Postmortem Donor Eyes Research Articles

Insights into human choroid melanocyte and stromal cells

Aims/Purpose: The human choroid is a complex highly vascular pigmented tissue between Bruch's membrane and the sclera. Its dynamic nature is important for eye homeostasis and for in pathology including macular degeneration and myopia. The stroma comprises heterogeneous cell populations including fibroblasts, melanocytes, immune cells, smooth muscle cells and neurons within a loose collagenous extracellular matrix. We used RNA transcriptome and multimarker immunolabelling of human choroidal melanocytes and tissue to further characterize adult choroidal melanocytes.Methods: Human choroidal melanocytes were cultured (n = 6 postmortem donor eyes, <18 h delay, UNSW HREC), and used between passage (P) 2 to 5 for RNA transcriptome and multimarker immunofluorescence labelling and confocal microscopy. Sections and flatmounts from central choroid were also compared. Immunolabelling included melanocyte (Tyrosinase, TRYP1, DCT, MelanA) and stromal cell (S1004A, vimentin, α‐smooth muscle actin, CD45) markers.Results: Cultured melanocytes displayed mostly bipolar morphology with heterogeneous eumelanin density related to donor eye iris colour. Whole transcriptome findings showed enriched melanogenesis genes (high level TYR, PMEL, MITF, TYRP1; MLANA; low level DCT1, MC1R) but not genes reported for RPE (e.g. RPE65), pericytes (e.g. ACTA2) or fibroblasts (e.g. IGF2) confirming melanocyte culture quality. We also found expression for proposed Schwann cell precursors PLP1 and SOX10. Immunolabelling confirmed overall expression of melanogenesis proteins, and subpopulations expressing PLP1 and Sox10, in culture and in choroidal tissue.Conclusions: Melanocytes and stromal fibroblast are among the most numerous and complex heterogeneous populations within the choroid and our study provides further details on characteristics markers and topography. A possible role for Schwann cell precursors in renewing adult melanocyte populations is intriguing and continues to be investigated.

Immunohistochemical features of cells in peripheral microcystoid retinal degeneration

Peripheral microcystoid retinal degeneration (PMD) is an age-related, benign condition in which the peripheral retina develops small holes and undergoes cystic degeneration. This paper demonstrates neuronal alterations in PMD, as studied by immunohistochemistry in postmortem donor eyes (age: 76–89 years; N = 6 donors). In all cases, the degeneration was located in the inferior temporal quadrant, creating holes in the far peripheral retina. There was thinning of the inner retinal layers and the outer plexiform layer (OPL) was patchy or inconspicuous. As a response, Müller cell processes showed increased vimentin immunoreactivity. None of the retinas examined expressed glial fibrillary acidic protein. Cone photoreceptor cells were significantly altered: compared to the adjoining cones that were short, those located in the cystoid retina underwent significant elongation of their inner segments, evident from calbindin immunolabeling, to maintain synaptic contacts with the remnant OPL. The latter consisted of small photoreceptor terminals and scanty processes from shrunken bipolar cells. Besides, cones and ganglion cells undergo oxidative stress, they showed immunoreactivity to 4-hydroxy 2-nonenal and nitrotyrosine. The level of superoxide dismutase-2 was relatively low in the PMD region than in adjacent area, suggesting that the former suffers from oxidative stress.

Evidence of complement dysregulation in outer retina of Stargardt disease donor eyes

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4−/−. These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype.

Histopathological changes in the anterior segment with anterior and posterior chamber intraocular lens

ObjectivePatients have shown a lowering of intraocular pressure (IOP) after cataract surgery. Histopathology studies have reported trabecular meshwork (TM) changes in pseudophakic eyes with posterior chamber intraocular lens (PCIOL) and have eluded to the mechanisms for IOP decrease. Unlike PCIOLs, TM histopathology changes after implantation of an anterior chamber intraocular lens (ACIOL) have not been studied, to our knowledge. Therefore, this study aims to examine the histopathological changes in both the TM and corneal endothelium among donor eyes with ACIOL, PCIOL, and phakic eyes. MethodsForty fixed postmortem donor eyes were obtained, sectioned, and embedded. Slides were stained with Masson's trichrome and CD31 vascular endothelial antibody, and further digitalized. Customized Medical Parachute TMAN software quantified the cellular components, the trabecular extracellular matrix (ECM), ECM fibrosis, and trabecular area. Schlemm's canal and corneal endothelium were quantified across the ACIOL, PCIOL, and phakic groups. ResultsCellular area component of the TM was lower in the ACIOLs and PCIOLs than in phakic eyes, but statistically significant only between PCIOL and phakic eyes (p = 0.0023). ECM area component, TM fibrosis score and TM lamellae area, ciliary process fibrosis, and CD31 expression in Schlemm's canal showed no differences (p = 0.40, 0.99, 0.10, 0.83, 0.45). Significantly lower corneal endothelial cells were seen in ACIOLs compared with both PCIOLs and phakic eyes (p = 0.0002). ConclusionsACIOLs and PCIOLs in our sample group showed that there is loss of cellular components in the TM compared with the phakic eyes, with PCIOLs displaying the least amount of TM cells statistically, in this cohort. The ACIOLs led to a greater loss of corneal endothelial cells than both PCIOLs and phakic eyes after cataract surgery. The endothelial cells in Schlemm's canal did not seem to be affected by the IOL placements. Therefore, this study illustrates that there are histopathological differences seen with the placements of ACIOLs in both TM and cornea.

Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.

Topography of Neurons in the Rod Pathway of Human Retina.

The objective of this study was to map the distribution and density of the three major components of the classical scotopic "night vision" pathway (rods, rod bipolar, and AII amacrine cells) in postmortem human retinas. Four postmortem donor eyes (male and female, aged 44-56 years) were used to cut vertical sections through the temporal horizontal meridian. The sections were processed for immunohistochemistry and imaged using high-resolution multichannel confocal microscopy. Rods, rod bipolar, and AII amacrine cells were counted along the temporal horizontal meridian. Two additional retinas were used for intracellular injections. Rod peak density is close to 150,000 cells/mm2 at 4 to 5 mm (15° to 20°) eccentricity, declining to below 70,000 cells/mm2 in peripheral retina. Rod bipolar density is lower but follows a similar distribution with peak density near 10,000 cells/mm2 between 2 and 4 mm (7° to 15°) eccentricity declining to below 4000 cells/mm2 in peripheral retina. The peak density of AII amacrine cells (near 4000 cells/mm2) is located close to the fovea, at 0.5- to 2 mm-eccentricity (2° to 7°) and declines to below 1000 cells/mm2 in the periphery. Thus, convergence between rods and AII cells increases from central to peripheral retina. Comparison with human psychophysics and ganglion cell density indicates that the spatial resolution of scotopic vision is limited by the AII mosaic at eccentricities below 15° and by the midget ganglion cell mosaic at eccentricities above 15°.

The Utah Protocol for Postmortem Eye Phenotyping and Molecular Biochemical Analysis.

PurposeCurrent understanding of local disease pathophysiology in AMD is limited. Analysis of the human disease-affected tissue is most informative, as gene expression, expressed quantitative trait loci, microenvironmental, and epigenetic changes can be tissue, cell type, and location specific. Development of a novel translational treatment and prevention strategies particularly for earlier forms of AMD are needed, although access to human ocular tissue analysis is challenging. We present a standardized protocol to study rapidly processed postmortem donor eyes for molecular biochemical and genomic studies.MethodsWe partnered with the Utah Lions Eye Bank to obtain donor human eyes, blood, and vitreous, within 6 hours postmortem. Phenotypic analysis was performed using spectral-domain optical coherence tomography (SD-OCT) and color fundus photography. Macular and extramacular tissues were immediately isolated, and the neural retina and retinal pigment epithelium/choroid from each specimen were separated and preserved. Ocular disease phenotype was analyzed using clinically relevant grading criteria by a group of four ophthalmologists incorporating data from SD-OCT retinal images, fundus photographs, and medical records.ResultsThe use of multimodal imaging leads to greater resolution of retinal pathology, allowing greater phenotypic rigor for both interobserver phenotype and known clinical diagnoses. Further, our analysis resulted in excellent quality RNA, which demonstrated appropriate tissue segregation.ConclusionsThe Utah protocol is a standardized methodology for analysis of disease mechanisms in AMD. It uniquely allows for simultaneous rigorous phenotypic, molecular biochemical, and genomic analysis of both systemic and local tissues. This better enables the development of disease biomarkers and therapeutic interventions.

Human choroidal melanocytes express functional Toll-like receptors (TLRs)

Toll-like receptors (TLRs) are a class of pattern recognition receptors that sense highly conserved pathogen associated antigenic determinants, triggering an innate immune response and subsequently instructing the adaptive immune system so that together, the pathogen can be eliminated. TLRs are widely distributed in human ocular tissues and cell types, and are active players in ocular inflammation. To date, the presence and function of TLRs on human choroidal melanocytes (HCMs), the most abundant choroidal cell type, have not been characterized. The current study investigated the in vitro and in situ expression and functional status of TLRs on HCMs. HCMs were isolated and cultured from post-mortem human donor eyes, and displayed characteristic melanocyte morphology and MART1 expression – a key melanocyte lineage marker up to passage 5 (P5). In vitro experiments used P1 to P4 HCMs from different donor eyes. Initial quantitative real-time PCR (qPCR) analysis revealed that HCMs (n = 3 donors) expressed specific mRNA transcripts for TLR1-10 and MYD88 (a key adaptor protein initiating the TLR signalling pathway). HCMs were stimulated with a set of synthetic TLR specific agonists and the secretion of pro-inflammatory cytokines, MCP-1 and IL-8, at 24 h measured by ELISA (n = 3 donors). The agonists Pam3CSK4 (TLR1/2), Poly I:C (TLR3), LPS (TLR4), Flagellin (TLR5), and FLS-1 (TLR2) induced a significant increase in the production of MCP-1 and IL-8, compared to untreated cells. Application of biotinylated Pam3CSK4 provided in vitro visualization of receptor-agonist interactions for TLR1/2. We confirmed that cultured HCMs (n = 3 donors) expressed TLR1-6 protein using immunocytochemistry and confocal microscopy. The expression and distribution of TLR 1–6 was also studied in human choroid and retinal pigment epithelium (RPE) sections (n = 3 eyes) using immunofluorescence and confocal microscopy. Strong TLR1-6 immunolabelling that co-localized with melanocyte-dense areas (and RPE) was consistently observed; intraluminal and blood vessel-related cells (including endothelial cells) also expressed several TLRs. Taken together these observations show for the first time that HCMs constitutively express a range of functional TLRs, and as such can contribute to choroidal responses during infection and inflammation.

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation

Background/aimsThe Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this...

Identification of AⅡ amacrine, displaced amacrine, and bistratified ganglion cell types in human retina with antibodies against calretinin.

Antibodies against calretinin are markers for one type of rod pathway interneuron (AⅡ amacrine cell) in the retina of some but not all mammalian species. The AⅡ cells play a crucial role in night-time (scotopic) vision and have been proposed as a target for optogenetic restoration of vision in retinal disease. In the present study we aimed to characterize the AⅡ cells in human retina. Postmortem human donor eyes were obtained with ethical approval and processed for calretinin immunofluorescence. Calretinin-positive somas in the inner nuclear and the ganglion cell layer were filled with the lipophilic dye DiI. The large majority (over 80%) of calretinin-immunoreactive cells is located in the inner nuclear layer, is immunopositive for glycine transporter 1, and shows the typical morphology of AⅡ amacrine cells. In addition, a small proportion of calretinin-positive cells in the inner nuclear layer and in the ganglion cell layer is glutamic acid decarboxylase-positive and shows the morphology of widefield amacrine cells (stellate, semilunar, and thorny amacrine cells). About half of the calretinin cells in the ganglion cell layer are bistratified ganglion cells resembling the small bistratified (presumed blue-ON/yellow-OFF) and the G17 ganglion cell previously described in primates. We conclude that in human retina, antibodies against calretinin can be used to identify AⅡ amacrine cells in the inner nuclear layer as well as widefield amacrine and small bistratified ganglion cells in the ganglion cell layer.

Activation of the Alternative Complement Pathway in Vitreous is Controlled by Genetics in Age-Related Macular Degeneration

To determine if the progression of age-related macular degeneration (AMD) is associated with complement activation in the eye. Immunohistochemistry and ELISAs were used to determine the distribution, concentration, and activation of the alternative pathway complement proteases factor B (FB) and factor D (FD) and the central complement protein C3 in genotyped human postmortem donor eyes graded as having no or minimal drusen (category 1; controls), large drusen (category 3), and large drusen with advanced AMD (category 4). C3, FB, and FD were present in vitreous and Bruch's membrane choroid (BM/C) interface of the macula of eyes in all tested AMD severity categories (n = 100). C3, FB, and FD were predominantly located to the choroidal vasculature and Bruch's membrane and, together with the serum proteins transferrin and albumin, elevated in BM/C extracts of category 4 eyes (n = 23) compared with category 1 eyes (n = 24). A significant increase in FB activation was found only in vitreous of category 4 eyes (n = 23) compared with category 1 eyes (n = 25). Genetic variants of complement factor H (CFH), C3, C2, and FB associated with increased risk of AMD were correlated with alternative pathway complement activation in vitreous, but not with complement proteins in BM/C protein extracts. Increased activation of the alternative complement pathway in vitreous was controlled by disease stage and genetic variation in the complement pathway, supporting a role for complement activation in AMD disease pathogenesis.

Histopathology and Functional Correlations in a Patient with a Mutation inRPE65, the Gene for Retinol Isomerase

Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.

Modulation of Nrf2-Dependent Antioxidant Functions in the RPE by Zip2, a Zinc Transporter Protein

To characterize the involvement of Zip2, a zinc transporter protein, in the antioxidant functions of cultured human retinal pigment epithelial (RPE) cells. The expression of zinc transporter proteins was determined by RT-PCR. Intracellular zinc concentration was assessed by staining with a zinc-sensitive dye followed by flow cytometry. Stable overexpression of the transporter protein Zip2 was achieved by transducing ARPE-19 cells with a retroviral vector containing the open reading frame of the human Zip2 gene. Activity of nuclear factor erythroid 2-related factor 2 (Nrf2) was measured using a dual luciferase assay after transient transfection of reporter plasmids containing the antioxidant response element (ARE). Glutamate-cysteine ligase (GCL) expression was measured by quantitative real-time RT-PCR. Cultured RPE cells could transport zinc with Zip2 as an influx transporter expressed in ARPE-19 cells and human RPE cells isolated from postmortem donor eyes. The mRNA level of Zip2 was influenced by intracellular and extracellular zinc concentrations. Overexpression of Zip2 resulted in increased Nrf2 activity, higher GCL expression, and increased glutathione synthesis. RPE cells can actively uptake zinc through the transporter Zip2, and the increased intracellular zinc upregulates the Nrf2-dependent antioxidant function.

Effect of bag-in-the-lens implantation on posterior capsule opacification in human donor eyes and rabbit eyes

To evaluate bag-in-the-lens implantation by studying the feasibility of implanting a new type of intraocular lens (IOL) and the occurrence of posterior capsule opacification (PCO) in human postmortem eyes and in eyes of living rabbits. Department of Ophthalmology, University of Antwerp, Belgium, and Netherlands Research Institute of Amsterdam, Amsterdam, The Netherlands. The IOL was implanted in 10 postmortem human donor eyes (in vitro study) and in 17 eyes of 10 rabbits (in vivo study). The postmortem capsular bags were cultured for 4 to 6 weeks, and the rabbits were killed 1 to 5 months after implantation. All capsular bags with the bag-in-the-lens were examined by light microscopy and scanning electron microscopy. The IOL design was highly effective in restricting lens epithelial cell (LEC) proliferation in the remaining lens bag in human donor eyes and in rabbit eyes. In eyes in which the capsules were not positioned well within the groove of the IOL, LEC proliferation and PCO occurred. Bag-in-the-lens implantation was highly effective in preventing PCO in vitro and in vivo provided the anterior and posterior capsules were secured properly in the peripheral groove of the IOL.

Induction of Tyrosinase Gene Transcription in Human Iris Organ Cultures Exposed to Latanoprost

Topical administration of latanoprost sometimes induces gradual iris darkening. The present study was undertaken to determine if latanoprost can increase transcription of the gene for tyrosinase, an important enzyme in the biosynthesis of melanin. Results from brown, hazel, and blue irides were compared. Iris tissue was isolated from 30 pairs of postmortem human donor eyes, and 2 iris segments from each eye were incubated in tissue culture medium supplemented with 200nM latanoprost acid or vehicle for 7 days. Tyrosinase messenger RNA (mRNA) was determined using real-time polymerase chain reaction analysis (TaqMan quantitative polymerase chain reaction). Results for tyrosinase mRNA were normalized according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. Tyrosinase mRNA expression was similar in blue and hazel irides, and ranged from 0.7-fold to 12.6-fold greater than GAPDH expression. In contrast, control brown iris culture tyrosinase expression ranged from 6.4-fold to 265-fold greater than GAPDH expression. Induction of tyrosinase mRNA by latanoprost was below threshold in all the blue iris cultures (n = 8 pairs), present in 1 of 9 hazel iris cultures, and present in 5 of 13 brown iris cultures. Mean induction in the responding hazel iris cultures was 1.40-fold. Mean induction among the responding brown iris cultures was 2.97-fold. These observations support the view that iris darkening associated with latanoprost treatment reflects induction of tyrosinase expression. Moreover, they suggest that the probability that latanoprost will increase tyrosinase expression is directly related to the magnitude of tyrosinase expression before treatments are initiated. The variability of iris darkening with latanoprost may reflect natural variation in the basal transcription of tyrosinase.

Interferon-gamma induces the expression of major histocompatibility antigens by human retinal glial cells

Retinal glial cells normally do not express major histocompatibility (MHC) molecules. However, the expression of MHC antigens by retinal glial cells under certain pathological conditions suggests that these cells may play a role in immune responses of the retina. At present, the identity of molecules that may regulate the expression of class I and class II MHC antigens by retinal glial cells has not been established. Since interferon-gamma (IFN-γ) induces MHC antigens in a variety of cell types, we examined the effect of this lymphocyte product on class I and class II antigen expression by human retinal glial cells in culture. Glial cells derived from post-mortem donor eyes were exposed to recombinant human IFN-γ. Cells with MHC class I or class II antigens were detected with the use of specific monoclonal antibodies that were labeled directly or indirectly with fluorescent probes. Fluorescence of labeled cells was assayed by flow cytometry. Exposure to IFN-γ increased the expression of class I and class II antigens by the retinal glial cells. Double labeling studies showed that 90% of the glial cells induced to express class II antigens also had class I antigens. Overall, the results show that IFN-γ can regulate the expression of MHC molecules by human retina glial cells in culture. These findings are consistent with the concept that glia may play a role in immunological disorders affecting the retina.

Detection of Lymphocytes in the Vitreous Gel of Patients With Retinitis Pigmentosa

We used monoclonal antibodies against specific T cell, B cell, and macrophage antigens to detect immune system cells in the vitreous gel of retinitis pigmentosa patients. Vitreous samples were obtained by biopsy from three patients and by dissection from eight postmortem donor eyes. All samples contained CD4, T helper and CD8, suppressor cells, activated T cells, some B cells, and macrophages. About one third of the cells were nonreactive. Vitreous gel from normal controls contained mostly nonreactive cells with a few macrophages and rare T cells. These observations further indicated the nature of the cellular reaction in retinitis pigmentosa and characterized the biomicroscopically visible particulate bodies characteristically seen in the vitreous gel of these patients.

Bruch's membrane age-related changes vary by region.

Aging changes known to occur in Bruch's membrane may be associated with drusen formation and retinal pigment epithelial mottling, which often have a peripheral as well as a macular distribution. Few details of peripheral Bruch's membrane aging changes have been reported. We conducted a histochemical investigation of Bruch's membrane in 31 postmortem donor eyes and an ultrastructural morphometric investigation of these regional changes in a subgroup of 22 eyes. The age of our donors ranged from 12 days to 80 years. When we reacted 5 micron paraffin embedded sections of chorio-retinal complex fixed in 2% glutaraldehyde-1% paraformaldehyde, we observed increased PAS positivity, and staining with Weigert's elastin and alcian blue at pH 2.5 in Bruch's membranes from donors over 46 years. In this older group, the macula showed increased histochemical reactivity for glycoconjugates, glycosaminoglycans, collagen and elastin as compared with the equator and periphery. Thickening of Bruch's membrane was first detected in the periphery in tissue from donors aged 10 to 45. Thickening of macular Bruch's membrane was first detected in tissue from donors older than 45. The major proportion of thickening, which appears to be due to the deposition of fibrillar and amorphous material, occurred earlier in the inner than the outer collagenous zone. The equatorial region was relatively spared. Our findings confirm and extend the observations of the timing, regional predeliction, and extent of Bruch's membrane aging changes.