Purpose To assess the contribution of corneal myofibroblasts to optical changes induced by photorefractive keratectomy (PRK) in a cat model. Methods First, we tested the transforming growth factor β (TGFβ)-dependence of feline corneal keratocyte differentiation into α-smooth muscle actin (αSMA)-positive myofibroblasts in vitro. Twenty-nine eyes from 16 cats were then treated with −10D PRK in vivo and divided into two post-operative treatment groups: (1) eyes that received 100μg anti-TGFβ antibody for 7 days, followed by 50μg Dexamethasone for another 7 days to inhibit myofibroblast differentiation; (2) eyes that received vehicle solution for 14 days (controls). Corneal thickness and reflectivity were measured by optical coherence tomography. Wavefront sensing was performed in the awake-behaving state pre-operatively and 2, 4, 8 and 12 weeks post-operatively. Wound healing was monitored using in vivo confocal imaging and postmortem αSMA immunohistochemistry. Results In culture, TGFβ caused cat corneal keratocytes to differentiate into αSMA-positive myofibroblasts, an effect that was blocked by co-incubation with anti-TGFβ antibody. In vivo, anti-TGFβ treatment post-PRK resulted in less αSMA immunoreactivity in the sub-ablation stroma, lower corneal reflectivity, less stromal re-growth and lower non-spherical, higher order aberration (HOA) induction than controls. However, there were no inter-group differences in epithelial regeneration or lower order aberration changes. Conclusions Anti-TGFβ treatment reduced feline myofibroblast differentiation in vitro and following PRK. It also decreased corneal haze and fine-grained irregularities in ocular wavefront after PRK, suggesting that attenuation of the differentiation of keratocytes into myofibroblasts can significantly enhance optical quality after refractive surface ablations.

Purpose To characterize phenotypic differences between anterior and posterior corneal keratocytes following stimulation with the pro-fibrotic agent transforming growth factor-beta1 (TGF-β1) in vitro. Methods Sixteen corneas from healthy felines were obtained immediately post-mortem. Lamellar dissection was performed to separate the anterior and posterior stroma at approximately 50% depth either manually (N=2) or using a Moria microkeratome (300μm head, N=14). Cells from the anterior and posterior stroma were cultured separately but under identical conditions. Using immunohistochemistry and Western Blot techniques, Ki-67 staining and relative expression of Thy-1, alpha smooth muscle actin (α–SMA), and fibronectin were assessed following stimulation with different TGF-β1 concentrations. In addition, anterior and posterior cells cultured in different concentrations of TGF-β1 were wounded with a razor blade and wound area and time to closure determined. Results Stimulation by all concentrations of TGF-β1 increased the proportion of Ki-67 positive cells in anterior and posterior cell cultures, but this increase was noted at earlier time-points in posterior cells as compared to anterior cells. Increasing TGF-β1 concentration also increased the relative expression of Thy-1, α-SMA and fibronectin in anterior and posterior fibroblasts. However, anterior cells expressed these fibrotic markers at lower TGF-β1 concentrations than posterior keratocytes. Following mechanical wounding, posterior cells closed the wound area faster than anterior cells at all concentrations of TGF-β1. Conclusions The present experiments show that anterior and posterior corneal keratocytes exhibit different sensitivities to the pro-fibrotic growth factor, TGF-β1. This heterogeneity of keratocyte response may impact wound closure following mechanical wounding.

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