Study design
Seven three-year-old rhesus macaques (14 eyes) were divided into two groups using a completely randomized design, with 4 monkeys in group A (8 eyes) and 3 monkeys in group B (6 eyes). The right eye of each rhesus macaque was used for the experiment, and the left eye was used as a control. In group A, the superior temporal quadrant of the right eye was irradiated with blue light (Fig. 1). In group B, the superior and inferior temporal quadrants of the right eye and the superior temporal quadrant of the left eye were irradiated with blue light (Fig. 2). The estimation of the sample size in each group for this study was performed based on other reports in the literature [9,10,11]. Both eyes of all the rhesus macaques were examined clinically before SXL and 1 week, 1 month, and 3 months postoperatively, and the examiners were blinded to the group allocation during the experiment.
Subjects
Seven healthy rhesus macaques (age, 3.0–3.5 years; weight, 4.3–6.0 kg) were obtained from the Experimental Animal Centre of the People’s Liberation Army Academy of Military Medical Sciences in China. They were raised in the laboratory animal breeding room of Capital Medical University. They were housed individually and provided free access to food and water throughout the study. The room temperature was maintained between 23 °C and 27 °C, and the relative humidity was maintained between 45 and 55%. The general condition of all macaques met the national health monitoring standards for primate laboratory animals. They underwent anterior-segment and fundus examinations before initiation of the experimental procedure to exclude potential health factors that might affect the IOP measurements. They showed no abnormities, and baseline measurements of the IOP showed no pathological findings. The use of experimental animals in this study was approved by the Institutional Animal Care and Use Committee of Capital Medical University (AEEI-2014–127), and all macaques included in the study were acquired and used in compliance with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research. After the experiment, the rhesus macaques continued to receive good care and were used in subsequent studies.
Surgery and SXL
All macaques were anaesthetized with a mixture of ketamine hydrochloride (10%; 20 mg/kg) and xylazine hydrochloride (5 mg/kg) injected intramuscularly into the buttocks. All surgical procedures were performed by one surgeon (Mengmeng Wang). All eyes underwent a 360-degree conjunctival peritomy, and Tenon’s capsule was dissected to expose the equatorial sclera in the corresponding quadrant. Two scleral traction wires were placed in front of the planned cross-linking site; these wires were pulled to rotate the eyeball and expose the irradiation zone. A 0.5% solution of riboflavin was instilled on the exposed scleral surface 20 min before irradiation. The diameter of the irradiation zone in the equatorial sclera in all animals was 10 mm. A collimated beam of blue light (460 nm) was generated with a beam illumination system (Beijing Obodi Optoelectronic Technology Co., Ltd., Beijing, China), which conformed to the national standards. The exposed sclera was irradiated from a distance of 5 cm, with a resulting surface area of irradiation of 22.5 mW/cm2; riboflavin solution was dripped onto the scleral surface every 20 min during irradiation. Blank irradiation zones were not irradiated, but the 0.5% riboflavin solution was administered in the same manner. Postoperatively, the preset sutures were removed, and the conjunctiva was sutured with absorbable surgical sutures. A 0.3% gatifloxacin eye gel was applied to all eyes to avoid infection, 4 times a day for 1 week.
IOP Measurements
The IOP measurements were performed before SXL and 1 week, 1 month, and 3 months after the SXL operation. After the induction of general anaesthesia, each macaque was secured in a seated position. An appropriately experienced examiner then measured the IOP of the experimental and control eyes in groups A and B using a Model 30 pneumatonometer (Reichert, America) and TonoLab rebound tonometer (iCare Finland Oy, Helsinki, Finland). When performing measurements using the Model 30 pneumatonometer, the operator held the probe and moved the sensor towards the eye gently to touch the corneal apex. As the membrane touched the cornea, the sensor handle was continuously moved towards the eye until an audible tone was generated, which indicated that the most accurate reading was obtained. The tone changed to a noticeably low pitch when the standard deviation among IOP readings was maintained below 1.0 mmHg for three seconds. Additionally, the instrument displayed the average IOP and standard deviation. When performing measurements using the TonoLab rebound tonometer, the IOP was assessed 5 times, and for each of these assessments, it was measured 6 times, and the mean IOP values were calculated.
Measurement of OPA
The OPA measurements were performed before SXL and 1 week, 1 month, and 3 months after the SXL operation. After the induction of general anaesthesia, the rhesus monkeys were placed in a sitting position. An appropriately experienced examiner then measured the OPA of the experimental and control eyes in groups A and B using the Model 30 pneumatonometer. When measuring the OPA in pulse tonometry mode, the operator held the probe in contact with the cornea. A changed tone was generated when the pneumatonometer sensed five ocular pulses, indicating that the instrument had enough samples to compute an average OPA; the test ended when the instrument sensed ten ocular pulses. The reading displayed was the average of all the detected pulses. The measurement was repeated 3 times, and the mean value of the 3 measurements was obtained.
Statistical Analysis
SPSS version 20.0 (SPSS, Chicago, IL, USA) was used to analyse the results. The IOP and OPA data are recorded as the mean ± standard deviation. The Shapiro–Wilk test was used to test normality. We evaluated differences in the IOP and OPA between the experimental and control eyes via the paired t test at the scheduled follow-up time points within both groups. P < 0.05 was considered statistically significant.