Myopia has reached almost epidemic proportions in the world, possibly affecting one-third of the world's population by 2020, and has become a major cause of visual impairment and blindness, especially in Asian countries.1–3 The prevalence of children and teenagers with myopia is 67.3% in China.4 Some people with myopia have pathological changes, such as progressive elongation of the eyeball and posterior staphyloma, which is probably caused by scleral biomechanical weakness and thinning.5 Until now, although many methods to resist myopic progression have been developed, including various posterior scleral reinforcement surgery,6,7 their efficiency is still controversial and no evidence shows that the internal structure of the weakened sclera can be regenerated by these means.
Corneal cross-linking (CXL) was initially demonstrated as a possible treatment for keratoconus in 1999,8 and was proven to be a successful therapy for stabilizing the cornea and preventing progression of keratoconus in 2003.9,10 In 2004, Wollensak and Spoerl9,11 from Germany were the first to apply scleral CXL using riboflavin/UVA and to propose scleral CXL as a treatment to heal progressive pathologic myopia similar to the cornea with progressive keratoconus. Initially, they found retinal damage after scleral CXL in the rabbit,12 which could be prevented in their long-term study by avoiding a drying effect on the sclera during the procedure.13 Other studies have confirmed the efficacy of scleral CXL in strengthening scleral tissue by creating cross-links,14,15 whereas reductions in dark-adapted electroretinography amplitudes and ultrastructural changes were described in nuclear and inner segments of photoreceptors in cross-linked rabbit albino eyes with no pigmented retinal pigment epithelium.16 Recently, the exploration of different devices and different methods of scleral CXL have been applied in animal models, and some of the studies suggested that scleral CXL could effectively prevent occlusion-induced axial elongation, which may be a potential method to control the pathologic process of myopia.17–19
Previous studies mainly focused on changes of scleral biomechanical properties and histopathological evaluation after scleral CXL to evaluate its efficacy and safety. However, in vivo biological parameters as visualized by OCT and OCTA were not included in these articles. On the basis of former research by our team, early changes of ocular biological parameters in rhesus monkeys after scleral CXL with riboflavin/UVA were observed and proved to be not affected by scleral CXL until 6 months postoperatively.20,21 Therefore, the current study was conducted in rhesus monkeys to investigate the long-term changes of in vivo biological parameters, scleral biomechanical properties, and histopathological findings after scleral CXL, aiming to estimate the safety and long-term scleral biomechanical stability after scleral CXL by riboflavin/UVA in primates.
Materials and Methods
The experimental study included 6 normal adolescent male rhesus macaque monkeys (Macaca mulatta) with a mean age of 3.0 ± 0.4 years (range: 2.8 to 3.5 years) and a mean weight of 4.9 ± 0.5 kg (range: 3.6 to 6.2 kg). 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. Before recruiting into the experiment, all rhesus monkeys were given a comprehensive ocular examination, including anterior segment and fundus examination, to exclude any ocular disease.
Table 1 outlines the CXL methods. Both eyes of the monkey were examined clinically before and 1 week and 1, 3, 6, and 12 months after the scleral CXL operation, including biomicroscopy, ophthalmoscopy, intraocular pressure measurements, A-scan ultrasonography, OCT, and OCTA. For all examinations and surgeries, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride (20 mg/kg body weight; ketamine 5%) and xylazine hydrochloride (0.2 mg/kg body weight), 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 eyelid speculum and the tear film was maintained by the frequent application of artificial tears (sodium hyaluronate 0.1%). Cycloplegia was achieved by topically instilling one to two drops of 5% tropicamide (tropicamide phenylephrine eye drops) at 5-minute intervals 30 minutes before performing retinoscopy, OCT, and OCTA.
Surgery and Scleral CXL Treatment
According to our previous scleral CXL laboratory technique,20,21 one eye of each rhesus monkey was randomly selected for the CXL procedure, and the contralateral eye served as the intra-individual control. Animals were anesthetized by the above-mentioned method. The cornea was anesthetized with one to two drops of 0.5% proparacaine hydrochloride before surgery. An eyelid speculum was placed into the fornix. The conjunctiva was incised in the upper anterior quadrant of the CXL eye with a pair of scissors. Sutures were placed in the treatment quadrant for towing extraocular muscles and holding the eyeball to allow better exposition of superior temporal equatorial sclera as the cross-linked area (Figure A, available in the online version of this article).
(A) Diagram of a rhesus 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 light
Photosensitizer solution containing 0.1% riboflavin (0.1% riboflavin, 20% dextran 500, Peschke D; PESCHKE Trade) was placed every 3 minutes onto the irradiation zone 20 minutes before and every 5 minutes during the 30-minute irradiation period. UVA irradiation (365 nm) was applied perpendicular to the sclera of CXL eyes by a UVA device (UV-X 1000; Avedro, Inc) with a surface irradiance of 3 mW/cm2 for 30 minutes (total dose of UVA, 5.4 J/cm2) at a distance of 5 cm from the sclera (Figure A). The control eyes were exposed and photosensitizer solution was applied without UVA irradiation (full CXL details following the standard convention are represented in Table 1). After surgery, the sutures around the muscles were removed and the conjunctiva was closed using polyglactin 7-0 (Ethicon, Inc). A 0.3% gatifloxacin eye gel was applied for 1 week postoperatively to avoid infection.
SD-OCT Scan Acquisition
Both eyes of each rhesus monkey were scanned using the Spectralis spectral-domain OCT device (software version 22.214.171.124; Heidelberg Engineering). The scan was performed on automatic real time for 30 frames including 768 A-scans. The automatic eye tracking technology maintains fixation on the retina. Only well-centered images with a signal strength of greater than 20 db were used for analysis.
Macular and retinal layer thickness were reported in an Early Treatment of Diabetic Retinopathy Study macular map (ETDRS),22 and the 1-, 3-, and 6-mm rings were considered for the analysis. The 1-mm ring was defined as central thickness. The intermediate and outer rings were divided into four zones designated as superior, nasal, inferior, and temporal. The numerical values recorded for each of the nine zones were used in the analysis of retinal thickness.
With enhanced depth imaging and manual caliper provided by the software, choroidal thickness values were measured from the outer border of the hyperreflective retinal pigment epithelium to the inner aspect of the sclera at seven locations: beneath the foveal center and at 500-µm intervals up to 1,500 µm temporal and nasal to the fovea center. The OCT image of choroidal thickness had been shown in our previous research.21
All images were taken by the same trained examiner and all measurements were repeated three times at each point.
OCTA Scan Acquisition
The structure of the fundus and the distribution of retinal vasculature in rhesus monkeys were similar to that of humans. In rhesus monkey retina, the central retinal vessels were sent out from the optic disc and divided into four branches to supply the four quadrants of the retina, and then the branches became thinner until they reached the peripheral part and the vessels in the macular area concentrated toward the central fovea.
In this study, both eyes of each rhesus monkey were scanned using the RTVue XR with AngioVue (software version 2015.100.0.3; Optovue, Inc), with a light source centered at 840 nm, a bandwidth of 50 nm, and an A-scan rate of 70,000 scans per second. Scans of macular areas were acquired and the scan size was 6 × 6 mm.
Flow density of retinal superficial vascular networks was calculated automatically using the above software and was defined as the average de-correlation value as previously described.23,24 Five zones divided by 1 and 3 mm ETDRS rings were considered for the analysis. The OCT angiographic image was showed in our previous research.21
All images were taken by the same trained examiner (XJ) and all measurements were repeated three times at each point.
Scleral Stress-Strain Measurement
To evaluate the biomechanical effect of the scleral CXL, the scleral stress-strain measurement was performed on 3 randomly selected rhesus monkeys in group 1 (n = 3) at 12 months postoperatively. After killing these rhesus monkeys with an overdose of pentobarbital, the whole eye globes were enucleated and a complete circular incision was applied 2 mm behind the limbus. The anterior eye segment was removed from the enucleated eyes. The posterior eye cups were turned around with the help of a forefinger and the retina and choroid were removed. In each eye, one equatorial strip (size: 14 × 2 mm) was dissected from the equatorial sclera of the superotemporal quadrant with the help of a scalpel similar to the study by Wollensak and Iomdina.13 The scleral strips from CXL eyes and control eyes were respectively measured. The scleral thickness was determined for the strips with the help of a laser displacement sensor (KEYENCE LK-G30 1-3-14; Higashi-Nakajima). Each strip was examined under the microscope and measured by a mechanical micrometer caliper for their widths.13 These strips were clamped vertically with a distance of 10 mm between the jaws of a commercially available microcomputer-controlled biomaterial tester (BOSE ElectroForce Series II 3330; Bose Corporation) (Figure B, available in the online version of this article). Each specimen was preloaded and preconditioned as described by us previously.16 Strain was increased linearly at a velocity of 1 mm/min. The parameters stress σ (MPa) and strain ∊ (%) of the samples were used for analysis.
Biomechanical test of scleral strips.
Rhesus monkeys in group 2 (n = 3) were killed using an overdose of pentobarbital at 12 months postoperatively. The eyes were immediately enucleated and fixed in a solution of formalin 10% for light microscopy. Six thick sections (5 µm) of each rhesus monkey eye were cut through the equatorial ocular in the superior temporal quadrant. Three of them were stained with hematoxylin–eosin. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed on the other three sections, according to the manufacturer's suggestions (In Situ Cell Death Detection Kit; Roche Biochemicals), to detect UVA induced cell death. TUNEL-positive color development (brown) was obtained using diaminobenzidine (Vector Laboratories) as a color substrate. Three light microscope images at a magnification of 400× were photographed from three different treatment areas of one ocular section. The thickness of outer nuclear layer and the apoptotic ratio of retinal cells were evaluated by Image-Pro Plus 6.0 (Media Cybernetics, Inc) to assess the damaged degree of the retina in cross-linked areas.
Statistical analysis was performed using SPSS for Windows software, version 19.0 (IBM-SPSS). The measurements were presented as mean ± standard deviation. The data of ocular biological parameters, scleral Young's modulus at 8.0% strain, and the apoptotic ratio between CXL and control eyes were compared using paired t tests. 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 ocular biological parameters between the two eyes of enrolled rhesus monkeys (each P > .05).21 No signs of inflammation were observed after scleral CXL and throughout the follow-up period. After surgery, all rhesus monkeys had normal corneas and anterior chambers and clear lenses. No vitreous or retinal lesion was observed clinically.
The retinal thickness and flow density of retinal superficial vascular networks in the macular area were summarized in Figures 1–2. No statistical difference was noted between CXL eyes and control eyes at 12 months postoperatively (each P > .05) in these parameters. The choroidal thickness was summarized in Figure 3, and there was no statistical difference between the two groups at 12 months postoperatively (each P > .05).
Thickness of central foveal retina of rhesus monkeys in different preoperative and postoperative periods in the scleral corneal cross-linking (CXL) and control groups.
Flow density of retinal superficial vascular networks in the central fovea of rhesus monkeys in different preoperative and postoperative periods in the scleral corneal cross-linking (CXL) and control groups.
Thickness of the subfoveal choroid of rhesus monkeys in different preoperative and postoperative periods in the scleral corneal cross-linking (CXL) and control groups (*P < .05).
With the help of a laser displacement sensor, the thickness of control scleral strips was determined to be 0.36 ± 0.18 mm. The cross-linked scleral strips were a little thinner than the control strips (0.34 ± 0.12 mm). However, there was no statistically significant difference between the control and CXL specimens (P > .05).
The stress-strain behavior curves and Young's modulus curves for scleral specimens obtained from CXL and control eyes are shown in Figure 4. The results revealed that the scleral specimens exhibited nonlinear stress-strain behavior with an initial low Young's modulus increasing gradually under higher stresses, which is similar to our previous research in rabbit eyes.16 At the same strain levels, the CXL group exhibited higher stress and Young's modulus than the control group. The scleral Young's modulus value at 8% strain in the CXL eyes corresponded to 183% of the control eyes. These corresponding values indicated significant differences between the CXL and control groups (P < .001).
(A) Stress-strain behavior curves of corneal cross-linking (CXL) and control scleral specimens at 12 months postoperatively to compare the biomechanical effect of scleral CXL. (B) Young's modulus curves of CXL and control scleral specimens at 12 months postoperatively to compare the biomechanical effect of scleral CXL. (The scleral Young modulus value at 8% strain in the CXL eyes corresponded to 183% of the control eyes.)
The histology of both CXL and control eyes at 12 months postoperatively was not distinguishable and showed no anatomic sign of photodamage. No sign of retinal necrosis, cystic degeneration, or hypocellularity of the nuclear layer was observed in these rhesus monkeys.
Apoptotic cells (stained by the TUNEL method described earlier) from the retina of the CXL and control eyes are shown in Figures 5–6. The corresponding ratios of apoptotic cells in the outer nuclear layer of the retina were 21.91% and 20.94% in CXL and control eyes, respectively, at 12 months postoperatively, and no statistical difference was noted between these two groups (P > .05).
The expression of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells in the outer nuclear layer of retinal sections in (A) corneal cross-linking (CXL) and (B) control eyes at 12 months postoperatively (hematoxylin–eosin stain, original magnification ×400). The black arrow shows the apoptotic cell in the retina.
The ratios of apoptotic cells in retina of rhesus monkeys at 12 months postoperatively. CXL = corneal cross-linking; + = positive control according to the manufacturer's protocols; − = negative control according to the manufacturer's protocols
The current study found that scleral CXL with riboflavin/UVA in rhesus monkeys eyes could strengthen the biomechanical properties of scleral tissue and maintain the stability for 12 months postoperatively, without influencing ocular biological parameters in vivo and histopathological changes in vitro.
The efficiency of this scleral CXL procedure was investigated by scleral stress-strain behavior curves. It was found that, at the same strain levels, CXL specimens showed higher Young's modulus than control specimens at 12 months postoperatively. This biomechanical outcome was constant with previous research,13–16 which proved the efficiency of this CXL technique in strengthening scleral tissue and validated the long-term scleral biomechanical stability of this procedure.
As for safety aspects, controversy exists regarding the safety of this treatment in different studies.16,25–27 Wang et al16 found that scleral CXL (365 nm, 3 mW/cm2, 30 minutes) might lead to apoptotic cells and ultrastructural changes in the retinal layers of rabbits. Zhang et al26 reported that retinal damage could occur when the rabbit eyes were irradiated for 50 and 60 minutes with 3 mW/cm2 at 365 nm. Another study showed no notable postoperative damage or apoptosis in rabbit retina and choroid after scleral CXL with an iontophoresis-assisted drug delivery system and accelerated UVA irradiation (10 mW/cm2, 9 minutes).18 Although accelerated CXL procedures were performed using shorter treatment times with higher UVA irradiations, the conventional CXL approach (3 mW/cm2, 30 minutes) was chosen to be applied in this study because it has been generally studied and applied in both in vivo experiments of scleral CXL13–16 and clinical practice of corneal CXL.28,29
In this study, biological parameters of the retina and choroid were investigated by SD-OCT and OCTA examinations, aiming to verify the safety of this scleral CXL technique in vivo in primates. Previous research had explained these biological parameter changes between CXL and control eyes at 6 months postoperatively, and preliminarily showed the safety of scleral CXL in vivo. In the current study, there was no statistical difference between CXL and control eyes in retinal and choroidal thickness of detected zones or in flow density of retinal superficial vascular networks at 12 months postoperatively.
Histopathological examination was applied in this study to evaluate the safety of this scleral CXL procedure in vitro. No side effect was found in any retinal layer of rhesus monkey eyes using the photomicrography with hematoxylin–eosin. In the TUNEL assay, although positive cells were detected in the retinal sections of CXL eyes, there was no significant difference in the corresponding ratios of retinal apoptotic cells between CXL and control eyes. To some extent, it indicates that UVA radiation in the scleral CXL procedure in this study would not cause retinal damage.
Potential limitations of this study should be mentioned. First, the sample size in this investigation was small, so further research in a larger sample is required. Second, parameters of retina and choroid measured in this research are limited to the posterior pole of the eyeball, because there has not been any OCT equipment providing the software for measurement in the equatorial region so far. Besides, although the posterior sclera would be just as important as equatorial sclera in the treatment of myopia, it is hard to expose and irradiate unless with the help of soft fiberoptics. Therefore, in this study, the equatorial sclera was chosen as the treatment area as described previously.13–16 Finally, these results might be valid for rhesus monkeys, but it has not been clarified whether the results are applicable in other species, including humans.
On the whole, UVA-CXL on the sclera of rhesus monkey eyes seems to be effective and safe. Further studies should be conducted to verify and modify the effect of this sclera procedure on actually preventing progression of pathologic myopia and axial elongation in clinical application.
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|Treatment target||1-cm diameter circle area in superior temporal equatorial sclera|
|Fluence (total) (J/cm2)||5.4|
|Soak time and interval (minutes)||20(q3)|
|Treatment time (minutes)||30|
|Epithelium status||There is no epithelium on the sclera|
|Chromophore||Riboflavin (Peschke D; PESCHKE Trade)|
|Light source||UV-X 1000 (Avedro, Inc)|
|Irradiation mode (interval)||Continuous|
|Protocol abbreviation in manuscript||S-CXL(3*30) (Standard)|