Abstract
Purpose :
Soft drusen is the leading risk factor for AMD progression and attributed to dysregulated outer retinal lipid transfer. The purpose of this study is to identify and determined the spatial distribution of lipids in soft drusen using cutting edge spatially resolved lipidomics methods of laser capture microdissection (LCM) coupled nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) and imaging mass spectrometry (IMS).
Methods :
Four AMD human donor eyes (> 80 years) with soft drusen and two normal eyes were fixed and subjected to ex vivo OCT and processed for 12 µm thick cryosections. The Soft drusen were characterized in neighbor sections stained with periodic acid Schiff hematoxylin and identified by the morphology and location between retinal pigment epithelium layer and Bruch’s membrane. Highly sensitive tools of imaging mass spectrometry (IMS) using 1,5-diaminonaphthalene (DAN) for negative ion mode and 2,5-dihydroxyacetophenone (DHA) for positive ion mode and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify lipids, respectively. For nano LC-MS/MS analysis, soft drusen content from central macula was isolated using laser capture microdissection (LCM). LC-MS/MS and MALDI-IMS data were interpreted using LipidMaps and LipostarMSI.
Results :
: Lipids in soft drusen of AMD human donor retinas were observed as glycerophospholipids including phosphatidylcholine (PC 42:9), phosphatidylethanolamine (PE O-34:2), phosphatidylserine (PS O-40:2 and O-34:4), phosphatidic acid (PA 32:3), Phosphatidylinositol (PI O-32:0) Lyso phosphatidylcholine (LysoPC 18:3), lysophosphatidylethanolamine (PE O-38:3), and ceramide (Cer 36:0;O4 and 34:1; O2) in negative ion mode; sphingomyelin lipids including SM(34:1, 34:2, 36:1, 36:2, 38:1, 40:1, 42:1, 42:2) in positive model.
Conclusions :
Soft drusen specific lipids were identified using IMS and nLC-MS/MS. An interpretable supervised machine learning approach is ongoing to review in an automatic and unbiased fashion hundreds of lipid signatures of soft drusen and non-soft drusen regions of human donor retina (Anderson et al, 2023). Further studies are required to understand the role of specific lipids and associated pathways in soft drusen formation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.