Application of FTIR microspectroscopy in studies on keratoconus

Abstract

Purpose : Keratoconus is a prevalent disease of still unknown etopathology, that causes non-inflammatory corneal ectasia and progressive deterioration of vision. Our study aims to asses the usefulness of Fourier transform infrared (FTIR) spectroscopy in studies on keratoconus etiopathology, which still has not been clearly under covered. In the course of the disease, both the composition and biomechanical properties of the cornea change, resulting in the change of corneal curvature into a cone. There are many reports on changes in the structure of molecules in corneal extracellular matter, especially- in corneal collagen, that may influence changes in corneal curvature. FTIR microspectroscopy is very sensitive in detecting conformational changes in proteins, because of the strong IR absorption of peptide carbonyl groups, as well as other molecules.

Methods : Human corneal sections from multiple radical keratectomies were collected, formalin-fixed and paraffin embedded and then cut into 9 µm thick slides. Corneal tissue not affected by the disease process served as a control group. Slides were analysed with microspectrometer FTIR Vertex 70 with focal plane array detector. The spectra were recorded in the range from 4000 cm-1 to 850 cm-1, with 4 cm-1 resolution, 64 sample scans and 64 background scans. The microscopic image of a corneal section slide and its exemplary spectra (Amide A+B) are shown in the Figure 1. The analysis of the spectral results was carried out on the basis of the data mining techniques of the OPUS 7.5 program (Bruker Optik, Germany)

Results : The performed spectral analyses allowed for the identification of quantitative differences in the biochemical composition of individual layers between cornea affected by keratoconus and physiological cornea. Molecular markers were identified as statistically significant differences between second negative derivative of difference in mean absorbance between physiological and keratoconic cornea in bands associated with structure of proteins, glycosaminoglycans and lipids.

Conclusions : Our results prove that FTIR microspectroscopy can be used in evaluation of keratoconus and further studies on its pathogenesis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.