Nine female Japanese White rabbits weighing 1.5–2.0 kg were used in the study. All animals were healthy and free of ocular disease. In six rabbits, the right eyes were treated with collagenase type II, and the left eyes were treated as the control group (n = 6 in each group). In the other three rabbits, KeraVio treatment was applied to both eyes after topical collagenase application (n = 6). All animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
A topical anaesthetic consisting of 0.4% oxybuprocaine hydrochloride was applied to the eyes. After epithelial debridement, corneal trephines were placed on the corneas. In the collagenase group, a 10 mg/mL collagenase type II solution (Worthington, Lakewood, NJ) in balanced salt solution and 15% dextran was applied to the surface of the corneas with corneal trephines. The solution was then removed with cotton swabs, and the corneas were rinsed with 0.9% sodium chloride solution. The control eyes were subjected to the same protocol but without collagenase type II in the applied solution. The KeraVio process began with the application of collagenase in the same manner as in the collagenase group. VL irradiation (375 nm) was applied using a single VL diode (Nitride Semiconductors Co., Ltd., Tokushima, Japan) with an irradiance of 0.31 mW/cm2 for 180 minutes at a distance of 60 cm from the cornea (total energy dose 3.3 J/cm2). KeraVio treatment using only VL irradiation was continued for 7 days (total energy dose 23.4 J/cm2).
Before surgery, the rabbit eyes were subjected to slit-lamp examinations to identify evidence of conjunctival injection, corneal infiltration and corneal stromal inflammation. These examinations were repeated every day during the 7-day study to assess ocular safety; an examination was carried out before collagenase treatment and repeated every day during the 7-day study. Corneal keratometry and the central corneal thickness (CCT) were recorded at baseline (the day before treatment) and 7 days after treatment using a handheld keratometer (Retinomax 3; Righton, Tokyo, Japan) and an ultrasound pachymeter (SP-100; Tomey, Nagoya, Japan), respectively, under topical anaesthesia. Three measurements were taken at each time point, and the mean value of each parameter was recorded along with the change in its value. The mean steep keratometry (Ks), corneal astigmatism, CCT and changes in each parameter were evaluated. The axial length was also recorded at 7 days after treatment using a digital calliper.
The rabbits were euthanized with an intravenous overdose of sodium pentobarbital on day 7. The corneas were harvested en bloc along the sclera. A 2- to 3-mm scleral rim was preserved, and the cornea was attached along a custom-made scale. Then, a 5-mm wide corneal strip was resected vertically along the cornea. The corneal strips were clamped vertically at a distance of 5 mm between the jaws. The CCT at day 7 in each cornea was used to calculate the cross-sectional area of the corneal strip. After the prepared corneal strip was placed on a computer-controlled electronic universal testing machine (TA, XTplusC Texture AnalyserTM, Stable Micro Systems, Ltd., London, UK), a fixture was applied to hold the corneoscleral limbus of the corneal strip during a uniaxial tensile test. For the actual measurement, the sample was stretched at a velocity of 1.8 mm/min up to a maximum force of 5 N. The elastic modulus was defined as the ratio of tensile stress (amount of force causing deformation per unit trans-sectional area of the corneal strip) to tensile strain (percentage change in the length caused by the stress). For the subsequent statistical analysis, the elastic modulus was consistently evaluated at 3%, 5%, and 10% strain.
Analyses were performed with Statistical Analysis Software (version 9.4; SAS Institute, Cary, NC). The outcome measures are reported as the mean ± standard deviation. A two-tailed paired t-test was used in the statistical analyses to compare the parameters between the two groups. A P value of < 0.05 was considered statistically significant.