Adjacent Retinal Pigment Epithelium Research Articles

The Retina-Based Visual Cycle.

The continuous function of vertebrate photoreceptors requires regeneration of their visual pigment following its destruction upon activation by light (photobleaching). For rods, the chromophore required for the regeneration of rhodopsin is derived from the adjacent retinal pigmented epithelium (RPE) cells through a series of reactions collectively known as the RPE visual cycle. Mounting biochemical and functional evidence demonstrates that, for cones, pigment regeneration is supported by the parallel supply with chromophore by two pathways-the canonical RPE visual cycle and a second, cone-specific retina visual cycle that involves the Müller glial cells in the neural retina. In this article, we review historical information that led to the discovery of the retina visual cycle and discuss what is currently known about the reactions and molecular components of this pathway and its functional role in supporting cone-mediated vision.

PROGRESSION OF PERIPAPILLARY AND MACULAR CHORIORETINAL ATROPHY IN MULTIFOCAL CHOROIDITIS IS ASSOCIATED WITH PERIATROPHIC INFLAMMATORY PLUMES.

To investigate peripapillary atrophy and macular chorioretinal scars in eyes affected by multifocal choroiditis and panuveitis. This retrospective cohort study reviewed the medical records, fundus photographs, and spectral-domain optical coherence tomographic scans of 31 eyes from 19 patients. Patients had a mean age of 45 years (range 24-69 years). The average follow-up duration was 7 years (range, 2.5-14.5 years), with 14 patients undergoing immunosuppressive treatment. In the group of 31 eyes, 20 showed peripapillary plumes of ill-defined hyperreflectivity at the termination border of the retinal pigment epithelium. These plumes, extending from bare Bruch membrane to the outer nuclear layer, sometimes undermined the adjacent retinal pigment epithelium. They responded to corticosteroid treatment and resembled the material under the retinal pigment epithelium in acute lesions. Among 20 eyes with these peripapillary inflammatory lesions, 16 (80%) experienced increased atrophy, in contrast to none in the eyes without these lesions ( P < 0.001). Similar patterns were observed at the edges of macular chorioretinal atrophy. This observation occurred in patients using immunosuppressive medication who were otherwise thought to be under adequate control. In patients with multifocal choroiditis and panuveitis, previously unrecognized plumes of smoldering inflammatory activity at the borders of chorioretinal atrophy appears to be linked to atrophy expansion. The recognition of this phenomenon may require a reappraisal of treatment of multifocal choroidopathies to help mitigate the expansion of atrophy in these eyes.

Hyperreflective Material Boundary Remodeling in Neovascular Age-Related Macular Degeneration: A Post Hoc Analysis of the AVENUE Trial

To describe the spatial and temporal characteristics of hyperreflective material (HRM) on spectral-domain OCT (SD-OCT) in neovascular age-related macular degeneration (nAMD) during antiangiogenic treatment and explore associations with best-corrected visual acuity (BCVA) and macular atrophy (MA). Retrospective regrading of SD-OCT-images from the multicenter, randomized controlled AVENUE trial (NCT02484690, conducted from August 2015 to September 2017). Treatment-naïve nAMD patients enrolled from 50 sites in the US. Retrospective regrading and secondary analysis. Spectral-domain OCT images from 207 study eyes that fit criteria for the present analysis were graded for HRM features, its evolution, and associated hypertransmission into choroid (HTC), a proxy for MA. The appearance of a well-defined hyperreflective inner boundary that separated persistent HRM from the neurosensory retina continuous with the adjacent retinal pigment epithelium layer was defined as hyperreflective material boundary remodeling (HRM-BR). Patterns of HRM composition/evolution were defined as follows: (1) no subretinal HRM at baseline, (2) fully resolved, (3) persistent with complete HRM-BR, or (4) partial/absent HRM-BR. Associations of HRM patterns with BCVA and HTC were analyzed. Predictive factors for complete HRM-BR were explored. Of 207 included eyes, subretinal HRM was present in 159 (76.8%) at baseline and persisted until month 9 in 118 (57.0%) eyes. Of these 118 eyes, 44.9% developed complete HRM-BR and had similar BCVA outcomes by month 9 compared with no/fully resolved subretinal HRM. Partial/absent HRM-BR had a strong negative association with BCVA outcome (-6.1 ETDRS letters; P= 0.016) and a higher frequency of intralesional HTC (69.2%) compared with eyes with complete HRM-BR (20.8%) at month 9. Older age (odds ratio [OR], 0.96; P= 0.054) and presence of intralesional HTC (OR, 0.06; P= 0.010) at baseline were associated with lower odds of complete HRM-BR at month9. In nAMD eyes under antiangiogenic treatment, complete HRM-BR occurred frequently and was associated with better BCVA than when HRM-BR was only partial/absent. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal pigment epithelium exhibits gene expression and phagocytic activity alterations when exposed to retinoblastoma chemotherapeutics

Retinoblastoma (Rb) is a rare malignant disorder affecting the developing retina of children under the age of five. Chemotherapeutic agents used for treating Rb have been associated with defects of the retinal pigment epithelium (RPE), such as hyperplasia, gliosis, and mottling. Herein, we have developed two pluripotent stem cell (PSC)-RPE models to assess the cytotoxicity of known Rb chemotherapeutics such as Melphalan, Topotecan and TW-37. Our findings demonstrate that these drugs alter the RPE by decreasing the monolayer barrier's trans-epithelial resistance and affecting the cells' phagocytic activity. Transcriptional analyses demonstrate an altered expression of genes involved in melanin and retinol processing, tight junction and apical-basal polarity pathways in both models. When applied within the clinical range, none of the drug treatments caused significant cytotoxic effects, changes to the apical-basal polarity, tight junction network or cell cycle. Together, our results demonstrate that although the most commonly used Rb chemotherapeutic drugs do not cause cytotoxicity in RPE, their application in vitro leads to compromised phagocytosis and strength of the barrier function, in addition to changes in gene expression that could alter the visual cycle in vivo. Our data demonstrate that widely used Rb chemotherapeutic drugs can have a deleterious impact on RPE cells and thus great care has to be exercised with regard to their delivery so the adjacent healthy RPE is not damaged during the course of tumor eradication.

Senescence-induced alteration of circadian phagocytic activity of retinal pigment epithelium cell line ARPE-19

Renewal of retinal photoreceptor outer segments is conducted through daily shedding of distal photoreceptor outer segment tips and subsequent their phagocytosis by the adjacent retinal pigment epithelium (RPE) monolayer. Dysregulation of the diurnal clearance of photoreceptor outer segment tips has been implicated in age-related retinal degeneration, but it remains to be clarified how the circadian phagocytic activity of RPE cells is modulated by senescence. In this study, we used the human RPE cell line ARPE-19 to investigate whether hydrogen peroxide (H2O2)-induced senescence in ARPE-19 cells alters the circadian rhythm of their phagocytic activity. After synchronization of the cellular circadian clock by dexamethasone treatment, the phagocytic activity of normal ARPE-19 cells exhibited significant 24-h oscillation, but this oscillation was modulated by senescence. The phagocytic activity of senescent ARPE-19 cells increased constantly throughout the 24-h period, which still exhibited blunted circadian oscillation, accompanied by an alteration in the rhythmic expression of circadian clock genes and clock-controlled phagocytosis-related genes. The expression levels of REV-ERBα, a molecular component of the circadian clock, were constitutively increased in senescent ARPE-19 cells. Furthermore, pharmacological activation of REV-ERBα by its agonist SR9009 enhanced the phagocytic activity of normal ARPE-19 cells and increased the expression of clock-controlled phagocytosis-related genes. Our present findings extend to understand the role of circadian clock in the alteration of phagocytic activity in RPE during aging. Constitutive enhancement of phagocytic activity of senescent RPE may contribute to age-related retinal degeneration.

Spatiotemporal Live-Cell Analysis of Photoreceptor Outer Segment Membrane Ingestion by the Retinal Pigment Epithelium Reveals Actin-Regulated Scission.

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term "phagocytosis." However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as "trogocytosis," in which one cell "nibbles" another cell.SIGNIFICANCE STATEMENT The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.

Regulation of the rhythmic diversity of daily photoreceptor outer segment phagocytosis in vivo.

Outer segment phagocytosis (OSP) is a highly-regulated, biological process wherein photoreceptor outer segment (OS) tips are cyclically phagocytosed by the adjacent retinal pigment epithelium (RPE) cells. Often an overlooked retinal process, rhythmic OSP ensures the maintenance of healthy photoreceptors and vision. Daily, the photoreceptors renew OS at their base and the most distal, and likely oldest, OS tips, are phagocytosed by the RPE, preventing the accumulation of photo-oxidative compounds by breaking down phagocytosed OS tips and recycling useful components to the photoreceptors. Light changes often coincide with an escalation of OSP and within hours the phagosomes formed in each RPE cell are resolved. In the last two decades, individual molecular regulators were elucidated. Some of the molecular machinery used by RPE cells for OSP is highly similar to mechanisms used by other phagocytic cells for the clearance of apoptotic cells. Consequently, in the RPE, many molecular regulators of retinal phagocytosis have been elucidated. However, there is still a knowledge gap regarding the key regulators of physiological OSP in vivo between endogenous photoreceptors and the RPE. Understanding the regulation of OSP is of significant clinical interest as age-related macular degeneration (AMD) and inherited retinal diseases (IRD) are linked with altered OSP. Here, we review the in vivo timing of OSP peaks in selected species and focus on the reported in vivo environmental and molecular regulators of OSP.

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the “eat me” signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.

High-Fat Diet Alters the Retinal Pigment Epithelium and Choroidal Transcriptome in the Absence of Gut Microbiota.

Relationships between retinal disease, diet, and the gut microbiome have started to emerge. In particular, high-fat diets (HFDs) are associated with the prevalence and progression of several retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy (DR). These effects are thought to be partly mediated by the gut microbiome, which modulates interactions between diet and host homeostasis. Nevertheless, the effects of HFDs on the retina and adjacent retinal pigment epithelium (RPE) and choroid at the transcriptional level, independent of gut microbiota, are not well-understood. In this study, we performed the high-throughput RNA-sequencing of germ-free (GF) mice to explore the transcriptional changes induced by HFD in the RPE/choroid. After filtering and cleaning the data, 649 differentially expressed genes (DEGs) were identified, with 616 genes transcriptionally upregulated and 33 genes downregulated by HFD compared to a normal diet (ND). Enrichment analysis for gene ontology (GO) using the DEGs was performed to analyze over-represented biological processes in the RPE/choroid of GF-HFD mice relative to GF-ND mice. GO analysis revealed the upregulation of processes related to angiogenesis, immune response, and the inflammatory response. Additionally, molecular functions that were altered involved extracellular matrix (ECM) binding, ECM structural constituents, and heparin binding. This study demonstrates novel data showing that HFDs can alter RPE/choroid tissue transcription in the absence of the gut microbiome.

Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis.

SUMMARYFumarate can be a surrogate for O2 as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O2 to H2O. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous 13C4-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O2 consumption by uncoupling adjacent RPE mitochondria.

Challenges and opportunities for modeling monogenic and complex disorders of the human retina via induced pluripotent stem cell technology.

The human retina is a highly structured and complex neurosensory tissue central to perceiving and processing visual signals. In a healthy individual, the close interplay between the neuronal retina, the adjacent retinal pigment epithelium and the underlying blood supply, the choriocapillaris, is critical for maintaining eyesight over a lifetime. An impairment of this delicate and metabolically highly active system, caused by genetic alteration, environmental impact or both, results in a multitude of pathological phenotypes of the retina. Understanding and treating these disease processes are motivated by a marked medical need in young as well as in older patients. While naturally occurring or gene-manipulated animal models have been used successfully in ophthalmological research for many years, recent advances in induced pluripotent stem cell technology have opened up new avenues to generate patient-derived retinal model systems. Here, we explore to what extent these cellular models can be useful to mirror human pathologies in vitro ultimately allowing to analyze disease mechanisms and testing treatment options in the cell type of interest on an individual patient-specific genetic background.

The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems.

Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

SummaryRecent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia.

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

Photoreceptor cells and RPE contribute to the development of diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness. It has long been regarded as vascular disease, but work in the past years has shown abnormalities also in the neural retina. Unfortunately, research on the vascular and neural abnormalities have remained largely separate, instead of being integrated into a comprehensive view of DR that includes both the neural and vascular components. Recent evidence suggests that the most predominant neural cell in the retina (photoreceptors) and the adjacent retinal pigment epithelium (RPE) play an important role in the development of vascular lesions characteristic of DR. This review summarizes evidence that the outer retina is altered in diabetes, and that photoreceptors and RPE contribute to retinal vascular alterations in the early stages of the retinopathy. The possible molecular mechanisms by which cells of the outer retina might contribute to retinal vascular damage in diabetes also are discussed. Diabetes-induced alterations in the outer retina represent a novel therapeutic target to inhibit DR.

Retinal pigment epithelial responses based on the irradiation density of selective retina therapy.

We evaluated the response of the retinal pigment epithelium (RPE) to high-density (HD) or low-density (LD)-selective retina therapy (SRT) with real-time feedback-controlled dosimetry (RFD) in rabbits. Sixteen eyes of 8 Chinchilla Bastard rabbits underwent SRT with RFD (527-nm wavelength, 1.7-μs pulse duration), using automatically titrated pulse energy, by using optoacoustic dosimetry or real-time reflectometry. Fifty-six 25-μJ SRT, including LD-SRT (1-spot or 2-spot-spacing) and HD-SRT (4-spot, 7-spot, or 9-spot-no-spacing), were applied per eye. Color fundus photography and fundus fluorescein angiography (FFA) were used to confirm SRT spots 1-h post-SRT. Light microscopy and scanning electron microscopy (SEM) were performed at 2-h, 3-day, 7-day, and 1-month post-treatment. We tested 896 spots irradiated by SRT with RFD and confirmed that SRT lesions were adequate, based on invisibility on fundoscopy and visibility on FFA. On SEM, at 2-h post-SRT, flattened RPE cells were observed in the center of the SRT lesion. While normal RPE cells were clearly observed between LD-SRT lesions, healthy RPE cells were rare in HD-SRT lesions at 2-h post-treatment. At 7-day post-SRT, SEM revealed completely restored LD-SRT lesions with small or large RPE cells with microvilli, whereas HD-SRT lesions were covered with RPE cells without microvilli. At 1-month post-SRT, SEM revealed restored RPE cells with microvilli in HD-SRT lesions. On light microscopy, both HD- and LD-SRT lesions were completely restored with adjacent RPE cells and spared photoreceptors at 1-month post-treatment. Although both HD- and LD-SRT lesions had recovered at 1-month post-SRT, LD-SRT lesions healed faster than HD-SRT lesions.

Application of subretinal fluid to close refractory full thickness macular holes: treatment strategies and primary outcome: APOSTEL study.

Persisting macular holes (PMH) after surgical release of any epiretinal traction of the vitreous and adjacent membrane may rely on secondary firm adhesions between the retracted retina and adjacent retinal pigment epithelium. Secondary application of subretinal (SR)-fluid may release these adhesions followed by an anatomical closure. Twelve surgeons applied in a consecutive case series SR-fluid in 41 eyes with PMH and reported retrospectively their initial surgical, anatomical and functional experience with this approach. The mean duration of the MH prior to SR-fluid application was 17months (6-96months). The mean age of the patients at the time of surgery was 72years (54-88). The mean preoperative aperture diameter of the opening was 1212μm (239-4344μm), base diameter 649 μm (SD 320 μm). The mean preoperative BCVA prior to surgery was 0.1 (0.01-0.3). All patients (41/41) complained about reduced BCVA and a significant central scotoma (negative scotoma) in their central field of vision. The secondary closure rate for our PMH was 85.36% (35 out of 41 eyes) at 6weeks after surgery. The postoperative BCVA improved to 0.22 (0.02-0.5). The application of SR-fluid was not associated with major intraoperative adverse effects. Remaining SR-adhesions may inhibit PMH closure. Their release by application of SR-fluid will lead to a fast and immediate anatomical closure in many cases without serious adverse events.

Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics.

PurposeThe development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina.MethodsIn the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells.ResultsThis method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved.ConclusionsAlthough different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates.Translational RelevanceWith this model, a variety of vision restoration methods can be tested in the non-human primate.

Closure of Persisting Full Thickness Macular Holes by Subretinal Fluid Application: Technical Approach and Surgical Considerations

Firm adhesions between the retina and adjacent retinal pigment epithelium (RPE) may prevent the closure of macular holes (MH) after chromovitrectomy. Controlled application of subretinal (SR) fluid with BSS may release these adhesions leading to closure of the retracted retina in large and or refractory macular holes. For a standardized procedure, it is recommended to exclude residues of epiretinal membranes on the retinal surface preoperatively at OCT or intraoperatively by means of vital dyes. Intraoperatively, a perfluorocarbon (PFO) bubble is placed above the MH and lowers the infusion bottle of 20 mmHg. Subsequently, SR-fluid blebs are applied in the upper, temporal and inferior quadrants with a subretinal 41-gauge cannula. After removing decalin bubble, the SR-detachment is enlarged toward the foveal center. This is essential to achieve a complete detachment of the outer macular edges from the RPE. The MH can be closed by a temporary gas endotamponade. With a standardized procedure, the operation can be carried out safely and with minimal effort. Additional measures, such as care for bubble-free SR-fluid sands or machine assistance, were added. In a pilot study, experienced VR surgeons performed the SR-fluid application safely and without complications. The preoperative diameter of the MH was 1150 µm (651 - 2350 µm). The secondary closure rate for our PMH was 80.9%. SR-adhesions seem to have a previously unnoticed component in persistent macular holes. An SR-fluid application can be carried out quickly, safely and with minimal material effort. The initial results show a high secondary closure rate.

The retina and retinal pigment epithelium differ in nitrogen metabolism and are metabolically connected

Defects in energy metabolism in either the retina or the immediately adjacent retinal pigment epithelium (RPE) underlie retinal degeneration, but the metabolic dependence between retina and RPE remains unclear. Nitrogen-containing metabolites such as amino acids are essential for energy metabolism. Here, we found that 15N-labeled ammonium is predominantly assimilated into glutamine in both the retina and RPE/choroid ex vivo [15N]Ammonium tracing in vivo show that, like the brain, the retina can synthesize asparagine from ammonium, but RPE/choroid and the liver cannot. However, unless present at toxic concentrations, ammonium cannot be recycled into glutamate in the retina and RPE/choroid. Tracing with 15N-labeled amino acids show that the retina predominantly uses aspartate transaminase for de novo synthesis of glutamate, glutamine, and aspartate, whereas RPE uses multiple transaminases to utilize and synthesize amino acids. Retina consumes more leucine than RPE, but little leucine is catabolized. The synthesis of serine and glycine is active in RPE but limited in the retina. RPE, but not the retina, uses alanine as mitochondrial substrates through mitochondrial pyruvate carrier. However, when the mitochondrial pyruvate carrier is inhibited, alanine may directly enter the retinal mitochondria but not those of RPE. In conclusion, our results demonstrate that the retina and RPE differ in nitrogen metabolism and highlight that the RPE supports retinal metabolism through active amino acid metabolism.