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. 2015 Sep;113:Blodi.

The Age-Related Macular Degeneration Complex: Linking Epidemiology and Histopathology Using the Minnesota Grading System (The Inaugural Frederick C. Blodi Lecture)

Timothy W Olsen 1, Alexander R Bottini 1, Pia Mendoza 1, Hans E Grossniklausk 1
PMCID: PMC5108344  PMID: 27895380

Abstract

Purpose

To describe the histopathologic findings of the four stages of age-related macular degeneration (AMD) as defined by the Age-Related Eye Disease Study (AREDS) using the Minnesota grading system (MGS).

Clinical Relevance

There are no animal models for AMD. Eye banks enable access to human tissue with AMD. The level of AMD (grades 1 through 4) as defined by AREDS is determined ex vivo using the MGS. The AREDS has the largest collection to date of prospectively gathered data on the natural history of AMD. Since the MGS uses the same clinical criteria as AREDS, the addition of histopathologic findings of graded tissue confirms important pathophysiology at each stage of AMD.

Methods

Four eye bank eyes were graded according to the MGS. Only the right eyes were dissected for phenotype grading. The fellow (left) eyes were fixed for histopathologic study. The eyes were serially sectioned (7 μm) through the macula. Individual slides were examined, and a two-dimensional reconstruction of the topography of the macula was created for each donor. Selected, unstained slides were used for immunohistochemical staining. In one donor, portions of tissue were obtained for transmission electron microscopic (TEM) processing.

Results

Donor 1 had a rare hard, nodular druse (MGS1). Donor 2 had intermediate confluent drusen (MGS2). Donor 3 had numerous intermediate drusen (MGS3) in the right eye. Histopathology of the fellow left showed basal laminar deposits (BLamD), soft drusen, and an area of occult choroidal neovascularization underlying the retinal pigment epithelium (RPE) with endothelial cells (CD31-positive). Donor 4 also had MGS 3 along with reticular pseudodrusen (RPD). Histologic and TEM examination demonstrated diffuse BLamD, thickening of Bruch’s membrane, hard drusen, and focal nodules underlying the RPE that corresponded to the RPD. EM examination demonstrated both BLamD and electron-dense material located just external to the elastic layer of Bruch’s membrane.

Conclusion

Eye bank eyes graded using the MGS serve as an important link to the phenotypic and epidemiologic data from the AREDS. Thus, the MGS serves as a system to study the histopathology at each stage of AMD to better understand the relevant pathophysiologic changes in disease progression.

INTRODUCTION AND BACKGROUND

Frederick C. Blodi, MD, was a legendary figure in ophthalmology. A trained ophthalmic pathologist, Dr Blodi served as Professor and Chairman of the Department of Ophthalmology and Visual Sciences at the University of Iowa from 1967 until 1983. Dr Blodi was born near Vienna, Austria, and first trained in Europe until World War II, when he was eventually imprisoned for activities against the Third Reich. Following liberation by the Allies, he moved to the United States and was married to his wife, Ottie, in 1946. Through years of hard work, Dr Blodi earned an international reputation as a leader in our field. This study was presented as the inaugural lecture in honor of Dr Frederick C. Blodi, his family, Barbara and Christopher Blodi, both ophthalmologists specializing in retina, and his legacy1 at the 151st meeting of the American Ophthalmologic Society.

Age-related macular degeneration (AMD) is a complex disorder in terms of the etiology, associated genetics, and the biologic and molecular pathways that are involved in its pathogenesis. AMD is a leading cause of irreversible blindness and is one of the causes of visual impairment in those persons over 50 years of age.2–12 Worldwide, the aging demographic is increasing both in developed countries and, rather dramatically, in developing countries. According to the United Nations, the percentage of the world’s population over the age of 60 will triple from 1950 to 2050.13

Brown and associates14,15 have convincingly demonstrated that when visual acuity is affected by AMD, there is significant impairment in the quality of life as measured by time trade-off utility analysis. Thus, AMD is common, it is increasing as our global population ages, and those affected have a very poor quality of life.

The best natural history data for AMD is found in the Age-Related Eye Disease Study (AREDS).16–20 Both AREDS and AREDS2 have study subjects, with ages ranging from 50 to 85 at enrollment, multiplied by years of observation, that total over 36,000 person-years.17,20,21 The original AREDS compared placebo treatment to the use of antioxidant vitamins with zinc. The study demonstrated the benefits of supplementation with oral antioxidant vitamins plus zinc for those individuals with clinical features consistent with AREDS grade 3 or greater.22 The clinical characterization and phenotype analysis were carefully classified using characteristic funduscopic features.23

In 2004, we described the Minnesota Grading System (MGS) using eye bank eyes to replicate the classification from AREDS.24 While the MGS is a noninterventional model for studying human AMD, the true disease is studied, as opposed to an animal model. Animal models have major deficiencies that include the lack of a true macula, an insufficient lifespan to see true aging changes that are relevant to humans, and the fact that most are created by genetic manipulation (ie, gene knockout). We have used the MGS to study detailed biochemical, proteomic, and molecular changes that occur in the macular tissues at each specific stage of AMD.25–34 The study of other detailed genetic or cellular mediated pathways in AMD are also possible using the MGS.35–37 We refer to the MGS as the only true model of AMD, albeit noninterventional.

Another advantage of the MGS is the ready availability of eye bank tissue and the existing systems for prompt procurement of fresh human tissue to use for biologic study. Since eye bank donor eyes are primarily used in corneal transplantation, posterior segments are often available for study, research, and further analysis. We have demonstrated support for an oxidative mechanism in the pathogenesis of AMD.26–31 Using the MGS at the subcellular level, we demonstrated the influence and impact of both mitochondrial protein changes and mitochondrial DNA damage that were present in more advanced stages of AMD.33,34 The mitochondrial DNA repair mechanisms, as reported by other laboratories, also confirmed our suspicion that the mitochondria play a central role in AMD progression.38,39

Herein, we seek to describe the histopathology of AMD tissue, graded using the MGS, to examine the macular anatomy of phenotypic features that correspond to the stereoscopic fundus images. The AREDS grading system defines what appears to be a common phenotypic pathway of macular aging that originates from multiple complex genetic polymorphisms, oxidative injury, mitochondrial damage, or other mechanisms. Defining the ultrastructure and histologic features of the MGS will help to enable yet another approach to studying this complex aging disorder.

METHODS

The Emory University Institutional Review Board approved the conduct of this study and determined that this project was exempt from human subjects or clinical investigation criteria.

We acquired human cadaveric eye tissue in cooperation with the staff at the Lions Eye Institute for Transplantation and Research (LEITR) in Tampa, Florida. All eyes were obtained in compliance with the rules and regulations that govern the procurement of donor tissue post mortem and with the expressed consent of the donor or donor family. A LEITR staff member identified eye bank donors with a known history of AMD. Both eyes from each donor were processed within 8 hours of death.

Both globes from each donor pair were cut circumferentially at the pars plana to remove the anterior segment and enable an unobstructed view of the macula and peripheral retina. A 1000-μm-diameter ruby sphere was placed over the optic nerve head for size reference,24 and high-resolution color images of the optic nerve and macula were taken using a dissecting microscope (Nikon SMZ1500) with a digital camera (Nikon DS-Fi1).24 The neurosensory retina of the right eye from each pair was dissected and removed to expose the underlying retinal pigment epithelium (RPE). The macula and optic nerve of the right eye were then viewed using stereoscopic images with direct, tangential, indirect, and transscleral illumination.

The acquired digital images were processed digitally using Adobe Illustrator software (CS6 version 16.0.0). A modified grading template from the AREDS was centered on the fovea, with size reference to the 1000-μm-diameter ruby sphere, then superimposed on the images to facilitate grading according to the MGS.24

The images from the right eye only were graded (by author T.W.O.) both before and after dissection and removal of the neurosensory retina, and the MGS grade for the pair was based on the right eye only. The fellow (left) eye was assumed to be at or near the same grade. The presence of reticular pseudodrusen, calcified drusen, and basal laminar drusen was also noted. In the fellow (left) eye, any specific abnormalities of the macula, as viewed through a more opaque, undissected neurosensory retina, were noted.

The entire left globe was placed in 10% formalin, and the right eye was snap frozen in liquid nitrogen. The left eye specimens were then sent to the L. F. Montgomery Ocular Pathology Laboratory at Emory University for histopathologic processing and evaluation, whereas tissues from the right eye were dissected and stored at −80°C. The left eye was removed from the 10% formalin, dehydrated through graded alcohols and xylene, then embedded in paraffin. Serial 7-μm horizontal sections were cut through the entire region of the macula and mounted on glass slides. Following a repeating sequence, three of the sections were stained using hematoxylin-eosin, periodic acid–Schiff, and Masson trichrome. Two of the sections were left unstained for future use. For each eye, approximately 600 to 700 slides were processed.

Two-dimensional reconstruction of the histopathologic features of the left eyes was performed according to methods described previously.40,41 Slides were reviewed on an Olympus BH-2 microscope. A scaled measuring reticle was calibrated through the 4×, 10×, and 40× objectives using a hemocytometer for reference. Each slide was carefully examined for the presence of pathologic features. The presence or absence of drusen was noted and mapped, and drusen were assessed as hard or soft. Any peripapillary drusen, which can accumulate around the optic nerve head, were also noted. Pigment abnormalities, including hypopigmentation or hyperpigmentation of the RPE, as well as geographic atrophy, were recorded and mapped. Photoreceptor damage was assessed. Bruch’s membrane was reviewed for areas of thickening or discontinuity as well as for basal laminar deposits. When present, choroidal neovascularization (CNV) was noted as well as the CNV category—type I (sub-RPE), type II (trans-RPE), or both. Finally, any apparent disciform scarring was noted.

When a pathologic feature was discovered, the relative location as compared to the temporal edge of the optic nerve head was determined using a measuring reticle. AMD features were plotted with the number of the serial section along the y-axis data and the distance from the optic nerve edge on the x-axis data. For each slide, data and measurements were stored to a spreadsheet and graphed using Plot2 (Version 2.0) and then edited using Image J (Version 1.48). Graphs were rotated in order to place the fovea into a correct anatomical position relative to the optic nerve. On the 2-dimensional (2D) graphs, major grid lines represent 2000 μm and minor grid lines represent 500 μm.

When CNV was noted on histopathologic review, unstained sections adjacent to areas of CNV were sent for immunohistochemical staining. Sections were transferred to Emory Medical Laboratory services, where staining was done with CD68 (a macrophage marker), CD31 (an endothelial cell marker), CD3 (a T-cell marker), and CD20 (a B-cell marker).

RESULTS

MGS TISSUE GRADING

The four donors, three female and one male, were evaluated. The donors, referred to as donor 1, 2, 3, and 4, were 64, 83, 90, and 79 years of age, respectively, and globes were processed within 8 hours from the time of death. The MGS grades of the four donors’ right eyes were MGS 1, 2, 3, and 3, respectively (Figures 1 through 4). Donor 3 was later found to have MGS level 4. The drusen in the right eye for donor 3 were best visualized using the transillumination mode (Figure 3, top center). The undissected fellow (left) macular image from donor 3 (Figure 3, top right) has an unusual peripapillary opacity combined with hard exudates that were present near the fovea; these findings were suggestive of CNV, but not confirmatory. In donor 4, reticular pseudodrusen were noted in the dissected images of the right eye (Figure 4, top left), best detected in the superior and temporal outer grid fields.

FIGURE 1.

FIGURE 1

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Donor 1. Top left, Color postmortem fundus image following dissection and removal of the neurosensory retina with an overlying grid template. A ruby sphere is located over the optic nerve as a size reference. No drusen are seen or detected within the grading template. Therefore, this eye was graded as MGS 1. Artifactual pigmentary disturbance surrounds the optic nerve. Top right, Section is through the left macula with artifactual separation of the retinal pigment epithelial (RPE) cells from underlying Bruch’s membrane (hematoxylin-eosin, ×25). Bottom left, Section taken elsewhere in the macula demonstrates a single, small hard druse (arrow) that disrupts the RPE cell layer. Note that some of the RPE cells are adherent to the neurosensory retina, and other segments remain attached to the underlying Bruch’s membrane (periodic acid–Schiff, ×25). Bottom right, A 2-dimensional mapping of the left macula. On the grid, each major hash mark indicates 2000 μm and each smaller hash mark 500 μm. The optic nerve is the shaded image to the left (grey) with peripapillary atrophy (turquoise), and the small circle represents the center of the fovea. A single small hard druse is noted (red).

FIGURE 2.

FIGURE 2

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Donor 2. Top left, Color postmortem fundus image following dissection and removal of the neurosensory retina with an overlying grid template. A ruby sphere is located over the optic nerve as a size reference. Rare drusen are detected within the grading template. Note the pigmentary changes present in the superior and temporal portions of the grading template. This pair was graded as MGS 2. Artifactual pigmentary disturbance is seen surrounding the optic nerve and also in the center and inner temporal grading subfields. These are known artifacts because they were not present prior to dissection of the neurosensory retina. Top right, Section taken through the foveal center demonstrates intact retinal pigment epithelium (RPE) and Bruch’s membrane with artifactual detachment of the neurosensory retina (periodic acid–Schiff, ×25). Middle left, Section taken from elsewhere in the macula demonstrating confluent drusen (darker blue) between the intact RPE cell layer (arrow) and Bruch’s membrane (arrowheads) (Masson trichrome, ×100). Middle right, Section taken in the parafoveal region demonstrates a pigmented choroidal nevus (dark cells in the choroid) (periodic acid–Schiff, ×25). Bottom, A 2-dimensional mapping of the left macula. On the grid, each major hash mark indicates 2000 μm and each smaller hash mark 500 μm. The optic nerve is the shaded image to the left (grey) with peripapillary atrophy (turquoise), and the small circle represents the center of the fovea. There is a small area of drusen (green), consistent with MGS 2, and the small choroidal nevus (brown) is also mapped.

FIGURE 3.

FIGURE 3

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Donor 3. Top left, Color postmortem fundus image following dissection and removal of the neurosensory retina with an overlying grid template. A ruby sphere is located over the optic nerve as a size reference. Several intermediate drusen are detected within the grading template. Note the area of depigmentation present in the nasal, outer subfield of the grading template. Therefore, this pair was graded as MGS 3. Top center, Color postmortem, transillumination image of image at left. Note the easier-to-detect drusen and pigmentary changes in the macula. The grading template has been removed. The transillumination image supports the numerous intermediate drusen consistent with MGS 3. Top right, Color postmortem fundus image prior to the histopathologic analysis of the eye. There is an overlying grid template. A ruby sphere is located over the optic nerve as a size reference. Hard exudates are present in the nasal inner and outer subfields. Also, the peripapillary opacification that appears to be present under the retinal vessels is suggestive of a peripapillary choroidal neovascular lesion. Middle row left, Section is through the left fovea of donor 3 with artifactual separation of the neurosensory retina from underlying Bruch’s membrane. Endothelial-lined vascular channels filled with erythrocytes (arrow) are located between an intact retinal pigment epithelial (RPE) cell layer and Bruch’s membrane (hematoxylin-eosin, ×25). Middle row center, Section is through the left macula with a higher-power view of the image seen at left and also supporting the presence of endothelial-lined channels between the RPE (arrows) and Bruch’s membrane (arrowheads) (hematoxylin-eosin, ×100). Middle row right, Channels between the RPE and Bruch’s membrane stain positively for CD31 (arrows) (CD31 stain, ×100). Bottom, A 2-dimensional mapping of the left macula. On the grid, each major hash mark indicates 2000 μm and each smaller hash mark 500 μm. The optic nerve is the shaded image (grey) to the left and the small circle represents the center of the fovea. There is an extensive, subfoveal choroidal neovascular complex (red) with breaks in Bruch’s membrane (black), and basal laminar deposits (turquoise) surround this area. A disciform scar is present (purple) near the superior border of the optic nerve and suggests a peripapillary choroidal neovascular process.

FIGURE 4.

FIGURE 4

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Donor 4. Top left, Color postmortem fundus image following dissection and removal of the neurosensory retina with an overlying grid template. A ruby sphere is located over the optic nerve as a size reference. Numerous, ill-defined, reticular yellowish drusen are detected in the outer rings of the template, especially superior and temporal. There are numerous indistinct intermediate and some small hard drusen. Therefore, this pair was graded as MGS 3 with reticular pseudodrusen. Top middle, Section taken through the fovea of the left eye. There is artifactual detachment of the neurosensory retina and a small segment of the retinal pigment epithelial (RPE) cell layer. Thickening of Bruch’s membrane is present through the fovea (arrows) (periodic acid–Schiff, ×25). Top right, A higher-power section taken through the left macula of the image at left shows thickening of Bruch’s membrane that extends down between the vessels of the choriocapillaris and in some areas appears to encase the vessels of the choriocapillaris (periodic acid–Schiff, ×100). Bottom left (arrow heads), Section is through another area of the left fovea of the image at top middle (arrows) showing a small nodular druse (arrow) (hematoxylin-eosin, ×100). Bottom middle, Section through the macula from an area of reticular pseudodrusen. The RPE monolayer appears intact. In addition to basal laminar deposits (***) there is electron-dense material (^^^) causing thickening of the pillars of Bruch’s membrane (white arrow) as it extends toward the choriocapillaris (transmission electron microscopy, ×1900). Bottom right, A 2-dimensional mapping of the left macula. On the grid each major hash mark indicates 2000 μm and each smaller hash mark 500 μm. The optic nerve is the shaded image (grey) with peripapillary drusen (turquoise) to the left, and the small circle represents the center of the fovea. Extensive patchy or reticular areas of thickening of Bruch’s membrane (blue) are seen along with a few hard drusen (brown).

HISTOPATHOLOGY AND ELECTRON MICROSCOPY

Donor 1 had mostly normal histologic and anatomical findings with an artifactual detachment of the RPE from Bruch’s membrane (Figure 1, top right). There was a single small druse (Figure 1, bottom left) in the macula, creating a slight disruption of the RPE layer.

Donor 2 also had normal anatomy in the macula (Figure 2, top right) with an area of confluent drusen seen elsewhere in the macula (Figure 2, middle left). Incidentally, a choroidal nevus was also detected in the macula (Figure 2, middle right).

Donor 3 had a thin layer of CNV that extended into the subfovea (Figure 3, middle row left). On higher-power images, the small, blood-filled neovascular channels were seen between Bruch’s membrane and the RPE layer (Figure 3, middle row center). Choroidal neovascular endothelium was positively stained for the endothelial cell marker CD31 (Figure 3, middle row right).

Donor 4 was noted to contain reticular pseudodrusen. Histopathologic evaluation in the macular area showed thickened areas of Bruch’s membrane (Figure 4, top center). A higher-power view demonstrated thickening of Bruch’s membrane that was present between the vessels of the choriocapillaris and appeared to encase the vessels (Figure 4, top right), whereas in other locations, nodular areas of thickening emerged (Figure 4, bottom left). On transmission electron microscopy, the RPE cells appear intact with basal laminar deposits located between the basement membrane of the RPE and Bruch’s membrane. Additionally, there are areas of electron-dense material external to the elastic layer of Bruch’s membrane with wide-spaced collagen and vacuoles.

TWO-DIMENSIONAL MAPPING

Donor 1 had an unremarkable map with only a single, small area of hard drusen noted (Figure 1, bottom right).

Donor 2 had slightly more soft drusen detected, which is consistent with MGS level 2 and also with AREDS grading. A nevus is mapped as an incidental finding (Figure 2, bottom).

Donor 3 had more extensive findings that were mapped in Figure 3, bottom. The findings in the left eye revealed subfoveal CNV that elevated the MGS 3 grade from the right eye to MGS 4 in the left. In AREDS, the pair would be graded as AREDS 4; thus the normal eye is at high risk for progression. This type of disparity in grading related to both laterality reinforces the importance of seeing the bare RPE in order to determine the proper grade for the pair. There were complex areas of CNV as delineated, defects detected in Bruch’s membrane, basal laminar deposits, and disciform scarring.

Donor 4 had areas of hard drusen detected in a small surface area combined with a more extensive area of thickening in Bruch’s membrane that corresponded to the reticular pseudodrusen, and changes were mapped (Figure 4, bottom right).

DISCUSSION

In the study of AMD, animal models are simply inadequate to accurately examine the complexities and pathogenic mechanisms involved. Most animals do not have a macula, most do not live long enough to manifest AMD features, and most of the detailed clinical phenotype of AMD has not been accurately replicated.

The AREDS has a very extensive, prospectively collected database on eyes with AMD that have been followed for over 10 years.18,19,22,42,43 The MGS enables the study of eyes that are graded according to funduscopic features described by the AREDS.23 Thus, the MGS donor tissue represents a helpful model to assist us in examining biochemical, molecular, and histopathologic features in addition to the ultrastructural aspects of AMD. Also, by applying the epidemiologic data from AREDS, the risk of progression from a particular phenotype as detected on MGS grading of eye bank tissue can be accurately risk-assessed.

Many current studies examine eyes with AMD, yet do not accurately classify the stage of AMD. In such studies, conclusions may apply to early, mid, or later stages of AMD. Post mortem, the retina becomes opaque and limits a detailed analysis of the features required to accurately grade the tissue. A more careful evaluation of the macula prior to histologic, biochemical, or molecular analysis is critical for understanding the mechanism involved at each specific stage of AMD. This study used carefully graded tissue from one eye (right) of a donor pair; tissue from the fellow (left) eye was used for careful histopathologic analysis, 2D mapping of the pathology, and in one case, electron microscopy to study the ultrastructure.

We have collected four donor pairs, each representing the four levels of AMD and graded according to the MGS.24 In the AREDS, eyes with grades 1 and 2 did not benefit from the use of antioxidant vitamins with zinc and also had a low risk for transition to more advanced vision loss.22 However, AREDS grade 3 eyes were at much higher risk for progression. The details of progression are largely based on drusen area, pigmentary changes, and the presence of noncentral geographic atrophy.42 In donor eyes that had MGS grade 1 or 2, we were able to identify minor changes in the macula with the appearance of a few drusen (see section on “Two-dimensional Mapping”) Otherwise, the histologic features of these eyes were essentially normal. The low risk of MGS 1 and 2 is consistent and well supported in our proteomic studies that use the MGS system.25,26,28–31 In addition, the minimal changes noted in the mitochondrial DNA at MGS 1 and 2, along with the status of the mitochondrial DNA repair mechanisms, are also supportive.33,38,39

On the other hand, we found that donors 3 and 4 had more profound alterations in the macula of the fellow eye. Donor 3 was determined to be MGS 3 based on extensive intermediate drusen, corresponding to higher-risk AREDS 3. The histopathology of the fellow eye revealed extensive choroidal neovascularization along with the presence of a type 1 (sub-RPE) occult subfoveal CNV. Looking at the undissected fellow left eye (Figure 3, top right), there is the suspicion of CNV based on the presence of an opaque, subretinal lesion and hard exudates near the fovea. Therefore, the pair would be considered to be MGS4, as the pair is graded based on the higher level of the two eyes. During histopathologic examination, we found sub-RPE CNV present with confirmed endothelial-lined channels located above Bruch’s membrane. The CNV extends from a peripapillary region into the subfoveal region. This CNV complex is also associated with numerous discontinuities of Bruch’s membrane (Figure 3, middle row center), noted on 2D mapping, a feature that is consistent with the fluorescein angiographic appearance of an occult CNV.40

Donor 4 is also very interesting (MGS 3 with high-risk drusen), based largely on the presence of reticular pseudodrusen. This clinical feature is associated with a high risk for advancement to end-stage AMD.44 Multimodal imaging, as well as histopathologic studies of pseudodrusen, suggests that the deposits are located in the subretinal space and at the apex of the RPE and are also referred to as subretinal drusenoid deposits.45–48 While the single case presented in this series is inadequate to make definitive statements, at least the histologic findings suggest that the abnormality is located at the level of Bruch’s membrane. Although we were not able to detect subretinal deposits in our case, it’s possible that the tissue processing was insufficient to preserve the deposits or simply that they were not present. Curcio and associates46 did not use the MGS system, and their macular images of opaque retina had the appearance of pseudodrusen, yet such changes may also represent postmortem changes in the neurosensory retina. A reticular pseudodrusen appearance has also been associated with pseudoxanthoma elasticum as well as Sorsby fundus dystrophy; both suggest the pathology would be located at the level of damaged Bruch’s membrane, rather than in the subretinal space.49,50 Interestingly, in 1976, Sarks15 reported the results of 378 eyes studied by pathologic examination and suggested that thickening of Bruch’s membrane with encroachment on the choriocapillaris with eventual hyalinization extending into the intercapillary pillars was a prominent feature. Such changes described in Sarks’ work suggest that thickening and sclerosis of Bruch’s membrane lead to more advanced AMD. These changes are nearly identical to the histologic findings in donor 4 (Figure 4, top middle and right).

CONCLUSION

Dr Frederick Blodi was an accomplished ocular pathologist, and we have chosen to dedicate this work to him. Our study demonstrates a new methodology that incorporates histopathology to the MGS and thus linked to the AREDS to improve our understanding of the pathogenesis of AMD by using human donor eye bank eyes. We believe that this is the best model to study the pathogenesis of the human condition of AMD and leverages the natural history of AMD from the AREDS. We hope that future analysis, with additional cases and subsequent histopathology of various features seen on MGS grading, will help to enhance our understanding of the pathogenesis of AMD.

ACKNOWLEDGMENTS

Funding/Support: This work was supported by NEI RO1 EY022097, NIA RO1 AG025392, and a departmental core NEI P30 EY006360, all from National Institutes of Health; The Lions Institute Transplantation and Research (LEITR), Tampa Bay, Florida; and an unrestricted departmental grant from Research to Prevent Blindness, New York, New York.

Financial Disclosures: None.

Author Contributions: Design of the study (T.W.O., A.R.B., H.E.G.); Conduct of the study (T.W.O., A.R.B., P.M., H.E.G.); Collection of data (T.W.O., A.R.B., P.M., H.E.G.); Study management (T.W.O.); Data analysis (T.W.O., A.R.B., P.M., H.E.G.); Data interpretation (T.W.O., A.R.B., P.M., H.E.G.); Manuscript preparation (T.W.O., A.R.B.); Manuscript review (T.W.O., A.R.B., H.E.G.); Manuscript approval (T.W.O.).

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