Phototoxicity of low doses of light and influence of the spectral composition on human RPE cells.

Light is known to induce retinal damage affecting photoreceptors and retinal pigment epithelium. For polychromatic light, the blue part of the spectrum is thought to be the only responsible for photochemical damage, leading to the establishment of a phototoxicity threshold for blue light (445 nm). For humans it corresponds to a retinal dose of 22 J/cm2. Recent studies on rodents and non-human primates suggested that this value is overestimated. In this study, we aim at investigating the relevance of the current phototoxicity threshold and at providing new hints on the role of the different components of the white light spectrum on phototoxicity. We use an in vitro model of human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelial (iRPE) cells and exposed them to white, blue and red lights from LED devices at doses below 22 J/cm2. We show that exposure to white light at a dose of 3.6 J/cm2 induces an alteration of the global cellular structure, DNA damage and an activation of cellular stress pathways. The exposure to blue light triggers DNA damage and the activation of autophagy, while exposure to red light modulates the inflammatory response and inhibits autophagy.

The blue light hazard and its use on the evaluation of photochemical risk for domestic lighting. An in vivo study.

BACKGROUND: Nowadays artificial light highly increases human exposure to light leading to circadian rhythm and sleep perturbations. Moreover, excessive exposure of ocular structures to photons can induce irreversible retinal damage. Meta-analyses showed that sunlight exposure influences the age of onset and the progression of Age-related macular degeneration (AMD), the leading cause of blindness in people over fifty-year old. Currently, the blue-light hazard (BLH) curve is used in the evaluation of the phototoxicity of a light source for domestic lighting regulations. OBJECTIVES: Here, we analyze the phototoxicity threshold in rats and investigate the role played by the light spectrum, assessing the relevance of the use of the BLH-weighting to define phototoxicity. METHODS: We exposed albino rats to increasing doses of blue and white light, or to lights of different colors to evaluate the impact of each component of the white light spectrum on phototoxicity. Cellular mechanisms of cell death and cellular stress induced by light were analyzed. RESULTS: Our results show that the phototoxicity threshold currently accepted for rats is overestimated by a factor of 50 when considering blue light and by a factor of 550 concerning white light. This is the result of the toxicity induced by green light that increases white light toxicity by promoting an inflammatory response. The content of green in white light induces 8 fold more invasion of macrophages in the retina than the content of blue light. Moreover, the use of BLH-weighting does not evaluate the amount of red radiations contained in white light that mitigates damage by inhibiting the nuclear translocation of L-DNase II and reducing by 33% the number of TUNEL-positive cells. DISCUSSION: These findings question the current methods to determine the phototoxicity of a light source and show the necessity to take into account the entire emission spectrum. As current human phototoxicity thresholds were estimated with the same methods used for rats, our results suggest that they might need to be reconsidered.

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.

Multimodal imaging in early onset non-neovascular pigment epithelial detachments.

PURPOSE: To describe multimodal imaging of two cases of bilateral non-vascularized pigment epithelial detachments (PED) in young patients with a long-term follow-up. METHODS: A complete ophthalmological examination was performed at each follow-up visit including best corrected visual acuity (BCVA), intraocular pressure, slit lamp examination, spectral domain optical coherence tomography (SD-OCT), fluorescein and indocyanine green angiography, OCT angiography. RESULTS: Multimodal imaging of two women presenting avascular PED, aged 43 and 57, respectively, was described. In both patients, SD-OCT revealed a high central macular hyporeflective elevation corresponding with PED. Both patients showed a choroidal layer thicker than 420 µm. Fluorescein and indocyanine green angiography didn't show any choroidal neovascularization either at early or late frames. Cross-sectional and en face optical coherence tomography angiography (OCTA) didn't show any flow beneath the PED. During the follow up period one eye showed a retinal pigment epithelium tear and all eyes showed the presence of apical sub-retinal fluid and hyperreflective material on the top of the PED. None of the two patients showed any sign of atrophy during the follow-up period. CONCLUSION: The peculiar characteristics of the presented cases suggest that specific pathogenetic mechanisms, not necessarily related to age related macular degeneration, may play a key role in the development of these lesions. Whether early onset of such drusenoid PED is a specific entity resulting from a genetic deficit of lipid transporters in the RPE is unknown. Further genetic and metabolic studies should be conducted.

Spironolactone Eyedrop Favors Restoration of Corneal Integrity after Wound Healing in the Rat.

Abnormal corneal wound healing can compromise corneal transparency and lead to visual impairment. Mineralocorticoid receptor antagonists (MRA) are promising candidates to promote corneal remodeling with anti-inflammatory properties and lack gluococorticoids-associated side effects. In this preclinical study, a new polymer-free hydroxypropyl-gamma-cyclodextrin-based eyedrop containing 0.1% spironolactone (SPL), a potent but non-water-soluble MRA, was investigated for its ocular surface tolerance and efficacy in a rat model of corneal wound healing. SPL eyedrops were stable for up to 9 months at 4 °C. The formulation was well-tolerated since no morphological changes or inflammatory reactions were observed in the rat cornea after multiple daily instillations over 7 days. SPL eyedrops accelerated rat corneal wound healing, reduced corneal edema and inflammation, enhanced epithelial integrity, and improved nerve regeneration, suggesting restoration of corneal homeostasis, while potassium canrenoate, an active and soluble metabolite of SPL, had no effect. SPL eyedrops could benefit patients with impaired corneal wound healing, including that secondary to glucocorticoid therapy. Repurposing known drugs with known excipients will expedite translation to the clinic.

Evaluation of Ocular and Systemic Oxidative Stress Markers in Ocular Rosacea Patients.

Purpose: To investigate oxidative stress markers in tears and serum of patients with ocular rosacea and to examine their association with both ocular surface parameters and cutaneous rosacea subtypes. Methods: This prospective study includes rosacea patients with ocular involvement and healthy controls. We performed ophthalmological examination of all participants and collected tear breakup time (TBUT), Schirmer, Meibomian gland dysfunction (MGD) and Ocular Surface Disease Index (OSDI) scores. We quantified the total antioxidant status (TAS), total oxidant status (TOS), and arylesterase (ARE) levels from tear and serum samples, and calculated the oxidative stress index (OSI). We also classified patients into phymatous, erythematotelangiectatic, papulopustular subtypes. Results: We included 90 ocular rosacea patients and 30 healthy controls. Oxidative stress (TOS, OSI) levels were significantly higher (P < 0.01) and antioxidant levels (TAS, ARE) were significantly lower (P < 0.01) in both tear and serum samples of ocular rosacea patients as compared to controls. We found a significant positive correlation between the tear and serum values regarding oxidative stress parameters (P < 0.05). Besides, OSI was negatively correlated with TBUT and positively correlated with MGD score (meiboscore) and OSDI (P < 0.05). The Schirmer score was not correlated with OSI. No difference was found between the cutaneous subtypes with respect to TAS, TOS, ARE, and OSI results. Conclusions: In this study, we identified oxidative stress markers in the serum and tears of ocular rosacea patients and showed their correlation with clinical signs of MGD, suggesting that oxidative stress contributes to ocular rosacea pathogenesis and that oxidative stress could be an indicator of MGD severity.

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.

In Vitro and In Vivo Safety of Hyaluronic Acid-Decorated Microparticles for Intravitreal Injection of Palmitoylethanolamide, Citicoline, or Glial-Cell-Derived Neurotrophic Factor.

The treatment of posterior eye segment diseases through intravitreal injection requires repeated injections of an active molecule, which may be associated with serious side effects and poor patient compliance. One brilliant strategy to overcome these issues is the use of drug-loaded microparticles for sustained release, aiming at reducing the frequency of injections. Therefore, the aim of this work was to assess the safety features of poly(lactic-co-glycolic acid) (PLGA)-based, hyaluronic acid-decorated microparticles loaded with palmitoylethanolamide (PEA), citicoline (CIT), or glial-cell-derived neurotrophic factor (GDNF). Microparticles were prepared by double emulsion-solvent evaporation and fully characterized for their technological features. Microparticles possessed a satisfactory safety profile in vitro on human retinal pigment epithelial (ARPE-19) cells. Interestingly, the administration of free GDNF led to a loss of cell viability, while GDNF sustained release displayed a positive effect in that regard. In vivo results confirmed the safety profile of both empty and loaded microparticles. Overall, the outcomes suggest that the produced microparticles are promising for improving the local administration of neuroprotective molecules. Further studies will be devoted to assess the therapeutic ability of microparticles.

Ocular steroidome in human eyes and in eyes with complex central serous chorioretinopathy (CSCR).

The exact link between systemic and ocular endogenous corticoids (steroidome) is unclear and whether the ocular steroidome is altered in CSCR eyes is unknown. The aims of this study were to analyze the human steroidome in the aqueous humor as a function of age, sex and time of the day, to correlate systemic and ocular steroidome and to analyze the ocular steroidome in long lasting complex inactive CSCR. Based on our results, we present two CSCR cases treated by the combination of oral mineralocorticoid antagonist and glucocorticoids drops. In a cross-sectional study, aqueous humor (AH) was collected between 8am and 6 pm from 50 unaffected individuals (25 men and 25 women) and from 14 patients with chronic CSCR, during cataract surgery. In addition, simultaneous serum and AH were collected from 27 individuals undergoing cataract surgery and, simultaneous AH and vitreous were collected from 9 patients undergoing cataract and vitrectomy to estimate corticoids levels in the different compartments. The steroidome was determined using a LC-MS/MS method that quantifies 13 endogenous corticoids from the gluco, mineralocorticoid and androgen pathways. In AH and vitreous, the highest corticoid level is reached by cortisol (F), that represents less than 10% of F serum level. The cortisol levels in the serum did not correlate with ocular cortisol levels. Serum and ocular cortisone (E) levels correlate, although less than 5% of circulating E reaches the eye. The only mineralocorticoids measured in the AH were corticosterone (B) and its inactive form, the 11-desoxycorticosterone (A). There was no influence of circadian rhythm on cortisol ocular levels and there was no correlation between the age or the sex and the level of F, E, A, and B. In eyes with chronic inactive CSCR, the levels of the active glucocorticoid form F was lower than in control eyes and the F/E ratio was reduced by 50% but the B/A ratio was higher indicating imbalance towards active mineralocorticoids. Base on this observation, we propose to combine an antagonist of the mineralocorticoid receptor together with topical glucocorticoids in two CSCR patients, resistant to all other treatments, with favorable outcome. Our results indicate that the ocular psteroidome is highly regulated suggesting a local metabolism of ocular corticoids. In eyes with long-lasting complex inactive CSCR, the steroidome analysis shows lower active glucocorticoids and higher active mineralocorticoids.

POTENTIAL TREATMENT FOR PERIPAPILLARY PACHYCHOROID SYNDROME.

PURPOSE: The purpose of this study was to describe the effect of topical prednisolone on intraretinal fluid in patients with peripapillary pachychoroid syndrome. METHODS: We selected 11 consecutive patients (17 eyes) with a diagnosis of peripapillary pachychoroid syndrome, who were treated with topical prednisolone (Pred Forte [PF] 10 mg/mL) three times daily for 4 weeks. We tapered off PF among patients who demonstrated a reduction of intraretinal fluid. RESULTS: Of the included 17 eyes, the average follow-up before PF treatment ranged from 6 to 192 months, during which patients experienced no apparent reduction of intraretinal fluid. The baseline mean best-corrected VA (BCVA) was 0.6 (20/33) Snellen. The median subfoveal and peripapillary choroidal thickness were 430 µm and 202 µm, respectively. All patients showed an initial reduction of intraretinal fluid after 4 weeks of topical prednisolone. Six patients (35%) experienced a prolonged reduction of intraretinal fluid when the dosage was reduced to once daily. On tapering off PF, four eyes (24%) experienced a recurrence of intraretinal fluid. Four eyes (24%) experienced elevated intraocular pressure above 26 mmHg. In two eyes, PF was discontinued, on which intraretinal fluid reappeared. The BCVA seemed to be improved in 9 eyes (53%) and remained equal in 4 eyes (24%). CONCLUSION: In this case series of patients with peripapillary pachychoroid syndrome, we observed a reduction of peripapillary intraretinal fluid after treatment with topical prednisolone for 4 weeks in all 17 eyes. The disappearance of intraretinal fluid seemed to concede with a slight improvement in BCVA for some cases. Thus, topical prednisolone may prove to be a viable treatment option in peripapillary pachychoroid syndrome.

INDOCYANINE GREEN ANGIOGRAPHY OF TYPE 1 MACULAR NEOVASCULARIZATION IN AGE-RELATED MACULAR DEGENERATION AND CENTRAL SEROUS CHORIORETINOPATHY REVEALS DIFFERENT DISEASE MECHANISMS.

PURPOSE: To assess the rate of late phase hyperfluorescent plaque (LPHP) in Type 1 macular neovascularization (MNV) in central serous chorioretinopathy (CSCR) and age-related macular degeneration (AMD) and to evaluate its prognostic value. METHODS: Retrospective study including Type 1 MNV in AMD and CSCR, from 2012 to 2020. Eyes with a late indocyanine green angiography image (>20 minutes) and clear visualization of MNV on optical coherence tomography angiography (OCTA) were included. Quantitative and qualitative parameters on optical coherence tomography and best-corrected visual acuity were recorded at baseline and after three monthly antivascular endothelial growth factor injections. RESULTS: Eighty-three eyes were included, 35 with CSCR and 48 with AMD. Patients in the CSCR group were significantly younger than in the AMD group (61.3 ± 10.4 vs. 80.2 ± 6.8 years, respectively, P < 0.001), predominantly male (68.6% CSCR vs. 35.4% AMD; P = 0.003), and with a thicker choroid (379 ± 93.3 µ m vs. 204.2 ± 93.2 µ m; P < 0.001). Type 1 MNV in CSCR showed fewer LPHP compared with AMD (31.4% vs. 77.1%; P < 0.001). The baseline visual acuity was lower in patients with LPHP (0.37 ± 0.22 vs. 0.27 ± 0.28 logarithm of the minimum angle of resolution, P = 0.03). On multivariate analysis, AMD was associated with the presence of LPHP ( P < 0.001). No significant difference in the response to antivascular endothelial growth factor was observed. CONCLUSION: Leakage of macromolecules from MNV and accumulation in the retinal pigment epithelium and/or in the stroma imaged by the LPHP is less common in eyes with Type 1 MNV in CSCR than in AMD. Late phase indocyanine green angiography imaging offers an insight into the metabolism of the dye and the environment surrounding the neovascular membrane.

High Levels of C-Reactive Protein with Low Levels of Pentraxin 3 as Biomarkers for Central Serous Chorioretinopathy.

Purpose: To investigate the association between the 2 acute phase proteins, C-reactive protein (CRP) and pentraxin 3 (PTX3) with central serous chorioretinopathy (CSCR), as PTX3 is a glucocorticoid-induced protein. Design: Cross-sectional multicenter study. Participants: Patients with CSCR compared with age- and sex-matched healthy participants. Methods: Patients with CSCR from 3 centers in Europe were included in the study. The clinical form of CSCR was recorded. Blood samples from patients with CSCR and healthy participants were sampled, and high-sensitivity CRP and PTX3 levels were measured in the serum. Main Outcome Measures: C-reactive protein and PTX3 serum level comparison between patients with CSCR with age- and sex-matched healthy participants. Results: Although CRP levels were higher in patients with CSCR (n = 216) than in age- and sex-matched controls (n = 130) (2.2 ± 3.2 mg/l vs. 1.5 mg/l ± 1.4, respectively, P = 0.037), PTX3 levels were lower in patients with CSCR (10.5 ± 19.9 pg/ml vs. 87.4 ± 73.2 pg/ml, respectively, P < 0.001). There was no significant difference in CRP or PTX3 levels between patients with acute/recurrent and chronic CSCR. Conclusions: In patients with CSCR, high CRP and low PTX3 levels suggest a form of low-grade systemic inflammation together with a lack of glucocorticoid pathway activation, raising new hypotheses on the pathophysiology of CSCR. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Congenital aniridia beyond black eyes: From phenotype and novel genetic mechanisms to innovative therapeutic approaches.

Congenital PAX6-aniridia, initially characterized by the absence of the iris, has progressively been shown to be associated with other developmental ocular abnormalities and systemic features making congenital aniridia a complex syndromic disorder rather than a simple isolated disease of the iris. Moreover, foveal hypoplasia is now recognized as a more frequent feature than complete iris hypoplasia and a major visual prognosis determinant, reversing the classical clinical picture of this disease. Conversely, iris malformation is also a feature of various anterior segment dysgenesis disorders caused by PAX6-related developmental genes, adding a level of genetic complexity for accurate molecular diagnosis of aniridia. Therefore, the clinical recognition and differential genetic diagnosis of PAX6-related aniridia has been revealed to be much more challenging than initially thought, and still remains under-investigated. Here, we update specific clinical features of aniridia, with emphasis on their genotype correlations, as well as provide new knowledge regarding the PAX6 gene and its mutational spectrum, and highlight the beneficial utility of clinically implementing targeted Next-Generation Sequencing combined with Whole-Genome Sequencing to increase the genetic diagnostic yield of aniridia. We also present new molecular mechanisms underlying aniridia and aniridia-like phenotypes. Finally, we discuss the appropriate medical and surgical management of aniridic eyes, as well as innovative therapeutic options. Altogether, these combined clinical-genetic approaches will help to accelerate time to diagnosis, provide better determination of the disease prognosis and management, and confirm eligibility for future clinical trials or genetic-specific therapies.

In Vivo Retinal Pigment Epithelium Imaging using Transscleral Optical Imaging in Healthy Eyes.

Objective: To image healthy retinal pigment epithelial (RPE) cells in vivo using Transscleral OPtical Imaging (TOPI) and to analyze statistics of RPE cell features as a function of age, axial length (AL), and eccentricity. Design: Single-center, exploratory, prospective, and descriptive clinical study. Participants: Forty-nine eyes (AL: 24.03 ± 0.93 mm; range: 21.9-26.7 mm) from 29 participants aged 21 to 70 years (37.1 ± 13.3 years; 19 men, 10 women). Methods: Retinal images, including fundus photography and spectral-domain OCT, AL, and refractive error measurements were collected at baseline. For each eye, 6 high-resolution RPE images were acquired using TOPI at different locations, one of them being imaged 5 times to evaluate the repeatability of the method. Follow-up ophthalmic examination was repeated 1 to 3 weeks after TOPI to assess safety. Retinal pigment epithelial images were analyzed with a custom automated software to extract cell parameters. Statistical analysis of the selected high-contrast images included calculation of coefficient of variation (CoV) for each feature at each repetition and Spearman and Mann-Whitney tests to investigate the relationship between cell features and eye and subject characteristics. Main Outcome Measures: Retinal pigment epithelial cell features: density, area, center-to-center spacing, number of neighbors, circularity, elongation, solidity, and border distance CoV. Results: Macular RPE cell features were extracted from TOPI images at an eccentricity of 1.6° to 16.3° from the fovea. For each feature, the mean CoV was < 4%. Spearman test showed correlation within RPE cell features. In the perifovea, the region in which images were selected for all participants, longer AL significantly correlated with decreased RPE cell density (R Spearman, Rs = -0.746; P < 0.0001) and increased cell area (Rs = 0.668; P < 0.0001), without morphologic changes. Aging was also significantly correlated with decreased RPE density (Rs = -0.391; P = 0.036) and increased cell area (Rs = 0.454; P = 0.013). Lower circular, less symmetric, more elongated, and larger cells were observed in those > 50 years. Conclusions: The TOPI technology imaged RPE cells in vivo with a repeatability of < 4% for the CoV and was used to analyze the influence of physiologic factors on RPE cell morphometry in the perifovea of healthy volunteers. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

"En Face" Spectral-Domain Optical Coherence Tomography versus Multifocal Electroretinogram in Hydroxychloroquine Retinopathy Screening.

INTRODUCTION: The performance of "en face" optical coherence tomography (OCT) in screening for chloroquine (CQ) or hydroxychloroquine (HCQ) retinopathy has not been largely explored. The aim of this study was to determine the concordance of "en face" OCT with multifocal electroretinography (mfERG) in screening for CQ/HCQ retinopathy. METHODS: This is a prospective cohort study conducted at the Rothschild Foundation Hospital, Paris, between August 2016 and February 2021. Patients taking HCQ were followed up over 2 consecutive years and received an "en face" OCT and a mfERG on each visit. RESULTS: A total of 91 patients (182 eyes) were analyzed. mfERG and "en face" OCT were concordant in 147 eyes (86.3%). Cohen's kappa coefficient for concordance between mfERG and "en face" OCT was considered weak with a value 0.61 (95% CI: 0.50-0.72). The sensitivity and specificity of "en face" OCT were 70% (95% CI: 59-79%) and 91% (95% CI: 83-96%), respectively, relatively to mfERG. Proportion of abnormal R2/R5 and R3/R5 ratios did not differ between patients with normal and abnormal "en face" OCT (p = 0.2). DISCUSSION: "En face" OCT and mfERG have low concordance and cannot be used interchangeably as each investigation evaluates a different facet of CQ/HCQ retinopathy. "En face" OCT could be used as a complement in screening for CQ/HCQ retinal toxicity if the anomalies detected on "en face" OCT are confirmed by B-scan OCT sections.

Toll-like receptor-4 expression and oxidative stress in ocular rosacea.

Purpose: To investigate systemic and ocular toll-like receptor (TLR)-4 expression and its association with oxidative stress markers in ocular rosacea (OR). Methods: This prospective study included 40 patients with rosacea with ocular involvement and 20 healthy volunteers. Tear break-up time (TBUT), Schirmer test, meibomoscore, and ocular surface disease index (OSDI) scores were estimated for all participants. TLR-4 expression in conjunctival epithelium and peripheral blood mononuclear cells was quantified using real-time polymerase chain reaction (RT-PCR). In the tears and serum samples of all participants, antioxidant status (TAS), total oxidant status (TOS), and arylesterase (ARE) activation levels were measured using a fully automated spectrophotometric method, and the oxidative stress index (OSI) was calculated. Results: TLR-4 expression levels and oxidative stress status (TOS and OSI values) were significantly higher (p < 0.01), and antioxidant status (TAS and ARE values) were significantly lower (p < 0.01) in both ocular and blood samples of patients with OR compared with those in controls. A significant positive correlation was found between the ocular and blood values in all parameters (p < 0.05). According to the clinical associations of these results, we found negative correlations between TLR-4, OSI, and TBUT and between TLR-4 and Schirmer, whereas a positive correlation was observed between TLR-4, OSI, and meiboscore and between TLR-4, OSI, and OSDI (p < 0.05). No correlation was found between the OSI and Schirmer results (p = 0.92). Conclusions: TLR-4 and oxidative stress both play important roles in OR pathophysiology and are closely related to clinical findings.

Neuroprotective Effects of Transferrin in Experimental Glaucoma Models.

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.

Nestin contributes to laser choroidal and retinal neovascularization.

Purpose: Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells. Methods: We used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF). Results: In the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels. Conclusions: Our results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.

The Use of Polymer Blends in the Treatment of Ocular Diseases.

The eye is an organ with limited drug access due to its anatomical and physiological barriers, and the usual forms of ocular administration are limited in terms of drug penetration, residence time, and bioavailability, as well as low patient compliance. Hence, therapeutic innovations in new drug delivery systems (DDS) have been widely explored since they show numerous advantages over conventional methods, besides delivering the content to the eye without interfering with its normal functioning. Polymers are usually used in DDS and many of them are applicable to ophthalmic use, especially biodegradable ones. Even so, it can be a hard task to find a singular polymer with all the desirable properties to deliver the best performance, and combining two or more polymers in a blend has proven to be more convenient, efficient, and cost-effective. This review was carried out to assess the use of polymer blends as DDS. The search conducted in the databases of Pubmed and Scopus for specific terms revealed that although the physical combination of polymers is largely applied, the term polymer blend still has low compliance.