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Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective or modify photoreceptor degeneration in concentric retinitis pigmentosa.

Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at OcularGeneExpression.org/singlecell. With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The complement membrane attack complex (MAC) shows increased abundance in the choriocapillaris during normal aging and is especially prevalent in age-related macular degeneration (AMD). While perivascular MAC accumulation occurs in the choroid, it is not well understood whether similar deposition occurs in other aging tissues. In this study we examined the abundance of MAC across multiple human tissues. For studies on fixed tissues, paraffin sections were obtained from six human donor eyes and a commercially available tissue array containing 19 different tissues. Immunohistochemical labeling was performed using antibodies directed against the MAC and intercellular adhesion molecule-1 (ICAM-1), as well as the lectin Ulex europaeus agglutinin-I (UEA-I). The choriocapillaris was the only tissue with high levels of the MAC, which was not detected in any of the 38 additional samples from 19 tissues. ICAM-1 was abundantly expressed in the majority of tissues evaluated, and UEA-I labeled the vasculature in all tissues. A second experiment was performed using unfixed frozen sections of RPE-choroid and 7 extraocular tissues, which confirmed the relatively limited localization of the MAC to the choriocapillaris. In comparison to other tissues assessed, the restricted accumulation of MAC in the choriocapillaris may, in part, explain the specificity of AMD to the neural retina, RPE and choroid, and the relative absence of systemic pathology in this disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sub-retinal implantation of foreign materials is becoming an increasingly common feature of novel therapies for retinal dysfunction. The ultimate compatibility of implants depends not only on their in vitro chemical compatibility, but also on how well the mechanical properties of the material match those of the native tissue. In order to optimize the mechanical properties of retinal implants, the mechanical properties of the mammalian retina itself must be carefully characterized. In this study, the compressive moduli of eye tissues, especially the retina, were probed using a dynamic mechanical analysis instrument in static mode. The retinal compressive modulus was lower than that of the sclera or cornea, but higher than that of the RPE and choroid. Compressive modulus remained relatively stable with age. Conversely, apparent retinal softening occurred at an early age in mice with inherited retinal degeneration. Compressive modulus is an important consideration for the design of retinal implants. Polymer scaffolds with moduli that are substantially different than that of the native tissue in which they will ultimately reside will be less likely to aid in the differentiation and development of the appropriate cell types in vitro and will have reduced biocompatibility in vivo., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Data from human genetics, histopathology, and animal models reveal a major role for the complement system in the development of age-related macular degeneration (AMD). Genetic variations in the complement factor H (CFH) gene are associated with an elevated risk of AMD. In this study we sought to determine whether eyes from donors with a high-risk genotype (homozygosity for the histidine allele at codon 402) exhibit altered levels of membrane attack complex (MAC) in the choroid, compared to eyes with a low risk genotype (homozygosity for tyrosine). Proteins were extracted from the RPE/choroid of 18 donors (10 low risk and 8 high risk) and levels of MAC were assessed using an ELISA assay. Eyes from donors homozygous for the histidine allele showed 69% higher levels of MAC than those homozygous for the tyrosine allele (p < 0.05), independent of whether the eyes showed signs of early AMD. Our results provide evidence that high-risk CFH genotypes may affect AMD risk by increased deposition of MAC around the aging choriocapillaris., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD) is a common degenerative disease resulting in injury to the retina, retinal pigment epithelium and choriocapillaris. Recent data from histopathology, animal models and genetic studies have implicated altered regulation of the complement system as a major factor in the incidence and progression of this disease. A variant in the gene SERPING1, which encodes C1INH, an inhibitor of the classical and lectin pathways of complement activation, was recently shown to be associated with AMD. In this study we sought to determine the localization of C1INH in human donor eyes. Immunofluorescence studies using a monoclonal antibody directed against C1INH revealed localization to photoreceptor cells, inner nuclear layer neurons, choriocapillaris, and choroidal extracellular matrix. Drusen did not exhibit labeling. Genotype at rs2511989 did not appear to affect C1INH abundance or localization, nor was it associated with significant molecular weight differences when evaluated by Western blot. In a small number of eyes (n = 7 AMD and n = 7 control) AMD affection status was correlated with increased abundance of choroidal C1INH. These results indicate that C1INH protein is present in the retina and choroid, where it may regulate complement activation.

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.

Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma.

Best macular dystrophy (BMD) is an autosomal dominant retinopathy caused by mutations in the VMD2 gene that encodes a chloride channel in the basolateral membrane of the retinal pigment epithelium (RPE). BMD patients were studied using optical coherence tomography (OCT) to understand the disease process in the macula leading to vision loss. BMD patients (ages 5-61), representing four families with known VMD2 mutations, were included. OCT scans were recorded in the central retina and longitudinal reflectivity profiles were analysed. The central retina in BMD showed different OCT abnormalities at or near the level of the highly reflective deep retinal band termed the outer retina-choroid complex (ORCC). Two types of ORCC change were noted to occur either separately or together: (1) splitting with or without intervening hyporeflective areas; and (2) elevation. Longitudinal study of a BMD patient indicated that such abnormalities were dynamic and changed in type and degree with time. The pathogenetic sequence in BMD may begin with defective fluid transport across the RPE secondary to the channelopathy in the basolateral membrane. In the macula, this leads to an abnormal interface with adjacent structures at both apical and basal surfaces of the RPE. The disease process results in detachments of the neurosensory retina, such as in central serous chorioretinopathy, and sub-RPE pathology resembling some stages of age-related macular degeneration, with eventual loss of photoreceptors, inner retina and central vision., (Copyright 2003 Elsevier Science Ltd.)

Choroideremia is an incurable X-linked retinal degeneration caused by mutations in the gene encoding Rab escort protein-1. A group of clinically defined and genotyped patients were studied to determine: (1) the degree of rod and cone dysfunction and structural abnormality in the central retina and the level of macular pigment; and (2) the response of macular pigment and foveal vision to a 6 month trial of supplementation with oral lutein (at 20 mg per day). Rod and cone-mediated function was measured with dark-adapted static perimetry; in vivo retinal structure was determined with optical coherence tomography; and macular pigment optical density was measured with heterochromatic flicker photometry. In this cohort of patients (ages 15-65 years), both rod- and cone-mediated central function declined with age as did central retinal thickness. Macular pigment levels did not differ between patients and male control subjects. Supplementation of oral lutein in a subset of patients led to an increase in serum lutein and macular pigment levels; absolute foveal sensitivity did not change. It is concluded that macular pigment density can be augmented by oral intake of lutein in patients with choroideremia. There was no short-term change in the central vision of the patients on the supplement, but long-term influences of lutein supplementation on disease natural history warrant further study., (Copyright 2002 Elsevier Science Ltd.)