Neuron-Epithelial Cell Fusion in Mouse Cornea Revealed Using Anterograde Labeling

Abstract

Purpose : The general understanding of corneal innervation is that stromal nerves penetrate the epithelial basal lamina and innervate the epithelium. Using serial block-face scanning electron microscopy (SBF-SEM), we found evidence of a novel alternative neuron-epithelial interaction whereby 42.8% of central corneal neurons fuse with basal epithelial cells. While SBF-SEM provides excellent ultrastructural data, neuron-epithelial fusion is only visible at high magnification and consequently, limiting the field of view to microns. Light microscopic detection of fusion events using fluorescence-labeling would allow the entire surface of the cornea to be examined (8mm²). DiI is a commonly used membrane tracer that can only pass from one cell to another if they are joined by a shared plasma membrane. The purpose of this study was to determine if the fluorescent membrane tracer DiI could be used to identify sites of neuron-epithelial cell fusion in the mouse cornea.

Methods : C57BL/6J mice were euthanized and dissected to reveal the trigeminal ganglia. DiI was applied directly to one trigeminal ganglion, the head placed in 2% paraformaldehyde, and subjected to 28 hours of microwave-assisted labeling, after which the eyes were enucleated and corneal flat-mounts prepared. Images were taken on a DeltaVision epifluorescence microscope. Fusion was defined as a DiI labeled stromal nerve terminating at a basal epithelial cell where the DiI fluorescence extended from the neuron and was continuous with the epithelial cell.

Results : The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion. Consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. Fusion events were more common in the central and mid-peripheral regions of the cornea with approximately 3-5% of the basal epithelial cells being labeled. DiI labeling in the peripheral paralimbal region was much less (0.33%).

Conclusions : The data show DiI identified sites of neuron-epithelial cell fusion. DiI serves as a neuronal tracer because it diffuses along the plasma membrane. DiI cannot pass from the neuron to another cell unless their plasma membranes are contiguous and this only occurs at sites of neuronal-epithelial fusion. This technique makes it possible to study neuron-epithelial cell fusion at the level of the light microscope.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.