Bruch's membrane of the brachymorphic mouse.

Objective To investigate the imaging features of the retinal pigment epithelium (RPE)-Bruch's membrane (BM)-choriocapillaris complex in central serous chorioretinopathy (CSC) with spectral-domain optical coherence tomography (Spectralis OCT). Methods This was a noninterventional, observational study. One hundred and eleven eyes of 109 consecutive patients (male 89, female 20) diagnosed with CSC were included. All the patients underwent a full ophthalmologic examination, including the visual acuity, slit-lamp microscope, pre-set lens, fundus photography, Spectralis OCT and fundus fluorescein angiography (FFA). Indocyanine green angiography (ICGA) was performed on some patients. The RPE-BM-choriocapillaris complex, which manifested as a high-reflecting band in Spectralis OCT, was carefully checked. Based on the visibility of BM, it was classified into two types: a visible type or invisible type. The visible type was then classified into an obvious type or occult type. If visible, BM appears thinner and more reflective than the RPE-BM-choriocapillaris complex. Meantime, the pigment epithelial detachment (PED) was classified into different types corresponding to the BM categories. The number of different categories were counted, measured and recorded as percentages. Results The visualization of BM manifested in all 11 eyes (100%), but differed to the extent for which it was visible. Of the 111 eyes, the BM was obvious in 99 eyes (89.8%). In the other 12 eyes (10.2%) it was difficult to discriminate, but there were still distinguishable differences from normal RPE-BM-choriocapillaris complexes. The visualization of BM was more obvious and wider in the 27 chronic CSC eyes than in the 84 acute CSC eyes when accompanied by the atrophy of choriocapillaris and RPE. BM was discriminated or visualized at all of the tracking leakage points demonstrated on FFA images. Conclusion BM is sandwiched between the RPE and choriocapillaris, and it cannot be distinguished in the normal high-reflecting band of the complex in a Spectralis OCT image. Visualization of BM in the RPE-BM-choriocapillaris complex demonstrates a detachment not only between BM and the RPE, but also perhaps between BM and the choriocapillaris. This discovery may offer new morphological and pathological evidence for central serous chorioretinopathy. Key words: Tomography,optical coherence; Chorioretinis,serous,central; Retinal detachment; Bruch's membrane; Capillaris,choroid

PURPOSE To determine the mechanism of reattachment of harvested human retinal pigment epithelium (RPE) to RPE-derived extracellular matrix and Bruch's membrane. METHODS Confluent first-to third-passage human RPE were harvested from tissue culture and plated onto RPE-derived extracellular matrix or human Bruch's membrane exoplants denuded of cells by treatment with 0.02 N ammonium hydroxide. The authors measured RPE reattachment to uncoated surfaces or surfaces precoated with extracellular matrix proteins (fibronectin, laminin, vitronectin, or type IV collagen), antibodies to extracellular matrix-proteins, or the synthetic peptide RGDS (arginine-glycine-aspartate-serine). Some RPE were pretreated with anti-beta 1 integrin antibodies before plating onto either substrate. RESULTS Coating the surface of either RPE-derived extracellular matrix or Bruch's membrane with fibronectin, laminin, vitronectin, or type IV collagen increased the RPE attachment rate. Exposing RPE to anti-beta 1 integrin antibodies or RGDS or precoating the surface with antibodies to fibronectin, laminin, vitronectin, or type IV collagen decreased the RPE attachment rate to both surfaces. The RPE attachment rate to Bruch's membrane was lower when the exoplants were harvested from the macula of older (age, 70 to 90 years) versus younger (age, 30 to 40 years) persons (52.4 +/- 3.6% versus 64.3 +/- 3.5%, respectively; P < 0.05). CONCLUSIONS The attachment of cultured human RPE cells to human Bruch's membrane or to RPE-derived extracellular matrix is mediated by an interaction between the beta 1-subunit of integrin on the RPE surface and ligands in the extracellular matrix that include laminin, fibronectin, vitronectin, and type IV collagen. The lower rate of RPE reattachment to the macula from older human cadaveric eyes may have implications for studies aimed at RPE transplantation in elderly persons.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

PurposeLittle is known about the development of Bruch's membrane (BrM), the structure separating and supporting the retina and choroid, nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies, both in the development of accurate in vitro models, and effective RPE transplant strategies. Here, we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE.MethodsThe presence of laminin, elastin, fibronectin, and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR).ResultsIn human fetal retina, laminin and fibronectin were present from 6 PCW, type IV collagen from 8 PCW, elastin from 12 PCW, type I collagen by 17 PCW, and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW, becoming distinct by 17 PCW. In hPSC-derived RPE cultures, basement membranes containing laminin and fibronectin were present from week 1, type IV collagen from week 2, and type I collagen from week 4. Type III collagen was present at all timepoints, although not localized as a basement membrane. Elastin was absent at all timepoints.ConclusionsBrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development, excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease, and inform cell therapy approaches, as de novo BrM-like membrane has the potential to support transplanted donor RPE.

Trypsin digestion of retinal pigment epithelium is a technique that bares Bruch's membrane, to allow topographical examination by scanning electron microscopy. Twenty-five human eyes were examined. The zonula occludens of the pigment epithelium was clearly seen as a surface feature, but attachment plaques at the sides and base were not visible. The adhesion between the pigment epithelium and the basal lamina was stronger than between the basal lamina and the rest of Bruch's membrane. Surface features of the basal lamina, inner collagenous zone, elastic layer, and outer collagenous zone were seen in a way that previously required an artist's representation constructed from microscopic sections.

ObjectiveTo compare the visibility and accessibility of the outer retina in neovascular age-related macular degeneration (nAMD) between 2 OCT devices. MethodsIn this prospective, cross-sectional exploratory study, differences in thickness and loss of individual outer retinal layers in eyes with nAMD and in age-matched healthy eyes between a next-level High-Res OCT device and the conventional SPECTRALIS OCT (both Heidelberg Engineering GmbH, Heidelberg, Germany) were analyzed. Eyes with nAMD and at least 250 nL of retinal fluid, quantified by an approved deep-learning algorithm (Fluid Monitor, RetInSight, Vienna, Austria), fulfilled the inclusion criteria. The outer retinal layers were segmented using automated layer segmentation and were corrected manually. Layer loss and thickness were compared between both devices using a linear mixed-effects model and a paired t test. ResultsNineteen eyes of 17 patients with active nAMD and 17 healthy eyes were included. For nAMD eyes, the thickness of the retinal pigment epithelium (RPE) differed significantly between the devices (25.42 μm [95% CI, 14.24–36.61] and 27.31 μm [95% CI, 16.12–38.50] for high-resolution OCT and conventional OCT, respectively; p = 0.033). Furthermore, a significant difference was found in the mean relative external limiting membrane loss (p = 0.021). However, the thickness of photoreceptors, RPE integrity loss, and photoreceptor integrity loss did not differ significantly between devices in the central 3 mm. In healthy eyes, a significant difference in both RPE and photoreceptor thickness between devices was shown (p < 0.001). ConclusionCentral RPE thickness was significantly thinner on high-resolution OCT compared with conventional OCT images explained by superior optical separation of the RPE and Bruch's membrane.

We have used the electron microscopic immunogold technique to localize precisely various extracellular matrix components in human retinal vessels and Bruch's membrane. Collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein were localized in basement membranes of retinal capillaries. In addition to the capillary basement membrane components, collagen types I and III and fibronectin were found in the basement membranes of retinal arterioles and venules. The basement membranes of choriocapillaries and retinal pigment epithelial cells in Bruch's membrane also showed a similar distribution of the retinal capillary basement membrane components. Both the inner and outer collagenous layers of Bruch's membrane contained collagen types I and III along with fibronectin, whereas collagen type VI was mostly limited to the central elastic lamina. The precise localization and distribution of various extracellular matrix components in human retinal vessels and Bruch's membrane may be important for understanding their normal function as well as their alteration in disease.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for "dry," or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch's membrane pathology is part of this process. Alterations in human Bruch's membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch's membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch's membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch's membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

To develop a reproducible surgical technique for the induction of choroidal neovascularization (CNV) in the subretinal space of porcine eyes and to analyse the resulting CNV clinically and histologically. Two different modifications of a surgical technique previously described were compared with the original method. In ten porcine eyes retinal pigment epithelial (RPE) cells were removed using a silicone tipped cannula, in ten porcine eyes Bruch's membrane was perforated once with a retinal perforator without prior RPE removal and in ten eyes RPE removal was followed by a single perforation of Bruch's membrane. Fifteen of the eyes, five from each group, were enucleated 30 minutes after surgery, while the remaining eyes were enucleated after 14 days. Prior to enucleation, at day 14, fundus photographs and fluorescein angiograms were obtained. Eyes were examined by light microscopy and by immunohistochemical staining. In addition to these 30 eyes, two eyes underwent surgery with the purpose of subsequent scanning electron microscopic (SEM) examination. In eyes enucleated immediately after surgery neuroretinas overlying the induced lesions were intact without apparent atrophy of cells regardless of the surgical technique applied. The process of RPE removal was found to induce breaks in Bruch's membrane and both the size and the number of breaks varied between eyes. CNV membranes were identified in 15 of 15 eyes enucleated after 14 days. CNV membranes induced by perforation of Bruch's membrane without prior RPE removal were significantly thicker than membranes from eyes undergoing both RPE removal and Bruch's perforation (p = 0.03) and also thicker than membranes from eyes with only RPE-removal (p < 0.01). CNV membranes from eyes with perforation of Bruch's membrane without prior RPE removal had a higher cellular content and were more richly vascularized and also exhibited the highest propensity to leak in fluorescense angiograms. All three surgical techniques were capable of inducing CNV, but the one applying perforation of Bruch's membrane without RPE removal was easier to reproduce and involved fewer variables than the techniques utilizing RPE removal. The presence of RPE cells seems to affect both the morphology and cellular composition of induced CNV.

Cell monolayers that form a barrier between two structures play an important role for the maintenance of tissue functionality. In the anterior portion of the eye, the corneal endothelium forms a barrier that controls fluid exchange between the aqueous humor of the anterior chamber and the corneal stroma. This monolayer is central in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). FECD is a common corneal disease, in which corneal endothelial cells deposit extracellular matrix that increases the thickness of its basal membrane (Descemet's membrane), and forms excrescences (guttae). With time, there is a decrease in endothelial cell density that generates vision loss. Transplantation of a monolayer of healthy corneal endothelial cells on a Descemet membrane substitute could become an interesting alternative for the treatment of this pathology. In the back of the eye, the retinal pigment epithelium (RPE) forms the blood-retinal barrier, controlling fluid exchange between the choriocapillaris and the photoreceptors of the outer retina. In the retinal disease dry age-related macular degeneration (dry AMD), deposits (drusen) form between the RPE and its basal membrane (Bruch's membrane). These deposits hinder fluid exchange, resulting in progressive RPE cell death, which in turn generates photoreceptor cell death, and vision loss. Transplantation of a RPE monolayer on a Bruch's membrane/choroidal stromal substitute to replace the RPE before photoreceptor cell death could become a treatment alternative for this eye disease. This review will present the different biomaterials that are proposed for the engineering of a monolayer of corneal endothelium for the treatment of FECD, and a RPE monolayer for the treatment of dry AMD.

To search for the histological correlate of peripheral 'cobblestones' in highly myopic eyes. The histomorphometric investigation included histologic sections of enucleated eyes of Caucasian patients. Using light microscopy, we measured the thickness of the retina, Bruch's membrane (BM) and choriocapillaris. The study included 50 eyes (mean age:60.6 ± 18.7 years;axial length:26.5 ± 3.8 mm), with cobblestone regions detected in 7 eyes. BM thickness and choriocapillaris thickness in the cobblestone region were thinner (1.1 ± 0.2 µm versus 2.4 ± 0.8 µm; p < 0.001 and 1.6 ± 0.5 µm versus 2.6 ± 1.9 µm; p = 0.02, respectively), and just outside of the cobblestone region they were thicker (3.3 ± 0.6 µm versus 2.4 ± 0.8 µm; p = 0.005 and 5.7 ± 1.6 µm versus 2.6 ± 1.9 µm; p = 0.002, respectively) than in corresponding regions of eyes without cobblestones. Within the group of eyes with cobblestones, BM thickness (1.1 ± 0.2 mm versus 3.3 ± 0.6 mm; p < 0.001), choriocapillaris thickness (1.6 ± 0.5 mm versus 5.7 ± 1.6 mm; p < 0.001) and choriocapillaris density (48±15 µm/300 µm versus 159 ± 66 µm/300 µm;PP=0.002) were significantly lower in the cobblestone region than just outside of the cobblestone region. The cobblestone regions were characterized by firm adhesion of disorganized retina with thinned BM, few retinal pigment epithelium (RPE) islands within cobblestone regions, and absence of regional scleral or overall choroidal thinning. BM was mono-layered within, and double-layered outside of cobblestone regions, with the inner layer missing within the cobblestone region (except for the RPE islands). Peripheral cobblestone regions in highly myopic eyes are characterized by marked BM thinning with absence of an inner BM layer, almost complete RPE absence, choriocapillaris thinning and firm connection of a disorganized retina to BM. These findings may help elucidating the process of axial elongation in myopic eyes.

Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression.

ABSTRACTNon-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

An electro-conductive hybrid scaffold as an artificial Bruch's membrane

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect