Characterizing molecular tissue may provide insight into new treatments for high-risk AMD factors.

Imaging mass spectrometry (IMS) allows the location and quantity of proteins and lipids to be mapped across tissue sections, and thus is able to characterize tissues on an almost cellular scale.

These features ensure that IMS is able to provide biologically meaningful insight through the imaging data, revealing the structures of peptides and other biomolecules in a spatially resolved manner.

Researchers at Vanderbilt University Medical Center, in collaboration with the University of Alabama at Birmingham (UAB), are taking advantage of these insights to understand the human eye and, specifically, to characterize retinal deposits that form with age and help identify who is  high-risk for age-related macular degeneration (AMD).

Using IMS to localize modified lens protein and peptides that are cataract specific is a technique pioneered at the Vanderbilt Mass Spectrometry Research Center by Stanford Moore Chair in Biochemistry Richard Caprioli, Ph.D., and adapted for ocular tissues by Kevin Schey, Ph.D., a professor of biochemistry and ophthalmology and visual sciences.

“There are different types of retinal deposits, and we don’t understand their origin,” Schey explained. “If we can understand the composition, we can understand their mechanism of formation – where these deposits are coming from, what cells are forming them. Understanding how they’re formed will hopefully lead to new treatments.”

A Workflow for Retinal Imaging

Working with the UAB laboratory of Christine Curcio, Ph.D., through a Research to Prevent Blindness Catalyst Award, Schey and colleagues are using donor eyes from the Alabama Eye Bank to examine ex vivo retinal tissues.

The team’s first paper, published in 2020, displayed the capability of IMS in a normal eye. Their latest findings, presented at the 2022 annual meeting for the Association for Research in Vision and Ophthalmology, are the result of their work studying human donor eyes with retinal deposits.

“Our goal is to define the molecular composition of the deposits, which we know are a mixture of proteins and lipids,” Schey said. “Lipid transport is key, and when you get dysfunction, you get accumulation of lipid transporter proteins and the lipids they carry.”

To facilitate acquisition and imaging of the retina tissue, the collaborating labs have developed an IMS workflow.

“If we can understand the composition, we can understand their mechanism of formation – where these deposits are coming from, what cells are forming them. Understanding how they’re formed will hopefully lead to new treatments.”

At the University of Alabama at Birmingham, researchers describe the pathology and acquire ex vivo optical coherence tomography images, then prepare the eyes by fixing them. The tissues then come to Vanderbilt, where they undergo multi-modal imaging: autofluorescence, matrix-assisted laser desorption ionization (MALDI) IMS, post-acquisition autofluorescence, and post-acquisition hematoxylin and eosin staining. IMS and microscopy data are registered, and a Vanderbilt-developed algorithm aligns all the images.

“It takes experts across several disciplines – ophthalmology, biochemistry, advanced imaging and biomedical informatics – to prepare and analyze the IMS data,” Schey noted.

Tracking Molecular Signals

Using IMS to distinguish the individual retinal layers – for example, Henle fiber, photoreceptor, and retinal pigment epithelium (RPE) layers – the researchers look for deposits that are risk factors for AMD based on evidence that macular retinal deposits contain unique molecular compositions – lipids that are distinct from photoreceptors and RPE cells and distinct from peripheral regions.

 “We used IMS to examine the composition of subretinal drusenoid deposits (SDD) and discovered the same as well as different lipid signals in central and peripheral deposits,” Schey said. “To our knowledge, this is the first report of peripheral SDD.”

Informing Disease Mechanisms

Schey says it may be years until their work is translated to the clinic, but it could be transformational for researchers.

“With this molecular knowledge, we can build our hypotheses on how cataracts and retinal deposits may be forming. It is our hope that the IMS technology will help us discover biomarkers or enable new molecular tests.”

To expand their work in molecular mapping, the Vanderbilt researchers are participating in the NIH-funded Human BioMolecular Atlas Program. The goal is to create an open-access, 3D molecular guide to the human eye.

About the Expert

Kevin Schey, Ph.D.

Kevin Schey, Ph.D., is a professor of biochemistry and ophthalmology and visual sciences at Vanderbilt University Medical Center. His focus includes method and instrument development in proteomics analysis and application of proteomics technologies in ocular development and disease.