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

the subbasal and subepithelial nerve plexuses produce, in general, less serious and more short-term damage to the corneal innervation than do deep or penetrating incisions that affect major stromal nerve bundles.

Refractive Surgery

In laser-assisted in situ keratomileusis (LASIK), a mechanical or femtosecond laser microkeratome is used to create a circular flap of corneal epithelium and superficial stroma with a hinge at one end; the flap is then folded back and an excimer laser is used to remove several tens of microns of stroma from beneath the flap. The microtome severs subbasal and subepithelial nerves all around the flap margin except at the hinge, and the excimer laser destroys additional nerves in the anterior stromal bed. Regenerating nerve fibers emerge first as neurites from the subbasal plexus at the hinge and, to a lesser extent, elsewhere around the flap margin, followed by a second generation of neurites originating from the transected stromal nerve trunks. Abnormal proteoglycans present at the LASIK scar interface between the stromal bed and the overlying flap are postulated to impede successful reinnervation from the anterior stromal plexus. Regeneration is slow and incomplete, with subbasal and stromal nerve densities returning to less than half of preoperative levels by 12 months after LASIK and failing to reach preoperative levels even 3–5 years later. In addition, the architecture and morphology of the regenerated subbasal nerve plexus are often abnormal. Long-term studies are needed to determine whether subbasal nerve density will eventually return to pre-LASIK levels.

Although corneal reinnervation after LASIK surgery is incomplete, the corneal sensitivity, as determined by Cochet–Bonnet esthesiometry, in most cases returns to preoperative levels by 6–12 months and the short-term clinical outcome in most patients is excellent. Most patients experience a decrease in corneal sensitivity and mild-to-moderate dry eye after LASIK surgery that lasts for only a few days; however, approximately 5% of LASIK patients suffer long-term dry eye symptoms that may be related at least in part to impaired corneal reinnervation and interruption of the neural circuits that drive reflex tear production. Some patients with chronic dry eye after LASIK experience persistent and aberrant pain sensations that likely reflect sensitization and altered responsiveness of the immature, regenerating nerves.

In photorefractive keratectomy (PRK), the corneal epithelium is removed and an excimer laser is used to ablate the most anterior portion of the corneal stroma. Nerve regeneration and recovery of corneal sensitivity under these conditions, in which a flap is not cut in the cornea, reportedly occurs more quickly than following LASIK surgery; nevertheless, subbasal nerve density, architecture, and corneal sensitivity remain depressed for up to 1–2 years postoperatively.

Cataract Surgery

Penetrating perilimbal incisions, such as those performed for cataract surgery, are curvilinear incisions that are a few millimeters in length, which transect small numbers of major stromal nerve trunks near their sites of entry into the cornea. Neural regeneration proceeds slowly through a combination of nerve regrowth and collateral sprouting from adjacent, uninjured stromal nerves. The success of nerve regeneration varies from individual to individual; some nerve trunks fail to regenerate and, the ones that do regenerate, often contain diminished numbers of axons or form tangled masses of disorganized nerves in previously denervated areas of the stroma. Since the advent of phacoemulsification, the incision sizes used in cataract surgery have decreased steadily to as little as 1 mm and the risk of significant injury to the corneal innervation has been minimized.

Penetrating Keratoplasty

PK requires a full-thickness, 360 corneal incision that cuts all corneal nerves and results in complete denervation of the transplanted cornea. Nerve regeneration proceeds from the peripheral recipient cornea into the donor cornea very slowly and, even many years later, the innervation density of the grafted tissue remains far less than that of the host, peripheral cornea (Figure 7). Stromal nerves, in particular, regenerate very poorly after PK, perhaps because when the graft is introduced Schwann cell channels in the donor cornea are misaligned with the stromal nerve stumps in the host cornea. This contrasts to the perlimbal incisions used in cataract surgery, where nerve trunks and Schwann cell channels on opposing sides of the incision remain closely aligned. The limited nerve regeneration that does take place following PK derives mostly from small numbers of subbasal nerve fibers that elongate through epithelial bridges at the graft margin to enter directly into the donor basal epithelium. Many of the regenerated subbasal nerves are shorter than normal and exhibit atypical orientations and morphologies. Even several decades after surgery, median subbasal nerve density and corneal sensitivity in clear grafts remain significantly reduced compared to healthy corneas, and in about one-half of cases no subbasal nerves are visible by routine in vivo confocal microscopy. The return of sensation to the donor tissue is highly variable and, in many cases, hypoesthesia persists for many years after initial surgery. Fortunately, a return to normal innervation levels is not necessary for corneal clarity. Transplanted corneas can have severe nerve deficits and hypoesthesia for many years but remain clear; nevertheless, it is theorized that the impaired sensory innervation after PK may contribute to the relatively high frequency of epithelial complications observed after this procedure.

Mechanisms of Nerve Regrowth

The mechanisms that stimulate and direct neurite outgrowth from injured and intact areas of the corneal innervation following local nerve injury are incompletely understood and, in most cases, these mechanisms are unable to restore the corneal innervation completely to its preoperative density, architecture, and sensory function. Growth factors expressed by corneal epithelial cells and released at the wound site following injury may play important roles. Nerve growth factor (NGF), a small protein that promotes the growth, survival, and maintenance of peripheral sensory nerve fibers, is constitutively expressed in corneal epithelial cells and is upregulated after epithelial wounding. Topical NGF administration stimulates corneal nerve regeneration and recovery of corneal sensitivity after PRK and LASIK in rabbits, and promotes healing of neurotrophic ulcers in clinical patients. Development of additional molecules that promote regeneration of injured nerves and restore the functional integrity of the ocular surface after corneal surgery is needed.

See also: Cornea Overview; Corneal Nerves: Function; Refractive Surgery; Refractive Surgery and Inlays.

Further Reading

Auran, J. D., Koester, C. J., Kleiman, N. J., et al. (1995). Scanning slit confocal microscopic observation of cell morphology and

movement within the normal human anterior cornea. Ophthalmology 102: 33–41.

Calvillo, M. P., McLaren, J. W., Hodge, D. O., and Bourne, W. M. (2004). Corneal reinnervation after LASIK: Prospective 3-year longitudinal

study. Investigative Ophthalmology and Visual Science 45: 3991–3996.

deLeeuw, M. A. and Chan, K. Y. (1989). Corneal nerve regeneration. Correlation between morphology and restoration of sensitivity. Investigative Ophthalmology and Visual Science 30: 1980–1990.

Jones, M. A. and Marfurt, C. F. (1998). Peptidergic innervation of the rat cornea. Experimental Eye Research 66: 421–435.

Marfurt, C. F. (2000). Nervous control of the cornea. In: Burnstock, G. and Sillito, A. (eds.) Nervous Control of the Eye, pp. 41–92. Amsterdam: Harwood Academic Publishers.

Marfurt, C. F. (2009). Peptidergic innervation of the cornea: Anatomical and functional considerations. In: Troger, J. and Kieselbach, G. (eds.) Neuropeptides in the Eye, pp. 22–37. Trivandrum, India: Research Signpost.

Mu¨ller, L. J., Pels, L., and Vrensen, G. F. J. M. (1996). Ultrastructural organization of human corneal nerves. Investigative Ophthalmology and Visual Science 37: 476–488.

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

Niederer, R. L, Perumal, D., Sherwin, T., and McGhee, C. (2007). Corneal innervation and cellular changes after corneal transplantation: An in vivo confocal microscopy study. Investigative Ophthalmology and Visual Science 48: 621–626.

Oliveira-Soto, L. and Efron, N. (2001). Morphology of corneal nerves using confocal microscopy. Cornea 20: 374–384.

Patel, D. V. and McGhee, C. (2005). Mapping of the normal human corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy. Investigative Ophthalmology and Visual Science 46: 4485–4488.

Patel, D. V. and McGhee, N. J. (2009) In vivo confocal microscopy

of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: A review. British Journal of Ophthalmology 93: 853–860.

Ro´zsa, A. J. and Beuerman, R. W. (1982). Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain 14: 105–120.

Tervo, T., Vannas, A., Tervo, K., and Holden, B. A. (1985). Histochemical evidence of limited reinnervation of human corneal grafts. Acta Ophthalmologica 63: 207–214.

Zander, E. and Weddell, G. (1951). Observations of the innervation of the cornea. Journal of Anatomy 85: 68–99.

Glossary

Dysesthesias – Unpleasant abnormal sensations, spontaneous or evoked, such as burning, dryness, itching, electric shock, and pins and needles caused by the lesions of the peripheral or central nervous system. Ectopic activity – The abnormal generation of propagated nerve impulses in areas of the sensory neuron membrane that are normally not spontaneously excitable, often generated by peripheral sensory axons or cell bodies of injured sensory ganglion neurons.

Ion channels – Pore-forming proteins located in the cell membrane of all living cells that selectively regulate the flow of ions (cations or anions) into and out of the cell. The ion channel switches between open and closed when the protein undergoes a conformational change, helping to establish and control a voltage gradient (membrane potential) across the plasma membrane.

Nerve impulse – The electrical signal conducted along the axon of neurons by which information is conveyed within the nervous system.

Neuropathic pain – Pain of pathological origin resulting of the abnormal functioning of the peripheral and/or central neural pathways involved in the detection of noxious stimuli.

Sensory afferents – Peripheral branches of sensory neurons located in the dorsal root or trigeminal ganglia that innervate body tissues and carry sensory information to the brain. When covered with a myelin sheath (myelinated or A fibers), they conduct nerve impulses at rapid speeds (over 3 m s–1), while unmyelinated (or C) fibers lack the myelin sheath and have conduction velocities below 2 m s–1.

Sensory receptors – The terminal portion of peripheral sensory axons specialized in the transduction of physical or chemical forces into a discharge of short-lasting ( 1 ms) membrane depolarizations, referred to as nerve impulses, which travel rapidly along the axon.

Signal transduction pathways – The binding of extracellular signaling molecules (or ligands) to cell-surface receptors triggers ordered sequences of biochemical reactions carried out by enzymes activated by second messengers that finally result in a cellular event as, for instance, the opening or closing of specific ion channels.

The cornea is innervated by different functional types of sensory afferent fibers that are selectively activated by physical and chemical stimuli. They give rise to conscious sensations of variable quality that are referred to the eye and/or the periocular region.

The cell bodies of corneal sensory afferents, most of which are of small or medium size, are located in the ipsilateral trigeminal ganglion and reach the cornea through the ciliary nerves. Sensory axons enter the cornea grouped into a variable number of radially oriented nerve bundles that branch extensively and form the sub-basal plexus below the basal epithelial cells. From this plexus, naked single fibers ascend vertically between the epithelial cells ending at variable depths. Immunocytochemical staining of the cell soma and axons of ocular sensory neurons reveals the presence of neuropeptides, principally substance P (SP) and calcitonin gene-related peptide (CGRP). Neuropeptides participate in neurogenic inflammation and promote corneal healing and/or maintenance of epithelial integrity either alone or in combination with growth factors.

Functional Properties of Corneal

Sensory Receptors

Functional studies of corneal sensory fibers have been performed recording electrophysiologically in single corneal nerve fibers nerve impulses evoked by physical and chemical stimuli applied to the corneal surface. The majority of corneal sensory nerve fibers, about 70%, are polymodal nociceptors (Figure 1). They are activated by near-noxious mechanical energy, heat, chemical irritants, and by a large variety of endogenous chemical mediators released by damaged corneal tissue, resident inflammatory cells, or those which leak from vessels in the periphery of the cornea (the limbus). Some of the polymodal nociceptor fibers belong to the group of thin myelinated (A-delta) nerve fibers, but most of them are unmylelinated C fibers. Polymodal nociceptors respond to their natural stimuli with a continuous, irregular discharge of nerve impulses that persist as long as the stimulus is maintained. They have a firing frequency roughly proportional to the intensity of the stimulating force. Therefore, the impulse discharge of polymodal nociceptors not only signals the presence of a noxious stimulus, but also encodes its intensity and duration in a certain degree. Corneal polymodal nociceptors have a mechanical threshold slightly lower than mechanonociceptors

(Figure 2B(a)) and, when stimulated with heat, they begin to fire at temperatures over 39–40 C (Figure 2B(b)). A fraction of polymodal fibers (c. 50%) also increase their firing rate when the corneal temperature is reduced below 29 C. Many chemical agents known to excite polymodal nociceptors of other territories also activate ocular nociceptors. Acidic solutions (of pH 5.0–6.5), or gas jets containing increasing concentrations of CO2 (that in contact with the aqueous corneal surface forms carbonic acid and drops the local pH), evoke an impulse discharge in corneal polymodal nociceptors (Figure 2B(c)). Polymodal nociceptors are likely the origin of unpleasant sensations evoked by near-noxious and injurious chemical, thermal, and mechanical stimuli acting on the cornea.

Approximately 15–20% of the axons innervating the cornea (all thin myelinated) respond only to mechanical forces in the order of magnitude close to that required to damage corneal epithelial cells. Accordingly, they belong to the mechanonociceptor type (Figure 2A). Fibers of this class of receptor fire only one or a few nerve impulses in response to brief or sustained indentations of the corneal surface and, often, also when the stimulus is removed. Thus, they are phasic sensory receptors that signal the

presence of the stimulus and, in a very limited degree, its intensity and duration. The threshold force required to activate mechanonociceptors is apparently low (c. 0.6 mN), far below the force that activates mechanonociceptor fibers in the skin. However, this intensity might be sufficient to damage unkeratinized corneal epithelium. Mechanonociceptors in the cornea are probably responsible for the acute, sharp sensation of pain produced by touching the corneal surface. The after-sensations of pain elicited by noxious stimuli are probably explained by the more sustained activity exhibited by polymodal nociceptors.

Another category of corneal nerve fibers that represents 10–15% of the total population is cold-sensitive thermal receptors. These are A-delta and C fibers that discharge spontaneously at rest and increase their firing rate when the normal temperature of the corneal surface (c. 33 C) is reduced while they are transiently silenced upon warming. They also increase their firing rate as soon as the temperature of the cornea drops because of evaporation at the corneal surface, application of cold solutions, or blowing of cold air on the cornea. Cold receptor fibers are able to detect and encode as a change in impulse

A

(a)

(b)

47 C

35 C

(c)

B

frequency and small temperature variations of 0.1 C or less, thus allowing for the perception of decreases in corneal temperature as a conscious sensation of innocuous cooling.

Finally, it has been suggested that the cornea possesses mechanically insensitive, silent nociceptors, that is, nerve terminals that are not activated by mechanical or thermal stimuli when the tissue is intact, but, in the case of local inflammation, become responsive to these exogenous stimuli as well as to a variety of endogenous chemicals. Although the experimental evidence for their presence in the cornea is only indirect, they have been identified in virtually all other somatic tissues. Thus, it seems likely that such nociceptors also exist in the cornea. Figure 1 presents a schematic diagram of the firing response to mechanical, thermal, and chemical stimuli of the various functional types of sensory endings identified electrophysiologically in the cornea.

The detection of stimuli by corneal receptor terminals, as occurs with sensory receptors of other tissues of the body, depends on membrane signaling proteins which convert the stimulus energy into a conformational change, leading to an alteration in ionic permeability and an electrical depolarization of the membrane of the nerve terminal, the generator potential. This potential change in the peripheral nerve endings gives rise to propagated nerve impulses in more proximal portions of the axon which travel centripetally to the brain. In nociceptors, most transduction molecules are ion channels that are

Figure 2 A and B: Impulse response to different stimulus modalities of corneal sensory fibers. A: Transient discharge evoked in a mechanonociceptor fiber by a sustained mechanical indentation. B: Response of a polymodal nociceptor fiber to

(a) mechanical indentations of increasing amplitude (80 and 150 mm); (b) stepwise heating of the corneal surface from 34 to 47 C; and (c) application of a drop of 10-mM acetic acid to the corneal surface. In all cases, upper traces depict the nerve impulse recordings and lower traces the stimulus waveform; time scales: A and B(a) = 1 s; B(b) = 15 s; and B(c) = 0.5 s.

C and D: Sensitization to heat of corneal polymodal nociceptors. C: Peristimulus histograms of a corneal unit showing the first (upper) and second (lower) response to two identical stepwise heatings separated by 3 min. Each bar indicates the number of impulses evoked per second and the lower trace shows the stimulus waveform. E: Mean stimulus response relation of eight corneal units in response to the first (filled circles) and the second (open circles) stepwise heating. Each point represents the mean number of impulses evoked at the temperature indicated in the abscissa. The bars indicate the standard error of mean. A and B reproduced from Belmonte, C. Gallar, J. The primary nociceptive neuron: A nerve cell with many functions. In: Rowe M. J. (ed.) Somatosensory processing: From single neuron to brain imaging. Sydney, NSW: Harwood Academic Publishers. C and D reproduced from Belmonte, C. and Giraldez, F. (1981). Responses of cat corneal sensory receptors to mechanical and thermal stimulation. Journal of Physiology 321: 355–368.

directly gated by the stimulus or open by intracellular messenger systems that can, in turn, be activated by a variety of membrane proteins.

Several classes of ion channels have been associated with the transduction of the various forms of energy and the production of the generator potential at nociceptor nerve terminals. A channel protein named transient receptor potential type vanilloid 1 (TRPV1) receptor, a nonselective cation channel with pronounced permeability for Ca2+ ions, is the main molecular substrate for the ability of polymodal nociceptors to respond to acid, heat, certain irritant chemicals, and inflammatory mediators. Another ion channel of the same family – transient receptor potential cation channel, subfamily A, member 1 (TRPA1) – also exhibits a prominent sensitivity to pungent chemicals. Thus, these channels serve as molecular integrators for multiple types of stimuli. The nature of the molecular entities involved in the transduction of mechanical forces at nociceptor endings is still uncertain. Stretch-activated ion channels have been identified in the membrane of mammalian sensory neurons. Extracellular matrix attachments have been proposed as the mechanism involved in the transmission of external mechanical forces to the neuronal surface and, subsequently, to ion channels activated or inactivated by stretch. Finally, TRPM8 and leak K+ channels have been suggested as cold sensors in cold thermoreceptor corneal fibers. The depolarization of sensory nerve terminals in the cornea secondary to ion channel opening has a variable duration. In the case of rapidly adapting corneal mechanosensory endings, only one or few nerve impulses are generated. Thus, they serve primarily to signal the presence of a stimulus. Polymodal nociceptors and cold receptors encode the intensity and the time course of the stimulus in their firing rate and duration. They therefore provide information to the brain on both the modality and the characteristics of the stimulus (Figure 1).

Response of Corneal Receptors to Local Inflammation

One of the most prominent features of polymodal nociceptors is that sustained or repetitive stimulation tends to augment their response to new noxious stimuli. This phenomenon, referred to as sensitization, is also present in corneal nociceptors. Sensitization is characterized by a reduction of the impulse firing threshold such that impulse discharges are now evoked by intensities of the stimulus within the innocuous range. Moreover, noxious stimuli elicit a stronger and more sustained impulse discharge. Finally, sensitized corneal nociceptors often develop an irregular, low-frequency ongoing activity of nerve impulses in the absence of stimulation (Figures 2D and 2E). This spontaneous activity, coupled with the enhanced

responsiveness to stimulating forces, is the peripheral substrate of the augmented pain sensitivity and the presence of pain sensation in the absence of stimulus, exhibited by injured and/or inflamed corneas.

The sensitization of corneal nociceptors occurs as a result of the local release by injury of a large variety of inflammatory mediators from damaged cells and from resident and invasive cells of the immune system. Endogenous chemicals include H+ and K+ ions, adenosine and adenosine triphosphate (ATP), serotonin, histamine, platelet-activating factor, bradykinin, prostaglandins, leukotrienes, tromboxanes, interleukins, tumor-necrosis factor, and nerve growth factor (NGF). These mediators are responsible for local inflammatory reactions in the cornea and conjunctiva (vasodilatation, plasma extravasation, and cell migration), and act on membrane channels and receptor proteins at corneal nociceptor nerve endings, provoking short-term changes in their opening probability. TRPV1 and TRPA1 channels at the membrane of polymodal nociceptors are direct or indirect targets for many of the inflammatory mediators released upon corneal injury. The enhanced ion flow and increased membrane excitability resulting of ion channel activation create the augmented impulse firing and reduced threshold that characterize the process of sensitization of injured corneal nerve terminals (Figures 2D and 2E). Inflammatory mediators use different signaling pathways to exert their effects on channel activity. For instance, prostaglandin E2, ATP, and adenosine or 5-hydroxytryptamine (5-HT) activate the protein kinase A (PKA) signaling pathway, whereas other mediators such as bradykinin act on protein kinase C to produce sensitization. The sensitization of corneal nociceptors develops within minutes of an acute corneal injury and normally fades when inflammation subsides. Long-lasting corneal inflammation, as occurs, for instance, in chronic dry eye, may produce more permanent changes in nociceptive terminals, including modified expression of the existing receptor molecules and expression of new ones possibly mediated by growth factors, in particular NGF.

The nerve impulses evoked by noxious stimuli in corneal nociceptors not only travel centripetally to the brain, but also invade antidromically other nonstimulated peripheral branches of the parent axon, causing the release of neuropeptides contained into their peripheral endings. CGRP and SP released by depolarized nociceptor endings contribute to local inflammatory responses and amplify the proinflammatory effect of other endogenous mediators. This neurogenic inflammation mediated by the sensory nerves affects noninjured areas of the cornea and conjunctiva and explains the extension of inflammation to distant, intact tissues (conjunctiva, iris, and ciliary body) following a limited corneal lesion.

Effects of Injury on Corneal Nerves

After ocular surgery, accidental trauma, and nerve injury secondary to certain ocular or systemic diseases, corneal sensory nerves may become damaged at different points of their trajectory. It is well established that peripheral axotomy changes the morphology and functional properties of nerve fibers projecting to the cornea substantially. Denervated areas are invaded by outgrowths of adjacent noninjured nerve fibers and sprouts of the injured axons. Some of terminal stumps of severed axons are entrapped by scar tissue and form microneuromas. The expression of genes that encode ion channels (in particular sodium and potassium channels) and receptor proteins by primary sensory neurons is also modified by peripheral nerve injury. This alters nerve excitability and favors the development of ectopic discharges and abnormal responsiveness in damaged neurons, giving rise to peripheral neuropathic pain.

Sensitivity of the Intact Cornea

Clinical exploration of the sensitivity of the cornea or conjunctiva to mechanical stimulation is normally performed by gently touching the ocular surface with a wisp of cotton, and observing the blink reflex or comparing the subjective sensation with that evoked in the fellow eye. A more quantitative approach is obtained using a calibrated hair of variable length (the Cochet–Bonnet esthesiometer). The Belmonte noncontact gas esthesiometer uses an air jet of adjustable flow and temperature that contains CO2 in a variable concentration to reduce local pH, which allows separate mechanical, thermal, or chemical stimulation applied to a restricted area of the cornea or the conjunctiva. Using these procedures, changes in normal corneal sensitivity in relation to age, sex, pregnancy, iris color, and use of contact lenses; pathological changes associated to ocular surgery; corneal pathologies such as herpes virus infections, keratitis, iritis, uveitis, and glaucoma; or systemic diseases such as fibromyalgia or diabetes have been detected in multiple clinical and experimental studies.

Studies with the Belmonte esthesiometer have determined that the quality of the sensation evoked from the intact cornea and conjunctiva depends on the modality of the stimulus applied. Furthermore, electrophysiological recordings of nerve impulse activity in experimental animals applying mechanical, thermal, and acidic stimuli to the cornea with the Belmonte esthesiometer showed that each of them excited, in a variable degree, the various functional subpopulations of corneal nerve fibers. In humans, the sensations produced by suprathreshold mechanical, chemical, and heat stimulation of the cornea

possessed, in each case, a distinct quality that allowed the psychophysical identification of the stimulus modality, but always included a component of irritation. In contrast, the application of cold pulses to a corneal spot that moderately decreased (1–3 C) the local temperature evoked almost exclusively an innocuous cooling sensation, which acquired an additional component of irritation when more pronounced temperature reductions were applied. The psychophysical characteristics of sensations evoked by stimulation of the bulbar conjunctiva are quite similar to those of the cornea, except that sensitivity was comparatively lower and light mechanical stimuli were felt as nonirritating.

Sensitivity of the Injured Cornea

Following surgical procedures for cataract removal, retinal detachment, and glaucoma, and particularly after surgery performed to correct refractive defects (radial keratotomy, photorefractive keratectomy, laser-assisted in situ keratomileusis, and keratoplasty), nerves directed to the cornea are usually damaged in a degree that, for photorefractive surgery, depends on the extent of the corneal lesion. Severed nerves start to regenerate soon after injury, but only a part of them succeed in penetrating the injured corneal tissue, and corneal innervation remains disorganized and reduced in number, months and even years after surgery. In parallel with the morphological changes, functional alterations have been described. Axotomized corneal neurons and nerve fibers innervating the injured cornea exhibit an altered threshold to their natural stimuli and abnormal responsiveness to mechanical, chemical, and thermal stimuli.

As a result of the disturbances in peripheral innervation, corneal sensitivity to mechanical stimulation is impaired, with a remarkable increase in threshold of the denervated areas that takes months to recover, and may never return to normal values. Transplanted corneas or implanted lenticules in epikeratophakia remain totally anesthetic for years, or recover, at best, a very limited mechanical sensitivity usually restricted to the periphery of the transplant. After laser-assisted in situ keratomileusis (LASIK) surgery, corneal sensitivity to mechanical and chemical stimulation measured with the Belmonte esthesiometer remained significantly below control 3 and 6 months post-LASIK, becoming close to normal only 2 years postsurgery. However, at that time, a few patients with deep photo ablations presented severe sensory impairments.

In parallel with the hypoesthesia just described, spontaneous pain sensations and dysesthesias may appear following refractive surgery. The lower sensitivity of the cornea to external corneal stimulation is explained by the reduced number and the altered threshold of corneal

sensory nerve fibers after surgery. The additional development of unpleasant dryness sensations and/or pain referred to the eye in some patients is attributable to the spontaneous firing and abnormal responsiveness of some of the injured corneal nerve fibers that were unable to fully recover their transducing capacities and exhibit abnormal excitability after injury. These disturbances seem to affect mainly polymodal nociceptor and cold corneal nerve fibers. Thus, these two functional subpopulations of corneal nerves appear to be the main peripheral source of abnormal sensations following corneal surgery.

See also: Corneal Nerves: Anatomy.

Further Reading

Belmonte, C. (2007). Eye dryness sensations after refractive surgery. Impaired tear secretion or ‘phantom’ cornea? Journal of Refractive Surgery 23: 598–602.

Belmonte, C., Acosta, M. C., and Gallar, J. (2004). Neural basis of sensation in intact and injured corneas. Experimental Eye Research 78: 513–525.

Belmonte, C. and Tervo, T. T. (2005). Pain in and around the eye. In: McMahon, S. and Koltzenburg, M. (eds.) Wall and Melzack’s Textbook of Pain, 5th edn., pp. 887–901. London: Elsevier.

Belmonte, C. and Viana, F. (2007). Transduction and encoding of noxious stimuli. In: Schmidt, R. F. and Willis, W. (eds.) Encyclopedia of Pain, vol. 3, pp. 2515–2528. Berlin: Springer.

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.

Glossary

Apoptosis – The most common form of physiological (as opposed to pathological) cell death. Apoptosis is an active process requiring metabolic activity by the dying cell; often characterized by shrinkage of the cell, cleavage of the DNA into fragments that give a so-called laddering pattern on gels and by condensation and margination of chromatin. Cytoskeleton – General term for the internal components of animal cells which give them structural strength and motility: plant cells and bacteria use an extracellular cell wall instead. The major components of cytoskeleton are the microfilaments (of actin), microtubules (of tubulin), and intermediate filament systems in cells. Diabetic retinopathy – Damage to the retina caused by diabetes mellitus.

Dystrophic epidermolysis bullosa – A rare disorder caused by a mutation in the keratin gene and is characterized by the presence of extremely fragile skin and recurrent blister formation. Filopodia – A thin protrusion from a cell, usually supported by microfilaments; may be functionally the linear equivalent of the leading lamella.

Idiopathetic pulmonary fibrosis – A chronic, progressive interstitial lung disease with an unknown cause.

Phagocytosis – Uptake of particulate material by a cell (endocytosis).

Phenotype – The characteristics displayed by an organism under a particular set of environmental factors, regardless of the actual genotype of the organism.

Recurrent corneal erosion – A disorder of the eye characterized by failure of the epithelial cells to attach to the basement membrane.

Corneal Epithelial Wound Healing:

Introduction

The main function of the cornea is to prevent infectious invasion and to retain a smooth, optically clear surface to

transmit light into the retina. The cornea has evolved a complex wound healing process for dealing with any insults to this tissue. The overall goal of corneal wound healing, as in most healing processes, is to repair the damaged tissue to resemble the unwounded tissue as closely as possible. In the cornea, this is especially important, as a failure to recapitulate an unwounded state can lead to visual impairments and a lower quality of life. The process of corneal wound healing is multifaceted involving many cell bound and secreted factors which work together to induce large-scale gene expression and phenotypic alterations in order to coordinate cell migration, proliferation, and survival rates for optimum regeneration and restoration of tissue function. While the process of corneal epithelial wound healing is well understood on the macro-molecular level (as will be discussed), there remain many micromolecular aspects to be elucidated if we are to completely understand this process.

Corneal Epithelial Wound Healing:

Phases of Wound Healing Process

The corneal epithelial wound healing process can be described as having three overlapping phases: the lag phase, the migration phase, and the proliferation/ restratification phase (Figure 1). The lag phase occurs immediately following any disruption of the corneal epithelium such as a scratch or puncture. During this phase, polymorphonuclear (PMN) cells from the tear fluid clear damaged and necrotic tissue from the wound area by phagocytosis, resulting in a microscopic enlargement of the wound area. These PMN cells generally disappear shortly after complete resurfacing of the wound by the epithelial cells though they can persist in cases of infection. Concurrently, a provisional matrix composed mainly of fibrin and fibronectin is secreted by the epithelial cells and the lacrimal gland and is laid down over the wound area. The provisional matrix acts as a substrate for migrating epithelial cells as well as an activator of integrin receptors on the cell surface to keep intact intracellular migration signals, mediated by the small GTPase proteins Rho and ROCK.

Also, during this phase, the epithelial cells must prepare themselves for the migratory phase. For cells proximal to the wound edge, this includes a dramatic alteration

in their gene expression profile including a secession of proliferation, and an alteration in cytokine and protease secretion, an alteration of the cytoskeleton including formation of lamellapodia and fillipodia, and an alteration of cell–cell and cell–matrix adhesions. Cells farther from the wound continue to proliferate to replace cells that enter into migratory phase. Additionally, during this phase, loss of superficial keratocytes due to apoptosis can be observed directly beneath the wound even when the basement membrane (BM) remains intact immediately following wounding. In a nonpenetrating wound, these stromal keratocytes are slowly replaced by proliferation of the surviving keratocytes without fibroblast activation or myofibroblast differentiation. In penetrating wounds, secreted factors induce fibroblast activation and proliferation and myofibroblast differentiation of the remaining keratocytes as will be discussed.

The second phase of corneal wound healing is the migratory phase. It is important that the wound resurfaces quickly, as prolonged or chronic wound healing scenarios may lead to degradation of the BM due to an increased proteolytic profile. This may in turn lead to ulceration of the underlying stroma, activation of the keratocytes into myofibroblasts, scarring, and possible visual impairments. The migratory phase is characterized by the centripetal migration of a continuous monolayer of nondividing corneal epithelial cells (CECs) over the wound and usually lasts between 24 and 36 h depending on the size of the wound and animal species. Cells at the wound edge flatten out and migrate while those more distal to the wound are

induced to proliferate to replace the migratory cells. Cell migration rates vary by species, with rabbit CECs migrating at around 64 mm day–1 and mouse CECs at around 17 mm day–1. During this phase, epithelial cells must be anchored enough to the underlying provisional matrix so that they are not removed casually (blinking, rubbing, etc.), but if the attachments are too strong, the cells will be unable to migrate. Laminin-5 has been implicated as a major determinant in the adhesion of epithelial cells to the underlying matrix. In the unwounded cornea, processed laminin-5 is present and helps hemidesmosomes in epithelial cells adhere strongly to the collagen VII anchoring fibrils. However, following wounding, the presence of the processed form becomes secondary to that of the unprocessed form which is not bound by cells as strongly. Unprocessed laminin-5 is deposited in the provisional matrix and, along with other provisional matrix components such as fibronectin, promotes the migration of epithelial cells rather than stationary adherence.

It has been proposed that epithelial cell migration is driven by an adhesion/deadhesion cycle that is regulated by two things: (1) a careful ratio of adherence molecules for attachment and proteolytic enzymes that degrade these attachments and (2) intracellular extension and contractile signals that alter the actin cytoskeleton morphology. Migratory cells first extend lamellapodia and fillopodia forward and form temporary focal adhesion complexes with the provisional matrix. Focal adhesions at the tail end of the cell are removed by a combination of proteolytic enzymes that cleave the complex and