Impact of topical anti-fibrotics on corneal nerve regeneration in vivo

Abstract

Recent work in vitro has shown that fibroblasts and myofibroblasts have opposing effects on neurite outgrowth by peripheral sensory neurons. Here, we tested a prediction from this work that dampening the fibrotic response in the early phases of corneal wound healing in vivo could enhance reinnervation after a large, deep corneal injury such as that induced by photorefractive keratectomy (PRK). Since topical steroids and Mitomycin C (MMC) are often used clinically for mitigating corneal inflammation and scarring after PRK, they were ideal to test this prediction. Twenty adult cats underwent bilateral, myopic PRK over a 6 mm optical zone followed by either: (1) intraoperative MMC (n = 12 eyes), (2) intraoperative prednisolone acetate (PA) followed by twice daily topical application for 14 days (n = 12 eyes), or (3) no post-operative treatment (n = 16 eyes). Anti-fibrotic effects of MMC and PA were verified optically and histologically. First, optical coherence tomography (OCT) performed pre-operatively and 2, 4 and 12 weeks post-PRK was used to assess changes in corneal backscatter reflectivity. Post-mortem immunohistochemistry was then performed at 2, 4 and 12 weeks post-PRK, using antibodies against α-smooth muscle actin (α-SMA). Finally, immunohistochemistry with antibodies against βIII-tubulin (Tuj-1) was performed in the same corneas to quantify changes in nerve distribution relative to unoperated, control cat corneas. Two weeks after PRK, untreated corneas exhibited the greatest amount of staining for α-SMA, followed by PA-treated and MMC-treated eyes. This was matched by higher OCT-based stromal reflectivity values in untreated, than PA- and MMC-treated eyes. PA treatment appeared to slow epithelial healing and although normal epithelial thickness was restored by 12 weeks-post-PRK, intra-epithelial nerve length only reached ∼1/6 normal values in PA-treated eyes. Even peripheral cornea (outside the ablation zone) exhibited depressed intra-epithelial nerve densities after PA treatment. Stromal nerves were abundant under the α-SMA zone, but appeared to largely avoid it, creating an area of sub-epithelial stroma devoid of nerve trunks. In turn, this may have led to the lack of sub-basal and intra-epithelial nerves in the ablation zone of PA-treated eyes 4 weeks after PRK, and their continuing paucity 12 weeks after PRK. Intra-operative MMC, which sharply decreased α-SMA staining, was followed by rapid restoration of nerve densities in all corneal layers post-PRK compared to untreated corneas. Curiously, stromal nerves appeared unaffected by the development of large, stromal, acellular zones in MMC-treated corneas. Overall, it appears that post-PRK treatments that were most effective at reducing α-SMA-positive cells in the early post-operative period benefited nerve regeneration the most, resulting in more rapid restoration of nerve densities in all corneal layers of the ablation zone and of the corneal periphery.