gms | German Medical Science

Background: Lipofuscin in the retinal pigment epithelium (RPE) and drusen deposits on Bruch’s membrane (BrM) are utilized to diagnose ocular aging and age-related macular degeneration (AMD). However, the molecules responsible for these signals and the pathophysiological relevance of these features are not understood. Therefore, we developed a multimodal molecular imaging approach to determine which molecules underlie the clinically observed lesions.

Methods: Flat mounted human RPE/choroid and BrM/choroid tissues of various ages were first imaged for brightfield and autofluorescence (AF), then imaging mass spectrometry was employed to determine the distributions of lipophilic small molecules in the range of m/z 200-1500 across the sample with a spatial resolution of 300 µm. The molecular images were then co-registered with AF images and Pearson correlations were determined. Principal component analysis (PCA) was used to identify specific molecular patterns.

Results: RPE autofluorescence did not correlate with known bisretinoid compounds. However, we have identified a molecule (m/z 536) that exhibited significant co-localization with lipofuscin AF. Compared to the RPE, PCA analysis of BrM had the ability to distinguish tissues of different ages and disease states. Hundreds of small molecules changed consistently with age in BrM samples, but only 3 showed positive correlation. Of these molecules, we have identified anhydroretinol (268 m/z) as co-localizing with autofluorescent drusen deposits on aging BrM.

Conclusion: These multimodal molecular imaging experiments link diagnostically relevant clinical features to the underlying molecules in AMD. Known bisretinoids were absent from BrM and their localization in the RPE questions a detrimental role in aging and AMD. On the other hand, contrary to the RPE, the molecular analysis of BrM was able to predict the AMD status of tissues. Multimodal molecular imaging is an important new tool for the identification of the molecular underpinnings of age- and disease-related changes in ocular tissue.