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 6mm 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,

How scars shape the neural landscape: Key molecular mediators of TGF-β1’s anti-neuritogenic effects

“…The increased expression of TGF-β1 after injury plays positive roles by regulating the immune response, modulating neuronal phenotype and regulating expression of growth factors important for neuronal repair [12][13][14]. However, TGF-β1 also stimulates the formation of fibrotic scars at the lesion site; this can be problematic for nerve regeneration, as scar-forming cells can inhibit regrowth, plasticity and recovery of damaged neurons [15][16][17][18]. Scar formation is particularly detrimental in the cornea, where it decreases transparency and the ability to see.…”

A Novel CD147 Inhibitor, SP-8356, Attenuates Pathological Fibrosis in Alkali-Burned Rat Cornea

“…However, only SP-8356 depleted α-SMA (+) myofibroblast population in injured cornea whereas PA slightly decreased the number of myofibroblasts and α-SMA expression ( Figure 2). Similarly, Hindman et al and Kim et al [38,39] reported that PA decreased the number of myofibroblast in damaged cornea, but this reducing effect disappeared when PA treatment is discontinued. In addition, Hill et al [40] reported that corticosteroid is ineffective to eliminate myofibroblast population in damaged cornea.…”

“…Presently, topical corticosteroids are frequently used to prevent corneal haze in clinical fields and PA is one popular topical steroid [5,[35][36][37][38]. Thus, we used PA as a positive control drug.…”

How scars shape the neural landscape: key molecular mediators of TGF-β1’s anti-neuritogenic effects

“…The increased expression of TGF-β1 after injury plays positive roles by regulating the immune response, modulating neuronal phenotype and regulating expression of growth factors important for neuronal repair [12-14]. However, TGF-β1 also stimulates the formation of fibrotic scars at the lesion site; this can be problematic for nerve regeneration, as scar-forming cells can inhibit regrowth, plasticity and recovery of damaged neurons [15-18]. Scar formation is particularly detrimental in the cornea, where it decreases transparency and the ability to see.…”

“…Although corneal nerves are part of the peripheral nervous system and able to regenerate, wounding of the cornea often results in abnormal reinnervation, with surprisingly serious and long-lasting effects [23-30]. We recently showed that corneal myofibroblasts, which differentiate largely from stromal keratocytes in and around the wound site [31], inhibit the regrowth of corneal nerves into the wound area [16, 18]. Importantly, this effect was reproduced in vitro : when corneal myofibroblasts were co-cultured with neurons derived from the ND7/23 cell line, they inhibited neurite outgrowth [16].…”