Synchrotron‐based FTIR microspectroscopy of human primary retinal pigmented epithelial cells as a model for age‐related macular degeneration

Abstract

AbstractPurpose: To investigate the characteristics of untreated and treated human retinal pigmented epithelial cells (hRPEs) as an ex vivo model for age‐related macular degeneration (AMD) using high resolution synchrotron radiation‐based Fourier Transform Infrared (FTIR) microspectroscopy and multivariate analysis.Methods: hRPEs obtained from cadavers were treated by autophagy ‐inducer rapamycin (Rap), and ‐inhibitor bafilomycin A1 (Baf), as well as hydrogen peroxide (H2O2) to induce oxidative stress, in an ex vivo model for studying AMD. FTIR measurements were performed at the MIRAS beamline of the ALBA Synchrotron, Barcelona, Spain.Results: The FTIR spectra consisted of several bands arising from the vibration of different groups belonging to proteins, lipids and nucleic acids, indicating the rich biochemical composition of the hRPEs. In the protein region, the amide I band of the treated groups (Rap, Baf, H2O2) was different from the control untreated group, indicating an overall protein disordering under such treatments. In the DNA region, two spectral areas appeared to be modified upon treatment, indicating different modifications of the DNA conformational changes or rearrangements attributed to the distortions of the DNA double helix in the presence of Rap, Baf or H2O2. The same treatments could also induce several modifications in the lipid spectral region, which can affect a wide range of biological processes. The H2O2 treated group showed higher prevalence for the lipid peroxidation, a process generated by the effect of several reactive oxygen species.Conclusions: The present study shows that high resolution FTIR can be used to evaluate the complete bio−/macro‐molecular spectra of hRPEs such as proteins, lipids and nucleic acids, which can be used as an ex vivo model for AMD, thus giving possibilities for developing and testing new treatment modalities.