The confocal scanning microscope was invented in 1955 by Professor Marvin Minsky.1 In 1988, it provided the first images of the epithelial cells, keratocytes, and endothelial cells in the living human cornea.2 In the 1990s, in vivo confocal microscopy (IVCM) was being used to characterize the human corneal nerves, which were referred to as the subbasal nerves. IVCM can be used to diagnose disease in patients presenting with various ocular and nonocular symptoms.3–9Figure 1 shows examples of the corneal nerves from healthy human (Figs. 1A–C) and mouse corneas (Fig. 1D); these images emphasize the sites where stromal nerves enter the epithelium. Although a powerful clinical tool, IVCM is not without limitations, as described by Tervo et al10 and De Silva et al.11 Imaging the corneal epithelial basal axons is relatively straightforward, but the apical extensions cannot be readily visualized. The curved surface of the cornea can also limit resolution, particularly in the periphery. IVCM uses reflected light to obtain images; backscatter from disrupted extracellular matrix can affect the quality of the images obtained. In addition, the cost of the IVCM device is substantial and, unlike optical coherence tomography, it is still not readily available in many outpatient ophthalmology clinics or practices.
To make full use of IVCM as a clinical tool requires an appreciation of the knowledge gained from human and animal studies using fixed tissues stained to reveal the corneal nerves at a high resolution.12–14 In 1983, Rozsa et al15 described reinnervation of the sensory nerves in the rabbit cornea after different types of corneal injury, including incisions and keratectomies. The method used to study the morphology of the nerves was state of the art at that time: gold chloride staining using bright-field microscopy. In images shown of rabbit corneas 60 days after radial keratectomy, Rozsa et al15 referred to disorganized epithelial nerves as “neuromas,” as shown in Figure 2A. Figures 2B–E show the IVCM images we acquired from people with diabetes. Since that time, several authors have used the term “microneuroma” or “neuroma” to describe anomalous nerve features typically associated with corneal injury and disease.6,16,17 Here, we propose that, in some contexts, the terms neuroma and microneuroma may be misleading, given that similar nerve morphologies are evident in healthy corneas, with no reported pathology. In addition, we believe there is a need for a standardized terminology to accurately describe corneal nerve anatomy in healthy corneas and anomalies seen in patients (Table 1).
The online Merriam Webster's Dictionary lists the first use of the term neuroma in 1829 to describe “a tumor growing from a nerve and consisting of nerve fibers.” 18 In medicine, the term is no longer limited to tumors. Although the online medical dictionary19 states that neuroma is a “general term for any neoplasm derived from cells of the nervous system,” it then describes several types of noncancerous neuromas, including acoustic neuromas, amputation neuromas, neuroma cutis, plexiform neuromas, and traumatic neuromas. Traumatic neuromas19 arise from trauma to the nerves and consist of tangles of nerves, Schwann cells, and perineural fibroblasts and are found in the dermis or other interstitial tissues where they can persist and cause pain and discomfort.
In 1982, corneal sensory nerve fibers were believed to be located beneath the corneal epithelial basement membrane.20 They were called the subbasal nerves; the network the nerves assume was referred to as the subbasal nerve plexus. Nerve terminals branched apically from the subbasal nerves.20 Since then, improved imaging procedures have shown that the subbasal nerves actually localize within the basal cell layer and are enclosed by the cell membranes of the corneal epithelial basal cells.13,21 To minimize confusion regarding where these sensory nerves localize within the cornea, in 2017, the term intraepithelial corneal nerves (ICNs) was proposed21 to include the subbasal nerves and nerve terminals. This terminology has yet to be broadly adopted in the literature. Here, we propose that once the sensory nerves enter the epithelium and travel within the basal epithelial cell layer above the basement membrane, they be referred to not as the subbasal nerves but as the intraepithelial corneal basal nerves (ICBNs); the ICBNs branch and extend between the epithelial cells toward the apical squames forming the intraepithelial corneal nerve terminals (ICNTs). Together, the ICBNs and ICNTs comprise the ICNs, as indicated in Table 1.
The sites where the corneal stromal nerves exit the stroma, penetrate the basement membrane, and enter the basal epithelial cell layer have complex morphologies. The stromal nerves, like other nerves of the peripheral nervous system, have 2 types of neural crest–derived cells, namely the Schwann cells and endoneurial fibroblasts, associated with them. The Schwann cells can be of 2 types: myelinating and nonmyelinating. Studies have shown that the myelinating Schwann cells in the cornea are restricted to the periphery12,14; in the central cornea, the nonmyelinating Schwann cells wrap their plasma membranes around each axon. The endoneurial fibroblasts secrete collagen around the axons to insulate individual axons from one another and increase their ability to resist mechanical forces without severing. As the stromal nerves penetrate the epithelial basement membrane, they shed associated Schwann cells, endoneurial fibroblasts, and the matrix proteins they secrete.
Al-Aqaba et al14 have used scanning electron microscopy and transmission electron microscopy to demonstrate the unique anatomical features of the junction between the stromal nerves and ICBNs in human corneas. They observed bulb-like structures; a stromal nerve entered the stromal side of the bulb and a thinner ICBN exited the epithelium. The bulbs did not stain with antibodies against βIII-tubulin or contain the nuclei but were positive for the neuronal enzyme acetylcholinesterase. Based on these observations, the authors concluded that the bulbs contained the cytoplasm and/or matrix surrounding the Schwann cells and/or endoneurial fibroblasts and stromal axons that terminate at the epithelial basement membrane. Other cell types that could contribute to the bulbs are dendritic cells, which have been shown to be present at the sites where stromal nerves enter the epithelium.22 A recent study by Courson et al23 also demonstrated bundles at the sites of stromal nerve penetration into the epithelium in the mouse cornea using serial block-face scanning electron microscopy. The morphology of these sites suggests to the authors that some stromal nerves fuse with the corneal epithelial cells. Before accepting this view, it is necessary to determine the contributions of the other cell types present at these sites, including the nonmyelinating Schwann cells, endoneurial cells, corneal epithelial cells, and immune cells, and the potential involvement of collagen produced by the endoneurial fibroblasts and extracellular matrix in the epithelial basement membrane. Reports from studies of the human4,11,24 cornea indicated more of these sites within the periphery; Al-Aqaba et al24 reported an average of 185 per cornea. To promote consistency in the reporting of corneal anatomical observations in both clinical and basic science literature, we propose a new terminology to describe these nonpathological entry points as the corneal stromal–epithelial nerve penetration sites (CSENPS) (Table 1).
The stromal nerves navigate past the epithelial basement membrane in mice and the epithelial basement membrane and Bowman layer in human and primate corneas. Figure 3A, B shows en face confocal microscopy images of whole flat-mounted mouse corneas immunostained to colocalize βIII-tubulin (axons), sdc3 (Schwann cells), and LN332 (basement membrane) to highlight these CSENPS. The hemidesmosomes that maintain epithelial adhesion to the stroma are displaced. Hemidesmosomes located in the basal cell membrane link α6β4 integrin to LN332 in the basement membrane and type VII collagen in the anterior stroma within the adhesion complexes25; when the stromal nerves push the adhesion complexes aside at the CSENPS, mechanical strain is transferred to the anterior basement membrane and stromal matrix. Syndecan-3 (sdc3) is a heparan sulfate proteoglycan expressed by the glial cells in both central and peripheral nervous systems.26 The mouse stromal nerves have been shown previously to express sdc3.27 Some stromal nerve axons may terminate at the CSENPS, whereas others enter the epithelium. We propose that these factors contribute to the complex morphologies seen at the stromal nerve exit sites and give rise to hyperreflective structures in the IVCM images. A complete understanding of the contribution of stromal matrix deformation and each cell type contributing to the morphology of the CSENPS is not yet possible. It will require additional high-resolution transmission electron microscopy imaging and the ability to define each cell type involved.
Once the axons enter the epithelium and branch, they extend parallel to the basement membrane. These long, extended axon bundles have been called leashes.28–30 There are examples in the literature where the so-called microneuromas8,17 appear similar to the sites of stromal nerve penetration into the basal epithelium in healthy31,32 and diabetic corneas, as shown in Figures 2D–E.
A schematic image showing the CSENPS is shown in Figure 3C. Once the nerve has penetrated the basement membrane, it aligns parallel to the basal surface of the corneal epithelial cells and branches into thinner nerve fibers, referred to as leashes, each of which consists of varying numbers of individual axons. Given the improvements in imaging hardware and platforms, it is anticipated that the CSENPS in the healthy and diseased mammalian corneas will soon be more accurately resolved. Complex as they may be, it is likely that some of the structures being reported in the clinical literature are not pathological microneuromas or neuromas but physiological phenomena, such as the CSENPS.
If, as a scientific community, we can agree to term these features as the CSENPS in healthy corneas, then this raises the question as to what to call the sites where we observe disordered nerves within the corneal epithelium, including those presented in Figure 2. Using terms such as axon tangles, clumps, bulges, varicosities, and hyperreflective sites more accurately describe these structures.33 Standardization of the terminology used to classify corneal epithelial nerve features (Table 1) will lead to more consistent reporting and improved data collection to support an increased understanding of the etiology and potential functional significance of these neural phenomena. Epithelial cell injury and pathologies, including dystrophies, barrier defects, dry eye disease, ocular allergy, and exposure keratopathy, will impair the ability of the corneal epithelial cells to provide support for the ICNs. The relationship between corneal epithelial cells and sensory nerves that innervate them is similar to that between the Schwann cells and other peripheral nerves: they are interdependent. Pathology in one will lead to pathology in the other. When tangles persist over time in the corneal epithelium, but the stromal nerves seem normal, corneal epithelial cell pathology should be considered as a cause. We suggest that the terms microneuroma and neuroma should thus be strictly reserved for pathologies involving verifiable tumors or tangles of the stromal nerves with keratocytes and the Schwann cells.
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