An in vitro investigation into the impact of corneal rinsing on riboflavin/UVA corneal cross-linking

Background Corneal cross-linking (CXL) using riboflavin and ultraviolet-A light (UVA) is a treatment used to prevent progression of keratoconus. This ex vivo study assesses the impact on CXL effectiveness, as measured by tissue enzymatic resistance and confocal microscopy, of including a pre-UVA corneal surface rinse with balanced salt solution (BSS) as part of the epithelium-off treatment protocol. Methods Sixty-eight porcine eyes, after epithelial debridement, were assigned to six groups in three experimental runs. Group 1 remained untreated. Groups 2-6 received a 16-min application of 0.1% riboflavin/Hydroxypropyl methylcellulose (HPMC) drops, after which Group 3 was exposed to 9 mW/cm(2) UVA for 10 min, and Groups 4-6 underwent corneal surface rinsing with 0.25 mL, 1 mL or 10 mL BSS followed by 9 mW/cm(2) UVA exposure for 10 min. Central corneal thickness (CCT) was recorded at each stage. Central 8.0 mm corneal buttons from all eyes were subjected to 0.3% collagenase digestion at 37 degrees C and the time required for complete digestion determined. A further 15 eyes underwent fluorescence confocal microscopy to assess the impact of rinsing on stromal riboflavin concentration. Results Application of riboflavin/HPMC solution led to an increase in CCT of 73 +/- 14 mu m (P < 0.01) after 16 min. All CXL-treated corneas displayed a 2-4 fold greater resistance to collagenase digestion than non-irradiated corneas. There was no difference in resistance between corneas that received no BSS rinse and those that received a 0.25 mL or 1 mL pre-UVA rinse, but each showed a greater level of resistance than those that received a 10 mL pre-UVA rinse (P < 0.05). Confocal microscopy demonstrated reduced stromal riboflavin fluorescence after rinsing. Conclusions All protocols, with and without rinsing, were effective at enhancing the resistance to collagenase digestion, although resistance was significantly decreased, and stromal riboflavin fluorescence reduced with a 10 mL rinse. This suggests that a 10 mL surface rinse can reduce the efficacy of CXL through the dilution of the stromal riboflavin concentration.