB microenvironment. For this reason, we developed an ex vivo mouse lung vibratome slice model for confocal live-cell imaging of physiological reactions in the NEB microenvironment and surrounding epithelial cells (De Proost et al, 2008;Pintelon et al, 2005).…”

A Mechanism by Which Propofol Induces Cytotoxicity

“…Mitochondrial membrane potential (∆Ψm) was assessed using 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolocarbocyan ine iodide (JC-1; Molecular Probes, OR, USA) [13,14]. Cells were loaded for 30 minutes with 1 µM JC-1 at 37°C.…”