Investigative Ophthalmology & Visual Science

PurposeMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in many inflammatory and fibrotic diseases; however, its role in primary open-angle glaucoma (POAG) and trabecular meshwork (TM) dysfunction remains unknown. In this study, we investigated whether MIF-CD74 signaling regulates TM pathobiology through modulation of the transcription factor, Blimp-1, and downstream cytoskeletal reorganization and extracellular matrix (ECM) remodeling. MethodPrimary human TM cells (HTMC) were exposed to glaucomatous stressors, including TGF-{beta}2, rMIF, or a pro-inflammatory milieu. Expression of MIF, its receptor CD74, and Blimp-1 was measured by qPCR and immunoblotting. ECM proteins and phosphorylated myosin-light chain (pMLC) were evaluated by immunofluorescence staining. In vivo, MIF-CD74 and Blimp-1 expression were examined in the TM/anterior segment (AS) tissue of Tg.CreMYOCY437H and lentiviral (LV)-TGF-{beta}2-induced ocular hypertension (OHT) mouse models. Functional involvement of MIF signaling in TM pathobiology was examined using the irreversible MIF inhibitor 4-IPP and the immunomodulatory metabolites agmatine and thiamine. ResultsGlaucomatous stressors significantly upregulated MIF and CD74 expression with concomitant suppression of Blimp-1 in HTMC. Similarly, TM/AS tissue from both OHT models (Tg.CreMYOCY437H and LV-TGF-{beta}2) demonstrated increased MIF-CD74 expression accompanied by reduced Blimp-1 levels. Activation of MIF-CD74 signaling triggered pro-inflammatory and cell death pathways and promoted ECM remodeling, characterized by increased fibrotic protein expression and enhanced RhoA/ROCK-mediated MLC phosphorylation, indicating modulation of TM contractility. Pharmacological inhibition of MIF attenuated inflammatory signaling, reduced ECM deposition and cytoskeletal remodeling, and suppressed RhoA/ROCK/MLC activation, restoring a protective TM phenotype. ConclusionOur findings identify MIF-CD74 signaling as a previously unrecognized regulator of TM dysfunction in POAG. MIF-mediated suppression of Blimp-1 mechanistically links inflammatory signaling to cytoskeletal contractility and fibrotic ECM remodeling, key determinants of aqueous humor outflow resistance. Targeting the MIF-CD74/Blimp-1 axis may represent a novel therapeutic strategy to restore TM homeostasis and reduce intraocular pressure in glaucoma.

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

PurposeThe inner wall of Schlemms canal (SC) is a mechanosensitive endothelial monolayer that provides resistance to conventional aqueous humor drainage, a process dependent on pore formation. This study examined how microtubule (MT) stability affects SC cell mechanobiology, transcellular pore formation, and aqueous humor outflow dynamics. MethodsMT stability in cultured SC cells from normal and glaucomatous human donors was manipulated pharmacologically. Changes in MT acetylation, phosphorylated myosin light chain, and F-actin were assessed by immunofluorescence and immunoblotting. GEF-H1 was knocked down using siRNA. Cellular stiffness was measured by atomic force microscopy. Transcellular pore formation was quantified using an established pore formation assay. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. ResultsMT stabilization in normal SC cells decreased actomyosin contractility and cellular stiffness, whereas MT destabilization increased contractility and stiffness; these effects involved the MT-associated Rho guanine nucleotide exchange factor GEF-H1. MT stability was also mechano-responsive to substrate stiffness. Furthermore, SC cells derived from glaucomatous donors exhibited reduced MT stability compared with normal SC cells. MT stabilization increased transcellular pore formation in both normal and glaucomatous SC cells. In ex vivo mouse eyes, paclitaxel perfusion to stabilize MTs significantly increased outflow facility relative to contralateral control eyes. ConclusionsOur data suggest that MT stability influences SC cell contractility, stiffness, and transcellular pore formation and can alter aqueous humor outflow facility. These findings identify MT-dependent cytoskeletal remodeling as an important contributor to the biomechanics of the conventional outflow pathway and suggest that MT-associated pathways may represent potential targets for improving outflow function in glaucoma.

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

Porcine organotypic retinal explant cultures are widely used to study retinal neurodegeneration under controlled conditions, but the biological process that occurs in the retinal explant over time due to preparation-induced injury and culture are not well understood. Here, we generated a time-resolved transcriptomic reference for porcine neural retinal explants-maintained ex vivo for 10 days. Global expression profiles are strongly separated by culture time, with Day 0 clearly distinct from cultured samples and at Day 7 and Day 10 showing the highest similarity, indicating a transition toward a later stabilized state. Across the time course, 3,187 genes were differentially expressed relative to Day 0, with the largest shifts occurring at an early stage of culture (Day 1-Day 3). Pathway-level analyses revealed coordinated remodeling involving inflammatory signaling, and metabolic/bioenergetic changes, including reduced mitochondrial and oxidative phosphorylation-related programs at later time points. Here, we provide a time-resolved transcriptomics reference dataset for cultured porcine retinal explants. These data can build a foundation to interpret data generated in this model, differentiate changes inherent to the explant culture from treatment-specific effects and to select appropriate experimental windows for mechanistic studies of retinal degeneration.

Age-related macular degeneration (AMD) is a common, complex disease affecting older individuals that can lead to severe vision loss. It is characterized by early anatomical changes in the retina, retinal pigment epithelium (RPE), and choroid, especially in the central (macular) region. AMD can progress to severe atrophy and/or pathologic angiogenesis that leads to visual decline. Over 30 genetic loci have been identified as contributing to AMD risk; however, the mechanisms by which genetic variants affect pathology has not been thoroughly explored. In this report we examined single-nucleus gene expression in the retina, RPE and choroid of 88 individuals categorized by AMD stage, as well as 37 previously published samples. Genotyping was performed on 1.8 million SNPs, with additional SNPs imputed, on each donor to identify expression quantitative trait loci (eQTLs). We found that two AMD-risk loci (PILRB and ARMS2/HTRA1) affected the expression of PILRB and HTRA1, respectively. The risk allele of PILRB was associated with increased PILRB RNA in cones, fibroblasts, choroidal macrophages, and RPE, whereas the HTRA1 risk locus was associated with decreased HTRA1 RNA in the RPE. We also identified an age-related decrease in complement inhibitors in the choriocapillaris, a tissue susceptible to complement mediated damage in AMD.

PurposeHyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have previously shown that HOS damages mitochondria and impairs cell metabolism in corneal epithelial cells. Small extracellular vesicles (sEVs) are cell-derived lipid envelopes that are present in all body fluids, including tears. Prior studies suggest that sEV release and composition may be linked with changes in cell metabolism. In this study, we tested the effects of HOS on sEV release and composition, and found that sEV cargo may reflect early, underlying changes in dry eye disease. MethodsTelomerase-immortalized human corneal epithelial (hTCEpi) cells were treated with 450 mOsm NaCl for five days to induce chronic HOS. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. Particle number was determined using Nanoparticle Tracking Analysis (NTA). Mass spectrometry was used to assess the sEV proteome, and selected proteins were validated by immunoblot. Proteome pathways were analyzed using KEGG and CORUM. ResultsPathway analysis revealed an increase in metabolic proteins and proteasome components in sEV cargo released from hTCEpi cells exposed to HOS. These proteins were increased more than fourfold in HOS-sEVs. Examination of proteins involved in the endosomal pathway and NTA further confirmed an increase in HOS-sEV release. ConclusionOur findings suggest a potential mechanism whereby corneal epithelial cells exposed to HOS retain proteins involved in maintaining tissue integrity, while simultaneously releasing unneeded proteins involved in cell metabolism. The presence of metabolic proteins in sEVs may serve as early indicators of dry eye disease.

Oxidative stress is proposed to be a driver of age-related diseases. Age-related macular degeneration is one such disease, where the retinal pigment epithelium (RPE) is affected early in the disease. Vasculature damage also occurs, sometimes preceding RPE damage. To model some aspects of dry AMD, we used the NaIO3 mouse model of oxidative damage. Disruption of the deep retinal vascular plexus, disorganization and death of capillaries within the choriocapillaris, and marked electroretinographic decline were observed. AAV overexpressing the transcription factor, NRF2, which induces anti-oxidation enzymes and represses inflammation, was tested for protection of damage. The BEST1 promoter limited expression to the RPE. The RPE, photoreceptors, and vascular architecture in both retinal and choroidal compartments were protected. Conditioned medium from RPE-choroid explants, infected by AAV8/BEST1-NRF2, was sufficient to transfer partial protection in vivo, indicating that NRF2 induces a protective secreted factor(s). Analysis of RNA-seq data identified growth differentiation factor 15 (GDF15) as a candidate downstream mediator. Injection of recombinant GDF15 reproduced key protective phenotypes in vivo, whereas Gdf15-deficiency attenuated NRF2-mediated rescue. Pharmacologic inhibition of TGF-{beta} receptor signaling diminished NRF2 associated protection, supporting involvement of this signaling pathway. In a laser-induced choroidal neovascularization model, intravitreal GDF15 injection reduced fluorescein leakage and lesion size. These findings support a model in which NRF2 activation in the RPE induces expression of GDF15, which is capable of protecting the RPE, photoreceptors, and the retinal and choroidal vasculature. NRF2 and GDF15 have therapeutic potential for ocular diseases, as well as for other diseases with vascular pathology.

ObjectiveTo determine whether non-axon optic nerve morphometric features correlate with clinical visual function as strongly as the traditional axon count gold standard. DesignCross-sectional histological analysis with longitudinal clinical correlation. SubjectsEighteen mice from three strains: C57BL/6J (n=6), BXD51 (n=6), and DBA/2J (n=6). MethodsLeft eye (OS) optic nerves from mice euthanized at 12 months of age were resin-embedded and stained with p-phenylenediamine. Bright-field cross-sectional images were segmented using an AxonDeepSeg-based workflow to generate axon, myelin, whole nerve, and glial coverage masks for morphometric quantification. Seven morphometrics were extracted: axon count (nAx), axon density (AxDen), glial coverage area ratio (GliaR), mean solidity (Sol), mean axon diameter (AxDiam), mean myelin area (MyArea), and mean axon-myelin area (AxMyArea). Morphometrics were correlated with longitudinal clinical data collected at 1, 3, 6, 9, and 12 months, including visual acuity (VA), contrast threshold, intraocular pressure (IOP), and pattern electroretinography P50 and N95 amplitudes (PERG P50 and N95). Main Outcome MeasuresPearson correlation coefficients were used to assess associations between morphometric features and clinical measures, and Fisher z-transformed meta-analytic correlations were used to aggregate these associations across ages. ResultsVA and contrast threshold demonstrated strong correlations with GliaR that matched or exceeded nAx. Meta-analysis across ages revealed GliaR correlated with VA (r = -0.84, p = 4.49 x 10-21) and contrast threshold (r = 0.86, p=7.55 x 10-23), comparable to nAx correlations with VA (r = 0.80, p=8.13x10-17) and contrast threshold (r = -0.80, p= 1.74x10-16). Structure-function relationships shifted with age: at 6 months, GliaR had the strongest correlation with contrast threshold (r = 0.96), while at 12 months, AxDiam became the dominant correlate of both VA (r = 0.77) and contrast threshhold (r = -0.74). IOP, PERG P50, and PERG N95 exhibited weak correlations with all morphometrics (|r| < 0.27). ConclusionsNon-axon morphometrics, particularly glial coverage area ratio, correlate with visual function as strongly as traditional axon count. Automated optic nerve assessment should incorporate glial and other non-axon features. Further, stage-aware biomarker selection may better capture structure-function relationships in glaucoma.

PurposePitx2-associated developmental glaucoma is characterized by anterior segment dysgenesis, ocular hypertension, and optic neuropathy. Its consequences for corneal sensory innervation remain poorly understood. We investigated whether this disease alters corneal nerve structure and sensory function in a sex-dependent manner. MethodsMale and female Pitx2egl1/egl1 and Pitx2+/+ mice were examined at 1 and 3 months. Ocular phenotyping included intraocular pressure, fundus imaging, visual evoked potentials, and optic nerve ultrastructure. RNA sequencing of corneas and trigeminal ganglia was performed at 3 months. Corneal innervation was assessed by {beta}III-tubulin immunofluorescence and volumetric quantification of nerve fibers. Corneal sensitivity was measured using Von Frey filaments. ResultsPitx2egl1/egl1 mice developed progressive ocular hypertension, fundus abnormalities, reduced visual evoked potential amplitudes, and optic nerve degeneration, supporting the model as early-onset glaucoma. Baseline sex-related transcriptional differences were limited in both cornea and trigeminal ganglia. In contrast, Pitx2 mutation induced sex-dependent molecular responses. Female corneas showed broader transcriptional changes enriched in inflammatory, stress-response, and tissue-remodeling pathways, whereas male corneas showed a more restricted response involving metabolic and homeostatic processes. Similar sex divergence was observed in trigeminal ganglia. Corneal nerve fiber volume was reduced in both sexes at 3 months but not at 1 month, whereas reduced sensitivity was detected only in mutant males. ConclusionsThis study identifies sexual dimorphism as a component of Pitx2-associated developmental glaucoma. Furthermore, our findings suggest that glaucoma affects the corneal sensory system beyond optic nerve pathology, highlighting a potentially overlooked dimension of disease relevant to ocular surface monitoring and patient management. HighlightsO_LIDisruption of the cornea-trigeminal ganglion axis with coordinated molecular and functional alterations in Pitx2-associated early-onset glaucoma C_LIO_LISex-dependent modifications in both cornea and trigeminal ganglion responses to early-onset glaucoma C_LIO_LIProgressive corneal neurodegeneration in early-onset glaucoma C_LI

PurposeMachine learning approaches for automated quantification of optic nerve histology have emerged as potential tools for objective assessment of axonal injury in experimental glaucoma models. However, the generalizability of these models to independent datasets remains unclear. Guided by a scoping review of the literature, this study performed independent validation testing of publicly available models on a novel rat optic nerve dataset to assess their generalizability. MethodsWe conducted a scoping review following PRISMA-ScR guidelines. PubMed, EMBASE, Scopus, and Cochrane CENTRAL were searched from 2000 through 2025. Two reviewers independently screened records and extracted data on model characteristics and performance metrics. Additionally, we performed independent validation of three models (AxoNet, AxonDeepSeg, AxoNet 2.0) on a novel rat optic nerve dataset comprising 57 images with 9,514 manually annotated axons. Because AxonDeep is not publicly available, we instead evaluated AxonDeepSeg, a separate publicly available deep learning-based tool that, while not previously applied to optic nerve tissue, is widely used for nerve fiber segmentation. ResultsFrom 2,036 records, four manuscripts describing three deep learning models met inclusion criteria. Published correlation coefficients between model predictions and reference counts ranged from 0.959 to 0.99. On independent validation, performance was reduced: AxoNet 2.0 achieved the highest correlation (r = 0.89), followed by AxonDeepSeg (r = 0.86) and AxoNet (r = 0.79). Segmentation quality metrics revealed high precision (>0.94) but low recall (0.18 to 0.27), with Dice coefficients of 0.29 to 0.40, substantially below published benchmarks of 0.81. ConclusionsDeep learning models for optic nerve histology demonstrate strong within-study performance but show meaningful performance decrements when applied to independent datasets. The observed generalizability gap (correlations 0.07 to 0.182 points below published values) demonstrates the need for standardized validation datasets and multi-center testing before widespread adoption of these tools.

PurposeRetinal ganglion cells (RGCs) are essential for visual signal transmission, yet they are vulnerable to injury and degeneration. Gene modulation in RGCs using adeno-associated virus (AAV) offers a promising avenue for neuroprotection and regeneration, but promoters lack sufficient RGC specificity, limiting precision needed for preclinical studies. This study aims to identify novel promoter-enhancer combinations (PECs) to achieve gene expression preferentially in RGCs. MethodsWe evaluated existing transcriptomic data to identify Neuritin 1(Nrn1) as a gene with highly restricted RGC expression in the retina. Synthetic PECs derived from human and mouse Nrn1 loci were incorporated into AAV2 vectors driving expression of a nuclear-targeted reporter GreenLantern. AAVs were delivered via intravitreal injection into C57BL6/J mice, and transduction efficiency and RGC specificity were evaluated in both young and aged retinas and those subjected to intraorbital optic nerve crush (ONC), using immunohistochemistry and quantitative analysis of RBPMS+ cells. ResultsWe found that AAV2 with a human Nrn1 PEC drives gene expression in RGCs. Quantitative analysis revealed that over 83% of transduced cells were RBPMS-positive, indicating robust RGC selectivity and significantly outperforming ubiquitous promoters. Notably, the Nrn1 PEC retained strong and selective transgene expression in RGCs in aged mice and following ONC, demonstrating its resilience under aged and injury conditions. ConclusionThe Nrn1 PEC enables efficient and injury-resilient gene expression in RGCs, addressing a key limitation in cell-specific targeting. This AAV-incorporated PEC offers a robust platform for evaluating neuroprotective interventions and accelerates translational development of gene therapies for glaucoma and other optic neuropathies.

PurposeAlanine transaminases (ALT), encoded by the GPT gene, catalyzes the reversible conversion of pyruvate and glutamate to alanine and alpha-ketoglutarate, thereby correlating carbohydrate and amino acid metabolism. However, its role in the human neural retina remains unclear. This study aimed to explore the expression, localization, and metabolic function of ALT in the human neural retina and its potential involvement in retinal diseases. MethodsALT1 and ALT2 expression and localization were examined in the retinas of healthy and diabetic retinopathy (DR) donors via immunoblotting and immunofluorescence. ALT function was assessed in ex vivo human retinal explants using pharmacological inhibition with beta-chloro-L-alanine (BCLA), followed by the analyses of enzyme activity, tissue injury, and transcriptomic responses. Stable-isotope tracing with 13C-and 15N-labelled substrates combined with GC-MS was used to define ALT-dependent carbon and nitrogen fluxes in macular and peripheral retinas. Redox level (NADPH/NADP+) was also evaluated under tert-butyl hydroperoxide-induced oxidative stress. ResultsALT1 and ALT2 were both expressed in the human neural retina, with prominent localization in Muller glia and photoreceptor inner segments. ALT1 displayed a diffuse cytoplasmic distribution, whereas ALT2 demonstrated a punctate pattern consistent with mitochondrial localization. In DR retinas, ALT1 expression was spatially disorganized and heterogeneous, while ALT2 remained comparatively preserved. Inhibition of ALT with BCLA markedly reduced ALT activity without causing overt cytotoxicity or major transcriptional changes. Isotope tracing demonstrated that retinal ALT predominantly channels pyruvate-derived carbon into alanine, whereas alanine was minimally contributed to pyruvate production under basal conditions. ALT inhibition suppressed alanine synthesis and release, redirected nitrogen flux towards glutamate, glutamine, and aspartate, and uncovered distinct metabolic adaptations in macular but not peripheral retinas. Under oxidative stress, ALT inhibition induced the decrease of NADP+/NADPH ratio and LDH release, indicating improved redox balance and reduced tissue injury. ConclusionsALT is previously unrecognized as a regulator of carbon and nitrogen partitioner in the human neural retina, contributing to redox homeostasis under stress. The altered distribution of ALT1 in DR retina and the protective metabolic effects of ALT inhibition suggest ALT as a potential contributor to retinal metabolic vulnerability and a candidate therapeutic target in retinal diseases.

Open-angle glaucoma (OAG) affects approximately 57.5 million individuals worldwide and is characterized by the progressive loss of retinal ganglion cells (RGC) and irreversible optic nerve damage resulting from chronic ocular hypertension. Intraocular pressure (IOP) is the only major modifiable risk factor in OAG and clinical treatments necessarily aim to lower IOP in order to preserve RGCs and prevent vison loss. Pharmacological therapies, such as prostaglandin analog containing eye drops, are known to be effective at reducing IOP, but are critically undermined by poor patient compliance and are unable to control for potentially damaging diurnal fluctuations in IOP, leading to vision loss even in patients diagnosed early. Herein we evaluate the effectiveness of a long-acting, single use, prostaglandin-based recombinant adeno-associated virus (rAAV)-mediated IOP-lowering gene therapy treatment in glaucomatous DBA/2J mice and demonstrate that sustained IOP reduction leads to preservation of both optic nerve anatomy and function in end-stage glaucomatous disease. One Sentence SummaryIOP-lowering gene therapy provides partial anatomical and functional rescue in glaucomatous mouse model following single dose treatment

The mammalian retina lacks meaningful regenerative capacity, and degeneration usually leads to irreversible vision loss. Although lower vertebrates regenerate retinal neurons through Muller glia, this capacity has generally been considered absent in humans. Using long-term organotypic retinal cultures from 39 adult donors, we show that defined humoral cues alone are sufficient to unlock a latent neurogenic program in human Muller cells. FGF-2 treatment and GSK-3 inhibition induced robust proliferation across both peripheral and central retina, with 79.09 {+/-} 6.32% of dividing cells identified as Muller glia, some completing multiple cell cycles. Single-cell transcriptomics revealed activation of progenitor-like and neuronal differentiation pathways, whereas immunohistochemistry demonstrated expression of early and late neuronal markers spanning all major retinal lineages. Newly generated cells expressed markers of cone, rod, bipolar, horizontal, amacrine, and ganglion cell identities, together with evidence of early synaptogenesis. These findings reveal an intrinsic regenerative potential in adult human Muller glia, with implications for future vision-restoration strategies in degenerative retinal disease. SummaryAdult human Muller glia retain an intrinsic capacity for proliferation and neural lineage commitment independent of donor age or gender. In long-term organotypic cultures of human donor retina, defined humoral cues, without genetic manipulation, induce Muller glia proliferation and the onset of neuronal differentiation. These findings reveal intrinsic neurogenic potential in human Muller glia and provide a human-relevant platform for retinal regeneration studies.

Subretinal fibrosis underlies the end-stage pathogenesis of retinal diseases including age- related macular degeneration (AMD). It can disrupt retinal structure and eventually lead to legal blindness by generating contractile force, fibrotic scarring, subretinal hemorrhage, and retinal detachment. Myofibroblasts are the predominant cells critically involved in subretinal fibrosis, however, the cellular contribution to myofibroblasts remains unclear. Here we demonstrate that multiple cell lineages, including macrophages, endothelial cells (EC), retinal pigment epithelial (RPE) cells and pericytes, significantly contribute to myofibroblasts in a laser-induced subretinal fibrosis model. We found microRNA miR-24 is significantly downregulated in the plasma of wet AMD patients. Overexpression of miR-24 represses epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EndMT), and the resulting fibrosis by regulating TGF- {beta}/SMAD3 and PAK4/LIMK2/MRTF pathways. Consistently, a combination of SMAD3 and MRTF inhibitors show superior efficacy to individual inhibitors in repressing fibrosis in vitro and laser-induced subretinal fibrosis in vivo. Together, these suggest the contribution of multiple cell-types in myofibroblast transformation in subretinal fibrosis, and repression of miR-24-regulated TGF-{beta}/SMAD3 and PAK4/LIMK2/MRTF pathways in multiple cell types holds therapeutic potential for treating subretinal fibrosis in AMD and other fibrotic disorders.

Aim: To evaluate the potential of a three-dimensional microscope combining Laser scanning confocal imaging and white-light interferometry for quantitative topographic characterisation of Descemet's membrane (DM) in Fuchs endothelial corneal dystrophy (FECD). Methods: Descemet's membranes were collected from 38 FECD patients undergoing endothelial keratoplasty and 4 healthy donors. After flat-mounting on glass slide and drying, specimens were analysed using the VK-X3000 system (KEYENCE). Entire samples were reconstructed by image stitching at low magnification (x10) in white-light interferometry mode (0.01nm axial resolution). Higher magnifications (x20-x150) in confocal mode (12nm axial resolution) enabled detailed structural analysis. Three-dimensional height maps were generated to calculate standardised surface roughness parameters. Guttae and other DM features were classified according to spatial organisation and elevation profiles. Results: White-light interferometry enabled full-field mapping of whole 8mm diameter DMs with nanometric vertical resolution (~2 hours/sample). Surface roughness (Sa) was higher in FECD than in controls (median{+/-}IQR: 0.571{+/-}0.259 m vs 0.239{+/-}0.161 m ; p = 0.0018). In FECD, three zones were identified: central (guttae buried in the posterior fibrillar layer; Sa 0.442 {+/-} 0.112 m), paracentral (large uncovered guttae; Sa 0.562{+/-}0.170 m ; p = 0.0423), and outer zone (no confluent guttae; Sa 0.261{+/-}0.143 m ; p < 0.0001). Confocal 3D imaging revealed radial striae, embossments and furrows in the DM, confluent central guttae, and fused or buried structures. Conclusions: Combining white-light interferometry and confocal microscopy enables label-free, high-resolution surface characterisation of DM in FECD, providing quantitative metrics to compare histological subtypes and supporting the predominance of radial structural organisation.

Pentosan polysulfate (PPS) is a semisynthetic sulfated polysaccharide that was approved by the United States Food and Drug Administration (FDA) for treatment of interstitial cystitis (IC). A 2018 study by our group described a vision-threatening macular toxicity associated with long-term use of PPS. However, given the relatively recent characterization of PPS maculopathy, we have limited knowledge of its pathophysiology. The present study therefore investigated the pathophysiology of PPS maculopathy in a cell culture model, assessing impacts of PPS exposure on morphology and mitochondrial function. We treated ARPE-19 cells with increasing doses of PPS and investigated both mitoprotective and cytoprotective mechanisms, mitochondrial reactive oxygen species production (ROS) and respiration, cellular structure, and retinal pigment epithelium (RPE) dysfunction through phagocytosis assays. We found that PPS increased mitochondrial superoxide accumulation and that increased doses of PPS impaired basal and maximal respiration in a Seahorse assay without the expected response of increases in the cellular energy sensor pAMPK. PPS exposure disrupted mitochondrial and cell protective mechanisms against ROS accumulation as assessed through examination of mitochondrial biogenesis markers PGC-1 and SIRT1 and autophagy markers LC3 and p62. PINK1 expression increased with increasing duration of exposure to PPS. Further, we found that PPS led to functional and structural changes to RPE cells, which exhibited an increase in cell aspect ratio and impaired phagocytosis with higher doses of PPS. Lastly, we found an increase in cell death in response to higher doses of PPS, evident through ethidium homodimer cell viability assays. Taken together, our study shows PPS exposure has profound effects on RPE viability and function through impairment of mitochondrial respiration and mito- and cyto-protective mechanisms and highlights mitochondrial insult as a potential focus of future PPS research.

Thyroid eye disease (TED) is a debilitating condition characterized by orbital fibroblast (OF) activation and excessive hyaluronic acid (HA) accumulation within the retro-ocular space. While IGF-1R blockade with teprotumumab has significantly advanced TED management, incomplete clinical responses and disease relapse underscore the need to identify alternative targets. In this study, we used high-throughput RNA sequencing to map the transcriptomic landscape in TED OFs compared with non-TED OF controls. Our analysis identified robust enrichment of pathways critical to the TED phenotype, including PI3K-AKT signaling, the platelet-derived growth factor (PDGF) pathway, and extracellular matrix remodeling. We validated several key upregulated mediators that may contribute to orbital remodeling, including FOXC2, HGF, MET, and HMGA2, alongside the downregulation of the Wnt antagonist SFRP2. By employing a computational drug-repositioning approach, we identified the multi-kinase inhibitor sorafenib, which targets VEGFR, PDGFR, and RAF, as a potent candidate to neutralize the TED-specific gene signature. Functional assays demonstrated that sorafenib dose-dependently inhibited PDGF-induced AKT phosphorylation and significantly attenuated HA synthesis in primary TED OFs. These results define a persistent, receptor tyrosine kinase-driven program in the TED orbit and suggest that multi-kinase inhibition represents a viable therapeutic strategy for refractory TED. HighlightsO_LIThyroid eye disease (TED) orbital fibroblasts exhibit a transcriptomic signature characterized by elevated PI3K/AKT, angiogenic, and growth factor signaling. C_LIO_LIComputational drug prediction identifies sorafenib as a candidate to reverse the TED gene signature. C_LIO_LISorafenib dose-dependently inhibits AKT activation and hyaluronic acid production in TED orbital fibroblasts. C_LI

PurposeAtoh7 is a transiently expressed developmental transcription factor that gives rise to the seven major retinal cell types. Despite this broad lineage, Atoh7 is only required for retinal ganglion cell (RGC) genesis and survival, even though a significant portion of RGCs are Atoh7 negative based on lineage tracing in mice, suggesting a cell nonautonomous role for Atoh7 in the genesis and survival of all RGCs. Atoh7 function is conserved in zebrafish, yet the full retinal lineage, including the RGC population, has remained unidentified. Therefore, we sought to determine the atoh7 retinal lineage in wild type and atoh7 mutant zebrafish retinas. MethodsWe generated atoh7:iCre transgenic zebrafish and in combination with the established ubi:Switch lineage trace permanently labeled cells that represent the atoh7 lineage. A combination of in vivo live imaging and immunohistochemical techniques were used to validate atoh7:iCre transgene expression and the atoh7 lineage in embryonic, larval, and adult retinas as well as the adult brain. ResultsThe atoh7:iCre;ubi:Switch transgene combination successfully recapitulated the onset of endogenous atoh7 expression and transgene fluorophores persisted into adulthood labeling the atoh7 lineage. Most notably, we determined 79% of total RGCs in the wild type retina come from atoh7+ progenitor cells, a greater number than reported in the mouse retina. In atoh7 mutant retinas, we confirmed a complete loss of RGCs and observed a statistically significant increase in the proportion of atoh7+/Pax6+ amacrine cells, as well as an increase in the total number of Prox1+ bipolar cells. Interestingly, we discovered atoh7+ cells located outside the eye in other areas of the central nervous system. ConclusionsThese data demonstrate the presence of atoh7 positive and negative retinal cell types in the zebrafish retina, including RGCs, highlighting the potential to study survival mechanisms of atoh7 negative RGCs and fate switch paradigms using zebrafish retinal development models.