Optical Effects of Anti-TGFβ Treatment after Photorefractive Keratectomy in a Cat Model

Abstract

purpose. To assess the contribution of corneal myofibroblasts to optical changes induced by photorefractive keratectomy (PRK) in a cat model.

methods. The transforming growth factor (TGF)-β-dependence of feline corneal keratocyte differentiation into α-smooth muscle actin (αSMA)-positive myofibroblasts was first tested in vitro. Twenty-nine eyes of 16 cats were then treated with −10 D PRK in vivo and divided into two postoperative treatment groups that received either 100 μg anti-TGFβ antibody for 7 days, followed by 50 μg dexamethasone for another 7 days to inhibit myofibroblast differentiation, or vehicle solution for 14 days (control eyes). Corneal thickness and reflectivity were measured by optical coherence tomography. Wavefront sensing was performed in the awake-behaving state before surgery and 2, 4, 8, and 12 weeks after surgery. 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 coincubation with anti-TGFβ antibody. In vivo, anti-TGFβ treatment after PRK resulted in less αSMA immunoreactivity in the subablation stroma, lower corneal reflectivity, less stromal regrowth, and lower nonspherical higher order aberration induction than in control eyes. However, there were no intergroup differences in epithelial regeneration or lower order aberration changes.

conclusions. Anti-TGFβ treatment reduced feline corneal myofibroblast differentiation in vitro and after PRK. It also decreased corneal haze and fine-grained irregularities in ocular wavefront after PRK, suggesting that attenuation of the differentiation of keratocytes into myofibroblast can significantly enhance optical quality after refractive surface ablations.

Increasing awareness of the potential risk of iatrogenic keratectasia after laser in situ keratomileusis (LASIK) has led to a renewed interest in photorefractive keratectomy (PRK) and prompted the development of advanced surface ablation techniques like laser subepithelial keratomileusis (LASEK) ¹ and epi-LASIK. ² One major disadvantage of surface ablations over LASIK is the more pronounced wound-healing response, whose functional consequences include a longer visual rehabilitation period, regression, and haze. Since these side effects can significantly limit the treatment of higher myopia (reviewed in Refs. ^{3 4 5} ), several attempts have been made to decrease their occurrence through preservation of the epithelial layer (e.g., LASEK and Epi-LASIK) and pharmacologic modulation of wound healing, as proposed in the early days of PRK. ⁶ Topical steroids have been used widely, ⁷ but their effects on haze and refractive regression remain controversial. ^{8 9 10 11} Mitomycin C, a cytostatic agent originally introduced for chemotherapy of malignant tumors, has also been shown to attenuate wound healing after PRK, particularly in cases of higher susceptibility to regression and haze (i.e., those involving the treatment of higher myopia). ^{12 13 14 15} However, safety concerns and side effects have been associated with the use of steroids (elevation of intraocular pressure, cataract induction, and delayed epithelial healing) and mitomycin C (cytotoxic, possibly mutagenic, and limited data on long-term keratocyte integrity), prompting the search for alternatives. 

One means of modulating corneal wound healing is via the inhibition of transforming growth factor (TGF)-β, ^{16 17} a multifunctional cytokine released by the lacrimal gland, the corneal epithelium, and conjunctival cells. ¹⁸ It promotes keratocyte proliferation, ^{19 20} migration, ²¹ differentiation into myofibroblasts that express α-smooth muscle actin (αSMA; reviewed in Ref. ²² ) and the deposition of extracellular matrix proteins. ¹⁹ TGFβ has been shown to play a crucial role in the development of haze after PRK, so that application of anti-TGFβ antibodies to the eye reduces both corneal reflectivity (haze) and fibrosis after PRK in rabbits. ^{16 17} However, stromal regrowth still occurred, suggesting that at least in the rabbit, stromal regeneration may be controlled by TGFβ-independent mechanisms. ¹⁷  

Other than on haze, the optical consequences of blocking TGFβ after PRK ^{16 17} have never yet been examined. The cat model used in the present study is unique in allowing simultaneous investigation of biological and optical aspects of corneal wound healing after PRK. ^{23 24 25} However, before testing the in situ effects of anti-TGFβ treatment in the cat, we first measured the response of feline corneal keratocytes to TGFβ stimulation in vitro, to verify that they behaved similarly to keratocytes from rabbits, pigs, and humans. In vivo experiments were then performed to test the hypothesis that blocking TGFβ activity in the feline eye after PRK (1) decreases transformation of corneal keratocytes into contractile myofibroblasts, (2) decreases haze (corneal reflectivity) by reducing the incidence of reflective myofibroblasts in the ablation optical zone, (3) decreases refractive regression by slowing keratocyte proliferation and the generation of new extracellular matrix in the stroma, and (4) decreases the induction of higher order aberrations (HOAs) by decreasing the fine-grained, contractile influence of myofibroblasts on the corneal surface. 

Materials and Methods

All animal procedures were conducted according to the guidelines of the University of Rochester Committee on Animal Research (UCAR), the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the NIH Guide for the Care and Use of Laboratory Animals. 

Cell Culture Experiments

Corneal keratocytes were isolated from four normal, adult domestic short-hair cats, as previously described. ^{22 26 27 28} Cells were plated on both 1-mm collagen IV-coated glass coverslips and six-well collagen IV-coated tissue culture plates (VWR International, West Chester, PA). Cells were seeded at a density of 10⁵ cells per well and cultured in 1× penicillin-streptomycin, gentamicin, 1× Dulbecco’s modified Eagle’s medium/nutrient mix F-12 (DMEM/F-12) (1×) liquid, 1:1, containing l-glutamine, but no HEPES buffer or phenol red (Invitrogen, Carlsbad, CA). When the first batch of cells approached 80% confluence, they were exposed to recombinant human TGFβ (Calbiochem, San Diego, CA) ranging in concentration from 0 to 10 ng · mL⁻¹ to assess whether this factor caused them to differentiate into αSMA-positive myofibroblasts. The optimal dose at which TGFβ induced strong αSMA expression at 72 hours was 1 ng · mL⁻¹. New sets of primary feline corneal keratocytes were then incubated with a combination of 1 ng · mL⁻¹ TGFβ and neutralizing mouse monoclonal anti-TGFβ antibody (Clone 1D11, blocks all subtypes of TGFβ; R&D Systems, Minneapolis, MN) ranging in concentration from 0 to 2 ng · mL⁻¹. After 72 hours in culture, the experiment was stopped to assess what dose of antibody blocked TGFβ-dependent induction of myofibroblast differentiation, as measured via αSMA expression. All cell culture experiments were repeated three times. 

Immunofluorescence.

Cultured cat corneal keratocytes plated on coverslips were rinsed once in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde in 0.1 M PBS for 6 minutes at room temperature (25°C). They were then permeabilized by incubation with 0.2% Triton X-100 in 0.1 M PBS (Sigma-Aldrich, St. Louis, MO) for 6 minutes at 25°C before incubation for 1 hour at 37°C with 2 μg · mL⁻¹ of mouse monoclonal anti-αSMA antibody (clone 1A4; Sigma-Aldrich). After primary antibody was washed off, the coverslips were incubated with anti-mouse IgG tagged with AlexaFluor 488 (2 μg · mL⁻¹; Invitrogen-Molecular Probes, Eugene, OR). Finally, the cells were double-stained with propidium iodide (0.1 μg · mL^-1, Invitrogen, Carlsbad, CA) to identify all nuclei. Coverslips were then mounted onto slides and imagined with a 63× objective on a confocal microscope (LSM510; Carl Zeiss Meditec, Inc., Dublin, CA). 

Western Blot Analysis.

Cells were collected from cultures at various time points (0, 24, 48, 72, and 120 hours) by incubating them for 5 minutes in 0.05% trypsin with EDTA 4 Na (1×, Invitrogen) and spinning them into a pellet before lysis for 10 minutes at 4°C. The samples were then spun again at 14,000 rpm for 10 minutes at 4°C, the supernatant was collected, and the protein concentration was determined by measuring absorbance at 280 nm with a spectrophotometer. The samples were adjusted to 10 μg/well, boiled for 5 minutes with 4× sample buffer (Invitrogen), then loaded and run on a 1% Bis-Tris gel (Invitrogen) before being stained for 1 hour in Coomassie blue (Bio-Rad, Hercules, CA) to verify equal protein loading for each well. After they were destained overnight, the gels were then transferred to nitrocellulose membrane (Invitrogen) and electrophoresed for 1 hour at 25 V. The membranes were blocked overnight at room temperature in 5% condensed milk (Nestle-Carnation, Wilkes-Barre, PA) and incubated for 2 hours at room temperature in mouse monoclonal anti-αSMA antibody (10 μg · mL⁻¹; Sigma-Aldrich). After the membranes were washed with PBS, they were incubated with goat anti-mouse HRP conjugate (the Jackson Laboratory, Bar Harbor, ME). Equal amounts of Western Dura reagents A and B (Pierce, Rockland, IL) were placed on the blot and incubated for 5 minutes at room temperature, after which the blot was developed (Gel Doc system; Bio-Rad, Hercules, CA). 

In Vivo Experiments

A list of treatments administered and measurements collected in living cats is provided in Table 1 . 

PRK Surgery.

Twenty-nine eyes from 16 normal, domestic short hair cats (felis cattus) underwent a conventional spherical PRK for −10 D with a 6-mm optical zone (OZ) and a 1.55-mm transition zone, resulting in a total treatment zone 9.1 mm in diameter and a central ablation depth of 168 μm (Planoscan 4.14; Bausch & Lomb). PRK was performed by one of two surgeons (SM, JB) in cats under topical (proparacaine 0.5%; Falcon, Fort Worth, TX) and surgical (ketamine, 5 mg · kg⁻¹; medetomidine hydrochloride 0.08 mg · kg⁻¹) anesthesia, with a laser (Technolas 217; Bausch & Lomb). The ablation was centered to the pupil, which was constricted with two drops of pilocarpine 3% (Bausch & Lomb). 

Anti-TGFβ Treatment.

After PRK, 13 eyes received a topical administration of anti-TGFβ antibody (Clone 1D11, blocks all subtypes of TGFβ; R&D Systems). Eleven of these eyes received 50 μg of anti-TGFβ twice daily, a dose that was determined to be optimal based on experiments in a separate set of cats (including two from this cohort; data not shown) and on results from the literature. ^{16 17} The contralateral eyes were treated with vehicle solution (RefreshCelluvisc; Allergan, Irvine, CA) to serve as the control. An additional three eyes from three cats were later added to the control group when a power analysis revealed the need for additional animals to attain statistical significance. For the first application immediately after PRK, both treatment and vehicle solutions were held in place on the cornea using a saturated, sterile, gelatin sponge (Surgifoam; Ethicon, Piscataway, NJ) for 2 minutes. Each eye then received a drop of triple antibiotic solution (Neomycin, Polymyxin B Sulfate, Gramicidin Ophthalmic Solution USP; Bausch & Lomb). For the next 7 days, treatment eyes received 1 drop of 1 mg · mL⁻¹ anti-TGFβ antibody and 1 drop of antibiotic solution twice daily. Control eyes received one drop of vehicle solution and one drop of antibiotic solution twice daily. During the second postoperative week, treatment eyes received 1 drop of 0.1% (1 mg · mL⁻¹) dexamethasone suspension (Maxidex; Alcon, Fort Worth, TX) per day. In case of delayed epithelial closure, the first week’s treatment was continued until the epithelium closed (usually a couple of extra days). Pain in the early postoperative period was treated with IV flunixin meglumine 1.1 mg · kg⁻¹ (Banamine; Schering-Plough Animal Health, Kenilworth, NJ) three times/day for 3 days. Four eyes were excluded from this study: two received a nonoptimal dose of anti-TGFβ antibody (as part of dose-response trials), one had a severely decentered optical zone, and one developed an ulcer and a sequestrum. 

Optical Coherence Tomography.

A custom-built, 1310-nm anterior segment optical coherence tomographer (OCT) was used to image corneas before and 2, 4, 8, and 12 weeks after PRK. The animals were lightly anesthetized, treated with eye gel (GenTeal; Novartis, East Hanover, NJ) and placed in a head-restraint device to hold the head stable. ²⁴ The OCT recorded a video stream of the central 10 mm of cornea at a rate of eight frames per second. Twenty-five corneal images extracted from the video stream were analyzed and averaged per eye and time-point. Custom software was used to measure and calculate the normalized intensity profile across each corneal image, as reported previously. ^{24 29 30} Briefly, a rectangular analysis area 105 μm wide and spanning the entire thickness of the cornea and gel layer was positioned 1 mm nasal to the middle of each corneal image (light gray rectangles in 1 2 3 Fig. 4A ). This nasal location was chosen to ensure that the same region of cornea was analyzed in all eyes and that it lay outside the area of saturated reflectivity associated with the specular reflection. A profile of pixel intensities was generated across the vertical extent of each analysis area, as previously described. ^{24 29 30} To compensate for fluctuations in laser strength, we normalized each profile by dividing the mean pixel intensity at each vertical pixel location by the mean pixel intensity in the analyzed region of cornea. Finally, the area under the normalized intensity curve was computed for the most superficial 20% of the stroma and expressed as a cumulative intensity value in Figure 4C . The normalized backscatter intensity profiles were also used to estimate the thickness of the epithelium and stroma from each OCT image, by measuring the vertical difference between intensity peaks within each analysis area. ^{24 29 30}  

In Vivo Confocal Imaging.

Confocal imaging of the central cornea was performed in two animals before and 2, 4, 8, and 12 weeks after PRK. After OCT imaging, the anesthetized cats were imaged with the Heidelberg Retina Tomographer with the Rostock Cornea Module (Heidelberg Engineering, Dossenheim, Germany). A drop of eye gel (GenTeal; Novartis) was placed on each cornea and on the contact cap. Correct alignment was attained, and after the focus was set on the epithelium, several automated scans, each 58 μm in depth, were performed until the endothelium became visible. Scans were recorded as digital video sequences and stored on a PC for analysis. 

Wavefront Analysis in Awake-Fixating Cats.

Cats were trained to fixate on single spots of light presented on a computer monitor as previously described. ^{23 24} Wavefront measurements were performed in each eye before surgery and 2, 4, 8, and 12 weeks after PRK with a custom-built Hartmann-Shack wavefront sensor. The wavefront sensor was aligned to the visual axis of one eye while the other eye fixated a spot on the computer monitor. ²³ At least 10 spot array patterns were collected per imaging session per eye. From each spot array pattern, wavefront errors (WFEs) were calculated using a 2nd- to 10th-order Zernike polynomial expansion according to the published standards for reporting aberration data of the eye. ³¹ The measurements were centered on the ablation OZ by shifting a 6-mm centroiding area (analysis pupil) manually to find the wavefront that yielded the most negative C₂ ⁰ value (i.e., the maximum treatment effect). ²⁵ For calculation of preoperative WFEs, the analysis pupil was shifted according to the mean offset relative to the pupil center. 

Lower-order aberration (LOA) Zernike coefficients were converted into dioptric power vectors (M, J ₀, J ₄₅), where M is the spherical equivalent; J ₀ = 0°/90° and J ₄₅ = 45°/135° are the astigmatic components. Higher order aberrations (HOAs) were broken down into coma RMS (the RMS of all coma terms C _n ^±1); spherical aberration RMS (the RMS of all coefficients C _n ⁰); and the RMS of the residual, noncoma, nonspherical aberrations (the RMS value of all remaining HOA C _n ^{≥ ±2}). Best theoretical image quality was assessed (Visual Optics Laboratory [VOL]-Pro 7.14; Sarver and Associates, Carbondale, IL), to calculate the best corrected visual Strehl ratio based on the optical transfer function (BCVSOTF) ³² for a simulated endpoint of the subjective refraction. 

Immunohistochemistry.

Two cats were killed at 2 weeks after PRK, five at 4 weeks after PRK and four at 12 weeks after PRK for the purpose of performing corneal histology. Two animals were kept for long-term optical follow-up and no ex vivo histology was performed on their corneas. Two separate, normal, adult cats were also killed to serve as nonsurgical control subjects. After the cats were euthanized, the corneas were excised and drop fixed in 1% paraformaldehyde in 0.1 M PBS (pH 7.4) for 10 minutes. They were then transferred to 30% sucrose in 0.1 M PBS and stored at 4°C for 2 days. After they were embedded in OCT Compound (Tissue Tek; Sakura Finetek, Zoeterwoude, The Netherlands), serial, 20-μm-thick cross-sections were cut on a cryostat (2800 Frigocut E; Leica, Nussloch, Germany), mounted on microscope slides and stored in a −20°C freezer until ready to stain. 

Slides containing three corneal sections each were air dried and rinsed in 0.1 M PBS. Two of the sections on each slide were incubated overnight at 4°C with 2 μg · mL⁻¹ mouse monoclonal anti-αSMA antibody (clone 1A4; Sigma-Aldrich). The third section was incubated with 0.1 M PBS as a negative control. After washing off the primary antibody with 0.1 M PBS, the sections were then reacted with anti-mouse IgG tagged with AlexaFluor 488 (2 μg · mL⁻¹; Invitrogen-Molecular Probes), followed by propidium iodide (0.1 μg · mL⁻¹; Invitrogen), to label the cell nuclei. The double-labeled sections were imaged with a fluorescence microscope (AX70; Olympus, Lake Success, NY), and photomicrographs were collected via a high-resolution video camera interfaced with a computer (ImagePro software; MediaCybernetics, Bethesda, MD). 

Statistical Analysis

Intergroup differences in reflectivity, corneal thickness, and wavefront aberrations were compared with a paired (or two-sample), two-tailed Student’s t-test. If data were not normally distributed according to a Kolmogorov-Smirnov-Lillefors test, a Wilcoxon-Mann-Whitney U test was used instead. Finally, where appropriate, a 2 (condition) × 4 (postoperative time point) mixed factorial ANOVA was also performed. A probability of error of P < 0.05 was considered statistically significant. All statistical tests were performed with commercial software (SPSS 11.0; SPSS Inc., Chicago, IL). 

Results

Cell Culture Experiments

Primary cultures of cat corneal keratocytes failed to express αSMA and exhibited a dendritic morphology typical of quiescent corneal keratocytes. When TGFβ was added to the serum-free medium, feline keratocytes altered their morphology, retracting some of their dendritic processes, multiplying, and acquiring a distinct myofibroblastic phenotype, which was accompanied by intracellular expression of αSMA after 72 hours of culture (Fig. 1A) . The minimum concentration of TGFβ required for this differentiation and for strong αSMA expression at 72 hours was 1 ng · mL⁻¹, as confirmed by immunofluorescence and Western blot analysis (Figs. 1A 1C) . Addition of anti-TGFβ antibodies to the incubation medium blocked expression of αSMA in a dose-dependent manner, as shown by immunofluorescence (Fig. 1B)and Western blot (Fig. 1D) . 

In Vivo Experiments

Clinical Course and Slit Lamp Findings after PRK.

Of the 10 eyes treated with 1 mg · mL⁻¹ anti-TGFβ twice daily, only one exhibited slightly delayed epithelial healing, which required us to start dexamethasone treatment 2 days late. All other eyes showed uneventful follow-up, with full epithelial closure by day 7. 

In Vivo Confocal Imaging.

Preoperative examination showed a regular epithelium and a syncytium of quiescent keratocytes in all eyes (Figs. 2A 2E 3A 3E) . One week after PRK, the epithelium, though usually closed in eyes treated with anti-TGFβ, appeared hyperplastic and edematous. The superficial stroma was reflective, though hypocellular, with some inflammatory cell infiltrates. The first layer of identifiable keratocytes was found at a depth of 50 to 100 μm below the epithelium. In deeper layers, a fine fiber network and spindle-shaped migratory fibroblasts were visible between the keratocytes. In control eyes, migratory fibroblasts appeared in the hypocellular stroma immediately below the moderately reflective epithelial-stromal interface, and keratocytes became identifiable at depths of only 20 to 30 μm below the epithelium. In all eyes, the posterior corneal layers were quiescent, with only occasional migratory fibroblasts observed. The endothelium was inconspicuous in both treatment and control eyes. 

Two weeks after PRK, there was increased reflectivity at the epithelial-stromal interface, which was more prominent in control than in treated eyes (Figs. 2B 2F) . The number of migratory fibroblasts increased, with some sprouting processes in both groups. Below the hypocellular zone (10 to 20 μm below the epithelial-stromal interface), activated, reflective keratocytes with distinct cell processes were clustered (Fig. 3B) . In control eyes, the epithelial-stromal interface appeared to contain more pronounced cellular elements (Fig. 2F) —likely to be myofibroblasts—and the layer below the epithelial-stromal interface was populated by activated keratocytes, which appeared more densely packed than in treated eyes (Fig. 3F) . The entire layer of activated cells and increased reflectivity was ∼90 μm thick in both control and anti-TGFβ-treated eyes. 

Three and 4 weeks after PRK, both treatment and control eyes showed further increases in the reflectivity of the epithelial-stromal interface relative to their preoperative state. Once again, this reflectivity appeared greater in control eyes (Figs. 2C 2G). Eyes treated with anti-TGFβ contained some bright stromal cells below the epithelium, but they were less dense than in control eyes and appeared separated by large, optically clear spaces (Figs. 3C 3G) . The subepithelial layer of activated cells and increased reflectivity now measured ∼120 μm in treated eyes and ∼90 μm in control eyes. 

Six, 8, and 12 weeks after PRK, the epithelial-stromal interface and the underlying stroma became progressively more similar between treated and control eyes. Both groups displayed decreased interface reflectivity, a layer of stellate, activated, subepithelial keratocytes that were arranged in an irregular fashion (Figs. 2D 2H)and a denser, deeper meshwork of cells (Figs. 3D 3H) . Twelve weeks after PRK, the subepithelial layer of activated cells and increased reflectivity measured ∼80 μm thick in both anti-TGFβ-treated and control eyes. Apart from the presence of migrating fibroblasts, which became progressively fewer, the posterior stromal layers did not change significantly throughout the observation time and the endothelium remained inconspicuous at all time points. 

Corneal Backscatter Reflectivity.

OCT-derived preoperative reflectivity of the anterior 20% of the cornea was relatively low and did not differ significantly between cat eyes destined for anti-TGFβ treatment or control groups (Figs. 4A 4B 4C) . PRK increased reflectivity in the anterior 20% of the stroma of both groups (P < 0.05; Figs. 4A 4B 4C ). However, this increase was significantly lower in treated than in control eyes, with a peak difference at 4 weeks after PRK (P < 0.05; Figs. 4A 4B 4C ). 

Corneal Thickness Changes.

Before surgery, the mean central corneal thickness of treatment eyes was 587 ± 55 μm (control eyes: 582 ± 40 μm), the mean stromal thickness was 537 ± 67 μm (control eyes: 538 ± 52 μm), and the mean epithelial thickness was 67 ± 15 μm (control eyes: 59 ± 8 μm). There were no significant intergroup differences in any of these measures before surgery (P > 0.05; Figs. 4D 4E ). The epithelial layer was scraped off during PRK, but 2 weeks later, central epithelial thickness had not only regenerated, but in the control group, it was 36 ± 37 μm thicker than it had been before surgery (P < 0.005). This effect was not observed in eyes treated with anti-TGFβ until 4 weeks after PRK, after which, epithelial thickness returned to preoperative values in both groups (Fig. 4D) . An ANOVA showed no significant effect of treatment or postoperative time on epithelial thickness. PRK removed approximately 168 μm of central stroma. Two weeks later, the central stroma had regenerated half that loss in control eyes, but was still 141 ± 31 μm thinner than before surgery in treated eyes (P < 0.01; Fig. 4E ). An ANOVA revealed a main effect of treatment (F _1,11 = 4.96, P = 0.048) across the entire postoperative period with control eyes maintaining a significantly thicker stroma after surgery than eyes treated with anti-TGFβ (Fig. 4E) . 

Ex Vivo Histology.

Normal, unoperated cat corneas exhibited a classic histologic structure and a complete absence of αSMA staining within the stroma (Fig. 5) . Two and 4 weeks after PRK, the subablation zone was identifiable by the absence of a clear Bowman’s layer, and the expression of αSMA below the epithelium. In control eyes, the band of αSMA immunoreactivity was thick and continuous, whereas in eyes treated with anti-TGFβ, it formed a much thinner layer, interrupted by αSMA-negative zones (Fig. 5) . Qualitative inspection of PI-staining revealed an apparent decrease in keratocyte density after PRK relative to surgical corneas. However, among PRK-treated corneas, the density of subepithelial stromal cells always appeared higher in control corneas than in corneas treated with anti-TGFβ. Twelve weeks after PRK, stromal αSMA immunoreactivity decreased relative to its levels 4 weeks after PRK in both treatment groups. However, PI staining continued to show apparently higher cell density in the subablation zone of control eyes (Fig. 5) . 

Wavefront Analysis.

Before surgery, there were no statistically significant differences in lower or higher order wavefront aberrations between the two experimental groups (P > 0.05; Figs. 6 7 ). Two weeks after PRK, the mean change in spherical equivalent was 4.79 ± 0.86 D in the anti-TGFβ group and 5.04 ± 2.11 D in the control group (Table 2 ; Fig. 6A ). Similar amounts of astigmatism (J ₀ and J ₄₅) were induced in the two groups after PRK (P > 0.05; Table 2 , Figs. 6B 6C ). 

With respect to HOA, control eyes exhibited a dramatic and significant increase in total higher order RMS (HOA RMS) 2 weeks after PRK relative to preoperative levels (P < 0.05, Fig. 7A ). This increase was statistically greater in controls than in eyes treated with anti-TGFβ (P < 0.05, Table 2 , Fig. 7A ). An ANOVA revealed a main effect of post-PRK time (F _3,21 = 3.38, P = 0.037), as well as a significant interaction of time with treatment group for HOA RMS (F _3,21 = 4.71, P = 0.011). Controls also exhibited marked, significant increases in coma, spherical and residual HOA RMS at 2 weeks after PRK, (P < 0.05, Fig. 7B 7C 7D ). These aberrations then decreased, with the exception of spherical aberration, which remained high (Fig. 7C) . Eyes that received anti-TGFβ treatments did not exhibit the peak increase in HOA observed in control eyes at 2 weeks after PRK (Table 2 , Fig. 7 ). Coma RMS, residual HOA RMS, and total HOA RMS remained close to preoperative levels throughout the post-PRK period examined. Only spherical aberration approximated levels attained in controls, particularly at later time points. A consequence of the greater magnitude of HOA in control eyes was a greater irregularity of the wavefront relative to anti-TGFβ treated eyes. This peaked 2 weeks after surgery, but was still clearly visible at later time points (Fig. 8) . 

Discussion

Several major new findings emerged from the present study. First, cat corneal keratocytes reacted similarly to keratocytes from other species when exposed to TGFβ and its blocking antibodies. Second, after PRK, myofibroblast differentiation in the feline eye could be decreased by topical application of antibodies against TGFβ. Third, decreasing myofibroblast differentiation after PRK significantly decreased nonspherical, higher-order aberrations without affecting refractive outcome. In short, the present experiments identify, for the first time, a specific contribution of myofibroblast activity to changes in ocular optics after laser refractive surgery. 

Comparative Behavior of Feline Keratocytes in Culture

Given that earlier studies had suggested some important interspecies differences in corneal wound healing, a fundamental finding in the present study was that primary feline corneal keratocytes exhibited a dendritic morphology, lacked αSMA immunoreactivity, and reacted to the administration of TGFβ in a manner that was consistent with that of rabbit, human, and bovine keratocytes. ^{27 33 34 35} Furthermore, our cell culture experiments did not support the concept that differences in corneal wound healing that might be observed in our cat model of PRK would be related to differential TGFβ regulation of feline keratocyte differentiation into αSMA-positive myofibroblasts. Having established this behavioral baseline in vitro, we were able to proceed with in vivo experiments in which the TGFβ regulatory pathway was manipulated after PRK in cats. Specifically, we tested the hypothesis that myofibroblasts, through their contractile ability, are significant contributors to the induction of higher (rather than lower) order ocular aberrations after PRK. 

Methodological Issues

Unlike prior experiments with neutralizing antibodies against TGFβ in rabbits, we used a prolonged, sequential treatment approach in our cat experiments, with neutralizing antibodies for the first postoperative week and dexamethasone for a second week (after epithelial closure). Although dexamethasone reduces TGFβ₁ and -β₂ mRNA levels in healing tissues, ³⁶ it was not administered during the first week after PRK for two reasons (1) because steroids can impair epithelial closure ³⁷ and (2) because epithelial disruption is a major source of signals for myofibroblast differentiation. ^{38 39} Once the epithelium closed, dexamethasone was administered for 1 week to provide a more upstream, prolonged anti-TGFβ effect than antibody application alone. ³⁶ However, differentiation between antibody and steroid action was not the purpose of our study. Thus, control eyes received only vehicle solution, and we did not include a third, steroid-only treatment group. 

Structural Effects of Anti-TGFβ Treatment after PRK

In vivo confocal imaging and postmortem histology revealed anti-TGFβ treatment after PRK to reduce (but not eliminate) the differentiation of cat stromal keratocytes into αSMA-positive myofibroblasts in the subablation zone. A similar effect was previously demonstrated in rabbit models of lamellar keratectomy ¹⁶ and PRK. ¹⁷ The fact that myofibroblast differentiation could not be completely suppressed is not surprising, since it is influenced by both TGFβ-dependent and independent mechanisms. ²⁶ Furthermore, although topically administered anti-TGFβ antibodies accumulate in the cornea to some extent, ¹⁶ our application pattern would have created two concentration peaks per day. In contrast, TGFβ was likely released from the injured cornea in a more sustained manner. ^{18 39 40 41} Unfortunately, repeated administration of eye drops to awake-behaving cats caused significant stress in the animals, precluding more frequent anti-TGFβ treatment. Despite this technical limitation, anti-TGFβ treatment delayed the repopulation of the anterior stroma after PRK. Whether it did so by inhibiting keratocyte migration, proliferation or increasing keratocyte death remains to be determined. The likely effects of anti-TGFβ treatment on the stromal extracellular matrix were not examined in the present study, although some of the differences noted between the thickness of the subablation αSMA positive layer and the thickness of the area of high reflectivity observed on confocal images at 2 weeks after PRK could be due to both reactive myofibroblasts and the presence of abnormal extracellular matrix. Immunohistochemistry for αSMA, on the other hand, recognizes only the cellular component of this reaction, and when used alone, is likely to underestimate the extent of “abnormal” or “reactive” stroma in a given piece of tissue. 

OCT imaging showed higher reflectivity in the anterior 20% of the stroma in control corneas relative to the treated group throughout the follow-up period, confirming previous results with anti-TGFβ treatment in rabbits, which was also shown to decrease reflectivity and haze in the cornea. ^{16 17 42}  

Whereas the epithelial layer regenerated to approximately the same extent in both treatment groups, anti-TGFβ treatment decreased stromal regrowth throughout the postoperative period in the cat. This contrasts with previous results in rabbits treated with the 1D11 anti-TGFβ antibody, albeit only three times a day for 3 days after PRK. ¹⁷ In rabbits, this treatment decreased haze and αSMA expression in the subablation zone, but did not impede stromal regrowth. ¹⁷ It is conceivable that the difference in treatment regimen, and to a lesser extent, in method of measuring stromal thickness (OCT in the present study versus confocal microscopy through focusing in Møller-Pedersen et al. ¹⁷ ), can explain our different results. However, the possibility remains that cats and rabbits differ in the TGFβ-dependence of cellular mechanisms controlling their stromal regrowth. 

Effects of Anti-TGFβ Treatment on Ocular Wavefront Aberrations after PRK

The present study showed for the first time, that cat eyes treated with anti-TGFβ after PRK exhibit less nonspherical wavefront aberrations than control eyes. While cats exhibit a general undercorrection relative to humans when PRK is performed over a 6-mm OZ, ²⁴ PRK-induced HOAs in the feline model are comparable in magnitude and type to those induced by this procedure in humans. ^{24 25 43} While HOA RMS, coma RMS, and residual HOA RMS peaked in the early post-PRK period, levels of these HOA remained close to preoperative levels in eyes treated with anti-TGFβ. Only the amounts of spherical aberration and lower order aberrations induced by PRK were similar in control and anti-TGFβ-treated eyes after surgery. Thus, myofibroblast contractility appears to alter corneal shape in an irregular, non-radially symmetric manner. A possible explanation for this is the development of local irregularities in corneal curvature due to small-scale, myofibroblastic contraction of the stroma ⁴⁴ that remains uncompensated by epithelial filling-in. However, anti-TGFβ treatment did not mitigate the induction of spherical aberration, suggesting this to be a phenomenon dominated by factors that do not significantly implicate myofibroblast differentiation. Previous work suggested that loss of laser ablation efficiency in the corneal periphery and changes in corneal biomechanics may play a more important role in the induction of spherical aberration after laser refractive surgery. ⁴⁵ The present data show that myofibroblast differentiation and contractility are unlikely to contribute significantly to this phenomenon. 

Implications for Clinical Practice

The present experiments demonstrated that immediate pharmacologic modulation of corneal wound healing after PRK with agents whose main effect is to block TGFβ activity decreases keratocyte differentiation into contractile myofibroblasts. Of novel interest is that the decreased incidence of myofibroblasts in the post-PRK cornea decreased HOA induction and caused faster optical rehabilitation after PRK. In contrast to treatment with mitomycin C, which decreases repopulation of the acellular zone after PRK via nonspecific, cytotoxic effects, ¹⁵ the treatment used in our study was aimed directly at the transformation of keratocytes into myofibroblasts. Although steroids indirectly block myofibroblast differentiation by downregulating TGFβ mRNA in the eye, they bear the risk of nonspecific side effects, such as delayed re-epithelialization ³⁷ and increased intraocular pressure (reviewed in Ref. ⁴⁶ ). Thus, treatment with anti-TGFβ antibodies in the early postoperative phase after PRK has the advantage of an immediate myofibroblast blocking action without the side effects of steroids or mitomycin C. Clinically, a pharmacologic treatment that shortens recovery time after PRK is highly desirable because it potentially mitigates the main problems associated with this procedure: wound-healing-associated delay of visual recovery and optical instability. On the other hand, such treatment is expensive, requires special handling to avoid antibody degradation, and is only partially effective. Ongoing research in our laboratory is focusing on alternative, nonimmunogenic substances to specifically block other aspects of myofibroblast function via both TGFβ- and non-TGFβ-regulated pathways. 

In conclusion, reduction of myofibroblast differentiation after PRK decreases HOA induction and increases retinal image quality. By improving visual outcome, pharmacologic modulation of corneal wound healing with myofibroblast-blocking agents could strengthen the value and feasibility of surface ablation procedures as an alternative to lamellar procedures like LASIK. 

 Supported by National Institutes of Health Grant R01 EY015836 (KRH), R01 EY017123 (RPP), Core Grant P0EY01319F to the Center for Visual Science, grants from Bausch & Lomb Inc., and the University of Rochester’s Center for Electronic Imaging Systems, a NYSTAR-designated Center for depithelial-stromal interfacegnated Center for Advanced Technology, Deutsche Forschungsgemeinschaft (DFG) Grant Bu2163/1-1 (JB), and an unrestricted grant to the University of Rochester’s Department of Ophthalmology from the Research to Prevent Blindness Foundation, Inc.

 Submitted for publication May 10, 2008; revised August 26 and September 22, 2008; accepted December 8, 2008.

 Disclosure: J. Bühren, None; L. Nagy, None; J.N. Swanton, None; S. Kenner, None; S. MacRae, Bausch & Lomb (C, F), R.P. Phipps, None; K.R. Huxlin, Bausch & Lomb (F)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

In Vivo Experiment: Eyes, Treatments, and Measures

	PRK-Treated Eyes (n) and Test
		Exclusion from Study	OCT	In Vivo Confocal	Histology	Wavefront Sensing	Total
Anti-TGFβ treatment	3	10	2	8	4	13
Control	1	15	2	10	7	16

Figure 1.

 

Cell culture results. (A) Photomicrographs of feline corneal keratocytes cultured for 72 hours in serum-free defined medium supplemented with different concentrations of TGFβ. Cells are double-labeled with propidium iodide (PI) and antibodies against αSMA (green). Note the strong αSMA immunoreactivity of cells exposed to 1 and 5 ng · mL⁻¹ TGFβ. (B) Western blot analysis of feline corneal keratocytes cultured for 72 hours in serum-free defined medium supplemented with different concentrations of TGFβ. The left-most blot is a Coomassie stain demonstrating equal protein load in each lane. Lanes 1, 2, 3, 4, and 5 were loaded with keratocytes exposed to 0, 0.1, 1, 5 and 10 ng · mL⁻¹ of TGFβ, respectively. Lane 6 was loaded with pure αSMA protein as a control. The right blot shows anti-αSMA staining of the same blot, and demonstrates increasing amounts of αSMA protein in keratocytes cultured with increasing concentrations of TGFβ. (C) Photomicrographs of feline corneal keratocytes cultured for 72 hours in serum-free defined medium containing 1 ng · mL⁻¹ TGFβ and supplemented with different concentrations of anti-TGFβ antibodies. Staining was as in (A). Note the strong αSMA immunoreactivity of cells cultured with 1 ng · mL⁻¹ TGFβ and up to 0.5 ng · mL⁻¹ of anti-TGFβ. At anti-TGFβ concentrations of 1 or 1.5 ng · mL⁻¹, αSMA expression was strongly inhibited. (D) Western blot analysis of feline corneal keratocytes cultured for 72 hours in serum-free defined medium containing 1 ng · mL⁻¹ TGFβ and different concentrations of anti-TGFβ antibodies. The left blot is a Coomassie stain demonstrating equal protein load in each lane, as well as a reference ladder (left lane). Lanes 1, 2, 3, 4, and 5 were loaded with keratocytes cultured for 72 hours in serum-free defined medium containing 1 ng · mL⁻¹ TFGβ and 2.0, 1.5, 1, 0.1, or 0 ng · mL⁻¹ of anti-TGFβ antibody, respectively. Lane 6 was loaded with pure αSMA protein as a control. The right blot shows anti-αSMA staining of the same Western blot, and demonstrates decreasing amounts of αSMA protein in keratocytes cultured with increasing concentrations of anti-TGFβ antibody.

Figure 2.

 

In vivo confocal imaging of the epithelial-stromal interface. (A–D) Images of the right eye (OD) of one cat treated with anti-TGFβ/dexamethasone after PRK. Images were collected before surgery, 2, 4, and 12 weeks after surgery, and demonstrate the radical change in reflectivity in this corneal region over time. (E–H) Images of the left eye (OS) of the same cat, which only received vehicle solution after −10-D PRK. Images were also collected before surgery, and then at 2, 4, and 12 weeks after PRK. Note the well-organized, regular syncytium of quiescent keratocytes before surgery and the disrupted, reactive and strongly reflective activated keratocytes and myofibroblasts that replace it after surgery. Note also that the control eye exhibits much stronger reflectivity and cellularity than the anti TGFβ-treated eye, especially at 2 and 4 weeks after PRK.

Figure 3.

 

In vivo confocal imaging 20 μm below the epithelial-stromal interface. (A–D) Images of the right eye (OD) of the cat in Figure 2 , which was treated with −10-D PRK and anti-TGFβ dexamethasone after surgery, and demonstrating the radical change in reflectivity in this corneal region over time. (E–H) Images of the left eye (OS) of the same cat, which received only vehicle solution after the −10-D PRK. Images were also collected before surgery, and then at 2, 4, and 12 weeks after PRK. The well-organized, regular syncytium of quiescent keratocytes before surgery contrasted with the less regular, reactive, and strongly reflective activated keratocytes and myofibroblasts that replace it after surgery, particularly in the control (left) eye. Indeed, the control eye exhibited much stronger reflectivity and cellularity than the anti-TGFβ-treated eye, especially at 2 and 4 weeks after PRK. There were spindle-shaped migratory fibroblasts (arrow) and the cluster of reflective activated keratocytes in the right eye at 2 weeks after PRK. Clustering was still present in anti-TGFβ-treated eyes 4 weeks after PRK, but was not seen in control eyes. As is evident in this set of images, control eyes exhibited greater cell density and greater reflectivity at this corneal depth than did eyes treated with anti-TGFβ/dexamethasone, at all postoperative time points.

Figure 4.

 

Effect of anti-TGFβ treatment on corneal reflectivity and thickness, as measured with optical coherence tomography (OCT). (A) OCT images of the left (OS) and right (OD) corneas of a cat before and 4 weeks after −10-D PRK. Note the greater reflectivity in the subablation stroma of the control eye (arrows) relative to the eye that received anti-TGFβ treatment after PRK. The rectangles superposed over the corneal images indicate the location and approximate size of the areas analyzed for reflectivity and thickness. These analysis areas were located 1 mm nasal to the center of each cornea and well outside the zone of the specular reflex. (B) Plot of normalized backscattered light intensity obtained from the rectangular analysis areas in 25 OCT images of the left and right corneas of the cat shown in (A) versus central stromal depth, expressed as a percentage of the total stromal depth: 100% indicates the stromal-endothelial boundary, whereas 0% indicates the stromal-epithelial boundary. Reflectivity profiles are shown for both eyes of this cat before surgery and 4 weeks after PRK. Note that the curves were relatively flat for both eyes before surgery, but that stromal reflectivity increased after PRK. However, the eye that received anti-TGFβ treatments exhibited lower anterior stromal reflectivity than the control eye. (C) Plot of the area under the reflectivity curve (see examples of these in B) for the anterior 20% of the corneal stroma as a function of time. Eyes that received anti-TGFβ treatment after PRK exhibited lower mean reflectivity of the anterior relative to the posterior stroma compared with control eyes at all postoperative time points. (D) Central epithelial thickness for control and anti-TGFβ eyes as a function of time. The first points indicate preoperative values, followed by values on performing PRK (arrow). There was no significant difference between the two treatment groups before or after surgery. (E) Central stromal thickness for control and anti-TGFβ eyes as a function of time. The first points indicate preoperative values, followed by values on performing PRK (arrow). Note the consistently thinner stromas in anti-TGFβ treated eyes across post-PRK time points. All graphs show means and standard errors of the mean.

Figure 5.

 

Effect of anti-TGFβ treatment on αSMA expression and stromal cell density. Photomicrographs of corneal sections from normal, nonsurgical cat eyes and pairs of eyes that underwent PRK and received either vehicle or anti-TGFβ treatment after surgery. These cats were killed at 2, 4, and 12 weeks after PRK and sections of their corneas were double-labeled with antibodies against αSMA to label myofibroblasts and with propidium iodide (PI) to label cell nuclei. Note the absence of αSMA staining in the nonsurgical cat cornea, in contrast with the significant αSMA expression in the subablation stroma of eyes that underwent PRK. The band of αSMA expression was significantly thicker and more continuous in control eyes than in contralateral eyes treated with anti-TGFβ. It was also most intense at 2 and 4 weeks after PRK, becoming almost absent in the stroma by 12 weeks after PRK. PI staining also revealed an area of increased cellularity under the ablation zone in all postoperative eyes, although cell density appeared consistently higher in control eyes relative to eyes treated with anti-TGFβ.

Figure 6.

 

Effect of anti-TGFβ treatment on lower-order ocular aberrations. (A) Spherical equivalent M. (B) 0°/90° astigmatic component J ₀. (C) 45°/135° astigmatic component J ₄₅. All graphs are plotting values expressed as dioptric power vectors M, J ₀, and J ₄₅ for a 6-mm pupil diameter as a function of postoperative time. All values are the mean ± SEM. On the horizontal axis, P is preoperative time. Note the lack of significant effect of anti-TGFβ treatment on changes in lower order aberrations induced by PRK.

Figure 7.

 

Effect of anti-TGFβ treatment on higher order ocular aberrations. (A) Total HOA RMS. (B) Coma RMS (RMS of all C _n ^±1). (C) Spherical aberration (SA) RMS (RMS of all C _n ⁰). (D) Residual HOA RMS (RMS of all C _n ^{≥ ±2}). All graphs plot the mean ± SEM as a function of postoperative time; P, preoperative time. *Significantly greater change from before surgery in controls than eyes that received anti-TGFβ treatment at that particular time-point (P < 0.05). Note the peak in HOA, coma, spherical aberration and residual HOA exhibited by control eyes 2 weeks after PRK. Except for spherical aberration RMS, anti-TGFβ-treated eyes maintained preoperative levels of HOA after PRK.

Table 2.

 

Treatment Effects at 2 Weeks after PRK

	Anti-TGFβ (n = 4)	Control (n = 7)
M (D)	4.79 ± 0.86 (4.04 to 5.68)	5.04 ± 2.11 (2.56 to 7.65)
J 0 (D)	0.09 ± 0.09 (−0.03 to 0.20)	0.27 ± 0.48 (−0.38 to 0.84)
J 45 (D)	0.20 ± 0.13 (0.05 to 0.37)	0.30 ± 0.52 (−0.20 to 1.14)
Total HOA RMS (μm)	0.20 ± 0.18* (0.036 to 0.362)	0.82 ± 0.32 (0.477 to 1.232)
Coma RMS (μm)	0.08 ± 0.28, † (−0.13 to 0.46)	0.48 ± 0.22 (0.17 to 0.71)
Spherical aberration RMS (μm)	0.21 ± 0.07 (0.139 to 0.279)	0.41 ± 0.40 (−0.136 to 1.001)
Residual HOA RMS (μm)	0.06 ± 0.1* (−0.025 to 0.201)	0.49 ± 0.27 (0.196 to 0.874)
6th- to 10th-order RMS (μm)	0.01 ± 0.01 (−0.022 to 0.297)	0.10 ± 0.09 (0.014 to 0.235)
Log BCVSOTF	−0.27 ± 0.34 (−0.65 to 0.18)	−0.67 ± 0.27 (−1.15 to −0.46)

 Results are shown as change of refraction. VSOTF, HOAs for a 6-mm pupil diameter relative to baseline. All data are expressed as the mean ± SD (minima and maxima in parentheses). Data in bold indicate statistically significant differences between control eyes and those that received anti-TGFβ treatment. BCVSOTF, visual Strehl ratio, based on the optical transfer function (simulation for best spectacle correction); total RMS, RMS of 2nd- to 5th-order aberrations; HOA RMS, RMS of 3rd- to 5th-order aberrations; coma RMS, RMS of 3rd- and 5th-order coma terms; residual HOA RMS, RMS of all noncoma, nonspherical HOA.

*   P < 0.01 (intergroup differences Student’s t-test).

†   P < 0.05 (intergroup differences Student’s t-test).

Figure 8.

 

Anti-TGFβ treatment causes spatial smoothing of higher order optical aberrations after PRK. Wavefront error maps (for a 9-mm pupil diameter) in the right and left eye of the same cat at 8 weeks after PRK, demonstrating the greater spatial irregularity of the wavefront in the control eye (bottom) compared with the eye treated with anti-TGFβ after PRK. While this effect was greatest at 2 weeks after PRK, it was also maintained over the longer term.