Effect of irradiation time on riboflavin–ultraviolet-A collagen crosslinking in rabbit sclera

LABORATORY SCIENCE

Effect of irradiation time on riboflavin–ultraviolet-A collagen crosslinking in rabbit sclera Yali Zhang, MD, Changxin Zou, MD, Lei Liu, MD, Lijun Cao, MD, Xinchang Xia, MD, Zhaona Li, MD, Ming Hu, MD, Haiqun Yu, MD, Guoying Mu, MD

PURPOSE: To determine the effect of the duration of irradiation on the biomechanical parameters of combined riboflavin–ultraviolet-A (UVA) collagen crosslinking (CXL) in rabbit sclera. SETTING: Department of Ophthalmology, Provincial Hospital affiliated with Shandong University, Shandong, China. DESIGN: Experimental study. METHODS: Thirty-six New Zealand rabbits were divided into 6 groups based on the duration of irradiation (10, 20, 30, 40, 50, or 60 minutes). After the application of riboflavin 0.1% drops (without dextran) as a photosensitizer, the animals were irradiated with 3 mW/cm2 UVA at 365 nm. Only the left eye of each rabbit was treated. All the animals were humanely killed 24 hours postoperatively. One eye in each treated group was used for light microscopy. The other treated eye and all control eyes were prepared for biomechanical testing. The biomechanical parameters were ultimate stress, Young modulus, and the physiological modulus. RESULTS: The eyes irradiated for 10 or 20 minutes did not differ significantly from the control eyes. Stress–strain measurement of scleral strips irradiated for 40 minutes or longer showed a significant increase in the ultimate stress, Young modulus, and the physiological modulus. There was a significant increase in the physiological modulus of scleral strips irradiated for 30 minutes or longer. Eyes that were irradiated for 50 minutes and 60 minutes had retinal damage. CONCLUSIONS: Riboflavin–UVA CXL can lead to a noticeable increase in the biomechanical stiffness of the sclera. The physiological modulus is the most sensitive tool to measure stiffness. In this study, the optimum duration of irradiation was 40 minutes. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2013; 39:1184–1189 Q 2013 ASCRS and ESCRS

Collagen crosslinking (CXL) using the photosensitizer riboflavin and ultraviolet A (UVA) is a new treatment for progressive keratoconus.1,2 Experiments performed in 1998 showed that CXL treatment with riboflavin and UVA irradiation as well as weak glutaraldehyde or Karnovsky solutions can stiffen the cornea.3 Since then, several studies have shown that riboflavin–UVA CXL can be used not only to stiffen the cornea1,4 but also to treat iatrogenic keratectasia,5 corneal melting,6 and keratitis.7 The treatment parameters (riboflavin 0.1% and surface UVA [370 nm] irradiance at 3 mW/cm2 for 30 minutes) have been confirmed to not damage corneal 1184

Q 2013 ASCRS and ESCRS Published by Elsevier Inc.

endothelial cells and other intraocular tissues in eyes with a corneal thickness of more than 400 mm.8,9 Scleral CXL can be applied to increase scleral rigidity in patients with progressive myopia,10 which is associated with thinning of the cornea. In 2004, Wollensak and Spoerl10 found that the CXL induced by riboflavin combined with UVA in treated porcine and human sclera can lead to a significant increase in the stress on the tissue and in Young modulus. The Young modulus reflects the ability of a material to return to its original shape after being under stress due to a certain force. The CXL parameters used in this study (riboflavin 0.1%, 370 nm UVA, 3 mW/cm2 0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2013.02.055

CROSSLINKING: SCLERAL CXL AT DIFFERENT IRRADIATION TIMES

irradiance for 30 minutes) were chosen based on previous reports of corneal CXL. In 2005, CXL was applied in rabbits with a surface irradiance of 4.2 mW/cm2 for 30 minutes (7.6 J/cm2).11 These conditions led to an increase of 227.9% in ultimate stress and an increase of 464.7% in the Young modulus in vivo. These effects were associated with serious damage to the retina. In 2009, Wollensak and Iomdina12 reduced the surface irradiance used for scleral CXL to 3 mW/cm2 for 30 minutes (5.4 J/cm2). The authors showed that the degree of stress and the Young modulus increased significantly over the long term. Histological examination did not show tissue damage. Because of the differences in fiber composition and arrangement between the cornea and the sclera, the use of combined riboflavin–UVA treatment may not be as effective in the sclera as it has proved to be in the cornea. Therefore, the present study was designed to determine the optimum duration of irradiation when administering surface irradiance of 3 mW/cm2 in the in vivo rabbit model. MATERIALS AND METHODS Study Design This study involved 36 New Zealand white rabbits weighing 2.0 to 2.5 Kg. All animals were healthy and free of ocular disease. The animals were divided into 6 groups depending on the duration of UVA irradiation (10, 20, 30, 40, 50, or 60 minutes). One treated eye from each group was fixed in 4% neutral-buffered formalin for light microscopy (LM). The other treated eyes were prepared for biomechanical testing. In each rabbit, the upper nasal quadrant was used as the CXL treatment area. The control data were pooled to yield reliable mean values. The animals used in this study were treated in accordance with the guidelines proposed by the Shandong University Animal Experimentation Ethic Committee.

1185

injected intramuscularly. Oxybuprocaine hydrochloride eyedrops were instilled in the left eyes before the procedures. In each rabbit, only the left eye was exposed to UVA radiant energy. The conjunctiva was incised in the upper and nasal quadrants of the left eye. Two sutures (polyester 5-0 [Dacron]) were placed in the sclera of the treatment quadrant 2.0 mm behind the limbus and used as reins to move and hold the globe. Using the sutures, the globe was rotated to expose the equatorial and posterior sclera of the upper nasal quadrant. Starting 15 minutes before the treatment, riboflavin-5phosphate 0.1% solution (without dextran) was dropped onto the sclera at 3-minute intervals to facilitate deep penetration of riboflavin into the sclera. The riboflavin 0.1% solution was instilled into the treatment area at 3-minute intervals during the irradiation. The eyes were irradiated with UVA (365 nm, UV-X 1000 system, IROC Innocross AG Co. Ltd.) for 10, 20, 30, 40, 50, or 60 minutes at an irradiance of 3 mW/cm2 5 cm from the sclera. Before every treatment, the desired surface irradiance of 3 mW/cm2 was controlled with a calibrated UVA meter at a 5 cm distance. The treatment area was 9.0 mm  9.0 mm and included the anterior, equatorial, and posterior sclera, starting 2.0 mm behind the limbus (Figure 1). After irradiation, the scleral sutures were removed. Levofloxacin eyedrops were applied, and the conjunctiva was closed using 7-0 absorbable sutures.

Specimen Preparation All the rabbits were humanely killed 24 hours postoperatively with an overdose of sodium pentobarbital that was injected intravenously. The globes used for biomechanical measurements were bisected, and the retina and choroid were removed. Two 3.0 mm  12.0 mm scleral strips were dissected sagitally from the treatment area. The control strips were taken from the contralateral right eyes at the same position. The scleral thickness of the strips was measured using mechanical micrometer calipers.

Collagen Crosslinking Treatment As premedication, 1.5 mL of ketamine hydrochloride 10.0% and 1.0 mL of promethazine hydrochloride were Submitted: August 14, 2012. Final revision submitted: February 6, 2013. Accepted: February 18, 2013. From the School of Medicine, Shandong University (Zhang), the Department of Ophthalmology (Zhang, Liu, Cao, Xia, Li, Hu), the Second People’s Hospital of Jinan, the Material Evidence Authentication Center of Jinan Traffic Police Department (Zou), and the Department of Ophthalmology (Yu, Mu), Provincial Hospital affiliated with Shandong University, Jinan, Shandong, China. Corresponding author: Guoying Mu, MD, Department of Ophthalmology, Provincial Hospital affiliated with Shandong University, No.324 Jingwu Weiqi Road, Jinan, Shandong, 250021, China. E-mail: [email protected].

Figure 1. Photomicrographs of irradiated and nonirradiated eyes. The left eye was not irradiated. The right eye was irradiated, as reflected by visible UVA- and riboflavin-induced fluorescence in the sclera.

J CATARACT REFRACT SURG - VOL 39, AUGUST 2013

1186

CROSSLINKING: SCLERAL CXL AT DIFFERENT IRRADIATION TIMES

Biomechanical Measurements Stress–strain measurements were performed for 30 sclera specimens and all 30 contralateral controls. The 3.0 mm  12.0 mm scleral strips were clamped vertically with a distance of 4.0 to 6.0 mm between the jaws of the biomaterial tester (Instron 5544-C8024, Instron Co. Ltd.). To ensure accurate and consistent results, the specimens were loaded and unloaded under a constant velocity of loading (2.0 mm/min) for 7 cycles. The strain was then increased linearly at a rate of 2.0 mm/min until the scleral specimens ruptured. Ultimate stress was obtained at the tearing point. The Young modulus was calculated as the slope of the stress–strain graph at 8% strain. The physiologic level of intraocular pressure (p) in an adult rabbit globe is approximately 2.1 kPa; the radius of the adult rabbit eye is approximately 11.0 mm. The load (F) on a sclera with a width (w) of 3.0 mm is approximately 0.035 N, as calculated according to the following formula: F Z p  r  w/2. Therefore, the tensile load corresponding to the calculated modulus of elasticity (0.035 to 0.06 N) was considered to be the physiological modulus.13

and control eyes. The stress–strain measurement of the scleral strips irradiated for 40 minutes or longer had a significant increase in ultimate stress, Young modulus, and the physiological modulus in comparison with untreated control eyes (Table 1). There was a significant increase in the physiological modulus of scleral strips irradiated for 30 minutes or longer (Table 1). Histological examination showed a moderate and severe acute inflammatory infiltrate of neutrophils in the irradiated area of the sclera. The retinal cells and pigment epithelium were not damaged in the eyes that were irradiated for 40 minutes or less (Figure 2). Eyes irradiated for 50 or 60 minutes had retinal damage, including irregularities in cell morphology, nuclei that were reduced in quantity, edema, and hyperemia (Figure 3).

Pathology Evaluation

DISCUSSION

The rabbit globes to be used for LM were fixed in 4% neutral-buffered formalin for 4 hours. The globes were then bisected and fixed in 4% neutral-buffered formalin for 24 hours to fix the retina. The specimens were then embedded in paraffin; 4 mm thin paraffin sections were stained with hematoxylin–eosin. The specimens were evaluated using a light microscope at 200-fold and 400-fold magnification.

Our study found an increase in scleral stiffness after CXL treatment by riboflavin and UVA. Irradiation for 30 minutes increased ultimate stress by 29.6%, Young modulus by 35.1%, and the physiological modulus by 33.6%. The physiological modulus increased at durations of 30 minutes or more. Irradiation of 50 minutes or more resulted in damage to the retina. Thus, CXL using the combination of riboflavin and UVA is effective and the optimum duration of irradiation for the sclera is 30 to 40 minutes (riboflavin 0.1%; 365 nm UVA; 3 mW/cm2 irradiance). The stress value and Young modulus are the major biomechanical indicators used to evaluate scleral strength. The sclera is a viscoelastic material rather than an elastic material, which leads to substantial

Statistical Evaluation The data for ultimate stress and the Young modulus were compared between the crosslinked treatment group and the untreated group using the Student t test.

RESULTS The 36 rabbits were treated successfully. For the control strips, the mean ultimate stress was 6.12 MPa G 2.92 (SD), the mean Young modulus was 6.96 G 4.63 MPa, and the mean physiological modulus was 4.62 G 1.89 MPa. There was no significant difference between eyes that were irradiated for 10 or 20 minutes Table 1. The biomechanical parameters with different irradiated times.

Time

Ultimate Stress (MPa)

Young Modulus (MPa)

Physiological Modulus (MPa)

10 min 20 min 30 min 40 min 50 min 60 min

7.00 G 2.15 7.13 G 2.58 7.93 G 2.05 8.62 G 3.70* 11.74 G 2.24* 9.89 G 3.46*

6.31 G 3.42 5.88 G 3.86 9.40 G 4.59 13.86 G 5.78* 14.43 G 4.72* 16.09 G 5.59*

4.25 G 1.64 5.00 G 2.73 6.17 G 2.23* 10.09 G 5.89* 11.88 G 6.25* 10.07 G 5.84*

*P!.05; significant difference between the treated scleral strips and the control strips

Figure 2. Photomicrograph of an eye irradiated for 40 minutes. The retinal cells were intact. The choroid vessels were hyperemic. Inflammation was noted in the sclera.

J CATARACT REFRACT SURG - VOL 39, AUGUST 2013

CROSSLINKING: SCLERAL CXL AT DIFFERENT IRRADIATION TIMES

1187

Figure 3. Photomicrograph of an eye irradiated for 50 minutes. The retinal organization was deficient. The number of cells in the retina was decreased. The layers of the retinal cells were disordered. The choroidal vessels were severely hyperemic.

Figure 4. Stress–strain curves. The curve is not a straight line. The ultimate strain was less than 50%.

stress–strain measurement variability.14 The sclera can be regarded as a 2-layered structure; the outer layer is the treated area and the inner layer is the untreated area. In our study, we considered the Young modulus at 8% strain as well as the physiological value of this modulus. The physiological modulus is a sensitive indicator of biomechanical strength. Because the stress– strain curve is not a straight line (Figure 4), the anterior and posterior parts of the curve affect the value of Young modulus. Previous studies15,16 found that the stress–strain relationship was nonlinear and exhibited hysteresis when cycled through a pattern of increasing, then decreasing stress. In some studies,11,12,17 Young modulus is calculated at 50% strain; however, the ultimate strain in this experiment was less than 50% (Figure 4). Thus, there was a deviation in the gradient of the stress–strain graph. However, within the physiological scale, the stress–strain curves resemble a straight line. Therefore, the physiological modulus can be used as an accurate index of biomechanical status. In our study, we considered that when the irradiated time is 30 minutes or longer, CXL can induce significant increases in the mechanical rigidity in rabbit sclera, even though the increases in ultimate stress and Young modulus at 8% strain were statistically insignificant. Riboflavin-5-phosphate (MW, 456 Da) should penetrate the sclera easily because the sclera is permeable to molecules as large as 150 000 Da.18 Sufficient riboflavin-5-phosphate was instilled in the treated area when CXL was initiated. We therefore postulate that the duration of irradiation affects the biomechanical parameters of the tissue. With prolonged irradiation, more UVA can penetrate scleral tissues. Some

studies19,20 have found that in the context of corneal CXL through the use of combined riboflavin–UVA treatment (riboflavin 0.1%, 370 nm UVA, 3 mW/cm2 irradiance), the anterior 300 mm portion of the corneal stroma is effectively crosslinked. The sclera is not as transparent as the cornea, so the penetration of UVA in the sclera is poorer than in the cornea. Therefore, when the duration of irradiation was 40 minutes, the ultimate stress and Young modulus increased significantly more in the treated eyes than in the control eyes. A previous study21 has shown that with a dose density of 5.4 J/cm2, the Bunsen-Roscoe reciprocity law is valid for illumination intensities up to 40 to 50 mW/cm2 and illumination times of more than 2 minutes, and this theory has been verified by the comparable Young modulus increase between a standard CXL with 3 mW/cm2 and 30 minutes and a rapid CXL with 9 mW/cm2 and 10 minutes.22 The irradiation of 5.4 J/cm2 UVA induces an enhancement of corneal stiffness along with keratocyte or endothelium death in rabbits23 and partial keratocyte damage in human corneas.24 Crosslinking with the same dose density induces comparable changes in collagen density and diameter in the cornea and sclera.25 Also, the irradiation of 5.4 J/cm2 UVA induces an increase in Young modulus of sclera without leading to observable tissue damage,12 while 7.6 J/cm2 UVA has been verified to induce severe injury to retinal and retinal pigment epithelium cells in the rabbit.11 Considering the possible effective dose–density range and dose-dependent relationship between tissue damage and radiant exposure, we used dose densities from 1.8 to 10.8 J/cm2 by using 3 mW/cm2 UVA combined with varied irradiation time in this study. Our research shows that scleral

J CATARACT REFRACT SURG - VOL 39, AUGUST 2013

1188

CROSSLINKING: SCLERAL CXL AT DIFFERENT IRRADIATION TIMES

CXL can be safely and effectively performed with riboflavin 0.1%, 365 nm UVA, and 3 mW/cm2 irradiance for 40 minutes. Further studies should evaluate the use of more intensive irradiation combined with reduced exposure time. Future studies will be necessary to elucidate the optimum irradiance density and exposure duration by measuring the UVA transmission in the sclera. Wollensak et al.11 found a scleral temperature elevation of only 1.5 C after the use of 4.2 mW/cm2 UVA combined with riboflavin for 30 minutes to induce CXL. Another study14 also found that an increase in temperature from 37 to 41 C had no significant effect on the reaction to stress in human and pig scleras. This study did not consider the change in temperature in the treated area. To keep the sclera from drying out and increase the permeability of the tissue to riboflavin, riboflavin was instilled at 3-minute intervals. This procedure may have further decreased the temperature of the sclera; we did not find histological signs of tissue shrinkage or coagulation induced by the elevation in temperature even though the duration of irradiation was 60 minutes. The penetration of UVA through scleral tissue is limited. One form of CXL involves the use of blue light (wavelength 465 nm), which penetrates more deeply than UVA. In combination with riboflavin 0.5%, blue-light scleral CXL (465 nm at 26 mW/cm2 for 20 minutes) showed an impressive stiffening effect in rabbit eyes.26 Histological evaluation showed no retinal damage. This riboflavin with blue-light approach represents a new modality for strengthening scleral tissue. Further studies should be performed to compare scleral CXL with riboflavin–UVA versus riboflavin–blue light. In progressive myopia, axial elongation and reduced scleral thickness result in staphylomas, which are most frequently found at the posterior sclera.27 Due to the architecture of the scleral fiber bundles, the posterior sclera is more elastic than other regions.14 Notably, glycation-induced CXL is increased in diabetic patients. These patients typically have a reduced axial length and rarely show axial myopia.28 Therefore, CXL can be performed in patients with progressive myopia; the posterior sclera should be the main target of the treatment. In this study, we controlled the CXL activity in a particular area by applying a UVA light spot with a specific diameter (9.0 mm) onto the exposed tissue. The riboflavin solution was not restricted to a particular area in our study, which is consistent with corneal CXL. In our experiment, even if 2 scleral sutures would be able to rotate and hold the globe, exposing the posterior sclera in vivo is difficult without damaging the optic nerve and blood vessels. The target area was therefore designed

to include equatorial sclera and parts of the posterior sclera because this area could be irradiated reliably; this is the primary limitation of this study. The globe is spherical; therefore, the treated area was not a plane surface, which led to an uneven pattern of irradiation. This was another limitation of the study. Further studies addressing these difficulties and evaluating the feasibility of whole-globe examinations are needed. In conclusion, riboflavin–UVA CXL led to a significant increase in the biomechanical stiffness of the sclera. The physiological modulus is the most sensitive value for the measurement of biomechanical parameters. The optimum duration of irradiation was 40 minutes. WHAT WAS KNOWN  In 2004, combined riboflavin–UVA CXL was used in the sclera and the parameters were chosen based on previous reports of corneal CXL.  In 2005, CXL was applied in rabbits with a surface irradiance of 4.2 mW/cm2 for 30 minutes (7.6 J/cm2). These conditions led to serious damage to the retina.  In 2009, Wollensak and Iomdina reduced the surface irradiance to 3 mW/cm2 for 30 minutes (5.4 J/cm2). The authors showed that the degree of stress and Young modulus increased significantly over the long term. Histological examination did not show tissue damage.  Young modulus is the major biomechanical indicator used to evaluate scleral strength, which is often measured in 8% strain or 50% strain. WHAT THIS PAPER ADDS  Combined riboflavin–UVA CXL was applied at different irradiated time (10, 20, 30, 40, 50, or 60 min) in the in vivo rabbit model, with a surface irradiance 3 mW/cm2. The pathological evaluation was also considered to obtain the proper treatment effectiveness, which had a high stiffening effect and low toxicity on rabbit sclera.  In this study, not only Young modulus in 8% strain but also Young modulus in the physiological scope were considered.

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
E, Frueh BE, Trazza S, Epstein D. Two-year 2. Vinciguerra P, Albe corneal cross-linking results in patients younger than 18 years with documented progressive keratoconus. Am J Ophthalmol 2012; 154:520–526

J CATARACT REFRACT SURG - VOL 39, AUGUST 2013

CROSSLINKING: SCLERAL CXL AT DIFFERENT IRRADIATION TIMES

3. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res 1998; 66:97–103 4. Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-Ainduced cross-linking. J Cataract Refract Surg 2003; 29:1780– 1785 5. Salgado JP, Khoramnia R, Lohmann CP, Winkler von Mohrenfels C. Corneal collagen crosslinking in post-LASIK keratectasia. Br J Ophthalmol 2011; 95:493–497 6. Al-Sabai N, Koppen C, Tassignon MJ. UVA/riboflavin crosslinking as treatment for corneal melting. Bull Soc Belge Ophtalmol 2010; 315:13–17. Available at: http://www.ophthalmologia.be/ download.php?dof_idZ755. Accessed February 28, 2013 7. Makdoumi K, Mortensen J, Crafoord S. Infectious keratitis treated with corneal crosslinking. Cornea 2010; 29:1353–1358 €rl E, Reber F, Pillunat L, Funk R. Corneal 8. Wollensak G, Spo endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res 2003; 35:324–328 9. Wollensak G, Spoerl E, Wilsch M, Seiler T. Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit. J Cataract Refract Surg 2003; 29:1786–1790 10. Wollensak G, Spoerl E. Collagen crosslinking of human and porcine sclera. J Cataract Refract Surg 2004; 30:689–695 11. Wollensak G, Iomdina E, Dittert D-D, Salamatina O, Stoltenburg G. Cross-linking of scleral collagen in the rabbit using riboflavin and UVA. Acta Ophthalmol Scand 2005; 83:477–482. Available at: http://onlinelibrary.wiley.com/doi/10. 1111/j.1600-0420.2005.00447.x/pdf. Accessed February 28, 2013 12. Wollensak G, Iomdina E. Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet A (UVA). Acta Ophthalmol 2009; 87:193–198. Available at: http://onlinelibrary.wiley.com/doi/10.1111/j.1755-3768.2008. 01229.x/pdf. Accessed February 28, 2013 13. Weiyi C, Wang X, Wang C, Tao L, Li X, Zhang Q. An experimental study on collagen content and biomechanical properties of sclera after posterior sclera reinforcement. Clin Biomech 2008; 23(suppl 1):S17–S20 14. Curtin BJ. Physiopathologic aspects of scleral stress-strain. Trans Am Ophthalmol Soc 1969; 67:417–461. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310350/pdf/ taos00032-0433.pdf. Accessed February 28, 2013 15. Battaglioli JL, Kamm RD. Measurements of the compressive properties of scleral tissue. Invest Ophthalmol Vis Sci 1984; 25:59–65. Available at: http://www.iovs.org/content/25/1/59. full.pdf. Accessed February 28, 2013 16. Thornton IL, Dupps WJ, Roy AS, Krueger RR. Biomechanical effects of intraocular pressure elevation on optic nerve/lamina cribrosa before and after peripapillary scleral collagen crosslinking. Invest Ophthalmol Vis Sci 2009; 50:1227–1233. Available at: http://www.iovs.org/content/50/3/1227.full.pdf. Accessed February 28, 2013 17. Wollensak G, Iomdina E. Long-term biomechanical properties of rabbit cornea after photodynamic collagen crosslinking. Acta

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

1189

Ophthalmol 2009; 87:48–51. Available at: http://onlinelibrary. wiley.com/doi/10.1111/j.1755-3768.2008.01190.x/pdf. Accessed February 28, 2013 Ambati J, Canakis CS, Miller JW, Gragoudas ES, Edwards A, Weissgold DJ, Kim I, Delori FC, Adamis AP. Diffusion of high molecular weight compounds through sclera. Invest Ophthalmol Vis Sci 2000; 41:1181–1185. Available at: http://www.iovs.org/ content/41/5/1181.full.pdf. Accessed February 28, 2013 Wollensak G, Spoerl E, Wilsch M, Seiler T. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea 2004; 23:43–49 Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-riboflavin cross-linking of the cornea. Cornea 2007; 26:385–989 Wernli J, Schumacher S, Spoerl E, Mrochen M. The efficacy of corneal cross-linking shows a sudden decrease with very high intensity UV-light and short treatment time. Invest Ophthalmol Vis Sci 2013; 54:1176–1180 Schumacher S, Oeftiger L, Mrochen M. Equivalence of biomechanical changes induced by rapid and standard corneal cross-linking, using riboflavin and ultraviolet radiation. Invest Ophthalmol Vis Sci 2011; 52:9048–9052. Available at: http:// www.iovs.org/content/52/12/9048.full.pdf. Accessed February 28, 2013 Wollensak G, Iomdina E, Dittert D-D, Herbst H. Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA. Cornea 2007; 26:600–605 Wollensak G. Histological changes in human cornea after crosslinking with riboflavin and ultraviolet A [letter]. Acta Ophthalmol 2010; 88(2):e17–e18. Available at: http://onlinelibrary.wiley. com/doi/10.1111/j.1755-3768.2008.01474.x/pdf. Accessed February 28, 2013 Choi S, Lee S-C, Lee H-J, Cheong Y, Jung G-B, Jin K-H, Park H-K. Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light. Lasers Med Sci 2012 Nov 23. [Epub ahead of print] Iseli HP, Spoerl E, Wiedemann P, Krueger RR, Seiler T. Efficacy and safety of blue-light scleral cross-linking. J Refract Surg 2008; 24:S752–S755 McBrien NA, Gentle A. Role of the sclera in the development and pathological complications of myopia. Prog Retin Eye Res 2003; 22:307–338 Pierro L, Brancato R, Robino X, Lattanzio R, Jansen A, Calori G. Axial length in patients with diabetes. Retina 1999; 19:401–404

J CATARACT REFRACT SURG - VOL 39, AUGUST 2013

First author: Yali Zhang, MD School of Medicine, Shandong University, Shandong, China