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Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

Published: May 26th, 2023



1Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham Heersink School of Medicine, 2Department of Ophthalmology, University Hospital of Zurich, 3Vitreous Retina Macula Consultants of New York, 4Department of Ophthalmology, New York University Grossman School of Medicine, 5Advancing Sight Network, 6Department of Ophthalmology, University Hospital Bonn
* These authors contributed equally

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged ≥80 years old, with a death-to-preservation time (DtoP) of ≤6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30° macula cube, 30 µm spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 ± 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Fifteen years of managing neovascular age-related macular degeneration (AMD) with anti-VEGF therapy under the guidance of optical coherence tomography (OCT) has offered new insights into the progression sequence and microarchitecture of this prevalent cause of vision loss. A key recognition is that AMD is a three-dimensional disease involving the neurosensory retina, retinal pigment epithelium (RPE), and choroid. As a result of the OCT imaging of trial patients and the fellow eyes of treated clinic patients, the features of pathology beyond those seen by color fundus photography, a clinical standard for decades, are now recognized. These include intraretinal neovascul....

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The institutional review board at the University of Alabama at Birmingham approved the laboratory studies, which adhered to Good Laboratory Practices and Biosafety Level 2/2+. All US eye banks conform to the 2006 Uniform Anatomical Gifts Act and US Food and Drug Administration. Most US eye banks, including Advancing Sight Network, conform to the medical standards of the Eye Bank Association of America.

The Table of Materials lists the supplies and equipment. Supplement.......

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Table 1 shows that during 2016-2017, 184 eyes from 94 white non-diabetic donors >80 years of age were recovered. The mean death-to-preservation time was 3.9 h (range: 2.0-6.4 h). Of the 184 eyes reviewed, 75 (40.2%) had certain AMD. The following categories were identified: Unremarkable (39.7%), Questionable (11.4%), Early-Intermediate AMD (22.8%), Atrophic (7.6%), Neovascular (9.8%), Other (8.7%), and Unknown/Not Recorded/Not Gradable (<1%). Figure 2,

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Using a population-based screening approach during a 16 month period in the pre-COVID era, it was possible to procure 75 donor eyes with AMD. All were recovered with a short DtoP and staged using OCT-anchored MMI. The age criterion (>80 years) is outside the typical age range for tissue recoveries intended for transplantable corneas. Despite the advanced age, our criteria resulted in eyes at all stages of AMD. Many RPE phenotypes are common to all AMD stages, and some are exclusive to neovascular AMD

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We thank Heidelberg Engineering for the instrumentation and the design of the original eye holder, Richard F. Spaide MD for the introduction to OCT-based multimodal imaging, Christopher Girkin MD for facilitating access to clinical imaging devices, and David Fisher for Figure 1. The recovery of the human donor eyes for research was supported by National Institutes of Health (NIH) grants R01EY06019 (C.A.C.), P30 EY003039 (Pittler), R01EY015520 (Smith), R01EY027948 (C.A.C., T.A.) R01EY030192 (Li), R01EY031209 (Stambolian), and U54EY032442 (Spraggins), IZKF Würzburg (N-304, T.A.), the EyeSight Foundation of Alabama, the Intern....

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NameCompanyCatalog NumberComments
Beakers, 250 mLFisher# 02-540K
Bottles, 1 L, Pyrex Fisher# 10-462-719storage for preservative
Bunsen burner or heat sourceEisco# 17-12-818To melt wax
Camera, digitalNikon D7200D7200
Computer and storageAppleiMac Pro; 14 TB external hard driveImage storage
Container, insulatedFisher# 02-591-45For wet ice
Containers, 2 per donor, 40 mLFisherSameco Bio-Tite  40 mL # 13-711-86For preservative
Crucible, quartz 30 mLFisher# 08-072DHold globe for photography
Cylinder, graduate, 250 mLFisher# 08-549G
Disinfectant cleaning supplies
Eye holder with lens and mounting bracketcontact J. Messingerjeffreymessinger@uabmc.educustom modification of Heidelberg Engineering original design
Face Protection MasksFisher# 19-910-667
Forceps, Harmon FixRoboz # RS-8247
Forceps, Micro AdsonRoboz # RS-5232
Forceps, TissueRoboz# RS-5172
Glass petri dish, KimaxFisher# 23064
Gloves Diamond GripFisher# MF-300
Gowns GenProFisher# 19-166-116
Image editing softwareAdobePhotoshop 2021, Creative Suite
KimWipesFisher# 06-666
Lamps, 3 goosenecksSchott Imaging# A20800
Microscope, stereoNikonSMZ 1000for dissection
Microscope, stereoOlympus SZX9color fundus photography
Paraformaldehyde, 20% EMS# 15713-Sfor preservative; dilute for storage
pH meterFisher # 01-913-806
Phosphate buffer, Sorenson’s, 0.2 M pH 7.2 EMS# 11600-10
Ring flashB & H Photo VideoSigma EM-140 DG 
Ruby bead, 1 mm diameterMeller Optics# MRB10MD
Safety Glasses 3MFisher# 19-070-940
Scanning laser ophthalmoscopeHeidelberg EngineeringHRA2
Scissors, curved springRoboz# RS-5681
Sharps containerFisher# 1482763
Shutter cord, remoteNikonMC-DC2
Spectral Domain OCT deviceHeidelberg EngineeringSpectralis HRA&OCT
Stainless steel ball bearing, 25.4 mm diameterMcMaster-Carr# 9529K31
Tissue marking dye, blackCancer Diagnostics Inc# 0727-1
Tissue slicer bladesThomas Scientific# 6767C18
Trephine, 18-mm diameterStratis Healthcare# 6718L
TV monitor (HDMI) and cord for digital cameraB&H Photo VideoBH # COHD18G6PROBfor live viewing and remote camera display features
Wax, pink dentalEMS # 72670
Wooden applicatorsPuritan# 807-12

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