Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

mammals maintain an epithelial stem cell population at the periphery of the cornea at an area called the corneoscleral junction or limbus. At this location, the CESCs are adjacent to a rich environment, often called a niche, which includes both melanocytes that produce melanin and absorb excess light to protect the stem cells from DNA damage, and blood vessels that release growth factors as well as immune cells. The idea that the limbus is a unique and distinct environment is supported by the fact that the composition of the basement membrane beneath the limbal basal cells is unique. It has different relative ratios of collagens and laminins compared to either the conjunctival or corneal basement membrane. Data characterizing the microanatomical relationships among the cells that make up the limbal niche are shown schematically in Figure 3(a). Different integrin heterodimers have been studied as potential markers for the CESCs. Figures 3(b) and 3(c) show the localization of integrins in the human and mouse limbus respectively. One integrin, a9b1, is only expressed in the unwounded adult cornea at the limbus; detailed studies in the mouse using bromodeoxyuridine to study label retaining cells have shown that a9b1 is not expressed on the stem cells themselves but is expressed on the cells that are the immediate progeny of the stem cells, which have been called transit amplifying cells. The CESCs have been shown to express high levels of a6b4- and b1-family integrins.

The Palisades of Vogt

The palisades of Vogt were first described over 90 years ago but were named by Vogt in 1921. They can be seen at the corneal periphery at the limbus by a clinician using an ophthalmoscope as shown in Figures 4(a)–(c). A scanning electron micrograph of the palisades of Vogt is shown in Figure 4(d). The palisades of Vogt consist of ridges of stromal tissue covered by epithelial cells. After noting their loss during progression of certain corneal disease states, recent studies have shown that the palisades of Vogt become increasingly less prominent as the cornea ages and disappear entirely in patients with conditions that are associated with CESC deficiency.

As discussed above and shown in Figure 3, the CESCs reside in the corneoscleral junction. These adult stem cells are small and relatively undifferentiated. Recently, in vivo confocal micrographs taken of human corneas as a function of age from 10to 80-year olds show that the epithelial cells within the palisades of Vogt are smaller than those of the central cornea, that the palisades disappear with aging, and that the sizes of the corneal epithelial cells get larger as the palisades get smaller and disappear. These data are consistent with the hypothesis that the CESCs are located within the palisades of Vogt and that the aging cornea displays a progressive reduction in both

the palisades and the stem-like cells that reside there. While palisades of Vogt have not been reported in nonhuman corneas, it is likely that similar structures are present but are not as morphologically distinct as those in the young human cornea.

Bowman’s Layer Is an Acellular Zone Located Immediately under the Corneal Epithelial Basement Membrane

Bowman’s layer is usually considered the second of the five layers of the cornea. It is a specialized acellular layer immediately beneath the epithelial basal cell basement membrane; in humans it is 12–18-mm thick and it begins at the corneoscleral junction. It is composed of an amorphous collection of collagen fibers intertwined with a rich collection of fine sensory nerve axons that mostly run parallel to the corneal surface. First observed in the chicken cornea, anatomically distinct Bowman’s layers have been widely reported to be present only in avian, primate, and cat corneas. It can be seen in Figure 1(a). Rabbits and mice were thought to lack a Bowman’s layer. More recent studies suggest that Bowman’s layers are found in mammalian, avian, and even fish corneas, but their thickness and composition vary significantly. The function of Bowman’s layer has remained controversial primarily because there are no true markers for Bowman’s layer and the lack of consensus regarding which species possess true Bowman’s layers; a better understanding of what constitutes a Bowman’s layer is necessary before its function can be determined. The most likely function for Bowman’s layer is to enhance corneal strength and/or integrity perhaps by providing a mesh-like environment through which the thicker more uniform anterior stromal collagen lamellae insert into the type VII collagen containing adhesion complex. This insertion would link and stabilize the corneal epithelial basal cell adhesion complex in the anterior corneal stroma. A secondary function for Bowman’s could be to facilitate nerve innervation of the ocular surface by providing a space through which axons can move during development.

The Collagenand Proteoglycan-Rich Corneal Stroma with Its Stromal Cells, Descemet’s Membrane, and the Corneal Endothelial Cells are all Vital to the Health of the Cornea

The third layer of the cornea is the stroma; it is transparent, compact, and fibrous and serves as a structural support. The clarity of the corneal stroma is due to the regularity of the diameter of the various collagen fibers that comprise it and the packing and specialized arrangement of these sheets, called lamellae, relative to one another. The collagen fiber spacing within and between lamellae is regulated by

TDC: Terminal differentiated cells PMC: Post-mitotic cells

l TAC: Late transient amplifying cells eTAC: Early transient amplifying cells

SC: Stem cells

MC: Mesenchymal cells Bo: Bowman’s membrane

α6

β1

proteoglycans, including lumican, decorin, and bilycan. The proteoglycans bind and organize water molecules and constrain the spacing of the collagen fibers. The cells that produce and maintain the collagens and proteoglycans of the stromal matrix are mesenchymal cells of neural crest origin and are frequently referred to as corneal keratocytes. During wound healing, these cells become activated to become myofibroblasts and they function to facilitate wound closure.

Between the stroma and the corneal endothelial cells there is a specialized homogenous basement membrane known as Descemet’s membrane. The single layers of flattened cells that make up the corneal endothelium are primarily responsible for secreting the extracellular matrix proteins that make up Descemet’s membrane. They are absolutely essential for a healthy cornea. The corneal endothelial cells have numerous channel proteins in their plasma membrane that pump ions and excess water in and out of the cornea to maintain its hydration state. The clarity of the corneal stroma is readily lost if the stroma begins to dry out or desiccate. Dessication disrupts the organization of the collagen molecules by removing the ordered water molecules associated with the proteoglycans. The endothelial

cells also pump nutrients from the aqueous humor into the corneal stroma. Nutrients diffuse across Descemet’s membrane and then passively diffuse outward to nourish the corneal stromal and epithelial cells. Because the cornea is avascular, it requires the corneal endothelial cells to provide its nourishment. It is likely that Descemet’s membrane acts as a sponge to trap nutrients delivered by the corneal endothelial cells. This would provide a sustained release of those nutrients to the rest of the cornea. In the healthy cornea, the corneal endothelial cells are more metabolically active compared to the corneal epithelium and corneal stromal cells. The generation of nutrient and ion gradients is energy dependent and these cells are constantly synthesizing channel and ion pump proteins. In contrast, in the unwounded cornea, the corneal stromal cells and the corneal epithelial cells are fairly quiescent and primarily utilize glycolysis to generate energy.

The Avascular Cornea

The healthy central cornea is avascular to permit light to fall on the retina without distortion; blood and

lymphatic vessels are present only at the corneal periphery at the limbus. Developing a better understanding of the mechanisms underlying the maintenance corneal avascularity is of extreme importance to cell biologists and many excellent studies have made progress in this area. We now know that the avascular status of the healthy cornea is maintained by the production by the corneal epithelial and stromal cells of several antiangiogenic factors, including angiostatin and endostatin. After corneal injury, corneal cells transiently produce angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF) disrupting the normal balance of proand anti-angiogenic factors and allowing for the ingrowth of blood vessels from the limbus. After healing is complete, these transient vessels undergo pruning and the avascular nature of the cornea is restored. However, prolonged inflammation or recurrent epithelial erosions can compromise corneal avascularity.

The clarity of the cornea and its accessibility has given rise to several widely used assays to assess the mechanisms underlying angiogenesis. One is called the corneal pocket assay. This assay has been used by researchers to identify factors that inhibit new blood vessel growth primarily as a tool to learn how to inhibit the growth of tumor cells in various different types of cancer. A small pellet is inserted into an incision made on the cornea surface. If the pellet contains pro-angiogenic growth factors such as FGF or VEGF, blood vessels will sprout from the limbus and grow toward the pellet. The number of vessels that form and the speed with which they move toward the pellet can be quantified, and drugs that inhibit or enhance angiogenesis can be tested. A second procedure used for similar types of studies of angiogenesis involves placing a suture on the cornea. If the suture is placed at a regular distance from the limbus then the ingrowth of vessels from the limbal vasculature can be measured and the ability of various drugs to enhance or inhibit angiogenesis can also be assessed.

See also: Artificial Cornea; Conjunctival Goblet Cells; Contact Lenses; Corneal Dystrophies; Corneal Endothelium: Overview; Corneal Epithelium: Response to Infection; Corneal Epithelium: Transport and Permeability;

Corneal Epithelium: Wound Healing Junctions, Attachment to Stroma Receptors, Matrix Metalloproteinases, Intracellular Communications; Corneal Imaging: Clinical; Corneal Nerves: Anatomy; Corneal Nerves: Function; Corneal Scars; The Corneal Stroma; Cornea Overview; Defense Mechanisms of Tears and Ocular Surface; Dry Eye: An Immune-Based Inflammation; Imaging of the Cornea; Ocular Mucins; Refractive Surgery and Inlays; Regulation of Corneal Endothelial Cell Proliferation; Regulation of Corneal Endothelial Function; Stem Cells of the Ocular Surface; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.

Further Reading

Argu¨eso, P., Balaram, M., Spurr-Michaud, S., et al. (2002). Decreased levels of the goblet cell mucin MUC5AC in tears of patients with Sjo¨gren syndrome. Investigative Ophthalmology and Visual Science

43: 1004–1011.

Dupps, W. J., Jr. and Wilson, S. E. (2006). Biomechanics and wound healing in the cornea. Experimental Eye Research 83: 709–720.

Gipson, I. K. (2007). The ocular surface: The challenge to enable and protect vision: The Friedenwald lecture. Investigative Ophthalmology and Visual. Science 48: 4390–4398.

Li, W., Hayashida, Y., Chen, Y. T., and Tseng, S. C. (2007). Niche regulation of corneal epithelial stem cells at the limbus. Cell Research 17: 26–36.

Mu¨ller, L. J., Marfurt, C. F., Kruse, F., and Tervo, T. M. (2003). Corneal nerves: Structure, contents and function. Experimental Eye Research 76: 521–542.

Pajoohesh-Ganji, A., Pal-Ghosh, S., Simmens, S. J., and Stepp, M. A. (2006). Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse. Stem Cells 24: 1075–1086.

Ruberti, J. W. and Zieske, J. D. (2008). Prelude to corneal tissue engineering – gaining control of collagen organization. Progress in Retinal and Eye Research 27: 549–577.

Sakimoto, T., Rosenblatt, M. I., and Azar, D. T. (2006). Laser eye surgery for refractive errors. Lancet 367: 1432–1447.

Stepp, M. A. (2006). Corneal integrins and their functions. Experimental Eye Research 83: 3–15.

Stepp, M. A. and Zieske, J. D. (2005). The corneal epithelial stem cell niche. Ocular Surface 3: 15–26.

Taneri, S., Zieske, J. D., and Azar, D. T. (2005). Evolution, techniques, clinical outcomes, and pathophysiology of LASEK: Review of the literature. Survey of Ophthalmology 49: 576–602.

Wilson, S. E., Chaurasia, S. S., and Medeiros, F. W. (2007). Apoptosis in the initiation, modulation and termination of the corneal wound healing response. Experimental Eye Research 85: 305–311.

Glossary

Chemotropic guidance – A process whereby a chemical substance, usually secreted by some target cell, attracts growing axons.

Cochet–Bonnet esthesiometer – A hand-held instrument that uses the pressure transmitted by a nylon monofilament of known diameter and variable length to measure the mechanical sensitivity of the cornea.

Iridectomy – A surgical procedure that removes a small piece of the iris, most often for

treatment of closed-angle glaucoma or melanoma of the iris.

Laser-assisted in situ keratomileusis (LASIK) –

A form of refractive laser eye surgery designed to change the shape of the cornea.

Neurite – Any cytoplasmic extension from a neuron; the term is used frequently to describe developing or regenerating axons that have not yet attained their mature adult form.

Neurotrophic epitheliopathy – The minor degenerative changes of the corneal epithelium caused by damage or functional impairment of the corneal innervation, especially as occurs after LASIK surgery.

Neurotrophic keratitis – A serious degenerative condition of the cornea, affecting especially the corneal epithelium, caused by impairment of

the corneal sensory innervation. It often manifests as corneal epithelial defects, ulceration, melting, and diminished wound healing.

Phacoemulsification – A procedure in which a cataractous lens is broken up (emulsified) by ultrasound and aspirated from the eye; an intraocular lens is then inserted.

Receptive field – The area of the cornea

(or other body part) supplied by a single sensory nerve fiber.

Trabeculectomy – A surgical procedure that removes part of the trabeculum of the eye in order to facilitate drainage of aqueous humor. It is performed for the relief of increased intraocular pressure associated with glaucoma.

Trophic substances – The molecules that promote the growth, differentiation, and survival of specific cell populations.

Origins of Corneal Nerves

The cornea is richly supplied by sensory neurons located in the ophthalmic region of the trigeminal ganglion (Figure 1). Although the entire sensory innervation of the mammalian cornea is derived from a relatively small number ( 50–450) of neurons, each neuron supports as many as 200–3000 individual corneal nerve endings. The sensory nerves reach the eye through the nasociliary branch of the ophthalmic nerve. In humans, the nasociliary nerve branches typically into two long ciliary nerves, one nasal and the other temporal, which course directly to the posterior pole of the eye, and a communicating branch carrying sensory fibers to the ciliary ganglion. Five to ten short ciliary nerves, carrying a mixture of sensory and autonomic fibers, emerge from the anterior pole of the ciliary ganglion and together with the long ciliary nerves pierce the posterior globe in the vicinity of the optic nerve. After penetrating the sclera, the nerves enter the suprachoroidal space and course anteriorly toward the cornea. While in transit to the anterior eye segment, the fibers branch repeatedly and eventually form a series of prominent, evenly spaced nerve bundles that approach the corneoscleral limbus uniformly from all directions.

Some mammalian corneas also receive a modest sympathetic innervation from the superior cervical ganglion; however, the existence and magnitude of this contribution vary widely among species. In rabbits and cats, corneal sympathetic nerves may constitute as much as 10–15% of the total corneal innervation, while in humans and other primates corneal sympathetic fibers are exceedingly rare or absent. Avery sparse parasympathetic innervation has been reported in rat and cat corneas; however, the meager nature of this innervation and its absence from most mammalian corneas suggest that they are likely of little physiologic significance.

Architecture of the Corneal Innervation

Limbal Plexus

Prior to entering the cornea, the nerve bundles traverse the limbus and contribute fibers to the limbal, or pericorneal plexus, a dense, ring-like meshwork of nerve fibers that completely surrounds the peripheral cornea. The three-dimensional structure of the limbal plexus varies considerably in anatomical complexity according to species and contains complex mixtures of sensory, sympathetic, and parasympathetic nerves. Most limbal

nerves supply functional innervation to the limbal vasculature; however, occasional fibers travel through the limbal stroma unrelated to blood vessels and may provide sensory or autonomic innervation to resident cells.

Stromal and Subepithelial Plexuses

Most nerves enter the peripheral cornea in a series of radially directed major stromal nerve bundles, each containing as many as several dozen axons, arranged at regular intervals around the limbal circumference. On average, 60–80 major stromal nerves supply innervation

to the human cornea, while 12–20 bundles supply rabbit, cat, and dog corneas. Additional numbers of smaller nerve fascicles enter the peripheral cornea slightly superficial to the main stromal bundles and provide limited innervation to the perilimbal and peripheral cornea. At their point of entry in the peripheral cornea, most corneal nerves are unmyelinated C-fibers; however, perhaps as many as 30% are finely myelinated A-delta fibers that shed their myelin sheaths within a millimeter or so after entering the cornea. The axons then continue into the stroma as flattened, ribbon-like fascicles surrounded only by Schwann cell cytoplasm and basal lamina.

Soon after entering the peripheral cornea, the major stromal bundles branch repeatedly to form elaborate, tree-like arbors whose distal branches anastomose extensively with neighboring branches to form a dense anterior stromal plexus. The plexus occupies approximately the anterior 25–50% of the corneal stroma, depending on the species, and consists of complex admixtures of seemingly randomly organized small and medium-sized nerve bundles and individual axons with straight, curvilinear, and tortuous trajectories. The plexus becomes increasingly denser and anatomically complex in the anterior direction. The most superficial layer of the anterior stromal plexus in humans and other large mammals is especially dense and is referred to as the subepithelial plexus (SEP). In contrast, the posterior half of the corneal stroma and the endothelium are, with rare exceptions, devoid of nerve fibers.

A small but indeterminate number of nerve fibers in the anterior stromal and SEPs appear to terminate in the stroma, usually as slightly bulbous terminal swellings or free nerve endings. At the electron microscopic level, the stromal fibers and their terminals resemble morphologically free nerve endings found in the corneal epithelium, suggesting that stromal nerve endings may subserve undetermined sensory transduction functions. Some stromal fibers form intimate relationships with keratocytes and are occasionally enwrapped in keratocyte cytoplasmic extensions that may provide a morphological substrate for reciprocal functional interactions.

Individual stromal axons entering at the corneoscleral limbus may travel as much as three-quarters of the way across the cornea before ending and, as a result of repetitive branching, possess receptive fields that range in size from less than 1 mm2 to as much as 50 or more square millimeters and may cover up to 20% or more of the corneal surface. The receptive fields are generally round, oval, or wedge-shaped in outline and often extend several millimeters beyond the cornea onto the adjacent limbus and bulbar conjunctiva. The large sizes and extensive overlap between adjacent receptive fields, coupled with convergent mechanisms in the central nervous system, explain why stimulation of the corneal surface is poorly localized.

Subbasal Nerve Plexus

The subbasal nerve plexus comprises the densest part of the corneal innervation and is so-named because the nerves that comprise this plexus travel in the subnuclear part of the basal epithelium or between the basal epithelial cells and their basal laminae. Estimates of subbasal nerve fiber density in the central human cornea are expressed conventionally as the total length of all nerve fibers and branches per square millimeter, and range from

15–27 mm mm–2 (by in vivo confocal microscopy) to 40–55 mm mm–2 (by immunohistochemical staining of corneal whole mounts).

Approximately 400–500 widely spaced stromal nerves penetrate Bowman’s membrane, mainly in the peripheral and intermediate cornea, to give origin to the human subbasal plexus. Additional subbasal nerves enter the peripheral cornea directly from the limbal plexus. Relatively few stromal nerves penetrate Bowman’s membrane in the central 2 mm of the human cornea; hence, the latter region receives most of its epithelial innervation from long subbasal nerves that invade the central cornea from more peripheral origins.

Immediately after penetrating Bowman’s membrane, each stromal nerve bends at a 90 angle and branches simultaneously into 2–20 thinner nerve fascicles termed subbasal nerves. Subbasal nerves course in the horizontal plane, roughly parallel to one another and to the epithelial basal lamina, for 1–2 mm in rats and up to 6 mm or more in humans. The neuroanatomical structure thus formed by a stromal axon branching into multiple, parallel subbasal daughter fibers is termed an epithelial leash, a unique, two-dimensional sensory nerve specialization found only in the cornea (Figure 2). At the electron microscopic level, human subbasal nerves average 1.5 mm in diameter and may contain up to 40 individual unmyelinated axons (Figure 3). Immediately upon entering the basal epithelium, the axons shed their Schwann cell investments and continue as naked axon cylinders. Subbasal nerves in adjacent leashes interconnect frequently with one another. As a result, the boundaries between individual leashes are soon lost and a composite subbasal nerve plexus is formed.

When viewed in its entirety, the subbasal nerve plexus forms a gentle spiral or whorl-like pattern on the curved corneal surface (Figure 4). The center of the spiral, often called the vortex, is located in human corneas approximately 2–3 mm inferior and nasal to the corneal apex. As a consequence of this arrangement, subbasal nerves in the superior and apical human cornea are oriented vertically, whereas subbasal nerves in other corneal regions may be oriented horizontally or obliquely, consistent with their geographical locations within the whorl-like plexus. The mechanisms that govern the formation and maintenance of this spiral-like pattern remain uncertain; however, it has been established that basal epithelial cells and subbasal nerves migrate centripetally in tandem. According to one theory, basal epithelial cells derived from stem cells in the corneoscleral limbus migrate centripetally in a whorl-like fashion toward the corneal apex in response to chemotrophic guidance, electromagnetic cues, and population pressures; the subbasal nerves, occupying cytoplasmic invaginations or narrow intercellular spaces between adjacent columns of migrating cells, are pulled along and undergo compensatory horizontal elongation. Alternatively, subbasal nerves may develop whorl-like,

Figure 3 Electron micrograph of a subbasal nerve from a human cornea. The nerve, cut in cross section, contains eight individual unmyelinated axons. M, mitochondria. Calibration bar is 1 mm. Reproduced from figure 5c in Muller, L. J., et al. (2003). Corneal nerves: Structure, contents and function. Experimental Eye Research 76: 521–542. With kind permission from Elsevier.

curvilinear orientations independent of epithelial cell dynamics and may, in turn, provide a structural scaffold that patterns and directs epithelial cell migration.

Intraepithelial Nerve Terminals

As the subbasal nerves course horizontally through the basal epithelium, they give rise to a profusion of thin,

Superior

Nasal

Figure 4 Architecture of the subbasal nerve plexus in the human cornea. The area illustrated is 6.5 mm in diameter and centered on the corneal apex. Subbasal nerves converge in a gentle, whorl-like pattern on a region approximately 2–3 mm inferonasal to the corneal apex known as the vortex.

occasionally beaded terminal axons that ascend vertically or obliquely, often with a modest amount of additional branching, into the more superficial epithelial layers before ending (Figure 5). In some mammalian corneas,

additional epithelial nerves originate as single axons directly from the SEP. The end of each intraepithelial fiber is tipped by a conspicuous, bulbous terminal expansion. At the ultrastructural level, these expansions contain abundant small clear vesicles, varying numbers of large dense-cored vesicles, mitochondria, glycogen particles, neurofilaments, and neurotubules. Morphologically, they resemble nociceptor nerve endings described in other tissues. The neurochemical content of the small clear vesicles is uncertain but may include excitatory amino acids, while the large, dense-cored vesicles likely contain substance P (SP), calcitonin gene-related peptide (CGRP), and/or other neuropeptides.

The terminals are located throughout all layers of the corneal epithelium but are especially numerous in the basal and wing cell layers. Some terminals may extend to within a few microns of the corneal surface. The epithelial cell membranes facing the nerve terminals often demonstrate numerous invaginations and, in many cases, the epithelial cell cytoplasm totally surrounds the nerve ending. The intimate morphological relationship thus formed between nerve terminal and epithelial cell does not constitute a true synapse, but may nevertheless provide a favorable environment for the exchange of diffusible substances and receptor-mediated interactions. The intimate contacts may also allow nerve endings to sense osmotic changes in epithelial cell shape and volume brought about by dessication of the ocular surface. When osmotic stimulation reaches threshold, the nerve fibers fire and activate brainstem circuits that promote reflex tear production and blinking in an effort to maintain the physiologic integrity of the ocular surface.

The innervation density of the corneal epithelium is probably the highest of any surface epithelium, and the central corneal epithelium of humans and rabbits contains approximately 5000–8000 nerve terminals per square millimeter. Both nerve terminal density and corneal sensitivity are greatest in the central cornea and decrease progressively in the peripheral direction. The richness of the corneal epithelial innervation provides a nociceptive detection system of unparalleled sensitivity and it is hypothesized that injuries to individual epithelial cells may be sufficient to trigger pain perception. Both corneal sensitivity and nerve density decrease progressively as a function of age and may contribute to the pathogenesis of dry eye disease in some elderly patients.

Corneal Nerve Neurochemistry

Corneal sensory nerves help maintain a healthy ocular surface by activating brainstem circuits that stimulate reflex tear production and blinking, and by releasing trophic substances, including numerous neuropeptides, which promote corneal epithelial physiologic renewal and wound repair. Corneal nerves contain, in varying proportions, the same neuropeptides that are expressed in other ocular nerves. Each corneal fiber population (sensory, sympathetic, and parasympathetic) maintains a distinctive phenotypic signature; however, the chemical coding is complex and most fibers likely express combinations of neuropeptides rather than individual markers. To date, 12 different neuropeptides have been detected by radioimmunoassay or immunohistochemistry in the mammalian cornea.

Corneal sensory nerves may contain one or more of six different neuropeptides. Two of these peptides, CGRP and SP, are found in especially high percentages of corneal nerves and remain the most studied and well characterized of the corneal peptidergic nerves (Figure 6).

CGRP and SP are expressed in approximately 30–60% and 10–20% of corneal sensory nerves, respectively, and have been found in all mammalian corneas investigated to date. The vast majority of nerves that contains CGRP also contain SP and the two peptides probably co-localize in the same vesicles. Other neuropeptides expressed in more limited numbers of corneal sensory nerves include: neurokinin A (a member of the tachykinin family) secretoneurin (a member of the chromogranin/secretogranin family), pituitary adenylate cyclase-activating peptide (PACAP, a member of the vasoactive intestinal polypeptide (VIP)-glucagon-secretin super family), and galanin. The extent to which these peptides coexist with CGRP and SP or represent distinct populations of corneal sensory nerves remains to be determined.

Despite the richness of the corneal peptidergic innervation, it does not fully account for the known density of the corneal sensory innervation and it is therefore likely that many corneal sensory nerves do not express neuropeptides. Immunohistochemical investigations of rat corneal sensory nerves suggest that up to 40% of rodent corneal nerves do not express known neuropeptides. Most of the so-called nonpeptidergic nerves express a cell-surface glycoconjugate that binds the plant isolectin Bandaireae simplicifolia IB4 and contain the enzyme, fluoride-resistant acid phosphatase (FRAP). The neurotransmitters expressed by this prominent population of corneal IB4-positive nerves remain to be determined; however, excitatory amino acids such as aspartame and glutamate, or unknown neuropeptides that remain to be characterized, are likely candidates.

Corneal autonomic nerves, which constitute in most species only a small percentage of corneal nerve fibers, are also neurochemically diverse. Corneal sympathetic nerves express (in addition to noradrenalin) serotonin and neuropeptide Y (NPY), while corneal parasympathetic nerves express VIP, met-enkephalin, NPY, and galanin.

Functional roles of most neuropeptides in the mammalian cornea remain unclear and knowledge of their physiological roles has not kept pace with the results of immunohistochemical analyses. An important exception to this statement is SP, which exerts essential trophic functions on the corneal epithelium. SP receptors are present on corneal and limbal epithelial cells and it is thought that, under resting physiologic conditions, SP released from corneal sensory nerve terminals promotes corneal epithelial maintenance and physiological renewal by activating cellular pathways that stimulate epithelial cell proliferation, migration, and adhesion. In addition, topical application of SP has been reported to accelerate the rate of corneal epithelial wound healing in both experimental animal models and clinical patients with persistent corneal epithelial defects. Damage to corneal sensory nerves by surgery or trauma deprives the corneal epithelium of SP and other essential nerve-derived trophic substances and is associated with a variety of ocular

surface disorders, including dry eye disease, epitheliopathy, and neurotrophic keratitis.

Even less is known of the functional roles of the cornea’s limited autonomic innervation. In animal models, corneal sympathetic nerves act through adrenergic mechanisms to modulate corneal epithelial cell ion transport processes, epithelial cell proliferation and mitosis, and cell migration during epithelial wound healing. In the corneoscleral limbus, NPY augments the vasoconstrictor effects of noradrenalin and exerts strong angiogenic effects.

Corneal Nerve Remodeling

Corneal subbasal nerves and their intraepithelial terminals are dynamic structures that undergo morphological rearrangements continuously under normal physiologic conditions. Time-lapse, in vivo confocal microscopic examination of living human eyes reveals that subbasal nerves slide centripetally in tandem with their neighboring basal epithelial cells at rates of 10–20 mm day–1 and that this nerve elongation occurs through the addition of new nerve material near the site of nerve penetration at Bowman’s membrane. As subbasal nerves cannot elongate indefinitely, it is hypothesized that the distal nerve segments eventually degenerate or slough into the tear film. Intraepithelial nerve terminals also undergo continuous remodeling through combinations of long-term, nervedirected reconfigurations and passive, short-term reorganization in response to outward migrations of differentiating epithelial cells.

Corneal Nerve Regeneration after Ocular Surgery

Corneal nerves are transected during a variety of corneal and anterior-segment surgical procedures, including refractive surgery, perlimbal incisions performed for cataract surgery, iridectomy and trabeculectomy, and penetrating keratoplasty (PK; corneal transplantation). Corneal nerves depend for their survival on axoplasmic transport of essential substances from their parent nerve cell bodies in the trigeminal ganglion; thus, surgical procedures that interrupt corneal nerve fibers cause rapid degeneration of the distal axons, decreased corneal sensitivity, and compromised functional integrity of the ocular surface.

Corneal nerves are capable of regeneration; however, it is a slow, imperfect process and the regeneration that takes place after most corneal surgeries is characterized by reduced nerve density, alterations in nerve architecture, and diminished corneal sensitivity. The more proximally the nerves are cut, the more delayed and incomplete the regeneration process will be. Thus, surgical disruption of