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&#160;EY003039 (Pittler), R01EY015520 (Smith), R01EY027948 (C.A.C., T.A.) R01EY030192 (Li), R01EY031209 (Stambolian), and U54EY032442 (Spraggins), IZKF Wu&#776;rzburg (N-304, T.A.), the EyeSight Foundation of Alabama, the International Retinal Research Foundation (C.A.C.), the Arnold and Mabel Beckman Initiative for Macular Research (C.A.C.), and Research to Prevent Blindness AMD Catalyst (Schey).

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...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

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 (&#62;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<sup class="xref"...

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 neovascularization (type 3 macular neovascularization1, formerly angiomatous proliferation), subretinal drusenoid deposits (SDDs, also called reticular pseudodrusen)2, multiple pathways of RPE fate3,4, and intensely gliotic M&#252;ller cells in atrophy5,6.
Model systems lacking maculas (cells and animals) recreate some slices of this complex disease7,8,9. Further success in ameliorating the burden of AMD could come from the discovery and exploration of primary pathology in human eyes, understanding the unique cellular composition of the macula, followed by translation to model systems. This report portrays a three decade collaboration between an academic research laboratory and an eye bank. The goals of the tissue characterization methods described herein are two-fold: 1) to inform evolving diagnostic technology by demonstrating the basis of fundus appearance and imaging signal sources with microscopy, and 2) to classify AMD specimens for targeted (immunohistochemistry) and untargeted molecular discovery techniques (imaging mass spectrometry, IMS, and spatial transcriptomics) that preserve the cone-only fovea and rod-rich para- and perifovea. Such studies could accelerate the translation to clinical OCT, for which a progression sequence and longitudinal follow-up are possible through eye-tracking. This technology, which is designed to monitor treatment effects, registers scans from one clinic visit to the next using retinal vessels. Linking eye-tracked OCT to laboratory results obtained with destructive techniques could provide a new level of prognostic value to molecular findings.
In 1993, the research laboratory captured color photographs of postmortem fundus on film10. This effort was inspired by the superb photomicroscopy and histology of the human peripheral retina by Foos and colleagues11,12,13&#160;and the extensive AMD clinicopathologic correlations by Sarks et al.14,15. Starting in 2009, ex vivo multimodal imaging (MMI) anchored on spectral domain OCT was adopted. This transition was inspired by the similar efforts of others16,17&#160;and especially by the realization that so much of the ultrastructure described by the Sarks was available in three dimensions, over time, in the clinic18,19. The goal was to acquire eyes with attached maculas in a reasonable time frame for well-powered studies of cellular-level phenotypes in the retina, RPE, and choroid. The intent was to move beyond &#34;per eye&#34; statistics to &#34;per lesion type,&#34; a standard influenced by the &#34;vulnerable plaque&#34; concepts from cardiovascular disease20,21.
The protocol in this report reflects experience with nearly 400 pairs of donor eyes accessioned in several streams. In 2011-2014, the Project MACULA website of AMD histopathology was created, which includes layer thicknesses and annotations from 142 archived specimens. These eyes were preserved from 1996-2012 in a glutaraldehyde-paraformaldehyde fixative for high-resolution epoxy-resin histology and electron microscopy. All the fundi had been photographed in color when received and were reimaged by OCT just prior to histology. An eye holder originally designed for optic nerve studies22&#160;was used to accommodate an 8 mm diameter full-thickness tissue punch centered on the fovea. OCT B-scans through the foveal center and a site 2 mm superior, corresponding to histology at the same levels, were uploaded to the website, plus a color fundus photograph. The choice of the OCT planes was dictated by the prominence of AMD pathology under the fovea23&#160;and the prominence of SDDs in rod-rich areas superior to the fovea24,25.
Starting in 2013, eyes imaged with OCT-anchored MMI during life were available for direct clinicopathologic correlations. Most (7 of 10 donors) involved patients at a retina referral practice (author: K.B.F.), which offered an advanced directive registry for patients interested in donating their eyes after death for research purposes. The eyes were recovered and preserved by the local eye bank, transferred to the laboratory, and prepared in the same way as the Project MACULA eyes. Pre-mortem clinical OCT volumes were seamlessly read in the laboratory, thus aligning the pathology features seen during life with the features seen under the microscope26.
Starting in 2014, prospective eye collection began by screening for AMD in donor eyes without a clinical history but preserved during a defined time limit (6 h). For this purpose, the eye holder was modified to accommodate a whole globe. This reduced the chance of detachment around the cut edges of the previously used 8 mm punch. The eyes were preserved in 4% buffered paraformaldehyde for immunohistochemistry and transferred to 1% the next day for long-term storage. In 2016-2017 (pre-pandemic), 184 eyes from 90 donors were recovered. The statistics and images in this report are generated from this series. During the pandemic era (2020 lockdowns and aftermath), prospective collections for transcriptomics and IMS collaborations continued at a reduced pace, essentially using the 2014 methods.
Other methods for donor eye assessment are available. The Minnesota Grading System (MGS)27,28&#160;is based on the AREDS clinical system for color fundus photography29. The limitations of this method include the combining of atrophic and neovascular AMD into one stage of &#34;late AMD&#34;. Further, the MGS entails the removal of the neurosensory retina before the photo-documentation of the RPE choroid. This step dislodges SDDs to varying degrees30,31&#160;and removes the spatial correspondence of the outer retina and its support system. Thus, efforts to link metabolic demand and signaling from the retina to pathology in the RPE-choroid may be impeded. The Utah System implemented MMI using ex vivo color photography and OCT to categorize eyes destined for dissection into regions for RNA and protein extractions32. Although preferable to whole eyecup extractions, the 3 mm diameter area at the highest risk for AMD progression33,34&#160;represents only 25% of a 6 mm diameter fovea-centered punch. Thus, techniques that can localize findings in reference to the fovea, such as serial sectioning for immunohistochemistry, are advantageous.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beakers, 250 mL</td>
			<td>Fisher</td>
			<td># 02-540K</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottles, 1 L, Pyrex&#160;</td>
			<td>Fisher</td>
			<td># 10-462-719</td>
			<td>storage for preservative</td>
		</tr>
		<tr>
			<td>Bunsen burner or heat source</td>
			<td>Eisco</td>
			<td># 17-12-818</td>
			<td>To melt wax</td>
		</tr>
		<tr>
			<td>Camera, digital</td>
			<td>Nikon D7200</td>
			<td>D7200</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer and storage</td>
			<td>Apple</td>
			<td>iMac Pro; 14 TB external hard drive</td>
			<td>Image storage</td>
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			<td>Container, insulated</td>
			<td>Fisher</td>
			<td># 02-591-45</td>
			<td>For wet ice</td>
		</tr>
		<tr>
			<td>Containers, 2 per donor, 40 mL</td>
			<td>Fisher</td>
			<td>Sameco Bio-Tite&#160; 40 mL # 13-711-86</td>
			<td>For preservative</td>
		</tr>
		<tr>
			<td>Crucible, quartz 30 mL</td>
			<td>Fisher</td>
			<td># 08-072D</td>
			<td>Hold globe for photography</td>
		</tr>
		<tr>
			<td>Cylinder, graduate, 250 mL</td>
			<td>Fisher</td>
			<td># 08-549G</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfectant cleaning supplies&#160;&#160;</td>
			<td></td>
			<td></td>
			<td>https://www.cardinalhealth.com/en/product-solutions/medical/infection-control/antiseptics.html</td>
		</tr>
		<tr>
			<td>Eye holder with lens and mounting bracket</td>
			<td>contact J. Messinger</td>
			<td>jeffreymessinger@uabmc.edu</td>
			<td>custom modification of Heidelberg Engineering original design</td>
		</tr>
		<tr>
			<td>Face Protection Masks</td>
			<td>Fisher</td>
			<td># 19-910-667</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Harmon Fix</td>
			<td>Roboz&#160;</td>
			<td># RS-8247</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Micro Adson</td>
			<td>Roboz&#160;</td>
			<td># RS-5232</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Tissue</td>
			<td>Roboz</td>
			<td># RS-5172</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass petri dish, Kimax</td>
			<td>Fisher</td>
			<td># 23064</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves Diamond Grip</td>
			<td>Fisher</td>
			<td># MF-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Gowns GenPro</td>
			<td>Fisher</td>
			<td># 19-166-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Image editing software</td>
			<td>Adobe</td>
			<td>Photoshop 2021, Creative Suite</td>
			<td></td>
		</tr>
		<tr>
			<td>KimWipes</td>
			<td>Fisher</td>
			<td># 06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamps, 3 goosenecks</td>
			<td>Schott Imaging</td>
			<td># A20800</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, stereo</td>
			<td>Nikon</td>
			<td>SMZ 1000</td>
			<td>for dissection</td>
		</tr>
		<tr>
			<td>Microscope, stereo</td>
			<td>Olympus&#160;</td>
			<td>SZX9</td>
			<td>color fundus photography</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 20%&#160;</td>
			<td>EMS</td>
			<td># 15713-S</td>
			<td>for preservative; dilute for storage</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher&#160;</td>
			<td># 01-913-806</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer, Sorenson&#8217;s, 0.2 M pH 7.2&#160;</td>
			<td>EMS</td>
			<td># 11600-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring flash</td>
			<td>B &#38; H Photo Video</td>
			<td>Sigma EM-140 DG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruby bead, 1 mm diameter</td>
			<td>Meller Optics</td>
			<td># MRB10MD</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Glasses 3M</td>
			<td>Fisher</td>
			<td># 19-070-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning laser ophthalmoscope</td>
			<td>Heidelberg Engineering</td>
			<td>HRA2</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, curved spring</td>
			<td>Roboz</td>
			<td># RS-5681</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharps container</td>
			<td>Fisher</td>
			<td># 1482763</td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter cord, remote</td>
			<td>Nikon</td>
			<td>MC-DC2</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral Domain OCT device</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA&#38;OCT</td>
			<td>https://www.heidelbergengineering.com/media/e-learning/Totara-US/files/pdf-tutorials/2238-003_Spectralis-Training-Guide.pdf</td>
		</tr>
		<tr>
			<td>Stainless steel ball bearing, 25.4 mm diameter</td>
			<td>McMaster-Carr</td>
			<td># 9529K31</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue marking dye, black</td>
			<td>Cancer Diagnostics Inc</td>
			<td># 0727-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer blades</td>
			<td>Thomas Scientific</td>
			<td># 6767C18</td>
			<td></td>
		</tr>
		<tr>
			<td>Trephine, 18-mm diameter</td>
			<td>Stratis Healthcare</td>
			<td># 6718L</td>
			<td></td>
		</tr>
		<tr>
			<td>TV monitor (HDMI) and cord for digital camera</td>
			<td>B&#38;H Photo Video</td>
			<td>BH # COHD18G6PROB</td>
			<td>for live viewing and remote camera display features</td>
		</tr>
		<tr>
			<td>Wax, pink dental</td>
			<td>EMS&#160;</td>
			<td># 72670</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden applicators</td>
			<td>Puritan</td>
			<td># 807-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo oct-based multimodal imaging of human donor eyes for research into age-related macular degeneration

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 &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;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&#176; macula cube, 30 &#181;m 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 &plusmn; 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.

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

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

This protocol enables new treatments and preventions for age-related macular degeneration or AMD. This is the largest disease with a central neurodegeneration and yet we think it's the most approachable because of the precise structure of the eye and the availability of cellular-level clinical imaging of the retina. The neovascular complications of the underlying disease of AMD have been treated successfully for 15 years in the 15%of patients who have it.Recently, the FDA approved the first drug for the end stage of the underlying disease. Single-cell RNA sequencing has revealed the molecular repertoire of the 100-plus cell types in the retina and supporting choroidal vasculature. Eye-tracked optical coherence tomography or OCT has enabled us to glimpse a detailed cellular-level progression sequence in the clinic.Existing cell and animal model systems for AMD research are non-predictive of human pathology and progression risk. This impedes the development of new treatments and preventions. Our lab contributed many top-level findings about AMD, including the early loss of broad photoreceptors required for night vision.It was also observed that better preserved maculas with attached neurosensory retinas are directly relevant to clinical OCT imaging. Many parts of AMD pathology became visible with clinical OCT which shows cross-sectional views of the retina and choroid. The OCT results of human donor eyes can be directly compared to findings in clinical imaging.If laboratories could emulate clinical imaging standards by using OCT to characterize eyes before assays and laboratory findings like gene expression or proteomics and leverage the longitudinal timeline of clinical imaging to focus on the most informative risk indicators.

Table 1 shows that during 2016-2017, 184 eyes from 94 white non-diabetic donors &#62;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 (&#60;1%). Figure 2, <strong class=...

Watch this Scientific Journal Video about Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration at JoVE.com

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 ...