November 2012
Volume 53, Issue 12
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Cornea  |   November 2012
Bevacizumab and Rapamycin Can Decrease Corneal Opacity and Apoptotic Keratocyte Number following Photorefractive Keratectomy
Author Notes
  • From the Department of Ophthalmology and Biomedical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea. 
  • Corresponding author: Jae Yong Kim, Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, #388-1 Pungnap-2-dong, Songpa-gu, Seoul, Korea 138-736; jykim2311@amc.seoul.kr
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7645-7653. doi:10.1167/iovs.12-10494
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      Kyoung Sub Lee, Dong-Ah Ko, Eun-Soon Kim, Myoung Joon Kim, Hungwon Tchah, Jae Yong Kim; Bevacizumab and Rapamycin Can Decrease Corneal Opacity and Apoptotic Keratocyte Number following Photorefractive Keratectomy. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7645-7653. doi: 10.1167/iovs.12-10494.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: We investigated the effects of bevacizumab and rapamycin on central corneal opacity and apoptotic keratocyte number after photorefractive keratectomy (PRK) followed by ultraviolet B (UV-B) irradiation.

Methods.: A total of 60 right eyes of Sprague-Dawley rats in four groups (n = 15 each) underwent PRK ablation to 80 μm with a 3-mm zone. Sponges soaked with 0.02% mitomycin C (MMC), 2.5% bevacizumab, 0.01% rapamycin, and balanced saline solution were applied for 2 minutes to these eyes in the MMC, bevacizumab, rapamycin, and control groups, respectively. At 3 weeks after PRK, all right eyes were exposed to 100 mJ/cm2 UV-B irradiation. Biomicroscopy was used to determine the amount of haze, and TUNEL staining for apoptosis and histology were performed at 3, 6, and 12 weeks.

Results.: Contrary to the results at 3 weeks, central corneal haze, and apoptotic keratocyte and keratocyte number decreased significantly in the MMC, bevacizumab, and rapamycin groups compared to the control group, and the keratocyte number was lower in the MMC group than the bevacizumab and rapamycin groups at 6 weeks (all P < 0.05). At 12 weeks, the apoptotic keratocyte number was lower in the MMC, bevacizumab, and rapamycin groups than the control group, and the keratocyte number was significantly lower in the MMC than the rapamycin and control groups (all P < 0.05).

Conclusions.: Intraoperative bevacizumab and rapamycin administration decreases central corneal haze and apoptotic keratocyte number after PRK. Bevacizumab and rapamycin may be safe alternatives to MMC during refractive surgery to prevent postoperative corneal opacity less affecting the keratocyte number.

Introduction
The early disappearance of keratocytes following epithelial injury due to trauma or an operation is mediated by apoptosis. 13 The remaining keratocytes are proliferative and may lead to the appearance of myofibroblasts. 4,5 During these processes, inflammatory cells have been observed in the corneal stroma. This recovery process contributes to the reorganization of the corneal stromal layer and reconstruction of the corneal epithelial cells. Adverse effects, including opacity and myopic regression, following refractive surgery are correlated with this apoptotic response and the reconstruction of the keratocytes in the area of cell death. 2 Under these conditions, the keratocyte response after surgery is a subject of some interest because corneal wound healing shows individual variability. 69 Modulating the process of keratocyte proliferation and stromal remodeling has become the main focus of efforts to reduce adverse effects. Among these processes, early cell apoptosis is the most important event for the modulation of the remodeling process. There still is no complete method for suppressing cell apoptosis. 
Mitomycin C (MMC) is the most popular corneal healing process–modulating drug at present and suppresses the fast growth of cells by blocking DNA synthesis via alkylation. This agent is used in glaucoma-filtering, pterygium, and refractive surgery. MMC also is used to treat carcinoma in situ of the conjunctiva and cornea. Kim et al. reported previously that MMC is a potent inhibitor of photorefractive keratectomy (PRK)–induced corneal haze. 10,11 MMC currently is used widely to suppress corneal opacity and regression after refractive surgery, 1214 but there still are concerns regarding corneal edema, thinning, perforation, and conjunctival injection after the use of this compound. 1517  
Bevacizumab is an anti-VEGF drug that originally was permitted for use during cancer treatment by the US Food and Drug Administration. 18 In ophthalmology settings, bevacizumab is used to treat age-related macular degeneration, proliferative diabetic macular edema, and other vascular ocular diseases, such as central serous chorioretinopathy. 1922  
Rapamycin, discovered from Streptomyces hygroscopicus in 1975, is a macrolide antibiotic agent that has antifungal and immunosuppressive effects. In rat experiments, Olsen et al. found that rapamycin reduced the rejection of corneal allograft and neovascularization. 23 In an experimental model, rapamycin demonstrates no cellular toxicity at concentrations that suppress cell proliferation and regulate neovascularization. 24 In our present study, the modulating effects of rapamycin and bevacizumab on the wound-healing process following PRK were evaluated. The effects of rapamycin and bevacizumab on keratocytes in the special environment that resulted from ultraviolet B (UV-B) irradiation following PRK also were studied. 
Materials and Methods
Surgical Methodology
The animals used in our study were treated in accordance with the ARVO Animal Statement for the Use of Animals in Ophthalmic and Vision Research. We divided 60 male 4-week-old, 250 to 300 g weighed Sprague Dawley rats into 4 groups (Fig. 1). The rats in each of the 4 groups (n = 15 each) were anesthetized using an intramuscular injection of a mixture of 10 mg/kg zolazepam (Zoletil; Yuhan Corp., Seoul, Korea) and 10 mg/kg xylazine hydrochloride (Rumpun; Bayer, Inc., Frankfurt, Germany). Proparacaine (Alcaine; Alcon-Couvreur, Puurs, Belgium) hydrochloride eye drops were instilled into each right eye, and a speculum was used to open the eyelids. PRK (3-mm optical zone, 80 μm deep) was performed on the right eye of each rat using a flying spot excimer laser (Technolas 217z; Bausch & Lomb Surgical, Munchen, Germany). The left eyes of the rats in all 4 groups were left untreated. 
Figure 1. 
 
Flow diagram of experimental procedures.
Figure 1. 
 
Flow diagram of experimental procedures.
Immediately following PRK, a sponge soaked with 0.02% MMC (Kyowa, Inc., Tokyo, Japan) was placed onto each exposed corneal bed of the treated right eyes in the MMC group for 2 minutes, then each eye was irrigated vigorously with a 30 mL balanced salt solution (BSS; Alcon Laboratories, Inc., Fort Worth, TX). 11 In the same manner, 2.5% bevacizumab (Avastin; Roche Diagnostics, Basel, Switzerland) was applied to the eyes in the bevacizumab group and 0.01% rapamycin (Sigma-Aldrich, Inc., St. Louis, MO) was applied to the eyes in the rapamycin group. For the control group, only the BSS washout was performed. The 0.3% ofloxacin ointment (Tarivid; Santen Pharmaceutical, Co., Osaka, Japan) was applied for 3 days onto each right eye in all 4 groups. 
At 3 weeks after PRK, 10 rats from each of the 4 groups were anesthetized, as described previously, and positioned in a standard dermatologic UV-light chamber (Fig. 1). The right eyes were kept open and exposed to UV-B (wavelength range 290–315 nm, work distance 15 cm) with a total equivalent energy of 100 mJ/cm2 for 1 to 2 minutes. Central stromal haziness was assessed biomicroscopically using the Fantes scale every 3 weeks thereafter. 25  
Tissue Processing and Sections
Five rats in each group were killed at 3, 6, and 12 weeks, and their right eyes were enucleated (Fig. 1). The harvested corneas were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned into 5-μm slices. Sections were double-stained with hematoxylin and eosin. 
TUNEL Assay
The number of apoptotic cells in each central cornea was determined using the In Situ Cell Death Detection Kit (Roche Diagnostics) and the fluorescein simplified TUNEL assay. Fluorescein can be used to quantify cell death (apoptosis) by labeling the DNA strand breaks in individual cells, allowing their detection by fluorescence microscopy. The assay used an optimized terminal transferase (TdT) to label free 3′-OH ends in the genomic DNA with fluorescein-dUTP. Photographs were obtained using a confocal laser scanning system (TSC-SP2; Leica, Heidelberg, Germany) at high magnification (×400) to count the TUNEL-positive keratocytes around the central corneal PRK wound. 
Transmission Electron Microscopy (TEM)
Central corneal sections were obtained using a flat cutter and fixed overnight at 4°C in 2.5% glutaraldehyde, then washed twice with PBS for 5 minutes. Each cornea was bisected and a 1-mm strip was obtained from its center, fixed in 1% OsO4 in phosphate buffer for approximately 90 minutes at room temperature, washed twice in a phosphate-buffered fixative vehicle, and then dehydrated using a graded ethanol series. The transition from 100% ethanol to epoxy was mediated by 2 changes of propylene oxide, and a pure epoxy medium was used for infiltration and embedding. The fragments were mounted on flat molds and hardened at 80°C overnight before sectioning. Both 600 to 1000 nm and 60 to 80 nm TEM sections were cut into polyvinyl butyral-coated grids (Pioloform; Sigma-Aldrich) and stained with saturated aqueous uranyl acetate and lead citrate. Samples then were evaluated by TEM (JEM1200 EX2; Jeol LTD, Tokyo, Japan). 
Cell Counting and Statistical Analysis
Five specimens from each group were used to count the number of cells at each time point. Cells were counted in 5 nonoverlapping central stromal fields by a single observer (KSL) at ×400 magnification. To reduce possible investigator bias, this procedure was performed in a blinded fashion on serially numbered slides. Using SPSS 18.0 software (SPSS Inc., Chicago, IL), ANOVA was used to compare the 4 groups and three time durations, followed by the Tukey honestly significant difference test between means. 
Results
Corneal Haziness
Corneal haze did not differ in each group (P > 0.05, Fig. 2), as time passed. Corneal haze was observed in the ablation area of all treated rats at 3 weeks after PRK and did not differ between the study groups at this time point (P = 0.07, Fig. 2). However, the corneal haze of the control group was significantly more severe than those of the other groups at 6 weeks (all P <0.05, Figs. 2, 3). At 12 weeks after PRK, corneal haze did not differ between the 4 groups (P = 0.29, Fig. 2). 
Figure 2. 
 
Biomicroscopy photographs demonstrating postoperative corneal opacity of rats in mitomycin C (A), bevacizumab (B), rapamycin (C), and control (D) groups at 6 weeks after PRK.
Figure 2. 
 
Biomicroscopy photographs demonstrating postoperative corneal opacity of rats in mitomycin C (A), bevacizumab (B), rapamycin (C), and control (D) groups at 6 weeks after PRK.
Figure 3. 
 
Quantitation of central corneal haze at 3, 6, and 12 weeks after PRK. Error bars: standard deviation of the mean. At 6 weeks, the corneal haze of the control group was significantly more severe than those of the other groups (all P < 0.05). **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 3. 
 
Quantitation of central corneal haze at 3, 6, and 12 weeks after PRK. Error bars: standard deviation of the mean. At 6 weeks, the corneal haze of the control group was significantly more severe than those of the other groups (all P < 0.05). **, *Significant differences between those groups at a particular time (P < 0.05).
Evaluation of Apoptosis
In the MMC, bevacizumab, and rapamycin groups, apoptotic keratocyte number was significantly higher at 3 weeks than at 6 and 12 weeks (all P < 0.05, Fig. 4), but it was not in the control group. At 3 weeks, apoptotic keratocytes were detected in the central corneas of all of the PRK-treated rats, but there was no significant difference in terms of the number of apoptotic keratocytes in each of the 4 groups (P = 0.08, Figs. 4, 5). At 6 and 12 weeks, more apoptotic keratocytes were observed in the control group than in the MMC, bevacizumab, and rapamycin groups (all P < 0.001). 
Figure 4. 
 
Quantitation of apoptotic keratocytes in the central stroma after PRK. Error bars: standard deviation of the mean. **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 4. 
 
Quantitation of apoptotic keratocytes in the central stroma after PRK. Error bars: standard deviation of the mean. **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 5. 
 
Immunolocalization of apoptotic keratocytes in the central cornea of mitomycin C (A), bevacizumab (C), rapamymark asancin (E), and control (G) groups using TUNEL staining at 12 weeks after PRK, and merged images with PI staining (B, D, F, H), respectively. There were significantly more apoptotic cells (white arrows in G) in the control group than in the mitomycin C, bevacizumab, and rapamycin groups (P < 0.05, cells ×400 field). Scale Bar = 25 μm.
Figure 5. 
 
Immunolocalization of apoptotic keratocytes in the central cornea of mitomycin C (A), bevacizumab (C), rapamymark asancin (E), and control (G) groups using TUNEL staining at 12 weeks after PRK, and merged images with PI staining (B, D, F, H), respectively. There were significantly more apoptotic cells (white arrows in G) in the control group than in the mitomycin C, bevacizumab, and rapamycin groups (P < 0.05, cells ×400 field). Scale Bar = 25 μm.
Histological Findings
In the MMC group, keratocyte number was significantly higher at 3 weeks than at 6 and 12 weeks (all P < 0.001, Fig. 6). In the bevacizumab group, it decreased as time passed (all P < 0.05, Fig. 6). In the rapamycin groups, it was significantly higher at 3 and 6 weeks than at 12 weeks (P = 0.02 and 0.04, Fig. 6). In the control group, it was significantly higher at 6 weeks than at 3 and 12 weeks (P = 0.008 and 0.006, Fig. 6). At 3 weeks, light microscopy did not show a statistically significant difference in terms of the number of keratocytes among the 4 groups (P = 0.72, Fig. 6). In all PRK-treated rats, morphologic changes, including epithelial irregularities and irregular scar tissue, were evident. At 6 weeks, the number of keratocytes was significantly lower in the MMC, bevacizumab, and rapamycin groups than in the control group (all P < 0.001), and lower in the MMC group than in the bevacizumab and rapamycin groups (all P < 0.001). At 12 weeks, the number of keratocytes was significantly lower in the MMC group than in the rapamycin and control groups (P = 0.001 and < 0.001, respectively), and lower in the bevacizumab group than in the control group (P = 0.001). 
Figure 6. 
 
Quantitation of keratocytes in the central cornea after PRK. Error bars: standard deviation of the mean. ***, **,*Significant differences between those groups at a particular time (P < 0.05). +Significant difference between bevacizumab and control groups.
Figure 6. 
 
Quantitation of keratocytes in the central cornea after PRK. Error bars: standard deviation of the mean. ***, **,*Significant differences between those groups at a particular time (P < 0.05). +Significant difference between bevacizumab and control groups.
TEM Findings
At 3 weeks after PRK, the dying stromal cells had shrunk and chromatin condensation was observed in some of the dead cells that had undergone apoptosis (Figs. 7A–D). An irregular epithelial basement membrane, and the infiltration of many polymorphonuclear leukocytes and monocytes were observed in the central corneas of all of the rats in each group. In addition, their collagen bundles had become irregular. At 12 weeks, there were fewer keratocytes or inflammatory cells in the central corneas of the rats in the MMC, bevacizumab, and rapamycin groups than in the control group (Figs. 7E–H). The collagen bundles were more irregular in the control group than in the MMC, bevacizumab, and rapamycin groups; however, consistency of the epithelial basement membrane was observed. In the central corneal stroma of the rats in the control group, relatively patent but some irregular epithelial basement membrane was found and the collagen fibers were more irregular than in the other groups (Fig. 7H). In addition, relatively large amounts of rough endoplasmic reticulum (RER) were observed in the myofibroblasts of the control group. 
Figure 7. 
 
TEM at the central cornea of rat. in mitomycin C (A, E), bevacizumab (B, F), rapamycin (C, G), and control (D, H) groups at 3 and 12 weeks, respectively, PRK. Dying stromal cell shrinkage (#), chromatin condensation (*), irregular epithelial basement membrane, and polymorphonuclear leukocyte (arrow) and monocyte (arrowhead) infiltration were observed in the central corneas of all of rats in each group at 3 weeks after PRK. At 12 weeks, there were fewer keratocytes or inflammatory cells in the central corneas of the rats in the MMC, bevacizumab, and rapamycin groups than in the control group (EH). The collagen bundles were more irregular in the control group than in the MMC, bevacizumab, and rapamycin groups; however, consistency of the epithelial basement membrane was observed. In the central corneal stroma of the rats in the control group, relatively patent but some irregular epithelial basement membrane was found, and the collagen fibers were more irregular than in the other groups (H). In addition, relatively large amounts of RER were observed in the myofibroblasts of the control group. Magnification ×8000.
Figure 7. 
 
TEM at the central cornea of rat. in mitomycin C (A, E), bevacizumab (B, F), rapamycin (C, G), and control (D, H) groups at 3 and 12 weeks, respectively, PRK. Dying stromal cell shrinkage (#), chromatin condensation (*), irregular epithelial basement membrane, and polymorphonuclear leukocyte (arrow) and monocyte (arrowhead) infiltration were observed in the central corneas of all of rats in each group at 3 weeks after PRK. At 12 weeks, there were fewer keratocytes or inflammatory cells in the central corneas of the rats in the MMC, bevacizumab, and rapamycin groups than in the control group (EH). The collagen bundles were more irregular in the control group than in the MMC, bevacizumab, and rapamycin groups; however, consistency of the epithelial basement membrane was observed. In the central corneal stroma of the rats in the control group, relatively patent but some irregular epithelial basement membrane was found, and the collagen fibers were more irregular than in the other groups (H). In addition, relatively large amounts of RER were observed in the myofibroblasts of the control group. Magnification ×8000.
Discussion
This intent of our study was to identify substances that may be used to prevent corneal opacity safely from arising after PRK and potentially replace MMC. Our results showed that rapamycin and bevacizumab can lower the post-PRK corneal opacity to a level similar to that of conventional MMC. It also was confirmed by histologic examination that the number of apoptotic keratocytes decreased in both groups compared to the control group. Moreover, the number of keratocytes was higher in the bevacizumab and rapamycin groups than in the MMC group at 6 weeks after PRK. Therefore, these 2 medications demonstrated potential for use as safe alternatives to MMC for the prevention of corneal opacity following photorefractive surgery, less affecting the number of keratocytes. 
Epithelial damage and surgical wounds induce apoptosis and the reorganization of the surrounding keratocytes. It is well known that part of an activated keratocyte is transformed into a myofibroblast after passing through the fibroblast phase during this process, and the extracellular matrix, including collagen, accumulates in the keratocytes, thereby causing changes that result in corneal opacity and increased thickness. It is believed that the recovery process is involved in myopic regression and corneal opacity following PRK. 2628 In other words, corneal opacity arises from the recovery process following corneal defects or PRK, and LASIK also is related to apoptosis. 2,2931 This conclusion is supported by previous findings indicating that apoptosis is increased in patients with keratoconus and corneal dystrophy, which may influence opacity, thinning, and epulosis in the cornea. 32,33  
With respect to apoptosis, the results at 3 weeks after PRK in our present analysis revealed that apoptosis occurs at a level similar to that in the control group. However, the results at 6 and 12 weeks indicated significantly less apoptosis in the MMC, rapamycin, and bevacizumab groups compared to the control group. Kim et al. investigated that MMC induced apoptosis in cultured corneal keratocytes through the caspase pathway, 34 and when applied immediately after PRK, MMC increased keratocyte apoptosis; however, at later times, it suppressed keratocyte apoptosis, keratocyte proliferation, and postoperative opacity after UV-B irradiation. 11  
There has been a paucity of studies directly showing the effect of the bevacizumab on keratocytes. Administration of bevacizumab increased apoptotic vascular cells in the brain arteriovenous malformation mouse model 35 and induced a 2- to 3-fold increase in human umbilical endothelial cell apoptosis following radiation in vitro. 36 However, Yoeruek et al. reported that bevacizumab at concentrations used clinically did not induce apoptosis or necrosis in human corneal endothelial cells in vitro. 37 Additionally, bevacizumab reduced the formation of nitric oxide (NO) in the action mechanism of VEGF, suppressed vascularization, and then reduced vascular permeability. 38,39 Bevacizumab also has an antiproliferative effect on keratocytes as well as fibroblasts, which can improve chances of successful filtration operation. 40,41 On the other hand, it has been studied that the operation on cells by rapamycin is made by suppressing mammalian target of rapamycin (mTOR). 4245 The mTOR is a phosphatidylinositol kinase-related kinase that has a pivotal role in adjusting growth and survival of a cell. 46,47 Rapamycin also controls the movement of cells through mTOR signals. 48 Based on basic research regarding rapamycin, a clinical study proves that it has an effect on control of corneal vascularization and transplant rejection. 24,49,50 Rapamycin has less toxicity and smaller available capacity with better effectiveness than drugs, like 5-fluorouracil and MMC. 17,51,52 Rapamycin generally induces apoptosis in various cells including rhabdomyosarcoma cells, 44 umbilical vein endothelial cells, 53 T-lymphocyte, 54 non–small cell lung cancer cells, 55 and so on. From our results, we assumed that bevacizumab and rapamycin would increase the apoptosis in the early postoperative period after PRK, and reduce the apoptosis in later time by the similar pattern of MMC. 11  
MMC is a drug that is used widely to prevent corneal opacity and regression following PRK. It is recognized that MMC controls the active increase in the number of keratocytes, which arises from the apoptosis of these cells followed by a series of recovery processes following PRK, and therefore suppresses the separation processes of myofibroblasts and decreases corneal opacity. 11 However, there are concerns that the use of this drug may cause long-term adverse effects, such as corneal trepanation and edema. 56 Our previous study reported that the number of keratocytes decreased to a greater extent in the MMC group than in the group that did not receive treatment following PRK. 11 It was noteworthy that the results at 6 weeks in our current analysis showed that the number of keratocytes had decreased more in the MMC group than in the bevacizumab or rapamycin groups. This finding could render us to speculate that rapamycin and bevacizumab cause fewer serious adverse effects, such as corneal perforation. 
Regarding the TEM findings, no difference was revealed among the 4 groups at 3 weeks after PRK. The MMC, rapamycin, and bevacizumab groups at 12 weeks showed collagen bundles that were more normal in appearance than the control group. On the other hand, it has been pointed out that MMC treatment will decrease the number of matrix cells continuously in the front corneal matrix; hence, thinning of the cornea might become a problem at long-term follow-up examinations. 11 However, such long-term side effects of rapamycin and bevacizumab would likely be less significant because the amount of thinning of the corneal matrix cells was less than that observed following MMC treatment. 
UV-B exposure during post-PRK stromal healing was known to exacerbate and prolong the stromal healing response, increase the severity of subepithelial haze, and decrease the endothelial cell count. 33,57,58 UV irradiation following PRK could induce more profound corneal haze and keratocyte apoptosis than PRK alone. 10,11,33 UV irradiation was used to maximize injury due to corneal haze and apoptosis of corneal cells in a manner similar to that of PRK in our study. However, there is a paucity of reports showing the effect of UV-B exposure on the rat post-PRK cornea. Even though rabbits were used as subjects in the previous study, 11 we postulated that rats could be used as subjects in the present study with the same protocol as previous study because Chen et al. reported the usefulness of the rat PRK model with long-term follow-ups. 59  
In conclusion, intraoperative application of bevacizumab and rapamycin decreased corneal haze and the number of apoptotic keratocytes following PRK with UV-B irradiation in a rat model. Therefore, bevacizumab and rapamycin may be safer options than MMC following refractive surgery for the prevention of postoperative corneal opacity less affecting the number of keratocytes. 
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Footnotes
 Presented as a poster at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2012.
Footnotes
 Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (NRF-2010-0025662). The authors alone are responsible for the content and writing of this paper.
Footnotes
 Disclosure: K.S. Lee, None; D.-A. Ko, None; E.-S. Kim, None; M.J. Kim, None; H. Tchah, None; J.Y. Kim, None
Figure 1. 
 
Flow diagram of experimental procedures.
Figure 1. 
 
Flow diagram of experimental procedures.
Figure 2. 
 
Biomicroscopy photographs demonstrating postoperative corneal opacity of rats in mitomycin C (A), bevacizumab (B), rapamycin (C), and control (D) groups at 6 weeks after PRK.
Figure 2. 
 
Biomicroscopy photographs demonstrating postoperative corneal opacity of rats in mitomycin C (A), bevacizumab (B), rapamycin (C), and control (D) groups at 6 weeks after PRK.
Figure 3. 
 
Quantitation of central corneal haze at 3, 6, and 12 weeks after PRK. Error bars: standard deviation of the mean. At 6 weeks, the corneal haze of the control group was significantly more severe than those of the other groups (all P < 0.05). **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 3. 
 
Quantitation of central corneal haze at 3, 6, and 12 weeks after PRK. Error bars: standard deviation of the mean. At 6 weeks, the corneal haze of the control group was significantly more severe than those of the other groups (all P < 0.05). **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 4. 
 
Quantitation of apoptotic keratocytes in the central stroma after PRK. Error bars: standard deviation of the mean. **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 4. 
 
Quantitation of apoptotic keratocytes in the central stroma after PRK. Error bars: standard deviation of the mean. **, *Significant differences between those groups at a particular time (P < 0.05).
Figure 5. 
 
Immunolocalization of apoptotic keratocytes in the central cornea of mitomycin C (A), bevacizumab (C), rapamymark asancin (E), and control (G) groups using TUNEL staining at 12 weeks after PRK, and merged images with PI staining (B, D, F, H), respectively. There were significantly more apoptotic cells (white arrows in G) in the control group than in the mitomycin C, bevacizumab, and rapamycin groups (P < 0.05, cells ×400 field). Scale Bar = 25 μm.
Figure 5. 
 
Immunolocalization of apoptotic keratocytes in the central cornea of mitomycin C (A), bevacizumab (C), rapamymark asancin (E), and control (G) groups using TUNEL staining at 12 weeks after PRK, and merged images with PI staining (B, D, F, H), respectively. There were significantly more apoptotic cells (white arrows in G) in the control group than in the mitomycin C, bevacizumab, and rapamycin groups (P < 0.05, cells ×400 field). Scale Bar = 25 μm.
Figure 6. 
 
Quantitation of keratocytes in the central cornea after PRK. Error bars: standard deviation of the mean. ***, **,*Significant differences between those groups at a particular time (P < 0.05). +Significant difference between bevacizumab and control groups.
Figure 6. 
 
Quantitation of keratocytes in the central cornea after PRK. Error bars: standard deviation of the mean. ***, **,*Significant differences between those groups at a particular time (P < 0.05). +Significant difference between bevacizumab and control groups.
Figure 7. 
 
TEM at the central cornea of rat. in mitomycin C (A, E), bevacizumab (B, F), rapamycin (C, G), and control (D, H) groups at 3 and 12 weeks, respectively, PRK. Dying stromal cell shrinkage (#), chromatin condensation (*), irregular epithelial basement membrane, and polymorphonuclear leukocyte (arrow) and monocyte (arrowhead) infiltration were observed in the central corneas of all of rats in each group at 3 weeks after PRK. At 12 weeks, there were fewer keratocytes or inflammatory cells in the central corneas of the rats in the MMC, bevacizumab, and rapamycin groups than in the control group (EH). The collagen bundles were more irregular in the control group than in the MMC, bevacizumab, and rapamycin groups; however, consistency of the epithelial basement membrane was observed. In the central corneal stroma of the rats in the control group, relatively patent but some irregular epithelial basement membrane was found, and the collagen fibers were more irregular than in the other groups (H). In addition, relatively large amounts of RER were observed in the myofibroblasts of the control group. Magnification ×8000.
Figure 7. 
 
TEM at the central cornea of rat. in mitomycin C (A, E), bevacizumab (B, F), rapamycin (C, G), and control (D, H) groups at 3 and 12 weeks, respectively, PRK. Dying stromal cell shrinkage (#), chromatin condensation (*), irregular epithelial basement membrane, and polymorphonuclear leukocyte (arrow) and monocyte (arrowhead) infiltration were observed in the central corneas of all of rats in each group at 3 weeks after PRK. At 12 weeks, there were fewer keratocytes or inflammatory cells in the central corneas of the rats in the MMC, bevacizumab, and rapamycin groups than in the control group (EH). The collagen bundles were more irregular in the control group than in the MMC, bevacizumab, and rapamycin groups; however, consistency of the epithelial basement membrane was observed. In the central corneal stroma of the rats in the control group, relatively patent but some irregular epithelial basement membrane was found, and the collagen fibers were more irregular than in the other groups (H). In addition, relatively large amounts of RER were observed in the myofibroblasts of the control group. Magnification ×8000.
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