Investigative Opthalmology & Visual Science

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.

PurposeThe human 10q26 locus is a major genetic risk factor for age-related macular degeneration (AMD). Fine mapping by linkage and large-scale genome-wide association studies (GWAS) has narrowed this region to a 30-kb interval encompassing the ARMS2 and HTRA1 genes. However, the causative gene(s), risk variants, and underlying pathogenic mechanisms remain unresolved. MethodsLong non-coding RNA (lncRNA) candidates within the ARMS2-HTRA1 region were identified using human postmortem retinal RNA-seq data and public databases (NCBI, Ensembl). Candidate transcripts were validated by RT-PCR and Sanger sequencing. Published single-cell RNA-seq datasets were analysed to define cell type-specific expression, and RNA levels were compared between AMD and non-AMD donor retinas. Additionally, expression changes were assessed in human iPSC-derived retinal pigment epithelium (RPE) cells exposed to cigarette smoke extract (CSE) and paraquat (PQT). ResultsWe identified and validated a lncRNA, HTRA1-AS1, and its transcript variants (ENST00000647969.1) within the ARMS2 locus. HTRA1-AS1 overlaps ARMS2 and is transcribed in the antisense orientation. It is predominantly expressed in rod photoreceptors, Muller glia and Choroid/RPE, and its retinal expression was significantly reduced in AMD compared with controls (43 AMD donors vs. 44 controls, p = 0.007). By contrast, HTRA1 mRNA showed no significant difference (p = 0.121). Furthermore, ENST00000647969.1, HTRA1-AS1 and ARMS2 expression increased dramatically, up to 101-fold, 8-fold and 75-fold, respectively, in induced pluripotent stem cells (iPSC)-derived RPE cells following cigarette smoke extract (CSE)-induced oxidative stress but showed no significant change after paraquat treatment. ConclusionThese findings suggest that HTRA1-AS1, a dysregulated lncRNA within the ARMS2 locus, may act as a non-coding element contributing to transcriptional mis-regulation underlying AMD pathogenesis.

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.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSPurposeC_ST_ABSTo characterize photoreceptor layer thinning in clinically unremarkable regions adjacent to the atrophic lesion in early-stage ABCA4 disease eyes. Methods27 patients with confined atrophic lesions ([&le;]3.5mm in diameter) were included. Two pathogenic alleles were confirmed by sequencing of the ABCA4 locus. Multimodal imaging included color fundus photography, short wavelength-autofluorescence (SW-AF) and near infrared-autofluorescence (NIR-AF) imaging. Total receptor+ (TREC+) thickness was segmented in spectral domain-optical coherence tomography (SD-OCT) scans in patient eyes (n=27) along with age-matched healthy control eyes (n=20). Results{micro}age of the study cohort was 24.1 years and 15/27 (55.6%) patients harbored genotypes consisting of the p.(Gly1961Glu) variant in ABCA4. Atrophic lesions in the central macula ranged from 0.61 to 3.13 mm in diameter ({micro} = 1.73,{sigma} = 0.70). Six patients had mild RPE mottling adjacent to the lesion on NIR-AF. The atrophic lesion corresponded to a disruption of photoreceptor-attributable bands on SD-OCT while all layers were visibly intact outside the lesion. TREC+ thickness in patient eyes were <0.15 mm or below 4{sigma} of normal control eyes immediately adjacent to the lesion edge and gradually normalized to within {+/-} 2{sigma} at{approx} 1.2 mm eccentricity from the fovea. ConclusionA uniform subclinical perilesional zone (SPZ) of photoreceptor thinning extends around the perimeter of early-stage atrophic lesions in ABCA4 disease. This region spatially maps to known regions of vision loss and more accurately approximates the extent of photoreceptor abnormality compared to the disease changes visible on standard fundus imaging. Translational relevanceSemi-automated segmentation of SD-OCT scans identifies a consistent subclinical biomarker relevant to early photoreceptor degeneration in ABCA4 disease.

PurposeSubretinal drusenoid deposits (SDDs) are a distinct entity in age-related macular degeneration (AMD) and associated with photoreceptor impairment during progression. Their early impact on photoreceptors remains incompletely understood. This study examined photoreceptor reflectivity during the phase when SDDs were not clinically detectable on optical coherence tomography (OCT) using adaptive optics scanning laser ophthalmoscopy (AOSLO). DesignLongitudinal observational study. ParticipantsPatients with intermediate AMD. MethodsSix eyes of four patients with intermediate AMD and predominantly SDDs underwent multimodal imaging 3-4 times over 3.5 years. Individual SDDs were graded using an OCT-based 3-stage system at each time point. Cross-sectional retinal structure and photoreceptor reflectivity at the location where the new SDDs developed during follow-up were evaluated using OCT and AOSLO. Main Outcome MeasuresPhotoreceptor reflectivity change prior to and during SDD development. ResultsForty-eight retinal locations where new dot-type SDDs developed during follow-up were identified. AOSLO revealed reduced photoreceptor reflectivity in these regions before OCT demonstrated clinically evident deposits (stage [&ge;] 1) between the ellipsoid zone and the retinal pigment epithelium at the corresponding sites. The mean time to development of stage 1, stage 2, and stage 3 SDDs was 11.78 {+/-} 5.01, 17.40 {+/-} 6.08, and 18.72 {+/-} 4.08 months, respectively. ConclusionsHigh-resolution adaptive optics confocal imaging enables detection of photoreceptor optical property alterations at a stage when SDDs are not yet evident on OCT. This finding underscores the exceptional sensitivity of photoreceptors to minimal structural or functional perturbations during SDD formation and defines an early window for potential intervention.

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.

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.

IMPORTANCEGenes and lifestyle factors influence progression to advanced age-related macular degeneration (AAMD). However, the impact of genetic and behavioral factors on age when this transition occurs has not been evaluated prospectively. OBJECTIVETo determine whether genetic and environmental factors are associated with age of progression to AAMD and to quantify the effect on age. DESIGN, SETTING, AND PARTICIPANTSLongitudinal progression to AAMD was based on the severity scale in the Age-Related Eye Disease Study database. Progression was defined as an eye that transitioned from non-advanced dry AMD without any evidence of geographic atrophy (GA) (levels 1-8) to any GA or evidence of neovascularization (NV) or both (levels [&ge;]9) during 13 years follow up. Genotypes were determined from DNA samples. MAIN OUTCOME AND MEASURESA stepwise selection of genetic variants with the eye as the unit of analysis, using age as the time scale, yielded 11 genetic variants associated with overall progression, adjusting for sex, education, smoking history, BMI, baseline severity scale, and AREDS treatment. Multivariate analysis was also performed to calculate the effect of genetic and behavioral factors on age of progression. RESULTSAmong 5421 eyes, 1206 progressed. Genetic variants associated with progression to AAMD were in the complement, immune, inflammatory, lipid, extracellular matrix, DNA repair and protein binding pathways. Three of these variants were significantly associated with earlier age of progression, adjusting for other covariates: CFH R1210C (P=0.019) with 4.7 years earlier age at progression among carriers of this mutation, C3 K155Q (P=0.011) with 2.44 years earlier for carriers, and ARMS2/HTRA1 A69S (P=0.012) with 0.67 years earlier per allele. Subjects who were smokers (P<. 001) or had high BMI (P=0.006) also had an earlier age at progression (4.1 years and 1.4 years, respectively). CONCLUSIONSCarriers of rare variants in the complement pathway and a common risk allele in ARMS2/HTRA1 develop advanced AMD at an earlier age, and unhealthy behaviors including smoking and higher body mass index lead to earlier age of progression to AAMD. KEY POINTSO_ST_ABSQuestionC_ST_ABSAre genetic and modifiable factors associated with earlier age of developing advanced AMD? FindingsIn this prospective analysis of 5421 eyes with non-advanced AMD at baseline, 1206 developed advanced stages of AMD. Smoking together with a higher BMI led to 5.5 years earlier progression to advanced disease, and genetic burden, determined by rare and common variants, lowered age of progression by up to an additional 6.0 years, adjusting for all covariates. MeaningModifiable factors alter age when advanced AMD and associated visual loss occurs, and genetic susceptibility impacts age of this transition. Results underscore the importance of both nature and nurture on earlier progression to advanced disease leading to a longer duration of disease and treatment burden. Submitted as ARVO abstract December, 2019: abstract citation: Widjajahakim R, Rosner B, Seddon J; Rare and Common Genetic Variants and Behavioral Modifiable Factors Are Associated with Earlier Age of Progression to Advanced AMD. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2436).

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.

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.

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.

PurposeTo identify optical coherence tomography angiography (OCTA)-derived biomarkers correlating with diabetic retinopathy (DR) severity and characterize longitudinal retinal microvascular changes across DR stages. MethodsIn this 3-year prospective study, we analyzed OCTA images from 328 eyes of 164 adults with type II diabetes and 33 eyes from 17 healthy controls. Patients were categorized as no DR, mild, moderate, or severe nonproliferative DR (NPDR), or proliferative DR (PDR) at baseline. Bilateral OCTA scans were obtained at each visit and processed with a validated pipeline to extract seven microvascular indices from the superficial and deep capillary plexuses: vessel density, skeleton density, acircularity index, average vessel caliber, foveal avascular zone (FAZ) area, FAZ perimeter, and fractal dimensions. Linear regression quantified changes over time, and intergroup comparisons were made using ANOVA and post-hoc testing. ResultsPatients with severe NPDR showed a significant annual decrease in vessel density and skeleton density in the deep capillary plexus compared to healthy controls, indicating progressive ischemia. Acircularity index increased significantly in severe NPDR compared to mild NPDR, suggesting worsening macular ischemia. In the superficial plexus, severe NPDR patients exhibited significantly greater annual vessel caliber narrowing than mild NPDR. Conversely, skeleton density in PDR increased relative to severe NPDR, possibly reflecting neovascular remodeling. ConclusionsLongitudinal OCTA analysis reveals stage-specific microvascular changes in DR. These dynamic biomarkers may provide early indicators of DR progression and offer a quantitative foundation for individualized monitoring and timely intervention strategies. Translational RelevanceOCTA-derived biomarkers offer a noninvasive tool for detecting early DR changes and guiding personalized care strategies.

Recent studies have suggested an association between GLP-1 receptor agonist (GLP-1RA) treatment and increased risk of non-arteritic anterior ischemic optic neuropathy, particularly within the first year of treatment. This study investigated potential mechanisms by examining retinal perfusion changes in both humans and mice during GLP-1RA therapy. We first compared six patients who underwent fluorescein angiography during GLP-1RA treatment with three patients after treatment completion. Two cases in a during-treatment group showed increased arteriovenous transit time. To validate these findings, we treated wild-type mice with liraglutide and measured retinal perfusion parameters before, during, and after treatment. Mice exhibited delayed injection-to-retina time and increased arteriovenous transit time during treatment, and decreased scotopic b-wave responses and increased hypoxia-related gene expression indicating retinal hypoxia, with recovery after treatment discontinuation. Investigation of mechanisms revealed that GLP-1RA-treated mice had significantly reduced intracranial pressure (ICP) at day 7, corresponding with the timing of retinal perfusion delays, and there was a trend toward a negative correlation between ICP and injection-to-retina time. Reduced ICP may affect ophthalmic vessels in the subarachnoid space, contributing to delayed retinal perfusion. These findings suggest that ocular circulation may be particularly vulnerable to GLP-1RA-related hemodynamic effects, independent of systemic cardiovascular changes. These results indicate that careful monitoring of ocular circulation may be warranted in individuals using GLP-1RA, and suggest the need for further investigation of temporal ICP changes in human patients.

BackgroundAdvances in mass spectrometry are providing new insights into the role of metabolomics in the aetiology of many diseases. Studies in retinopathy of prematurity (ROP), for instance, overlooked the role of metabolic alterations in disease development. Here, we employed comprehensive metabolic profiling and gold-standard metabolic analysis to explore major metabolites and metabolic pathways significantly affected in early stages of pathogenesis toward ROP. MethodsThis is a multicentre, retrospective case-control study. We collected serums from 57 ROP cases and 57 strictly baseline matched non-ROP controls. Non-targeted ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) from Metabolon, Inc. was used to detect the metabolites in serum samples. Machine learning was used to unravel most affected metabolites and pathways in ROP development. ResultsCompared to non-ROP controls, we found a significant metabolic perturbation in the ROP serums, featured with an increase in lipid, nucleotide, carbohydrate metabolites and a lower level of peptides. Machine leaning helped to distinguish a cluster of metabolic pathways (glycometabolism, redox homeostasis, lipid metabolism and arginine pathway) that were strongly related to the development of ROP. In addition, we found that the severity of ROP was related to the level of creatinine and ribitol. ConclusionIn the current study, our results suggested a strong link between metabolic profiling and retinal neovascularization during ROP pathogenesis. These findings provided an insight into identifying novel metabolic biomarkers for ROP diagnosis and prevention.

ObjectiveTo determine the profile of inflammation-related proteins and complement system factors in serum of CRB1-associated inherited retinal dystrophies (CRB1-IRDs). DesignA case-control study. Subjects, Participants, and/or ControlsA cohort of 30 Dutch CRB1-IRD patients and 29 Dutch healthy controls (HC) (Cohort I), and a second cohort of 123 CRB1-IRD patients from 14 countries and 1292 controls (Cohort II) were used in this study. MethodsQuantitative 370-plex targeted proteomics in blood plasma and genotyping of the single nucleotide variant (SNV) rs7535263 in the CFH gene. Main Outcome MeasuresPlasma concentrations of inflammation-related proteins and the genotype of the SNV rs7535263. ResultsCRB1-IRD patients showed increased plasma levels of complement system and coagulation cascade proteins compared to healthy controls. Complement Factor I [CFI], Serpin Family D1 [SERPIND1], and Complement Factor H [CFH] were significantly elevated (q<0.05, adjusted for age and sex), which correlated (Pearsons correlation coefficient >0.6) with higher levels of plasma Complement Component 3 [C3] (q = 0.064). The most enriched pathway in patients was the "Complement cascade" (R-HSA-166658, Padj = P = 3.03 x 10-15). An analysis of the genotype of CFH variant rs7535263, which is in close physical proximity to the CRB1 gene and is associated with other retinal conditions by influencing plasma complement levels, revealed significantly skewed allele distribution specifically in Dutch patients (A allele of rs7535263, odds ratio (OR) [95%CI = 2.85 [1.35-6.02], P = 6.19 x 10-3), but not in a global case-control cohort (P = 0.12). However, CRB1 missense variants that are common in patients display strong linkage disequilibrium (LD) with rs7535263 in CFH in the UK Biobank (D = 0.97 for p.(Cys948Tyr); D = 1.0 for p.(Arg764Cys)), indicating that genetic linkage may influence plasma complement factor levels in CRB1-IRD patients. After accounting for the CFH genotype in the proteomic analyses, we also detected significantly elevated plasma levels of Complement Factor H Related 2 [CFHR2] in CRB1-IRD patients (q = 0.04). ConclusionsCRB1-IRDs are characterized by changes in plasma levels of complement factors and proteins of the innate immune system, which is influenced by common functional variants in the CFH-CFHR locus. This indicates that innate immunity is implicated in CRB1-IRDs.

Fluorescence lifetime imaging ophthalmoscopy permits in vivo assessment of retinal metabolism but has remained limited by insufficient cellular resolution in the human eye. Here we present adaptive optics-enhanced fluorescence lifetime imaging ophthalmoscopy (AOFLIO), a method for single-cell-resolved, in vivo structural and metabolic imaging of the human retinal pigment epithelium (RPE). Through real-time correction of ocular wavefront aberrations, precisely synchronized adaptive optics reflectance and lifetime image acquisition via a phase-locked loop-based timing architecture, and sub-pixel photon registration that localizes individual autofluorescence photons with high spatial precision, AOFLIO directly resolves the RPE cell mosaic and measures autofluorescence decay using the same photons, enabling direct structural-functional correlation at the single-cell level. We demonstrate single-cell RPE lifetime mapping in healthy subjects and reveal altered metabolic signatures and fine characterization of RPE metabolic in age-related macular degeneration. AOFLIO establishes a platform for cellular-scale metabolic imaging in the living human eye.

Neuroinflammation is a critical factor in multiple sclerosis (MS), but in vivo monitoring of its cellular dynamics remains a significant challenge. The retina, derived from CNS tissue, provides a unique, non-invasive window into these processes. High-resolution retinal imaging techniques, such as adaptive optics scanning laser ophthalmoscopy (AOSLO), enable the observation of cellular dynamics in the retina, advancing our understanding of neuroinflammation in MS. In this prospective cohort study, we used custom-modified AOSLO to examine the retinal ganglion cell layer in 51 MS patients with different phenotypes: relapsing-remitting MS (RMS) with recent optic neuritis (ON) (N=31), RMS without recent ON (N=12), and progressive MS (N=8), alongside nine healthy controls. We identified immune infiltrates for the first time, likely lymphocytes and microglial cells, near the retinal vascular plexus, with the highest densities observed in ON-RMS patients (93.6%). These cells were sometimes detected weeks before clinical ON onset, and their density declined post-ON, though at varying rates. Infiltrates were more frequently found in MS patients than in controls, even outside acute ON episodes. The cells showed minimal movement and often interacted with vessels, suggesting migratory behavior. Our results suggest that AOSLO imaging can detect subtle retinal inflammatory changes not captured by conventional clinical systems, offering a promising tool for monitoring neuroinflammation in MS and other neurodegenerative diseases. These findings support the potential of high-resolution retinal imaging as a non-invasive biomarker for tracking neuroinflammation and therapeutic responses in patients with MS.

Abnormal visual experience during an early critical period of visual cortex development can lead to a neurodevelopmental disorder of vision called amblyopia. A key feature of amblyopia is interocular suppression, whereby information from the amblyopic eye is blocked from conscious awareness when both eyes are open. Suppression of the amblyopic eye is thought to occur at an early stage of visual processing and to be absolute. Using a binocular rivalry paradigm, we demonstrate that suppressed visual information from the amblyopic eye remains available for binocular integration and can influence overall perception of stimuli. This finding reveals that suppressed visual information continues to be represented within the brain even when it is blocked from conscious awareness by chronic pathological suppression. These results have direct implications for the clinical management of amblyopia.

The fovea is the small depression at the neurosensory retina that underlies high-resolution central vision. It is vulnerable to disease and disruption of its architecture causes visual disability. Foveal morphology varies significantly across individuals. The molecular causes and functional consequences of this anatomical diversity are incompletely understood. Here, we extracted six foveal morphological parameters from Optical Coherence Tomography (OCT) images of 39,521 UK Biobank participants. We found notable variability in foveal morphology and detected significant links with sex and genetic ancestry. Genome-wide association studies identified 161 lead loci across the six foveal morphological parameters, implicating genes involved in pigmentation (e.g., TYR, TSPAN10, GPR143) and patterning (e.g., FGFR2, PTPRD, CYP1A1). Heritability estimates ranged from 29-43%. Foveal pit volume was associated with future risk of age-related macular degeneration (HR=1.1, p=0.0004), a finding supported by Mendelian randomization. These results establish foveal morphology as a highly heritable trait with notable influence over retinal disease risk.

Macular corneal dystrophy (MCD) is classified as corneal stromal dystrophy. In this study, we retrospectively reviewed the surgical outcomes of 118 MCD patients receiving surgical treatment in the past 30 years and found patients receiving penetrating keratoplasty had the lowest recurrence rate 13.75%, compared with 40.91% patients receiving deep anterior lamellar keratoplasty and 25% receiving phototherapeutic keratectomy. Transcriptomic analysis in human corneal single-cell sequencing atlas found the MCD pathogenic gene CHST6 was abundant in corneal endothelium rather than other cell types. CHST6 protein showed a similar expression pattern to its mRNA. The mouse homologous gene Chst5 was 120-fold higher in corneal endothelium than in the epithelial and stromal layers. Mice with specifically Chst5 knockdown in the endothelial layer by microinjection of the adeno-associated virus serotype 9 - shRNA plasmids into the anterior chamber, rather than Chst5 knockdown into the stroma, showed MCD-like phenotypes. Corneal opacification and abnormally larger collagen fibrils were observed in the endothelial Chst5 knockdown mice. The same corneal characteristics were observed after overexpressing human CHST6 mutant R50H in the mouse endothelium. These observations indicating the pathogenesis of MCD is more related to the corneal endothelium rather than the stroma. Significance StatementOur study gave evidence that corneal endothelium contributing more to the macular corneal dystrophy (MCD) development, rather than other cell types in the cornea. We proposed penetrating keratoplasty might serve as a more proper surgical treatment for MCD according to the recurrence rate analysis. We also provided a novel method to construct MCD mouse model.