Carl F. Marfurt

Corneal nerves: structure, contents and function

This review provides a comprehensive analysis of the structure, neurochemical content, and functions of corneal nerves, with special emphasis on human corneal nerves. A revised interpretation of human corneal nerve architecture is presented based on recent observations obtained by in vivo confocal microscopy (IVCM), immunohistochemistry, and ultrastructural analyses of serial-sectioned human corneas. Current data on the neurotransmitter and neuropeptide contents of corneal nerves are discussed, as are the mechanisms by which corneal neurochemicals and associated neurotrophins modulate corneal physiology, homeostasis and wound healing. The results of recent clinical studies of topically applied neuropeptides and neurotrophins to treat neurotrophic keratitis are reviewed. Recommendations for using IVCM to evaluate corneal nerves in health and disease are presented.

Anatomy of the human corneal innervation

The anatomy of the human corneal innervation has been the subject of much investigation; however, a comprehensive description remains elusive. The purpose of the present study was to provide a detailed description of the human corneal innervation using a novel approach involving immunohistochemically stained anterior-cornea whole mounts. Sixteen donor corneas aged 19-78 years were cut with a 6.0 mm trephine into a central plug and two peripheral rims. Each specimen was sectioned serially on a cryostat to produce several 100 microm-thick stromal sections and a 100-140 microm-thick anterior-cornea whole mount that contained the entire corneal epithelium and much of the anterior stroma. The corneal innervation was stained with a primary antibody against beta neurotubulin and subjected to rigorous quantitative and qualitative analyses. The results showed that a mean of 71.3 +/- 14.3, uniformly spaced, main stromal nerve bundles entered the cornea at the corneoscleral limbus. The bundles averaged 20.3 +/- 7.0 microm in diameter, were separated by a mean spacing of 0.49 +/- 0.40 mm, and entered the cornea at a mean distance of 293 +/- 106 microm from the ocular surface. Each stromal bundle gave rise through repetitive branching to a moderately dense midstromal plexus and a dense subepithelial plexus (SEP). The SEP was comprised of modest numbers of straight and curvilinear nerves, most of which penetrated Bowmans membrane to supply the corneal epithelium, and a more abundant and anatomically complex population of tortuous, highly anastomotic nerves that remained largely confined in their distribution to the SEP. SEP density and anatomical complexity varied considerably among corneas and was less dense and patchier in the central cornea. A mean of 204 +/- 58.5 stromal nerves penetrated Bowmans membrane to supply the central 10 mm of corneal epithelium (2.60 nerves/mm(2)). The density of Bowmans membrane penetrations was greater peripherally than centrally. After entering the epithelium, stromal nerves branched into groups of up to twenty subbasal nerve fibers known as epithelial leashes. Leashes in the central and intermediate cornea anastomosed extensively to form a dense, continuous subbasal nerve plexus, while leashes in the peripheral cornea demonstrated fewer anastomoses and were less complex anatomically. Viewed in its entirety, the subbasal nerve plexus formed a gentle, whorl-like assemblage of long curvilinear subbasal fibers, 1.0-8.0 mm in length, that converged on an imaginary seam or gentle spiral (vortex) approximately 2.51 +/- 0.23 mm inferonasal to the corneal apex. Mean subbasal nerve fiber density near the corneal apex was 45.94 +/- 5.20 mm/mm(2) and mean subbasal and interconnecting nerve fiber diameters in the same region were 1.51 +/- 0.74 microm and 0.69 +/- 0.26 microm, respectively. Intraepithelial terminals originated exclusively as branches of subbasal nerves and terminated in all epithelial layers. Nerve terminals in the wing and squamous cell layers were morphologically diverse and ranged in total length from 9 to 780 microm. The suprabasal layers of the central corneal epithelium contained approximately 605.8 terminals/mm(2). The results of this study provide a detailed, comprehensive description of human corneal nerve architecture and density that extends and refines existing accounts. An accurate, detailed model of the normal human corneal innervation may predict or help to understand the consequences of corneal nerve damage during refractive, cataract and other ocular surgeries.

The TFOS International Workshop on Contact Lens Discomfort: Report of the Subcommittee on Neurobiology

This report characterizes the neurobiology of the ocular surface and highlights relevant mechanisms that may underpin contact lens-related discomfort. While there is limited evidence for the mechanisms involved in contact lens-related discomfort, neurobiological mechanisms in dry eye disease, the inflammatory pathway, the effect of hyperosmolarity on ocular surface nociceptors, and subsequent sensory processing of ocular pain and discomfort have been at least partly elucidated and are presented herein to provide insight in this new arena. The stimulus to the ocular surface from a contact lens is likely to be complex and multifactorial, including components of osmolarity, solution effects, desiccation, thermal effects, inflammation, friction, and mechanical stimulation. Sensory input will arise from stimulation of the lid margin, palpebral and bulbar conjunctiva, and the cornea.

Age-Related Changes in Rat Corneal Epithelial Nerve Density

PURPOSE To determine the effect of aging on corneal epithelial nerve density in an animal model. METHODS Corneal whole mounts from rats aged 6, 12, 18, and 24 months were stained immunohistochemically with antisera against the pan-neuronal marker neurotubulin. Epithelial nerve terminals and subbasal nerves in standardized 1-mm(2) central and peripheral zones from each cornea were drawn using a drawing tube attached to a light microscope. Images were scanned, and nerve densities were calculated as the percentage of each 1-mm(2) area occupied by nerves. The diameters of subbasal nerves in 6- and 24-month old animals were measured. Subbasal nerve vortices were analyzed qualitatively with reference to location, morphologic appearance, and directionality. RESULTS Epithelial nerve terminal density decreased by approximately 50% between 6 and 24 months. The rate of decline was roughly linear and similar in both central and peripheral cornea. In contrast, subbasal nerve density increased by more than 50% between 6 and 24 months in both central and peripheral cornea. The mean diameter of corneal subbasal nerves decreased approximately 30% (0.384 microm vs. 0.271 microm) between 6 and 24 months. The morphologic appearance and directionality of the subbasal nerve vortex demonstrated considerable interanimal variability and did not correlate with age. CONCLUSIONS Rat corneal nerve terminal density decreases, but corneal subbasal nerve density increases, as a function of age. The age-related loss of nerve terminal density seen in the rat cornea is in keeping with the decreased corneal sensitivity reported in elderly humans and may contribute to the pathogenesis of dry eye disease in aged persons.

Sympathetic nerve fibers in rat orofacial and cerebral tissues as revealed by the HRP-WGA tracing technique: a light and electron microscopic study

Horseradish peroxidase-wheat germ agglutinin injected into the superior cervical ganglion is taken up by the neuronal perikarya and transported anterogradely in large quantities into peripheral fibers and axonal terminals in a variety of orofacial and cerebral tissues, including the iris, cornea, pineal gland, facial skin and the adventia of cerebral and facial blood vessels. Subsequent histochemical processing according to the tetramethylbenzidine procedure produces a unique, highly visible intraaxonal marker that makes the identification and 3-dimensional tracing of the sympathetic nerve fibers clear and unambiguous at both the light and electron microscopic levels.

Stabilization of tetramethylbenzidine (TMB) reaction product at the electron microscopic level by ammonium molybdate

The ability to use the tetramethylbenzidine (TMB) method for studying neuronal connections at the electron microscopic level is often difficult because the conditions of osmification and dehydration used in processing the tissue may result in significant loss and/or decreased electron density of the reaction product. In the present study, we report that stabilization of TMB reaction product with 5% ammonium molybdate (AM) prior to osmificating the tissue results in the formation of TMB-AM crystals that are many times more electron dense and resistant to ethanol extraction than non-stabilized TMB crystals. The nature of the chemical interaction that underlies the stabilization of TMB by AM is uncertain, but it may involve the formation of an insoluble salt between molybdic ions and the TMB polymer. The use of this simple procedure increases the sensitivity of the TMB procedure at the electron microscopic level and may be used to label neuronal pathways in the peripheral and central nervous systems with equal success.

Uptake and transneuronal transport of horseradish peroxidase-wheat germ agglutinin by tooth pulp primary afferent neurons

Horseradish peroxidase-wheat germ agglutinin (HRP-WGA) applied to proximal stumps of tooth pulp primary afferent neurons in rats was taken up and transported transneuronally to neurons in the ipsilateral trigeminal brainstem nuclear complex. The results of this study suggest that HRP-WGA transport may be a novel means of labeling both primary and higher order neurons that transmit tooth pulp sensory information in the rat and may be used to investigate the fine structure and synaptic contacts of central nervous system neurons that receive tooth pulp afferent input.

Origins of the renal innervation in the primate, macaca fascicularis

The origins of the renal efferent and afferent nerves in 5 cynomolgus monkeys (Macaca fascicularis) were studied by using the retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase-wheat germ agglutinin (HRP-WGA). The cut ends of the right renal nerves were soaked for 30-45 min in solutions consisting of 15% HRP and 1% HRP-WGA. Three or four days later the animals were killed and the tissues examined for the presence of retrogradely labeled neurons, HRP-filled cells were observed, with rare exceptions, only in ganglia ipsilateral to the side of tracer application. Renal efferent neurons (4648-14565 cells per animal) were found in relatively equal numbers in prevertebral and paravertebral (sympathetic chain) ganglia. Labeled prevertebral cells were distributed among the renal (52%), aorticorenal (32%) and superior mesenteric (16%) ganglia, whereas labeled paravertebral neurons were mainly located in chain ganglia T11-L3, with 94% of these located in L1-3. Labeled renal sensory neurons (31-543 per animal) constituted less than 5% of all labeled cells and were found in ipsilateral dorsal root ganglia T10-L3, with (80%) in T12 and L1. The labeled sensory neurons ranged from 18-64 microns in diameter (X = 32.4 microns). With the exception of a single cell in one animal, no labeled neurons were observed in the nodose ganglia. Many parallels were observed between the organization of the renal plexuses of macaques and humans, suggesting the utility of the non-human primate as an experimental model for functional studies of renal innervation in humans.

Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis

OBJECTIVE To describe clinical, in vivo confocal microscopic, histopathologic, and immunohistochemical features of a dog with metaherpetic corneal disease that developed subsequent to a protracted episode of canine herpesvirus-1 (CHV-1) dendritic ulcerative keratitis. CASE DESCRIPTION A 7-year-old, spayed-female, Miniature Schnauzer was treated for bilateral CHV-1 dendritic ulcerative keratitis. Following resolution of ulcerative keratitis, sectoral peripheral superficial corneal gray opacification, vascularization, and pigmentation slowly migrated centripetally to the axial cornea of both eyes. Corneal sensitivity measured with a Cochet-Bonnet esthesiometer was dramatically and persistently reduced. In vivo corneal confocal microscopic examination revealed regions of epithelium with a conjunctival phenotype. In these areas, the surface epithelium was thin, disorganized, and composed of hyper-reflective epithelial cells. Goblet cells and Langerhans cells were frequent, and the subbasal nerve plexus was completely absent or markedly diminished. Histopathologic abnormalities in the globes were restricted to the superficial cornea and included sectoral corneal conjunctivalization, increased anterior stromal spindle cells, and vascularization. Immunohistochemical evaluation of the corneas with anti-neurotublin antibody demonstrated attenuation of the epithelial and subbasal nerve plexuses with marked stromal hyperinnervation and increased numbers of morphologically abnormal neurites. CONCLUSIONS Similar to herpes simplex virus keratitis in humans, CHV-1 ulcerative keratitis may be associated with the development of chronic degenerative corneal disease in dogs. In the described dog, this chronic corneal disease included progressive corneal opacification because of partial limbal stem cell deficiency and neurotrophic keratitis. Long-term monitoring of dogs following resolution of active CHV-1 keratitis may be indicated, particularly when ulcerations persist for an extended period.

Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques: An Alternate Approach to Investigate HIV-Induced PNS Damage

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings.