Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist

Abstract

Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1-expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3-5 days, with normal nociceptive responses returning by 5-7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1-expressing fibers, extended at least to the corneal-scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post-operative or post-injury ophthalmic pain.

Figure 1

RTX suppression of CAP eye wipe response 1 day after treatment was dose-dependent (A). Doses of 0.02 and 0.2 µg had no significant effect, while doses of 1 µg and above reduced or eliminated eye wipe response. Stars indicate statistical difference from the contralateral eye using a paired t-test; number of stars indicates the p-value (one star, p<0.05; 2 stars, p<0.01; 3 stars, p<0.001). A single dose of RTX applied to the corneal surface ablates the eye wipe response to 0.02% CAP for several days (B). Contralateral eyes received no treatment prior to the CAP eye wipe test. By day 7, response in RTX-treated eyes normalized. Animals treated with 0.02 and 0.2 µg RTX were only assessed at 2 hr and 1 day, as no effect was observed. As determined by 2-way ANOVA, stars indicate statistical difference between 2 µg RTX and vehicle and pound signs indicate statistical difference between 20 µg RTX and vehicle. Lidocaine pretreatment does not interfere with RTX therapeutic action (C). Animals received RTX alone, lidocaine alone, or lidocaine pretreatment 5 to 10 minutes prior to RTX (n=5 per group). The eye wipe test was performed one day later. RTX suppressed eye wipe response, both with and without lidocaine pretreatment. Lidocaine alone had no effect on eye wipe response 24 hr after application. Lidocaine did blunt the nociceptive stimulation that occurred with acute application of RTX. Treatment with 20 µg RTX spares corneal blink response to mechanical stimulation with von Frey hairs (D). 20 µg RTX or 20 µL 2% lidocaine was applied topically onto the cornea 24 hr and 10 min, respectively, before testing, with the contralateral eye receiving no treatment (n=4 per group). Von Frey hairs were touched to the corneal surface to assess blink response. Each hair was touched 5 times to each eye. RTX had no significant effect on the sensitivity of blink response, while lidocaine significantly suppressed response.

Figure 2

RTX-treated and untreated corneas were histologically indistinguishable using H&E and PAS staining. Corneas were acquired 24 hr after treatment with 2 µg RTX, post-fixed in 10% formalin, and embedded in methyl methacrylate. Sections were stained with H&E or PAS and evaluated by a masked observer. “E” indicates the epithelial layer and “S” indicates the stromal layer. (A) H&E untreated; (B) PAS untreated; (C) H&E 2 µg RTX; (D) PAS 2 µg RTX. Scale bar = 200 µm. (E) RT-PCR shows TRPV1 transcripts are expressed in trigeminal ganglia (TG) but not the cornea, using GPDH as a normalizing control. Thus, RTX likely has no direct effect on resident corneal cells.

Figure 3

Corneal wound healing rates were not significantly different for eyes treated with 20 µg RTX or vehicle (A). The epithelial layer was removed from an area of the cornea by applying a circle of filter paper soaked in n-heptanol to the cornea for 30 s, followed by copious saline flushing. The wound was visualized at 0, 10, 15, 22, 32, 37, and 42 hr by applying fluorescein solution to the cornea under UV light and the width of the wound was measured with digital calipers in the dorsal-ventral and medial-lateral dimensions. 2-way ANOVA identified no significant difference between average wound widths of RTX- and vehicle-treated eyes at any time point. The area of debridement contracted over time as it was reepithelialized from the edge, and complete reepithelialization occurred by 42 hours in all eyes (B, C, and D). Scale bar: 2 mm.

Figure 4

Immunohistochemistry shows selective, long-lasting, reversible removal of CGRP-positive fibers from the rat cornea following topical RTX. Staining of the cornea with the panneuronal marker β-tubulin III (green) reveals large fiber bundles in the stroma (arrows) giving rise to a sub-basal plexus of parallel fibers at the interface of the epithelial and stromal cell layers (stars) and terminating in the superficial epithelium (plus sign) (A,B,C). Red line (A) denotes the boundary between the epithelial and stromal cell layers as seen from a cross-sectional perspective. Scale bars: A – 25 µm; B - 500 µm; C - 100 µm. (D, E, F) CGRP staining (red), a surrogate for TRPV1, is removed following RTX. Vehicle-treated eyes showed strong CGRP staining in stromal fiber bundles (D). 24 hr after treatment with 20 µg RTX, CGRP staining was significantly reduced (E), with most stromal fiber bundles showing no CGRP staining. CGRP staining partially returned by 12 d following RTX treatment (not shown), and had returned completely when assessed at four months (F). CGRP-positive nociceptors returned along bundle tracts. Scale bar is 25 µm.