Argon fluoride excimer laser photoablation results in precise removal of corneal tissue with minimal damage to adjacent structures. 1–3 It is associated with a relatively mild epithelial and stromal wound healing response. 4–6 Stromal healing after excimer wounds involves the stimulation, migration, and proliferation of stromal keratocytes and collagen deposition in the treatment area. 6 This can result in the formation of a subepithelial scar that is clinically visible as corneal haze. Pharmacologic modulation of wound healing may reduce the development of corneal haze after excimer photokeratectomy. 7,8
Mitomycins are a group of antitumor antibiotics that covalently bind to DNA after reductive activation. Mitomycin C (MMC), isolated from Streptomyces caespitosus, is a clinically useful member of this group. It inhibits fibroblast function by a dose-dependent inhibition of fibroblast proliferation. 9 MMC has antiproliferative effects at concentrations below those cytotoxic to human keratocytes. 10 The use of MMC has been investigated for the prevention of corneal haze after excimer photokeratectomy. Talamo et al. 7 used topical MMC drops (0.5 mg/mL) after photorefractive keratectomy in rabbits, and Schipper et al. 11 applied a sponge soaked with MMC (0.4 mg/mL) after stromal ablation for 5 minutes. Both studies have reported reduced scar formation in eyes treated with MMC. However, no difference was found in subjectively assessed corneal haze between groups. Majmudar et al. 12 have reported that in patients with subepithelial scarring after refractive corneal surgery, a 2-minute intraoperative application of MMC (0.2 mg/mL) after epithelial debridement and mechanical scar removal successfully prevents recurrence of subepithelial fibrosis. Although these studies recognize the potential of MMC in preventing the development of stromal scarring and in reducing preexisting stromal scarring, further studies are needed to define the optimal method of application and dosage before its routine use in patients undergoing excimer laser surgery. In this study, we compared the effect of MMC on the development of corneal haze after excimer keratectomy by applying it intraoperatively over the ablation area in a discoid or annular fashion.
Assessment of corneal haze development and modulation, in most studies, has been hampered by the use of subjective methods to grade haze severity. The most commonly used method grades subepithelial haze in increments of 0.5 on a scale from 1 to 4. 13,14 Assessment of haze severity with such methods may be biased and imprecise. Objective methods of measuring corneal haze have not been widely developed. 15,16 Therefore, the evaluation of corneal haze currently depends primarily on subjective examination methods. We have developed a scatterometer, based on a simple adaptation of a slit-lamp biomicroscope, that detects light scattering from the ablated region of the cornea. 17 This instrument has been tested on rabbits and humans and found to yield reproducible results. 18–20 In our experiments, we have used the scatterometer to assess the magnitude of corneal haze.
We report herein the effect of intraoperative MMC on corneal light scattering after excimer laser phototherapeutic keratectomy using the scatterometer for objective measurements of corneal light scattering.
MATERIALS AND METHODS
Twenty-four eyes of 12 pigmented rabbits were used for this study. Rabbits were treated in accordance with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. Before ablation the rabbits were anesthetized with an intramuscular injection of xylazine (40 mg/kg body weight) and ketamine (7 mg/kg body weight). A topical anesthetic (0.05% proparacaine hydrochloride) and 1% atropine sulfate were applied to each eye. The corneas were examined and photographed with a Zeiss photo slit lamp (Carl Zeis Optical Inc., Chester, VA, U.S.A.), and baseline scatterometry measurements were made as described below. After this preexamination, ablations were performed using the 20/20 argon fluoride excimer laser (VISX, Inc., Sunnyvale, CA, U.S.A.) operating at 193 nm. Laser fluence was set at 160 mJ/cm2, and the pulse repetition frequency was 5 Hz. After carefully drying the surface with a sterile cellulose sponge, the epithelium was ablated in the central 6-mm diameter area to a depth of 40 μm.
Animals were then divided into three groups depending on the method of intraoperative MMC application over the exposed stromal surface. In experimental group 1 (n = 8), a 5-mm diameter filter paper disc soaked with MMC was applied for 1 minute. In group 1A (n = 4), the MMC concentration was 0.5 mg/mL, and in group 1B, (n = 4), it was 0.25 mg/mL. In experimental group 2 (n = 8), an annular filter paper having an outer diameter of 5 mm and an inner diameter of 3 mm, soaked with MMC was applied for 1 minute. In group 2A (n = 4) the MMC concentration was 0.5 mg/mL ,and in group 2B, (n = 4), it was 0.25 mg/mL. In control group 3 (n = 8), the vehicle (balanced salt solution [BSS]) was applied. The same application (method and dose) was not given to both eyes of the same rabbit.
Immediately after MMC (experimental groups) or BSS (control group) application, the cornea was irrigated with sterile balanced salt solution. The cornea was dried and 6-mm diameter phototherapeutic ablation was performed in all groups (experimental and control) to a depth of 100 μm. MMC (experimental groups) or BSS (control group) was thus applied before stromal ablation after removing the epithelium. Immediately after surgery, the eyes were again irrigated with sterile BSS, and topical erythromycin ointment 0.3% and atropine 1% were applied. At various intervals during the investigation (13 months for group 1, 8 months for group 2, and 8 and 13 months for the group 3), the rabbits were anesthetized with xylazine and ketamine hydrochloride as described previously, and 1% atropine was instilled to dilate their pupils. The corneas were then examined and photographed with a Zeiss photo slit lamp, and scatterometry measurements were obtained.
The scatterometer measures back-scattered light from a defined region of the cornea under standardized illumination conditions. Corneal light scattering is related to the degree of stromal scarring after excimer laser ablations. Details of the scatterometer, schematics, and its operation have been described previously. 17–20 Briefly, the scatterometer consists of an appropriately modified slit-lamp biomicroscope, with a fiber optic pickup, a bandpass filter for wavelength selection, and a photomultiplier detector. The fiber optic is located at the image plane of the slit lamp's objective and therefore acts as a field stop. The slit was adjusted so that its projected width on the cornea was 2.0 mm and the fiber optic detected radiation emanating from a 1.7-mm diameter area centered in the illuminated slit. A filter with a 550-nm peak transmission and 50-nm bandpass was used. The angle between incident and scattered radiation was fixed at 60° (a 120° scattering angle), and the slit-lamp was aligned so that scattering was measured in a direction that was perpendicular to the cornea at the point of interest, which was normally the center of the treated area. This configuration ensured against detecting any light that was specularly reflected from the curved anterior surface of the cornea and also was insensitive to ambient light. Darkening the room during measurements was sufficient to avoid interference from stray light. All corneal haze measurements were referenced to scattering from a block of optical grade Spectralon (Labsphere, North Sutton, NH, U.S.A.) under the same illumination conditions. Spectralon has the highest diffuse reflectance (>99% over the visible spectrum) of any known material and served as a standard scatterer for the measurements. A scattering index (SI) was calculated by ratioing the normalized scattering at any session to the average normalized baseline scattering of 100 normal rabbit corneas; thus an SI of 1 represents normal scattering. The results were analyzed for statistical significance by Student t test.
The mean preoperative corneal light SI values ranged from 0.87 to 1.19, with no statistically significant differences between groups. After excimer keratectomy, corneal light scattering increased in all groups. However, differences were noticed in the rate and magnitude of light scattering. The least amount of postoperative increase in light scattering was seen in group 2 (annular MMC). In the control group, the mean light SI increased from a preoperative value of 0.87 ± 0.18 (mean ± standard deviation) to a well-defined peak at 4 weeks (5.23 ± 3.89). The mean light scattering decreased gradually thereafter to 1.38 ± 0.41 at 8 months and 1.41 ± 0.53 at 13 months.
In group 1 (MMC discs), the increase in light scatter was sustained (Fig. 1). In group 1A (0.5 mg/mL MMC discs), the mean light scatter progressively increased from the preoperative value of 1.04 ± 0.35 to 10.85 ± 4.73 at 10 weeks. A statistically significant difference (p = 0.06) in mean SI was seen at 6 weeks between group 1A (8.95 ± 5.62) and the control group (3.74 ± 2.59). At 10 weeks, the difference in mean SI between group 1A (10.85 ± 4.73) and the control group (1.95 ± 1.98) was statistically significant (p = 0.03). At 13 months, the mean light scattering decreased considerably to 2.09 ± 1.32 (differences not statistically significant). In group 1B (0.25 mg/mL MMC disc), the mean light scatter index increased gradually from 0.91 ± 0.08 preoperatively to a peak at 4 weeks (8.22 ± 2.07). The mean SI was 5.34 ± 2.13 and 7.26 ± 6.56 at 6 and 10 weeks, respectively. At 13 months, differences in mean light scatter between this group (2.03) and the control group (1.41 ± 0.53) were not statistically significant.
In group 2 (annular MMC), light scattering showed minimal increase over time (Fig. 2). In group 2A (0.5 mg/mL MMC), the SI increased from a preoperative value of 1.19 ± 0.49 to a peak of 2.51 ± 0.39 at 1 week and then decreased to 1.46 ± 0.5 at 10 weeks. The difference in mean SI between this group (2.36 ± 2.86) and the control group (4.5 ± 1.45) was statistically significant (p = 0.01) at 2 weeks. The mean SI in group 2A was consistently lower than in the control group during the entire 8 months of observation; the differences at other time points were not statistically significant. In group 2B (0.25 mg/mL MMC), the SI increased from a preoperative value of 0.91 ± 0.21 to a peak of 4.8 ± 3.4 at 2 weeks. The mean light scattering decreased to 1.02 at 8 months. Although the light scattering in this group was consistently lower than that in the control group, the differences were not statistically significant.
We evaluated the effect of intraoperative MMC application on corneal light scattering after excimer keratectomy and observed that the mean light scattering after annular MMC application was lower than in a control group. In contrast, the mean light scattering after MMC disc application was higher than in the control group. Annular application of 0.5 mg/mL MMC for 1 minute was the best strategy to prevent an increase in corneal light scattering after excimer keratectomy. The beneficial effect of MMC appeared to be relatively transient in this rabbit model, with regression of haze to near baseline levels at 8 months (group 2) and 13 months (group 1).
In this study, MMC was applied before stromal ablation (as opposed to applying after stromal ablation on a thinned cornea) to limit potential endothelial toxicity. The corneal scarring after excimer keratectomy is typically subepithelial and under the ablation bed. Therefore, keratocyte inactivation in this area (as opposed to inactivation in deeper stroma) theoretically would be effective. By applying MMC before stromal ablation, we may have limited deeper MMC diffusion while allowing MMC to inactivate keratocytes in the vicinity of the ablation bed and therefore reducing potential endothelial toxicity. All ablations were performed with a VISX 20/20 laser. Although smoother ablations may have been achieved with the next generation of excimer lasers, it probably would not have made a significant difference in the outcome. In this study, we evaluated the effect of MMC on corneal scarring by objectively measuring corneal light scattering. Because the outcome measure (SI) was based on objective measurements obtained using the scatterometer in a standardized fashion, the observer bias characteristic of subjective measurements (slit lamp corneal haze grading) may have been reduced in our study although the design was not double-masked. The scatterometer averages the focal variations in corneal haze to give a single reading representative of the average haze in the ablated area. In a previous study, we evaluated the relationship of light scattering to clinical haze grading and visual acuity in patients undergoing phototherapeutic keratectomy. Corneal light scattering showed a stronger positive correlation with logMAR visual acuity than with clinical haze grading. 18
The dose of MMC used to prevent corneal scarring after excimer keratectomy that has been reported in previously published studies ranges from 0.4 to 0.5 mg/mL. Although we have also successfully prevented the development of corneal haze using similar MMC doses applied to the corneal midperiphery (group 2), our study shows that corneal light scattering may increase if MMC is applied to the central cornea (group 1). In group 1, MMC may have diffused deeper into the central cornea and produced endothelial toxicity. This may explain the absence of regression of haze toward baseline as in the other groups.
In this study, we did not investigate the effect of MMC on preexisting corneal subepithelial scarring (secondary to a previous corneal refractive surgery procedure). Therefore, we cannot conclude that this method of application and dose of MMC will be useful for treating corneal scarring caused by a previous refractive surgery procedure. Majmudar et al. 12 have recently reported successful treatment of preexisting subepithelial fibrosis using intraoperative application of a 6-mm circular sponge soaked with MMC (0.2 mg/mL). Although with the advent of excimer lasers with smoother ablation profiles the incidence of visually significant corneal haze has decreased, deeper ablations, which are required to treat moderate and high myopia, and phototherapeutic keratectomy may still benefit from modulation of wound healing to reduce corneal scarring.
The wound healing response after excimer keratectomy is generated by stromal cells in the vicinity of the ablated surface. Previously inactive keratocytes become activated. They migrate beneath the ablation bed and deposit collagen. 6,21 In vivo confocal microscopy studies have shown that the repopulation of keratocytes in the anterior stroma after corneal wounding depends on keratocyte migration from the peripheral anterior layers of the stroma. 22 Using a vital fluorescent probe, Owens-Kratz et al. 23 have also reported centripetal migration of peripheral keratocytes. Intraoperative MMC application allows the drug to reach areas of stromal cells that may contribute to the wound healing response. In group 2, MMC may have caused inhibition of keratocytes underlying the annular zone of application, thus surrounding the visual axis with a ring of inactive keratocytes. MMC-induced nonresponsiveness of peripheral keratocytes may have minimized their activation, centripetal migration, and subsequent collagen deposition.
Intraoperative scleral application of MMC has been used to promote successful outcome of glaucoma filtration surgery, and postoperative topical therapy with MMC drops has been used to prevent recurrence of surgically excised pterygiums. 24–26 The local side effects of such treatment include superficial punctate keratopathy, recurrent corneal epithelial defects, crystalline keratopathy, iridocyclitis, and scleral melting. Idiosyncratic reactions resulting in serious sight-threatening complications have been reported after administration of a relatively large cumulative dose of MMC. 27 The potential corneal toxicity is related to cumulative doses, and this limits the use of MMC drops for prolonged after surgery. To minimize complications, the lowest possible therapeutic concentration should be applied for the shortest effective period, ensuring minimal corneal contact, particularly when epithelial defects are present. In this study, we did not vary the time of MMC application because a 1-minute exposure can cause long-term inhibition of fibroblasts in the treated area and is nearly as effective as longer exposure. 25,28,29 The ability of a brief exposure of MMC to inhibit fibrosis may be the result of sustained tissue binding and possible modulation of cell migration and extracellular matrix production. 29
Corneal wound healing and scar remodeling in rabbits is different than that in humans. Data from several studies show that corneal scars in rabbits eventually become less opaque. During wound healing in rabbit corneas, the stromal fibroblasts initially deposit collagen as a matted meshwork of fibrils tangential to the cell surface. The organization of the collagenous matrix approaches the normal lamellar appearance after 2 years and the scar appears less opaque. 30 X-ray diffraction studies have shown that there is a significant decrease in the intermolecular spacing within collagen fibrils in the scar tissue for as long as 6 weeks, but by 21 months, the spacing returns to normal. 31 The gradual reduction in the spread of interfibrillar spacings may explain the progressive decrease in the light scattering to near baseline levels in all groups.
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