Indirectly assessing changes in corneal properties with OCT speckle after crosslinking in porcine eyes

The aim of this study was to assess the effect of the standard crosslinking (CXL) procedure on corneal properties and subsequent changes in collagen bonds formation using optical coherence tomography (OCT) corneal speckle statistics and vibrational spectroscopy. Porcine eyes with intact corneal epithelium were randomly selected to one of the four study groups: (1) untreated eyes moistened with phosphate-buffered saline (PBS); (2) eyes after the epithelial debridement and riboflavin application; (3) eyes after CXL procedure according to the Dresden protocol; and (4) eyes after corneal epithelial debridement, regularly moistened with PBS. Before and after this selection, each eyeball was subjected to the constant intraocular pressure of 20 mmHg. Then, ocular biometry was performed and the central cornea was imaged using spectral-domain OCT. Following this, a nonparametric approach to speckle modeling (the Contrast Ratio (CR)) was utilized within the region of interest for each B-scan covering the central corneal stroma. To verify whether the CXL performed ex-vivo results in formation of new bonds in the cornea, Fourier Transform Infrared Attenuated Total Reflectance (ATR-FTIR) spectra of dried corneas, dissected from examined eyeballs, were collected and analyzed. Corneal epithelium removal alone or with the riboflavin application leads to a statistically significant decrease in the CR median value (the Wilcoxon signed-rank test, p < 0.05). However, the most pronounced change in CR median value, which decreases with the increased number of scatterers, was shown after the complete CXL procedure including riboflavin soaking and UVA irradiation (the Wilcoxon signed-rank test, p = 0.004). Analysis of ATR-FTIR spectra revealed influence of UVA irradiation on collagen matrix. The study has shown the increased dehydration accompanied by almost no alteration of collagen native triple-helical structure. Significant changes have been observed for bands related to collagen crosslinks. Specifically, the predominant changes occurred in the sugar region from 1150 to 975 cm^-1, as well as in the absorbance of carbonyl groups. Furthermore, the ratio of two Amide I components at approximately 1660 cm^-1 and 1675 cm^-1 decreased after UVA irradiation. Together, these results provided the evidence for the creation of new corneal crosslinks. In conclusion, this study clearly indicates that the UVA exposure causes the substantial difference in optical scattering occurring in corneal stroma as a result of the induced biochemical changes at the molecular level in this tissue assessed with ATR-FTIR. The proposed speckle-based methodology brings a new insight into the development of OCT technology useful in an indirect assessment of some collagen changes.