Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma.

Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the retinal ganglion cells (RGCs), leading to irreversible vision loss. Nowadays, the traditional therapeutic approach to glaucoma consists of lowering the intraocular pressure (IOP), which does not address the neurodegenerative features of the disease. Besides animal models of glaucoma, there is a considerable need for in vitro experimental models to propose new therapeutic strategies for this ocular disease. In this study, we elucidated the pathological mechanisms leading to neuroretinal R28 cell death after exposure to glutamate and hydrogen peroxide (H2O2) in order to develop new therapeutic approaches for oxidative stress-induced retinal diseases, including glaucoma. We were able to show that glutamate and H2O2 can induce a decrease in R28 cell viability in a concentration-dependent manner. A cell viability of about 42% was found after exposure to 3 mM of glutamate and about 56% after exposure to 100 µM of H2O2 (n = 4). Label-free quantitative mass spectrometry analysis revealed differential alterations of 193 and 311 proteins in R28 cells exposed to 3 mM of glutamate and 100 µM of H2O2, respectively (FDR < 1%; p < 0.05). Bioinformatics analysis indicated that the protein changes were associated with the dysregulation of signaling pathways, which was similar to those observed in glaucoma. Thus, the proteomic alteration induced by glutamate was associated with the inhibition of the PI3K/AKT signaling pathway. On the other hand, H2O2-induced toxicity in R28 cells was linked to the activation of apoptosis signaling and the inhibition of the mTOR and ERK/MAPK signaling pathways. Furthermore, the data show a similarity in the inhibition of the EIF2 and AMPK signaling pathways and the activation of the sumoylation and WNT/ß-catenin signaling pathways in both groups. Our findings suggest that the exposure of R28 cells to glutamate and H2O2 could induce glaucoma-like neurodegenerative features and potentially provide a suitable tool for the development of new therapeutic strategies for retinal diseases.

Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

The prone position in COVID-19 impacts the thickness of peripapillary retinal nerve fiber layers and macular ganglion cell layers.

The prone position reduces mortality in severe cases of COVID-19 with acute respiratory distress syndrome. However, visual loss and changes to the peripapillary retinal nerve fiber layer (p-RNFL) and the macular ganglion cell layer and inner plexiform layer (m-GCIPL) have occurred in patients undergoing surgery in the prone position. Moreover, COVID-19-related eye problems have been reported. This study compared the p-RNFL and m-GCIPL thicknesses of COVID-19 patients who were placed in the prone position with patients who were not. This prospective longitudinal and case-control study investigated 15 COVID-19 patients placed in the prone position (the "Prone Group"), 23 COVID-19 patients not in the prone position (the "Non-Prone Group"), and 23 healthy, non-COVID individuals without ocular disease or systemic conditions (the "Control Group"). The p-RNFL and m-GCIPL thicknesses of the COVID-19 patients were measured at 1, 3, and 6 months and compared within and between groups. The result showed that the Prone and Non-Prone Groups had no significant differences in their p-RNFL thicknesses at the 3 follow-ups. However, the m-GCIPL analysis revealed significant differences in the inferior sector of the Non-Prone Group between months 1 and 3 (mean difference, 0.74 µm; P = 0.009). The p-RNFL analysis showed a significantly greater thickness at 6 months for the superior sector of the Non-Prone Group (131.61 ± 12.08 µm) than for the Prone Group (118.87 ± 18.21 µm; P = 0.039). The m-GCIPL analysis revealed that the inferior sector was significantly thinner in the Non-Prone Group than in the Control Group (at 1 month 80.57 ± 4.60 versus 83.87 ± 5.43 µm; P = 0.031 and at 6 months 80.48 ± 3.96 versus 83.87 ± 5.43 µm; P = 0.044). In conclusion, the prone position in COVID-19 patients can lead to early loss of p-RNFL thickness due to rising intraocular pressure, which is independent of the timing of prone positioning. Consequently, there is no increase in COVID-19 patients' morbidity burden.

Retinal Nerve Fiber Layer Damage Assessment in Glaucomatous Eyes Using Retinal Retardance Measured by Polarization-Sensitive Optical Coherence Tomography.

Purpose: To assess the diagnostic performance and structure-function association of retinal retardance (RR), a customized metric measured by a prototype polarization-sensitive optical coherence tomography (PS-OCT), across various stages of glaucoma. Methods: This cross-sectional pilot study analyzed 170 eyes from 49 healthy individuals and 68 patients with glaucoma. The patients underwent PS-OCT imaging and conventional spectral-domain optical coherence tomography (SD-OCT), as well as visual field (VF) tests. Parameters including RR and retinal nerve fiber layer thickness (RNFLT) were extracted from identical circumpapillary regions of the fundus. Glaucomatous eyes were categorized into early, moderate, or severe stages based on VF mean deviation (MD). The diagnostic performance of RR and RNFLT in discriminating glaucoma from controls was assessed using receiver operating characteristic (ROC) curves. Correlations among VF-MD, RR, and RNFLT were evaluated and compared within different groups of disease severity. Results: The diagnostic performance of both RR and RNFLT was comparable for glaucoma detection (RR AUC = 0.98, RNFLT AUC = 0.97; P = 0.553). RR showed better structure-function association with VF-MD than RNFLT (RR VF-MD = 0.68, RNFLT VF-MD = 0.58; z = 1.99; P = 0.047) in glaucoma cases, especially in severe glaucoma, where the correlation between VF-MD and RR (r = 0.73) was significantly stronger than with RNFLT (r = 0.43, z = 1.96, P = 0.050). In eyes with early and moderate glaucoma, the structure-function association was similar when using RNFLT and RR. Conclusions: RR and RNFLT have similar performance in glaucoma diagnosis. However, in patients with glaucoma especially severe glaucoma, RR showed a stronger correlation with VF test results. Further research is needed to validate RR as an indicator for severe glaucoma evaluation and to explore the benefits of using PS-OCT in clinical practice. Translational Relevance: We demonstrated that PS-OCT has the potential to evaluate the status of RNFL structural damage in eyes with severe glaucoma, which is currently challenging in clinics.

Outer Retinal Thickness Is Associated With Cognitive Function in Normal Aging to Intermediate Age-Related Macular Degeneration.

Purpose: Research on Alzheimer's disease (AD) and precursor states demonstrates a thinner retinal nerve fiber layer (NFL) compared to age-similar controls. Because AD and age-related macular degeneration (AMD) both impact older adults and share risk factors, we asked if retinal layer thicknesses, including NFL, are associated with cognition in AMD. Methods: Adults ≥ 70 years with normal retinal aging, early AMD, or intermediate AMD per Age-Related Eye Disease Study (AREDS) nine-step grading of color fundus photography were enrolled in a cross-sectional study. Optical coherence tomography (OCT) volumes underwent 11-line segmentation and adjustments by a trained operator. Evaluated thicknesses reflect the vertical organization of retinal neurons and two vascular watersheds: NFL, ganglion cell layer-inner plexiform layer complex (GCL-IPL), inner retina, outer retina (including retinal pigment epithelium-Bruch's membrane), and total retina. Thicknesses were area weighted to achieve mean thickness across the 6-mm-diameter Early Treatment of Diabetic Retinopathy Study (ETDRS) grid. Cognitive status was assessed by the National Institutes of Health Toolbox cognitive battery for fluid and crystallized cognition. Correlations estimated associations between cognition and thicknesses, adjusting for age. Results: Based on 63 subjects (21 per group), thinning of the outer retina was significantly correlated with lower cognition scores (P < 0.05). No other retinal thickness variables were associated with cognition. Conclusions: Only the outer retina (photoreceptors, supporting glia, retinal pigment epithelium, Bruch's membrane) is associated with cognition in aging to intermediate AMD; NFL was not associated with cognition, contrary to AD-associated condition reports. Early and intermediate AMD constitute a retinal disease whose earliest, primary impact is in the outer retina. Our findings hint at a unique impact on the brain from the outer retina in persons with AMD.

TGFß Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage.

Purpose: Mutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ocular anterior segment dysgenesis (ASD) and glaucoma. We previously showed that transforming growth factor beta (TGFß) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFß signaling ameliorated ASD, supporting a role for the TGFß pathway in disease pathogenesis. Here, we tested whether altered TGFß signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice. Methods: To test the role of TGFß signaling in glaucoma-relevant phenotypes, we genetically reduced TGFß signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFß ligands in Col4a1+/G1344D mice. We performed slit-lamp biomicroscopy and optical coherence tomography for qualitative and quantitative analyses of anterior and posterior ocular segments, histological analyses of ocular tissues and optic nerves, and intraocular pressure assessments using rebound tonometry. Results: Col4a1+/G1344D mice showed defects of the ocular drainage structures, including iridocorneal adhesions, and phenotypes consistent with glaucomatous neurodegeneration, including thinning of the nerve fiber layer, retinal ganglion cell loss, optic nerve head excavation, and optic nerve degeneration. We found that reducing TGFß receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice. Conclusions: Our results suggest that elevated TGFß signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

Racial Differences in Diagnostic Accuracy of Retinal Nerve Fiber Layer Thickness in Primary Open-Angle Glaucoma.

Purpose: To evaluate the diagnostic accuracy of retinal nerve fiber layer thickness (RNFLT) by spectral-domain optical coherence tomography (OCT) in primary open-angle glaucoma (POAG) in eyes of African (AD) and European descent (ED). Design: Comparative diagnostic accuracy analysis by race. Participants: 379 healthy eyes (125 AD and 254 ED) and 442 glaucomatous eyes (226 AD and 216 ED) from the Diagnostic Innovations in Glaucoma Study and the African Descent and Glaucoma Evaluation Study. Methods: Spectralis (Heidelberg Engineering GmbH) and Cirrus (Carl Zeiss Meditec) OCT scans were taken within one year from each other. Main Outcome Measures: Diagnostic accuracy of RNFLT measurements. Results: Diagnostic accuracy for Spectralis-RNFLT was significantly lower in eyes of AD compared to those of ED (area under the receiver operating curve [AUROC]: 0.85 and 0.91, respectively, P=0.04). Results for Cirrus-RNFLT were similar but did not reach statistical significance (AUROC: 0.86 and 0.90 in AD and ED, respectively, P =0.33). Adjustments for age, central corneal thickness, axial length, disc area, visual field mean deviation, and intraocular pressure yielded similar results. Conclusions: OCT-RNFLT has lower diagnostic accuracy in eyes of AD compared to those of ED. This finding was generally robust across two OCT instruments and remained after adjustment for many potential confounders. Further studies are needed to explore the potential sources of this difference.

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Purpose: Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Methods: Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Results: Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. Conclusions: To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Quantifying Putative Retinal Gliosis in Preclinical Alzheimer's Disease.

Purpose: Neuroinflammation plays a significant role in the pathology of Alzheimer's disease (AD). Mouse models of AD and postmortem biopsy of patients with AD reveal retinal glial activation comparable to central nervous system immunoreactivity. We hypothesized that the surface area of putative retinal gliosis observed in vivo using en face optical coherence tomography (OCT) imaging will be larger in patients with preclinical AD versus controls. Methods: The Spectralis II instrument was used to acquire macular centered 20 × 20 and 30 × 25-degrees spectral domain OCT images of 76 participants (132 eyes). A cohort of 22 patients with preclinical AD (40 eyes, mean age = 69 years, range = 60-80 years) and 20 control participants (32 eyes, mean age = 66 years, range = 58-82 years, P = 0.11) were included for the assessment of difference in surface area of putative retinal gliosis and retinal nerve fiber layer (RNFL) thickness. The surface area of putative retinal gliosis and RNFL thickness for the nine sectors of the Early Treatment Diabetic Retinopathy Study (ETDRS) map were compared between groups using generalized linear mixed models. Results: The surface area of putative retinal gliosis was significantly greater in the preclinical AD group (0.97 ± 0.55 mm2) compared to controls (0.68 ± 0.40 mm2); F(1,70) = 4.41, P = 0.039; Cohen's d = 0.61. There was no significant difference between groups for RNFL thickness in the 9 ETDRS sectors, P > 0.05. Conclusions: Our analysis shows greater putative retinal gliosis in preclinical AD compared to controls. This demonstrates putative retinal gliosis as a potential biomarker for AD-related neuroinflammation.

POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells.

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON-OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2-/- mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

Fundus Tessellation and Parapapillary Atrophy, as Ocular Characteristics of Spontaneously High Myopia in Macaques: The Non-Human Primates Eye Study.

Purpose: This study aimed to evaluate the ocular characteristics associated with spontaneously high myopia in adult nonhuman primates (NHPs). Methods: A total of 537 eyes of 277 macaques with an average age of 18.53 ± 3.01 years (range = 5-26 years), raised in a controlled environment, were included. We measured ocular parameters, including spherical equivalent (SE), axial length (AXL), and intraocular pressure. The 45-degree fundus images centered on the macula and the disc assessed the fundus tessellation and parapapillary atrophy (PPA). Additionally, optical coherence tomography (OCT) was used to measure the thickness of the retinal nerve fiber layer (RNFL). Results: The mean SE was -1.58 ± 3.71 diopters (D). The mean AXL was 18.76 ± 0.86 mm. The prevalence rate of high myopia was 17.7%. As myopia aggravated, the AXL increased (r = -0.498, P < 0.001). Compared with non-high myopia, highly myopic eyes had a greater AXL (P < 0.001), less RNFL thickness (P = 0.004), a higher incidence of PPA (P < 0.001), and elevated grades of fundus tessellation (P < 0.001). The binary logistic regression was performed, which showed PPA (odds ratio [OR] = 4.924, 95% confidence interval [CI] = 2.375-10.207, P < 0.001) and higher grades of fundus tessellation (OR = 1.865, 95% CI = 1.474-2.361, P < 0.001) were independent risk characteristics for high myopia. Conclusions: In NHPs, a higher grade of fundus tessellation and PPA were significant biomarkers of high myopia. Translational Relevance: The study demonstrates adult NHPs raised in conditioned rooms have a similar prevalence and highly consistent fundus changes with human beings, which strengthens the foundation for utilizing macaques as an animal model in high myopic studies.

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models.

BACKGROUND: Retinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR). METHODS: An in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted. RESULTS: API treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission. CONCLUSION: API protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

CTRP 9 mitigates the apoptosis and unfolded protein response of OGD/R-induced retinal ganglion cells by regulating the AMPK pathway.

C1q/TNF-related protein-9 (CTRP9) has been reported to play roles in several types of retinal diseases. However, the role and the potential mechanism of CTRP9 in glaucoma are still incompletely understood. The expression of CTRP9 in OGD/R-induced retinal ganglion cells (RGCs) was detected by quantitative real-time polymerase chain reaction and western blot assay. Cell proliferation was identified by cell counting Kit-8 assay. Flow cytometry, enzyme-linked immunosorbent assay and western blot assay were performed to assess cell apoptosis. Unfolded protein response (UPR), endoplasmic reticulum (ER) stress and the AMPK pathway were evaluated by western blot assay. The data showed that the expression of CTRP9 was significantly downregulated in OGD/R-induced 661W cells. OGD/R treatment reduced cell viability, promoted cell apoptosis and activated the UPR and ER stress. The overexpression of CTRP9 reversed the effects of OGD/R on 661W cell viability, apoptosis, the UPR and ER stress, as well as the AMPK pathway. However, Compound C, an inhibitor of AMPK signaling, reversed the protection of CTRP9 overexpression against injury from OGD/R in 661W cells. In summary, the results revealed that CTRP9 abated the apoptosis and UPR of OGD/R-induced RGCs by regulating the AMPK pathway, which may provide a promising target for the treatment of glaucoma.

Convolutional neural networks develop major organizational principles of early visual cortex when enhanced with retinal sampling.

Primate visual cortex exhibits key organizational principles: cortical magnification, eccentricity-dependent receptive field size and spatial frequency tuning as well as radial bias. We provide compelling evidence that these principles arise from the interplay of the non-uniform distribution of retinal ganglion cells, and a quasi-uniform convergence rate from the retina to the cortex. We show that convolutional neural networks outfitted with a retinal sampling layer, which resamples images according to retinal ganglion cell density, develop these organizational principles. Surprisingly, our results indicate that radial bias is spatial-frequency dependent and only manifests for high spatial frequencies. For low spatial frequencies, the bias shifts towards orthogonal orientations. These findings introduce a novel hypothesis about the origin of radial bias. Quasi-uniform convergence limits the range of spatial frequencies (in retinal space) that can be resolved, while retinal sampling determines the spatial frequency content throughout the retina.

Comparison of Brn3a and RBPMS Labeling to Assess Retinal Ganglion Cell Loss During Aging and in a Model of Optic Neuropathy.

Purpose: Retinal ganglion cell (RGC) loss provides the basis for diagnosis and stage determination of many optic neuropathies, and quantification of RGC survival is a critical outcome measure in models of optic neuropathy. This study examines the accuracy of manual RGC counting using two selective markers, Brn3a and RBPMS. Methods: Retinal flat mounts from 1- to 18-month-old C57BL/6 mice, and from mice after microbead (MB)-induced intraocular pressure (IOP) elevation, are immunostained with Brn3a and/or RBPMS antibodies. Four individuals masked to the experimental conditions manually counted labeled RGCs in three copies of five images, and inter- and intra-person reliability was evaluated by the intraclass correlation coefficient (ICC). Results: A larger population (approximately 10% higher) of RGCs are labeled with RBPMS than Brn3a antibody up to 6 months of age, but differences decrease to approximately 1% at older ages. Both RGC-labeled populations significantly decrease with age. MB-induced IOP elevation is associated with a significant decrease of both Brn3a- and RBPMS-positive RGCs. Notably, RGC labeling with Brn3a provides more consistent cell counts than RBPMS in interpersonal (ICC = 0.87 to 0.11, respectively) and intra-personal reliability (ICC = 0.97 to 0.66, respectively). Conclusions: Brn3a and RBPMS markers are independently capable of detecting significant decreases of RGC number with age and in response to IOP elevation despite RPBMS detecting a larger number of RGCs up to 6 months of age. Brn3a labeling is less prone to manual cell counting variability than RBPMS labeling. Overall, either marker can be used as a single marker to detect significant changes in RGC survival, each offering distinct advantages.

Extracellular vesicle encapsulated nicotinamide delivered via a trans-scleral route provides retinal ganglion cell neuroprotection.

The progressive and irreversible degeneration of retinal ganglion cells (RGCs) and their axons is the major characteristic of glaucoma, a leading cause of irreversible blindness worldwide. Nicotinamide adenine dinucleotide (NAD) is a cofactor and metabolite of redox reaction critical for neuronal survival. Supplementation with nicotinamide (NAM), a precursor of NAD, can confer neuroprotective effects against glaucomatous damage caused by an age-related decline of NAD or mitochondrial dysfunction, reflecting the high metabolic activity of RGCs. However, oral supplementation of drug is relatively less efficient in terms of transmissibility to RGCs compared to direct delivery methods such as intraocular injection or delivery using subconjunctival depots. Neither method is ideal, given the risks of infection and subconjunctival scarring without novel techniques. By contrast, extracellular vesicles (EVs) have advantages as a drug delivery system with low immunogeneity and tissue interactions. We have evaluated the EV delivery of NAM as an RGC protective agent using a quantitative assessment of dendritic integrity using DiOlistics, which is confirmed to be a more sensitive measure of neuronal health in our mouse glaucoma model than the evaluation of somatic loss via the immunostaining method. NAM or NAM-loaded EVs showed a significant neuroprotective effect in the mouse retinal explant model. Furthermore, NAM-loaded EVs can penetrate the sclera once deployed in the subconjunctival space. These results confirm the feasibility of using subconjunctival injection of EVs to deliver NAM to intraocular targets.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Sustained Retinal Defocus Increases the Effect of Induced Myopia on the Retinal Astrocyte Template.

The aim of this article is to describe sustained myopic eye growth's effect on astrocyte cellular distribution and its association with inner retinal layer thicknesses. Astrocyte density and distribution, retinal nerve fiber layer (RNFL), ganglion cell layer, and inner plexiform layer (IPL) thicknesses were assessed using immunochemistry and spectral-domain optical coherence tomography on seventeen common marmoset retinas (Callithrix jacchus): six induced with myopia from 2 to 6 months of age (6-month-old myopes), three induced with myopia from 2 to 12 months of age (12-month-old myopes), five age-matched 6-month-old controls, and three age-matched 12-month-old controls. Untreated marmoset eyes grew normally, and both RNFL and IPL thicknesses did not change with age, with astrocyte numbers correlating to RNFL and IPL thicknesses in both control age groups. Myopic marmosets did not follow this trend and, instead, exhibited decreased astrocyte density, increased GFAP+ spatial coverage, and thinner RNFL and IPL, all of which worsened over time. Myopic changes in astrocyte density, GFAP+ spatial coverage and inner retinal layer thicknesses suggest astrocyte template reorganization during myopia development and progression which increased over time. Whether or not these changes are constructive or destructive to the retina still remains to be assessed.

Retinal Neurodegeneration in an Intraocular Pressure Fluctuation Rat Model.

Increased intraocular pressure (IOP) is the most important risk factor for glaucoma. The role of IOP fluctuation, independently from elevated IOP, has not yet been confirmed in glaucoma. We investigated the effects of IOP fluctuation itself on retinal neurodegeneration. Male rats were treated with IOP-lowering eyedrops (brinzolamide and latanoprost) on Mondays and Thursdays (in the irregular instillation group) or daily (in the regular instillation group), and saline was administered daily in the normal control group for 8 weeks. The IOP standard deviation was higher in the irregular instillation group than the regular instillation group or the control group. The degree of oxidative stress, which was analyzed by labeling superoxide, oxidative DNA damage, and nitrotyrosine, was increased in the irregular instillation group. Macroglial activation, expressed by glial fibrillary acidic protein in the optic nerve head and retina, was observed with the irregular instillation of IOP-lowering eyedrops. Microglial activation, as indicated by Iba-1, and the expression of TNF-α did not show a significant difference between the irregular instillation and control groups. Expression of cleaved caspase-3 was upregulated and the number of retinal ganglion cells (RGCs) was decreased in the irregular instillation group. Our findings indicate that IOP fluctuations could be induced by irregular instillation of IOP-lowering eyedrops and this could lead to the degeneration of RGCs, probably through increased oxidative stress and macrogliosis.

Anatomical Features can Affect OCT Measures Used for Clinical Decisions and Clinical Trial Endpoints.

Purpose: To understand the association between anatomical parameters of healthy eyes and optical coherence tomography (OCT) circumpapillary retinal nerve fiber layer (cpRNFL) thickness measurements. Methods: OCT cpRNFL thickness was obtained from 396 healthy eyes in a commercial reference database (RDB). The temporal quadrant (TQ), superior quadrant (SQ), inferior quadrant (IQ), and global (G) cpRNFL thicknesses were analyzed. The commercial OCT devices code these values based on percentiles (red, <1%; yellow, ≥1% and <5%), after taking age and disc area into consideration. Four anatomical parameters were assessed: fovea-to-disc distance, an estimate of axial length, and the locations of the superior and the inferior peaks of the cpRNFL thickness curve. Pearson correlation values were obtained for the parameters and the thickness measures of each of the four cpRNFL regions, and t-tests were performed between the cpRNFL thicknesses coded as abnormal (red or yellow, <5%) versus normal (≥5%). Results: For each of the four anatomical parameters, the correlation with the thickness of one or more of the TQ, SQ, IQ, and G regions exceeded the correlation with age or disc area. All four parameters were significantly (P < 0.001) associated with the abnormal cpRNFL values. The significant parameters were not the same for the different regions; for example, a parameter could be negatively correlated for the TQ but positively correlated with the SQ or IQ. Conclusions: In addition to age and disc area, which are used for inferences in normative databases, four anatomical parameters are associated with cpRNFL thickness. Translational Relevance: Taking these additional anatomical parameters into consideration should aid diagnostic accuracy.