Pathologic myopia is a serious worldwide and sight-threatening refractive disorder. The prevalence of pathologic myopia was reported to be 0.9% to 3.1%, and the prevalence of visual impairment attributable to pathologic myopia ranged between 0.1% and 1.4%.1 Scleral biomechanical weakness and thinning are the main factors in the pathogenesis of this disease. Several methods have been developed for the treatment of pathologic myopia,2 such as injections of a polymeric composition forming a foamed gel under Tenon's capsule and posterior scleral reinforcement surgery to provide scleral support and reduce the progression of axial elongation,3 for the purpose of scleral strengthening. Nevertheless, these methods do not alter the internal structure of the sclera.
Corneal cross-linking (CXL) was initially introduced by Spoerl and Seiler4 as a possible treatment for keratoconus. Moreover, corneal CXL by application of riboflavin and ultraviolet-A (UV-A) light became an established and increasingly common treatment to stabilize the cornea and prevent keratoconus progression.5 This technique was also proven to be effective in strengthening the scleral tissue.6 Moreover, the safety of scleral CXL was partially estimated by histological measurements in a previous study; the effective dose of UV-A irradiation (365 nm, 3 W/cm2, 30 minutes) was reasonably controlled and there were no structural changes to the retina or retinal pigment epithelium layer in cross-linked rabbit eyes.7 However, in our previous study about cross-linked rabbit eyes, the ultrastructural changes occurred in the nuclear and inner segments of the photoreceptor.8
Previous studies mainly used the rabbit as the experimental animal. Therefore, to investigate the safety of scleral CXL and lay the foundation for clinical application of this new technique, this study experimented with rhesus monkeys. Because of the similarities between the anatomy of human and rhesus monkey eyes, it is reasonable to expect that data derived from monkeys can be generalized to humans.9
Materials and Methods
A total of 6 normal adolescent male rhesus monkeys (Macaca mulatta) with a mean age of 3 years (range: 2.8 to 3.5 years) and a mean weight of 4.9 kg (range: 3.6 to 6.2 kg) were studied. The animal production license number is SCXK (Beijing) 2010-0019. All rearing and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Capital Medical University (AEEI-2014-127) and were in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The rhesus monkeys involved in this study were all purchased from the Experimental Animal Center of the Military Medical Science Academy. The monkeys were housed and monitored at the large animal room of Capital Medical University according to the National Standard of China, which met the laboratory animal institutions' general requirements for quality and competence (GB/T 27416-2014).
On examination, all monkeys had not undergone any intervention and had not been involved in any study. Both eyes of the monkey were examined clinically before scleral CXL and 1 week, 1 month, and 3 months postoperatively. For all examinations and surgeries, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride (20 mg/kg body weight; ketamine 5%, Gutian Fujian Pharmaceutical Co., Ltd., Nanping, China) and xylazine hydrochloride (0.2 mg/kg body weight; Shengda Animal Pharmaceutical Co., Ltd., Dunhua, China), with repeated injections of ketamine (10 mg/kg) as needed during the examination. While the measurements were being taken, the eyelids were gently held apart by an eye speculum and the tear film was maintained by the frequent application of artificial tears (sodium hyaluronate eye drops; Santen-China, Beijing, China). Cycloplegia was achieved by topically instilling one to two drops of 0.5% tropicamide and 0.5% phenylephrine (tropicamide phenylephrine eye drops; Santen-China) at 5-minute intervals 30 minutes before performing retinoscopy and electroretinography (ERG).
Scleral CXL Treatment
According to our previous scleral CXL laboratory technique,8 each rhesus monkey had one eye randomly selected to receive riboflavin/UV-A scleral CXL in the temporal quadrant of the equatorial sclera and the contralateral eye served as an intra-individual control. To perform the riboflavin/UV-A scleral CXL, the animals were anesthetized by the above-mentioned method. The cornea was anesthetized with one to two drops of 0.5% proparacaine hydrochloride (Alcaine; Alcon Systems Inc., St. Louis, MO). An eyelid speculum was placed into the fornix. The conjunctiva was incised in the upper anterior quadrant of the scleral CXL eye using a pair of scissors. The superior rectus muscle and the temporal rectus muscle were displayed and fixed by 5-0 sutures to allow better exposition of the sclera and easier manipulation of the eye position during scleral treatment. Using the scleral sutures, the eyeball was rotated to expose the equatorial sclera of the superior temporal quadrant as the cross-linked area (Figure 1A). Photosensitizer solution containing 0.1% riboflavin (0.1% riboflavin, 20% dextran 500, Peschke D; PESCHKE Trade, Huenenberg, Switzerland) was instilled every 3 minutes on the exposed sclera to ensure a plain penetration of riboflavin into the scleral stroma (Figure 1B).
(A) Schematic elevation views of a monkey eyeball showing the scleral corneal cross-linking locations. (B) The eye was fixed and manipulated by sutures to protrude the eye and the cornea was covered by a piece of tinfoil to avoid irradiation. UVA = ultraviolet-A
After 20 minutes of riboflavin soaking time, UV-A irradiation (365 nm) was applied on the scleral CXL eye using a UV-A device (UV-X 1000; Avedro Inc., Waltham, MA) with a surface irradiance of 3 mW/cm2 for 30 minutes (total dose of UV-A: 5.4 J/cm2) at a distance of 5 cm from the sclera. Riboflavin drops were alternately applied every 5 minutes during the entire irradiation period to avoid excessive photobleaching of the fluorophore and drying of the eye. During the process of UV-A irradiation, the cornea was covered by a piece of tinfoil to shield it from UV-A irradiation (full CXL details following the standard convention are represented in Table 1). After irradiation, the sutures around the muscles were removed and the conjunctiva was closed using a polyglactin 7-0 suture (Ethicon Inc., Livingston, Scotland, United Kingdom). Finally, both eyes were treated with application of gatifloxacin eye gel (Shenyang Sinqi Pharmaceutical Co., Ltd., Shenyang, China) into the conjunctival fornix and the cornea to avoid infection. The animals were monitored until they awakened.
The biometry measurements were performed before scleral CXL and 1 week, 1 month, and 3 months after the scleral CXL operation.
The intraocular pressure (IOP) was measured using the TonoVet rebound tonometer (Icare Finland Oy, Helsinki, Finland) at approximately 9 AM. According to the manufacturer's recommended procedures, the equipment was programmed to average the IOP of six consecutive, acceptable measurements and produce a reading of the mean IOP. Five readings (each a mean of six measurements; a total of 30 separate measurements) were obtained from each eye, and the mean IOP was calculated and reported. The monkeys were positioned in a seated posture in the process of measuring IOP.
The axial dimensions of the eye, including anterior chamber and vitreous chamber depths, lens thickness, and the sum of these (axial length) were measured by A-scan ultrasonography and implemented with a 10-MHz transducer (ODM-1000A; MEDA Co., Ltd. Tianjin, China). Monkeys were in the supine position with the head immobilized by a head holder. The device was programmed to average the values of eight acceptable measurements and produce a reading of the mean. Three readings were recorded and averaged.
The refractive status of each eye, both the spherical and cylindrical components, was measured by an experienced investigator using a streak retinoscope (YZ24; 66 Vision Tech Co., Ltd., Suzhou, China) and recorded. Refractive errors were reported as the spherical equivalent in diopters.
The central corneal thickness was measured by optical coherence tomography (MasterOCT; Shenzhen MOPTIM Imaging Technique Co., Ltd., Shenzhen, China). The image was taken by the same trained examiner and measurements were repeated three times.
The full-field ERG was recorded by the RETI-port system (Roland Consult, Brandenburg, Germany). All procedures were performed according to the recommendations of the International Society for Clinical Electrophysiology of Vision (ISCEV).10 According to the recommendations, an ISCEV Standard ERG includes the following responses: dark-adapted 0.01 ERG, dark-adapted 3.0 ERG, dark-adapted 3.0 oscillatory potentials, light-adapted 3.0 ERG, and light-adapted 3.0 flicker ERG. Monkeys were anesthetized and dark adapted for 30 minutes before recording dark-adapted ERGs. The cornea was anesthetized with one to two drops of 0.5% proparacaine hydrochloride. Then the contact lens electrode (ERG-jet; Fabrinal, La Chaux-de-Fonds, Switzerland) was placed into the cornea to record retinal signals and one drop of 0.2% carbomer (Carbomer Eye Gel; Dr. Gerhard Mann GmbH, Berlin, Germany) was put between the cornea and electrode. The reference and ground electrodes (Needle Electrode; Roland Consult, Brandenburg, Germany) were subcutaneously inserted into the orbital rim and forehead, respectively. In addition, monkeys were adapted to a background luminance of 25 cd/m2 for 10 minutes before recording light-adapted ERGs. The electroretino-gram of the scleral CXL and control eyes were recorded simultaneously in the same examination.
Statistical analysis was performed using SPSS for Windows software (version 18.0; SPSS, Inc., Chicago, IL). The measurements were presented as mean ± standard deviation. Paired t tests were used to evaluate potential ocular biological parameter differences between cross-linked and untreated control eyes in different pre-operative and postoperative periods. In all tests, a P value of less than .05 was considered statistically significant.
At the initial measurement, there were no statistically significant differences in IOP, spherical equivalent refractive errors, axial dimensions of any ocular compartment, and amplitude of the ERG components between the two eyes of the monkeys (paired t tests, P values ranged from .206 for IOP to .860 for depth of vitreous chamber).
No signs of inflammation were observed after scleral CXL and throughout the follow-up period. After surgery, all monkeys had normal corneas, anterior chambers, and clear lenses. No vitreous or retinal lesion was observed clinically.
Figure 2 and Table A (available in the online version of this article) summarize the IOP, spherical equivalent refractive errors, and axial dimensions of ocular components in different preoperative and postoperative periods. There were no statistically significant differences between the control and cross-linked specimens in the different periods for IOP, refractive state, total axial length, and axial dimensions of the anterior chamber, crystalline lens, vitreous chamber, and central corneal thickness (each P > .05).
The (A) intraocular pressure (IOP), (B) spherical equivalent, (C) depth of anterior chamber, (D) thickness of crystaline lens, (E) depth of vitreous chamber, (F) overall axial length, and (G) central corneal thickness comparison for the cross-linked (CXL) and control eyes at different time periods. D = diopters
IOP, Refractive Error, and Ocular Components Comparison for the Cross-Linked (n = 6) and Control (n = 6) Eyes
Standard Full-Field ERGS
The amplitude of the ERG components is summarized in Figure 3 and Table B (available in the online version of this article). No obvious changes in waveform of the standard full-field ERGs were observed in the control and cross-linked specimens. In the dark-adapted 0.01 ERG, dark-adapted 3.0 ERG, light-adapted 3.0 ERG, and amplitudes of the a-wave and b-wave, there were no statistically significant differences between the control and cross-linked specimens in the different periods (each P > .05).
The (A) b-wave of the dark-adapted 0.01 electroretinogram (ERG),(B) a-wave of the dark-adapted 3.0 ERG, (C) b-wave of the dark-adapted 3.0 ERG, (D) a-wave of the light-adapted 3.0 ERG, and (E) b-wave of the light-adapted 3.0 ERG comparison for the cross-linked (CXL) and control eyes at different time periods.
Amplitude of the ERG Components Comparison for Cross-Linked (n = 6) and Control (n = 6) Eyes
Using our previous study as a base,6,8 this study successfully applied the scleral CXL treatment to non-human primates and demonstrated that this technique was a reliable ophthalmologic surgery procedure. Qiao-Grider et al.9 found that the rhesus monkey eye matured approximately three times faster than the human eye. According to this theory, a 3-year-old rhesus monkey eye was equivalent to a 9-year-old human eye. This age was comparable to the stage of human adolescence and also a high incidence of myopia.11
In the current study, the results showed that scleral CXL had no effect on the rhesus monkeys' IOP and ocular components postoperatively. The fact that IOP in monkeys were stable and normally distributed in different periods (Figure 2A) agrees with previous reports in normal monkeys.12,13 As represented in Figures 2B–2G, the available longitudinal data show that the refractive error and axial ocular components stabilized in the early postoperative period. In the studies by Qiao-Grider et al.9 and Bradley et al.,14 the refractive state of the rhesus monkey stabilized at approximately age 1.5 years and developed rates of different segments of the eye slowed down at approximately age 3 years, which were similar to our results. It was further proved that the scleral CXL might not affect the development of normal eyes in the early postoperative period.
In the second part of the current study, the full-field ERG was performed to investigate the retinal function of rhesus monkeys preoperatively and postoperatively. The full-field ERG was the sum of the electrical responses of neurons and non-neuronal cells in the retina recorded by the corneal electrode when the retina is stimulated by full-field flash, representing the cells from the photoreceptor to the retinal cells. The a-wave of the ERG originated from the photoreceptor cell layer of the retina, which represents the electrical activity of the receptors and reflects the potential changes of the retinal photoreceptors. The b-wave of the ERG originated from the outer plexiform layer and inner nuclear layer, mainly reflecting the function of the inner retina.15 To evaluate the retinal function of rhesus monkeys objectively, we mainly analyzed results from dark-adapted 0.01 ERG response (a rod-driven response of on-bipolar cells), dark-adapted 3.0 ERG response (combined responses arising from photoreceptors and bipolar cells of both the rod and cone systems, rod dominated), and light-adapted 3.0 ERG response (responses of the cone system; the a-wave arises from cone photoreceptors and off-cone bipolar cells; the b-wave comes from on- and off-cone bipolar cells).10 In addition, compared to the authors' previous research,8 this study adopted contact lens electrodes instead of gold ring electrodes as the active electrodes connected to the positive input for recording ISCEV Standard full-field ERGs, which could provide the highest amplitude and most stable recordings.10 The ERGs of both eyes were recorded at the same time to reduce the error caused by repeated measurements.
According to the data analysis of ERG, there was no significant difference in the waveform and amplitude of ERG between the control and cross-linked specimens. The results showed that the scleral CXL had no significant effect on the conducting function of the optic nerve in rhesus monkeys in the early postoperative period, but the long-term effect was still to be observed and verified.
In addition, some limitations of the current study should be noted. First, the sample size in this investigation was small and the scope was limited, so further research in a larger sample is required. Second, the scleral CXL field was not observed by electron microscopy, which could prove the safety of the scleral CXL from the perspective of histology. Third, the full-field ERG, which mainly reflected the whole retinal nerve conduction function, could not focus on the retinal function changes in the scleral CXL field.
The scleral CXL technique in the current study was the first to be successfully applied to primates. The parameters of ocular components and retinal function of rhesus monkeys were observed in the early postoperative stage of scleral CXL, which confirmed its safety and provided an objective and reliable experimental basis for further clinical application.
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- Avetisov ES, Tarutta EP, Iomdina EN, Vinetskaya MI, Andreyeva LD. Nonsurgical and surgical methods of sclera reinforcement in progressive myopia. Acta Ophthalmol Scand. 1997;75:618–623. doi:10.1111/j.1600-0420.1997.tb00617.x [CrossRef]
- Spoerl E, Seiler T. Techniques for stiffening the cornea. J Refract Surg. 1999;15:711–713.
- Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:620–627. doi:10.1016/S0002-9394(02)02220-1 [CrossRef]
- Wang M, Zhang F, Qian X, Zhao X. Regional biomechanical properties of human sclera after cross-linking by riboflavin/ultraviolet A. J Refract Surg. 2012;28:723–728. doi:10.3928/1081597X-20120921-08 [CrossRef]
- 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. doi:10.1111/j.1755-3768.2008.01229.x [CrossRef]
- Wang M, Zhang F, Liu K, Zhao X. Safety evaluation of rabbit eyes on scleral collagen cross-linking by riboflavin and ultra-violet A. Clin Exp Ophthalmol. 2015;43:156–163. doi:10.1111/ceo.12392 [CrossRef]
- Qiao-Grider Y, Hung LF, Kee CS, Ramamirtham R, Smith ER 3rd, . Normal ocular development in young rhesus monkeys (Macaca mulatta). Vision Res. 2007;47:1424–1444. doi:10.1016/j.visres.2007.01.025 [CrossRef]
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|Treatment target||Strengthening sclera|
|Fluence (total) (J/cm2)||5.4|
|Soak time and interval (minutes)||20 (q5)|
|Treatment time (minutes)||30|
|Chromophore||Riboflavin (Peschke D; PESCHKE Trade, Huenenberg, Switzerland)|
|Light source||UV-X 1000 (Avedro Inc., Waltham, MA)|
|Irradiation mode (interval)||Continuous Pulsed|
|Protocol abbreviation in manuscript||S-CXL(3*30) (Standard)|
IOP, Refractive Error, and Ocular Components Comparison for the Cross-Linked (n = 6) and Control (n = 6) Eyesa
|Parameter||Baseline||1 Week||1 Month||3 Months|
|IOP (mm Hg)||16.93 ± 3.60||18.23 ± 2.64||−1.452||.206||17.70 ± 2.38||19.57 ± 2.60||−2.412||.061||18.80 ± 2.83||17.67 ± 2.25||1.048||.343||18.60 ± 4.00||18.33 ± 4.34||0.437||.680|
|Refractive state (D)||1.20 ± 1.16||0.70 ± 1.11||0.877||.43||1.22 ± 1.39||0.73 ± 1.54||0.291||.786||0.95 ± 1.46||0.50 ± 1.17||1.095||.335||0.83 ± 1.14||0.25 ± 1.16||0.65||.33|
|ACD (mm)||3.53 ± 0.29||3.42 ± 0.37||0.675||.529||3.44 ± 0.21||3.31 ± 0.34||1.058||.338||3.49 ± 0 .36||3.56 ± 0.39||−0.973||.375||3.47 ± 0.22||3.48 ± 0.28||−0.077||.941|
|Crystalline lens thickness (mm)||3.82 ± 0.16||3.91 ± 0.26||−0.558||.601||3.74 ± 0.12||3.86 ± 0.13||−1.852||.123||3.87 ± 0.27||3.86 ± 0.28||0.459||.665||3.92 ± 0.08||3.84 ± 0.24||0.987||.369|
|VCD (mm)||12.22 ± 0.56||12.23 ± 0.58||−0.186||.860||12.52 ± 0.62||12.77 ± 1.38||1.062||.337||12.44 ± 0.49||12.42 ± 0.58||0.504||.636||12.44 ± 0.55||12.51 ± 0.63||−1.559||.180|
|Overall AL (mm)||19.63 ± 0.62||19.55 ± 0.63||1.305||.249||19.71 ± 0.73||19.59 ± 0.77||1.382||.226||19.80 ± 0.56||19.84 ± 0.66||−0.591||.580||19.83 ± 0.52||19.83 ± 0.72||0.069||.947|
|CCT (μm)||533.31 ± 48.01||542.58 ± 65.2||−0.831||.444||549.50 ± 38.14||546.50 ± 36.42||0.169||.872||521.83 ± 43.46||532.06 ± 59.12||−1.117||.315||525.34 ± 21.12b||527.25 ± 22.29b||−0.138||.899|
Amplitude of the ERG Components Comparison for Cross-Linked (n = 6) and Control (n = 6) Eyesa
|Parameter||Baseline||1 Week||1 Month||3 Months|
|Dark-adapted 0.01 ERG|
| b-wave (μV)||176.83 ± 48.45||169.83 ± 52.02||0.844||.437||138.17 ± 33.62||140.75 ± 37.44||−0.273||.796||136.27 ± 57.00||145.05 ± 64.85||−1.119||.314||169.00 ± 28.77||181.33 ± 45.19||−1.358||.233|
|Dark-adapted 3.0 ERG|
| a-wave (μV)||208.33 ± 37.94||196.50 ± 45.33||1.107||.319||146.67 ± 25.07||144.18 ± 27.53||0.355||.737||150.50 ± 37.91||163.60 ± 54.26||−1.037||.347||179.33 ± 34.17||198.67 ± 35.15||−1.747||.141|
| b-wave (μV)||402.50 ± 57.29||385.17 ± 69.93||0.816||.452||290.00 ± 67.81||284.17 ± 56.07||0.379||.720||302.0 ± 100.75||289.00 ± 93.53||0.718||.505||354.50 ± 62.18||385.00 ± 87.05||−2.027||.098|
|Light-adapted 3.0 ERG|
| a-wave (μV)||28.70 ± 5.38||27.13 ± 6.88||1.032||.349||18.47 ± 6.18||18.39 ± 6.04||0.074||.944||20.51 ± 10.10||21.56 ± 9.52||−0.522||.624||25.62 ± 8.47||26.77 ± 10.51||−0.745||.490|
| b-wave (μV)||120.55 ± 27.95||118.02 ± 33.25||0.373||.725||77.85 ± 30.16||79.90 ± 32.32||−0.61||.569||93.10 ± 41.93||90.80 ± 34.03||0.321||.761||112.73 ± 36.78||117.33 ± 45.33||−0.94||.390|