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J. R. Beattie, J. V. Glenn, N. Frizzell, S. Thorpe, M. E. Boulton, J. J. McGarvey, A. W. Stitt; Advanced Glycation Endproducts on Bruch’s Membrane Can Be Detected by Raman Microscopy and Correlate With Chronological Age. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2178.
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Advanced glycation endproducts (AGEs) accumulate in the retina and are implicated in diabetes and age-related pathology. Conventional methods of AGE adduct quantification require fixing or homogenisiation. Here, we investigate the potential of Raman microscopy for ex vivo evaluation of AGEs in Bruch’s membrane (BM) from unfixed post-mortem eyes.
BM-Choroid were peeled from donor eye-cups (n=40) and either snap-frozen or formalin-fixed, paraffin embedded and sectioned. Immunoreactivity for AGE epitopes in BM was analysed in the sections using confocal microscopy. AGE adducts were quantified from homogenized tissue using reverse-phase HPLC and GC/MS. For parallel Raman analysis, BM-Choroid was flat-mounted on slides and analysed using a Raman confocal microscope. Spectral signals unrelated to the parameters of interest were removed using Orthogonal Signal Correction (OSC). An independant test set was used to ensure reliability of predictions based on the cross-validated model created from the training set.
AGE adducts accumulated in BM with age and to a lesser extent in the underlying choroid. Quantitative analysis showed the AGEs pentosidine and CML occurred at significantly higher levels in patients >70 years old (P<0.05-0.01). Defined spectral "fingerprints" were obtained for AGEs and these were observed in the ex-vivo clinical samples using confocal Raman microscopy. The OSC-Raman dataset permitted modeling of the age of donor and pentosidine content with validation correlation coefficients of R2 = 0.705 and 0.881, respectively.
AGE adducts significantly accumulate in ageing BM. Raman spectroscopy is a valid approach for analysis of these pathogenic adducts and the changes with chronological age. Raman spectral analysis may non-invasively monitor AGEs and, with further development, this approach may have diagnostic potential in the in living eyes.
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