Physiologically relevant in vitro models of the human outer retina are required to better elucidate the complex interplay of retinal tissue layers and investigate their role in retinal degenerative disorders. Materials currently used to mimic the function of Bruch\'s membrane fail to replicate a range of important structural, mechanical, and biochemical properties. Here, we detail the fabrication of a surface-functionalized, fibrous collagen I membrane. We demonstrate its ability to better replicate a range of important material properties akin to the function of human Bruch\'s membrane when compared with a commonly utilized synthetic polyethylene terephthalate alternative. We further reveal the ability of this membrane to support the culture of the ARPE-19 cell line, as well as human pluripotent stem cell-derived RPE-like cells and human umbilical vein endothelial cells. This material could provide greater physiological relevance to the native Bruch\'s membrane than current synthetic materials and further improve the outcomes of in vitro outer retinal models.

UNASSIGNED: A micrometer scale hyporeflective band within the retinal pigment epithelium basal lamina - Bruch\'s membrane complex (RPE-BL-BrM) was topographically measured in aging and age-related macular degeneration (AMD).
UNASSIGNED: In a prospective cross-sectional study, 90 normal eyes from 76 subjects (range = 23-90 years) and 53 dry AMD eyes from 47 subjects (range = 62-91 years) were enrolled. Isotropic volume raster scans over 6 mm × 6 mm (500 × 500 A-scans) were acquired using a high-resolution (2.7 µm axial resolution) spectral-domain optical coherence tomography (SD-OCT) prototype instrument. Six consecutive optical coherence tomography (OCT) volumes were computationally motion-corrected and fused to improve feature visibility. A boundary regression neural network was developed to measure hyporeflective band thickness. Topographic dependence was evaluated over a 6-mm-diameter Early Treatment Diabetic Retinopathy Study (ETDRS) grid.
UNASSIGNED: The hyporeflective band thickness map (median of 4.3 µm and 7.8 µm in normal and AMD eyes, respectively) is thicker below and radially symmetric around the fovea. In normal eyes, age-associated differences occur within 0.7 to 2.3 mm from the foveal center (P < 0.05). In AMD eyes, the hyporeflective band is hypothesized to be basal laminar deposits (BLamDs) and is thicker within the 3-mm ETDRS circle (P < 0.0002) compared with normal eyes. The inner ring is the most sensitive location to detect age versus AMD-associated changes within the RPE-BL-BrM. AMD eyes with subretinal drusenoid deposits (SDDs) have a significantly thicker hyporeflective band (P < 0.001) than those without SDDs.
UNASSIGNED: The hyporeflective band is a quantifiable biomarker which differentiates AMD from aging. Longitudinal studies are warranted. The hyporeflective band may be a useful biomarker for risk stratification and disease progression.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd-/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and lipid deposits in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Furthermore, the RPE of Prcd-/- mice exhibit a 1.7-fold increase in the expression of lipid transporter gene ATP-binding cassette transporter A1 (Abca1). Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd-/- mice exhibited age-related pathological features such as lipofuscin accumulation, Bruch\'s membrane (BrM) deposits and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd-/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd-/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.

UNASSIGNED: To examine the prevalence of Bruch\'s membrane defects (BMDs) and subretinal proliferations (SRPs) in highly myopic eyes with myopic macular atrophy (myopic macular degeneration [MMD] stage 4) and myopic patchy atrophies (MMD stage 3) in three ethnically different cohorts recruited in a population-based manner.
UNASSIGNED: The Ural Eye and Medical Study (UEMS) and Beijing Eye Study (BES) included individuals aged 40+ years, and the Ural Very Old Study (UVOS) examined individuals aged 85+ years. Main outcome measures were the prevalence of BMDs and SRPs.
UNASSIGNED: Among 5794 UEMS participants, 19 eyes had MMD stage 4, with 17 (89%) eyes showing a foveal BMD; two eyes could not fully be explored. All 19 eyes showed localized SRPs. Among 21 eyes with MMD stage 3, BMD and SRP prevalence was 9 of 21 (44%) and 7 of 21 (33%), respectively. Among 930 UVOS participants, 17 eyes had MMD stage 4, with 16 (94%) eyes showing foveal BMDs and SRPs; one eye could not be assessed. Among 18 eyes with MMD stage 3, BMD and SRP prevalence was 3 of 18 (17%) and 2 of 18 (11%), respectively. Among 3468 BES participants, 8 eyes had MMD stage 4, with all eyes showing foveal BMDs and SRPs. Among 14 eyes with MMD stage 3, BMD and SRP prevalence was 10 of 14 (71%) and 7 of 21 (33%), respectively.
UNASSIGNED: All eyes with assessable myopic macular atrophy showed foveal BMDs associated with SRPs, while patchy atrophies could be differentiated into those with BMDs and SRPs and those without BMDs and without SRPs. Independent of the MMD stage, the prevalences of BMDs and SRPs were highly significantly associated with each other.

UNASSIGNED: The purpose of this study was to assess the choroidal thickness and the Bruch\'s membrane opening size and their relationship to visual acuity in eyes with myopic macular degeneration (MMD).
UNASSIGNED: This was a population-based, cross-sectional study. Patients over the age of 30 years with high myopia (spherical equivalent ≤-5 diopters [D]) were recruited. The eyes were grouped according to the International Meta-Analysis for Pathologic Myopia (META-PM) classification based on fundus photographs and diffuse atrophy was subdivided into peripapillary diffuse choroidal atrophy (PDCA) or macular diffuse choroidal atrophy (MDCA). Swept-source optical coherence tomography imaging was performed and then the subfoveal choroidal thickness (SFCT) and Bruch\'s membrane opening diameter (BMOD) were measured.
UNASSIGNED: Of the 470 study participants recruited, 373 patients (691 eyes), with a mean age of 42.8 ± 7.2 years, were eligible for the study and included in the analysis. There was no significant difference in SFCT between MDCA and patchy atrophy (M3) groups (P = 1.000), and the BMOD enlarged significantly from no myopic macular lesions to M3 (the P values of multiple comparison tests were all <0.005). Simple linear regression analysis showed that BMOD correlated positively with age (P < 0.001) and axial length (P < 0.001). Multiple linear regression analysis showed that best corrected visual acuity (BCVA) was significantly correlated with age (P = 0.041), axial length (P = 0.001), and BMOD (P = 0.017), but not with SFCT (P = 0.231).
UNASSIGNED: The significant variation of BMOD among MMD groups and the correlation between BMOD and BCVA in MMD eyes suggest that BMOD may be an imaging biomarker for monitoring MMD.

UNASSIGNED: The purpose of this study was to examine characteristics of lamina cribrosa (LC) configuration in highly myopic (HM) eyes.
UNASSIGNED: Participants from the Beijing Eye Study 2011, free of optic nerve or retinal diseases, were randomly selected to examine LC depth (LCD) and LC tilt (LCT) using three-dimensional optical coherent tomography images of the optic nerve head (ONH). LCD and LCT were measured as the distance and angle between the LC plane with two reference planes, including the Bruch\'s membrane opening (BMO) plane and the peripapillary sclera (PPS) plane, respectively. Each parameter was measured in both horizontal and vertical B-scans.
UNASSIGNED: The study included 685 individuals (685 eyes) aged 59.6 ± 7.7 years, including 72 HM eyes and 613 non-HM eyes. LCD measurements showed no significant differences between HM eyes and non-HM eyes in both horizontal (LCD-BMO = 421.83 ± 107.86 µm for HM eyes vs. 447.24 ± 104.94 µm for non-HM eyes, P = 0.18; and LCD-PPS = 406.39 ± 127.69 µm vs. 394.00 ± 101.64 µm, P = 1.00) and vertical directions (LCD-BMO = 435.78 ± 101.29 µm vs. 450.97 ± 106.54 µm, P = 0.70; and LCD-PPS = 401.62 ± 109.9 µm vs. 379.85 ± 110.35 µm, P = 0.35). However, the LCT was significantly more negative (tilted) in HM eyes than in non-HM eyes horizontally (LCT-BMO = -4.38 ± 5.94 degrees vs. -0.04 ± 5.86 degrees, P < 0.001; and LCT-PPS = -3.16 ± 5.23 degrees vs. -0.94 ± 4.71 degrees, P = 0.003), but not vertically (P = 1.00).
UNASSIGNED: Although LCD did not differ significantly between HM and non-HM eyes, LCT was more negative in HM eyes, suggesting that the temporal or inferior side of the LC was closer to the reference plane. These findings provide insights into morphological and structural changes in the LC and ONH between HM and non-HM eyes.

The optic nerve head (ONH) is a complex structure wherein the axons of the retinal ganglion cells extrude from the eyeball through three openings: 1) the Bruch\'s membrane opening (BMO) in the retinal layer, 2) the anterior scleral canal opening in the anterior scleral layer, and 3) the lamina cribrosa (LC). Eyeball expansion during growth induces an offset among openings, since the expansion affects the inner retinal and outer scleral layers differently: the posterior polar retinal structure is preserved by the preferential growth in the equatorial region, whereas no such regional difference is observed in the scleral layer. The various modes and extents of eyeball expansion result in diverse directionality and amount of offset among openings, which causes diverse ONH morphology in adults, especially in myopia. In this review, we summarize the ONH changes that occur during myopic axial elongation. These changes were observed prospectively in our previous studies, wherein LC shift and subsequent offset from the BMO center could be predicted by tracing the central retinal vascular trunk position. This offset induces the formation of γ-zone parapapillary atrophy or externally oblique border tissue. As a presumptive site of glaucomatous damage, the LC/BMO offset may render the LC pores in the opposite direction more vulnerable. To support such speculation, we also summarize the relationship between LC/BMO offset and glaucomatous damage. Indeed, LC/BMO offset is not only the cause of diverse ONH morphology in adults, but is also, potentially, an important clinical marker for assessment of glaucoma.

The role of mast cells in physiologic and pathological processes extends far beyond the allergy processes: they are involved in wound healing, chronic inflammation, and tumor growth. This short article emphasizes the role played by mast cells in age-related macular degeneration (AMD). Mast cells can induce angiogenesis and are present around Bruch\'s membrane during the early and late stages of choroidal neovascularization in AMD. Proteolytic enzymes released by mast cells lead to thinning of the choroid in AMD as well as degradation of vascular basement membranes and Bruch\'s membrane, which in turn could result in retinal pigment epithelial death and choriocapillaris degeneration in geographical atrophy and exudative AMD.

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch\'s membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch\'s membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young\'s modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

We investigated three-dimensional (3D) eyeball protrusion and its association with the offset between the lamina cribrosa (LC) and Bruch\'s membrane opening (BMO). 3D-MRI scans were taken from 93 subjects (186 eyes). An ellipsoid was fitted along the posterior 2/3 contour of each eyeball. Eyeball asymmetry with focal bulging was determined by the existence of an adjacent outward protrusion/reciprocal inward depression pair, and the angular deviation of the outermost protruded point (OPP) was measured from the nasal side of the fovea-BMO axis. The LC/BMO offset was evaluated by measuring the central retinal vascular trunk (CRVT) location from the BMO center: (1) the angular deviation and (2) the offset index as the ratio between the CRVT-BMO center distance and the BMO radius in the same direction. Seventy-nine eyes (42%) were classified as having eyeball asymmetry, which had a more superior LC/BMO offset (P < 0.001) and a larger offset index (P = 0.002). In those eyes, the angular deviation of the OPP showed a significant correlation with that of the LC/BMO offset (r = -0.724, P < 0.001), as did protrusion depth with the offset index (r = 0.291, P = 0.009). The presence of eyeball asymmetry was associated with superior LC/BMO offset (P = 0.004) and larger offset index (P = 0.009). Superior LC/BMO offset was associated with older age (P < 0.001), shorter axial length (P < 0.001) and inferior location of OPP (P < 0.001). The location and extent of focal bulging were closely associated with those of LC/BMO offset. This indicates that focal bulging during expansion might be associated with diverse directionality of LC/BMO offset.