Sensory and Sympathetic Nerve Sprouting in the Rat Cornea Following Neonatal Administration of Capsaicin

Abstract

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6-8 weeks both the CGRP- and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become "hyper-reinnervated." The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 +/- 0.49 microns and 34.3 +/- 0.72 microns), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70-80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant "hyper-reinnervation" is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.

Innervation of TRPV1-, PGP-, and CGRP-immunoreactive nerve fibers in the subepithelial layer of a whole mount preparation of the rat cornea

“…The paucity of these nerve fibers in the central cornea does not necessarily indicate the absence of fibers because it has previously been shown to be richly innervated with peptidergic nerve fibers 17) . The innervation pattern of PGP-and CGRP-IR nerve fibers in the present study is in accord with that observed in previous reports 5,6,[15][16][17]19) . Dense innervation of PGP-IR nerve fibers was demonstrated in whole mount corneas from rat pups (age, <14 days) 6) and cryostat-sections of canine 19) corneas.…”

“…The innervation pattern of PGP-and CGRP-IR nerve fibers in the present study is in accord with that observed in previous reports 5,6,[15][16][17]19) . Dense innervation of PGP-IR nerve fibers was demonstrated in whole mount corneas from rat pups (age, <14 days) 6) and cryostat-sections of canine 19) corneas. In these studies, the nerve fibers formed a network from the corneal center to the corneoscleral limbus, comprising a complex anastomotic meshwork of small-, medium-, and large-diameter nerve bundles.…”

“…To date, there have been many reports concerning corneal nerve fibers stained by various methods including enzyme detection [1][2][3][4][5][6][7][8][9] , gold impregnation 7,10 -13) , and neuropeptides 5,6,8,14 -20) , lectin IB4 15,20) or capsaicin receptor TRPV1 20,21) localization. Most analyses have been performed on whole mount preparations and paraffin or cryostat sections of the cornea from various mammals.…”

Capsaicin-resistant, Nonspecific Acetylcholinesterase (NsAchE) Reactive Nerve Fibers in the Rat Cornea: A Quantitative and Developmental Study

“…They also demonstrated a significant increase in the intraepithelial nerve fiber density at 7-9 weeks after treatment (100 mg/kg, i.p) compared with controls (Ogilvy et al, 1991). In spite of the complete disappearance of CGRP-ir subepithelial nerve fibers by 48 h in the capsaicintreated corneas (150 mg/kg, i.p), they increased markedly between 3 and 6 weeks (Marfurt et al, 1993). Thus, it is a common finding that the corneal nerve fibers first decrease and later regenerate (sprouting) during survival periods.…”

“…Thus, the reduction of wiping is closely related to the decrease of capsaicin-sensitive, corneal PGP-ir nerve fibers. Marfurt et al (1993) reported the decrease of PGP-ir subepithelial corneal nerves of the rat in the whole mount preparations 5, 7 and 10 days later of neonatal capsaicin injection. They argued that the remained nerve fibers may be sympathetic or capsaicin-insensitive corneal nerves.…”