Abstract
The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8–10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ≤ 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.
Highlights
The cornea is the most highly innervated tissue in the mammalian body [1]
Using serial block-face scanning electron microscopy (SBF-SEM) we were able to image conventional nerve penetration through the epithelial basal lamina, where a stromal nerve bundle containing multiple axons passes through the epithelial basal lamina to form a leash point whereby the nerve bundle gives rise to multiple smaller axonal projections which extend between epithelial cells and give rise to the sub-basal and epithelial plexuses
Nerve bundles involved in fusion contain axons whose plasma membrane is fused and continuous with that of a basal epithelial cell such that the axoplasm comes into direct contact with the cytoplasm of the fused epithelial cell (Fig 3B)
Summary
The cornea is the most highly innervated tissue in the mammalian body [1]. The nerves of the cornea provide autonomic responses such as tearing and blinking and assist in maintaining corneal epithelial homeostasis through the release of trophic factors [2]. Sympathetic innervation comes from nerve fibers originating in the superior cervical ganglion while sensory information is transmitted from the corneal epithelium to cell bodies located in the trigeminal ganglion, [3,4,5,6]. It is well-established that corneal stromal nerves enter the cornea in the peripheral stroma and travel horizontally before branching to give rise to vertical axons that penetrate the epithelial basal lamina [7, 8]. The mechanisms by which corneal nerve patterning is regulated are not well established
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