Purpose. 145.07 A.U. in group C. The difference in fluorescence was statistically significant between groupings A and B1 (= 0.001) and groups B2 and C (< 0.0001). Conclusions. Ultrasound treatment increased the entry of topical riboflavin into the corneal stroma despite the presence of a previously intact epithelial barrier. This approach may offer a means of achieving clinically useful concentrations of riboflavin within the cornea with minimum epithelial damage, thereby improving the risk profile of corneal cross-linking procedures. = 15); group B C ultrasound-treated epithelium-on eyes (= 31); group C C untreated epithelium-off eyes (= 16). In order to allow a depth-related fluorescence comparison among these groups, group B was subdivided in two groups: B1 C confocal analysis done with epithelium maintained (= 15); and B2 C confocal analysis done after removal of epithelium (= 16). In a second part of the experiment, we assessed the temperature variation in ultrasound-treated and untreated corneas. Treatment Eyes in group A were placed in a solution of 0.1% riboflavin (Sigma-Aldrich) for 45 minutes without receiving any additional treatment. Eyes in group B were treated with continuous-wave ultrasound 880 kHz at 1 W/cm2, applied to the central cornea for the first 6 minutes, and then remained in the riboflavin solution for an additional 39 minutes (total 45 minutes). Eyes in group C had the epithelium removed with a surgical blade (N24; Feather, Osaka, Japan) before placement in the riboflavin solution, where they were left for 45 minutes. A water bath system was used to maintain the temperature at 34C during the experiment (Fig. buy AEBSF HCl 1). After the 45 minute immersion, all eyes were removed from the riboflavin solution, corneas were excised, epithelium was removed from corneas in group B2, and a fluorescent analysis using confocal microscopy was immediately performed in all samples. The 458-nm wavelength was chosen as the excitation wavelength, and the emission was collected from wavelengths 560 to 615 nm through a Zeiss LD Plan-Neofluar 40/0.6 objective (Carl Zeiss, Jena, Germany) on a Zeiss LSM510 confocal microscope (Carl Zeiss). The microscope excitation and detection settings were selected based on pilot data and fixed for all experiments (pinhole = 1.0 airy units; optical section thickness = 3 m; detector gain = 691; amplifier gain = 1; laser transmission 10%). The buy AEBSF HCl experiment was repeated more than three times, always with at least one eye of each group. The anterior corneal surface was marked as the starting point (0-m depth), and mean fluorescence of the entire image field (440 m2) was measured in a 12 bit dynamic range in arbitrary Mouse monoclonal to OTX2 units, at 100-, 150-, 200-, and 250-m depth in the cornea. The results were compared between groups A and B1 (with epithelium) and between groups B2 and C (without epithelium). Figure 1.? Two cadaveric rabbit eyes submerged in riboflavin 0.1% solution (= 4) and at room temperature (24C) in group E (= 6). All eyes in group D and three buy AEBSF HCl eyes from group E were treated for 6 minutes with continuous wave ultrasound 880 Khz at 1 W/cm2; the remaining three eyes from group E were left untreated as controls. To assess the variation in corneal temperature, a thermocouple (HYP1; Omega, Stamford, CT) was inserted in the superficial corneal stroma of the preheated eyes immediately after its submersion in buy AEBSF HCl riboflavin solution (T0) and after 6 minutes (T6) with or without ultrasound treatment. Statistical Analysis The numerical data were initially entered into a spreadsheet (Microsoft Excel XP; Microsoft, Redmond, WA) and then exported to the R statistical package (version 2.12; R Foundation for Statistical Computing, Vienna, Austria). The difference in the mean vectors between groups was assessed using the Two-sample Hotelling test. An alpha level of less than or equal to 0.05 was chosen as the.