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.

Keywords: Epithelium; Laser refractive surgery; Prednisolone acetate; Stroma; Sub-basal layer; Wound healing.

Figure 1.. Immunohistochemical staining and analysis of feline corneas.

A. Monochrome photographs of triple-stained, intact, central, feline corneas showing expression of (from left to right) α-SMA, Tuj-1, and DAPI. The last image is a composite of the first three. Note sparse, thick cords of Tuj-1 positive corneal nerves in the anterior stroma, the almost continuous sub-basal nerves right under the epithelium and the thin nerve endings visible between epithelial cells. B. Tracing of an entire cat corneal cross section (unoperated) performed in Neurolucida, and illustrating with differential color-coding, the different layers in which corneal nerve densities were analyzed. Insets show higher power views of the central, mid-peripheral and peripheral regions of the cornea.

Figure 2:. OCT imaging methods and analysis.

A. Magnified OCT image of the central cornea of a cat in the present study. Backscatter reflectivity was estimated over 4 lines drawn perpendicular to the cornea’s surface as detailed in the text. Yellow and red segments on each line denote the approximate extent of the anterior and posterior stromal segments (respectively) over which intensity was computed in each image. Epi=epithelium. B. Sample reflectivity profile generated across 4 lines from the epithelium to the endothelium. Values represent the mean and standard deviation of the relative pixel intensity at each location across the cornea’s depth. Yellow and red data points indicate the approximate location of the anterior and posterior thirds of the cornea.

Figure 3.. Fibrosis post-PRK: effect of post-operative treatments.

A. OCT images taken preoperatively and 2 weeks after PRK in a cat eye from each treatment group. Note the markedly increased anterior stromal reflectivity in the post-operative eyes, which appears less intense but more extensive in depth for the PA-treated eye (which also appears swollen), and thinner in the MMC-treated eye. B. Plot of normalized backscatter intensity pre-operatively and 2 weeks after PRK in the anterior and posterior thirds of the cornea of all cats and treatment groups examined. Intensity increased in all eyes post-operatively, but did so dramatically more in the anterior than the posterior cornea. Note also that untreated corneas exhibited a greater increase in reflectivity than either the PA or MMC-treated eyes at 2 weeks post-PRK (see text for statistics). C-K. Immunohistochemical staining of feline corneas at different times post-PRK showing expression of α-SMA (green), Tuj-1 (red), and DAPI (blue). Scale bar = 100 μm for all photos.

Figure 4.. Sample tracings of immuno-stained corneal sections from the untreated group at different times post-PRK.

The 3 columns show illustrative tracings from the peripheral, mid-peripheral and central corneas of 3 different cat eyes. Red and orange: Tuj-1 positive corneal nerves in stroma and epithelial layers, respectively. Green: regions positive for α-SMA. In all cases, the epithelium is shown at the top of each image.

Figure 5.. Sample tracings of immuno-stained corneal sections from the PA-treated group at different times post-PRK.

The 3 columns show illustrative tracings from the peripheral, mid-peripheral and central corneas of 3 different cat eyes. Red, orange and purple nerves: Tuj-1 positive corneal nerves in stroma, epithelium and acellular zones, respectively. Green: regions positive for α-SMA. Blue: acellular zones. In all cases, the epithelium is shown at the top of each image.

Figure 6.. Sample tracings of immuno-stained corneal sections from the MMC-treated group at different times post-PRK.

The 3 columns show illustrative tracings from the peripheral, mid-peripheral and central corneas of 3 different cat eyes. Red, orange and purple nerves: Tuj-1 positive corneal nerves in stroma, epithelium and acellular zones, respectively. Blue: acellular zones. In all cases, the epithelium is shown at the top of each image.

Figure 7.. Quantitative analysis of nerve distributions in central ablation zone and peripheral cat corneas post-PRK.

Grey lines and shaded box indicate mean ± standard error of the mean (SEM) of values obtained in normal, unoperated control corneas (n=4). NDIe: nerve density index in the epithelium. NDIs: nerve density index in the stroma. All data points are means ± SEM. * indicates statistically significant difference relative to control (unoperated) corneas at p<0.05 level, computed using two-tailed Student’s t-tests. Horizontal brackets over columns indicated statistically significant differences relative to untreated corneas (black bars) at p<0.05 level, computed using two-tailed Student’s t-tests. See text for additional descriptive statistics.