Investigative Opthalmology & Visual Science

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in ageing populations, with oxidative stress recognised as a key pathogenic driver. The dietary antioxidant and cytoprotectant, L-ergothioneine (ET), is avidly accumulated in many tissues, especially the eye. However its relationship to AMD has not been investigated. Here, we examined ETs distribution in ocular tissue and assessed circulating and intraocular ET levels in patients with neovascular AMD. Compared with ocularly-normal age-matched individuals, AMD patients exhibited significantly lower serum ET; elevated levels of ET metabolites, hercynine and ETSO, which may be generated by oxidative stress; and elevated levels of serum allantoin, a product of oxidative damage to urate in humans. Levels of ET in aqueous humour in AMD patients were marginally lower than cataractous patients who are already known to have significantly lower ET levels than healthy eyes. High ET levels were seen in human ocular tissues concentrating in regions vulnerable to oxidative injury, including the lens, retina, retinal pigment epithelium, and choroid, supporting a physiological protective role of ET in the eye. These findings identify the strong association between low ET levels and AMD, warranting further studies to determine whether ET supplementation can modify AMD risk or progression.

Background: Mitochondrial dysfunction is an emerging metabolic hallmark of age-related diseases, yet tools to directly profile mitochondrial pathways and test metabolic interventions in the living human eye remain limited. Multi-omics ocular liquid biopsy enables real-time proteomic and metabolomic profiling of the intraocular microenvironment, complementing systemic biomarkers and imaging surrogates. Here, we used this approach to define mitochondrial and tricarboxylic acid (TCA) cycle dysregulation in geographic atrophy (GA) and to assess whether oral -ketoglutarate (-KG) supplementation can modulate mitochondrial metabolites within the eye. Methods: Mitochondrial and TCA cycle-related proteins were profiled in aqueous humor (AH) samples from patients with GA using DNA-aptamer-based proteomics. In a phase 0 study, a second cohort undergoing sequential cataract surgery provided paired AH samples collected at first-eye surgery and at second-eye surgery after interim -KG supplementation. These samples underwent targeted metabolomic profiling using hydrophilic interaction liquid chromatography coupled with mass spectrometry. Results: In GA, 64 mitochondrial proteins were differentially expressed, including coordinated TCA-cycle deficiencies marked by reduced expression of enzymes regulating TCA entry and flux, including PDHB and DLST. In the phase 0 cohort, oral -KG supplementation significantly increased intraocular -KG levels and the -KG-to-succinate ratio (P < 0.05), with coordinated shifts across TCA intermediates consistent with enhanced TCA cycle flux. Conclusions: AH proteomics demonstrated mitochondrial pathway depletion in GA, consistent with reduced oxidative bioenergetic capacity. AH metabolomics provided first-in-human in vivo evidence that systemic -KG supplementation can modify intraocular metabolites and may enhance intraocular energy metabolism. These findings support ocular liquid biopsy as a precision-health framework for per-patient biomarker-guided metabolic trials in GA.

PurposeTo better understand the etiologic pathways in glaucoma, we aimed to identify pre-diagnostic plasma metabolites associated with glaucoma risk. MethodsIn a case-control study from the Nurses Health Study (NHS), NHSII and Health Professionals Follow-Up Study (HPFS), 599 incident primary open-angle glaucoma (POAG) cases (mean time between blood draw and diagnosis was 10.3 years) were 1:1 matched to 599 controls. Plasma metabolites were measured with LC-MS/MS at the Broad Institute (Cambridge, MA, USA); 367 metabolites from 17 metabolite classes passed quality control analyses. For comparison, in a cross-sectional study in the UK Biobank, 168 NMR metabolites (Nightingale, Finland; version 2020) were measured in serum samples from 2,238 prevalent glaucoma cases and 44,723 controls. Metabolites were probit-score transformed for normality; multiple logistic regression was used to identify metabolites associated with POAG in NHS/NHSII/HPFS and glaucoma in UK Biobank. In NHS/NHSII/HPFS, we also used Metabolite Set Enrichment Analysis to identify metabolite classes associated with POAG. All analyses adjusted for established glaucoma risk factors. False discovery rate (FDR) and number of effective tests (NEF) were used to adjust for multiple comparisons. ResultsNine metabolite classes were associated (FDR<0.05) with POAG in NHS/NHSII/HPFS: triglycerides, diglycerides, two lysophospholipids classes [lysophosphatidylcholines and lysophosphatidylethanolamines], and two phospholipid class [phosphatidylethanolamines and phosphatidylcholines] were positively associated, while cholesteryl esters, carnitines, and organic acids and derivatives were inversely associated with POAG risk; further adjustment for covariates minimally altered the results. These associations were particularly stronger for POAG with paracentral visual field loss. In the UK Biobank, notably, triglycerides and phospholipids (from which lysophospholipids are derived through hydrolysis), were confirmed to be associated (p<0.05) with higher glaucoma risk. Also, in the UK Biobank, the metabolites of tyrosine, glucose, and glutamine were positively associated (NEF<0.2) while 3-hydroxybutyrate, acetate, citrate, pyruvate, and lactate (the latter 4 being anionic organic acids) were inversely associated with glaucoma (NEF<0.05). ConclusionsHigher levels of glycerides (diglycerides and triglycerides) and phospholipids were adversely associated with glaucoma in both the NHS/NHSII/HPFS and the UK Biobank, suggesting that they play an important role in glaucoma pathogenesis. PRECISHigher glyceride and phospholipid levels in pre-diagnostic plasma was associated with glaucoma risk in three cohorts and were associated with prevalent glaucoma in the UK Biobank. Altered lipid metabolism may be etiologically important in glaucoma.

PurposeAge-related macular degeneration (AMD) is a leading cause of blindness in the developed world. One of the most genetically well-characterized degenerative diseases, genome-wide association studies (GWAS) have identified 52 independent common or rare AMD risk associated variants. While transcriptome-wide association analyses (TWAS) and expression quantitative trait loci (eQTL) efforts have characterized the effects of these AMD-associated genes on mRNA expression in retinal tissue, we aimed to characterize the AMD-associated transcriptional profiles of functionally distinct ocular tissues including the macular and extramacular regions of the retina and the retinal-pigment epithelium (RPE)/choroid. MethodsUsing publicly available microarray data (NCBI GEO accession: GSE29801) comprised of retinal and RPE/choroidal tissue samples from 142 AMD patients and 151 healthy individuals (118 retina and 175 RPE/Choroid samples), tissue-specific differential gene expression analyses were conducted. Transcriptome analyses were focused on 878 genes surrounding known AMD-associated loci. ResultsMany genes which contain clinically significant or causal variants identified via GWAS or TWAS/eQTL studies were significantly differentially expressed and display transcriptional heterogeneity across different subtypes of ocular tissue and retinal geography in AMD-associated tissues. ConclusionThese findings demonstrate the importance of spatial heterogeneity and tissue specificity in the mRNA expression of known AMD-associated genes. Genes known to harbor rare or causal AMD- associated variants are differentially expressed in functionally distinct ocular tissues of AMD patients, suggesting they might contribute to disease regardless of mutation status.

ObjectiveMitochondrial tricarboxylic acid (TCA) cycle is central to energy production and redox balance in the eye, which must sustain high metabolic activity to support vision. Retinal neurons, the retinal pigment epithelium (RPE), cornea, and lens each have distinct physiological roles and metabolic demands, yet the absolute concentrations of key TCA intermediates and their variation by tissue, sex, and time of day are not well-defined. MethodsTargeted gas chromatography-mass spectrometry was employed to quantify the absolute concentrations of TCA cycle metabolites in mouse ocular tissues collected at 10 AM and 2 PM to capture diurnal variations. Key metabolite ratios were subsequently calculated to provide insight into TCA cycle dynamics across eye tissues. ResultsThe retina showed the highest concentrations of TCA metabolites among all ocular tissues, particularly succinate, citrate, and malate, consistent with its high energy demands. The RPE/choroid demonstrated well-balanced intermediates with the highest -ketoglutarate (-KG)/Isocitrate ratio, reflecting its efficient mitochondrial oxidation and reductive carboxylation. Corneal metabolism was featured by dominant malate, especially in females, suggesting a metabolic adaptation for redox regulation and oxidative stress defense. The lens had uniformly low metabolite levels except for succinate, indicating minimal mitochondrial activity under physiologically low oxygen conditions. Notably, both the cornea and lens showed significant sex-dependent and diurnal variations in TCA cycle intermediates. ConclusionThis study demonstrates distinct tissue-specific mitochondrial metabolism in the eye, reflecting the unique functional and biochemical demands of each tissue. These metabolic signatures may underlie their susceptibility to mitochondrial dysfunction in various ocular diseases.

PurposeEndophthalmitis is a serious complication of intraocular surgery due to the risk of irreversible retinal damage. Because retinal cell populations are highly heterogenous, single-cell resolution is required to uncover the detailed mechanisms of infection response. Using a mouse model of bacterial endophthalmitis, we investigated transcriptional changes across resident and infiltrating cell types in the retina. MethodsA methicillin-sensitive strain of Staphylococcus aureus was isolated from a patient with endophthalmitis. Adult C57Bl/6J mice received an intravitreal injection of phosphate-buffer solution (PBS) with or without 5000 CFU S. aureus (n=3 per group). 24 hours later, retinas were isolated and single-cell suspensions were sent to the Penn Genomic Core for sequencing with an Illumina NovaSeq 6000. After standard pre-processing of the data, differential genes and pathways were identified for each cell type (adjusted p < 0.01, log2FC > 1 or < -1). ResultsOur analysis identified all expected retinal cell types, including a population of infiltrating neutrophils in the infected retinas. We surveyed genes known to be upregulated at the bulk-retina level in this model (e.g. Tlr2, Nlrp3, Il1b), and found that infiltrating cells mainly drove this expression. Several genes were altered across nearly all retinal cell types, including upregulation of Hsph1 and Stat3. Muller glia downregulated Gpx4 while upregulating Acsl4 and iron importers Tfrc, Zip14, and Dmt1. Top pathways for macrophages/microglia included chemotaxis, cell-cell adhesion, and wound healing. Vascular cells upregulated angiogenesis-related genes. Cellular respiration was a commonly affected pathway across several neuronal populations, with most genes decreasing. ConclusionsThis study advances our understanding of the pathobiology of bacterial endophthalmitis. Muller glia appear to be undergoing ferroptosis, potentially while activating a program to sequester iron away from bacteria. Decreased cellular respiration may indicate hypoxia among neurons. Our results reveal several trends in the retinal response to infection, including iron dysregulation and hypoxia. Understanding these cell-type-specific responses to endophthalmitis may help design therapies to combine with antibiotics.

The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. To better understand the properties of different retinal areas we conducted an untargeted metabolomics analysis on full thickness punches from three different regions (macula, temporal peri-macula and periphery) of primate retina. Half of all metabolites identified showed differential abundance in at least one comparison between the three regions. The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Furthermore, mapping metabolomics results from macula-specific eye diseases onto the region-specific distributions of healthy primate retina revealed differential abundance defining systemic metabolic dysregulations that were region specific, highlighting how our results may help to better understand the pathobiology of retinal diseases with region specificity.

IntroductionMyopia is a leading cause of visual impairment worldwide, whose pathogenesis remains poorly understood. Identification of its risk factors would benefit the management and treatment of myopia. MethodsWe comprehensively performed phenome-wide Mendelian randomization analysis (MR-PheWAS) to explore causal factors and potential therapeutic targets for myopia in participants from the UK Biobank study. ResultsOur PheWAS revealed that 55 robust associations (1 disease, 2 employment, 3 cognitive functions, 4 sex-specific factors, 4 mental health factors, 5 lifestyle and environmental factors, 10 sociodemographic factors, 12 physical measures and 14 ocular measures/conditions) were significantly causally correlated with myopia. ConclusionsThe results indicate that myopia may be influenced by several factors, including serum metabolic traits, fatty acid intake, fat-related indices, mental health, as well as some previously acknowledged risk factors. Future clinical trials are needed to verify our results.

PurposeSoft drusen and subretinal drusenoid deposits (SDD) aka reticular macular disease (RMD) characterize two pathways to advanced age-related macular degeneration (AMD). We propose these pathways are distinct diseases, with distinct genetic risks, serum risks and associated systemic diseases. Methods126 Subjects with AMD had: retinal imaging for RMD status, serum risks, genetic testing, and histories of cardiovascular disease (CVD) and stroke. Results62 subjects had RMD, 64 were nonRMD (drusen only), 51 had CVD or Stroke. RMD correlated significantly with: ARMS2 risk allele (p= 0.019); lower mean serum HDL (61{+/-}18 vs. 69{+/-}22 mg/dl, p= 0.038, t test); CVD and troke (34/51 RMD, p= 0.001).NonRMD correlated/trended with APOE2 (p= 0.032) and CETP (p= 0.072) risk alleles. 97 subjects total had some drusen, which correlated with CFH risk (p= 0.016). Multivariate independent risks for RMD were: CVD and Stroke (p= 0.008), and ARMS2 homozygous risk (p= 0.038). ConclusionThe RMD and soft drusen AMD pathways have distinct systemic associations, serum and genetic risks. RMD is associated with CVD and stroke, ARMS2 risk, and lower HDL; drusen with CFH risk and two lipid risk genes. These pathways appear to be distinct diseases leading to advanced AMD. Summary StatementTwo phenotypes of age-related macular degeneration, soft drusen and reticular macular disease (the combination of subretinal drusenoid deposits and choriocapillaris insufficiency), are shown here to have distinct systemic vascular, serum, and genetic risks. These findings support the concept that these phenotypes actually represent distinct disease processes.

Age-related macular degeneration (AMD) shows substantial clinical heterogeneity that remains unexplained despite extensive genetic and clinical characterization. We evaluated whether proteomic stratification could provide insight beyond clinical phenotype and genetic risk. We performed 384-plex plasma proteomics in a cohort of 215 individuals, including patients with early and late neovascular AMD, other complement-associated retinal diseases, and age-matched controls. Proteome-based reclassification identified four disease-overarching clusters. Neovascular AMD cases were partitioned almost exclusively between two clusters (30/36). Early AMD cases were predominantly assigned to one of these clusters (10/18), whereas only two localized to the other (2/18). Both AMD-associated clusters shared elevated levels of a protein module enriched for lipoprotein-related functions compared to the other clusters. However, the cluster containing both early and neovascular AMD cases showed higher levels of additional protein modules enriched for complement pathways and cellular stress-response pathways compared with the other AMD-associated cluster. Importantly, this molecular divergence in neovascular AMD could not be explained by genetic predisposition (i.e., 52-variant AMD genetic risk score), signatures of biological ageing, nor by other clinical features. Together, these findings support two proteomic endotypes of neovascular AMD with distinct involvement of cellular stress pathways.

PurposeAnti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for neovascular age-related macular degeneration (AMD), yet many patients exhibit persistent retinal degeneration, fibrosis, and incomplete therapeutic response. The molecular pathways underlying this incomplete response remain poorly understood. We sought to identify VEGF-independent signaling pathways active in the vitreous of anti-VEGF-treated AMD patients. MethodsWe performed multiplex antibody-based proteomic profiling of 1,000 human proteins in vitreous samples from patients with neovascular AMD receiving anti-VEGF therapy (n=8) and comparative controls (n=6). Differential protein expression was assessed using one-way ANOVA, followed by gene ontology and pathway enrichment analyses. Drug-target relationships were evaluated to identify potential opportunities for therapeutic repositioning. ResultsWe identified 107 differentially expressed proteins (p<0.05), including key regulators of immune signaling, angiogenesis, and metabolism. Notably, multiple components of cytotoxic lymphocyte pathways were dysregulated, including IL-21R, SIGLEC-7, CTLA4, and IL-2-associated signaling. Enrichment analyses revealed significant activation of pathways related to T-cell activation, interleukin signaling, and leukocyte-mediated cytotoxicity. These immune signatures persisted despite suppression of VEGF signaling. Several clinically available immunomodulatory agents--including abatacept, sirolimus, and dupilumab--targeted pathways identified in this dataset. ConclusionsAnti-VEGF-treated neovascular AMD exhibits persistent cytotoxic immune signaling in the vitreous, suggesting that VEGF-independent immune mechanisms may contribute to ongoing retinal damage and incomplete therapeutic response. These findings provide a rationale for combination therapeutic strategies targeting both angiogenic and immune pathways in AMD.

PurposePremature infants at risk of retinopathy of prematurity (ROP) miss placental transfer of carotenoids, lutein (L) and zeaxanthin (Z), during the third trimester. We previously demonstrated that prenatal L and Z supplementation raises carotenoid levels in infants at birth in the Lutein and Zeaxanthin in Pregnancy (L-ZIP) study (NCT03750968). Based on their antioxidant effects and bioavailability, we hypothesized that prenatal maternal supplementation with macular carotenoids would reduce risk of ROP. To test this hypothesis, we utilized "macular pigment mice" genetically engineered to take up L and Z into the retina in a model of oxygen-induced retinopathy (OIR). MethodsPregnant Bco2-/- mice were divided into nine experimental subgroups based on the type of supplementation (L, Z, or placebo) and on maternal supplementation start date corresponding to the three trimesters of human fetal development (E0, E11, and P1). Pups and nursing mothers were exposed to 75% O2 for 5 days (P7-12) and returned to room air for 5 days (P12-17). Pups were sacrificed at P12 and P17, and their retinas were analyzed for vaso-obliteration (VO) and intravitreal neovascularization (INV). ResultsPups of pregnant mice supplemented with L or Z had significant reductions in VO and INV areas compared to placebo. Prenatal carotenoid supplementation starting at E0 or E11 was significantly more protective against OIR than postnatal supplementation starting at P1. ConclusionPrenatal supplementation with L and Z was beneficial in a mouse OIR model. We recommend testing prenatal L and Z supplementation in future human clinical trials to prevent ROP.

PurposeTo characterize microscopic alteration of photoreceptors and RPE surrounding cuticular drusen in age-related macular degeneration (AMD) using multimodal imaging, including high resolution adaptive optics scanning laser ophthalmoscopy (AOSLO). MethodsEyes with early to intermediate AMD and predominantly cuticular drusen underwent color fundus photography, infrared reflectance, fundus autofluorescence, optical coherence tomography (OCT), and AOSLO. Cuticular drusen were identified using multimodal imaging and classified into three OCT-defined phenotypes. Cone photoreceptor reflectivity was assessed on AOSLO. A subset of eyes underwent longitudinal AOSLO and OCT imaging. ResultNineteen eyes from 12 subjects aged 70.3 {+/-} 5.8 years were studied. Six eyes had longitudinal follow-up imaging. A total of 3177 cuticular drusen were evaluated and classified into 3 types based on cross sectional OCT imaging. AOSLO revealed corresponding phenotype-dependent cone reflectivity alterations associated with the 3 types of cuticular drusen. Type 1: Maintained cone reflectivity overlying the drusen on a hyporeflective background. Type 2: Cone reflectivity loss overlying the cuticular drusen. Type 3: Cones are predominantly not visible over the cuticular drusen. Lesion diameters were 52.62 {+/-} 9.38 {micro}m (Type 1), 71.88 {+/-} 12.39 {micro}m (Type 2), and 124.72 {+/-} 20.94 {micro}m (Type 3). All lesions were accompanied by hypertransmission in the choroid on OCT. Longitudinal imaging showed that localized outer retinal reflectivity reduction on AOSLO preceded the detection of new cuticular drusen on OCT. ConclusionsCellular-resolution multimodal imaging demonstrates progressive, phenotype-specific disruption of the photoreceptor-RPE complex associated with cuticular drusen in AMD. Early AOSLO-detected reflectivity changes preceding OCT-visible lesions highlight the sensitivity of adaptive optics imaging for identifying early outer retinal alterations and for advancing understanding of the biogenesis of cuticular drusen.

ImportanceHigh-risk vascular diseases (HRVs) may remain undetected until catastrophe ensues. Detection from non-invasive retinal imaging would be highly significant. ObjectiveTo demonstrate that certain lesions of Age-Related Macular Degeneration (AMD) found on retinal imaging correlate with co-existing HRVs. DesignCross-sectional cohort study. Two years. Retinal image graders blinded to HRV status. Setting2 retina referral clinics. Participants151 consecutive AMD patients, ages 50-90, 97 females, 54 males, with lesions of soft drusen and/or subretinal drusenoid deposits (SDD). 12 others approached, 10 refused, 2 excluded. MethodsPatients were classified by retinal imaging into SDD (SDD present, +/- drusen) or nonSDD (soft drusen only), and by history into HRV (cardiac pump defect (myocardial infarction (MI), coronary artery bypass grafting (CABG), congestive heart failure (CHF)), valve defect, and carotid stroke) or nonHRV, with serum risk factors and medical histories. Main Outcome MeasuresCorrelations of HRV with SDD and other covariates (Univariate chi-square and multivariate regression). Performance of Machine Learning predicting HRV. Results75 SDD subjects; 76 nonSDD subjects; HRV prevalence 19.2% (29/151). O_LIHigh density lipoprotein (HDL) < 62 mg/Dl was found in 24/29 HRV, 42/122 nonHRV, OR 12.40, 95% Confidence Interval (CI) 5.125-30.014; p= 0.0002. C_LIO_LI15 Pump defects, 14/15 SDD, 8 Valve defects, 6/8 SDD (4 severe aortic stenosis), 6 carotid strokes, 5/6 SDD. Total HRVs 29, 25/29 SDD, OR 9.0, 95% CI 2.95-27.46; p= 0.000012. C_LIO_LIAdjusted multivariate correlations. HRV with SDD (p= 0.000333). SDD and HDL < 62 with HRV (p= 0.000098 and 0.021). C_LIO_LIMachine Learning prediction of HRVs from SDD status and HDL level: specificity 87.4%, sensitivity 77.4%, accuracy 84.9%; 95% CIs(%) 79.0-93.3, 58.0-90.4, 77.5-90.7, respectively. C_LI Conclusions and RelevanceHigh-risk vascular diseases were accurately identified in a cohort of AMD patients from the presence of characteristic deposits (SDDs) on imaging and HDL levels. The SDDs are directly consequent to inadequate ocular perfusion resulting from the systemic vasculopathies. Further validation in larger cohorts of both vasculopathic and AMD subjects could bring this system into widespread medical practice, to reduce mortality and morbidity from vascular disease, particularly in women, where undiagnosed cardiac disease remains a serious issue. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the relationship and driving mechanism between High Risk Vascular Diseases (HRVs) and Age-Related Macular Degeneration (AMD)? FindingsThe specific AMD lesions of Subretinal Drusenoid Deposits (SDDs) were found to be highly correlated with and directly consequent to the inadequate ocular perfusion resulting from the HRVs of severe cardiac pump insufficiency or valve defect, and carotid occlusion, These vasculopathies could be predicted from the presence of SDDs on spectral domain optical coherence tomography (SD-OCT) imaging and serum HDL. MeaningScreening for SDDs with SD-OCT imaging could reduce mortality and morbidity from severe vascular disease, particularly in women, where undiagnosed cardiac disease remains a serious issue.

The choroid is critical for maintaining vision and implicated in several ocular diseases, being the sole source of nutrients and waste removal for the outer retina. Genetic discovery can help elucidate the pathways through which choroidal features influence disease risk. Our meta-analysis of genome-wide association studies (n= 78,682 participants) identified 30 genomic regions, including 20 novel loci, associated with choroidal thickness. Findings suggest inflammatory and vascular processes drive choroidal thickness, with overlapping mechanisms shared with refractive error. Genome-wide independently significant SNPs accounted for 18.7% of the genetic variance in choroidal thickness. Mendelian randomisation analyses showed a causal effect of age-related macular degeneration on choroidal thickness, and suggest a bidirectional causal effect between choroidal thickness and primary angle-closure glaucoma. These findings provide insight into the shared genetic architecture and biological pathways linking choroidal thickness and related diseases.

PurposeMuller cell (MC) morphology and markers were investigated using histology and immunohistochemistry in an eye with clinically documented multifocal geographic atrophy (GA) and correlated with clinical images. MethodsThe donor was followed clinically for five years and last examined six years before death. The superior posterior pole retina was dissected and immunolabeled with antibodies against glial fibrillary acidic protein (GFAP; activated MCs and astrocytes) and glutamine synthetase (GS, MC) and Ulex Europaeus Agglutinin-1 lectin (blood vessels) before embedding for JB-4 cross section analysis. The inferior macula was cryopreserved. Cryosections were immunolabeled with MC homeostatic and activation markers. Transmission electron microscopy (TEM) of the fellow eye was used to study ultrastructure changes. ResultsGross examination demonstrated mottled retinal pigment epithelium (RPE) over presumably calcified drusen. In the submacular retina, MC processes surrounding both drusen and outer retinal pigmented lesions created a large subretinal membrane. Cryosection analysis demonstrated persistence of aquaporin 4 and GS in MCs with both proteins prominently expressed in the subretinal membrane. Increased MC S100B and GFAP expression were also observed in the atrophic area as well as the OJZ. Cryosection labeling and TEM confirmed the MC encasing calcified drusen and RPE debris as well as invading basal laminar deposits. ConclusionsThis multifocal GA case demonstrates how MC activation and structural changes surrounding individual drusen could coalesce, contributing to photoreceptor loss. MCs penetrating basal laminar deposits and encasing calcified drusen suggests that they are attempting to clear these and/or protect the retina from harmful contents.

Glucose-sensing ChREBP and MondoA are transcriptional factors involved in lipogenic, inflammatory, and insulin signaling pathways implicated in metabolic disorders; however, limited ocular studies have been conducted on these proteins. We aimed to investigate the potential role of ChREBP in pathogenesis of diabetic retinopathy (DR). We used diabetic human and mouse retinal cryosections analyzed by immunohistochemistry. qRT-PCR was performed to quantify gene expression. To explore the role of ChREBP in rods, we generated caChREBPRP mice with constitutively active (ca) ChREBP. These mice underwent retinal function testing, followed by proteomic analysis using LC-MS. Furthermore, ARPE-19 cells were infected with lentiviral particles expressing human ChREBP (ARPE-19ChREBP) and subjected to global proteomics. Our results demonstrate that both pro-teins were expressed across the retina, although with distinct distribution patterns: MondoA was more prominently expressed in cones, while ChREBP was broadly expressed throughout the retina. Elevated expression of both proteins was observed in DR. This may have contributed to rod photo-receptor degeneration as we observed diminished scotopic ERG amplitudes detected in caChREB-PRP mice at P35. The retinal proteomic landscape indicated a decline in KEGG pathways associated with phototransduction, amino acid metabolism, and cell adhesion. Furthermore, rod-specific ca-ChREBP induced TXNIP expression. Consistent with altered retinal proteomics, ARPE-19ChREBP cells displayed a metabolic shift toward increased glyoxylate signaling, sugar metabolism, and lysosomal activation. Our study demonstrates that ChREBP overexpression causes significant metabolic reprograming triggering retinal functional loss in mice

PurposeRod-mediated dark adaptation delays are among the earliest functional abnormalities in age-related macular degeneration (AMD), preceding photoreceptor loss. This study evaluated whether parafoveal fundus-tracked dark adaptometry detects earlier rod dysfunction than more eccentric mid-macula testing and assessed the diagnostic performance of dynamic and steady-state parameters across eccentricities. MethodsIn this cross-sectional study, 35 patients with predominantly early / intermediate AMD and 32 age-spanning controls underwent fundus-controlled dark adaptometry (S-MAIA-2; iCare/CenterVue) and multimodal imaging. After standardized bleaching, cyan stimuli were presented 2{degrees}, 4{degrees}, and 6{degrees} temporal to the fovea. Dark-adaptation curves were modeled to derive rod intercept time (RIT), final rod threshold (FT), and cone threshold (CT), each compared with age-adjusted normative data. Diagnostic accuracy was quantified using age-adjusted receiver operating characteristic (ROC) analyses. ResultsAmong 67 analyzed eyes, RIT was abnormal in 89% of AMD eyes at 2{degrees}, 77% at 4{degrees}, and 74% at 6{degrees}, whereas FT and CT were less frequently abnormal (29 to 51% and 17 to 23%, respectively). Median RIT at 2{degrees} reached 60 minutes, indicating incomplete recovery in many eyes. RIT achieved the highest diagnostic accuracy, with age-adjusted AUC values of 0.91 (95% CrI, 0.81-0.98) at 2{degrees}, 0.88 (0.77-0.96) at 4{degrees}, and 0.87 (0.76-0.95) at 6{degrees}. ConclusionsFundus-tracked dark adaptometry enables spatially precise assessment of parafoveal rod recovery. Parafoveal RIT prolongation represents the earliest and most frequent functional abnormality in AMD and demonstrates excellent diagnostic performance, supporting its potential as a sensitive functional biomarker for early disease and therapeutic trials.

PurposeTo compare the plasma metabolic profile of patients with a CRB1-associated inherited retinal degeneration (CRB1-IRD) with healthy controls (HCs). DesignA case-control study. MethodsPlasma concentration of 619 metabolites was measured with the MxP(R) Quant 500 Kit in 30 patients with a CRB1-IRD and 29 HCs. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in {micro}M ({micro}mol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls. ResultsOver-representation of pathways among metabolites associated strongest to CRB1-IRDs (P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes. ConclusionsLipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1-IRD.

PurposeTo identify genetic variants associated with glucocorticoid (GC)-induced intraocular pressure (IOP) change using genome-wide association study (GWAS) and whole exome sequencing (WES) analyses. Methods530 participants from the Fluocinolone Acetonide in Diabetic Macular Edema (FAME) trials were analyzed, with replication performed in an independent cohort of 588 participants from the Mass Eye and Ear/Retina Health Center (MEE/RHC). All participants were exposed to GC, primarily via intravitreal injection. IOP was measured at baseline and serially within 6 months following GC exposure. GWAS and rare variant gene burden analyses were applied, adjusting for covariates. ResultsGenetic associations for maximal IOP change within 6 months after GC exposure were evaluated. For the primary outcome across all ancestries in FAME, one variant, rs13425173 within the UBE2E3 locus reached genome-wide significance (P=2.88 X 10-8). In the FAME and MEE/RHC meta-analysis, variant rs1040227, also in the UBE2E3 locus, was significantly associated at the genome-wide level (P=2.88 X 10-8) and showed nominal significance in the MEE/RHC cohort (P=0.02). In the colocalization analyses, the significant FAME GWAS UBE2E3 locus was linked to expression regulation of this gene in six tissues including artery aorta. In gene-level analysis, UBE2E3 also demonstrated subthreshold significance (P=6 X 10-6). 532 FAME and 586 MEE/RHC participants were included in the WES gene burden analysis. One gene, MSTO1, passed false discovery rate correction for the primary outcome in FAME. ConclusionWe have identified genome-wide significant common variants associated with GC-IOP change, as well as genes and rare variants that may influence GC-induced IOP change.