Investigative Opthalmology & Visual Science

Purpose: Animal models of myopia typically induce monocular refractive shifts via form deprivation (FD) or lens-induced myopia (LIM), modeling susceptibility to myopia, but with potentially limited applicability to childhood myopia. Here we describe a novel, genetically diverse mouse model of naturally occurring refractive error (NORE) with three distinct refractive phenotypes: hyperopic, myopic, and intermediate. Methods: C57BL/6J mice were mated to 129S2/SvPasCrl mice to create F1 or F2 offspring. Refractive errors in male and female F1 (N=21) and F2 (N=101) mice were assessed on postnatal days (P) 28 and 42 using photorefractometry. In a subset of mice (N=30 - 40), corneal radius of curvature, axial ocular dimensions, retinal and visual function were assessed. Results: F2 mice were classified as NORE with either hyperopic (RE [&ge;] 0 diopters (D) at P28 and P42), myopic (RE<0D at P28 and P42) or intermediate (RE<0D at P28 and RE [&ge;] 0D at P42) refractions based on individual trajectories. All ocular parameters changed with age, with significantly slower growth in axial length and vitreous chamber depth in the intermediate versus myopic mice (p<0.05). Lens thickness was smaller in the myopic group at P28. Differences in refraction were not attributed to variances in retinal function or dopamine signaling. Conclusions: NORE mice represent a novel, genetically diverse wild-type mouse model that, unlike traditional models, does not require interventions such as FD or LIM to induce myopia. NORE mice provide a valuable tool for future investigations of genetic and environmental mechanisms and targeted therapeutic strategies for refractive errors.

PurposeAqueous humor outflow through the trabecular meshwork (TM) is segmental, demonstrating high flow (HF) and low flow (LF) regions. Here, we investigate transcriptomic differences between flow regions in naive mouse and non-glaucomatous human TM tissue to better understand intraocular pressure (IOP) regulation. MethodsHuman eyes (<6 hours postmortem) from two donors were perfused with a fluorescent tracer to identify HF and LF regions and fixed. In parallel, one pair of 8-month-old C57Bl/6J mouse eyes were similarly processed. Sagittal sections from HF and LF regions underwent whole-transcriptome spatial profiling. Differential expression and gene set variation analysis were conducted to identify transcript and pathway-level differences between HF vs. LF TM. Selected targets were validated by immunolabeling. ResultsGenes with relevance to TM outflow were identified as significantly differentially expressed in the human dataset, including ADAM metallopeptidase domain 15 (ADAM15), vimentin (VIM), chitinase 3-like 1 (CHI3L1), and several transcription factors (e.g., FOS, JUNB, ESR1). Pathways related to epigenetic modifications were also differentially enriched in human eyes. In both human and mouse eyes, myocilin was significantly upregulated in HF regions, despite greater protein labeling in LF regions of human eyes. In both species, rho-kinase signaling pathways showed increased enrichment in LF regions, while cell stress pathways, and TNF- signaling were increased in HF TM. ConclusionsHF regions maintain a more active stress response that facilitates greater outflow, whereas LF regions exhibit more matrix accumulation and contractility. This characterization of segmental flow regions can inform future studies to target trabecular outflow and lower IOP.

It is clear that the human retina and its underlying retinal pigment epithelium and choroid (RPE/choroid) form an interdependent metabolic ecosystem, but how metabolism differs between the cone-rich macula and rod-rich periphery remains unclear. Using targeted metabolomics, we quantified 133 metabolites in paired macular and peripheral neural retina and RPE/choroid explants from human donor eyes following short-term culture to restore metabolic activity. Distinct metabolic differences were identified between retinal regions and between tissues. Compared with the peripheral retina, the macula showed metabolic features consistent with greater glycolytic activity, increased NADH availability and higher levels of the neurotransmitter-associated metabolites N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG), consistent with increased energetic and neuronal activity. Compared with peripheral RPE/choroid, the macular RPE/choroid had higher levels of the flavin cofactor FAD together with NAD-related metabolites, including NAD, NADP and NAAD. Comparisons between the neural retina and RPE/choroid further showed that the neural retina was primarily associated with energy production and neurotransmission, whereas the RPE/choroid was associated with cofactor metabolism, nucleotide salvage and lipid metabolism. These findings are consistent with metabolic coupling between the neural retina and RPE/choroid. The macula has metabolic features consistent with high energetic demand, providing a potential metabolic basis for its selective vulnerability in macular disease.

Cone photoreceptors are essential for daylight vision, and their degeneration has more profound visual consequences than rod loss in retinal degenerative diseases. Metabolic dysfunction is closely associated with cone degeneration, however the relatively small population of cones in mice and humans have limited our understanding of cone-specific metabolism. Here, we leveraged cone-dominant and cone-degeneration mouse models including Nrl-/-, Cnga3-/-, and high-dose Triiodothyronine (T3) treatment to investigate cone-specific metabolism and their metabolic impacts on the retinal pigment epithelium (RPE). Across models, cone-dominant retinas consistently showed lower pyruvate abundance alongside increases in glutathione, purines, pentose phosphate pathway intermediates, and one-carbon metabolites. Increases in several key amino acids were also associated with higher cone abundance, such as proline, arginine, alanine, valine, leucine, and hypotaurine. Strikingly, aminoadipate, an intermediate in lysine catabolism, was the most robustly changed metabolite in the retina, showing highly consistent increases across models. Relative cone increases were also associated with metabolic changes in the RPE/choroid. Like the retina, RPE/choroids showed consistent increases in aminoadipate, proline, and hypotaurine, as well as xanthosine and betaine, alongside decreases in uracil. Moreover, proteomic analysis of Nrl-/- mice showed decreases in many key metabolite transporters in the RPE/choroid, including carriers for glucose, lactate, aspartate, glutamate, serine, lysine, taurine, and proline. Collectively, these findings further our understanding of cone-specific metabolism and highlight potential cone-specific metabolic vulnerabilities in retinal degeneration.

Purpose: To assess the feasibility of quantitative fundus autofluorescence (FAF) measurement in early age-related macular degeneration (AMD) using the freely available ImageJ software, to characterize signal intensity across FAF patterns, and to evaluate interobserver reproducibility in pattern classification. Methods: Single-center, non-blinded, retrospective, consecutive-case analytical study. FAF images acquired with Spectralis OCT+HRA (Heidelberg Engineering) from patients with early dry AMD seen at a tertiary referral center between January 2010 and September 2016 were analyzed. A standardized 300x300-pixel region of interest (ROI) centered on the fovea was evaluated in ImageJ v2.0.0-rc54/1.51h (Fiji distribution). Mean, minimum, and maximum autofluorescence (AF) pixel intensity were recorded. Each image was independently classified according to the Bindewald classification system by two graders; a third senior grader adjudicated discordances. Cohen's kappa (k) was used to assess interobserver agreement. Results: Of 423 patients with available FAF studies, 107 had dry AMD; 45 met quality and diagnostic criteria for early AMD and were included in the quantitative analysis. Mean age was 73.47 +/- 8.1 years; 62.2% were female. Mean FAF intensity was 120.26 (range 74.76-160.79); mean minimum was 32.07 (range 3-63) and mean maximum was 205.80 (range 125-255). Seven of eight Bindewald patterns were identified; the stippled pattern was absent. The most frequent pattern was minimal changes (31.1%), followed by increased focal (24.4%) and patchy (15.6%). Reticular pattern showed the highest mean AF (143.8), while lace pattern showed the lowest (88.4). Interobserver agreement for Bindewald pattern classification was almost perfect (k = 0.969; 95% CI, 0.908-1.000; p < 0.001). Agreement for lesion extent was moderate (k = 0.531) and for foveal involvement was substantial (k = 0.622). Conclusions: Quantitative FAF evaluation of early AMD using ImageJ is feasible and reproducible. ImageJ represents a cost-free alternative for multimodal retinal image analysis, with potential for automated screening applications in resource-limited settings. Keywords: age-related macular degeneration; fundus autofluorescence; ImageJ; quantitative autofluorescence; image analysis; Bindewald classification; interobserver agreement

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

Col8a2Q455K/Q455K (Q455K) mice exhibit features of early-onset Fuchs endothelial corneal dystrophy (FECD), including decreased endothelial cell density (ECD) and guttae formation. Within the context of these clinical features, this study longitudinally evaluates ferroptosis in Q455K and wild-type (WT) mice using in vivo imaging, PCR and immunohistochemistry. Fifty-six Q455K and 56 WT mice were evaluated from 3 to 24 months of age with in vivo confocal microscopy; ECD and guttae were measured. Ferroptosis marker expression was determined with PCR and immunohistochemistry (IHC). Data were analyzed using two-way ANOVA with Tukeys post hoc test, Chi-square test and a paired t-test. The ECD significantly decreased in both groups from 3 to 24 months of age, but more markedly in Q455K (2285-/+317 to 1012-/+58 cells/mmSquare) versus WT mice (2714-/+139 to 2057-/+149 cells/mmSquare, P<0.0001). Guttae were observed exclusively in Q455K mice beginning at 3 months of age and increased over time (P=0.0003). The Q455K mice demonstrate guttae at the vertices of corneal endothelial cells rather than their centers (74.3% vs. 25.7%P<0.001). Expression of ferroptosis-related genes (Tfrc, Slc40a1, Ftl1, Gpx4) were significantly increased in the Q455K versus WT mice (P<0.05). Furthermore, corresponding protein expression (transferrin receptor 1, ferroportin, ferritin and glutathione peroxidase 4) was significantly elevated adjacent to guttae in Q455K versus WT mice (P<0.05). These findings implicate guttae in the initiation of ferroptosis as it relates to the pathophysiology of FECD and provide an optimal window for testing novel FECD therapies using this murine model, particularly those that target ferroptosis.

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

Abstract Purpose: To expand the clinical and mechanistic understanding of the +57C>T seed-region mutation in miR-184 causing EDICT (endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning) syndrome. Design: Cross-sectional analysis and laboratory confirmation Participants: 18 members of a four-generation family with known +57C>T miR-184 status Methods: We used optical biometry, corneal topography, and medical history to characterize the clinical phenotype. Carrier effects on ocular biometric measurements were estimated using polygenic linear mixed models incorporating a pedigree-derived kinship matrix, adjusted for age and sex. Patient-derived and control induced pluripotent stem cells (iPSCs) were generated and differentiated into corneal endothelial cells (CECs). Main Outcome Measures: Axial length, keratometry (in diopters), white-to-white corneal diameter, topography mapping, central and peripheral corneal thickness, and history of retinal detachment or corneal transplant were compared between mutation carriers and noncarriers, adjusting for age and sex. Cellular analysis was conducted with immunostaining (ZO-1, ATP1A1), morphometric quantification, qRT-PCR of endothelial markers, and transendothelial electrical resistance (TEER). Results: 10 of 18 family members were heterozygous for +57C>T, with retinal detachment occurring in 5/10 affected individuals compared to 0/8 unaffected individuals (p=0.04). Affected eyes had 2.2 mm shorter axial length (p=0.02), 9.3 D steeper mean keratometry (p=0.004), 1.6 mm smaller horizontal corneal diameter (p=0.0001), and 139-micrometer thinner central corneas (p=0.003). Mutant iPSC-derived CECs were associated with irregular borders, increased cell and nucleus area, widened intercellular gaps, disrupted ATP1A1 membrane localization, and reduced barrier function on TEER (all p<0.05). Gene expression analysis showed downregulation of COL4A1, COL4A3, and AQP1 with upregulation of COL8A1. Conclusions: The miR-184 +57C>T mutation produces a broad ocular phenotype that includes smaller, thinner corneas and microphthalmia. Mechanistically, it disrupts CEC junctional integrity, extracellular matrix and pump-related genes, supporting a role for miR-184 in coordinated anterior-posterior eye morphogenesis.

Glaucoma is an irreversible blinding disease that affects millions of individuals worldwide. Elevated intraocular pressure (IOP), regulated by the trabecular meshwork (TM) in the anterior eye, is the only modifiable risk factor. Human TM cells can be cultured from donor eyes, providing a precious resource for studying factors that induce or prevent glaucoma. The goal of this study was to produce datasets that define the molecular profile of human non-glaucomatous TM cells. Using 18 human TM cell strains cultured from non-glaucomatous individuals deposited from seven laboratories in the USA and UK, this study used transcriptomic, proteomic, lipidomic, and metabolomic analyses to characterize the molecular content of TM cells. The data herein provides the most comprehensive multiomic analyses of human TM cells to date and will be a useful resource for researchers and clinicians in the TM and glaucoma fields.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

Objective: To investigate the performance of anterior segment (AS) OCT quantitative imaging of anterior chamber inflammation in uveitis patients with diverse demographics. Design: Prospective cross-sectional study. Participants: 144 adult patients managed at a tertiary care service in the UK Methods: Repeated swept-source ASOCT imaging was performed pre- and post-pupil dilation (i.e. 4 scan sets). Inflammation was quantified using a validated human in the loop automated image analysis pipeline, Minuscule Cell Detection (MCD), which identified and counted putative inflammatory cells on AS-OCT. Main Outcome Measures: Test-retest variability of ASOCT and diagnostic accuracy of various ASOCT derived measurands (minimum, maximum, median counts per cross sectional image, and total counts across volume image sets per eye or MINCC, MAXCC, MEDCC and TOTCC) versus Standardization of Uveitis Nomenclature (SUN) grading system as assessed by a uveitis specialist. Results: A total of 281 eyes were included in the analysis. Median age was 48 years (IQR 36 to 64). Strong test-retest measurand reliability was demonstrated, with a 95% tolerance interval ratio 0.3 to 3.0. The best diagnostic performances for SUN activity were observed with the MINCC threshold of 3 particles (negative predictive value for clinical activity of 89.8%, 95% CI 83.0 to 94.1). Associations between ASOCT measurands and patient age (adjusted coefficient 7.5 additional particles, 95% CI 0.5 to 14.6, p<0.04 for age over 60 years versus under 44), and pigment load (52.8, 11.8 to 92.9, p<0.01 in eyes with AC pigment versus without) were noted. Conclusions: ASOCT assessment of anterior chamber inflammation in uveitis meets current recommendations for quantitative imaging biomarkers, demonstrating strong repeatability, linearity with clinical assessment scores and stability with pupil dilation and patient characteristics of ethnicity and lens status. The absence of variability in diagnostic indices across derived measurands suggests similar performance across different acquisition protocols. Further longitudinal cross-platform studies are needed to determine limitations of use.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

Background/Objective: Diabetic macular oedema (DMO) is a leading cause for visual impairment primarily managed with intravitreal anti-VEGF agents such as ranibizumab (RBZ). Levosulpiride (LSP), a prokinetic medication, was recently repositioned as a safe oral treatment for naive DMO. Here, we investigated the adjuvant effect of oral LSP in combination with intravitreal RBZ injections for treating persistent DMO. Subjects/Methods: Double-blinded, dual-centre, phase 2 trial in patients with centre-involving DMO randomly assigned to be orally treated with placebo (15 patients, 18 eyes) or LSP (18 patients, 19 eyes) along with 3 successive (4 weeks apart) RBZ intravitreal injections and a 24-week follow-up. Results: Baseline best-corrected visual acuity (BCVA) improved (p[&le;]0.04) at week 12 in both RBZ+placebo and RBZ+LSP, but improvement was maintained (p=0.009) at week 24 only in RBZ+LSP. In agreement, longitudinal changes from baseline in BCVA from weeks 12 to 24 defined superior (p=0.02) visual gains measured by the Area Under the Curve (AUC) in RBZ+LSP vs. RBZ+placebo. The baseline value of mean central foveal thickness (CFT) decreased (p[&le;]0.002) in both groups at week 12 and CFT reduction was significant (p=0.006) at week 24 only in RBZ+LSP. Also, longitudinal changes from baseline in CFT resulted in a higher AUC reduction (p[&le;]0.04) at weeks 4 to12 in RBZ+LSP vs. RBZ+placebo. No significant adverse side effects were detected. Conclusions: Adjunctive LSP showed functional and anatomical benefits over the first-line therapy with RBZ. Adjuvant properties may involve the LSP-induced intraocular upregulation and downregulation of vasoinhibin and VEGF, respectively. Larger clinical trials are warranted.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

Tear fluid is amenable to non-invasive and repeated collection, making it a practical specimen for evaluating exercise-related physiological responses. However, the immediate proteome-wide alterations in tear fluid following acute exercise have not been characterised. In this study, we performed quantitative proteomic profiling of paired tear samples from healthy female participants before and immediately after a single exercise session using data-independent acquisition liquid chromatography-tandem mass spectrometry (DIA-LC-MS/MS). Among the 3,173 identified proteins, 744 were significantly altered post-exercise, of which 484 were up-regulated and 260 down-regulated. Functional enrichment analysis revealed that up-regulated proteins were predominantly associated with translation and ribosome biogenesis, whereas down-regulated proteins were involved in glycan metabolism, lysosomal processing, and extracellular matrix organisation. Collectively, these findings indicate that acute exercise elicits a rapid and coordinated reconfiguration of the tear proteome. This investigation provides a molecular basis for understanding exercise-mediated modulation of tear composition and ocular surface homeostasis.

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-pecific Vhl knockout mouse (RodVhl), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type-enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod{Delta}Vhl mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic 13C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

Purpose: To investigate corneal subbasal nerve plexus alterations using in vivo corneal confocal microscopy (IVCM) in patients with Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) and Spastic Paraplegia Type 7 (SPG7). Methods: This cross-sectional pilot study included eight ARSACS patients, five SPG7 patients, and twenty age- and sex-matched healthy controls. All participants underwent neurological and ophthalmological examination followed by central corneal imaging using IVCM. Quantitative corneal nerve parameters were analyzed with automated software, and correlations with clinical severity scales were assessed. Results: The mean age was 34.2 +/- 3.4 years in controls, 34.5 +/- 0.7 years in the ARSACS group, and 38.2 +/- 3.5 years in the SPG7 group. Corneal nerve branch density (CNBD) and corneal nerve total branch density (CTBD) were significantly lower in ARSACS and SPG7 patients compared with healthy controls. CNFD, CNFL, CNFA, CNFW, and CNFrD were lower in ARSACS and SPG7 patients compared with healthy controls; however, these differences did not reach statistical significance. No statistically significant differences in IVCM parameters were detected between ARSACS and SPG7 patients. Spearman correlation analysis did not show significant correlations between corneal nerve parameters and FARS, SARA, ADL scores, or disease duration. Conclusion: IVCM revealed reduced corneal nerve branching parameters in patients with ARSACS and SPG7. These findings indicate involvement of the corneal subbasal nerve plexus and support the potential role of corneal confocal microscopy as a non-invasive ocular imaging modality for evaluating peripheral neural alterations in hereditary spastic ataxias.