BASIC INVESTIGATION
Corneal Collagen Fibril Changes After Ultraviolet A/Riboflavin Corneal Crosslinking Yu Xia, PhD, Baichen Liu, MD, Zhengjun Fan, MD, and Xiujun Peng, MD
Purpose: The aim was to investigate the changes in collagen type 1 and type 3 in rabbit corneas undergoing corneal crosslinking with ultraviolet A and riboflavin and to analyze the possible mechanisms of corneal haze formation.
Methods: After removal of the central epithelium, the right corneas of 60 New Zealand rabbits were crosslinked with riboflavin and ultraviolet A, and 10 additional rabbits were used as the control group. The animals were killed 3, 7, 15, 30, 90, and 180 days postoperatively. Collagen type 1 and type 3 were analyzed using picrosirius red stain by means of polarized light microscopy. The biochemical changes in collagen type 3 at the time points indicated above were determined by Western blot analyses. Results: Collagen type 3 was significantly increased 30 days after corneal crosslinking compared with that in the control cornea, gradually increased until reaching its maximum value 90 days after riboflavin and ultraviolet A crosslinking, and then decreased until it returned to the normal state 180 days after crosslinking. There were no significant changes in collagen type 1 over time after corneal crosslinking. In agreement with the picrosirius red staining results, the western blot analyses showed that collagen type 3 was detected 15 days after the crosslinking treatment and continued to be present. However, 180 days after the crosslinking treatment, collagen type 3 could not be found in the crosslinked corneas. Conclusions: These findings suggest that ultraviolet A/riboflavin crosslinking results in collagen type 3 synthesis and degradation, which may offer at least a partial explanation for the formation of corneal haze. Key Words: corneal crosslinking, corneal wound healing, picrosirius red staining, corneal haze (Cornea 2014;33:56–59)
C
orneal collagen crosslinking using ultraviolet A light (370 nm) and riboflavin was introduced in clinical practice to halt the progression of keratoconus.1,2 Although available data suggest that corneal crosslinking with riboflavin and ultraviolet Received for publication May 7, 2013; revision received September 22, 2013; accepted September 24, 2013. Published online ahead of print November 14, 2013. From the Department of Ophthalmology, PLA Navy General Hospital, Beijing, China. Supported by the China Postdoctoral Science Foundation (No. 2012M512129). Financial disclosures/conflicts of interest: None reported. Reprints: Xiujun Peng, Department of Ophthalmology, PLA Navy General Hospital, 6 Fucheng Road, Beijing 100048, China (e-mail: PXJ1@vip. sina.com). Copyright © 2013 by Lippincott Williams & Wilkins
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A (UVA) seems to be a promising approach to increase the biomechanical stabilities of the cornea, the long-term effects of the technique on corneal tissue and the complications after corneal collagen crosslinking have not been fully elucidated.3 Experimental studies have proven that riboflavin liberates free radicals and reactive oxygen species into the surrounding corneal stroma when it interacts with UVA by photosensitized oxidation.3 Reactive oxygen species then induce the formation of covalent bonds between the amino acids of collagen chains. At the same time, the liberation of the so-called reactive oxygen species causes photochemical damage to corneal tissue.4,5 Additionally, the UVA/riboflavin (UVAR) crosslinking procedure involves the mechanical removal of the corneal epithelium, which also results in corneal injury. The corneal wound-healing response to this photochemical and mechanical damage is of particular relevance to the corneal crosslinking induced by UVAR because it is a major determinant of the efficacy and safety of the crosslinking procedure. In most cases, corneal haze is noted after corneal UVAR crosslinking, but the mechanisms leading to corneal haze formation after UVAR crosslinking treatment have not been fully clarified.3 Previous studies have reported that a dense extracellular matrix is seemingly compatible with corneal haze formation during the corneal wound-healing process because the structure of the extracellular matrix and its organization in the stromal tissue are important to the maintenance of corneal transparency.6 Corneal collagen is the main extracellular matrix involved in the wound-healing response to corneal damage. Therefore, it is important to evaluate the exact alterations in the biochemical nature and structural characteristics of stromal collagen fibers over time after corneal crosslinking with UVAR. The aim of our study was to investigate the changes in collagen fibers after corneal crosslinking with UVAR to elucidate the possible mechanisms leading to the formation of corneal haze. To the best of our knowledge, this study is the first to investigate the possible changes in the corneal collagen fibers after crosslinking treatment to better understand the effects of UVAR crosslinking on corneal collagen fibers and the formation of corneal haze associated with corneal crosslinking.
MATERIALS AND METHODS Animals Seventy New Zealand white rabbits (PLA Navy General Hospital’s animal room, Beijing, China) weighing 2 to 2.5 kg each were used. Sixty rabbits were treated with Cornea Volume 33, Number 1, January 2014
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UVAR crosslinking in 1 eye, and 10 of these treated rabbits were killed 3, 7, 15, 30, 90, and 180 days after the corneal crosslinking treatment. Another group of 10 animals was killed without any intervention and served as normal controls. All the procedures in this research study were approved by the PLA Navy General Hospital’s Ethics Committee and conformed to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.
UVAR Crosslinking Procedure The animals were anesthetized by giving them an intramuscular injection of 35 mg/kg of ketamine hydrochloride (Pharmaceutical Co, China), and a local anesthetic containing 0.4% oxybuprocaine hydrochloride (Benoxil, Santen Pharmaceutical Co, Osaka, Japan) was applied to the corneas before the crosslinking treatment. A central region of the cornea was gently marked with a 9.0-mm surgical trephine, and the corneal epithelium was mechanically removed using a blunt hockey knife to allow for better diffusion of riboflavin into the stroma. The UVAR crosslinking treatment was performed according to a protocol similar to the technique reported previously by Wollensak et al.1 Before irradiation with UVA, a 0.1% riboflavin solution consisting of 10 mg of riboflavin 5-phosphate (Sigma– Aldrich Trading Co, Shanghai) dissolved in 10 mL of 20% dextran-T-500 solution (Sigma–Aldrich) was applied onto the debrided corneal surface every 5 minutes until the presence of riboflavin could be confirmed through slit-lamp microscopy. Because the application of riboflavin solution to the corneal surface every 5 minutes continued to ensure corneal saturation, the corneas were irradiated with ultraviolet A (370 nm, 3 mW/cm2 at a distance of 1 cm) for 30 minutes using a radiation device (UV-X Corneal Crosslinking System, Zurich, Switzerland). Eye drops containing a steroid and antibiotics (Dexamethasone/tobramycin Alcon Laboratories Inc) were administered to the eyes at the end of the treatment.
Picrosirius Red Staining At 3, 7, 15, 30, 90, and 180 days after corneal crosslinking treatment, 3 animals were killed with an intravenous overdose of sodium pentobarbital. Their eyes were enucleated, and the central corneal buttons with a diameter of 9 mm were trephined and fixed in 4% paraformaldehyde in 0.1 M phoshpate buffered saline for 24 hours. The tissue samples were embedded in paraffin, sectioned to a thickness of 5 mm, and stained for 1 hour in the picrosirius solution described by Junqueira et al7 (0.1% solution of Sirius Red in saturated aqueous picric acid; Sirius Red was obtained from Sinopharm Chemical Reagent Co, Shanghai, China). The stained sections were then washed for 2 minutes in 0.01 N HCI, dehydrated, cleared, and mounted in synthetic resin. The collagen type 1 and type 3 of the corneal sections were observed by polarization microscopy (LSM 510 META; Carl Zeiss Inc, Jena, Germany). The collagen type 1 and type 3 contents of the sections were quantitatively analyzed using an image analysis program (Motic 2013 Lippincott Williams & Wilkins
Corneal Collagen Fibril Changes after Cross-linking
Images Advanced 3.2 Software, Motic Corp). For each slide, a total of 10 fields were analyzed with a 20· objective lens.
Western Blot Analysis of Collagen Type 3 Seven central corneal buttons were frozen in liquid nitrogen 3, 7, 15, 30, 90, and 180 days after UVAR crosslinking. First, the cornea samples were minced using razor blades and further homogenized in a blender. The proteoglycans were then extracted in 50 mL of 0.5 M Naacetate (pH 6) at 4°C for 24 hours. After centrifugation at 14,000 rpm for 30 minutes, the pellet was resuspended in 20 vol of 0.5 M acetic acid with 10 mg/mL of pepsin (800–2500 U/mg; EC 3.4.23.1; Sigma, Shanghai, China) under gentle stirring for 48 hours at 0 to 4°C. After centrifugation at 14,000 rpm for 30 minutes, the supernatant with solubilized collagen was neutralized to pH 8.0 using NaOH to inactivate pepsin for 15 minutes. After centrifugation at 14,000 rpm for 30 minutes, the supernatant was dialyzed against 1.7 M NaCl, pH 7.5, in 0.5 M acetic acid at 4°C to precipitate collagen type 3.8 After collagen type 3 extraction, the same proportions of each extracted sample solution were mixed with sample buffer and heated to 70°C for 10 minutes, loaded onto a sodium dodecylsulfate–6% polyacrylamide running gel with a sodium dodecylsulfate–5% stacking gel on top, and subjected to electrophoresis. After the electrophoresis was performed, the proteins separated by the electrophoresis were transferred onto nitrocellulose membranes by electroblotting (Bio-Trans) using a semidry electrophoretic transfer unit. Any nonspecific binding was blocked with 1% casein in phoshpate buffered saline at 4°C overnight. After blocking, the membranes with immobilized antigens were incubated overnight with anticollagen type 3 antibody (1:1000 in 5% milk; Sigma)at 4°C under constant shaking. After five 5-minute washes, the membranes were incubated with antirabbit immunoglobulin–horseradish peroxidase conjugate (1:7500 in 5% milk) secondary antibody (Sigma) for 30 minutes. After intensive washing, the membranes were incubated with avidin-biotin-peroxidase complex for 30 minutes. The bound conjugates were visualized using diaminobenzidinenickel as the chromogen. The membranes were examined on an HP Scanjet G3110 scanner and analyzed using the Image Master Total Lab v2.01 program (Amersham Biosciences).
Statistics The statistical analyses were performed using SPSS for Windows (version 16.0, SPSS, Chicago, IL). The differences in corneal collagen type 1 and type 3 at each time point between the experimental group and the control group were compared using the Wilcoxon signed-rank test. The values are presented as the mean 6 SD. The differences between the means were considered statistically significant at P , 0.05.
RESULTS The observation of picrosirius red–stained histological sections by polarized light microscopy is an effective way of identifying collagen type 1 and type 3 in collagen fibers. When viewed under polarized light, collagen type 1 appears www.corneajrnl.com |
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predominantly red, and collagen type 3 appears predominantly green after picrosirius red staining. In this study, there were significant changes in collagen type 3 over time after corneal crosslinking with UVAR. Collagen type 3 started to significantly increase 30 days after the UVAR crosslinking treatment relative to that in the control (Wilcoxon signed-rank test, P , 0.05; Figs. 1A, B), increased progressively thereafter, and reached its maximum value, and then began to decline 90 days after the UVAR crosslinking treatment (Fig. 1C). Six months after the crosslinking treatment, collagen type 3 was similar to that observed in the control corneas (Fig. 1D). However, there was no statistically meaningful difference (86% 6 7% vs. 82% 6 11%, P = 0.178) in collagen type 1 30 days after the UVAR crosslinking between the crosslinked corneas and the controls. Moreover, no statistically significant changes were observed (Wilcoxon signedrank test, P . 0.05) in type 1 collagen over time after the UVAR crosslinking treatment. The western blot analysis of corneal collagen type 3 indicated that collagen type 3 protein could be faintly detected 15 days after UVAR crosslinking treatment, and it increased slightly in intensity, became apparent 30 days after treatment, and continued to be present 90 days after the crosslinking treatment. In contrast, collagen type 3 protein was not observed in the normal corneas.
DISCUSSION Collagen, which is the principal component of the stroma, plays an important role in the preservation of corneal transparency; therefore, direct visualization of collagen fibers
is crucial for analyzing the collagen composition of the stroma.8 In this study, the observation of picrosirius red– stained sections by polarized light microscopy demonstrated that there was a significant increase in collagen type 3 between 30 and 90 days after the UVAR crosslinking treatment. However, the relative amount of collagen type 1 did not show any significant changes over time after corneal crosslinking treatment. These results provide important evidence of the exact alterations that occur in the structural characteristics of stromal collagen fibers and enabled us to better understand the modifications that occur in corneal collagen fibers subjected to UVAR crosslinking treatment. In this study, collagen type 3 protein was identified by western blotting analysis 15 days after treatment and continued to be present 90 days after UVAR crosslinking treatment. This finding is well correlated with the morphologic changes in collagen type 3 after crosslinking treatment as observed through picrosirius red staining. The changes in collagen type 3 over time after UVAR crosslinking treatment may partially reflect a wound-healing response in the corneal tissue because collagen type 3 may be mainly produced during the corneal wound-healing process.6 Further, previous experimental results regarding gel electrophoretic collagen type 1 show that irradiation with UVA in the presence of riboflavin mainly causes collagen type 1 to covalently crosslink into high-molecular-weight polymers by photosensitized oxidation.9,10 These data clearly suggest that the changes in collagen fibers that occur after crosslinking treatment could be related not only to immediate crosslinking effects but also to later wound-healing responses.
FIGURE 1. A, Collagen type 1 and type 3 in the control group with picrosirius red staining under polarized light. Collagen type 1 is predominantly red, and collagen type 3 predominantly green when stained with picrosirius red and observed under polarized light. Scale bar = 100 mm. B, Collagen type 1 and type 3 30 days after UVAR crosslinking treatment. Increased amounts of green fibers, which represent collagen type 3, can be observed with picrosirius red staining under polarized light. The amount of red fibers, representing collagen type 1, did not significantly increase in the regions analyzed. Scale bar = 100 mm. C, Collagen type 1 and type 3 90 days after UVAR crosslinking treatment. Green fibers representing collagen type 3 are prevalent in the regions analyzed. Scale bar = 100 mm. D, Collagen type 1 and type 3 180 days after UVAR crosslinking treatment. Red fibers representing collagen type 1 are prevalent in the regions analyzed. Scale bar = 100 mm.
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In previous studies, corneas were observed to have developed a haze that peaked between 30 and 90 days after UVAR crosslinking and then diminished over time to reach the baseline 1 year after treatment.11–15 Our experimental results regarding the changes in collagen type 3 seem to be consistent with the presence of haze after UVAR crosslinking treatment. These results support the hypothesis that concomitant changes in corneal collagen fibers may be a risk factor for corneal haze formation after performing corneal crosslinking because corneal haze has been reported to be caused by abnormal collagen fiber density, diameter, and spacing. Riboflavin irradiated by UVA caused the production of reactive oxygen species that not only induced the formation of crosslinking bridges between the collagen proteins but also caused photochemical damage to corneal tissue.3,9 The corneal injury caused by the UVAR crosslinking procedure led to a woundhealing response, which has been described as an exceedingly complex cascade process involving keratocyte apoptosis, proliferation, and migration.6,16 After keratocyte apoptosis, proliferation, and migration, activated keratocytes get transformed into myofibroblasts.17 These myofibroblasts are the major determinants of corneal transparency because these cells are associated with the processes of stromal remodeling through the regeneration of collagen, glycosaminoglycans, and other extracellular matrix components.18 As suggested in the literature, the generation of a large number of collagen fibers, and the disordered extracellular matrix associated with myofibroblasts, is likely the primary factor causing the formation of corneal haze.6 In this case, collagen type 3 synthesis, deposition, and degradation in the stroma after UVAR crosslinking treatment may offer at least a partial explanation for the development of corneal haze. To the best of our knowledge, the previous study showed that keratocyte apoptosis began 12 to 24 hours after UVAR crosslinking.5,19 Mitosis surrounding the area of keratocyte apoptosis can be detected in stromal keratocytes by immunohistochemical testing for the antibody anti-Ki67, which is a marker of activated keratocytes.20 A study on rabbits demonstrated that myofibroblasts are generated in the stroma 1 to 4 weeks after corneal crosslinking by immunocytochemical analysis using an antibody against smooth muscle actin.21 In our study, the observed significant changes in stromal collagen fibers with time after the UVAR crosslinking treatment provide valuable evidence of the progressive synthesis and degradation of collagen molecules. Further, Wollensak et al22 reported a significant increase in the collagen fibril diameter after UVAR crosslinking treatment. These changes in corneal collagen seemed to be compatible with a microscopically detectable corneal haze because the production of disorganized collagen fibers, irregular spacing, and increased diameter of collagen fibrils all contribute to corneal haze formation. This study combined molecular biology with histology to enhance the understanding of the events associated with the wound-healing response to corneal crosslinking. It is essential to thoroughly elucidate the precise effects of corneal crosslinking treatment not only on the exposed cellular populations but also on the component molecules of the extracellular matrix. More studies are necessary to understand the biological and molecular processes associated with the corneal crosslinking 2013 Lippincott Williams & Wilkins
Corneal Collagen Fibril Changes after Cross-linking
procedure. The complete biological response to corneal crosslinking will be investigated in future studies featuring a longer follow-up.
REFERENCES 1. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:620–627. 2. Caporossi A, Baiocchi S, Mazzotta C, et al. Parasurgical therapy for keratoconus by riboflavin–ultraviolet type A rays induced cross-linking of corneal collagen: preliminary refractive results in an Italian study. J Cataract Refract Surg. 2006;32:837–845. 3. Wollensak G. Corneal collagen crosslinking: new horizons. Exp Rev Ophthalmol. 2010;5:201–215. 4. Wollensak G, Spoerl E, Reber F, et al. Keratocyte cytotoxicity of riboflavin/UVA treatment in vitro. Eye (Lond). 2004;18:718–722. 5. Wollensak G, Spoerl E, Wilsch M, et al. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea. 2004;23:43–49. 6. Netto MV, Mohan RR, Ambrósio R Jr, et al. Wound healing in the cornea a review of refractive surgery complications and new prospects for therapy. Cornea. 2005;24:509–522. 7. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979;11:447–455. 8. Newsome DA, Gross J, Hassell JR. Human corneal stroma contains three distinct collagens. Invest Ophthalmol Vis Sci. 1982;22:376–381. 9. Wollensak G, Redl B. Gel electrophoretic analysis of corneal collagen after photodynamic cross-linking treatment. Cornea. 2008;27:353–356. 10. Zhang Y, Conrad AH, Conrad GW. Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal crosslinking. J Biol Chem. 2011;286:13011–13022. 11. Mazzotta C, Traversi C, Baiocchi S, et al. Corneal healing after riboflavin ultraviolet-A collagen crosslinking determined by confocal laser scanning microscopy in vivo: early and late modifications. Am J Ophthalmol. 2008;146:527–533. 12. Mazzotta C, Balestrazzi A, Traversi C, et al. Treatment of progressive keratoconus by riboflavin-UVA-induced crosslinking of corneal collagen: ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea. 2007;26:390–397. 13. Raiskup F, Hoyer A, Spoerl E. Permanent corneal haze after riboflavin– UVA-induced cross-linking in keratoconus. J Refract Surg. 2009;25: 824–828. 14. Wollensak G, Iomdina E, Dittert DD, et al. Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA. Cornea. 2007;26:600–605. 15. Greenstein SA, Fry KL, Bhatt J, et al. Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J Cataract Refract Surg. 2010;36: 2105–2114. 16. Koller T, Mrochen M, Seiler T. Complication and failure rates after corneal cross-linking. J Cataract Refract Surg. 2009;35:1358–1362. 17. Wilson SE, Kim WJ. Keratocyte apoptosis: implications on corneal wound healing, tissue organization, and disease. Invest Ophthalmol Vis Sci. 1998;39:220–226. 18. Hassell JR, Birk DE. The molecular basis of corneal transparency. Exp Eye Res. 2010;91:326–335. 19. Dhaliwal JS, Kaufman SC. Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas. Cornea. 2009; 28:62–67. 20. Mencucci R, Marini M, Paladini I, et al. Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea. Clin Experiment Ophthalmol. 2010;38:49–56. 21. Esquenazi S, He J, Li N, et al. Immunofluorescence of rabbit corneas after collagen cross-linking treatment with riboflavin and ultraviolet A. Cornea. 2010;29:412–417. 22. Wollensak G, Wilsch M, Spoerl E, et al. Collagen fiber diameter in the rabbit cornea after collagen cross-linking by riboflavin/UVA. Cornea. 2004;23:503–507.
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Corneal Collagen Fibril Changes After Ultraviolet A/Riboflavin Corneal Crosslinking Yu Xia, PhD, Baichen Liu, MD, Zhengjun Fan, MD, and Xiujun Peng, MD
Purpose: The aim was to investigate the changes in collagen type 1 and type 3 in rabbit corneas undergoing corneal crosslinking with ultraviolet A and riboflavin and to analyze the possible mechanisms of corneal haze formation.
Methods: After removal of the central epithelium, the right corneas of 60 New Zealand rabbits were crosslinked with riboflavin and ultraviolet A, and 10 additional rabbits were used as the control group. The animals were killed 3, 7, 15, 30, 90, and 180 days postoperatively. Collagen type 1 and type 3 were analyzed using picrosirius red stain by means of polarized light microscopy. The biochemical changes in collagen type 3 at the time points indicated above were determined by Western blot analyses. Results: Collagen type 3 was significantly increased 30 days after corneal crosslinking compared with that in the control cornea, gradually increased until reaching its maximum value 90 days after riboflavin and ultraviolet A crosslinking, and then decreased until it returned to the normal state 180 days after crosslinking. There were no significant changes in collagen type 1 over time after corneal crosslinking. In agreement with the picrosirius red staining results, the western blot analyses showed that collagen type 3 was detected 15 days after the crosslinking treatment and continued to be present. However, 180 days after the crosslinking treatment, collagen type 3 could not be found in the crosslinked corneas. Conclusions: These findings suggest that ultraviolet A/riboflavin crosslinking results in collagen type 3 synthesis and degradation, which may offer at least a partial explanation for the formation of corneal haze. Key Words: corneal crosslinking, corneal wound healing, picrosirius red staining, corneal haze (Cornea 2014;33:56–59)
C
orneal collagen crosslinking using ultraviolet A light (370 nm) and riboflavin was introduced in clinical practice to halt the progression of keratoconus.1,2 Although available data suggest that corneal crosslinking with riboflavin and ultraviolet Received for publication May 7, 2013; revision received September 22, 2013; accepted September 24, 2013. Published online ahead of print November 14, 2013. From the Department of Ophthalmology, PLA Navy General Hospital, Beijing, China. Supported by the China Postdoctoral Science Foundation (No. 2012M512129). Financial disclosures/conflicts of interest: None reported. Reprints: Xiujun Peng, Department of Ophthalmology, PLA Navy General Hospital, 6 Fucheng Road, Beijing 100048, China (e-mail: PXJ1@vip. sina.com). Copyright © 2013 by Lippincott Williams & Wilkins
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A (UVA) seems to be a promising approach to increase the biomechanical stabilities of the cornea, the long-term effects of the technique on corneal tissue and the complications after corneal collagen crosslinking have not been fully elucidated.3 Experimental studies have proven that riboflavin liberates free radicals and reactive oxygen species into the surrounding corneal stroma when it interacts with UVA by photosensitized oxidation.3 Reactive oxygen species then induce the formation of covalent bonds between the amino acids of collagen chains. At the same time, the liberation of the so-called reactive oxygen species causes photochemical damage to corneal tissue.4,5 Additionally, the UVA/riboflavin (UVAR) crosslinking procedure involves the mechanical removal of the corneal epithelium, which also results in corneal injury. The corneal wound-healing response to this photochemical and mechanical damage is of particular relevance to the corneal crosslinking induced by UVAR because it is a major determinant of the efficacy and safety of the crosslinking procedure. In most cases, corneal haze is noted after corneal UVAR crosslinking, but the mechanisms leading to corneal haze formation after UVAR crosslinking treatment have not been fully clarified.3 Previous studies have reported that a dense extracellular matrix is seemingly compatible with corneal haze formation during the corneal wound-healing process because the structure of the extracellular matrix and its organization in the stromal tissue are important to the maintenance of corneal transparency.6 Corneal collagen is the main extracellular matrix involved in the wound-healing response to corneal damage. Therefore, it is important to evaluate the exact alterations in the biochemical nature and structural characteristics of stromal collagen fibers over time after corneal crosslinking with UVAR. The aim of our study was to investigate the changes in collagen fibers after corneal crosslinking with UVAR to elucidate the possible mechanisms leading to the formation of corneal haze. To the best of our knowledge, this study is the first to investigate the possible changes in the corneal collagen fibers after crosslinking treatment to better understand the effects of UVAR crosslinking on corneal collagen fibers and the formation of corneal haze associated with corneal crosslinking.
MATERIALS AND METHODS Animals Seventy New Zealand white rabbits (PLA Navy General Hospital’s animal room, Beijing, China) weighing 2 to 2.5 kg each were used. Sixty rabbits were treated with Cornea Volume 33, Number 1, January 2014
Cornea Volume 33, Number 1, January 2014
UVAR crosslinking in 1 eye, and 10 of these treated rabbits were killed 3, 7, 15, 30, 90, and 180 days after the corneal crosslinking treatment. Another group of 10 animals was killed without any intervention and served as normal controls. All the procedures in this research study were approved by the PLA Navy General Hospital’s Ethics Committee and conformed to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.
UVAR Crosslinking Procedure The animals were anesthetized by giving them an intramuscular injection of 35 mg/kg of ketamine hydrochloride (Pharmaceutical Co, China), and a local anesthetic containing 0.4% oxybuprocaine hydrochloride (Benoxil, Santen Pharmaceutical Co, Osaka, Japan) was applied to the corneas before the crosslinking treatment. A central region of the cornea was gently marked with a 9.0-mm surgical trephine, and the corneal epithelium was mechanically removed using a blunt hockey knife to allow for better diffusion of riboflavin into the stroma. The UVAR crosslinking treatment was performed according to a protocol similar to the technique reported previously by Wollensak et al.1 Before irradiation with UVA, a 0.1% riboflavin solution consisting of 10 mg of riboflavin 5-phosphate (Sigma– Aldrich Trading Co, Shanghai) dissolved in 10 mL of 20% dextran-T-500 solution (Sigma–Aldrich) was applied onto the debrided corneal surface every 5 minutes until the presence of riboflavin could be confirmed through slit-lamp microscopy. Because the application of riboflavin solution to the corneal surface every 5 minutes continued to ensure corneal saturation, the corneas were irradiated with ultraviolet A (370 nm, 3 mW/cm2 at a distance of 1 cm) for 30 minutes using a radiation device (UV-X Corneal Crosslinking System, Zurich, Switzerland). Eye drops containing a steroid and antibiotics (Dexamethasone/tobramycin Alcon Laboratories Inc) were administered to the eyes at the end of the treatment.
Picrosirius Red Staining At 3, 7, 15, 30, 90, and 180 days after corneal crosslinking treatment, 3 animals were killed with an intravenous overdose of sodium pentobarbital. Their eyes were enucleated, and the central corneal buttons with a diameter of 9 mm were trephined and fixed in 4% paraformaldehyde in 0.1 M phoshpate buffered saline for 24 hours. The tissue samples were embedded in paraffin, sectioned to a thickness of 5 mm, and stained for 1 hour in the picrosirius solution described by Junqueira et al7 (0.1% solution of Sirius Red in saturated aqueous picric acid; Sirius Red was obtained from Sinopharm Chemical Reagent Co, Shanghai, China). The stained sections were then washed for 2 minutes in 0.01 N HCI, dehydrated, cleared, and mounted in synthetic resin. The collagen type 1 and type 3 of the corneal sections were observed by polarization microscopy (LSM 510 META; Carl Zeiss Inc, Jena, Germany). The collagen type 1 and type 3 contents of the sections were quantitatively analyzed using an image analysis program (Motic 2013 Lippincott Williams & Wilkins
Corneal Collagen Fibril Changes after Cross-linking
Images Advanced 3.2 Software, Motic Corp). For each slide, a total of 10 fields were analyzed with a 20· objective lens.
Western Blot Analysis of Collagen Type 3 Seven central corneal buttons were frozen in liquid nitrogen 3, 7, 15, 30, 90, and 180 days after UVAR crosslinking. First, the cornea samples were minced using razor blades and further homogenized in a blender. The proteoglycans were then extracted in 50 mL of 0.5 M Naacetate (pH 6) at 4°C for 24 hours. After centrifugation at 14,000 rpm for 30 minutes, the pellet was resuspended in 20 vol of 0.5 M acetic acid with 10 mg/mL of pepsin (800–2500 U/mg; EC 3.4.23.1; Sigma, Shanghai, China) under gentle stirring for 48 hours at 0 to 4°C. After centrifugation at 14,000 rpm for 30 minutes, the supernatant with solubilized collagen was neutralized to pH 8.0 using NaOH to inactivate pepsin for 15 minutes. After centrifugation at 14,000 rpm for 30 minutes, the supernatant was dialyzed against 1.7 M NaCl, pH 7.5, in 0.5 M acetic acid at 4°C to precipitate collagen type 3.8 After collagen type 3 extraction, the same proportions of each extracted sample solution were mixed with sample buffer and heated to 70°C for 10 minutes, loaded onto a sodium dodecylsulfate–6% polyacrylamide running gel with a sodium dodecylsulfate–5% stacking gel on top, and subjected to electrophoresis. After the electrophoresis was performed, the proteins separated by the electrophoresis were transferred onto nitrocellulose membranes by electroblotting (Bio-Trans) using a semidry electrophoretic transfer unit. Any nonspecific binding was blocked with 1% casein in phoshpate buffered saline at 4°C overnight. After blocking, the membranes with immobilized antigens were incubated overnight with anticollagen type 3 antibody (1:1000 in 5% milk; Sigma)at 4°C under constant shaking. After five 5-minute washes, the membranes were incubated with antirabbit immunoglobulin–horseradish peroxidase conjugate (1:7500 in 5% milk) secondary antibody (Sigma) for 30 minutes. After intensive washing, the membranes were incubated with avidin-biotin-peroxidase complex for 30 minutes. The bound conjugates were visualized using diaminobenzidinenickel as the chromogen. The membranes were examined on an HP Scanjet G3110 scanner and analyzed using the Image Master Total Lab v2.01 program (Amersham Biosciences).
Statistics The statistical analyses were performed using SPSS for Windows (version 16.0, SPSS, Chicago, IL). The differences in corneal collagen type 1 and type 3 at each time point between the experimental group and the control group were compared using the Wilcoxon signed-rank test. The values are presented as the mean 6 SD. The differences between the means were considered statistically significant at P , 0.05.
RESULTS The observation of picrosirius red–stained histological sections by polarized light microscopy is an effective way of identifying collagen type 1 and type 3 in collagen fibers. When viewed under polarized light, collagen type 1 appears www.corneajrnl.com |
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predominantly red, and collagen type 3 appears predominantly green after picrosirius red staining. In this study, there were significant changes in collagen type 3 over time after corneal crosslinking with UVAR. Collagen type 3 started to significantly increase 30 days after the UVAR crosslinking treatment relative to that in the control (Wilcoxon signed-rank test, P , 0.05; Figs. 1A, B), increased progressively thereafter, and reached its maximum value, and then began to decline 90 days after the UVAR crosslinking treatment (Fig. 1C). Six months after the crosslinking treatment, collagen type 3 was similar to that observed in the control corneas (Fig. 1D). However, there was no statistically meaningful difference (86% 6 7% vs. 82% 6 11%, P = 0.178) in collagen type 1 30 days after the UVAR crosslinking between the crosslinked corneas and the controls. Moreover, no statistically significant changes were observed (Wilcoxon signedrank test, P . 0.05) in type 1 collagen over time after the UVAR crosslinking treatment. The western blot analysis of corneal collagen type 3 indicated that collagen type 3 protein could be faintly detected 15 days after UVAR crosslinking treatment, and it increased slightly in intensity, became apparent 30 days after treatment, and continued to be present 90 days after the crosslinking treatment. In contrast, collagen type 3 protein was not observed in the normal corneas.
DISCUSSION Collagen, which is the principal component of the stroma, plays an important role in the preservation of corneal transparency; therefore, direct visualization of collagen fibers
is crucial for analyzing the collagen composition of the stroma.8 In this study, the observation of picrosirius red– stained sections by polarized light microscopy demonstrated that there was a significant increase in collagen type 3 between 30 and 90 days after the UVAR crosslinking treatment. However, the relative amount of collagen type 1 did not show any significant changes over time after corneal crosslinking treatment. These results provide important evidence of the exact alterations that occur in the structural characteristics of stromal collagen fibers and enabled us to better understand the modifications that occur in corneal collagen fibers subjected to UVAR crosslinking treatment. In this study, collagen type 3 protein was identified by western blotting analysis 15 days after treatment and continued to be present 90 days after UVAR crosslinking treatment. This finding is well correlated with the morphologic changes in collagen type 3 after crosslinking treatment as observed through picrosirius red staining. The changes in collagen type 3 over time after UVAR crosslinking treatment may partially reflect a wound-healing response in the corneal tissue because collagen type 3 may be mainly produced during the corneal wound-healing process.6 Further, previous experimental results regarding gel electrophoretic collagen type 1 show that irradiation with UVA in the presence of riboflavin mainly causes collagen type 1 to covalently crosslink into high-molecular-weight polymers by photosensitized oxidation.9,10 These data clearly suggest that the changes in collagen fibers that occur after crosslinking treatment could be related not only to immediate crosslinking effects but also to later wound-healing responses.
FIGURE 1. A, Collagen type 1 and type 3 in the control group with picrosirius red staining under polarized light. Collagen type 1 is predominantly red, and collagen type 3 predominantly green when stained with picrosirius red and observed under polarized light. Scale bar = 100 mm. B, Collagen type 1 and type 3 30 days after UVAR crosslinking treatment. Increased amounts of green fibers, which represent collagen type 3, can be observed with picrosirius red staining under polarized light. The amount of red fibers, representing collagen type 1, did not significantly increase in the regions analyzed. Scale bar = 100 mm. C, Collagen type 1 and type 3 90 days after UVAR crosslinking treatment. Green fibers representing collagen type 3 are prevalent in the regions analyzed. Scale bar = 100 mm. D, Collagen type 1 and type 3 180 days after UVAR crosslinking treatment. Red fibers representing collagen type 1 are prevalent in the regions analyzed. Scale bar = 100 mm.
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2013 Lippincott Williams & Wilkins
Cornea Volume 33, Number 1, January 2014
In previous studies, corneas were observed to have developed a haze that peaked between 30 and 90 days after UVAR crosslinking and then diminished over time to reach the baseline 1 year after treatment.11–15 Our experimental results regarding the changes in collagen type 3 seem to be consistent with the presence of haze after UVAR crosslinking treatment. These results support the hypothesis that concomitant changes in corneal collagen fibers may be a risk factor for corneal haze formation after performing corneal crosslinking because corneal haze has been reported to be caused by abnormal collagen fiber density, diameter, and spacing. Riboflavin irradiated by UVA caused the production of reactive oxygen species that not only induced the formation of crosslinking bridges between the collagen proteins but also caused photochemical damage to corneal tissue.3,9 The corneal injury caused by the UVAR crosslinking procedure led to a woundhealing response, which has been described as an exceedingly complex cascade process involving keratocyte apoptosis, proliferation, and migration.6,16 After keratocyte apoptosis, proliferation, and migration, activated keratocytes get transformed into myofibroblasts.17 These myofibroblasts are the major determinants of corneal transparency because these cells are associated with the processes of stromal remodeling through the regeneration of collagen, glycosaminoglycans, and other extracellular matrix components.18 As suggested in the literature, the generation of a large number of collagen fibers, and the disordered extracellular matrix associated with myofibroblasts, is likely the primary factor causing the formation of corneal haze.6 In this case, collagen type 3 synthesis, deposition, and degradation in the stroma after UVAR crosslinking treatment may offer at least a partial explanation for the development of corneal haze. To the best of our knowledge, the previous study showed that keratocyte apoptosis began 12 to 24 hours after UVAR crosslinking.5,19 Mitosis surrounding the area of keratocyte apoptosis can be detected in stromal keratocytes by immunohistochemical testing for the antibody anti-Ki67, which is a marker of activated keratocytes.20 A study on rabbits demonstrated that myofibroblasts are generated in the stroma 1 to 4 weeks after corneal crosslinking by immunocytochemical analysis using an antibody against smooth muscle actin.21 In our study, the observed significant changes in stromal collagen fibers with time after the UVAR crosslinking treatment provide valuable evidence of the progressive synthesis and degradation of collagen molecules. Further, Wollensak et al22 reported a significant increase in the collagen fibril diameter after UVAR crosslinking treatment. These changes in corneal collagen seemed to be compatible with a microscopically detectable corneal haze because the production of disorganized collagen fibers, irregular spacing, and increased diameter of collagen fibrils all contribute to corneal haze formation. This study combined molecular biology with histology to enhance the understanding of the events associated with the wound-healing response to corneal crosslinking. It is essential to thoroughly elucidate the precise effects of corneal crosslinking treatment not only on the exposed cellular populations but also on the component molecules of the extracellular matrix. More studies are necessary to understand the biological and molecular processes associated with the corneal crosslinking 2013 Lippincott Williams & Wilkins
Corneal Collagen Fibril Changes after Cross-linking
procedure. The complete biological response to corneal crosslinking will be investigated in future studies featuring a longer follow-up.
REFERENCES 1. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:620–627. 2. Caporossi A, Baiocchi S, Mazzotta C, et al. Parasurgical therapy for keratoconus by riboflavin–ultraviolet type A rays induced cross-linking of corneal collagen: preliminary refractive results in an Italian study. J Cataract Refract Surg. 2006;32:837–845. 3. Wollensak G. Corneal collagen crosslinking: new horizons. Exp Rev Ophthalmol. 2010;5:201–215. 4. Wollensak G, Spoerl E, Reber F, et al. Keratocyte cytotoxicity of riboflavin/UVA treatment in vitro. Eye (Lond). 2004;18:718–722. 5. Wollensak G, Spoerl E, Wilsch M, et al. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea. 2004;23:43–49. 6. Netto MV, Mohan RR, Ambrósio R Jr, et al. Wound healing in the cornea a review of refractive surgery complications and new prospects for therapy. Cornea. 2005;24:509–522. 7. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979;11:447–455. 8. Newsome DA, Gross J, Hassell JR. Human corneal stroma contains three distinct collagens. Invest Ophthalmol Vis Sci. 1982;22:376–381. 9. Wollensak G, Redl B. Gel electrophoretic analysis of corneal collagen after photodynamic cross-linking treatment. Cornea. 2008;27:353–356. 10. Zhang Y, Conrad AH, Conrad GW. Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal crosslinking. J Biol Chem. 2011;286:13011–13022. 11. Mazzotta C, Traversi C, Baiocchi S, et al. Corneal healing after riboflavin ultraviolet-A collagen crosslinking determined by confocal laser scanning microscopy in vivo: early and late modifications. Am J Ophthalmol. 2008;146:527–533. 12. Mazzotta C, Balestrazzi A, Traversi C, et al. Treatment of progressive keratoconus by riboflavin-UVA-induced crosslinking of corneal collagen: ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea. 2007;26:390–397. 13. Raiskup F, Hoyer A, Spoerl E. Permanent corneal haze after riboflavin– UVA-induced cross-linking in keratoconus. J Refract Surg. 2009;25: 824–828. 14. Wollensak G, Iomdina E, Dittert DD, et al. Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA. Cornea. 2007;26:600–605. 15. Greenstein SA, Fry KL, Bhatt J, et al. Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J Cataract Refract Surg. 2010;36: 2105–2114. 16. Koller T, Mrochen M, Seiler T. Complication and failure rates after corneal cross-linking. J Cataract Refract Surg. 2009;35:1358–1362. 17. Wilson SE, Kim WJ. Keratocyte apoptosis: implications on corneal wound healing, tissue organization, and disease. Invest Ophthalmol Vis Sci. 1998;39:220–226. 18. Hassell JR, Birk DE. The molecular basis of corneal transparency. Exp Eye Res. 2010;91:326–335. 19. Dhaliwal JS, Kaufman SC. Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas. Cornea. 2009; 28:62–67. 20. Mencucci R, Marini M, Paladini I, et al. Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea. Clin Experiment Ophthalmol. 2010;38:49–56. 21. Esquenazi S, He J, Li N, et al. Immunofluorescence of rabbit corneas after collagen cross-linking treatment with riboflavin and ultraviolet A. Cornea. 2010;29:412–417. 22. Wollensak G, Wilsch M, Spoerl E, et al. Collagen fiber diameter in the rabbit cornea after collagen cross-linking by riboflavin/UVA. Cornea. 2004;23:503–507.
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