Reactive oxygen species accelerated glycation for oxygen-independent corneal cross-linking

Abstract

Purpose : Pentose initiated Maillard reaction is an oxygen-independent non-enzymatic crosslinking (CXL) mechanism, but is too slow for clinical applications. We hypothesize that glycation-mediated CXL can be accelerated with reactive oxygen species (ROS), which may make the process viable for treatment of keratoconus, and correction of refractive errors.

Methods : Fresh rabbit eyes were enucleated, and their epithelium removed. Corneal buttons (3mm diameter) were exposed to 20% ribose PBS for 1hr in groups A, B and C (n=3 each). A and B were then irradiated with femtosecond laser with 450mW at 1069nm, and 250mW at 400nm, respectively. C was exposed to 365nm UVA lamp for 1hr. Corneal anterior surface were pressed with a glass coverslip to minimize oxygen replenishment. D (n=3) was CXLed following the Dresden protocol as positive control. CXL efficacy was assessed with 5um nanoindentations to obtain the equilibrium modulus and the viscoelastic ratio.
Effective refractive power (Eff.RP) was assessed with corneal topography on eyeballs kept with 20mm H₂O intraocular pressure in group E (n=4) over a 10-hr period. Treated eyes were exposed to 3hrs of UVA lamp irradiation with corneas coverslip-flattened after ribose application. Paired controls received no light irradiation in all groups.

Results : The equilibrium modulus of ROS-ribose-CXLed corneal tissues were significantly increased (A: p=0.005; B: p<0.001; C: p<0.0001). Modulus change of tissues subjected to UVA light was similar to positive controls (D: p<0.0001), while the infrared and blue laser treated samples stiffened less. Only UVA-riboflavin CXLed tissues had a pronounced elastic load-relaxation response (p<0.001) (Fig.1). There was a significant Eff.RP drop over the 10hrs period for eyes simultaneously glycation-CXLed and mechanically loaded (p<0.0001) (Fig.2).

Conclusions : ROS-Glycation-CXL produced oxygen-independent corneal stiffening comparable to the Dresden protocol, but viscoelastic ratio differences suggest different reaction mechanisms. The sustained diopters drop suggests ROS-Glycation-CXL’s potential in corneal flattening for non-invasive vision correction. Further investigation is warranted to assess viability of transepithelial ROS-Glycation-CXL and ultrastructure changes of the stromal extracellular matrix.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. Equilibrium modulus and viscoelastic ratio from nanoindentation.

 

Figure 2. Eff.RP changes from corneal topography.