Serial block-face scanning electron microscopy reveals neuronal-epithelial cell fusion in the mouse cornea

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.

Transient Ascaris suum larval migration induces intractable chronic pulmonary disease and anemia in mice

“…Mouse lungs were processed for serial block-face scanning electron microscopy (SBF-SEM) as described previously in detail[ 57 ]. Briefly, excised lungs were inflated through the trachea with fixative (0.1M sodium cacodylate buffer containing 2.5% glutaraldehyde), post-stained with heavy metals (Fe, OsO4, uranyl acetate, lead) before dehydration through an acetone series and embedding in Embed 812 resin (Embed-812, Electron Microscopy Sciences, Hatsfield, PA) containing Ketjen Black (Ketjenblack EC600JD, Lion Specialty Chemicals Co., Tokyo).…”

BCLA CLEAR - Anatomy and physiology of the anterior eye

“…Corneal epithelial nerve fibres travel primarily in infoldings of the basal cell basal plasmalemma [62,65]; this is also where most epithelial nerve endings are located. At intervals, the epithelial nerve fibres ascend between cells to the squamous cell layers, where they terminate at multiple levels within the corneal epithelium (Fig.…”

Microvascular thrombosis: experimental and clinical implications

“…Fig 3 demonstrates the 3dimensional ultrastructure of a microvascular thrombus generated by our group using the photochemical injury model in a mouse cremaster venule (procedures approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine), using the relatively new imaging technique of serial block-face scanning electron microscopy. 116 Many of the experimental models of microvascular thrombosis described above result in platelet-rich thrombi, often with little evidence of fibrin by electron microscopy. 78 As described below, the structure of thrombi in clinical conditions associated with microvascular thrombosis varies considerably; for example, platelet-rich thrombi predominate in thrombotic thrombocytopenic purpura and fibrin-rich thrombi in hemolytic-uremic syndrome and DIC.…”