Jump to ContentJump to Main Navigation
Neurobiology of Nociceptors$

Carlos Belmonte and Fernando Cervero

Print publication date: 1996

Print ISBN-13: 9780198523345

Published to Oxford Scholarship Online: March 2012

DOI: 10.1093/acprof:oso/9780198523345.001.0001

Corneal nociceptors

Chapter:
(p.146) 6 Corneal nociceptors
Source:
Neurobiology of Nociceptors
Author(s):

Belmonte Carlos

Gallar Juana

Publisher:
Oxford University Press
DOI:10.1093/acprof:oso/9780198523345.003.0006

Abstract and Keywords

It is not surprising that the cornea possesses the most dense sensory innervation of the body. It has been estimated that the cornea contains 300–600 times more sensory endings than the skin and 20–40 times more than the tooth pulp. As discussed in this chapter, most of this innervation appears to be nociceptive in nature. This abundance in nociceptive terminals, together with the absence of blood vessels and the structural simplicity of the supporting tissues, makes the cornea a good model for analyzing the morphological and functional properties of peripheral nociceptors.

Keywords:   corneal nociceptors, corneal innervations, avascular tissue, polystratified epithelium, substantia propria, nociceptive terminals

Introduction

Early studies on corneal innervation and sensitivity played an important role in our present understanding of the neurobiology of peripheral pain. In 1894, von Frey stated that different modalities of cutaneous sensation were subserved by morphologically distinct populations of sensory fibres and that unspecialized free nerve terminals were the origin of pain. This early version of the'specificity theory’ of sensory discrimination was partly based on the observation that pain was the sole sensation that could be evoked from the cornea which was innervated exclusively by free nerve endings. von Frey’s theory was challenged by Weddell and colleagues (Weddell and Zander 1950; Zander and Weddell 1951; Lele and Weddell 1956, 1959), again using the cornea as a model. These authors concluded that different qualities of sensations were evoked by the stimulation of the human cornea with various forms of energy, in spite of the unspecialized morphological and functional characteristics of corneal afferents (Lele and Weddell 1956, 1959). The capacity of apparently identical nerve fibres to evoke different sensations was taken as a key argument to support the alternative‘pattern theory’ of sensory discrimination. According to this view, non-noxious and noxious stimuli were distinguished in the central nervous system by the different temporal course of nerve discharges evoked at non-specialized peripheral terminals. On the basis of their electrophysiological data obtained in corneal free nerve endings, it was proposed that ‘an effective stimulus will cause spatially as well as temporally dispersed pattern of activity to be transmitted to the central nervous system. Sensory discrimination may, therefore, be dependent upon the central analysis of the space-time pattern of this activity’ (Lele and Weddell 1956, 1959). About a decade later, the alternative possibility that the detection of injurious stimuli depends on a functionally specific population of sensory fibres, the nociceptors (Iggo 1963; Bessou and Perl 1969), gained progressive support (Light 1992; see Perl, Chapter 1, this volume). Nevertheless, the cornea remained an apparent and unexplored‘exception’ to this rule. It took another decade to prove experimentally that corneal sensory afferents are functionally similar to nociceptive fibres innervating the skin and other territories of the body (Giraldez et al. 1979; Belmonte and Giraldez 1981).

The cornea is an avascular tissue of ectodermal origin, with a very simple structure (Maurice 1962). The external surface is formed by a polystratified epithelium, while a single-layered endothelium covers the internal surface. The stroma or substantia propria, represents about 90 per cent of the tissue and consists of collagen and of large, flattened fibroblasts (keratocytes or stromal cells).

The cornea is the first optical medium for the transmission of the light to the retina. It also represents the outermost barrier for the defence of the eye against injury and infection. Therefore, it is not surprising that the cornea possesses the most dense sensory (p.147) innervation of the body: it has been estimated that the cornea contains 300–600 times more sensory endings than the skin and 20–40 times more than the tooth pulp (Rózsa and Beuerman 1982). As is discussed below, most of this innervation appears to be nociceptive in nature. This abundance in nociceptive terminals, together with the absence of blood vessels and the structural simplicity of the supporting tissues, makes the cornea a good model for analyzing the morphological and functional properties of peripheral nociceptors (Belmonte et al. 1994).

Corneal trigeminal neurones

The cornea is almost exclusively innervated by sensory fibres in trigeminal ganglion neurones (Arvidson 1977; Marfurt 1981; Lehtosalo 1984; Morgan et al. 1987). Using fluorescence techniques that label adrenergic structures, a few sympathetic fibres have been also reported in the cornea (Laties and Jacobowitz 1964; Ehinger 1966). The existence of a weak parasympathetic innervation of the cornea, suggested by old histological studies but denied later (Palkama et al. 1986), has been recently confirmed in the rat (Marfurt and Jones 1995).

In mammalian species (mouse, rat, guinea-pig, cat, and monkey), about 95 per cent of trigeminal neurones innervating the cornea are located in the anteromedial side (ophthalmic division) of the ganglion (Arvidson 1977; Morgan et al. 1978, 1987; Kuwayama et al. 1987; Marfurt and DelToro 1987; Marfurt and Echtenkamp 1988; Tusscher et al. 1988; Ichikawa et al. 1993; Passagia et al. 1993); the remaining 4 per cent are found in the maxillary region (Morgan et al. 1978, 1987; Marfurt and Echtenkamp 1988). Retrograde labelling studies have shown that the number of corneal trigeminal cells varies between 100 and 900 depending on the staining technique and the animal employed. The total number of neurones in the trigeminal ganglion has been estimated to be around 50 000 in the rat and cat (Dixon 1963; Gregg and Dixon 1973); thus, corneal neurones represent less than 2 per cent of this total. Most corneal sensory neurones are small (diameter of less than 20 gm; mean surface, 200–400 gm2) and with dark cytoplasm (type B, Duce and Keen 1977), while a small proportion (about 11 per cent) are medium-sized neurones, with clear cytoplasm (diameter, 20–33 gm; mean surface, 500–800 gm2) (Martin and Dolivo 1983; Sugimoto et al. 1988). Both small-and medium-sized cells belong to the group of primary sensory neurones with unmyelinated or thin myelinated peripheral axons.

About 40 per cent of trigeminal neurones innervating the cornea and conjunctiva are tachykinin-positive (Lehtosalo 1984; Lehtosalo et al. 1984; Ichikawa et al. 1993; LaVail et al. 1993). Among rat trigeminal cells, identified as ocular by retrograde labelling with cholera toxin introduced in the anterior chamber, 18.5 per cent were reactive to substance P (SP), 40.4 per cent to calcitonin gene-related peptide (CGRP), and 2.7 per cent to cholecystokinin (CCK) (Kuwayama et al. 1987). This relative incidence of peptides in ocular trigeminal cells is similar for sensory neurones in the rat ganglion as a whole (Lee et al. 1985); in this species, virtually all ocular SP-containing cells are also reactive to CGRP (Terenghi et al. 1985).

(p.148) Nerve pathways to the cornea

The peripheral axons of sensory neurones innervating the cornea leave the trigeminal ganglion as a part of the ophthalmic nerve and travel to the eye with the nasociliary nerve. According to morphological studies, a small proportion of corneal nerve fibres in monkeys and presumably also in humans reach the cornea through the maxillary nerve (Marfurt and ahtenkamp 1988). The ophthalmic nerve gives off the long ciliary nerves through the nasociliary branch and a sensory root to the ciliary ganglion, which in turn reach the eye through the short ciliary nerves (Attias 1912). Long and short ciliary nerves mix while travelling to the eye and pierce the sclera as 10–20 fine nerve trunks at the posterior pole of the eyeball, around the optic nerve; nevertheless there is a considerable anatomical variability among species in the number and point of entrance into the eye of the ciliary nerves (Grimes and von Sallmann 1960). In these nerve trunks, sensory fibres of small diameter (2–4 gm) and some of larger size (about 6 pm) are mixed with sympathetic axons supplied by neurones of the superior cervical ganglion, and with parasympathetic fibres originating in the ciliary and the pterygopalatine ganglia. These autonomic fibres innervate blood vessels, secretory epithelia, and muscle fibres within the eye and correspond to the vast majority of unmyelinated axons found in the ciliary nerves.

Nerve trunks entering the eye run anteriorly in the suprachoroidal space or the scleral substance to innervate scleral tissue, ciliary body, iris, and cornea. Most corneal fibres enter this tissue at the level of the sclera, forming large nerve bundles; a few fibres penetrate the cornea more superficially, some of them from nerve trunks running in the subconjunctiva and others from nerves in the episcleral tissue. These superficial fibres join with branches from the corneal scleral bundles and form at the transition zone between the conjunctiva and the cornea (the limbus) an episcleral pericorneal plexus (Zandell and Weddell 1951).

Pattern of neural organization in the cornea

Between 10 and 80 large nerve trunks, depending on the species, penetrate radially the collagenous stromal substantia propria at various sites around the corneal circumference. They represent the main neural supply to the cornea in non-primate species Thinner nerve bundles arising from the pericorneal plexus also enter the most superficial layers of the stroma (Zandell and Weddell 1951; Lim and Ruskell 1978; (Fig. 6.1 (A), (B)).

Corneal nerve strands contain thin myelinated and unmyelinated axons. Myelinated fibres lose their myelin sheath early (Zander and Weddell 1951). When approaching the central region, axons branch off repeatedly to form the subepithelial plexus (Fig. 6.1). Axon terminals ascend from this plexus toward the stroma—epithelium interface, losing the perineural sheath. No nerve terminals have been found innervating the corneal endothelium. Ascending nerve filaments give off collaterals that run for long distances, predominantly in one direction within the basal epithelial layer, offering the character¬istic appearance of a‘leash’. The leashes, formed by a few up to several dozen strands, run for long distances (0.2–2 5 mm) and produce extensions that ascend vertically, branching both vertically and horizontally, to reach the outer epithelial layers (Fig. 6.1 (B), (C)). They end as terminal enlargements or boutons between the wing cells, at a few (p.149) microns of the corneal surface. Axons in the subepithelial plexus also produce terminals that ascend vertically, with or without branching, and reach the most superficial layers of the cornea. Some fibres ending as naked terminals in the anterior and medium portions of the stroma have also been described (Zander and Weddell 1951; Whitear 1960).

Corneal nociceptors

Fig. 6.1(A) Representation of corneal innervation, showing the distribution of nerves in the different planes of the cornea. From R. Beuerman, in Maurice (1984). (B) Diagram illustrating the innervations of the corneal epithelium by ascending nerve filaments that show varicosities and end as terminal boutons at the level of wing cells. From RamOn y Cajal (1899). (C) Camera lucida drawing of the branching pattern of a parent axon in the epithelium of the mouse cornea. Scale bar, 0.1 mm. Courtesy of F. de Castro.

(p.150) In the limbus of cat, monkey, and man (Whitear 1960; Lim and Ruskell 1978), fibres that are finer and more numerous than the deep stromal trunks form the episcleral plexus. Branches of this plexus are more superficial than the deep, radial stromal nerves and penetrate the cornea at the basal epithelial cell layer and immediately below the epithelial basement membrane (Chan-Ling 1989). In monkey, it has been suggested that most of the innervation of the corneal epithelium is provided by the limbal subepithelial plexus (Lim and Ruskell 1978). In the limbal area adrenergic fibres are frequently found, most of them innervating the blood vessels and other penetrating for short distances within the corneal tissue (see below). Also, endings with morphological specializations (Krause corpuscles) have been described in the limbus (Lawrenson and Ruskell 1991).

Fine structure of corneal nerve terminals

Electron microscopy studies (Whitear 1960; Matsuda 1968; Hoyes and Barber 1976; T. Tervo and Palkama 1978a, b; Beckers et al. 1992, 1993; Müller et al. 1996) have confirmed that nerve bundles running in the stroma contain several fibres (10–20) wrapped in a Schwann cell. The fibre diameters vary between 0.15 and 2 gm. Axon terminals traversing the epithelium-stroma interface are devoid of Schwann cells and present varicosities along their trajectory as well as at branching points (Fig. 6.2). In the basal layer of the epithelium, nerve fibres are invaginated into the epithelial cells in deep grooves containing one to more than one dozen axons. In the more superficial cells, thin nerve terminals (0 25 μm or less) run between epithelial cells and sometimes in a groove. There, the epithelial cells are in as close a relationship to the nerve fibres as are the Schwann cells in nerve bundles (Whitear 1960). Nerve varicosities contain neurotubuli, neurofilaments, and dark mitochondria in abundance. Numerous clear and incidental vesicles with small granules were also seen (Matsuda 1968; Hoyes and Barber 1976; Beckers et al. 1992; Müller et al. 1996). Terminals containing granular vesicles are more often observed near the limbus and are rare in the central cornea. They disappear after sympathectomy, which suggests that they belong to the scarce adrenergic fibres found in the avascular cornea (T. Tervo and A. Palkama 1978a).

Density of corneal innervation

Data on the number of nerve fibres present in the cornea are scarce, because of the difficulty involved in their quantification. The number of parent axons entering the cornea has been estimated to be 1200 in the human, each of which will produce 50 fibres subserving the epithelium (Lele and Weddell 1956). In the stroma of the rat, an average number of 60 nerve bundles and its branches were measured per mm2 of cornea. The density was lower in the centre than in the periphery, possibly reflecting the penetration of the epithelium by stromal branches before they reach the central cornea (Ishida et al. 1984).

Rózsa and Beuerman (1982) reported a total of 6570± 1709 nerve endings in all layers of the epithelium in rabbits. The highest number of terminals was found in the wing cells layer, which almost double those present in the basal layer and the wing-superficial layer. Also, the innervation density was greater in the central cornea than in the periphery in agreement with the stromal nerve density data.

Corneal nociceptors

Fig. 6.2 Electron microscopy images of nerve fibres in the epithelium of the human cornea. (A) Cross-section through a nerve bundle, showing mitochondria (M), vesicles (arrowheads), microtubules (large arrow), and neurofilaments (small arrow). Magnification, 19 712 × (B) Cross-section of a single nerve fibre at the level of a bead, showing numerous mitochondria (M), glycogen particles (large arrow), and vesicles (small arrow). Magnification, 16 576 x. (C) Frontal section of a nerve filament that bifurcates at the points indicated by the arrowheads. The single fibre running in the upper part of the picture has a bead filled with mitochondria (large arrow). Magnification, 3000 x. (D) Frontal section of a single fibre, showing two beads filled with numerous dark mitochondria (M), glycogen particles (small arrows), and vesicles (large arrow). Magnification, 12 544 x. BC, Basal cell; BM, Bowman’s membrane. Courtesy of Linda J. Muller; photographs (B)—(D) taken from Muller et al. (1996).

(p.151) Histochemical heterogeneity of corneal nerves

The presence of SP-immunoreactive nerves in the cornea of various animal species including man is now well documented (Miller et al. 1981; K. Tervo et al. 1981; Stone et al. 1982; T. Tervo et al. 1982; Bynke et al. 1984; Lehtosalo 1984; Beckers et al. 1993. The differences in density of SP innervation among reports is prominent, possibly due to technical difficulties and to poor penetration of antibodies (K. Tervo 1981). Corneal SP fibres originate in peptidergic neurones present in the trigeminal ganglion, because they disappear by maxillary and ophthalmic neurotomy (K. Tervo et al. 1982; T. Tervo et al. 1983). CGRP-immunoreactive nerves have been observed in the stroma and epithelium of the mammalian cornea and are eliminated by sensory denervation (Colin and Kruger (p.152) 1986; Stone et al. 1986, 1987; Stone and McGlinn 1988; Beckers et al. 1992; see also Kruger and Halata, Chapter 2, this volume). Also, galanin-containing fibres, that disappear after combined superior cervical ganglion removal and intracranial transac¬tion of the ophthalmomaxillary nerve have been reported (Marfurt and Jones 1995). Finally, neurokinin A/neurokinin B immunoreactivity has been detected in corneal tissue (Beding-Barnekow et al. 1988).

Capsaicin, a toxin that depletes neuropeptides from sensory nerve terminals (see Buck and Burks 1986), eliminates SP and CGRP from the cornea as well as from corneal fibres immunoreactive to these peptides when administered neonatally (Gamse et al. 1981; Terenghi et al. 1986). However, the reduction in the number of corneal nerves varies greatly from one species to another and is often followed in later stages by hyper¬reinnervation (K. Tervo 1981; Bynke et al. 1984; Fujita et al. 1984; Olgilvy and Borges 1990; Marfurt et al. 1993). This effect appears to be due in part to sprouting of capsaicin¬resistant sensory nerves that include CGRP-containing sensory fibres (Marfurt et al. 1993).

As was mentioned above, the existence of a small number of corneal nerve fibres of sympathetic origin was repeatedly suggested by optical and electron microscopy studies (Laties and Jacobowitz 1964; Ehinger 1966; T. Tervo 1977; Terenghi et al. 1986; Marfurt and Ellis 1993). These fibres appear to be more abundant in prenatal stages (Ehinger and Sjoberg 1971; K. Tervo et al. 1978), but are present in adult corneas in a variety of animal species, including the human (Toivanen et al. 1987; Marfurt and Ellis 1993). Ocular adrenergic fibres apparently contain neuropeptide Y, because nerves immunor¬eactive to this peptide disappeared from the rat cornea after extirpation of the superior cervical ganglion (Stone 1986; Marfurt and Jones 1995).

In the rat, limbal and corneal vasoactive intestinal peptide (VIP)-and Met-enkepha¬lin-immunoreactive nerve fibres have also been detected. These fibres were not affected by sympathetic and sensory denervation. Moreover, retrograde transport of wheat germ agglutinin—horseradish peroxidase (WGA—HRP) from the central cornea labels neu¬rones in the ciliary ganglion and in the optic nerve sheath miniganglia, thus suggesing a parasympathetic origin for these corneal fibres (Marfurt and Jones 1995).

Development of corneal innervation

In mammals, invasion of corneal tissue by sensory nerves takes place at the early stages of embryonic development, although corneal innervation is not fully developed at birth (Lukas and Dolezel 1975; zanies et al. 1977; K. Tervo and T. Tervo 1981). Penetration of the epithelium by nerve processes occurs late in the fetal period and during the first postnatal days, as demonstrated by the increased number of neurites extending upward from the stroma toward the epithelium seen postnatally in the rat. Moreover, the electron microscopic appearance of corneal nerve terminals during the fetal period suggests that complete maturation of nerve endings is not achieved before birth (Ozanics et al. 1977; K. Tervo and T. Tervo 1981).

The time course of corneal innervation has been followed in detail in the developing avian cornea (Bee 1982; Bee et al. 1986, 1988). Initially (embronic day 5), nerves extend around the cornea to enclose it within a perilimbal ring. In a second phase (embryonic day 11), nerves penetrate radially into the midcorneal stroma, branch extensively, and (p.153) innervate the epithelium at embryonic day 12. Nerves display extensive homology of position within the developing nerve ring, indicating that they follow specific pathways around the cornea. When bifurcation points of radial nerves are joined to its nearest neighbour on a line parallel of the cornea, a series of regular, non-overlapping, concentric circles is revealed (Bee et al. 1986). This suggests that the position of intracorneal nerve branching is also designated by the intracorneal milieu (Bee et al. 1986). Furthermore, the rate of radial growth of the eye and cornea determines the extension of nerves into the stroma (Bee and Locke 1992).

Corneal sensory nerves appear to be dependent on nerve growth factor (NGF) for their maturation as occurs with primary sensory neurones innervating other ectodermal tissues. NGF is produced by peripheral tissues and taken up by tyrosin kinase A (trkA), a high-affinity receptor present in sensory nerve terminals (Meakin and Shooter 1992). In transgenic mice deficient in trkA receptor (Smeyne et al. 1994), a very low number of corneal fibres was found. Furthermore, the blinking response of these animals to noxious mechanical stimuli, to saline at 0°C or 60°C, and to 10 mM acetic acid or 33 mM capsaicin was severely reduced (de Castro et al. 1995). Conversely, transgenic mice displaying an overproduction of NGF exhibited a hyperinnervation with a high density of abnormally thick corneal nerves (Crouch et al. 1995). This suggests that most corneal nociceptive neurones are NGF-dependent, although there is a small proportion of them that apparently do not depend on NGF for their development and behave functionally as polymodal nociceptive neurones (see below).

Functional types of corneal sensory neurones

General properties

Tower (1940) was the first to record the electrical activity of ocular sensory units evoked by mechanical stimulation of the cat corneal surface. Since then electrophysiological studies on corneal sensory afferents have been relatively scarce. Recordings have been performed mainly in anaesthetized cats (Lele and Weddell 1959; Giraldez et al. 1979; Belmonte and Giraldez 1981; Belmonte et al. 1991; Pozo et al. 1992; Gallar et al. 1993; Chen et al. 1995a) or rabbits (Tanelian and Beuerman 1984). Other experimental models include ’in vitro’ preparations of the isolated and perfused cornea of rat (Mark and Maurice 1977) or rabbit (Tanelian and Maclver 1990; MacIver and Tanelian 1993a, b) as well as preparations of the excised eye globe of the cat (Lele and Weddell 1959) or rat (Trimarchi 1967), or of the excised mouse eye conected to its trigeminal ganglion (Lopez de Armentia et al. 1995).

Mechanical stimulation has been employed routinely to localize the corneal receptive area of nerve filaments displaying multiple-or single-unit activity. Each of the ciliary nerves containing corneal sensory fibres covers a quadrant or more of the cornea and a variable area of the adjacent conjunctiva, with a considerable overlap (Tower 1940; Lele and Weddell 1959; Mark and Maurice 1977; Giraldez et al. 1979). Single units dissected from these nerve trunks also have large receptive fields with a size of 50–200 mm2 in the cat (Tower 1940) and 5–20 per cent of the corneal surface in the rabbit (Tanelian and Beuerman 1984). Receptive fields extend in most cases to the adjacent episclera, and partially overlap with those of neighbouring units. Figure 6.3 (A) shows the relative incidence of sensory fibres of the cat’s cornea with small receptive fields restricted to the (p.154) cornea (about 30 per cent) or with large receptive fields that extend into the limbus and adjacent episclera (70 per cent). Pure corneal fibres had slightly slower conduction velocities and gave a smaller response to acidic stimulation but were otherwise functionally similar to corneoscleral fibres (Belmonte et al. 1991).

Corneal nociceptors

Fig. 6.3 Mechanical response of corneal nociceptors. (A) Location and relative incidence of the receptive area of single corneal mechanosensory units. (B) Sample record of the impulse response evoked by a square-wave indentation of the cornea. Note the postdischarge at the end of the stimulus. (C) Instantaneous frequency response of a single unit to two square-wave indentations of increasing amplitude. (D) Fatigue of the mechanical response of the unit represented in (C). The number of impulses evoked per stimulus (black circles) is plotted versus the stimulus order for three different series of 5-s square-wave indentations at the amplitudes indicated by each bar. The interval was 5 s between successive pulses of the same amplitude and 3 min between two series at different amplitudes. White circles represent the number of impulses fired during the interstimulus periods. From Giraldez (1979) and Belmonte and Giraldez (1981).

Spontaneous ongoing activity is always present in multiunit recordings of the ciliary nerves (Tower 1940; Lele and Weddell 1959; Belmonte et al. 1971; Mark and Maurice1977). As will be discussed later, the contribution to this ongoing activity of the various functional classes of corneal sensory fibres is different. Furthermore, spontaneous activity also appears associated with the occurrence of corneal injury (see below).

Mechanosensory units

The existence of corneal units that respond exclusively to mechanical forces has been reported in the corneas of the cat and rabbit (Lele and Weddell 1959; Tanelian and Beuerman 1984; Belmonte et al. 1991; MacIver and Tanelian 1993b). These units belong to the highest conduction velocity group of corneal fibres and give large-amplitude, fast (p.155) action potentials. Pure mechanosensory units represent about 30 per cent of the population of the thin myelinated fibres innervating the cat’s cornea (Lele and Wedell 1959; Belmonte et al. 1991). In this species, the mechanical threshold of corneal mechanosensory fibres ranges between 0.11 and 1.96 mN with a mean value of 0.64 mN, which corresponds to a corneal indentation of about 40 um. The mechanical threshold of these fibres in the rabbit is 3–4 times higher (mean, 2.2 mN with a range between 1.7 and 2.5 mN; Tanelian and Beuerman 1984; Maclver and Tanelian 1993b). Mechanosensory fibres are more easily excited by a moving stimulus than by a sustained indentation (Mosso and Kruger 1973; Belmonte and Giraldez 1981). When indentations of increasing force are applied, the impulse response is composed of an accelerating discharge of spikes, whose duration, latency, and instantaneous frequency are roughly proportional to the amplitude and velocity of the stimulus (Fig. 6.3). In most units, long-lasting mechanical pulses cause a complete adaptation of the response.

Within the receptive field of a mechanosensitive unit, there are differences in threshold between the centre and the periphery. In corneo-episcleral units of the cat, the threshold is usually lowest in the limbus and is 2–3 times higher as the stimulus is moved into the sclera (Belmonte et al. 1991). Using selective electrical stimulation, MacIver and Tanelian (1993a) showed that the receptive fields of mechanosensory units have an elongated shape that corresponds to the trajectory of the fibre. Stimuli moving parallel to the long axis of the receptive area produced maximal activation, while perpendicular stimuli were less effective. This organization may provide a certain degree of directional sensitivity to this type of fibre.

Polymodal units

The existence of a separate population of Aδ and C corneal units responding to mechanical forces, temperature changes, and chemical agents was established through the application of controlled mechanical, thermal, and chemical stimuli to the cat’s cornea while recording single-unit activity of AS and C ciliary nerve afferents (Giraldez et al. 1979; Belmonte and Giraldez 1981; Belmonte et al. 1991; Gallar et al. 1993; Chen et al. 1995a). This class of fibres is the most abundant type of corneal sensory unit found in this species. Corneal polymodal units have large receptive fields (about 25 mm2), that often cover the adjacent episclera. They are usually silent at rest but may fire occasional spikes at very low frequency (0.06/s in AS fibres; 0.1/s in C-fibres) in the absence of intended stimulation or of corneal damage.

Response to mechanical forces

Polymodal units respond to mechanical stimulation of the cornea with an irregular discharge of impulses as do pure mechanosensory fibres. However, polymodal afferents often show spontaneous activity and a slightly lower mechanical threshold than that of mechanosensory units. Also, in response to a sustained mechanical indentation, they give a tonic, irregular discharge that persists throughout the stimulus with a variable degree of adaptation and whose frequency is roughly proportional to the intensity of the applied force. They also show a postdischarge after high-intensity stimuli and fatigue when these are repeated at short intervals (Fig. 6.3). All these response characteristics— tonic discharge, fatigue, and long-lasting postdischarge—are more prominent in unmyelinated (C) than in thin myelinated (AS) polymodal units (Gallar et al. 1993).

(p.156) Response to temperature changes

Heating of the cornea excites both AS and C polymodal units when temperatures over 38¬39°C are attained. The response to a sudden, suprathreshold temperature elevation consists of an accelerating train of impulses, whose frequency reaches a peak and then decays gradually to a lower, maintained level (Fig. 6.4 (A)). During sustained heating of the cornea, this impulse discharge is irregular. Temperature increases between threshold and noxious levels are encoded by proportional elevations of the mean firing frequency of the impulse discharge (Fig. 6.4 (D)). Returning to basal temperature stops firing transiently. Never¬theless, when noxious thermal levels have been exceeded, activity resumes a few seconds later as an irregular, low-frequency background impulse discharge (Fig. 6.4 (B, C); Giraldez et al. 1979; Belmonte and Giraldez 1981; Belmonte et al. 1991; Gallar et al. 1993).

Corneal nociceptors

Fig. 6.4 Response of corneal nociceptors to heating. (A) Sample recording of the impulse discharge of a corneal unit evoked by a temperature elevation of the corneal surface to 45°C. The same unit (as judged by the shape of the action potential shown at higher speed) was also recruited by a mechanical indentation applied to the receptive area. (C) Instantaneous frequency of the impulse discharge evoked in a corneal unit by a stepwise heating. Note the postdischarge at the end of the stimulus, after a brief silent period. (B) Peristimulus time histograms obtained in the same corneal unit showing the first (upper) and the second (lower) response to two identical stepwise heat stimuli separated by 3 min. (D) Mean stimulus—response relation of a population of eight corneal units in response to the first (black circles) and the second (open circles) stepwise heating. Bars are SEM. In the inset, the same data are plotted in a log-linear scale. From Belmonte and Giraldez (1981).

(p.157) Cold is usually ineffective in activating corneal polymodal units. Only a small proportion of AS fibres were weakly excited in the cat by temperature decreases within the noxious range (Belmonte and Giraldez 1981). In fact, temperature decreases below 20°C tend to diminish or silence background activity of polymodal afferents.

Response to chemicals

In the cat’s cornea, afferent units exhibiting sensitivity to mechanical stimuli (and to heat, when this stimulus was tested) were classified as polymodal when they responded also to acid and/or hyperosmotic NaC1 (Fig. Fig. 6.5 (A), (C)). The usefulness of NaC1 in predicting the sensitivity of nociceptive terminals to other chemical agents, such as those acting as putative excitants during injury or inflammation (Handwerker 1991), is questionable. Possibly, the high concentration of extracellular Na ± depolarizes small nerve terminals directly, by altering the distribution of charges across the membrane. In fact, only a fraction of corneal fibres that respond to NaC1 are also excited by acid (Belmonte et al. 1991; Gallar et al. 1993).

Protons appear to be a more selective stimulus for nociceptive endings. In the skin, low-threshold mechanosensory fibres are not consistently excited by application of acid, but acid causes a sustained excitation of thin sensory fibres and strong pain (Lindahl 1961; Keele 1962; Steen et al. 1992). Local decreases in pH have been obtained in the cornea by topical application on the corneal surface of solutions of increasing concentrations of acetic acid (down to pH 4.5), or of a gas jet of CO2 whose combination with water produces carbonic acid locally (Chen et al. 1995a). About 60 per cent of corneal fibres exhibiting mechanosensitivity also respond to acidic stimulation after a short latency, with an immediate discharge of impulses (Fig. 6.5 A, B) whose frequency is nearly proportional to proton concentration (Belmonte et al. 1991). Furthermore, when CO2 stimulation is employed, in addition to fibres that respond immediately, a small number of units produces an impulse discharge that appeared after a latency of several seconds (Chen et al. 1995a).

The site of action of protons in corneal nerve endings has not been established. Buffered solutions of the same pH made with acetic acid (which penetrates the cell membrane easily) or with less permeable citric acid have comparable excitatory effects on corneal nerve endings, suggesting an extracellular binding site for protons, presumably a non¬selective cationic channel (Belmonte et al. 1991). It has been hypothesized that capsaicin and protons may act at the same membrane site of the nociceptive ending (Rang 1991; Bevan and Geppetti 1994). In the cornea, capsaicin has an excitatory effect on both AS and C corneal polymodal units (Fig. 6.5 (D); Green and Tregear 1964; Belmonte et al. 1991; Gallar et al. 1993; Chen et al. 1996). At low concentrations (10−7 M) this excitatory action is not followed by inactivation. When higher doses (up to 33 × 10−3 M) are used, a complete inactivation to subsequent applications of the toxin and to mechanical stimulation, heat, or acid was observed in C-fibres (Belmonte et al. 1988, 1991; Gallar et al. 1993), while M units retained their mechanosensitivity but lost the responsiveness to thermal and chemical stimulation (Fig. 6.8 (A) (C); Belmonte et al. 1988, 1991). These data suggest that, in corneal polymodal nociceptors, sensitivity to protons and to mechanical stimuli are subserved by separate transduction mechanisms. On the other hand, the response of corneal polymodal units to low concentrations of capsaicin but not to protons, was blocked by capsazepine, a chemical analogue of capsaicin that acts as a (p.158) competitive antagonist (Chen et al., 1996). This observation speaks in favour of a distinct mechanism for the excitatory effects of protons and capsaicin on corneal polymodal fibres (see Garcia-Hirschfeld et al. 1995).

Corneal nociceptors

Fig. 6.5 Chemical response of corneal nociceptors. Frequency curves obtained with the pooled data of individual polymodal units responding to topical application to the cornea of: (A) acetic acid, 10 mM (n = 31); (B) a 98.5 per cent CO2 pulse for 30 s (2= 48); (C) 616 mM NaCl(n= 22); (D) 0.33 mM capsaicin (n =14). Time-scales: 0.5s for (A) and (C); 4s for B; 5s for (D). From Gallar et al. 1993, Chen et al. 1995.

In addition to protons, other chemical agents that may be released during corneal injury and/or inflammation exert a direct excitatory effect on polymodal terminals. Bradykinin (BK) is a kinin produced during inflammation with an apparently prominent role in the genesis of pain. It excites consistently polymodal nociceptive endings of the skin, muscle, joints, and testis Similarly, in the cornea of the cat, topical BK (le M) evokes an impulse discharge in about 80 per cent of M and C-polymodal fibres. This response outlasts the stimulation period and has a long latency and a slow onset, compared with other chemical stimuli, such as acid. Although a certain degree of tachyphylaxis is present, it does not seem to be as prominent in corneal nociceptors as in other preparations (Belmonte et al. 1994).

In the skin, the excitatory effects on nociceptors of inflammatory exudates have been reproduced with a mixture of BK, 5-hydroxytryptamine (5-FIT), histamine, prosta-glandin E2 (PGE2), SP (all at le M), and K+ (7 × 10−3M), at a pH of 7.0 (called inflammatory soup (IS); Kessler et al. 1992). As expected, application of IS to the cornea also excites polymodal nociceptors, in a more consistent and vigorous manner than when BK alone was applied.

(p.159) SP and CGRP are contained in sensory nerve terminals of the cornea and are presumably released antidromically during nerve excitation (Unger 1990; also see below). Although SP seems to contribute to the local inflammatory reaction, no impulse activity was evoked in AS and C corneal polymodal nociceptive fibres by topical application of SP at concentrations up to 10−3 M. Moreover, responses to mechanical and chemical stimuli or sensitization to heat were not changed by pretreat¬ment with this neuropeptide. The SP antagonists spantide and SP-150 did not vary neural activity or responsiveness of corneal nociceptors to acidic stimulation (Rebollo and Belmonte 1988). CGRP was equally ineffective in modifying spontaneous or evoked discharges of corneal polymodal units (Belmonte et al. 1994).

Mechanoheat units

The term mechanoheat nociceptor has been employed to characterize cutaneous nociceptors that are presumably polymodal, but in which chemosensitivity was not systematically explored (Meyer et al. 1994). In the cornea of the cat a small proportion of AS units presenting mechanical and thermal sensitivity but failing to respond initially to chemicals have been described. These fibres exhibited a high mechanical threshold and developed sensitivity to acid after repeated noxious heating (Fig. 6.6; Belmonte et al. 1991). Similarly, AS‘bimodal afferents’, responding to high-intensity mechanical forces

Corneal nociceptors

Fig. 6.6 Response of a mechanoheat unit to mechanical, chemical, and thermal stimulation and development of chemosensitivity by repeated heating. (A) Response to stimulation with a von Frey hair (horizontal bar) and absence of firing after the application of a drop of 10 mM acetic acid on the receptive area (arrow). (B) First (upper) and a second (lower) impulse response to two identical stepwise heat stimuli separated by 3 min. The lower trace shows the stimulus waveform. (C) Response to 10 mM acetic acid applied after heating. Time-scales for (A) and (B), 10 s; for C, 5 s. From Belmonte et al. (1991).

(p.160) and to heat, but not to acetylcholine (ACh) have been reported in the rabbit cornea (MacIver and Tanelian 1993b). The comparatively high mechanical threshold of mechanoheat fibres of the cornea may indicate that they are in reality polymodal nociceptors whose endings are more deeply located into the epithelium or in the stroma (Lele and Weddell 1956). If this is true, mechanoheat units would represent the fraction of polymodal nociceptors with the highest chemical threshold, rather than a specific subpopulation of nociceptive fibres (Adriaensen et al. 1983; Belmonte et al. 1991).

Cold units

Changes in multiunit activity of corneal nerves induced by temperature reductions were described in early reports (Lele and Weddell 1959; Trimarchi 1967; Mark and Maurice 1977). Tanelian and Beuerman (1984), using a saline jet at controlled temperature, detected the existence in the rabbit cornea of sensory units conducting in the C range that responded to decreases in temperature. Similar units have been identified in the cat’s cornea, where their functional properties have been studied in detail (Gallar et al. 1993). Cold-sensitive units are unmyelinated and fire spontaneously at the resting temperature of the cornea (around 33°C), giving an irregular discharge of impulses (0.75/s in the cat). They respond to cooling steps with a vigorous impulse discharge during the temperature drop, whose frequency is roughly proportional to the magnitude of the corneal

Corneal nociceptors

Fig. 6.7 Response of‘cold’ nociceptive units. (A)—(C) Firing response of a single unit in response to: (A) a drop of isotonic saline a 23°C; (B) touching with an ice-cooled bar (arrow indicates the moment of stimulus application, which is maintained throughout the remaining recording time); and (C) blowing with an air jet at room temperature. (D) impulse discharge in a‘cold’ nociceptive unit in response to constant-velocity cooling pulses of increasing amplitude applied with a contact thermode. (E) Average response of eleven units stimulated as in (D). Open squares: Response during the ramp. Filled squares: response during the steady-state part of the temperature pulse. From Gallar et al. (1993).

(p.161) temperature reduction (Fig. 6.7 (D), (E)). However, sustained low temperatures gave similar impulse discharges irrespective of their value, indicating that these fibres do not encode steady-state Corneal temperatures. In accordance with these response character¬istics, cold units fire repeatedly when cold air is blown to the cornea, or when a drop of cold saline is applied (Fig. 6.7 (A)—(C)).

The receptive fields of corneal cold units (about 10 mm2) are smaller than those of polymodal units and are preferentially found in the periphery of the cornea. Cold fibres do not respond consistently to mechanical stimulation, but have a weak response to acid and to hypertonic NaCI and are inactivated by 0.33 mM capsaicin.

Other types of corneal sensory units

The existence in the rabbit cornea of a specific population of chemosensory fibres, insensitive to mechanical or thermal stimuli has been suggested by Tanelian (1991) and Maclver and Tanelian (1993b). The chemosensitivity of such corneal fibres was based upon their excitation by ACh and also by a large battery of drugs that, in principle, cannot be considered natural chemical stimuli for corneal nociceptors (glutamate, N¬methyl-D-aspartate (NMDA)), associated with an absence of response to subsequent mechanical stimulation or heat. Unfortunately, the authors do not provide information on the conduction velocity of the explored fibres, or on the magnitude and duration of the evoked impulse response; neither do they report whether several drugs were assayed in the same unit and what doses were employed. Therefore, it is difficult to exclude the possibility that depolarizations were still obtained through the excitation by a drug of the regenerative area of the axon, in spite of a blockade of the transducing region of the fibre to other stimuli. It has long been known that unmyelinated fibres (such as those of the cornea) are more susceptible to chemicals than the myelinated ones, because their regenerative region is more exposed (Paintal 1964). When responsiveness to CO2 was systematically explored in the cat’s cornea in a population of 62 AS and C fibres, only three sensory units initially sensitive to a jet of CO2 could not be recruited by mechanical stimulation of the receptive field (Chen and Belmonte, unpublished results). With the available information, it seems premature to conclude that either these or the ACh-sensitive units found in the rabbit cornea represent pure chemosensory afferents, similar to mechanically insensitive (MIAs) or'silent’ nociceptors described in other territories (Schaible and Schmidt 1985; Meyer et al. 1994).

Limbal units

As was noted above, the margin of scleral conjunctiva immediately adjacent to the cornea (that is, the limbus) is innervated by collaterals of corneal axons (sclerocorneal units). Therefore, stimulation of the limbal portion of their receptive field excites both mechanosensory and polymodal sclerocorneal units. In addition, a small number of low-threshold mechanosensory units (mechanical threshold about 0.1 mN) have been found in the cat’s eye. These units have a very small receptive field, almost restricted to the limbus, and a comparatively high conduction velocity (about 20 m/s). They adapt rapidly, giving only 2–4 impulses in response to a sustained stimulation and are insensitive to heat or cold (Gallar 1991). It can be speculated that nerve fibres with specialized terminals described in the limbus (Lawrenson and Ruskell 1991) are the morphological correlate of limbal, low-threshold mechanosensory units.

(p.162) Also, cold fibres with functional properties somewhat different from those found in the cornea have been described in the limbal border and adjacent sclera of the cat’s eye (Gallar et al. 1993). Such cold units have small receptive fields (of about 4 mm2) and fire rhythmically at rest (33°C). They exhibit a dynamic and a tonic component in the impulse response caused by a sustained temperature reduction, thus resembling cold afferents of other territories (Hensel et al. 1960). Limbal cold receptors belong either to the low range of the M or to the C-fibre type and, in contrast with corneal cold units, they are insensitive to chemical stimulation.

Relationship between the morphological and functional characteristics of corneal afferents

No morphological specialization has been found in corneal nerve terminals associated with the various functional classes of corneal afferents. Neither has it been feasible to correlate the functional types of corneal afferents with their neuropeptide content, while there is only a broad relationship between sensory modality and the size and myelinization of afferent fibres of the cornea: the mechanosensory units of cat and rabbit are always myelinated, while corneal‘cold’ sensitive units are unmyelinated in both species (Tanelian and Beuerman 1984; Belmonte et al. 1991; Gallar et al. 1993). Nevertheless, polymodal fibres of the cat can be either thin myelinated or unmyelinated; this is only reflected in minor differences in responsiveness, which appear to be mainly associated with the size of the branching tree and with the diameter of the axon terminals but not with the transducing properties of the ending’s membrane (Gallar et al. 1993).

On the other hand, the branching pattern and diameter of corneal afferents determine some of the functional characteristics of their receptive field. This has been demonstrated through the combination of epifluorescence microscopy of living nerve endings with electrophysiological recordings of single fibres (MacIver and Tanelian 1993a). Living nerve terminals originating in identified M fibres, ran horizotally after traversing the basal lamina and formed elongated horizontal endings. Excitability maps in these fibres were composed of two to three continuous ridges that ran over the entire length of the elongated nerve endings observed by microscopy. On the other hand, C fibres produced nerve endings that form short branching clusters within the epithelium and end between the most superficial epithelial cells. In accordance with this morphological arrangement, the excitability map of C fibres was composed of spots that corresponded to clusters of nerve terminals separated by insensitive areas, with a variable density and number of excitability peaks per fibre.

Transduction mechanisms in corneal nociceptors

Corneal nociceptors possibly share common transduction mechanisms with nociceptors of other territories, as suggested by their morphological and functional similarities. Molecular and cellular processes involved in the transduction by nociceptive terminals of different territories to the various modalites of stimuli are still unknown to a great extent (Belmonte et al. 1994). The same is true for corneal nociceptors, where the evidence, albeit indirect, suggests that separate mechanisms exists for the transduction of chemical and mechanical stimuli (see above). As was proposed for other nociceptors, it is (p.163) conceivable that non-selective cationic channels are responsible for the depolarization of corneal terminals by noxious chemical and mechanical stimuli (Shepherd 1991).

Modification by injury and inflammation of corneal nociceptor responses

Once the cornea is damaged, a cascade of events results in an enhanced responsiveness of nociceptors (sensitization). The main difference between the cornea and other territories is that the absence of blood vessels prevents the rapid access of blood-borne substances to the injured area. However, polymorphonuclear leucocytes (PMNs) and mononuclear cells appear in the edges of corneal wound as early as 2–3 h after injury (Bazan 1990). Also, as a consequence of the insult, several biochemical changes take place in corneal tissue including the formation of several mediators of inflammation. The metabolites of arachidonic acid (AA) are generated through two major pathways, cyclooxygenase and lipooxygenase. However, epoxidation of AA by a microsomal cytochrome P450¬dependent mixed-function oxidase system has been also described in the cornea (Schwartzman et al. 1985, 1987). The basal synthesis of eicosanoids in the cornea is greatly increased after injury. Cyclooxygenase metabolites augment in the stroma and epithelium 2 h after a cryogenic injury of the cornea. The main metabolites in the epithelium are PGF20, and PGE2, while activated keratocytes of the stroma produce mainly PGI2 and thromboxane A2 (TXA2). A rapid increase of the lipoxygenase metabolites, 5-HETE and 12-HETE, particularly in the epithelium has been detected after injury (Bazan 1990). Epoxyeicosatrienoid acids are generated in the cornea through the cytochrome P450 system. Finally, synthesis of platelet-activating factor (PAF) occurs soon after corneal lesioning.

Sensitization of corneal nociceptors

The lipid inflammatory mediators generated by corneal cells, together with those released by activated PMNs and macrophages, are expected to contribute to nociceptor sensitization, as occurs in other territories (Handwerker and Reeh 1991). However, experimental information about the effect of these substances on corneal nociceptors is very scarce.

Sensitization of corneal nociceptors in vivo is seen in the cat when repeated'staircase’ heating pulses are applied to the receptive field of corneal polymodal units (Belmonte and Giraldez 1981; Gallar et al. 1993). In about 70 per cent of the AS and C corneal polymodal units, repetition of heat pulses over 40–45°C led to a 2–3 fold increase of the number of impulses evoked by a given temperature and a decrease of threshold of about 3°C. Also, poststimulus background activity appears (Fig. 6.4 (C)). No changes in mechanical threshold are apparent in heat-sensitized units. A proportion of the polymodal units do not develop sensitization or become totally or partially inactivated after repeated heating; this phenomenon is more prominent in C polymodal units.

Calcium ions seem to be necessary for sensitization to heat. When corneal nociceptors were superfused with a calcium-free medium, firing activity evoked by acid was preserved, but responses to heat and sensitization disappeared (Chen et al. 1994; Belmonte et al. 1994). Whether this is an effect on the nerve ending itself, on the epithelial cell, or on both cannot be inferred from these experiments.

Corneal mechanoheat fibres are also sensitized by repeated heating. Under these (p.164) circumstances, they become responsive to topical application of acetic acid (Fig. 6.6). This observation lends support to the hypothesis that mechanoheat units are polymodal nociceptors with a weak chemical sensitivity (Belmonte et al. 1991).

Only a few of the known mediators of corneal inflammation have been tested for their sensitizing effect on nociceptive nerve fibres of the cornea. PGE2 (10’7 to 10−4 M) increased spontaneous activity of M and C polymodal nociceptors of the cat in a dose-dependent manner (Gallar and Belmonte 1990). Furthermore, responses to topical application of 10 mM acetic acid were significantly enhanced after PG administration, thus suggesting that endogenous PGE2 released during corneal injury may be one of the substances contributing to sensitization of corneal polymodal nociceptors. The role of leukotrienes, PAF, and other putative inflammatory mediators generated during corneal inflammation in the sensitization process is still unexplored.

Electrical activity in corneal afferents following injury

Acute mechanical injury of the cornea elicits a high-frequency discharge in corneal polymodal nociceptors that is followed by a long-lasting afterdischarge Immediately after damage, threshold and near-threshold responses were depressed, but renewal of the ongoing activity was often produced by mild periliminal stimulation. Similar effects were observed when a strong acid (0.1 N HC1) was applied to the corneal surface (Belmonte and Giraldez 1981). At longer periods of time after mechanical wounding of the rabbit cornea (up to 7 days), spontaneous corneal nerve activity was not found to be markedly increased, although mild mechanical or chemical stimuli of the wound margin would elicit spontaneous firing that persisted for several hours. Also, an abnormal respon¬siveness to mechanical, thermal, and chemical stimuli was noticed: the borders of the wound showed an enhanced response to mechanical stimuli, whereas sensitivity was decreased in the centre of the wound. Application of hypertonic NaC1 or 40°C saline evoked a vigorous impulse response that outlasted the stimulus, whereas cold saline silenced this activity (Beuerman et al. 1985). It is conceivable that this abnormal activity originated from neuromas of the injured axons, from sprouting nerve endings, or from sensitized intact terminals. Depending on the depth of a corneal wound, the magnitude of corneal nerve damage will be different (Beuerman and Kupke 1982) and, when the stroma is also affected, nerve trunks of the subepithelial plexus are presumably injured. Sprouting terminals arising from undamaged nerves as well as neuromas formed by severed axons are present in the margin of the lesioned area (see below). It would be interesting to determine the relative contribution of these fibres to nerve activity in damaged corneas. Also, more detailed studies are required to establish the responsiveness of the different subtypes of corneal nociceptors affected by injury.

Corneal sensory afferents may also be excited by tissue disturbances not accompanied by direct nerve damage. For instance, acute ocular hypertension evokes an activation of corneal sensory units (Zuazo et al. 1986). Also hypoxia and hypoglycaemia increase the spontaneous discharge of corneal unmyelinated fibres (MacIver and Tanelian 1992).

Plasticity and regeneration of corneal nerve fibres after injury

The continuous shedding of corneal epithelium cells influences the arrangement and dynamics of corneal nerve terminals. Examination of corneal nerve afferents innervating (p.165) a selected territory of the intact corneal surface of mice after staining the nerves with the fluorescent dye 4-Di-2-ASP showed an extensive rearrangement of nerve terminals with time. Over a period of 24 h, changes in the position and appearance of epithelial endings were clear, although an overall similarity to initial configuration was retained. After 1 week, the architecture of terminal arborizations bears no resemblance with the initial branching pattern. In contrast, stromal nerves maintained a constant position (Harris and Purves 1989).

Sensory nerves of the cornea contain 43 kDa growth-assoctiated protein (GAP-43), a protein that is expressed in developing and regenerating neurones. This is co-localized with nerve-cell adhesion molecule (N-CAM). Moreover, an increase in the GAP-43 content of the corneal epithelium was noted after 24–48 h in corneas subjected to mild alkali injury. These data are interpreted as indicative of a synthesis of these proteins in the soma of sensory neurones to be carried by axonal transport to the cornea, where they will be involved in the continuous remodelling of corneal nerves (Martin and Bazan 1992).

Rearrangement of corneal sensory innervation also occurs when nerves are damaged by corneal injury or after experimental manipulations. Several studies have been devoted to analysing the remodelling of stromal and epithelial corneal nerves following corneal injury caused by epithelial wounding or by various surgical procedures developed for treatment of a heterogeneous group of ocular diseases (cataract removal, refraction defects, corneal opacities) that involve perilimbal or intracorneal incisions (radial or circular, with or without removal of a corneal bouton).

Small epithelial wounds of the cornea are covered in about 48 h by basal cells that migrate from the wound margin over the denuded area. In this type of wound (for instance, those produced by mechanical abrasion or by application of n-heptanol, which destroys only epithelium and intraepithelial nerves), a few terminals arriving from the subepithelial plexus outside the wound penetrate the wound in 2 days. Newly formed terminals originating at long distances occur irregularly around the wound margin in a radial fashion. Their density at the wound margin increases with time (from 39 fibres/ mm on day two to 55 fibres/mm by day seven; Beuerman and Kupke 1982; de Leeuw and Chan 1989). These fibres result from sprouting of fibres of the subepithelial and deeper stromal nerves located outside the wound area. With time, regenerating axons progress at the basoepithelial level towards the wound centre, either as single axons or adopting a leash-like pattern oriented towards the centre of the wound. Partial regeneration of the epithelial nerves is completed after 3–4 weeks with no further improvements up to 10 weeks afer wounding (Beuerman and Kupke 1982; Beuerman and Rózsa 1984; de Leeuw and Chan 1989).

When stromal corneal wounds are performed, 24 hours later the wound margins are completely surrounded by long, large-calibre sprouts, originating in neighbouring intact axons, that course perpendicularly to the wound border. They reach a maximum density in about 72 h (54 terminals/mm) A second wave of regeneration takes place at around 7 days, now originating in transected axons of the subepithelial plexus. This process is accompanied by secondary degeneration of the collateral sprouts of intact axons (Rózsa et al. 1983; Beuerman and Rózsa 1984). A reduced nerve density in relation to the intact cornea was still observed months afterwards. Essentially similar results have been described in nerve lesions caused by photokeratectomy, in which an accurate excision (p.166) of the corneal superficial stroma is performed with an excimer laser to modify the refractive power of the cornea (Pallicaris et al. 1990). In this case though, there is an increase in density of reinnervation in comparison with manually debrided or control corneas, with wider and longer leashes and a thicker subepithelial plexus, so that nerve density was significantly higher than normal 35 days after excimer laser surgery (Ishikawa et al. 1994; Trabucchi et al. 1994). This was accompanied by a significant enhancement of corneal sensitivity to mechanical stimulation (Ishikawa et al. 1994).

When penetrating perilimbal incisions covering half of the cornea are performed, such as those used in cataract or glaucoma surgery, the reinnervation process is much slower and incomplete. Nerve fibres distal to the lesion degenerate. New fibres enter the denervated area through the scar tissue, with minor contribution of axons originating in the innervated cornea (Rózsa et al. 1983). An abnormal and incomplete pattern of reinnervation persists more than 2 years later. Thus, it appears that the more proximal the nerve lesion, the more delayed and incomplete is the regeneration process. Also, in pure corneal incisions, the depth of the cut appears to be important in determining the degree of regeneration; this is incompletely achieved when it exceeds 50 per cent of the total corneal thickness (Chan-Ling et al. 1987, 1990). Similarly, corneal transplants in humans often remain devoid of nerve fibres for years and are invaded very slowly and incompletely by a reduced number of axons when reinnervation takes place (T. Tervo et al. 1985). This is not solely due to misalignment of Schwann cell channels by introduction of grafted tissue, because limited reinnervation also happens in cats in which a penetrating circular keratotomy was made. The formation of scar tissue appears to be a major obstacle for normal reinnervation (Chan-Ling et al. 1990).

Trophic interactions between the cornea and its innervation

The cornea, like other peripheral target tissues, appears to contribute to the survival and development of its sensory and autonomic innervation. Convincing experimental evidence of this trophic dependence of sensory neurones on corneal tissue was first obtained by Chan and Haschke (1981, 1982, 1985). These authors showed that neurone survival and neurite outgrowth were promoted by corneal and conjunctival epithelial cells co-cultured with trigeminal neurones (see also Garcia-Hirschfeld et al. 1994). This effect was attributed to the secretion by the epithelial cell of an‘epithelial neurono¬trophic factor’ (ENF), which also stimulated protein synthesis in cultured trigeminal neurones. A 2–4 times increase in the secretion of this ENF was reported after corneal wounding; such elevation was not parallel to the epithelial regeneration taking place during the first week of the wound closure process, but to the regeneration of intraepithelial nerves, which took place about 3 weeks after wounding (Chan et al. 1987).

There is additional experimental evidence supporting the promoting effect of corneal tissue on neurite outgrowth of corneal trigeminal neurones. In vivo, epithelial implants placed into the corneal stroma attract nerve spouts from deep stromal nerves and from the subepithelial plexus. These growing neurites penetrate the implant and form nerve terminals around epithelial cells, similar to those observed in normal epithelium (Emoto and Beuerman 1987). Also, when an epithelial lesion is made in the centre of the cornea, the subepithelial nerve leashes that in the intact rabbit cornea are predominantly oriented towards the nasal-most limbus, change their direction towards the wound (p.167) margin, thus suggesting that a wound-derived factor is attracting the regenerating neurites (de Leeuw and Chan 1989) Finally, substance P appears in avian corneal nerves concomitantly with their penetration into the epithelium (Bee et al. 1988), thus suggesting that the signal for the production of this neuropeptide by corneal sensory neurones is given by corneal epithelium.

Molecules influencing the development of neurones belong to various classes of agents, including: the neurotrophin family NGF; brain-derived neurotrophic factor (BDNF); and neurotrophins -3, -4, and-5 (NT-3, NT-4, NT-5)); ciliary neurotrophic factor (CNTF); other growth factors (for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF)); cell adhesion molecules (for example, N-CAM, cadherins); and components of the extracellular matrix (for example, fibronectin, laminin, collagens, thrombospondin, tenascin) (Hagg et al. 1993; Korsching 1993). Therefore, with the available evidence, it is difficult to determine the correspondence of the ENF described by Chan and Haschke with any of the identified trophic molecules, although these authors suggested that it was not identical to NGF or to extracellular matrix proteins that promote neurite extension. As mentioned above. NGF seems to play an important role in the development of corneal innervation in prenatal stages (de Castro et al. 1995). Whether this is also true for the survival and regeneration of adult corneal sensory neurones remains to be determined.

An inverse trophic dependence, namely, of the corneal epithelium cells on their sensory innervation, was suspected for a long time after the clinical observation that damage of the corneal innervation in human patients, either for therapeutic purposes (Gasserian ganglionectomy) or by accident, led to the appearance of severe lesions in the corneal epithelium called keratitis neuroparalytica (Pannabecker 1944). Also, destruc¬tion of corneal sensory neurones in more controlled experimental conditions has shown that several disturbances appear in the corneal epithelium: impaired cell attachment and altered epithelium structure (Alper 1976; Beuerman and Schimmelpfennig 1980; Araki et al. 1994); decreased mitotic rate (Sigelman and Friedenwald 1954; Mishima 1957);increased permeability (Beuerman and Schimmelpfennig 1980); reduction of glycogen content (Gilbard and Rossi 1990); and delayed wound healing rate (Araki et al. 1994). These alterations are conducive to recurrent corneal erosion of denervated corneas (Alper 1976).

The existence of trophic influences of sensory neurones on corneal epithelial cells has been shown experimentally in co-cultures of trigeminal neurones with corneal epithelial cells. When co-cultured with trigeminal sensory neurones, corneal epithelial cells increase their mitotic rate and number (Garcia-Hirschfeld et al. 1994) and express type VII collagen, a component of anchoring fibrils that adhere epithelial cells of the cornea to their basement membrane (Baker et al. 1993).

Indirect evidence suggests that such neurotrophic influences may be mediated at least in part, by neuropeptides contained in corneal sensory terminals. During embryonic development, the opaque cornea becomes transparent, a process that depends on the pumping of water by the epithelium cells; this phenomenon takes place in parallel with the appearance of SP in the nerves of the avian cornea (Bee et al. 1988). Capsaicin administered neonatally destroys a large amount of small peptidergic neurones in the trigeminal ganglion including those innervating the cornea; in these animals, the loss of (p.168) an important part of SP-and CGRP-containing nerves is accompanied by corneal signs of neuroparalytic keratitis; the severity of corneal lesions goes in parallel with the reduction of peptide-containing nerves (Buck et al. 1983; Fujita et al. 1984; Ogilvy and Borges 1990; Marfurt et al. 1993). Moreover, the healing rate of corneal epithelium wounds is delayed after retrobulbar injection of capsaicin (Gallar et al. 1990), a manoeuvre that blocks axoplasmic transport of neuropeptides in corneal nerves (Bynke 1983), while the healing rate is promoted by exogenous application of SP (Reid et al. 1990) Finally, SP enhanced the mitotic rate of corneal epithelium cells in culture (as did their co-culture with trigeminal neurones), while CGRP reduced it (Garcia-Hirschfeld et al. 1994). Therefore, there is experimental support for the possibility that SP and CGRP contained in sensory afferents modulate the functional activity of epithelial cells in the cornea, perhaps through antagonistic effects, thus contributing to the integrity of the normal cornea.

As was mentioned above, a sparse sympathetic innervation of the cornea has also been demonstrated by histochemical methods. Adrenergic fibres are abundant at the per¬iphery of the cornea, where they innervate limbal blood vessels. However, their functional role in the avascular cornea is unknown. While sympathectomy modifies corneal mitotic rate and corneal ion transport (Butterfield and Neufeld 1977; Klyce et al. 1985), electrical stimulation of the cervical sympathetic nerve for prolonged periods of time delays the healing time of experimental corneal wounds (Perez et al. 1987). In vitro, corneal epithelial cells increase their mitotic rate when they are co-cultured for long periods of time with sympathetic neurones (Garcia-Hirschfeld et al. 1994). These results suggest that sympathetic neurones may also participate in the trophic maintenance of corneal epithelium cells. In sympathectomized animals, a marked increase in CGRP immunoreactivity was observed, while in tissue subjected to sensory denervation the reverse was found (Unger et al. 1988). Moreover, in capsaicin-treated animals, a hyperreinnervation of the cornea by sympathetic sprouts has been described (Marfurt et al. 1993). This would reflect a trophic competition between sensory and adrenergic fibres in the cornea, as occurs in other territories (Kessler et al. 1983).

`Efferent’ actions of corneal nociceptors

Corneal irritation and wounding is often accompanied by corneal oedema and also by miosis, aqueous humour flare, photophobia, conjunctival vasodilatation, lid oedema, and lacrimation. A part of these symptoms are attributable to neurogenic inflammation, caused by antidromic release by corneal nerves of neuropeptides (SP and CGRP) that enhance local inflammatory processes (Jancsó et al. 1966; Unger 1990). Topical application to the cornea of capsaicin or nitrogen mustard evokes this irritation response, which can be attenuated by denervation or blockade of corneal nerves with local anaesthetics or tetrodotoxin (Jampol et al. 1975; Szolcsdnyi et al. 1975; Camras and Bito 1980a, b; Gonzalez et al. 1993, 1995). Diltiazem, which blocks the responsiveness of corneal fibres to chemical irritants, also reduces corneal neurogenic inflammation (Pozo et al. 1992; Gonzalez et al. 1993, 1995; Gallar et al. 1995). Corneal nerve fibres branching into the perilimbal conjunctiva and the root of the iris (Zuazo et al. 1986) may become excited antidromically during corneal irritation and contribute to the intense conjunc¬tival and iridal reaction developed during corneal injury. Also, photophobia (pain (p.169) caused by light) following corneal injuries is possibly mediated by these fibres, which will cause neurogenic inflammation and sensitization of nociceptive endings of the iris (Mintenig et al. 1995).

Effect of drugs on corneal nociceptors

Topical anaesthetic agents are used routinely to obtain corneal anaesthesia. These include among others tetracaine, procaine, benoxinate, and proxymetacaine (propar¬acaine) (Ritchie and Greene 1985). These agents eliminate propagated action potentials through their well-known blocking effects on axonal Na ± channels (Hille 1994). Interestingly, it has been reported that, in the cornea, lidocaine at low doses reduced multiunit, tonic discharges elicited by a corneal injury, while nerve impulses evoked by supramaximal electrical shocks persisted (Tanelian and Maclver 1991). However, this evidence is still insufficient to conclude that lidocaine at low concentrations has, in addition to the conventional blockade of propagated impulses, an additional selective effect on the ionic channels involved in the transduction process.

In spite of their usefulness for acute superficial anaesthesia, local anaesthetics have serious toxic effects on corneal epithelium and cannot be employed for prolonged elimination of neural activity evoked by a corneal wound. Pain is the main clinical problem in acute corneal lesions. Moreover, the recent development of refractive surgery techniques, which include extensive damage to corneal nerves, has prompted the demand for topical drugs to reduce the intense pain caused by this surgery. Non-steroidal anti-inflammatory drugs (NSAIDs) and morphine have been claimed to attenuate corneal pain when applied topically (Peyman et al. 1994; Epstein and Laurence 1994). There is conflicting evidence about a decrease of mechanical sensitivity of the cornea caused by topical administration of these agents to humans (Gwon et al. 1994; Peyman et al. 1994; Szerenyi et al. 1994). Flurbiprofen, diclofenac, and indomethacin applied to the cornea of the cat reduced spontaneous activity of polymodal nociceptors and their response to chemical stimulation with CO2 (Chen et al. 1995b). The effect appeared several minutes after application of the drugs and augmented with time (about 1 h); thus, the possibility exists that the decrease in the excitability of nociceptors was caused by a reduction in the production of arachidonic acid metabolites. A minor increase of mechanical threshold was also noticed in these experiments, which suggests an additional non-specific anaesthetic effect of these drugs. Morphine (0.5–5 mg/ml), applied topically, has been reported to diminish corneal sensibility in humans (Peyman et al. 1994). In the cat, topical morphine at 0.05–5 mg/ml concentrations reduced the mechanical and chemical responsiveness of both polymodal and mechanonociceptive corneal fibres, thus indicat¬ing that this drug has an anaesthetic effect on corneal sensory fibres (Chen et al. 1995c). Diltiazem, a calcium antagonist, at high concentrations (1 mM) blocks chemical responsiveness to acid of corneal polymodal units, without apparently reducing their mechanosensitivity (Pozo et al. 1992; Fig. 6.8 (D) (E)). This drug also decreases behavioural signs of pain evoked by irritant chemicals applied to the rabbit’s eye (Gonzalez et al. 1993). However, its efficacy on human ocular pain remains to be established.

Corneal nociceptors

Fig. 6.8 The effects of capsaicin and diltiazem on the response ofAO polymodal nociceptors to mechanical, chemical, and thermal stimulation. (A—C) Left, impulse discharges evoked in an intact fibre by: (A) 10 mM acetic acid; (B) stepwise heating; and (C) mechanical stimulation during the period indicated by the horizontal bar. Right, response of the unit to the same stimuli, applied after a 5-min pretreatment with 0.33 mM capsaicin. (D) Impulse discharge evoked by application of a pH 4.5 solution (left) or a mechanical stimulation (right) before and after pretreatment with 1 mM diltiazem. (E) Left, change in total number of impulses elicited by acid during a 30-s period, before and after 1 mM diltiazem in nine polymodal units; right, average mechanical threshold in the same units, before and after diltiazem. Error bars are SEM. Time-scale in nerve recordings, 5 s. From Belmonte et al. (1991) and Pozo et al. (1992).

(p.170)

Relationship between corneal nerves and sensation

It is a common experience that touching the cornea causes a brisk sensation of pain. However, the question whether other qualities of sensation could be evoked from the cornea by lower-intensity mechanical stimuli or by temperature changes, which was at the centre of the controversy over specificity of pain sensations, has not been answered completely. Psychophysical studies on corneal sensation are complicated by the extreme apprehension produced in experimental subjects by the approximation of any device to the front of the eye. Furthermore, any noxious stimuli of the cornea causes immediate and unavoidable blinking, even before the unpleasantness of the stimulus is consciously felt. Sensations experienced as a result of corneal stimulation are very difficult to describe and to compare with those elicited by analogous stimuli applied to the skin or mucosae.

(p.171) The loss of corneal sensibility has serious consequences for the integrity of corneal tissues; for this reason, clinicians have been mainly preoccupied by gross disturbances of corneal sensitivity. This interest prompted the development of several instruments aimed at quantifying corneal sensibility to mechanical stimuli. The most commonly used corneal aesthesiometers (Boberg-Ans 1956; Cochet and Bonnet 1960) are based on von Frey’s principle that the force required to buckle a long hair when it is pushed axially against the corneal surface is constant and proportional to the diameter of the hair and its length (von Frey 1922). Other, more sophisticated instruments have been developed to define with greater accuracy the magnitude of the mechanical force required to evoke a corneal sensation (Schirmer 1963a; Larson 1970; Draeger 1984; Weinstein et al. 1992). These used various types of probes directly placed on the cornea or an air jet, to apply a controlled force on the corneal surface. In all instances, the objective was to determine threshold to punctate mechanical stimulation of the different regions of the cornea or of the bulbar conjunctiva and its variations in a number of normal or pathological conditions.

The results of the numerous studies dedicated to this problem are essentially similar (Draeger 1984): Application of a mechanical probe to the cornea evokes an unpleasant sensation of touching that, with high intensities, becomes a sharp, jabbing pain outlasting the stimulus. The centre of the cornea has a lower mechanical threshold than the periphery and even more than the conjunctiva, with minor regional differences between the superior and inferior regions of the cornea (Boberg-Ans 1956; Cochet and Bonnet 1960; Millodot 1973; Norn 1973). These variations in corneal sensitivity appear to correlate well with the density of corneal innervation (Millodot et al. 1978; Reozsa and Beuerman 1982; Chan-Ling 1989). Also, mechanical sensitivity of the cornea decreases with age and, surprisingly, is higher in blue-eyed subjects (Millodot 1975). It is reduced at night and during pregnancy and menstruation in women, possibly associated to the presence of corneal oedema (Millodot and Lamont 1974; Millodot 1977). It is also lower in contact lens wearers (Schirmer 1963b), presumably due to a certain degree of corneal oedema due to chronic hypoxia and perhaps also to peripheral or central adaptation to continuous subthreshold stimulation of mechanosensory nerve fibres (Poise 1978; Millodot and O’Leary 1980; Tanelian and Beuerman 1980).

Corneal sensitivity is altered in a variety of clinical circumstances in which corneal innervation is disturbed. These include diabetes (Schwartz 1974), herpes simplex, herpes zoster, corneal and scleral inflammation (Lyne 1977; Metcalf 1982), corneal wounds (Zander and Weddell 1951) and ocular surgery accompanied by damage to corneal nerves, such as cataract extraction, radial keratotomy, excimer laser keratectomy (Biermann et al. 1992; Campos et al. 1992), or corneal transplantation (Zorab 1971; Rao et al. 1985). In these cases, reduction of corneal sensitivity is correlated with the degree of nerve damage and with the success of the regeneration of corneal nerves in the injured tissue (de Leeuw and Chan 1989; Mathers et al. 1988). Nevertheless, as noted above, transient (1–3 months) corneal hypersensitivity has been detected in the centre of excimer laser wounds, in spite of the fact that no intraepithelial neurites had reached the central wound at this time (Ishikawa et al. 1994). This may reflect abnormal sensitivity to mechanical stimuli of regenerating nerve fibres entering the wounded area as well as sensitization of surrounding intact nerve fibres.

The abundant research on clinical alterations of corneal mechanosensitivity was not (p.172) accompanied by a parallel interest in corneal sensations evoked by other modalities of stimuli. Only a few papers have been devoted to this topic in the literature of the last 40 years.

Lele and Weddell (1956) used warmed and cooled copper cylinders in direct contact with the cornea, air jets at different temperatures, and infrared radiation to stimulate the cornea of human volunteers and claimed that subjects were able to distinguish the temperature (warm or cold) of the applied stimulus. Kenshalo (1960) re-examined the corneal sensibility to temperature by applying to the human cornea, conjunctiva, and forehead skin the bulb of a warmed or cooled laboratory thermometer. Sensations evoked from the cornea by temperatures ranging between 20°C and 55°C were always described in terms of irritation and not of temperature, although subjects reported changes in the quality of the sensation at certain points of the temperature continuum, namely, when descending below 31°C and when reaching temperatures over 42°C. In oral reports, high temperature was experienced as a very sharp and irritating sensation, not unlike the one experienced by heating the skin but without a thermal component. Sensation experienced with cold appears to be very different; it was described as rather sharp but possessing a quality different from high temperatures. Yet, subjects refused to identify this different quality as the cool they experienced by stimulation of the skin or the conjunctiva. These observations were confirmed by Beuerman and co-workers (Beuerman et al. 1977; Beuerman and Tanelian 1979). They applied a jet of warmed or cooled saline to the eye, which was submerged in a bath, and showed that only sensations of irritation were evoked when the stimulus was restricted to the cornea.

Controlled chemical stimulation of the cornea has rarely been attempted. Diluted capsaicin has been dropped into the eye to determine the threshold concentration necessary to elicit a sensation of irritation; that was established at 6.0 × 10−8M (Dupuy et al. 1988). Also, hypertonic saline has been applied to detect ophthalmic nerve impairment (Mandahl 1993). A more accurate method has been developed by applying to the cornea a jet of CO2 at different concentrations (between 10 and 90 per cent) (Chen et al. 1995a). With this technique, a sensation of stinging pain was evoked when a threshold concentration around 40 per cent CO2 was attained. Higher concentrations produced a more intense sensation, which was also proportional to the duration of the stimulus.

A tentative correlation can be made between the functional type of corneal units recruited by the different modalities of stimuli in animal experiments and the corre¬sponding evoked sensation in psychophysical experiments in humans. Irritation elicited by mechanical stimulation is presumably due to the excitation of mechanosensory and polymodal nociceptive fibres. Both types of units have quite similar mechanical thresholds. Thus, undefined sensations of contact elicited by borderline mechanical stimulation appear to be due to a low firing frequency in the fraction of these two classes of fibres having the lowest mechanical threshold. Only mechanosensory limbal fibres appear to be suited to discriminate very-low-intensity, non-noxious mechanical stimuli but, because of their small receptive field and fast adaptation properties, they seem to be limited to detecting rapid stimuli, such as those produced by the sliding of the upper lid over the front of the eye.

Heat recruits mechanoheat and polymodal units, while protons are expected to stimulate only polymodal fibres. In both cases, the sensation evoked is one of irrita (p.173) tion. A good correspondence exists between the firing frequency in polymodal units of the cat cornea and the intensity of the sensations experienced in humans, both for increasing CO2 concentrations and for corneal temperature elevations (Fig. 6.9). On the other hand, thermal stimulation with temperatures below 30°C recruits exclusively‘cold’ corneal units and evokes a different kind of unpleasant sensation, suggesting that activation of the specific population of cold nociceptors is responsible for the peculiar irritation sensation evoked by low corneal temperatures. In contrast, sensations of cooling evoked by low temperatures acting more extensively on the front of the eye are presumably mediated by the abundant specific cold receptors, present in the limbal and perilimbal conjunctiva.

Corneal nociceptors

Fig. 6.9 The relationship between the amplitude of the noxious stimulus, the impulse frequency of polymodal fibres, and corneal sensation. (A) The stimulus—response curve obtained by stepwise heating of the cornea in nine corneal polymodal units of the cat (Belmonte and Giraldez 1981) has been plotted together with the verbal response profiles for the same temperature values obtained in humans by Beuerman et al. (1977; Beuerman and Tanelian 1979). (B) The mean firing response evoked by CO2 pulses of increasing concentration in 13 corneal polymodal fibres (circles) has been plotted together with the mean sensation of irritation measured with a visual analogue scale (triangles), evoked by the same CO2 pulses in seven human volunteers. From Belmonte et al. 1994; Chen et al. 1995a.

Concluding remarks

The cornea of the eye is innervated by unspecialized terminals of trigeminal sensory neurones, which show close functional similarities with the various subclasses of (p.174) nociceptors described in other ectodermal tissues. Mechanonociceptors, polymodal nociceptors, and‘cold’ nociceptors have been identified electrophysiologically in the cornea. Distinct types of irritation sensations are evoked by noxious mechanical, chemical, and thermal stimulation of the human cornea. These differences in the quality of pain sensation might be associated with a variable activation of the various subpopulations of corneal nociceptive afferents.

Furthermore, significant trophic interactions appear to exist between corneal sensory neurones and corneal tissue. The corneal epithelium appears to be a source of neurotrophic factors for its trigeminal sensory neurones. On the other hand, there is evidence that afferent nerve fibres contribute to maintain the integrity of the intact cornea and also participate in the repair processes activated by injury, both enhancing the inflammatory reaction (neurogenic inflammation) and stimulating the recovery of damaged tissues (migration and mitosis of epithelium cells). Substance P and CGRP, two neuropeptides that are present in corneal sensory fibres and are released upon injury, may possibly contribute to these effects.

Corneal irritation and pain are common problems associated with many clinical situations (accidental injury, surgery, contact lens wearing). Their incidence justifies a deeper knowledge of peripheral neural mechanisms involved in their genesis. The cornea is, in addition, an excellent model for morphological, biochemical, and electrophysio¬logical studies of the peripheral terminals of nociceptive neurones. Its structural simplicity and the absence of blood vessels facilitates the experimental manipulation of the nociceptive endings in a comparatively uncomplicated environment and makes the cornea an attractive preparation for this type of study.

Acknowledgements

The authors wish to express their deep appreciation to Drs Jennifer Laird and Fernando Cervero for helpful criticism and correction of the manuscript. Supported by the Plan Nacional de InvestigaciOn Cientifica y Desarrollo TecnolOgico (SAF93–0267), Spain.

References

Bibliography references:

Adriaensen, H., Gybels, J., Handwerker, H.O., and van Hees, J. (1983). Response properties of thin myelinated (A-delta) fibers in human skin nerves. J. Neurophysiol. 49, 111–22.

Alper, M.G. (1976). The anesthesic eye: an investigation of change in the anterior ocular segment of the monkey caused by interrupting the trigeminal nerve at various levels along its course. Trans. Am. Ophthalmol. Soc. 72, 323–65.

Araki, K., Ohashi, Y., Kinoshita, S., Hayashi, K., Kuwayama, Y., and Tano, Y. (1994). Epithelial wound healing in the denervated cornea. Curr. Eye Res. 13, 203–11.

Arvidson, B. (1977). Retrograde axonal transport of horseradish peroxidase from cornea to trigeminal ganglion. Acta neuropathol., Berlin 38, 49–52.

Attias, G. (1912). Die Nerven der Hornhaut des Menschen. v. Graefe’s Arch. Ophthalmol. 83, 14–316.

Baker, K.S., Anderson, S.C., Romanowski, E.G., Thoft, R.A., and SundarRaj, N. (1993). Trigeminal ganglion neurons affect corneal epithelial phenotype. Influence on type VII collagen expression in vitro. Invest. Ophthalmol. Vis. Sci. 34, 137–144.

Bazan, H E P(1990). Response of inflammatory lipid mediators following corneal injury. In Lipid mediators in eye inflammation (ed. N.G. Bazan), pp. 1–11. Karger, Basel.

(p.175) Beckers, H.J.M., Klooster, J., Vrensen, G.F.J.M., and Lamers, W.P.M.A. (1992). Ultrastructural organization of trigeminal nerve endings in the rat cornea and iris. Invest, Ophthalmol. Vis. Sci. 33, 1979–86.

Beckers, H.J.M., Klooster, J., Vrensen, G.F.J.M., and Lamers, W.P.M.A. (1993). Substance P in rat corneal and iridal nerves: An ultrastructural immunohistochemical study. Ophthalmic Res. 25, 192–200.

Beding-Barnekow, Brodin, E., and Hakanson, R. (1988). Substance P, neurokinin A and neurokinin B in the ocular responses to injury in rabbits. Br. J. Pharmacol. 95, 259–67.

Bee, J.A. (1982). The development and pattern of innervation of the avian cornea. Develop. Biol. 92, 5–11.

Bee, J.A. and Roche, S.M. (1992). Reducing intraocular pressure by intubation elicits precocious development and innervation of the embryonic chick cornea. Invest. Ophthalmol. Vis. Sci. 33, 3469–78.

Bee, J.A. Hay, R.A., Lamb, E.M., Devore, J.J., and Conrad, G.W. (1986). Positional specificity of corneal nerves during development. Invest. Ophthalmol. Vis. Sci. 27, 38–43.

Bee, J.A., Kuhl, U., Edgar, D., and von der Mark, K. (1988). Avian corneal nerves: co-distribution with collagen type IV and acquisition of substance P immunoreactivity. Invest. Ophthalmol. Vis. Sci. 29, 101–7.

Belmonte, C. and Giraldez, F. (1981). Responses of cat corneal sensory receptors to mechanical and thermal stimulation. J. Physiol. 321, 355–68.

Belmonte, C., Simon, J., and Gallego, A. (1971). Effects of intraocular pressure changes on the afferent activity of the ciliary nerves. Exp. Eye Res. 12, 342–55.

Belmonte, C., Gallar, J., Pozo, M.A., and Rebollo, I. (1988). Effects of capsaicin on the neural discharge of corneal sensory fibres in the anaesthetized cat. J. Physiol. 401, 57P.

Belmonte, C., Gallar, J., Pozo, M.A., and Rebollo, I. (1991). Excitation by irritant substances of sensory afferent units in the cat’s cornea. J. Physiol. 437, 709–25.

Belmonte, C., Gallar, J., Lopez-Briones, L.G., and Pozo, M.A. (1994). Polymodality in noci¬ceptive neurons: Experimental models of chemotransduction. In Cellular mechanisms of sensory processing, NATO series, Vol. H 79 (ed. L. Urban), pp. 87–117. Springer-Verlag, Berlin.

Bessou, P. and Perl, E.R. (1969). Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J. Neurophysiol. 32, 1025–43.

Beuerman, R.W. and Kupke, K. (1982). Neural regeneration following experimental wounds of the cornea in the rabbit. In The structure of the eye (ed. J.G. Hollyfield), pp. 319–30. Elsevier North Holland, Amsterdam.

Beuerman, R.W. and Rózsa, A.J. (1984). Collateral sprouts are replaced by regenerating neurites in the wounded corneal epithelium. Neurosci. Lett. 44, 99–104.

Beuerman, R.W. and Schimmelpfennig, B. (1980). Sensory denervation of the rabbit cornea affects epithelial properties. Exp. Neurol. 69, 196–201.

Beuerman, R.W. and Tanelian, D.L. (1979). Corneal pain evoked by thermal stimulation. Pain 7, 1–14.

Beuerman, R.W., Maurice, D.M., and Tanelian, D.L. (1977). Thermal stimulation of the cornea. In Pain in the trigeminal region (ed. D. Anderson and B. Matthews), pp. 422–3. Elsevier, Amsterdam.

Beuerman, R.W., Rózsa, A.J., and Tanelian, D.L. (1985). Neurophysiological correlates of post traumatic acute pain. In Advances in pain research and therapy, Vol. 9 (ed. H.L. Fields, R. Dubner, and F. Cervero), pp. 73–81. Raven Press, New York.

Bevan, S. and Geppetti, P. (1994). Protons: small stimulants of capsaicin-sensitive sensory nerves. Trends Neurosci. 17, 509–12.

Biermann, H., Grabner, G., Baumgartner, I., and Reim, M. (1992). Zur Hornhautsensibilitdt nach Epikeratophakie. KIM. Mbl. Augenheilk. 201, 18–21.

Boberg-Ans, J. (1956). On the corneal sensitivity. Acta Ophthalmol. 34, 149–62.

(p.176) Buck, S.H. and Burks, T.F. (1986). The neuropharmacology of capsaicin: review of some recent observations. Pharmacol. Rev. 38, 179–226.

Buck, S.H., Walsh, J.H., Davis, T.P., Brown, M.R., Yamamura, H.1., and Burks, T.F. (1983). Characterization of the peptide and sensory neurotoxic effects of capsaicin in the guinea pig. J. Neurosci. 3, 2064–74.

Butterfield, L.C. and Neufeld, A.H. (1977). Cyclic nucleotides and mitosis in the rabbit cornea following superior cervical ganglionectomy. Exp. Eye Res. 25, 427–33.

Bynke, G. (1983). Capsaicin pretreatment prevents disruption of the blood-aqueous barrier in the rabbit eye. Invest. Ophthalmol. Vis. Sci. 24, 744–8.

Bynke, G., Hakanson, R., and Sundler, F. (1984). Is substance P necessary for corneal nocicep¬tion? Invest. Ophthalmol. Vis. Sci. 24, 744–8.

Campos, M., Hertzog, L., Garbus, J.J., and McDonnell, P.J. (1992). Corneal sensitivity after photorefractive keratectomy. Am. J. Ophthalmol. 114, 51–4.

Camras, C.B. and Bito, L.Z. (1980a). The pathophysiological effects of nitrogen mustard on the rabbit eye. I. The biphasic intraocular pressure response and the role of prostaglandins. Exp.Eye Res. 30, 41–52.

Camras, C.B. and Bito, L.Z. (1980b). The pathophysiological effects of nitrogen mustard on the rabbit eye. II. The inhibition of the initial hypertensive phase by capsaicin and the apparent role of substance P. Invest. Ophthalmol. Vis. Sci. 19, 423–8.

Chan, K.Y. and Haschke, R.H. (1981). Action of atrophic factor(s) from rabbit corneal epithelial culture on dissociated trigeminal neurons. J. Neurosci. 1, 1155–62.

Chan, K.Y. and Haschke, R.H. (1982). Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons. Exp. Eye Res. 35, 137–56.

Chan, K.Y. and Haschke, R.H. (1985). Specificity of a neuronotrophic factor from rabbit corneal epithelial cell cultures. Exp. Eye Res. 41, 687–99.

Chan, K.Y., Jones, R.R., Brak, D.H., Swift, J., Parker, J.A., and Haschke, R.H. (1987). Release of neuronotrophic factor from rabbit corneal epithelium during wound healing and nerve regeneration. Exp. Eye Res. 45, 633–46.

Chan-Ling, T. (1989). Sensitivity and neural organization of the cat cornea. Invest Ophthalmol.Vis. Sci. 30, 1075–82.

Chan-Ling, T., Tervo, K., Tervo, T., Vannas, A., Holden, B., and Erankii, L. (1987). Long-term neural regeneration in the rabbit following 180° limbal incision. Invest. Ophthalmol. Vis. Sci. 28, 2083–8.

Chan-Ling, T., Vannas, A., Holden, B.A., and O’Leary, D.J. (1990). Incision depth affects the recovery of corneal sensitivity and neural regeneration in the cat. Invest. Ophthalmol. Vis. Sci. 31, 1533–41.

Chen, X., Gallar, J., and Belmonte, C. (1994). Influence of calcium ions on excitation and sensitisation of corneal nociceptors. Eur. J. Neurosci. Suppl. 7, 133.

Chen, X., Gallar, J., Pozo, M.A., Baeza, M., and Belmonte, C. (1995a). CO2 stimulation of the cornea: a comparison between human sensation and nerve activity in polymodal nociceptive afferents of the cat. Eur. J. Neurosci. 7, 1154–63.

Chen, X., Gallar, J., and Belmonte, C. (1995b). Effects of nonsteroidal antiinflammatory drugs on chemical sensitivity of corneal nociceptors. Eur. J. Neurosci. Suppl. 8, 31.

Chen, X., Gallar, J., and Belmonte, C. (1995c). Effects of morphine on the response of pain corneal fibers to chemical and mechanical stimulation. JERMOV Meeting, Montpellier, France.

Chen, X., Belmonte, C., and Rang, H.P. (1996). Capsaicin and carbon dioxide act by distinct mechanisms on sensory nerve terminals in the cat cornea. Pain (submitted).

Cochet, P. and Bonnet, R. (1960). L’esthesie corneene. La Clin. Ophthalmol. 4, 3–27.

Colin, S. and Kruger, L. (1986). Peptidergic nociceptive axon visualisation in whole-mount preparations of cornea and tympanic membrane in rat. Brain Res. 398, 199–203.

Crouch, D.S., Davis, B.M., Albers, K.M., and Rickman, D.W. (1995). Nerve growth factor regulation of corneal sensory innervation. Invest. Ophthalmol. Vis. Sci. 36(suppl.), 574.

(p.177) de Castro, F., Silos-Santiago, I., Lopez de Armentia, M., Barbacid, M., and Belmonte, C. (1995). Corneal innervation in mice lacking nerve growth factor (NGF) receptor (trkA knockouts). Invest. Ophthalmol. Vis. Sci. 36 (suppl.), 574.

de Leeuw, M. and Chan, K.Y. (1989). Corneal nerve regeneration. Correlation between morphology and restoration of sensitivity. Invest. Ophthalmol. Vis. Sci. 30, 1980–90.

Dixon, A.D. (1963). Fine structure of nerve-cell bodies and satellite cells in the trigeminal ganglion. J. dent. Res:42, 990–9.

Draeger, J. (1984). Corneal sensitivity: measurement and clinical importance. Springer Verlag, Vienna.

Duce, I.R. and Keen, P. (1977). An ultrastructural classification of the neuronal cell bodies of the rat dorsal root ganglion using zinc iodide-osmium impregnation. Cell Tissue Res. 185, 263–77.

Dupuy, B., Thompson, H., and Beuerman, R.W. (1988). Capsaicin: a psychophysical tool to explore corneal sensitivity. Invest. Ophthalmol. Vis. Sci. 29(suppl.), 454.

Ehinger, B. (1966). Ocular and orbital vegetative nerves. Acta physiol. scand. 67(suppl.), 1–35. Ehinger, B. and Sjoberg, N.-O. (1971). Development of the ocular adrenergic nerve supply in man and guinea pig. Z. Zelllforsch. 118, 579–92.

Emoto, I. and Beuerman, R.W. (1987). Stimulation of neurite growth by epithelial implants into corneal stroma. Neurosci. Lett. 82, 140–4.

Epstein, R.L. and Laurence, E.O. (1994). Effect of topical diclofenac solution on discomfort after radial keratotomy. J. Cataract Refract. Surg. 20, 378–80.

Fujita, S., Shimizu, T., Izumi, K., Fukuda, T., Sameshima, M., and Ohba, N. (1984). Capsaicin induced neuroparalytic-like corneal changes in the mouse. Exp. Eye Res. 38, 165–75.

Gallar, J. (1991). CaracterizaciOn electrofisiOlogica de los receptores sensoriales del ojo del gato.Unpublished PhD thesis, University of Alicante, Spain.

Gallar, J. and Belmonte, C (1990). Modulation by prostaglandins of nervous activity in corneal nociceptors. Invest. Ophthalmol. Vis. Sci. 31(suppl.), 514.

Gallar, J., Pozo, M.A., Rebollo, I., and Belmonte, C (1990). Effects of capsaicin on corneal wound healing. Invest. Ophthalmol. Vis. Sci. 31, 1968–74.

Gallar, J., Pozo, M.A., Tuckett, R.P., and Belmonte, C (1993). Response of sensory units with unmyelinated fibres to mechanical, thermal and chemical stimulation of the cat’s cornea. J. Physiol. 468, 609–22.

Gallar, J., Garcia de la Rubia, P., Gonzalez, G.G., and Belmonte, C (1995). Irritation of the anterior segment by ultraviolet radiation: influence of nerve blockade and calcium antagonists. Curr. Eye Res. 14, 827–35.

Gamse, R., Leeman, S., Holzer, P., and Lembeck, F. (1981). Differential effects of capsaicin on the content of somatostatin, substance P and neurotensin in the nervous system of the rat. Naunyn¬Schmiedberg’s Arch. Pharmacol. 317, 140–8.

Garcia-Hirschfeld, J., Lopez-Briones, L.G., and Belmonte, C. (1994). Neurotrophic influences on corneal epithelial cells. Exp. Eye Res. 59, 597–605.

Garcia-Hirschfeld, J., Lopez-Briones, L.G., Belmonte, C., and Valdeolmillos, M. (1995). Intra¬cellular free calcium responses to protons and capsaicin in cultured trigeminal neurons. Neuroscience 67, 235–43.

Gilbard, J.P. and Rossi, S.R. (1990). Tear film and ocular surface changes in a rabbit model of neurotrophic keratitis. Ophthalmology 97, 308–12.

Giraldez, F. (1979). Estudio electrofisiolOgico de los receptores sensoriales de la cornea del gato. Unpublished PhD thesis. University of Valladolid, Spain.

Giraldez, F., Geijo, E., and Belmonte, C. (1979). Response characteristics of corneal sensory fibers to mechanical and thermal stimulation. Brain Res. 177, 571–6.

Gonzalez, G.G., Garcia de la Rubia, P., Gallar, J., and Belmonte, C. (1993). Reduction of capsaicin-induced ocular pain and neurogenic inflammation by calcium antagonists. Invest. Ophthalmol. Vis. Sci. 34, 3329–35.

(p.178) Gonzalez, G.G., Gallar, J., and Belmonte, C. (1995). Influence of diltiazem on the ocular irritative response to nitrogen mustard. Exp. Eye Res. 61, 205–12.

Gotz, R. (1972). Zwei neue Instrumente fiir die Untersuchung der Hornhautsensibilitat. Klin. Mbl. Augenheilk 161, 469–74.

Green, D.M. and Tregear, R.T. (1964). The action of sensory irritants on the cat’s cornea. J. Physiol. 175, 37P.

Gregg, J.M. and Dixon, A.D. (1973). Somatotopic organization of the trigeminal ganglion in the rat. Arch. oral Biol. 18, 487–98.

Grimes, P. and von Sallmann, L. (1960). Comparative anatomy of the ciliary nerves. Arch. Ophthalmol. 64, 81–91.

Gwon, A., Vaughan, E.R., Cheetham, J.K., and DeGryse, R. (1994). Ocufen (Flurbiprofen) in the treatment of ocular pain after radial keratotomy. CLAO J. 20, 131–8.

Hagg, T., Louis. J-C., and Varon, S. (1993). Neurotrophic factors and CNS regeneration. In Neuroregeneration (ed. A. Gorio), pp. 265–87. Raven Press, New York.

Handwerker, H.O. (1991). What peripheral mechanisms contribute to nociceptive transmission and hyperalgesia? In Towards a new pharmacology of pain (ed. A.I. Basbaum and J.M. Besson), pp. 5–19. John Wiley & Sons, New York.

Handwerker, H.O. and Reeh, P.W. (1991). Pain and inflammation. In Pain research and clinical management Proceedings of the 6th World Congress on Pain, Vol. 4, pp. 59–70. (ed. M.R. Bond, J.E. Charlton, and C.J. Wolf). Elsevier, Amsterdam.

Harris, L.W. and Purves, D. (1989). Rapid remodelling of sensory endings in the corneas of living mice. J. Neurosci. 9, 2210–14.

Hensel, H., Iggo, A., and Witt, I. (1960). A quantitative study of sensitive cutaneous thermo¬receptors with C afferent fibres. J. Physiol. 153, 113–26.

Hille, B. (1994). Ionic channels of excitable membranes. Sinauer, Sunderland, Massachusetts.

Hoyes, A.D. and Barber, P. (1976). Ultrastructure of corneal nerves in the rat. Cell Tissue Res. 172, 133–44.

Ichikawa, H., Milani, S., Hijiya, H., Nakago, T., Jacobowittz, D.M., and Sugimoto, T. (1933). Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat. Brain Res. 629, 231–8.

Iggo, A. (1963). An electrophysiological analysis of afferent fibres in primate skin. Acta Neuroveg. 24, 225–40.

Ishida, N., del Cerro, M., Rao, G.N., Mathe, M., and Aquavella, J.V. (1984). Corneal stromal innervation. A quantitative analysis of distribution. Ophthalmic Res. 16, 139–44.

Ishikawa, T., del Cerro, M., Liang, F-Q., Loya, N., and Aquavella, J.V. (1994). Corneal sensitivity and nerve regeneration after laser ablation. Cornea 13 225–31.

Jampol, L.M., Neufeld, A.H., and Sears, M.L. (1975). Pathways of the response of the eye to injury. Invest. Ophthalmol. Vis. Sci. 14, 184–9.

Jane* N., Jancsó-Gabor„ A., and Szoksanyi, J. (1966). The role of sensory nerve endings in neurogenic inflammation induced in human skin and in the eye and paw of the rat. Br. J. Pharmacol. Chemother. 33, 32–41.

Keele, C.A. (1962). The common chemical sense and its receptors. Arch. int. Pharmacodyn. 139, 547–57. Kenshalo, D.R.(1960). Comparison of thermal sensitivity of the forehead, lip, conjunctiva and cornea. J. appl. Physiol. 15, 987–91.

Kessler, J.A., Bell, W.O., and Black, I.O. (1983). Interactions between the sympathetic and sensory innervation of the iris. J. Neurosci. 3, 1301–07.

Kessler, W., Kirchhoff, C., Reeh, P.W., and Handwerker, H.O. (1992). Excitation of cutaneous afferent nerve endings in vitro by a combination of inflammatory mediators and conditioning effect of substance P. Exp. Brain Res. 91, 467–76.

Klyce, S.D., Beuerman, R.W., and Crosson, C.E. (1985). Alteration of corneal epithelial ion transport by sympathectomy. Invest. Ophthalmol. Vis. Sci. 26, 434–42.

Korsching, S. (1993). The neurotrophic factor concept: a re-examination. J. Neurosci. 13, 2739–48.

Gonzalez, G.G., Gallar, J., and Belmonte, C. (1995). Influence of diltiazem on the ocular irritative response to nitrogen mustard. Exp. Eye Res. 61, 205–12.

Gotz, R. (1972). Zwei neue Instrumente fiir die Untersuchung der Hornhautsensibilitat. Klin. Mbl. Augenheilk 161, 469–74.

Green, D.M. and Tregear, R.T. (1964). The action of sensory irritants on the cat’s cornea. J. Physiol. 175, 37P.

Gregg, J.M. and Dixon, A.D. (1973). Somatotopic organization of the trigeminal ganglion in the rat. Arch. oral Biol. 18, 487–98.

Grimes, P. and von Sallmann, L. (1960). Comparative anatomy of the ciliary nerves. Arch. Ophthalmol. 64, 81–91.

Gwon, A., Vaughan, E.R., Cheetham, J.K., and DeGryse, R. (1994). Ocufen (Flurbiprofen) in the treatment of ocular pain after radial keratotomy. CLAO J. 20, 131–8.

Hagg, T., Louis. J-C., and Varon, S. (1993). Neurotrophic factors and CNS regeneration. In Neuroregeneration (ed. A. Gorio), pp. 265–87. Raven Press, New York.

Handwerker, H.O. (1991). What peripheral mechanisms contribute to nociceptive transmission and hyperalgesia? In Towards a new pharmacology of pain (ed. A.I. Basbaum and J.M. Besson), pp. 5–19. John Wiley & Sons, New York.

Handwerker, H.O. and Reeh, P.W. (1991). Pain and inflammation. In Pain research and clinical management Proceedings of the 6th World Congress on Pain, Vol. 4, pp. 59–70. (ed. M.R. Bond, J.E. Charlton, and C.J. Wolf). Elsevier, Amsterdam.

Harris, L.W. and Purves, D. (1989). Rapid remodelling of sensory endings in the corneas of living mice. J. Neurosci. 9, 2210–14.

Hensel, H., Iggo, A., and Witt, I. (1960). A quantitative study of sensitive cutaneous thermo¬receptors with C afferent fibres. J. Physiol. 153, 113–26.

Hille, B. (1994). Ionic channels of excitable membranes. Sinauer, Sunderland, Massachusetts. Hoyes, A.D. and Barber, P. (1976). Ultrastructure of corneal nerves in the rat. Cell Tissue Res. 172, 133–44.

Ichikawa, H., Milani, S., Hijiya, H., Nakago, T., Jacobowittz, D.M., and Sugimoto, T. (1933). Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat. Brain Res. 629, 231–8.

Iggo, A. (1963). An electrophysiological analysis of afferent fibres in primate skin. Acta Neuroveg. 24, 225–40.

Ishida, N., del Cerro, M., Rao, G.N., Mathe, M., and Aquavella, J.V. (1984). Corneal stromal innervation. A quantitative analysis of distribution. Ophthalmic Res. 16, 139–44.

Ishikawa, T., del Cerro, M., Liang, F-Q., Loya, N., and Aquavella, J.V. (1994). Corneal sensitivity and nerve regeneration after laser ablation. Cornea 13225–31.

Jampol, L.M., Neufeld, A.H., and Sears, M.L. (1975). Pathways of the response of the eye to injury. Invest. Ophthalmol. Vis. Sci. 14, 184–9.

Jancsó N., Jancsó-Gabor„ A., and Szoksanyi, J. (1966). The role of sensory nerve endings in neurogenic inflammation induced in human skin and in the eye and paw of the rat. Br. J. Pharmacol. Chemother. 33, 32–41.

Keele, C.A. (1962). The common chemical sense and its receptors. Arch. int. Pharmacodyn. 139, 547–57. Kenshalo, D.R.(1960). Comparison of thermal sensitivity of the forehead, lip, conjunctiva and cornea. J. appl. Physiol. 15, 987–91.

Kessler, J.A., Bell, W.O., and Black, I.O. (1983). Interactions between the sympathetic and sensory innervation of the iris. J. Neurosci. 3, 1301–07.

Kessler, W., Kirchhoff, C., Reeh, P.W., and Handwerker, H.O. (1992). Excitation of cutaneous afferent nerve endings in vitro by a combination of inflammatory mediators and conditioning effect of substance P. Exp. Brain Res. 91, 467–76.

Klyce, S.D., Beuerman, R.W., and Crosson, C.E. (1985). Alteration of corneal epithelial ion transport by sympathectomy. Invest. Ophthalmol. Vis. Sci. 26, 434–42.

Korsching, S. (1993). The neurotrophic factor concept: a re-examination. J. Neurosci. 13, 2739–48. Carlos Belmonte and Juana Gallar179

(p.179) Kuwayama, Y., Terenghi, G., Polak, J.M., Trojanowski, J.Q., and Stone, R.A. (1987). A quantitative correlation of substance P-, calcitonin gene-related peptide-and cholecystoki¬nin-like immunoreactivity with retrogradely labelled trigeminal ganglion cells innervating the eye. Brain Res. 405, 220–6.

Larson, W.L. (1970). Electro-mechanical corneal esthesiometer. Br. J. Ophthalmol. 54, 342–7.

Laties, A. and Jacobowitz, D. (1964). A histochemical study of the adrenergic and cholinergic innervation of the anterior segment of the rabbit eye. Invest. Ophthalmol. Vis. Sci. 3, 592–600.

LaVail, J., Johnson, W.E., and Spencer, L.C. (1993) Immunohistochemical identification of trigeminal ganglion neurons that innervate the mouse cornea: relevance to intercellular spread of herpes simplex virus. J. comp. Neurol. 327, 133–40.

Lawrenson, J.G. and Ruskell, G.L. (1991). The structure of corpuscular nerve endings in the limbal conjunctiva of the human eye. J. Anat. 177, 75–84.

Lee, Y., Kawai, Y., Shiosaka, S., Takami, K., Kiyama, H., Hillyard, C.J., Girgis, S., MacIntyre, I., Emson, P.C., and Tohyama, M. (1985). Coexistence of calcitonin gene-related peptide and substance P-like in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis. Brain Res. 330R, 194–6.

Lehtosalo, J.I. (1984). Substance P-like immunoreactive trigeminal ganglion cells supplying the cornea. Histochemistry 80, 273–6.

Lehtosalo, J.I., Uusitalo, H., Stjernschantz, J., and Palkama, A. (1984). Substance P-like immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study. Histochemistry 80, 421–7.

Lele, P.P. and Weddell, G. (1956). The relationship between neurohistology and corneal sensibility. Brain 19, 119–54.

Lele, P.P. and Weddell, G. (1959). Sensory nerves of the cornea and cutaneous sensibility. Exp. Neurol. 1, 334–59.

Light, A.R. (1992). The initial processing of pain and its descending control: spinal and trigeminal systems. Pain and headache, Vol. 12 (ed. Ph.L. Gidenberg). Karger, Basel.

Lim, C H. and Ruskell, G.L. (1978). Corneal nerve access in monkey. Albrecht v. Graefes Arch. klin. exp. Ophthal. 208, 15–23.

Lindahl, 0. (1961). Experimental skin pain induced by injection of water-soluble substances in humans. Acta Physiol. Scand. suppl. 179, 1–89.

Lopez de Armentia, M., Gallar, J., and Belmonte, C. (1995). An in vitro preparation of the eye¬trigeminal ganglion of the mouse for intracellular recording of corneal neurons. Invest. Ophthalmol. Vis. Sci. 36(suppl.), 575.

Lukas, Z. and Dolezel, S. (1975). A histochemical study on the development of the innervation of the rabbit cornea. Folia Morpholog. 23, 272–6.

Lyne, A.J. (1977). Corneal sensation in scleritis and episcleritis. Br. J. Ophthalmol. 61, 650–4. MacIver, M.B. and Tanelian, D.L. (1992). Activation of C fibers by metabolic perturbations associated with tourniquet ischemia. Anesthesiology 76, 617–23.

MacIver, M.B. and Tanelian, D.L. (1993a). Free nerve ending terminal morphology is fiber type specific for A-delta and C fibers innervating rabbit corneal epithelium. J. Neurophysiol. 69, 1779–83.

MacIver, M.B. and Tanelian, D.L. (1993b). Structural and functional specialization of A-delta and C fiber free nerve endings innervating rabbit corneal epithelium. J. Neurosci. 13 4511–24.

Mandahl, A. (1993). Hypertonic saline test for ophthalmic nerve impairment. Acta Ophthalmol. 71, 556–9.

Marfurt, C.F. (1981). Somatotopic organization of the cat trigeminal ganglion as determined by the horseradish peroxidase technique. Anat. Rec. 201, 105–18.

Marfurt, C.F. and DelToro, D.R. (1987). Corneal sensory pathway in the rat: a horseradish peroxidase tracing study. J. comp. Neurol. 261, 450–9.

Marfurt, C.F. and Echtenkamp, S.F. (1988). Central projections and trigeminal ganglion location of corneal afferent neurons in the monkey, Macaca fascicularis. J. comp. Neurol. 272, 370–82.

(p.180) Marfurt, C.F. and Ellis, L.C. (1993) Immunohistochemical localization of tyrosine hydroxylase in the corneal innervation of five mammalian species, including human. J. comp. Neurol. 336, 517–31.

Marfurt, C.F. and Jones, M.A. (1995). Parasympathetic innervation of the rat cornea. Invest. Ophthalmol. Vis. Sci. 36 (Suppl.), 5576.

Marfurt, C.F., Ellis, L.C., and Jones, M.A. (1993). Sensory and sympathetic nerve sprouting in the rat cornea following neonatal administration of capsaicin. Somatosens. Mot. Res. 10, 377–98.

Mark, D. and Maurice. D. (1977). Sensory recording from isolated cornea. Invest. Ophthalmol. Vis. Sci. 16, 541–5.

Martin, R.E. and Bazan, N.G. (1992). Growth-associated protein GAP-43 and nerve cell adhesion molecule in sensory nerves of the cornea. Exp. Eye Res. 55, 307–14.

Martin, X. and Dolivo, M. (1983). Neuronal and transneuronal tracing in the trigeminal system of the rat using the herpes virus suis. Brain Res. 273, 253–76.

Mathers, W.D., Jester, J.V., and Lemp, M.A. (1988). Return of human corneal sensitivity after penetrating keratoplasty. Arch. Ophthalmol. 106, 210–15.

Matsuda, H. (1968). Electron microscopic study of the corneal nerve with special reference to the nerve endings. Acta soc. ophthalmol. Jap., 72, 860.

Maurice, D.M. (1962). The cornea and sclera. In The eye. Vegetative physiology and biochemistry, Vol. (ed. H. Dayson), pp. 289–368. Academic Press, New York.

Meakin, S.O. and Shooter, E.M. (1992). The nerve growth factor family of receptors. TrendsNeurosci. 15, 323–31.

Metcalf, J.F. (1982). Corneal sensitivity and neuro-histochemical studies of experimental herpetic keratitis in the rabbit. Exp. Eye Res. 35, 231–7.

Meyer, R.A., Campbell, J.N., and Raja, S.N. (1994). Peripheral neural mechanisms of nociception. In Textbook of pain (ed. P.D. Wall and R. Melzack), pp. 13–44. Churchill Livingstone, Edinburgh.

Miller, A., Costa, M., Furness, J.B., and Chubb, I.W. (1981). Substance P immunoreactive sensory nerves supply to the rat iris and cornea. Neurosci. Lett. 23, 243–9.

Millodot, M. (1973). Objective measurement of corneal sensitivity. Acta ophthalmol. 51, 325–34. Millodot, M (1975). Do blue-eyed people have more sensitive corneas than brown-eyed people? Nature 255, 151–2.

Millodot, M. (1977). The influence of pregnancy on the sensitivity of the cornea. Br. J. Ophthalmol. 61, 646–9.

Millodot, M. and Lamont, M. (1974). Influence of menstruation on corneal sensitivity. Br. J. Ophthalmol. 58, 752–6.

Millodot, M. and O’Leary, D.J. (1980). Effect of oxygen deprivation on corneal sensitivity. Actaopththalmol. 58, 434–9.

Millodot, M., Lim, C H., and Ruskell, G.L. (1978). A comparison of corneal sensitivity and nerve density in albino and pigmented rabbits. Ophthalmic Res. 10, 307.

Mintenig, G., Sanchez-Vives, MN., Martin, C., Gual, A., and Belmonte, C. (1995). Sensory receptorsin the anterior uvea of the cat’s eye. An in vitro study. Invest. Ophthalmol. Vis. Sci. 36, 1615–24.

Mishima, S (1957). The effects of denervation and stimulation of the sympathetic and the trigeminal nerve on the mitotic rate of the corneal epithelium in the rabbit. Jap. J. Ophthalmol. 1, 65–73.

Morgan, C.W., Nadelhaft, I., and deGroat, W.C. (1978). Anatomical localization of corneal afferent cells in the trigeminal ganglion. Neurosurgery 2, 252–7.

Morgan, C., Jannetta, P.J., and deGroat, W.C. (1987). Organization of corneal afferent axons in the trigeminal nerve root entry zone in the cat. Exp. Brain Res. 68, 411–16.

Mosso, J.A. and Kruger, L. (1973). Receptor categories represented in trigeminal nucleus caudalis. J. Neurophysiol. 36, 472–88.

Muller, L.J., Pels, L., and Vrensen, G.F.J.M. (1996). Ultrastructure of human corneal nerves. Invest. Ophthalmol. Vis. Sci. (in press).

Norn, M.S. (1973). Conjunctival sensitivity in normal eyes. Acta ophthalmol. 51, 58–66.

Ogilvy, C.S. and Borges, L.F. (1990). Changes in corneal innervation during postnatal develop¬ment in normal rats and in rats treated with capsaicin. Invest. Ophthalmol. Vis. Sci. 31, 1810–15.

(p.181) Ozanics, V., Rayborn, M., and Sagun, D. (1977). Observations on the morphology of the developing primate cornea: epithelium, its innervation and anterior stroma. J. Morph. et al. 153, 263–98.

Paintal, A.S. (1964). Effects of drugs on vertebrate mechanoreceptors. Pharmacol. Rev. 16, 341–80.

Palkama, A., Uusitalo, H., and Lehtosalo, J. (1986). Innervation of the anterior segment of the eye: with special reference to functional aspects. In Neurohistochemistry: modern methods and applications. (ed. P. Panula, H. Paivarinia, and S. Soinila), pp. 587–615. Alan R. Liss, New York.

Pallikaris, IG., Papatzanaki, M.E., and Frenschock, O. (1990). A comparative study of neural regeneration following corneal wounds induced by argon fluride laser and mechanical methods. Lasers and light Ophthalmol 3, 89–95

Papatzanaki, M.E., Georgiadis, A., and Frenschok, 0. (1990). A comparative study of neural regeneration following corneal wounds induced by argon fluoride excimer laser and mechanical methods. Lasers Light Ophthalmol. 3, 89–95.

Pannabecker, C.L. (1944). Keratitis neuroparalytica: corneal lesion following operations for trigeminal neuralgia. Arch. Ophthalmol. 32, 456–63.

Passagia, J.G., Benabid, A.L., and Chirossel, J.P. (1993). Approche de la topie corneenne dans le ganglion trigeminal du rat. Bull. Assoc. Anatomistes 77, 73–8.

Perez, E., LOpez-Briones, L.G., Gallar, J., and Belmonte, C. (1987). Effects of chronic sympathetic stimulation on corneal wound healing. Invest. Ophthalmol. Vis. Sci. 28, 221–4.

Peyman, G.A., Rahimy, M.H., and Fernandes, M.L. (1994). Effects of morphine on corneal sensitivity and epithelial wound healing: implications for topical analgesia. Br. J. Ophthalmol. 78, 138–41.

Polse, A. (1978). Etiology of corneal sensitivity changes accompanying contact lens wear. Invest. Ophthalmol. Vis. Sci. 78, 1202–6.

Pozo, M.A., Gallego, R., Gallar, J., and Belmonte, C. (1992). Blockade by calcium antagonists of chemical excitation and sensitization of polymodal nociceptors in the cat’s cornea. J. Physiol. 450, 179–89.

Ramon y Cajal, S. (1899). Textura del sistema nervioso del hombre y de los vertebrados. Vidal Leuca, Alicante.

Rang, H.P. (1991). The nociceptive afferent neurone as a target for new types of analgesic drug. Pain Research and Clinical Management (Proceedings of the 6th World Congress on Pain) 4, 119–27.

Rao, G., John, T., Ishida, N., and Aquavella, J.V. (1985). Recovery of corneal sensitivity in grafts following penetrating keratoplasty. Ophthalmology 92, 1408–11.

Rebollo, I. and Belmonte, C. (1988). Effects of substance P and SP-antagonists on the neural activity of corneal sensory fibers. Proceedings of the 8th International Congress of Eye Research, (ed. M.R. Bond, J.E. Charlton and C.J. Woolf), p. 110.

Reid, T.W., Murphy, C.J., Twahashi, C., Malfroy, B., and Mannis, M.J. (1990). The stimulation of DNA synthesis in epithelial cells by substance P and CGRP. Invest. Ophthalmol. Vis. Sci. 31(suppl.), 2.

Ritchie, J.M. and Greene, M.N. (1985). Local anesthetics. In Goodman and Gilman’s pharmaco¬logical basis of therapeutics (ed. A.G. Gilman, L.S. Goodman, T.W. Rall, and F. Murad), pp. 302–21. McMillan, New York.

Rózsa, A.J. and Beuerman, R.W. (1982). Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain 14, 105–20.

Rózsa, A.J., Guss, R.B., and Beuerman, R.W. (1983). Neural remodelling following experimental surgery of the rabbit cornea. Invest. Ophthalmol. Vis. Sci. 24, 1033–51.

Schaible, H-G. and Schmidt, R. (1985). Effect of an experimental arthritis on the sensory properties of the fine articular afferent units. J. Neurophysiol. 54, 1109–22.

Schirmer, K.E. (1963a). Assessment of corneal sensitivity. Br. J. Ophthalmol. 47, 488–92. Schirmer, K.E. (1963b). Corneal sensitivity and contact lenses. Br. J. Ophthalmol. 47, 493–5. Schwartz, D.E. (1974). Corneal sensitivity in diabetics. Arch. Ophthalmol. 91, 174–8. Schwartzman, M.L., Abraham, N.G., Masferrer, J., Dunn, M.W., and McGiff, J.C. (1985).Cytochrome P-450 dependent metabolism of arachidonic acid in bovine corneal epithelium. Biochem. Biophys. Res. Commun. 132, 343–51.

(p.182) Schwartzman, M.L., Balazy, M., Masferrer, J., Abraham, N.G., McGiff, J.C., and Murphy, R.C. (1987). 12(R)-hydroxyicosatetranoic acid: a cytochrome P450-dependent arachidonate meta¬bolite that inhibits Na K ±-ATPase in the cornea. Proc. natl Acad. Sci., USA 84, 8125–9.

Shepherd, G.M. (1991). Sensory transduction: entering the mainstream of membrane signalling. Cell 67, 845–51.

Sigelman, S. and Friedenwald, J.S. (1954). Mitotic and wound-healing activities of the corneal epithelium. Arch. Ophthalmol. 52, 46–57.

Smeyne, R.J., Klein, R., Schnapp, A., Long, L.K., Bryant, S., Lewin, A., Lira, S.A., and Barbacid, M. (1994). Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368, 246–9.

Steen, K.H., Reeh, P.W., Anton, F., and Handwerker, H.O. (1992). Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in the rat skin. J. Neurosci. 12, 86–95.

Stone, R.A. (1986). Neuropeptide Y and the innervation of the human eye. Exp. Eye Res. 42, 349–55. Stone, R.A. and McGlinn, A.M. (1988). Calcitonin gene-related peptide immunoreactive nerves in human and rhesus monkey eyes. Invest. Ophthalmol. Vis. Sci. 29, 305–10.

Stone, R.A., Laties, M.A., and Brecha, N.C. (1982). Substance P-like immunoreactive nerves in the anterior segment of the rabbit, cat and monkey eye. Neuroscience 7, 2459–68.

Stone, R.A., Kuwayama, Y., Terenghi, G., and Polak, J.M. (1986). Calcitonin gene-related peptide: occurrence in corneal sensory nerves. Exp. Eye Res. 43, 279–83.

Stone, R.A., Kuwayama, Y., and Laties, A.M. (1987). Regulatory peptides in the eye. Experientia 43, 791–800.

Sugimoto, T., Takemura, M., and Wakisaka, S. (1988). Cell size analysis of primary neurons innervating the cornea and tooth pulp of the rat. Pain 32, 375–81.

Szerenyi, K., Sorken, K., Garbus, J.J., Lee, M., and McDonnell, P.J. (1994). Decrease in human corneal sensitivity with topical diclofenac sodium. Am. J. Ophthalmol. 118, 312–15.

Szolcsdnyi, J., Jancsó-Gabor, A., and JoO, F. (1975). Functional and fine structural characteristics of the sensory neuron blocking effect of capsaicin. Naunyn-Schmiedebergs Arch. Pharmacol. 287, 157–69.

Tanelian, D.L. (1991). Cholinergic activation of a population of corneal afferent nerves. Exp.Brain Res. 86, 414–20.

Tanelian, D.L. and Beuerman, R.W. (1980). Recovery of corneal sensation following hard contact lens wear and the implication for adaptation. Invest. Ophthalmol. Vis. Sci. 19, 1391–4. Tanelian, D.L. and Beuerman, R.W. (1984). Responses of rabbit corneal nociceptors to mechanical and thermal stimulation. Exp. Neurol. 84, 165–78.

Tanelian, D.L. and Maclver, M.B. (1990). Simultaneous visualization and electrophysiology of corneal A-delta and C fiber afferents. J. Neurosci. Meth. 32, 213–22.

Tanelian, D.L. and Maclver, M.B. (1991). Analgesic concentrations of lidocaine suppress tonic A-delta and C fiber discharges produced by acute injury. Anesthesiology 74, 934–6.

Terenghi, G., Polak, J.M., Ghatei, M.A., Mulderry, P.K., Butler, J.M., Unger, W.G., and Bloom, S.R. (1985). Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye. J. comp. Neurol. 233, 506–16. Terenghi, G., Zhang, S.-Q., Unger, W.G., and Polak, J.M. (1986). Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in periperal tissues following chronic denervation. Histochemistry 86, 89–95.

Tervo, K. (1981). Effect of prolonged and neonatal capsaicin treatments on the substance P immunoreactive nerves in the rabbit eye and spinal cord. Acta ophthalmol. 59, 737–46. Tervo, K. and Tervo, T. (1981). The ultrastructure of rat corneal nerves during development. Exp. Eye Res. 33, 393–402.

Tervo, K., Tervo, T., and Palkama, A. (1978). Pre-and postnatal development of catecholamine¬containing and cholinesterase-positive nerves of the rat cornea and iris. Anat. Embryol. 154, 253–65.

(p.183) Tervo, K., Tervo, T., Eranko, L., and Eranko, 0. (1981). Substance P immunoreactive nerves in the rodent cornea. Neurosci. Lett. 25, 95–7.

Tervo, K., Tervo, T., Eranko, L., Ernnko, 0., Valtonen, S., and Cuello, C. (1982). Effect of sensory and sympathetic denervation on substance P immunoreactivity in nerve fibres of the rabbit eye. Exp. Eye Res. 34, 577–85.

Tervo, T. (1977). Consecutive demonstration of nerves containing catecholamine and acetylcho¬linesterase in the rat cornea. Histochemistry 50, 291–9.

Tervo, T. and Palkama, A. (1978a). Innervation of the rabbit cornea. A histochemical and electronmicroscopic study. Acta Anat. 102, 164–75.

Tervo, T. and Palkama, A. (1978b). Ultrastructure of the corneal nerves after fixation with potassium permanganate. Anat. Rec. 190, 851–60.

Tervo, T., Tervo, K., ErdnkO, L., Vannas, A., Cuello, C., and Ernnkti, 0. (1982). Substance P¬immunoreactive nerves in the human cornea and iris. Invest. Ophthalmol. Vis. Sci. 23, 671–4.

Tervo, T., Tervo, K., ErdnkO, L., Vannas, A., ErankO, 0., and Cuello, C. (1983). Substance P immunoreaction and acetylcholinesterase activity in the cornea and gasserian ganglion. Ophthalmic Res. 15, 280–8.

Tervo, T., Vannas, A., Tervo, K., and Holden, B.A. (1985). Histochemical evidence of limited reinnervation of human corneal grafts. Acta ophthalmol. 63, 207–10.

Toivanen, N., Tervo, T., Partanen, M., Vannas, A., and Hervanen, A. (1987). Histochemical demonstration of adrenergic nerves in the stroma of human cornea. Invest. Ophthalmol. Vis. Sci. 28, 398–400.

Tower, S. (1940). Unit for sensory reception in cornea. With notes on nerve impulses from sclera, iris and lens. J. Neurophysiol. 3, 486–500.

Trabucchi, G., Brancato, R., Verdi, M., Carones, F., and Sala, C. (1994). Corneal nerve damage and regeneration after excimer laser photokeratectomy in rabbit eyes. Invest. Ophthalmol. Vis. Sci. 35, 229–35.

Trimarchi, F. (1967). La sensibility corneale alle variazioni termiche. Ann. Oftal. 93, 592–8.

Tusscher, M.P.M. ten, Klooster, J., and Vrensen, G.F.J.M. (1988). The innervation of the rabbit’s anterior eye segment: a retrograde tracing study. Exp. Eye Res. 46, 717–30.

Unger, W.G. (1990). Review: mediation of the ocular response to injury. J. ocular Pharmacol. 6, 337–52.

Unger, W.G., Terenghi, G., Zhang, S.-Q., and Polak, J.M. (1988). Alteration in the histochemical presence of tyrosine hydroxylase and CGRP-immunoreactivities in the eye following chronic sympathetic or sensory denervation. Curr. Eye Res. 7, 761–9.

von Frey, M. (1894). Beitrnge zur Physiologie des Schmerzsinnes. In Berichte fiber die Verhandlun¬gen der keiniglich-söchsischen Gesellschaft der Wissenschaften zu Leipzig mathematische Klasse, pp. 185–96. Hirzel, Leipzig.

von Frey, M. (1922). Die Sensibilitdt der Hornhaut and Bindehaut des menschlichen Auges. Dtsch. med. Wschr. 48, 212–25.

Weddell, G. and Zander, E. (1950). A critical evaluation of methods used to demonstrate tissue neural elements, illustrated by reference to the cornea. J. Anat. 84, 168–99.

Weinstein, S., Drozdenko, R., and Weinstein, C. (1922). A new device for corneal esthesiometry: clinical significance and application. Clin. Eye Vision Care 4, 123–8.

Whitear, M. (1960). An electron microscope study of the cornea in mice, with special reference to the innervation. J. Anat. 94, 387–409.

Zander, E. and Weddell, G. (1951). Observations on the innervation of the cornea. J. Anat., London 85, 68–99.

Zorab, E.C. (1971). Corneal sensitivity after grafting. Proc. R. Soc. Med. 64, 117–18.

Zuazo, A., Ibafiez, J., and Belmonte, C. (1986). Sensory nerve responses elicited by experimental ocular hypertension. Exp Eye Res. 43, 759–69.