Diffusion of oxygen and riboflavin during corneal cross-Linking (CXL)

Corneal Cross-Linking (CXL) is a one hour therapeutic treatment involving epithelial removal, application of riboflavin drops for 30 min. followed by UV light exposure to the cornea for the treatment of keratoconus, ectasia and infection. The photochemical reaction mechanism during CXL effectively halts the progression of corneal disease. The three main components of successful CXL are 1) the diffusion of riboflavin, 2) the presence of molecular oxygen (O2) and the formation of reactive oxygen species, and 3) UV interactions during CXL which results in the biomechanical strengthening in the corneal stroma, thus halting disease progression. Recent applications such as reduced treatment time, epithelial by-passing and the combination of advanced CXL protocols as an alternative to refractive procedures have been investigated. The purposes of this study are to 1) investigate the diffusion of riboflavin using intra-stromal channels in order to determine the effective diffusion coefficients as compared to traditional axial diffusion with epithelium on or off, and 2) investigate O2 behaviours during CXL in order to better understand Type I and II photochemical reactions using an established luminescence quenching technique.Measurements of riboflavin diffusion using intra-stromal channels were created by means of a mechanical stromal instrument in whole-mounted post-mortem porcine eyes. The use of fluorescent imaging along with numerical modelling allows determination of effective diffusion coefficients under different conditions.Time-Correlated Single Photon Counting Phosphorescence Lifetime Imaging (TCSPC-PLIM) is an innovative and complex method for fluorescence and phosphorescence lifetime measurements. Phosphorescence sensitive O2 based probes in soluble form have the ability to detect O2 concentrations repeatedly and non-invasively in living biological tissue. This study investigated the use of O2 sensitive phosphorescent probes using three models: (i) O2-sensitive nanoparticles in soluble solution of riboflavin without collagen, (ii) collagen type-I gel with O2-sensitive nanoparticles and riboflavin, and (iii) porcine eyes stained with O2-sensitive nanoparticles and riboflavin. Several O2-sensitive nanoparticle probes were evaluated. One probe was chosen (SII-A), displaying sufficient brightness, photostability and efficient in-depth staining. Results showed O2 behaviour after UV- induced CXL in all samples measured, concluding TCSPC-PLIM to be a novel, effective method in measuring CXL.