Study Population
We registered the study protocol with the University Hospital Medical Information Network Clinical Trial Registry (000031162). We enrolled twenty eyes of 20 patients (9 men and 11 women) in this retrospective study. All patients received standard CXL treatment for progressive keratoconus at Kitasato university hospital, and completed a 3-month follow-up. The patients were recruited in a continuous cohort. Diagnosis of keratoconus was performed by one experienced clinician (K.K.) with evident findings characteristic of keratoconus (e.g., corneal topography with asymmetric bow-tie pattern with or without skewed axes), and at least one keratoconus sign (e.g., stromal thinning, conical protrusion of the cornea at the apex, Fleischer ring, Vogt striae, or anterior stromal scar) on slit-lamp examination.3 Progression of the disease was interpreted as an increase in the maximum keratometric reading of at least 1 diopter (D), or a deterioration of corrected visual acuity with an increase of astigmatism ≥1 D confirmed in at least 2 examinations during the preceding 6 to 12 months before treatment. Due to safety issues related to corneal endothelial cells, CXL was not done in eyes with thinner corneas (the thinnest corneal thickness less than 400 µm). We excluded eyes with pellucid marginal degeneration, other corneal diseases, and previous ocular trauma or surgery. We received written informed consent for the CXL treatment from all patients. This clinical chart review was approved by the Institutional Review Board of Kitasato University, and followed the tenets of the Declaration of Helsinki. Our Institutional Review Board waived the requirement for informed consent for this retrospective review. The data that support the findings of the present study are available from the corresponding author upon reasonable request.
Assessment of Corneal Endothelial Cells
Preoperatively and 3 months postoperatively, we quantitatively evaluated the ECD, the coefficient of variation in cell size (CV), and the percentage of hexagonal cells (HEX), in the peripheral regions (2, 4, 6, 8, 10, and 12-o’clock positions on a 6-mm arc) of the cornea, using a non-contact specular microscope (EM-3000TM, Tomey, Aichi, Japan). Patients were asked to fixate at each peripheral target, and photographs were taken of the peripheral corneal endothelium using the auto-alignment function. The system automatically measures the area of corneal endothelial cells (> 100 cells), and after than it automatically calculates the ECD, CV, and HEX within an area of 0.1 mm2 by the instrument’s built-in software for cell analysis.
Surgical Procedures of Corneal Cross-linking
The standard CXL technique was performed in accordance with the Dresden protocol.1 Topical anesthesia was administered, and the corneal epithelium at a central circular area of 8-mm in diameter was removed with a blunt spatula. Next, we topically administrated riboflavin 0.1% solution every 2 minutes for 30 minutes, and we used a slit-lamp microscope to confirm that adequate riboflavin had penetrated into the anterior chamber. UVA irradiation at a wavelength of 370 nm and a surface irradiance of 3 mW/cm2 was performed for 30 minutes with an OptoXLink corneal cross-linking systemTM (North Miami, FL, US). Riboflavin solution was administered every 5 minutes during the irradiation. We used topical steroidal and antibiotic medications 4 times daily for 2 weeks after treatment, with the dose being reduced gradually thereafter, with a soft contact lens until re-epithelialization.
Statistical Analysis
All statistical analyses were performed with a commercially available statistical software (Bellcurve for Excel, Social Survey Research Information Co, Tokyo, Japan). In order to compare the pre- and the post-surgical data in the peripheral regions, we performed the Wilcoxon signed-rank test. The results are shown as mean ± standard deviation, and a value of p<0.05 was considered statistically significant, unless otherwise indicated.