Second-Harmonic Reflection Imaging of Normal and Accelerated Corneal Crosslinking Using Porcine Corneas and the Role of Intraocular Pressure

Purpose: The aim of this study was to investigate morphological changes, using second-harmonic (SH) optical imaging, in the corneal stroma after normal- and high-intensity collagen crosslinking in postmortem enucleated porcine eyes at controlled intraocular pressures (IOPs). Methods: Reflection-mode SH optical imaging of the stroma was realized after standard collagen cross-linking (CXL) and accelerated crosslinking (AXL) of porcine corneas, and the results were compared with the results for untreated controls. Ultraviolet-X lamps (365 nm) were used during riboflavin crosslinking with intensities of 3, 10, 30, and 100 mW/cm for constant 5.4-J/cm exposure doses. The IOP was varied using saline solution injected through the optic nerve and monitored using a Schiotz tonometer. SH optical imaging was realized with an in-house build multiphoton microscope using an ultrafast dispersion-compensated Ti-sapphire laser. Results: SH reflection images of CXL and AXL porcine corneas obtained at IOPs of 8, 11, 16, and 26 mm Hg showed a similar fibrillar structure of collagen lamellae. All crosslinked corneas showed an increased fibrillar contrast in comparison with untreated baseline images. At low IOPs, strong variations in the scattering were observed that reduced with an increase in the IOP, when fibrils tended to straighten out. At low and normal IOPs, no significant difference between CXL and AXL results could be observed. At very elevated IOPs, however, the impact of AXL was found to alter the fibrillar structure of the collagen becoming less apparent in SH images when compared with that of CXL. Conclusions: We found a strong influence of the IOP on SH reflection imaging of postmortem porcine corneal stroma. CXL and AXL led to similar SH images indicative of a similar tensile strength. Only at very elevated IOPs (26 mm Hg) did the results for AXL deviate from those of CXL, suggesting an IOP-related threshold for reliable applications of AXL.