Identification of Structures Labeled by Indocyanine Green in the Rat Choroid and Retina Can Guide Interpretation of Indocyanine Green Angiography.

Purpose: Indocyanine green (ICG) is an albumin and lipoprotein binding dye absorbing in the far red used in angiography to visualize choroidal vessels (ICG angiography [ICGA]). To guide interpretation, ICG transport in the choroid, RPE, and retina of rats was studied. Methods: Two conditions were used: RPE/choroid organoculture, incubated for 45 minutes in DMEM medium, 1% fetal bovine serum containing 0.25 mg/mL ICG and RPE/choroid and neural retina flat-mounts at 1 and 6 hours after intravenous ICG injection. Early and late sequences of ICGA were recorded until 6 hours. Ultra-deep red confocal microscope was used to localize ICG in flat-mounts and immunohistochemistry was performed for caveolin-1, tryptase (mast cell marker), and tubulin ß3 (a nerve marker). Results: In the organoculture, ICG penetrated homogeneously in the cytoplasm and stained the membranes of the RPE. At 1 hour after intravenous injection, ICG appeared in fine granules in RPE, partly labeled with caveolin-1 and decreasing at 6 hours. At 1 hour and 6 hours, ICG was found in the retinal vessels, faintly in the inner retina, and in the photoreceptor outer segments at 6 hours. In the choroid, ICG colocalized with mast cells, immunostained with tryptase, and accumulated along the large tubulin ß3-labeled nerve bundles. The hypothesis was raised on the interpretation of late ICGA infrared photography in case of transthyretin amyloidosis with neuropathy. Conclusions: Beside being a vascular dye, ICG is transported from the vessels to the RPE toward the outer retina. It stains mast cells and large choroidal nerves. These observations could help the analysis of ICGA images.

The choroidal nervous system: a link between mineralocorticoid receptor and pachychoroid.

Central serous chorioretinopathy (CSCR) belongs to the pachychoroid spectrum, a pathological phenotype of the choroidal vasculature, in which blood flow is under the choroidal nervous system (ChNS) regulation. The pathogenesis of CSCR is multifactorial, with the most recognised risk factor being intake of glucocorticoids, which activate both the gluco- and the mineralocorticoid (MR) receptors. As MR over-activation is pathogenic in the retina and choroid, it could mediate the pathogenic effects of glucocorticoids in CSCR. But the role of MR signalling in pachychoroid is unknown and whether it affects the ChNS has not been explored. Using anatomo-neurochemical characterisation of the ChNS in rodents and humans, we discovered that beside innervation of arteries, choroidal veins and choriocapillaris are also innervated, suggesting that the entire choroidal vasculature is under neural control. The numerous synapses together with calcitonin gene-related peptide (CGRP) vesicles juxtaposed to choroidal macrophages indicate a neuro-immune crosstalk. Using ultrastructural approaches, we show that transgenic mice overexpressing human MR, display a pachychoroid-like phenotype, with signs of choroidal neuropathy including myelin abnormalities, accumulation and enlargement of mitochondria and nerves vacuolization. Transcriptomic analysis of the RPE/choroid complex in the transgenic mice reveals regulation of corticoids target genes, known to intervene in nerve pathophysiology, such as Lcn2, rdas1/dexras1, S100a8 and S100a9, rabphilin 3a (Rph3a), secretogranin (Scg2) and Kinesin Family Member 5A (Kif5a). Genes belonging to pathways related to vasculature development, hypoxia, epithelial cell apoptosis, epithelial mesenchymal transition, and inflammation, support the pachychoroid phenotype and highlight downstream molecular targets. Hypotheses on the imaging phenotype of pachychoroid in humans are put forward in the light of these new data. Our results provide evidence that MR overactivation causes a choroidal neuropathy that could explain the pachychoroid phenotype found in transgenic mice overexpressing human MR. In patients with pachychoroid and CSCR in which systemic dysautonomia has been demonstrated, MR-induced choroidal neuropathy could be the missing link between corticoids and pachychoroid.

Mineralocorticoid Receptor Pathway and Its Antagonism in a Model of Diabetic Retinopathy.

Diabetic retinopathy remains a major cause of vision loss worldwide. Mineralocorticoid receptor (MR) pathway activation contributes to diabetic nephropathy, but its role in retinopathy is unknown. In this study, we show that MR is overexpressed in the retina of type 2 diabetic Goto-Kakizaki (GK) rats and humans and that cortisol is the MR ligand in human eyes. Lipocalin 2 and galectin 3, two biomarkers of diabetes complications regulated by MR, are increased in GK and human retina. The sustained intraocular delivery of spironolactone, a steroidal mineralocorticoid antagonist, decreased the early and late pathogenic features of retinopathy in GK rats, such as retinal inflammation, vascular leakage, and retinal edema, through the upregulation of genes encoding proteins known to intervene in vascular permeability such as Hey1, Vldlr, Pten, Slc7a1, Tjp1, Dlg1, and Sesn2 but did not decrease VEGF. Spironolactone also normalized the distribution of ion and water channels in macroglial cells. These results indicate that MR is activated in GK and human diabetic retina and that local MR antagonism could be a novel therapeutic option for diabetic retinopathy.

Meteorin Is a Novel Therapeutic Target for Wet Age-Related Macular Degeneration.

The aim of this study was to evaluate the potential anti-angiogenic effect of MTRN (meteorin) in the laser-induced CNV rat model and explore its mechanisms of action. MTRN, thrompospondin-1, glial cell markers (GFAP, vimentin), and phalloidin were immuno-stained in non-human primate flat-mounted retinas and human retina cross sections. The effect of MTRN at different doses and time points was evaluated on laser-induced CNV at 14 days using in vivo fluorescein angiography and ex vivo quantification of CNV. A pan transcriptomic analysis of the retina and the RPE/choroid complex was used to explore MTRN effects mechanisms. In human retina, MTRN is enriched in the macula, expressed in and secreted by glial cells, and located in photoreceptor cells, including in nuclear bodies. Intravitreal MTRN administered preventively reduced CNV angiographic scores and CNV size in a dose-dependent manner. The highest dose, administered at day 7, also reduced CNV. MTRN, which is regulated by mineralocorticoid receptor modulators in the rat retina, regulates pathways associated with angiogenesis, oxidative stress, and neuroprotection. MTRN is a potential novel therapeutic candidate protein for wet AMD.

[Macular edema: Understand mechanisms to develop treatments]. / Les œdèmes maculaires - Mieux comprendre leurs mécanismes pour mieux les traiter.

Macular edema is an increase in volume of the central area of the retina, responsible for visual acuity. Visual symptoms handicap the lives of millions of patients with macular edema secondary to chronic and sometimes acute retinal disease. Proteins that neutralize the vascular endothelial growth factor (VEGF) pathway or glucocorticoids, at the cost of repeated intraocular injections over years, limit visual symptoms. A better understanding of why and how edema forms and how therapeutic molecules exert an anti-edematous effect will help prevent this disabling and blinding retinal complication from occurring.

Title: Les œdèmes maculaires - Mieux comprendre leurs mécanismes pour mieux les traiter. Abstract: L'œdème maculaire est une augmentation de volume de la macula, zone centrale de la rétine, responsable de l'acuité visuelle. Des symptômes visuels handicapent la vie de millions de patients atteints d'œdème maculaire secondaire à une maladie chronique et parfois aiguë de la rétine. Les protéines qui neutralisent la voie du facteur de croissance de l'endothélium vasculaire (VEGF) ou les glucocorticoïdes, au prix d'injections intraoculaires répétées pendant des années, limitent les symptômes visuels. Mieux comprendre pourquoi et comment l'œdème se forme et comment les molécules thérapeutiques exercent un effet anti-œdémateux permettra de mieux prévenir la survenue de cette complication rétinienne handicapante et cécitante.

[Anatomy of the retina]. / Anatomie de la rétine.

The neuroretina is a functional unit of the central nervous system that converts a light signal into a nerve impulse. Of neuroectodermal origin, derived from the diencephalon, the neuroretina is a layered tissue composed of six types of neuronal cells (two types of photoreceptors: cones and rods, horizontal, bipolar, amacrine and ganglion cells) and three types of glial cells (Müller glial cells, astrocytes and microglial cells). The neuroretina lays on the retinal pigmentary epithelium, that together form the retina. The existence of the internal and external blood-retinal barriers and intra-retinal junctions reflects the fineness of regulation of the retinal exchanges with the circulation and within the retina itself. The central zone of the human retina, which is highly specialized for visual acuity, has anatomical specificities. Recent imaging methods make it possible now to enrich our knowledge of the anatomical and functional characteristics of the retina, which are still imperfectly described.

TITLE: Anatomie de la rétine. ABSTRACT: La neurorétine est une unité fonctionnelle du système nerveux central assurant la conversion d'un signal lumineux en un influx nerveux. D'origine neuroectodermique, dérivée du diencéphale, la neurorétine est un tissu stratifié, composé de six types de cellules neuronales (deux types de photorécepteurs : les cônes et les bâtonnets ; les cellules horizontales, bipolaires, amacrines et ganglionnaires) et de trois types de cellules gliales (les cellules gliales de Müller, les astrocytes et les cellules microgliales). La neurorétine repose sur l'épithélium pigmentaire rétinien, l'ensemble constituant la rétine. L'existence des barrières hémato-rétiniennes interne et externe et des jonctions intra-rétiniennes rend compte de la finesse de la régulation des échanges de la rétine avec la circulation et au sein de la rétine elle-même. La zone centrale de la rétine humaine, la macula, zone hautement spécialisée pour assurer l'acuité visuelle, présente des spécificités anatomiques. Les méthodes d'imagerie récentes permettent d'enrichir nos connaissances sur les caractéristiques anatomiques et fonctionnelles de la rétine, qui restent encore imparfaitement décrites.

Mechanisms of FH Protection Against Neovascular AMD.

A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.

Glial cells of the human fovea.

Purpose: The exact cellular types that form the human fovea remain a subject of debate, and few studies have been conducted on human macula to solve this question. The purpose of this study was to perform immunohistochemistry on fresh human samples to characterize the glial cells that form the human fovea. Methods: Immunohistochemistry was performed using antibodies against proteins expressed in astrocytes or in retinal Müller glial cells or both types of cells on six human macula obtained from eyes enucleated for peripheral intraocular tumors and on two postmortem eyes from healthy donors. The posterior poles of the enucleated eyes were cryosectioned and stained with antibodies against the glial proteins GFAP, vimentin, CRALBP, glutamine synthetase, and connexin 43. Results: A population of cells positive for GFAP and negative for glutamine synthetase and CRALBP that express connexin 43 were identified at the roof of the foveal pit. These cells are distinct from the Müller cone cells described by Yamada and Gass, suggesting that another type of foveal glial cells, most likely astrocytes, are present in the human fovea. Conclusions: This study showed that in humans, astrocytic glial cells cover the foveal pit. Their roles in macula homeostasis and mechanisms of macular diseases disease remain to be determined.

Nogo-A-targeting antibody promotes visual recovery and inhibits neuroinflammation after retinal injury.

N-Methyl-D-aspartate (NMDA)-induced neuronal cell death is involved in a large spectrum of diseases affecting the brain and the retina such as Alzheimer's disease and diabetic retinopathy. Associated neurological impairments may result from the inhibition of neuronal plasticity by Nogo-A. The objective of the current study was to determine the contribution of Nogo-A to NMDA excitotoxicity in the mouse retina. We observed that Nogo-A is upregulated in the mouse vitreous during NMDA-induced inflammation. Intraocular injection of a function-blocking antibody specific to Nogo-A (11C7) was carried out 2 days after NMDA-induced injury. This treatment significantly enhanced visual function recovery in injured animals. Strikingly, the expression of potent pro-inflammatory molecules was downregulated by 11C7, among which TNFα was the most durably decreased cytokine in microglia/macrophages. Additional analyses suggest that TNFα downregulation may stem from cofilin inactivation in microglia/macrophages. 11C7 also limited gliosis presumably via P.Stat3 downregulation. Diabetic retinopathy was associated with increased levels of Nogo-A in the eyes of donors. In summary, our results reveal that Nogo-A-targeting antibody can stimulate visual recovery after retinal injury and that Nogo-A is a potent modulator of excitotoxicity-induced neuroinflammation. These data may be used to design treatments against inflammatory eye diseases.