Analysis of the Change Induced by Riboflavin and Ultraviolet Light on Corneal Collagen by Infrared Spectrometry

Ab s t r Ac t Aim: Corneal collagen cross-linking (CCL) is a procedure that exposes the cornea to ultraviolet light and/or riboflavin to halt the progression of corneal ectatic disease. Currently, most investigations using Fourier-transform infrared spectroscopy (FTIR) of corneal changes following CCL focus on corneal ultrastructure, and not on changes at the molecular level. The aim of this study was to investigate the temporal and spatial separation of corneal collagen linkages that underlie the success of CCL. Materials and methods: Controlled experimental trial. Pairs of donor globes from five patients (n = 10) were divided into interventional and control groups. Interventional group corneas (n = 5) were exposed to riboflavin 0.1% and ultraviolet-A (UVA) light according to the modified Dresden protocol, harvested, cryo-microtomed, and placed on glass slides. Control group corneas (n = 5) underwent cryo-microtoming without CCL. Molecular changes were imaged using the synchrotron mid-infrared beamline at the Canadian Light Source. Results: Fourier-transform infrared spectroscopy imaging of total protein, integrated area under the amide I band from 1,700 to 1,600 cm− 1, FTIR imaging of collagen triple helix structures, second-derivative intensity as 1,666 cm− 1, and FTIR imaging of aggregated proteins, secondderivative intensity as 1,625 cm− 1 detected no difference in intramolecular cross-links between the interventional and control corneas. The secondary structure of collagen was neither significantly altered nor was their evidence of aggregation or denaturation within the cornea. Conclusion: Our data suggest that intramolecular cross-linking does not play a major role in CCL and that it is more likely an increase in intermolecular linkages that accounts for increased corneal strength. Clinical significance: An increase in intermolecular linkages likely accounts for the increased corneal strength observed following CCL. We hope that these results will guide future work to optimize techniques for CCL.