Our pepsin digestion methodology has been published previously.
17,23 We chose this method as it facilitates relatively slow digestion rates. This has allowed us in our past studies to not only detect significant differences in the digestion rates in terms of disc diameter measurements and time to complete digestion but also to measure dry weights at 10 days, which has highlighted significant differences between treatment protocols that are not evident by just measuring disc diameters and time to digestion alone.
17,23 In addition, using this same protocol we have repeatedly shown that there are no differences in digestion times in nonirradiated corneas that have not received riboflavin drops and those soaked in riboflavin for 30 minutes.
17,23 This allowed us in this study to reduce the number of control groups and just use de-epithelialized, nontreated, nonirradiated corneas as a sole control group. In brief, following complete debridement of the corneal epithelium using a single-edged razor blade, the eyes were divided randomly into the five groups described in
Table 1, in which group 1 (no riboflavin administered and no UVA exposure) served as an untreated control and groups 2, 3, 4, and 5 received varying concentrations of riboflavin solution (0.05%, 0.1%, 0.2%, 0.3%, respectively) for a period of 30 minutes. All riboflavin solutions contained 20% dextran and were identical in formulation except for the riboflavin concentration. The riboflavin was applied using a 10-mm suction ring/container (J2294; E. Janach srl, Como, Italy) that was completely filled to its brim with the relevant riboflavin solution concentration after being placed and suctioned over the central cornea. Following the 30-minute riboflavin diffusion period, all eyes in groups 2, 3, 4, and 5 underwent irradiation using a CCL-VARIO corneal cross-linking UVA lamp (Peschke Meditrade GmbH, Huenenberg, Switzerland) with a wavelength of 365 nm and a 9.0-mm aperture, with an intensity of 9 mW/cm
2 for 10 minutes (total energy dose 5.4 J/cm
2). An accelerated treatment protocol was chosen, as we needed to treat 44 eyes in each experimental run and wished to use only one UVA irradiation device so that fluence levels and UV beam profile were consistent between treatments. If we had used the standard protocol of 3 mW/cm
2 for 30 minutes, it would have taken 22 hours to complete the treatments in radiation time alone. This would mean that the eyes being treated last would be well over 24 hours old since time from enucleation. Even though we treated one eye from each group sequentially to minimize any effects of some eyes being treated later than others, we did not want to include any eyes in which treatment occurred after 24 hours. This was undertaken to diminish any effects of natural decomposition, endothelial and epithelial cell death with subsequent changes in corneal hydration, and possible fungal/bacterial contamination. By using an accelerated treatment of 9 mW/cm
2 for 10 minutes, we could reduce the total irradiation time to just over 7 hours and treat all eyes with 12 hours.