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Purpose.: To quantitatively investigate the reflectivity and structure of the outer retinal layers in children with hyperopic anisometropic amblyopia by using swept-source optical coherence tomography (SS-OCT). Methods: Seventy-eight patients with amblyopia and 64 age-matched children with normal vision were included in this study. All participants underwent SS-OCT and detailed ophthalmic examinations. Longitudinal reflectance profile measurements were measured using Image J. The reflectivity of outer retinal layers was measured at the three selected positions: subfovea, 1 mm nasal to the fovea, and 1 mm temporal to the fovea. The reflectivity ratios were calculated by outer retinal layers divided by the nuclear layer (ONL) for normalization. Photoreceptor outer segment layer thickness was also measured. The results were compared between the amblyopia and normal controls. The possible effects of age, sex, and axial length on results were adjusted by generalized estimating equations. Results: Photoreceptor outer segment layer thickness was significantly greater in amblyopic eyes than in normal control eyes at all three regions (18.41 ± 1.83 vs. 16.84 ± 1.39, P < 0.001 at the fovea; 14.78 ± 1.34 vs. 14.19 ± 1.40, P = 0.030 at 1 mm nasal to the foveal; 14.92 ± 1.48 vs. 14.41 ± 1.32, P = 0.049 at 1 mm temporal to the fovea). The reflectivity ratio of outer segment/ONL was higher only at 1 mm nasal to the fovea (2.94 ± 0.61 vs. 2.70 ± 0.42, P = 0.02). Subfoveal OS thickness was positively correlated with choroidal thickness (r = 0.248, P = 0.018) but was not correlated with spherical equivalent, age, axial length, or logMAR visual acuity. Conclusion: Quantitative measurement of SS-OCT images revealed greater photoreceptor outer segments in both eyes of children with amblyopia than in normal control eyes. A thicker OS thickness is somehow related to amblyopia, and this may be a new useful diagnostic parameter for amblyopia. [ABSTRACT FROM AUTHOR]

Photoreceptors (PRs) and retinal pigment epithelial (RPE) cells form a functional unit called the PR-RPE complex. The PR-RPE complex plays a critical role in maintaining retinal homeostasis and function, and the quantification of its structure and topographical arrangement across the macula are important for understanding the etiology, mechanisms, and progression of many retinal diseases. However, the three-dimensional cellular morphology of the PR-RPE complex in living human eyes has not been completely described due to limitations in imaging techniques. We used the cellular resolution and depth-sectioning capabilities of a custom, high-speed Fourier domain mode-locked laser-based adaptive optics–optical coherence tomography (FDML-AO-OCT) platform to characterize human PR-RPE complex topography across the temporal macula from eleven healthy volunteers. With the aid of a deep learning algorithm, key metrics were extracted from the PR-RPE complex of averaged AO-OCT volumes including PR and RPE cell density, PR outer segment length (OSL), and PR/RPE ratio. We found a tight grouping among our cohort for PR density, with a mean (±SD) value of 53,329 (±8106) cells/mm2 at 1° decreasing to 8669 (±737) cells/mm2 at 12°. We observed a power function relationship between eccentricity and both PR density and PR/RPE ratio. We found similar variability in our RPE density measures, with a mean value of 7335 (±681) cells/mm2 at 1° decreasing to 5547 (±356) cells/mm2 at 12°, exhibiting a linear relationship with a negative slope of −123 cells/mm2 per degree. OSL monotonically decreased from 33.3 (±2.4) µm at 1° to 18.0 (±1.8) µm at 12°, following a second-order polynomial relationship. PR/RPE ratio decreased from 7.3 (±0.9) µm at 1° to 1.5 (±0.1) µm at 12°. The normative data from this investigation will help lay a foundation for future studies of retinal pathology. [ABSTRACT FROM AUTHOR]

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor outer segment (POS) phagocytosis occurs as a daily peak, roughly about 1 hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2) lack a daily peak in POS phagocytosis. By qPCR analysis, we found that core clock genes were rhythmic over 24 hours in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time points preceding and during the peak of POS phagocytosis. In contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time point. Finally, based on STRING analysis, we found a group of interacting genes that potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b, and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE. [ABSTRACT FROM AUTHOR]

Purpose: To quantitatively investigate the reflectivity and structure of the outer retinal layers on spectral-domain optical coherence tomography (SD-OCT) in commotio retinae. Methods: Nineteen patients with acute macular commotio retinae and 19 age-matched normal controls were examined using SD-OCT. Longitudinal reflectance profiles (LRP) were obtained using Image J. The reflectivity of outer retinal layers was measured at the fovea, 1 mm nasal to fovea and 1 mm temporal to fovea. The reflectivity ratios of outer retinal layers divided by the outer nuclear layer (ONL) were calculated for normalization. Photoreceptor outer segment layer thickness was also measured. The results were compared between the patients and controls. Results: The reflectivity ratio of ellipsoid zone/ONL and outer segment/ONL was higher in commotio retinae than in controls only at fovea (12.66 ± 4.73 vs 9.67 ± 3.34, p = 0.041; 7.70 ± 2.20 vs 3.73 ± 1.63, p < 0.001, respectively) but not at 1 mm nasal or temporal to the fovea. Photoreceptor outer segment layer thickness was significantly shorter in commotio retinae compared to controls at all three locations (19.64 ± 3.05 vs 25.16 ± 3.53, 16.95 ± 4.02 vs 20.00 ± 3.00, and 15.42 ± 3.22 vs 20.05 ± 2.48, respectively, all p < 0.05). Conclusions: Quantitative measurement of SD-OCT images revealed that shortening of photoreceptor outer segment is an additional, and potentially better, biomarker for commotio retinae on top of increased reflectivity. [ABSTRACT FROM AUTHOR]

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions. [ABSTRACT FROM AUTHOR]

The first step of our vision is initiated when photons are absorbed by the subcellular organelle of photoreceptor neuron called outer segment. Outer segment incorporates phototransduction proteins and structural constituents for its continuous renewal. The author and his colleagues recently developed a photoconversion technique, which allowed monitoring specific protein transport and renewal of outer segment structures. Moreover, quantitative microscopy was harnessed to understand the roles of transport signals in the prototypical ciliary GPCR, rhodopsin. This review summarizes our recent findings on the process of outer segment morphogenesis, membrane protein trafficking and compartmentalization in vertebrate rod photoreceptors. [ABSTRACT FROM AUTHOR]

Background: To evaluate the prognostic value of foveal microstructures as determined using spectral-domain optical coherence tomography (SD-OCT) in eyes with surgically closed macular holes (MHs). Methods: Thirty eyes of 30 patients that underwent successful vitrectomy for idiopathic MHs were studied. Best-corrected visual acuity (BCVA) and SD-OCT images of the fovea were examined preoperatively and at 2 weeks, 1, 3, and 6 months postoperatively. The SD-OCT characteristics evaluated included MH diameter, external limiting membrane (ELM) defect diameter, photoreceptor inner/outer segment (IS/OS) junction defect diameter, the presence or absence of subretinal fluid (SRF), central foveal thickness (CFT), and outer foveal thickness (the distance between the ELM and the inner border of the retinal pigment epithelium). The correlations between SD-OCT parameters and BCVA were analyzed. Results: The IS/OS junction defect diameter was most strongly correlated with BCVA at 2 weeks and 1 month postoperatively while outer foveal thickness showed the strongest correlation with BCVA at 3 and 6 months. Outer foveal thickness gradually increased over the follow-up period. Among the pre- and early postoperative quantitative parameters, the only significant predictors of BCVA at 6 months were the IS/OS junction defect diameter and outer foveal thickness at 2 weeks ( p = 0.012, p = 0.018, respectively). Disrupted ELM at 2 weeks was also associated with significantly poorer BCVA at 6 months ( p < 0.001). Conclusions: An increase in outer foveal thickness, indicating photoreceptor outer segment restoration, is an important factor for visual recovery after MH surgery. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelium (RPE) is the most active phagocyte in the human body, and this function is indispensable for vision. In this study, we introduce an easy and inexpensive way to quantify RPE phagocytosis by opsonizing FITC-labeled latex beads of a defined size with a crude mixture of photoreceptor outer segment fragments, using cells of porcine origin. After performance of the desired experiment, phagocytosis can easily be quantified by counting the ingested particles. With this protocol, we combine the advantage of a natural target with the advantages of the defined properties of latex beads. Moreover, in order to conduct these phagocytosis experiments, as laboratory equipment, in addition to cell culture equipment, only a centrifuge and a simple fluorescence microscope are needed. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To evaluate retinal thickness using spectral-domain optical coherence tomography (SD-OCT) with reduced speckle noise in eyes with polypoidal choroidal vasculopathy (PCV) compared to those with normal eyes and those with central serous chorioretinopathy (CSC). We retrospectively reviewed cases of foveal serous retinal detachment in 36 eyes of 36 patients with active CSC and 23 eyes of 23 patients with active PCV, and 44 eyes of 44 normal subjects. Patients were examined using SD-OCT with reduced speckle noise, and the thickness of the outer nuclear layer (ONL), photoreceptor inner segment (IS), and photoreceptor outer segment (OS) were measured. The ONL and IS were thicker in normal eyes than in eyes with CSC or PCV ( P < 0.001). The OS was significantly less thick in eyes with PCV than in normal eyes ( P < 0.001), whereas there was no significant difference between eyes with CSC and normal eyes. The thickness of IS and OS in eyes with PCV was related to fibrin or hemorrhage being present in the subretinal space. In eyes with PCV, best-corrected visual acuity at baseline correlated with IS thickness ( P = 0.023). Thinning of each photoreceptor layer was observed in the eyes of PCV patients as compared to that observed in the case of normal individuals. The differentiating factors between PCV and CSC, observed using SD-OCT, include the thinning of the OS in eyes with PCV, which makes SD-OCT helpful in differentiating PCV from CSC. More severe photoreceptor alterations were seen in PCV, because fibrin and hemorrhage were present in the subretinal space, which correlated with poorer vision. [ABSTRACT FROM AUTHOR]

Abstract: Complement activation is thought to be involved in the pathogenesis of age-related macular degeneration (AMD), in part because certain gene polymorphisms in complement factor H (CFH), an important regulator of the alternative complement activation pathway, are high risk factors for AMD. How CFH is regulated locally at the retina/choroid interface and how this contributes to AMD development remain unknown. In the present study, we have confirmed that CFH was detectable by immunohistochemistry in the choroid, and at low levels in the RPE cell and interphotoreceptor matrix, but appeared to be concentrated in dense patches in Bruch''s membrane. In vitro, cultured human and mouse RPE cells expressed high levels of CFH as evidenced by immunohistochemistry and western blot. Using a stabilized mouse RPE cell line, we confirmed that RPE cells constitutively synthesise CFH. Synthesis of CFH was not affected by a short-term (2h) photoreceptor outer segment (POS) treatment. However, long-term (24–48h) treatment of RPE cells with oxidised POS (ox-POS) but not normal POS (n-POS) markedly down-regulated CFH mRNA expression. Phagocytosis of both ox-POS and n-POS appeared to reduce intracellular CFH protein expression in RPE cultures. Synthesis of CFH by cultured RPE cells was also reduced at the mRNA level by the proinflammatory cytokines TNF-α and IL-6. Other cytokines tested including IFN-γ, IL-1α and IL-4 showed no effect on either CFH protein or mRNA levels. Our results support the view that RPE cells synthesise and express CFH and are probably a major local source of this protein at the retina/choroid interface, secreting CFH into the interphotoreceptor matrix as well as Bruch''s membrane. Prolonged phagocytosis of POS, particularly if modified by oxidative processes as occurs in inflammation, appears to markedly impair synthesis and secretion of CFH, with potential loss of important regulatory functions in counteracting the pro-inflammatory effects of activated complement. [Copyright &y& Elsevier]

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37°C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidationlxylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethyithiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelial (RPE) cells that underlie the retina ingest and metabolize thousands of lipid-rich photoreceptor outer segments (POS) every day. The scavenger receptor (SR) CD36 and integrin αvβ5 have been shown to participate in POS binding and internalization by RPE cells. The objective of the current study was to determine whether RPE cells express SRs other than CD36. Primary cultures of human RPE cells express both mRNA and protein for SR-BI and SR- BII. SR-BI and SR-BII mRNAs were detected by reverse transcription–polymerase chain reaction. SR-BI protein was detected by immune precipitation of [35S]methionine-labeled crude cellular extracts. SR-BII was detected by Western blotting of immune precipitated crude cellular extracts. SR-BI and SR-BII proteins were also detected by immunofluorescence staining of RPE cells in culture. The results suggest that these SRs may play a role in POS lipid binding and uptake by RPE cells in the eye. [Copyright &y& Elsevier]

Photoreceptor disease results in irreparable vision loss and blindness, which has a dramatic impact on quality of life. Pathogenic mutations in RP1L1 lead to photoreceptor degenerations such as occult macular dystrophy and retinitis pigmentosa. RP1L1 is a component of the photoreceptor axoneme, the backbone structure of the photoreceptor's light-sensing outer segment. We generated an rp1l1 zebrafish mutant using CRISPR/Cas9 genome editing. Mutant animals had progressive photoreceptor functional defects as determined by electrophysiological assessment. Optical coherence tomography showed gaps in the photoreceptor layer, disrupted photoreceptor mosaics, and thinner retinas. Mutant retinas had disorganized photoreceptor outer segments and lipid-rich subretinal drusenoid deposits between the photoreceptors and retinal pigment epithelium. Our mutant is a novel model of RP1L1-associated photoreceptor disease and the first zebrafish model of photoreceptor degeneration with reported subretinal drusenoid deposits, a feature of age-related macular degeneration. [ABSTRACT FROM AUTHOR]