Animals and Study Design

The right eyes of 10 male New Zealand white rabbits (with body weights of approximately 2.5 kg) were treated. All animals were healthy and free of clinically observable ocular disease. The right eyes were given the collagen cross-linking with a femtosecond laser. Postoperative medications included topical antibiotics (tobramycin 0.3% drops 3 times/day) and steroidal anti-inflammatory drugs (dexamethasone 0.1% drops 3 times/day) for 1 week.

Intrastromal Pocket Creation

The rabbits were premedicated with a subcutaneous injection of diazepam and atropine (1 mg). For general anesthesia, 1.5 mL of ketamine hydrochloride 10% (25 mg/kg body weight) was injected intramuscularly. For additional local anesthesia, oxybuprocaine 0.4% drops were instilled into the right eyes. The femtosecond laser (Visumax, Carl Zeiss Meditec AG, Germany) with the software profile (version 2.50) was used to create a stromal flap with an upper hinge at 80 μm corneal depth, 7 mm diameter centered at the pupil. The spot size was 35 μm and the energy density was 190 nJ at a repetition rate of 200 kHz. Both the spot distance and track distance were 5 μm.

After the flap creation, a mini-spatula was used to enter corneal stroma with a 10-degree side cut and bluntly dissect between the flap and the inferior stroma. Following the pocket creation, a 25-gauge air cannula was introduced through the cut to inject riboflavin. The duration of stromal pocket completion was less than 3 minutes.

Collagen Cross-Linking Procedure

A 0.1-mL dose of 0.1% riboflavin solution was administered with a 25-gauge air cannula into the intrastromal pocket until it was clearly visualized that the entire pocket was bright yellow and stromal infiltration of the riboflavin solution was completed. After 2 minutes, the UVA light exposure was started. An ultraviolet A irradiation source (UV-X; IROC AG, Zürich, Switzerland) of 370-nm wavelength was used to irradiate the corneal surface. The effective fluence at the corneal surface was calibrated at 3 mW/cm ² for a duration of 10 minutes with the distance between rabbit cornea and UVA light equals to 1 cm.

Preoperative and Postoperative Examinations

Animals were examined before the CXL treatment. Postoperative examinations were performed at postoperative day 1, week 1, week 2, and months 1, 3, and 6. The corneal topography and pachymetry were measured using a rotating Scheimpflug camera (Pentacam; Oculus, Wetzlar, Germany). The anterior segment image was acquired at the same time by the Pentacam. For the corneal pachymetry, the central corneal thickness (CCT) was manually measured at the 90-degree image by the same examiners. Three consecutive measurements were performed, from which the median data were adopted.

Histologic Methods

The micromorphologic examination was performed at postoperative month 6. Corneoscleral tissues were dissected from eye globes, followed by fixation in 4% formaldehyde for approximately 48 to 72 hours and then embedded in paraffin. The sections of 4-μm thickness were stained with hematoxylin-eosin (HE).

Statistical Analysis

Statistical analysis was performed using Microsoft Excel 2007 (Microsoft; Redmond, Washington, USA) and SPSS 13.0 software (SPSS Inc, Chicago, Illinois, USA). Comparisons between preoperative and postoperative data were performed by paired t tests. A P value < .05 was considered statistically significant.