Histologic and Immunohistochemical Characterization of GA-Like Pathology in the Rat Subretinal Sodium Iodate Model.

Purpose: Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration with multifactorial etiology and no well-established treatment. A model recapitulating the hallmarks would serve as a key to understanding the underlying pathologic mechanisms better. In this report, we further characterized our previously reported subretinal sodium iodate model of GA. Methods: Retinal degeneration was induced in rats (6-8 weeks old) by subretinal injections of NaIO3 as described previously. Animals were sacrificed at 3, 8 and 12 weeks after injection and eyes were fixed or cryopreserved. Some choroids were processed as flatmounts while other eyes were cryopreserved, sectioned, and immunolabeled with a panel of antibodies. Finally, some eyes were prepared for transmission electron microscopic (TEM) analysis. Results: NaIO3 subretinal injection resulted in a well-defined focal area of retinal pigment epithelium (RPE) degeneration surrounded by viable RPE. These atrophic lesions expanded over time. RPE morphologic changes at the border consisted of hypertrophy, multilayering, and the possible development of a migrating phenotype. Immunostaining of retinal sections demonstrated external limiting membrane descent, outer retinal tubulation (ORT), and extension of Müller cells toward RPE forming a glial membrane in the subretinal space of the atrophic area. TEM findings demonstrated RPE autophagy, cellular constituents of ORT, glial membranes, basal laminar deposits, and defects in Bruch's membrane. Conclusions: In this study, we showed pathologic features of a rodent model resembling human GA in a temporal order through histology, immunofluorescence, and TEM analysis and gained insights into the cellular and subcellular levels of the GA-like phenotypes. Translational Relevance: Despite its acute nature, the expansion of atrophy and the GA-like border in this rat model makes it ideal for studying disease progression and provides a treatment window to test potential therapeutics for GA.

Visualization of choroidal vasculature in pigmented mouse eyes from experimental models of AMD.

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO3) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H2O2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO3-injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases.

Loss of REP1 impacts choroidal melanogenesis and vasculogenesis in choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined the ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron and confocal microscopy. Significant pigmentary disruptions were observed, with lack of melanosomes in the choroid of chmru848 fish from 4 days post fertilisation (4dpf), and a reduction in choroidal blood vessel diameter and interstitial pillars suggesting a defect in vasculogenesis. Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month, with reduced eumelanin at 1 year. The choroid in CHM patients were also examined using spectral domain optical coherence tomography (SD-OCT) and OCT-angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, suggesting that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.

Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation following internal limiting membrane disruption.

Intercellular cytoplasmic material transfer (MT) occurs between transplanted and developing photoreceptors and ambiguates cell origin identification in developmental, transdifferentiation, and transplantation experiments. Whether MT is a photoreceptor-specific phenomenon is unclear. Retinal ganglion cell (RGC) replacement, through transdifferentiation or transplantation, holds potential for restoring vision in optic neuropathies. During careful assessment for MT following human stem cell-derived RGC transplantation into mice, we identified RGC xenografts occasionally giving rise to labeling of donor-derived cytoplasmic, nuclear, and mitochondrial proteins within recipient Müller glia. Critically, nuclear organization is distinct between human and murine retinal neurons, which enables unequivocal discrimination of donor from host cells. MT was greatly facilitated by internal limiting membrane disruption, which also augments retinal engraftment following transplantation. Our findings demonstrate that retinal MT is not unique to photoreceptors and challenge the isolated use of species-specific immunofluorescent markers for xenotransplant identification. Assessment for MT is critical when analyzing neuronal replacement interventions.

Machine learning-driven multifunctional peptide engineering for sustained ocular drug delivery.

Sustained drug delivery strategies have many potential benefits for treating a range of diseases, particularly chronic diseases that require treatment for years. For many chronic ocular diseases, patient adherence to eye drop dosing regimens and the need for frequent intraocular injections are significant barriers to effective disease management. Here, we utilize peptide engineering to impart melanin binding properties to peptide-drug conjugates to act as a sustained-release depot in the eye. We develop a super learning-based methodology to engineer multifunctional peptides that efficiently enter cells, bind to melanin, and have low cytotoxicity. When the lead multifunctional peptide (HR97) is conjugated to brimonidine, an intraocular pressure lowering drug that is prescribed for three times per day topical dosing, intraocular pressure reduction is observed for up to 18 days after a single intracameral injection in rabbits. Further, the cumulative intraocular pressure lowering effect increases ~17-fold compared to free brimonidine injection. Engineered multifunctional peptide-drug conjugates are a promising approach for providing sustained therapeutic delivery in the eye and beyond.

Clinicopathologic Findings in Three Siblings With Geographic Atrophy.

Purpose: Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly worldwide. Clinical imaging and histopathologic studies are crucial to understanding disease pathology. This study combined clinical observations of three brothers with geographic atrophy (GA), followed for 20 years, with histopathologic analysis. Methods: For two of the three brothers, clinical images were taken in 2016, 2 years prior to death. Immunohistochemistry, on both flat-mounts and cross sections, histology, and transmission electron microscopy were used to compare the choroid and retina in GA eyes to those of age-matched controls. Results: Ulex europaeus agglutinin (UEA) lectin staining of the choroid demonstrated a significant reduction in the percent vascular area and vessel diameter. In one donor, histopathologic analysis demonstrated two separate areas with choroidal neovascularization (CNV). Reevaluation of swept-source optical coherence tomography angiography (SS-OCTA) images revealed CNV in two of the brothers. UEA lectin also revealed a significant reduction in retinal vasculature in the atrophic area. A subretinal glial membrane, composed of processes positive for glial fibrillary acidic protein and/or vimentin, occupied areas identical to those of retinal pigment epithelium (RPE) and choroidal atrophy in all three AMD donors. SS-OCTA also demonstrated presumed calcific drusen in the two donors imaged in 2016. Immunohistochemical analysis and alizarin red S staining verified calcium within drusen, which was ensheathed by glial processes. Conclusions: This study demonstrates the importance of clinicohistopathologic correlation studies. It emphasizes the need to better understand how the symbiotic relationship between choriocapillaris and RPE, glial response, and calcified drusen impact GA progression.

Effects of experimental glaucoma in Lama1nmf223 mutant mice.

To identify changes in response to experimental intraocular pressure (IOP) elevation associated with the laminin α1 nmf223 mutation in mice. Laminin mutant (LM) mice (Lama1nmf223) and C57BL/6J (B6) mice in two age groups each (4-5 months and >1 year) underwent intracameral microbead injections to produce unilaterally elevated IOP. We assessed axonal transport block of immunofluorescently labeled amyloid precursor protein (APP) after 3 days and retinal ganglion cell (RGC) axon loss after 6 weeks. Light, electron and fluorescent microscopy was used to study baseline anatomic differences and effects of 3-day IOP elevation in younger LM mice. In younger mice of both LM and B6 strains, elevated IOP led to increased APP block in the retina, prelaminar optic nerve head (preONH), unmyelinated optic nerve (UON), and myelinated optic nerve (MON). APP blockade not significantly different between younger B6 and LM mouse strains. Older LM mice had greater APP accumulation in both control and glaucoma eyes compared to older B6, however, accumulation was not significantly greater in LM glaucoma eyes compared to LM controls. Axon loss at 6 weeks was 12.2% in younger LM and 18.7% in younger B6 mice (difference between strains, p = 0.22, Mann Whitney test). Untreated LM optic nerve area was lower compared to B6 (nerve area, p < 0.0001, t-test). Aberrant axon bundles, as well as defects, thickening and reduplication of pia mater, were seen in the optic nerves of younger LM mice. Axonal transport blockade significantly differed between old B6 and old LM mice in control and glaucoma eyes, and younger LM mice had abnormal axon paths and lower optic nerve area.

Topical Ketotifen Fumarate Inhibits Choroidal Mast Cell Degranulation and Loss of Retinal Pigment Epithelial Cells in Rat Model for Geographic Atrophy.

Purpose: This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model. Methods: Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function. Results: PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF. Conclusions: Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA. Translational Relevance: The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Bruch's Membrane and the Choroid in Age-Related Macular Degeneration.

A healthy choroidal vasculature is necessary to support the retinal pigment epithelium (RPE) and photoreceptors, because there is a mutualistic symbiotic relationship between the components of the photoreceptor/retinal pigment epithelium (RPE)/Bruch's membrane (BrMb)/choriocapillaris (CC) complex. This relationship is compromised in age-related macular degeneration (AMD) by the dysfunction or death of the choroidal vasculature. This chapter will provide a basic description of the human Bruch's membrane and choroidal anatomy and physiology and how they change in AMD.The choriocapillaris is the lobular, fenestrated capillary system of choroid. It lies immediately posterior to the pentalaminar Bruch's membrane (BrMb). The blood supply for this system is the intermediate blood vessels of Sattler's layer and the large blood vessels in Haller's layer.In geographic atrophy (GA), an advanced form of dry AMD, large confluent drusen form on BrMb, and hyperpigmentation (presumably dysfunction in RPE) appears to be the initial insult. The resorption of these drusen and loss of RPE (hypopigmentation) can be predictive for progression of GA. The death and dysfunction of CC and photoreceptors appear to be secondary events to loss in RPE. The loss of choroidal vasculature may be the initial insult in neovascular AMD (nAMD). We have observed a loss of CC with an intact RPE monolayer in nAMD, by making RPE hypoxic. These hypoxic cells then produce angiogenic substances like vascular endothelial growth factor (VEGF), which stimulate growth of new vessels from CC, resulting in choroidal neovascularization (CNV). Reduction in blood supply to the CC, often stenosis of intermediate and large blood vessels, is associated with CC loss.The polymorphisms in the complement system components are associated with AMD. In addition, the environment of the CC, basement membrane and intercapillary septa, is a proinflammatory milieu with accumulation of proinflammatory molecules like CRP and complement components during AMD. In this toxic milieu, CC die or become dysfunctional even early in AMD. The loss of CC might be a stimulus for drusen formation since the disposal system for retinal debris and exocytosed material from RPE would be limited. Ultimately, the photoreceptors die of lack of nutrients, leakage of serum components from the neovascularization, and scar formation.Therefore, the mutualistic symbiotic relationship of the photoreceptor/RPE/BrMb/CC complex is lost in both forms of AMD. Loss of this functionally integrated relationship results in death and dysfunction of all of the components in the complex.

Retinal Glial and Choroidal Vascular Pathology in Donors Clinically Diagnosed With Stargardt Disease.

Purpose: The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). Methods: Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis. Results: Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Müller cells formed pre- and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss. Conclusions: Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Müller cell membranes correlating with RPE loss. Müller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

A role for mast cells in geographic atrophy.

Mast cells (MCs) are the initial responders of innate immunity and their degranulation contribute to various etiologies. While the abundance of MCs in the choroid implies their fundamental importance in the eye, little is known about the significance of MCs and their degranulation in choroid. The cause of geographic atrophy (GA), a progressive dry form of age-related macular degeneration is elusive and there is currently no therapy for this blinding disorder. Here we demonstrate in both human GA and a rat model for GA, that MC degranulation and MC-derived tryptase are central to disease progression. Retinal pigment epithelium degeneration followed by retinal and choroidal thinning, characteristic phenotypes of GA, were driven by continuous choroidal MC stimulation and activation in a slow release fashion in the rat. Genetic manipulation of MCs, pharmacological intervention targeting MC degranulation with ketotifen fumarate or inhibition of MC-derived tryptase with APC 366 prevented all of GA-like phenotypes following MC degranulation in the rat model. Our results demonstrate the fundamental role of choroidal MC involvement in GA disease etiology, and will provide new opportunities for understanding GA pathology and identifying novel therapies targeting MCs.

Choriocapillaris dropout in early age-related macular degeneration.

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n = 7; mean age = 86.1) and from eyes with a Grade 2 clinical classification of early AMD (n = 7; mean age = 87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA ± standard deviation in submacula of control subjects was 78.1 ± 3.25%. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 ± 10.4% (p < 0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 ± 0.09 mm2 in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 ± 6.1 mm2; p < 0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging.

Ultrastructural analysis of submacular choriocapillaris and its transport systems in AMD and aged control eyes.

The choriocapillaris is the source of nutrients and oxygen for photoreceptors, which consume more oxygen per gram of tissue than any other cell in the body. The purpose of this study was to evaluate and compare the ultrastructure of the choriocapillaris and its transport systems in patients with and without age-related macular degeneration (AMD). Ultrastructural changes were also evaluated in subjects that were homozygous for polymorphisms in high risk CFH alleles (Pure 1) only or homozygous only for high risk ARMS2/HTRA1 (Pure 10) alleles. Tissue samples were obtained from the macular region of forty male (n = 24) and female (n = 16) donor eyes and prepared for ultrastructural studies with transmission electron microscopy (TEM). The average age of the aged donors was 74 ±â€¯7.2 (n = 30) and the young donors 31.7 ±â€¯11.25 (n = 10). There was no significant difference in average ages between the adult groups. TEM images of the capillaries in the choriocapillaris (CC) were taken at 4,000X and 25,000X and used to measure the area of endothelial cell somas, the number of fenestrations, and area of caveolae within the endothelial cells per length of Bruchs membrane (BrMb). The Student t-test and Wilcoxon sum rank test were used to determine significant differences. There was no significant difference between young subjects and aged controls in any of the morphological criteria assessed. There was a significant decrease in the number of fenestrations/mm of BrMb in atrophic areas of GA eyes (p = 0.007) when compared with aged control eyes. A significant increase was found in the caveolae area as a percent of the endothelial cell soma of capillaries from GA subjects as compared with the controls (p = 0.03). Loss of capillary segments in choriocapillaris was also evident, especially in areas of geographic atrophy and CNV. In eyes from patients with sequence variations, the capillary endothelial cells often appeared degenerative and exhibited atypical fenestrations and pericytes covering the blood vessels. Subjects that were homozygous for polymorphisms in high risk CFH alleles only had more fenestrations/mm of BrMb than subjects that were homozygous only for high risk ARMS2/HTRA1 alleles (p = 0.04), while the latter had greater caveolae area/endothelial cell area than the former (p = 0.007). This study demonstrated an attenuation of CC and a significant decline in the two major transport systems in CC endothelial cells in AMD. This may contribute to drusen deposition, nutrient transport, and vision loss in AMD subjects.

An Acute Injury Model for the Phenotypic Characteristics of Geographic Atrophy.

Purpose: Geographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3). Methods: Rats were given subretinal injections of NaIO3 (5 µg/µL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes. Results: Fundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days. Conclusions: The rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Ocular Histopathology and Immunohistochemical Analysis in the Oldest Known Individual with Autosomal Dominant Vitreoretinochoroidopathy.

PURPOSE: To assess the immunohistochemical and histopathological changes in a subject with Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC). DESIGN: Case study. PARTICIPANT: Ninety two year-old Caucasian male with ADVIRC. METHODS: The subject was documented clinically for 54 Years. The retina/choroid complex of the right eye was evaluated with cryosections stained with hematoxylin and eosin or periodic acid schiff reagent. Cryosections were also evaluated with immunofluorescence or alkaline phosphatase immunohistochemistry (IHC) using primary antibodies against bestrophin1, GFAP, PEDF, RPE65, TGFß, VEGF, and vimentin. The left retina and choroid were evaluated as flat mounts using immunofluorescence. UEA lectin was used to stain viable vasculature. MAIN OUTCOME MEASURES: The immunohistochemical and histopathological changes in retina and choroid from a subject with ADVIRC. RESULTS: The subject had a heterozygous c.248G>A variant in exon 4 of the BEST1 gene. There was widespread chorioretinal degeneration and atrophy except for an island of spared RPE monolayer in the perimacula/macula OU. In this region, some photoreceptors were present, choriocapillaris was spared, and retinal pigment epithelial cells were in their normal disposition. There was a Muller cell periretinal membrane throughout much of the fundus. Bestrophin-1 was not detected or only minimally present by IHC in the ADVIRC RPE, even in the spared RPE area. Beyond the island of retained RPE monolayer on Bruch's membrane (BrMb), there was migration of RPE into the neuro-retina, often ensheathing blood vessels and producing excessive matrix within their perivascular aggregations. CONCLUSIONS: The primary defect in ADVIRC is in RPE, the only cells in the eye that express the BEST1 gene. The dysfunctional RPE cells may go through epithelial/mesenchymal transition as they migrate from BrMb to form papillary aggregations in the neuro-retina, often ensheathing blood vessels. This may be the reason for retinal blood vessel nonperfusion. Migration of RPE from BrMb was also associated with attenuation of the choriocapillaris.

Mast Cell-Derived Tryptase in Geographic Atrophy.

Purpose: Our previous study demonstrated significantly more degranulating mast cells (MCs) in choroids from subjects with age-related macular degeneration compared to aged controls. This study examined the immunolocalization of tryptase, the most abundant MC secretory granule-derived serine protease, in aged control eyes and eyes with geographic atrophy (GA). Methods: Postmortem human eyes with and without GA were obtained from the National Disease Research Interchange. Tissue was fixed, cryopreserved, sectioned, and immunostained with a monoclonal antibody against tryptase. Sections were imaged on a Zeiss 710 Confocal Microscope. Results: In the posterior pole of all aged control eyes, tryptase was confined to choroidal MCs, which were located primarily in Sattler's layer. In eyes with GA, many MCs were located in the inner choroid near choriocapillaris and Bruch's membrane (BM). Tryptase was found not only in MCs but also diffusely around them in stroma, suggesting they had degranulated. In contrast with aged control eyes, eyes with GA also had strong tryptase staining in BM. Tryptase was observed within BM in regions of RPE atrophy, at the border of atrophy, and extending well into the nonatrophic region. Conclusions: Our results demonstrate that tryptase, released during choroidal MC degranulation, binds to BM in GA in advance of RPE atrophy. Tryptase activates MMPs that can degrade extracellular matrix (ECM) and basement membrane components found in BM. ECM modifications are likely to have a profound effect on the function and health of RPE and choroidal thinning in GA.

Subretinal Glial Membranes in Eyes With Geographic Atrophy.

Purpose: Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space. Methods: Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells. Results: Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry. Conclusions: In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.

Distribution and Quantification of Choroidal Macrophages in Human Eyes With Age-Related Macular Degeneration.

PURPOSE: Increasing evidence suggests a role for macrophages in the pathogenesis of age-related macular degeneration (AMD). This study examined choroidal macrophages and their activation in postmortem eyes from subjects with and without AMD. METHODS: Choroids were incubated with anti-ionized calcium-binding adapter molecule 1 (anti-IBA1) to label macrophages, anti-human leukocyte antigen-antigen D-related (anti-HLA-DR) as a macrophage activation marker, and Ulex europaeus agglutinin lectin to label blood vessels. Whole mounts were imaged using confocal microscopy. IBA1- and HLA-DR-positive (activated) cells were counted in submacula, paramacula, and nonmacula, and cell volume and sphericity were determined using computer-assisted image analysis. RESULTS: In aged control eyes, the mean number of submacular IBA1+ and HLA-DR+ macrophages was 433/mm2 and 152/mm2, respectively. In early AMD eyes, there was a significant increase in IBA1+ and HLA-DR+ cells in submacula compared to those in controls (P = 0.0015 and P = 0.008, respectively). In eyes with neovascular AMD, there were significantly more HLA-DR+ cells associated with submacular choroidal neovascularization (P = 0.001). Mean cell volume was significantly lower (P ≤ 0.02), and sphericity was significantly higher (P ≤ 0.005) in all AMD groups compared to controls. CONCLUSIONS: The average number of IBA1+ macrophages in submacular and paramacular choroid was significantly higher in early/intermediate AMD compared to that in aged controls. HLA-DR+ submacular macrophages were significantly increased in all stages of AMD, and they were significantly more round and smaller in size in the submacular AMD choroid, suggesting their activation. These findings support the concept that AMD is an inflammatory disease.

Histopathological Insights Into Choroidal Vascular Loss in Clinically Documented Cases of Age-Related Macular Degeneration.

IMPORTANCE: Age-related macular degeneration (AMD) is a multifactorial disease with genetic and environmental factors contributing to risk. Histopathologic changes underlying AMD are not fully understood, particularly the relationship between choriocapillaris (CC) dysfunction and phenotypic variability of this disease. OBJECTIVE: To examine histopathologic changes in the CC of eyes with clinically documented AMD. DESIGN, SETTING, AND PARTICIPANTS: The study was designed in 2011. Tissues were collected post mortem (2012-2016), and histopathological images were obtained from participants enrolled in AMD studies since 1988. Clinical records and images were collected from participants as standard protocol. Eyes without AMD (n = 4) and eyes with early (n = 9), intermediate (n = 5), and advanced stages of AMD (geographic atrophy, n = 5; neovascular disease, n = 13) were evaluated. Choroidal vasculature was labeled using Ulex europaeus agglutinin lectin and examined using confocal microscopy. MAIN OUTCOMES AND MEASURES: A standardized classification system was applied to determine AMD stage. Ocular records and images were reviewed and histopathologic analyses performed. Viability of the choroidal vasculature was analyzed for each AMD stage. RESULTS: All participants were white. Fourteen were male, and 16 were female. The mean age was 90.5 years among AMD patients and 88.5 years among control participants. Submacular CC dropout without retinal pigment eipthelial (RPE) loss was observed in all cases with early stages of AMD. Higher vascular area loss for each AMD stage was observed compared with control participants: 20.5% in early AMD (95% CI, 11.2%-40.2%; P < .001), 12.5% in intermediate AMD (95% CI, 2.9%-21.4%; P = .01), 39.0% loss in GA (95% CI, 32.1%-45.4%; P < .001), and 38.2% loss in neovascular disease where RPE remained intact (95% CI, 27.7%-47.9%; P < .001). Hypercellular, apparent neovascular buds were adjacent to areas of CC loss in 22.2% of eyes with early AMD and 40% of eyes with intermediate AMD. CONCLUSIONS AND RELEVANCE: Retinal pigment epithelial atrophy preceded CC loss in geographic atrophy, but CC loss occurred in the absence of RPE atrophy in 2 of 9 eyes with early-stage AMD. Given the cross-sectional nature of this study and the small number of eyes evaluated, definitive conclusions regarding this progression cannot be determined with certainty. We speculate that neovascular buds may be a precursor to neovascular disease. Hypoxic RPE resulting from reduced blood supply might upregulate production of vascular endothelial growth factor, providing the stimulus for neovascular disease.

Defective Angiogenesis and Intraretinal Bleeding in Mouse Models With Disrupted Inner Retinal Lamination.

PURPOSE: Abnormal retinal angiogenesis leads to visual impairment and blindness. Understanding how retinal vessels develop normally has dramatically improved treatments for people with retinal vasculopathies, but additional information about development is required. Abnormal neuron patterning in the outer retina has been shown to result in abnormal vessel development and blindness, for example, in people and mouse models with Crumbs homologue 1 (CRB1) mutations. In this study, we report and characterize a mouse model of inner retinal lamination disruption and bleeding, the Down syndrome cell adhesion molecule (Dscam) mutant, and test how neuron-neurite placement within the inner retina guides development of intraretinal vessels. METHODS: Bax mutant mice (increased neuron cell number), Dscam mutant mice (increased neuron cell number, disorganized lamination), Fat3 mutant mice (disorganized neuron lamination), and Dscam gain-of-function mice (Dscam(GOF)) (decreased neuron cell number) were used to manipulate neuron placement and number. Immunohistochemistry was used to assay organization of blood vessels, glia, and neurons. In situ hybridization was used to map the expression of angiogenic factors. RESULTS: Significant changes in the organization of vessels within mutant retinas were found. Displaced neurons and microglia were associated with the attraction of vessels. Using Fat3 mutant and Dscam(GOF) retinas, we provide experimental evidence that vessel branching is induced at the neuron-neurite interface, but that other factors are required for full plexus layer formation. We further demonstrate that the displacement of neurons results in the mislocalization of angiogenic factors. CONCLUSIONS: Inner retina neuron lamination is required for development of intraretinal vessels.