Keratoconus molecular alterations revealed by Fourier-transform infrared spectroscopy

In this study, we aimed to identify infrared spectral markers specific to keratoconic corneas obtained during penetrating keratoplasty, based on characteristic molecular alterations associated with the disease.

Corneal tissues obtained from patients with advanced keratoconus undergoing penetrating keratoplasty (n = 7) were analysed and compared with healthy corneal tissues (n = 4). Tissues were analysed with Fourier Transform Infrared Spectroscopy with a Focal Plane Array detector (FPA-FTIR). The spectra were recorded in the range from 4000 cm^-1 to 850 cm^-1.

In keratoconic corneas, spectrum of collagen has shown significant decrease both in its absolute (1204, p = 0.089), and relative (1204/1650, p = 0.011) contribution. Similar significant decrease could be seen in the absolute (1160, p = 0.020) and relative content of proteoglycans/polysaccharides and proteins (1160/1650, p = 0.021). We have identified non-significant decrease of relative content of phosphates compared to proteins (1238/1650, p = 0.089)). Common absorption band of collagen and proteoglycans was also increased relatively to the absorption band of phosphate group containing compounds, collagen and proteoglycans (1030/1082, p = 0.011).

This study demonstrates that FTIR microspectroscopy is a valuable tool for identifying spectral markers of corneal diseases. Significant differences in protein, proteoglycan and polysaccharide content were observed between keratoconic and healthy corneas. Notably, a reduced phosphate-to-protein ratio and decreased collagen and proteoglycan content were found in keratoconic samples. These changes reflect literature findings concerning ECM dysregulation driven by upregulated MMPs and altered collagen organization. Our findings highlight FTIR's potential for objective molecular characterization of corneal pathology and possibly-a tool in search of diagnostic biomarkers.