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The death of retinal ganglion cells (RGCs) is a key factor in the pathophysiology of all forms of glaucoma. RGC culture serves as a simple system for establishing and testing candidate therapies. This study aimed to explore the differentiation of primary retinal progenitor cells (RPCs) into RGC-like cells induced by low-dose cytarabine (Ara-C). RPCs were isolated from the retina of newborn rats and cultured in vitro. Different concentrations of Ara-C were added to the culture medium to induce the differentiation of RPCs into RGC-like cells. Differentiation efficiency was assessed through immunofluorescence staining and cell counting. The addition of Ara-C significantly increased the number of Brn3a/RBPMS double-positive cells. The RPC-RGCs induced displayed characteristic features of RGCs, with roughly 80.9 % ± 6.2 % of the cells positive for both TuJ1/NeuN and 77.5 % ± 4.9 % for Brn3a/RBPMS. The study demonstrates that the addition of Ara-C to primary cultures of rat RPCs can enhance their differentiation into RGC-like cells, providing a simple and rapid method for obtaining RGC-like cells with a relatively high purity. This method shows considerable promise for advancing glaucoma research and potential therapeutic strategies to restore vision after RGC loss., Competing Interests: Declaration of competing interest This study has neither been submitted for publication nor published in whole or part elsewhere. I acknowledge that both I and the other authors have read the Instructions to Authors and agree with its contents. Sources of financial support, corporate involvement, etc. for each author are enclosed in this manuscript. If accepted, the manuscript shall not be published elsewhere in the same form in either the same or any other language without the consent of the Editor and Publisher. Copyright transfer will be according to the principle of the editorial office. We declare that there are no conflicts of interest. And this manuscript is in accordance with the Authorship statement of ethical standards for manuscripts submitted to Biochemical and Biophysical Research Communications. I will take full responsibility for the data, the analyses and interpretation, and the conduct of the research. I had full access to all of the data and that I had the right to publish any and all data, separate and apart from the attitudes of the sponsor., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) is the leading cause of blindness among working-age adults, and inflammation and oxidative stress contribute to DR development. However, no effective treatments are currently approved for DR. Therefore, this study aimed to investigate the effects of SMTP-44D-a Stachybotrys microspora-derived compound with anti-inflammatory and antioxidant properties-on DR in in vivo and in vitro models. Diabetes was induced in rats using 60 mg/kg streptozocin, followed by treatment with SMTP-44D every second day. Retinal function was assessed using electroretinography every 2 months for 8 months. SMTP-44D prevented diabetes-induced b-wave amplitude reductions in electroretinogram and decreased retinal ganglion cell apoptosis. SMTP-44D also reduced the accumulation of advanced glycation end-products (AGEs), AGE receptors, and 8-hydroxydeoxyguanosine in the retina. Furthermore, when rat retinal Müller cells were cultured in DMEM medium containing 35 mM glucose (high glucose, HG) and treated with SMTP-44D for 24 h, SMTP-44D mitigated cell death, reactive oxygen species production, and inflammatory cytokine levels in the cells. These findings suggest that SMTP-44D exhibits significant antioxidant and anti-inflammatory effects, highlighting its potential as a therapeutic candidate for DR., Competing Interests: Conflict of interest The authors indicated no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hsiao-Hua Yu reports financial support was provided by National Science and Technology Council. Shih-Hwa Chiou reports financial support was provided by National Science and Technology Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: To characterize the presence of amyloid-beta (Aβ) in human glaucoma retina and to test the identification of retinal Aβ using a novel fluorescent Aβ-binding small molecule (AMDX-2011)., Methods: Postmortem human eyes with (n=4) and without (n=4) glaucoma were acquired from an eye bank. Retinas were dissected, flat-mounted, and fixed. Using the flat mounts, immunofluorescence was performed against Aβ, AMDX-2011 staining was conducted, and images were acquired using fluorescence microscopy., Results: Fluorescence microscopy demonstrated the presence of an Aβ signal that colocalized with AMDX-2011 staining in the glaucoma retina. Colabeled puncta appeared in all quadrants of the retina, including the retina temporal to the optic nerve. The puncta were mainly located within the inner layers of the retina. Glaucoma retinas had more colabeled puncta than control retinas in all locations ( P =0.002-0.02). Colabeled puncta were also larger in the superior quadrant of glaucoma compared with control retinas ( P =0.02)., Conclusions: Aβ was detected in human glaucomatous retina, and its distribution was mapped. AMDX-2011 identification of Aβ may lead to future diagnostic tests aimed at detecting Aβ in glaucoma patients., Competing Interests: Disclosure: J.P.: Amydis (Employee). S.K.: Amydis (Employee). A.T.: Amydis (Consultant). Z.M.: Amydis (Employee). S.S.: Amydis (Employee). P.V.: Amydis (Employee). S.S.: Amydis (Owner). A.S.: Amydis (Advisor). R.W.: Abbvie (Consultant), Alcon (Consultant), Allergan (Consultant), Amydis (Consultant), Balance (Consultant), Editas (Consultant), Equinox (Consultant), Eyenovia (Consultant), Iantrek (Consultant), Implandata (Consultant), IOPtic (Consultant), iStar Medical (Consultant), Santen (Consultant), TenPoint (Consultant), Topcon (Consultant), Toromedes (Patent, Owner), Zeiss-Meditec (Patent). A.H.: Allergan (Consultant), Amydis (Consultant), Celanese (Consultant), Diagnosys (Research Support), Equinox (Consultant), Glaukos (Consultant and Research Support), Heidelberg Engineering (Research Support), QLARIS (Consultant), Santen (Consultant), and Topcon (Consultant). The remaining authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Chronic innate immune activation in the central nervous system (CNS) significantly contributes to neurodegeneration in progressive multiple sclerosis (MS). Using multiple experimental autoimmune encephalomyelitis (EAE) models, we discovered that NLRX1 protects neurons in the anterior visual pathway from inflammatory neurodegeneration. We quantified retinal ganglion cell (RGC) density and optic nerve axonal degeneration, gliosis, and T-cell infiltration in Nlrx1 -/- and wild-type (WT) EAE mice and found increased RGC loss and axonal injury in Nlrx1 -/- mice compared to WT mice in both active immunization EAE and spontaneous opticospinal encephalomyelitis (OSE) models. To minimize the effects of Nlrx1 -/- on peripheral lymphocyte priming during EAE, we performed adoptive transfer experiments, in which activated myelin-specific T cells were transferred into lymphocyte-deficient Rag -/- or Nlrx1 -/- Rag -/- mice. In this model, we found more severe microgliosis and astrogliosis in the optic nerve of Nlrx1 -/- Rag -/- mice compared to Rag -/- mice, suggesting a regulatory role of NLRX1 in innate immune cells. Transcriptome analysis in primary astrocytes activated with LPS and IFNγ demonstrated that NLRX1 suppresses NF-κB activation and regulates mitochondrial oxidative phosphorylation in inflammatory reactive astrocytes. The novel pharmacologic NLRX1 activators NX-13 and LABP-66 decreased LPS-mediated gene expression of inflammatory cytokines and chemokines in mixed glial cultures. Moreover, treating EAE mice with oral LABP-66, compared to vehicle, after the onset of paralysis resulted in less anterior visual pathway neurodegeneration. These data suggest that pharmacologic NLRX1 activators have the potential to limit inflammatory neurodegeneration. This study highlights that NLRX1 could serve as a promising target for neuroprotection in progressive MS and other neurodegenerative diseases. Further studies are needed to better understand the cell-specific mechanisms underlying the neuroprotective role of NLRX1 in response to inflammation in the CNS., Competing Interests: Declarations. Ethical approval: All experimental protocols were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals and were approved by the Johns Hopkins Institutional Animal Care and Use Committee (protocol # MO22M158). Ethics approval and consent to participate: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Eph/ephrin signaling is crucial for organizing retinotopic maps in vertebrates. Unlike other EphAs, which are expressed in the embryonic ventral retina, EphA4 is found in the retinal ganglion cell (RGC) layer at perinatal stages, and its role in mammalian visual system development remains unclear. Using classic in vitro stripe assays, we demonstrate that, while RGC axons are repelled by ephrinB2, they grow on ephrinB1 stripes through EphA4-mediated adhesion. In vivo, retinal axons from EphA4-deficient mice from either sex show impaired arborization in the medial, but not lateral, regions of the superior colliculus that express ephrinB1. Gain-of-function experiments further reveal that ephrinB1-mediated adhesion depends on EphA4 tyrosine kinase activity but it is independent of its sterile alpha motif. Together, our findings suggest that EphA4/ephrinB1 forward signaling likely facilitates adhesion between retinal axon terminals and cells in the medial colliculus, contributing to the establishment of proper connectivity within the visual system., (Copyright © 2024 the authors.)

We developed ligandomics for the in vivo profiling of vascular ligands in mice, discovering secretogranin III (Scg3) as a novel angiogenic factor that selectively binds to retinal vessels of diabetic but not healthy mice. This discovery led to the development of anti-Scg3 therapy for ocular vasculopathies. However, in vivo ligandomics requires intracardial perfusion to remove unbound phage clones, limiting its use to vascular endothelial cells (ECs). To extend ligandomics to non-vascular cells, we investigated ex vivo ligandomics. We isolated ECs and retinal ganglion cells (RGCs) from diabetic and healthy mouse retinas by immunopanning. We quantified the binding of clonal phages displaying Scg3 and vascular endothelial growth factor (VEGF), confirming that their binding patterns to isolated diabetic versus healthy ECs matched in vivo patterns. Additionally, Scg3 and VEGF binding to isolated RGCs reflected their in vivo activity. These results support the feasibility of ex vivo ligandomics. We further mapped ligands binding to immunopanned diabetic and healthy ECs and RGCs by ligandomics, confirming that Scg3 was enriched with selective binding to diabetic ECs but not healthy ECs or diabetic/healthy RGCs. These findings demonstrate the feasibility of ex vivo ligandomics, which can be broadly applied to various cell types, tissues, diseases, and species.

Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed, human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient's iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1 -DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration.

Fatty acid binding proteins (FABPs) are a class of small molecular mass intracellular lipid chaperone proteins that bind to hydrophobic ligands, such as long-chain fatty acids. FABP5 expression was significantly upregulated in the N-methyl-d-aspartic acid (NMDA) model, the microbead-induced chronic glaucoma model, and the DBA/2J mice. Previous studies have demonstrated that FABP5 can mediate mitochondrial dysfunction and oxidative stress in ischemic neurons, but the role of FABP5 in oxidative stress and cell death in retina NMDA injury models is unclear. In this study, we found that FABP5 is significantly altered in a model of glutamate excitotoxicity and is regulated by Stat3. Inhibition of FABP5 alleviated oxidative stress imbalance and activation of NLRP3 inflammasome, reduced the release of inflammatory factors, and ultimately attenuated glutamate excitotoxicity-induced retinal ganglion cell loss. Meanwhile, caspase1 inhibitors could alleviate the retinal ganglion cell loss induced by glutamate excitotoxicity. In conclusion, FABP5 inhibition protects retina ganglion cells from excitotoxic damage by suppressing the ROS-NLRP3 inflammasome pathway. FABP5 maybe a promising new target for glaucoma diagnosis and treatment., (© 2025 Federation of American Societies for Experimental Biology.)

Signal transduction downstream of axon guidance molecules is essential for steering developing axons. Second messengers including cAMP are key molecules shared by a multitude of signaling pathways and are required for a wide range of cellular processes including axon pathfinding. Yet, how these signaling molecules achieve specificity for each of their downstream pathways remains elusive. Subcellular compartmentation has emerged as a flexible strategy to reach such a specificity. Here, we show that point contact-restricted cAMP signals control ephrin-A5-evoked axon repulsion in vitro by modulating focal adhesion kinase (FAK; also known as PTK2) phosphorylation and the assembly and disassembly rate of point contacts. Consistent with this, preventing point contact-specific cAMP signals in developing retinal ganglion cells in vivo alters the refinement of their terminal axonal arbor in the brain. Altogether, our study identifies point contacts as a compartment containing a local cAMP signal required for ephrin-A5-dependent axon guidance and highlights the crucial role of such subcellularly restricted second messenger signals in the wiring of neuronal circuits., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Background: Bushen-Huoxue-Mingmu-Formula (MMF) has achieved definite clinical efficacy. However, its mechanism is still unclear., Objective: Investigating the molecular mechanism of MMF to protect retinal ganglion cells (RGCs)., Methods: This study developed a pressurization-induced model of damaged RGCs, which were then treated with a serum supplemented with MMF. The effects of MMF on proliferation, apoptosis, adenosine 5'-triphosphate content, and mitochondrial structure of RGCs were investigated, and the underlying molecular mechanism was explored by RNA interference experiment., Results: In the pressurization-induced RGC injury model, apoptosis rate increased, cell proliferation decreased, adenosine 5'-triphosphate content reduced, mitochondrial structure was disrupted, BCL2-associated X, cleaved caspase-3, and microtubule-associated proteins light chain 3 II/I protein expression enhanced, B cell lymphoma-2 and p62 protein expression decreased, and the Pink1/Parkin pathway was activated. The stress-induced damage to RGCs was, however, reversible following MMF-mediated inhibition of the Pink1/Parkin pathway. Pink1 short-hairpin RNA downregulated Pink1 expression in RGCs, which led to outcomes that aligned with those observed with MMF intervention., Conclusions: MMF altered the expression of apoptosis- and autophagy-related proteins and possibly inhibited the Pink1/Parkin signaling pathway, which led to reduced pressurization-induced mitochondrial autophagy in RGCs. This preventive effect of MMF on RGCs can be potentially useful to preserve the viability of RGCs., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2025 the Author(s). Published by Wolters Kluwer Health, Inc.)

Ischemic retinopathies are the major causes of blindness, yet effective early-stage treatments remain limited due to an incomplete understanding of the underlying molecular mechanisms. Significant changes in gene expression often precede structural and functional alterations. Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are emerging as novel gene regulators, involved in various biological processes and human diseases. In this study, tsRNA-Gln-i-0095 was identified as a novel regulator, which was significantly upregulated in retinal ischemia/reperfusion (I/R) injury. Reducing the levels of tsRNA-Gln-i-0095 suppressed reactive gliosis, lowered inflammatory cytokine levels, and protected retinal ganglion cells from I/R injury. These effects led to reduced structural and functional damage, inhibited glial activation and inflammation, and enhanced neuronal function. Mechanistically, tsRNA-Gln-i-0095 downregulated the expression of NFIA and TGFBR2 through a miRNA-like mechanism. Collectively, this study highlights the potential of targeting tsRNA-Gln-i-0095 as a novel therapeutic approach to reduce retinal I/R injury and preserve visual function., Competing Interests: Declarations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Neuromodulators have major influences on the regulation of neural circuit activity across the nervous system. Nitric oxide (NO) has been shown to be a prominent neuromodulator in many circuits and has been extensively studied in the retina. Here, it has been associated with the regulation of light adaptation, gain control, and gap junctional coupling, but its effect on the retinal output, specifically on the different types of retinal ganglion cells (RGCs), is still poorly understood. In this study, we used two-photon Ca 2+ imaging and multi-electrode array (MEA) recordings to measure light-evoked activity of RGCs in the ganglion cell layer in the ex vivo mouse retina. This approach allowed us to investigate the neuromodulatory effects of NO on a cell type-level. Our findings reveal that NO selectively modulates the suppression of temporal responses in a distinct subset of contrast-suppressed RGC types, increasing their activity without altering the spatial properties of their receptive fields. Given that under photopic conditions, NO release is triggered by quick changes in light levels, we propose that these RGC types signal fast contrast changes to higher visual regions. Remarkably, we found that about one-third of the RGC types, recorded using two-photon Ca 2+ imaging, exhibited consistent, cell type-specific adaptational response changes throughout an experiment, independent of NO. By employing a sequential-recording paradigm, we could disentangle those additional adaptational response changes from drug-induced modulations. Taken together, our research highlights the selective neuromodulatory effects of NO on RGCs and emphasizes the need of considering non-pharmacological activity changes, like adaptation, in such study designs., Competing Interests: DG, MG, JO, TS, RA, ZZ, TS, OM, TE No competing interests declared, (© 2024, Gonschorek et al.)

Purpose: This study aimed to investigate the role of SIRT4 in retinal protection, specifically its ability to mitigate excitotoxic damage to Müller glial cells through the regulation of mitochondrial dynamics and glutamate transporters (GLASTs)., Methods: A model of retinal excitatory neurotoxicity was established in mice. Proteins related to mitochondrial dynamics, GLAST, and SIRT4 were analyzed on days 0, 1, 3, and 5 following toxic injury. The influence of SIRT4 on mitochondrial dynamics-related proteins and GLAST was examined by inducing SIRT4 overexpression through intraperitoneal injection of resveratrol or by using SIRT4 knockout (KO) mice. Additionally, the effects of upregulating and downregulating SIRT4 expression in rat Müller glial cell lines (rMC-1) were explored via lentiviral vector transfection to assess changes in mitochondrial morphology and GLAST expression., Results: After excitotoxic injury to the mouse retina, the retinal thickness and structure were disrupted, the number of retinal ganglion cells (RGCs) decreased, and Müller glial cells were activated by day 1. The levels of OPA1, GLAST, and SIRT4 proteins peaked on the first day after injury and then gradually decreased, indicating a synchronized dynamic trend. The upregulation of SIRT4 expression promoted OPA1 and GLAST protein expression, thereby alleviating retinal excitotoxic injury. Furthermore, the upregulation of SIRT4 expression promoted mitochondrial fusion and increased GLAST expression in rMC-1 cells, reducing cellular excitotoxic damage. Conversely, downregulation of SIRT4 had the opposite effect., Conclusions: SIRT4 plays a significant role in mitigating excitotoxic damage in the retina, modulating Müller glial cell injury by regulating mitochondrial dynamics and glutamate transporter expression, ultimately influencing retinal health.

Purpose: Stress granules (SGs) are cytoplasmic biocondensates formed in response to various cellular stressors, contributing to cell survival. Although implicated in diverse pathologies, their role in retinal degeneration (RD) remains unclear. We aimed to investigate SG formation in the retina and its induction by excessive LED light in an RD model., Methods: Rat retinas were immunohistochemically analyzed for SG markers G3BP1 and eIF3, and SGs were also visualized by RNA fluorescence in situ hybridization. Additionally, SGs were induced in primary retinal cell and eyeball cultures using sodium arsenite. Light exposure experiments used LED lamps with a color temperature of 5500 K and 200 lux intensity for short-term or two- to eight-day exposures., Results: SGs were predominantly detected in retinal ganglion cells (RGCs) and inner nuclear layer (INL) cells, with arsenite-induction verified in RGCs. SG abundance was higher in animals exposed to light for 2-8 days compared to light/dark cycle controls. RGCs consistently exhibited more SGs than INL cells, and INL cells more than outer nuclear layer (ONL) cells (Scheirer-Ray-Hare test: H = 13.2, P = 0.0103 for light condition, and H = 278.2, P < 0.00001 for retinal layer). These observations were consistent across four independent experiments, each with three animals per light condition., Conclusions: This study characterizes SGs in the mammalian retina for the first time, with increased prevalence after excessive LED light exposure. RGCs and INL cells showed heightened SG formation, suggesting a potential protective mechanism against photodamage. Further investigations are warranted to elucidate the role of SGs in shielding against light stress and their implications in retinopathies.

Purpose: Because chickens have excellent light perception properties, this study focused on investigating whether monochromatic light can cause photodamage in chicken retinal ganglion cells (RGCs)., Methods: Post-hatching day chickens were exposed to four different light-emitting diode light environments for five weeks, respectively, monochromatic blue light (480 nm), green light (560 nm), red light (660 nm), or white light (6000 K). The mechanisms through which monochromatic light influences the structure of the chicken retina were analyzed by detecting the morphological structure of the retina, gene and protein expression levels, and the ultrastructure of the optic nerve., Results: Blue light exposure for five weeks significantly impacted the thickness of the inner reticular layer, retinal synaptic function, and the number of RGCs in the chicken retina. Moreover, neurodegenerative disease characteristics, such as a reduction in the number of dendritic branches and the presence of myelin lesions, have also been observed in RGCs. The activation of the protein kinase RNA-like ER kinase-mediated unfolded protein response, along with the abnormal degradation of the autophagy substrate P62, accompanies these retinal pathological processes. Additionally, our findings indicate that the photosensitivity of OPN4 is linked to endoplasmic reticulum stress in RGCs, which may elucidate why chicken RGCs experience damage exclusively under blue light exposure., Conclusions: Blue light can damage the chicken retina through endoplasmic reticulum stress and autophagy, especially in RGCs. Our study enhances the understanding of the mechanisms underlying retinal photodamage and emphasizes the risk of retinal damage in low-illuminance blue light environments.

Purpose: Inflammatory processes have been involved in diabetic retinopathy (DR). Interleukin (IL)-17A, a pro-inflammatory cytokine, is associated with DR occurrence and development. However, mechanisms underlying the IL-17A impact on DR need further investigations. Herein, we show that exosomal miRNA delivers IL-17A signal from Müller cells (MCs) to retinal ganglion cells (RGCs) to facilitate DR., Methods: Exosomes isolated from primarily cultured MCs were identified and high-throughput sequencing was used to indicate differentially expressed miRNAs in the exosomes. Targeting relationship of miR-92a-3p and Notch-1 was determined by dual-luciferase reporter assay. MCs were treated with high glucose (HG), IL-17A, anti-IL-17A antibody, miR-92a-3p inhibitor, or/and miR-92a-3p mimic, and exosomes derived from MCs with the various treatments were applied to primary RGCs. DR mice were induced by streptozotocin (STZ) and subjected to intravitreal injection., Results: Expression of miR-92a-3p in exosomes derived from MCs with IL-17A treatment in either the absence or the presence of HG was upregulated, and the IL-17A effect was blocked by anti-IL-17A antibody. The exosomes derived from IL-17A-treated MCs downregulated Notch-1 expression in recipient RGCs and increased the neuronal death. These effects of IL-17A were suppressed by miR-92a-3p inhibitor but enhanced by miR-92a-3p mimic. In STZ mice, intravitreal administration of exosomes derived from IL-17A-treated MCs downregulated retinal Notch-1 expression and increased RGC apoptosis. These IL-17A effects were hindered by miR-92a-3p inhibitor., Conclusions: MC-released exosomal miR-92a-3p transmits IL-17A signal by inhibiting the target Notch-1 to accelerate DR progression. An intravitreal administration of exosomes containing miR-92a-3p inhibitor may become a potential therapeutic strategy for DR.

The nonvisual effects of light in humans are mainly conveyed by a subset of retinal ganglion cells that contain the pigment melanopsin which renders them intrinsically photosensitive (= intrinsically photosensitive retinal ganglion cells, ipRGCs). They have direct connections to the main circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and modulate a variety of physiological processes, pineal melatonin secretion, autonomic functions, cognitive processes such as attention, and behavior, including sleep and wakefulness. This is because efferent projections from the SCN reach other hypothalamic nuclei, the pineal gland, thalamus, basal forebrain, and the brainstem. The ipRGCs also directly impact the prefrontal cortex and the perihabenular nucleus (mood). In particular, light suppresses the secretion of melatonin in a dose-dependent manner, mainly depending on irradiance and spectral composition of light. There is evidence that exposure to light-emitting devices from luminaires and screens before bedtime can impact on sleep onset latency, sleep duration, and sleep quality. Likewise, light exposure during daytime modulates sleep architecture, duration, and sleep quality during the subsequent night. Therefore, the integration of acute, circadian, and long-term effects of light together influence sleep-wake quality and behavior in healthy individuals, as well as in patients with psychiatric or medical disorders., Competing Interests: Conflict of interest Both authors declare no conflict of interest. C.B. and M.M. are elected members of the Daylight Academy (www.DLA.org)., (Copyright © 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing of keratocytes from human cadaveric donors Basic Protocol 2: Reprogramming donor keratocytes into iPSCs Basic Protocol 3: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping Basic Protocol 4: Generation of 3D ROs Basic Protocol 5: Dissociation and culturing of RGCs from 3D ROs Support Protocol 1: Immunostaining for phenotypic characterization of cells Support Protocol 2: Sectioning of 3D ROs and immunostaining Support Protocol 3: Western blotting for cleaved CASP3 and THY1., (© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Excessive exposure to blue light can cause retinal damage. Hydrogen-rich saline (HRS), one of the hydrogen therapies, has been demonstrated to be effective in eye photodamage, but the effect on the expression of melanopsin in intrinsically photosensitive retinal ganglion cells (ipRGCs) is unknown. In this study, we used a rat model of light-induced retinal injury to observe the expression of melanopsin after HRS treatment and to determine the effect of HRS on retinal ganglion cell protection. Adult SD rats were exposed to blue light (48 h) and treated with HRS for 0, 3, 7, and 14 days. Real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were performed to find the expression of genes and proteins, respectively. The function of retinal ipRGCs was measured by pattern-evoked electroretinography (pERG). The number and morphological changes of melanopsin-positive ganglion cells in the retina were observed by immunofluorescence (IF). Acute blue light exposure caused a decrease in ipRGC function, decreased expression of melanopsin protein and the melanopsin-positive RGCs, and diminished immunoreactivity in dendrites. However, over time, melanopsin showed a tendency to self-recovery, with an increase in melanopsin protein expression and the number of melanopsin-positive RGCs, with incomplete recovery of function within two weeks. HRS treatment accelerated the recovery process, with a significant increase in melanopsin expression and the number of melanopsin-positive RGCs, and an improvement in the pERG waveform within two weeks., (© 2024 American Society for Photobiology.)

Extended glucocorticoid (GC) treatment can lead to ocular hypertension and induce iatrogenic open-angle glaucoma. GC-induced glaucoma mimics many clinical and pathological features of primary open-angle glaucoma (POAG), and therefore mouse models of GC-induced glaucoma are utilized to study pathophysiology of glaucoma. We have recently demonstrated that weekly periocular injections of dexamethasone-21-acetate (Dex-Ac) lead to robust and significant intraocular pressure (IOP) elevation, retinal ganglion cell (RGC) loss, and optic nerve degeneration in mice. Our mouse model exhibits signature features of POAG including significant IOP elevation due to reduced outflow facility, progressive optic nerve degeneration, and structural and functional loss of RGCs. Dex-induced IOP elevation is associated with increased aqueous outflow resistance due to trabecular meshwork (TM) dysfunction including excessive extracellular matrix deposition and induction of endoplasmic reticulum stress. Mouse model of Dex-induced glaucoma represents an ideal animal model to investigate both glaucomatous damage to the TM and RGC axons and to develop novel therapies. Here, we present a detailed protocol for developing this model in laboratory settings., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

IL-33 and its receptor ST2 play crucial roles in tissue repair and homeostasis. However, their involvement in optic neuropathy due to trauma and glaucoma remains unclear. Here, we report that IL-33 and ST2 were highly expressed in the mouse optic nerve and retina. Deletion of IL-33 or ST2 exacerbated retinal ganglion cell (RGC) loss, retinal thinning, and nerve fiber degeneration following optic nerve (ON) injury. This heightened retinal neurodegeneration correlated with increased neurotoxic astrocytes in Il33 -/- mice. In vitro, rIL-33 mitigated the neurotoxic astrocyte phenotype and reduced the expression of pro-inflammatory factors, thereby alleviating the RGC death induced by neurotoxic astrocyte-conditioned medium in retinal explants. Exogenous IL-33 treatment improved RGC survival in Il33 -/- and WT mice after ON injury, but not in ST2 -/- mice. Our findings highlight the role of the IL-33/ST2 axis in modulating reactive astrocyte function and providing neuroprotection for RGCs following ON injury., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© 2024. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

Besides an elevated intraocular pressure (IOP), advanced age is one of the most crucial risk factors for developing glaucoma. βB1-Connective Tissue Growth Factor (βB1-CTGF) high-pressure glaucoma mice were used in this study to assess whether glaucoma mice display more inflammatory and aging processes than age-matched controls. Therefore, 20-month-old βB1-CTGF and corresponding wildtype (WT) controls were examined. After IOP measurements, retinas were processed for (immuno-)histological and quantitative real-time PCR analyses. A significantly higher IOP and diminished retinal ganglion cell numbers were noted in βB1-CTGF mice compared to WT. An enhanced macrogliosis as well as an increased number of microglia/macrophages and microglia was detected in retinas of old glaucoma mice. Interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β2 were upregulated, suggesting an ongoing inflammation. Moreover, βB1-CTGF retinas displayed an increased senescence-associated β-galactosidase staining accompanied by a downregulation of Lmnb1 (laminin-B1) mRNA levels. Our results provide a deeper insight into the association between inflammation and high-pressure glaucoma and thus might help to develop new therapy strategies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetes is a major risk factor for compromised visual health, leading to retinal vasculopathy and neuropathy, both of which are hallmarks of neurovascular unit dysfunction. Despite the critical impact of diabetic retinopathy, the precise mechanism underlying neurovascular coupling and effective strategies to suppress neurovascular dysfunction remain unclear. In this study, the up-regulation of a tRNA-derived stress-induced RNA, 5'tiRNA-His-GTG, in response to diabetic stress is revealed. 5'tiRNA-His-GTG directly regulates Müller glia action and indirectly alters endothelial angiogenic effects and retinal ganglion cell (RGC) survival in vitro. Downregulation of 5'tiRNA-His-GTG alleviates diabetes-induced retinal neurovascular dysfunction, characterized by reduced retinal vascular dysfunction, decreased retinal neurodegeneration, and improved visually-guided behaviors in vivo. Mechanistically, 5'tiRNA-His-GTG acts as a key regulator of retinal neurovascular dysfunction, primarily by modulating arachidonic acid (AA) metabolism via the CYPs pathway. The 5'tiRNA-His-GTG-CYP2E1-19(S)-HETE signaling axis is identified as a key driver of retinal neurovascular dysfunction. Thus, targeting 5'tiRNA-His-GTG presents a promising therapeutic strategy for treating vasculopathy and neuropathy associated with diabetes mellitus. Modulating this novel signaling pathway can open up new avenues for intervention in diabetic retinopathy and its related complications., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Glaucoma is a common optic neuropathy characterized by degeneration of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP), that is, ocular hypertension, is the primary modifiable risk factor for glaucoma and the primary characteristic of most preclinical glaucoma models. Extensive genotype and phenotype diversity at relatively low cost and high accessibility makes laboratory mice an excellent preclinical model for glaucoma. The microbead occlusion model was introduced in 2010 as an inducible model of ocular hypertension in mice and is now one of the most extensively utilized models of rodent glaucoma. Subsequent modifications of the microbead model increased the magnitude and duration of IOP elevation, primarily through modification of injection materials. Despite its popularity, accessibility of the model is hindered by procedural consistency between users. Here we outline an updated and comprehensive protocol for execution of the microbead model that is focused on improving surgical and outcome measure consistency and on enabling single experimenter execution., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

miRNA, short non-coding RNA, are rapidly emerging as important regulators in cell homeostasis, as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC), whose axons connect the eye to the brain, are central nervous system cells of great interest, yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line, human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC, with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA's role in RGC function, as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Currently there are no effective treatments for an array of neurodegenerative disorders to a large part because cell-based models fail to recapitulate disease. Here we develop a reproducible human iPSC-based model where laser axotomy causes retrograde axon degeneration leading to neuronal cell death. Time-lapse confocal imaging revealed that damage triggers an apoptotic wave of mitochondrial fission proceeding from the site of injury to the soma. We demonstrate that this apoptotic wave is locally initiated in the axon by dual leucine zipper kinase (DLK). We find that mitochondrial fission and resultant cell death are entirely dependent on phosphorylation of dynamin related protein 1 (DRP1) downstream of DLK, revealing a mechanism by which DLK can drive apoptosis. Importantly, we show that CRISPR mediated Drp1 depletion protects mouse retinal ganglion neurons from degeneration after optic nerve crush. Our results provide a platform for studying degeneration of human neurons, pinpoint key early events in damage related neural death and provide potential focus for therapeutic intervention., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Glaucoma is a group of heterogeneous neurodegenerative diseases with abnormal energy metabolism and imbalanced neuroinflammation in the retina. Thioredoxin-interacting protein (TXNIP) is involved in glucose and lipid metabolism, and associated with oxidative stress and inflammation, however, not known whether to be involved in glaucoma neuropathy and its underlying mechanisms., Methods: To establish the chronic ocular hypertension (COH) mice model. Western blot, RT-PCR, immunofluorescence and F-VEP were used to detect neuroinflammation level, glial activation and RGCs survival in retina of wild type, TXNIP knockout and MCC950 treatment COH mice. Microglia high-pressure cultured model was constructed. Western blot, RT-PCR and immunofluorescence were used to investigate the proinflammatory cytokines secretion, glucose uptake and phenotype transformation in wild type, TXNIP knockout and overexpressed microglia combined with IL-17A treatment. Finally, we explored the possible underlying mechanisms using relevant pathway inhibitor interventions., Results: In this study, for the first time we reported that TXNIP expression was remarkably increased in experimental glaucomatous retina of chronic ocular hypertension (COH) mice, and it was mainly expressed in the ganglion cells layer (GCL). In addition, we found that ablation of TXNIP promoted retinal ganglion cells (RGCs) survival and alleviated visual function impairment in experimental glaucoma. Then, we explored the spatiotemporal consistency between glial activation and retinal inflammation levels in COH mice respectively with TXNIP-deficiency and under treatment of a thermo-containing protein domain 3 (NLRP3) inhibitor MCC950, and the results indicated that TXNIP probably mediated neuroinflammation in glaucomatous retina by activating microglia. Furthermore, upregulation of TXNIP was found in pressure-stimulated microglia, whereas silencing TXNIP facilitated microglial polarization trending towards M1 type and reduced glucose transporter-1 (Glut-1) expression on microglia under high pressure in vitro. Moreover, IL-17A was found to play a role in acting synergistically with TXNIP upon the regulation of microglia polarity transformation. Finally, knockout of TXNIP was revealed to promote PI3K phosphorylation, whereas inhibition of PI3K by LY294002 effectively suppressed Glut-1 expression, glucose uptake, and M1-like transformation tendency in microglia obtained from TXNIP-deficiency mice under high pressure stimulation., Conclusions: TXNIP is significantly involved in the inflammation-related neuropathy of experimental glaucoma and probably facilitates M1-like microglial transformation via PI3K/Akt pathway., Competing Interests: Declarations. Ethics approval and consent to participate: The Animal Care and Use Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine approved all animal experimental protocols. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration., Competing Interests: Declarations. Ethics approval and consent to participate: The study was conducted in compliance with the ARVO guidelines and approved by IACUC at the University of Pittsburgh. Consent for publication: Not applicable. Competing interests: KCC and JLG are co-inventors on a patent application submitted through Stanford University. The authors declare no other competing interests., (© 2024. The Author(s).)

Traumatic optic neuropathy (TON) has been regarded a vision-threatening condition caused by either ocular or blunt/penetrating head trauma, which is characterized by direct or indirect TON. Injury happens during sports, vehicle accidents and mainly in military war and combat exposure. Earlier, we have demonstrated that remote ischemic post-conditioning (RIC) therapy is protective in TON, and here we report that AMPKα1 activation is crucial. AMPKα1 is the catalytic subunit of the heterotrimeric enzyme AMPK, the master regulator of cellular energetics and metabolism. The α1 isoform predominates in immune cells including macrophages (Mφs). Myeloid-specific AMPKα1 KO mice were generated by crossing AMPKα1 Flox/Flox and LysM cre to carry out the study. We induced TON in mice by using a controlled impact system. Mice (mixed sex) were randomized in six experimental groups for Sham (mock); Sham (RIC); AMPKα1 F/F (TON); AMPKα1 F/F (TON+RIC); AMPKα1 F/F LysM Cre (TON); AMPKα1 F/F LysM Cre (TON+RIC). RIC therapy was given every day (5-7 days following TON). Data were generated by using Western blotting (pAMPKα1, ICAM1, Brn3 and GAP43), immunofluorescence (pAMPKα1, cd11b, TMEM119 and ICAM1), flow cytometry (CD11b, F4/80, CD68, CD206, IL-10 and LY6G), ELISA (TNF-α and IL-10) and transmission electron microscopy (TEM, for demyelination and axonal degeneration), and retinal oxygenation was measured by a Unisense sensor system. First, we observed retinal morphology with funduscopic images and found TON has vascular inflammation. H&E staining data suggested that TON increased retinal inflammation and RIC attenuates retinal ganglion cell death. Immunofluorescence and Western blot data showed increased microglial activation and decreased retinal ganglion cell (RGCs) marker Brn3 and axonal regeneration marker GAP43 expression in the TON [AMPKα1 F/F ] vs. Sham group, but TON+RIC [AMPKα1 F/F ] attenuated the expression level of these markers. Interestingly, higher microglia activation was observed in the myeloid AMPKα1 F/F KO group following TON, and RIC therapy did not attenuate microglial expression. Flow cytometry, ELISA and retinal tissue oxygen data revealed that RIC therapy significantly reduced the pro-inflammatory signaling markers, increased anti-inflammatory macrophage polarization and improved oxygen level in the TON+RIC [AMPKα1 F/F ] group; however, RIC therapy did not reduce inflammatory signaling activation in the myeloid AMPKα1 KO mice. The transmission electron microscopy (TEM) data of the optic nerve showed increased demyelination and axonal degeneration in the TON [AMPKα1 F/F ] group, and RIC improved the myelination process in TON [AMPKα1 F/F ], but RIC had no significant effect in the AMPKα1 KO mice. The myeloid AMPKα1c deletion attenuated RIC induced anti-inflammatory macrophage polarization, and that suggests a molecular link between RIC and immune activation. Overall, these data suggest that RIC therapy provided protection against inflammation and neurodegeneration via myeloid AMPKα1 activation, but the deletion of myeloid AMPKα1 is not protective in TON. Further investigation of RIC and AMPKα1 signaling is warranted in TON.

Degenerative retinal diseases can lead to blindness if left untreated. At present, there are no curative therapies for retinal diseases. Therefore, effective treatment strategies for slowing the progression of retinal diseases and thus improving patients' life standards are urgently needed. The present study aimed to assess the effect of sinusoidal electromagnetic field (EMF) (50 Hz, 1.3 mT) treatment for 15 and 30 min on spontaneously arising retinal pigment epithelial cells (ARPE-19) and retinal ganglion cells (RGC-5) and its short-term post-treatment significance. Our study indicated the beneficial impact of EMF treatment on the proliferative and migratory capacity of the tested cells. ARPE-19 and RGC-5 cells exposed to an EMF exhibited elevated levels of HO-1, increased N6-methyladenosine (m 6 A) and N5-methylcytosine (m 5 C) status mediated by METTL3 and NSUN2, respectively, and changes in levels of DNA damage repair factors, which may contribute to the regenerative properties of ARPE-19 and RGC-5 cells. Overall, this analysis showed that EMF (sinusoidal, 50 Hz, 1.3 mT) treatment may serve as a potential therapeutic strategy for retinal diseases.

Regeneration of injured central nervous system (CNS) axons is highly restricted, leading to permanent neurological deficits. The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells, plays critical roles in development, brain plasticity, and tissues regeneration. However, little is known about the role of Marcks in CNS axon regeneration. Here we show that Marcks overexpression promotes robust axon regeneration either before or after optic nerve crush, but insignificantly impacts neuronal survival. Notably, immunostaining and RNA sequencing demonstrate that Marcks overexpression does not affect known regeneration-associated genes and pathways. Furthermore, combining CNTF which activates the JAK-STAT3 pathway and Marcks overexpression further enhances axon regeneration. Finally, we demonstrate functionally essential effector domain (ED) of MARCKS has similar effects on inducing axon regeneration in RGCs. These results suggest that manipulating Marcks and its ED may become a therapeutic approach to promote axon regeneration after CNS injury., Competing Interests: Competing interests: The authors declare no competing interests. Ethical Approval and Consent to Participate: All animal experiments were approved by the Animal Committee of the Institute of Zoology, Chinese Academy of Sciences (Approval No. IOZ-IACUC-2021-126)., (© 2024. The Author(s).)

Disruption of calcium (Ca 2+ ) homeostasis in neurons is a hallmark of neurodegenerative diseases. Here, we investigate the mechanisms leading to Ca 2+ dysregulation and ask whether altered Ca 2+ dynamics impinge on neuronal stress and circuit dysfunction. Using two-photon microscopy, we show that ocular hypertension, a major risk factor in glaucoma, and optic nerve crush injury disrupt the capacity of retinal neurons to clear cytosolic Ca 2+ leading to impaired light-evoked responses. Gene- and protein expression analysis reveal the loss of the sarco-endoplasmic reticulum (ER) Ca 2+ -ATPase2 pump (SERCA2/ATP2A2) in injured retinal neurons from mice and patients with primary open-angle glaucoma. Pharmacological activation or neuron-specific gene delivery of SERCA2 is sufficient to rescue single-cell Ca 2+ dynamics and promote robust survival of damaged neurons. Furthermore, SERCA2 gene supplementation reduces ER stress, reestablishes circuit balance, and restores visual behaviors. Our findings reveal that enhancing the Ca 2+ clearance capacity of vulnerable neurons alleviates organelle stress and promotes neurorecovery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myopic eye growth induces mechanical stretch, which can lead to structural and functional retinal alterations. Here, we investigated the effect of lens-induced myopic growth on the distribution of retinal ganglion cells (RGCs), glial fibrillary acidic protein (GFAP) expression and intensity, and peripapillary retinal nerve fiber layer (ppRNFL) thickness in common marmosets ( Callithrix jacchus ) induced with myopia continuously for six months, using immunohistochemistry and spectral-domain optical coherence tomography. We also explored the relationship between cellular structural parameters and the photopic negative response (PhNR) using full-field electroretinography. Marmosets induced with myopia for six months developed axial myopia, had a thinner ppRNFL, reduced peripapillary ganglion cell (≈20%) and astrocyte density (≈42%), increased panretinal GFAP expression (≈42%) and nasal mid-periphery staining intensity (≈81%) compared to age-matched controls. Greater degrees of myopia and vitreous elongation were associated with reduced peripapillary RGCs and astrocyte density, and increased GFAP expression and intensity. These cellular structural changes did not show a significant relationship with the features of the PhNR, which remained unchanged. The outcomes of this study suggest that myopia induces a reorganization of the peripapillary inner retina at the cellular level that may not result in measurable functional repercussions at this stage of myopia development.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo in vivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The optical imaging agent TcapQ488 has enabled imaging of retinal ganglion cell (RGC) injury in vivo in rodents and has potential as an effective diagnostic probe for early detection and intervention monitoring in glaucoma patients. In the present study, we investigated TcapQ488 in non-human primates (NHPs) to identify labeling efficacy and early signals of injured RGC, to determine species-dependent changes in RGC probe uptake and clearance, and to determine dose-limiting toxicities. Doses of 3, 6, and 12 nmol of TcapQ488 were delivered intravitreally to normal healthy NHP eyes and eyes that had undergone hemiretinal endodiathermy axotomy (HEA) in the inferior retina. Post-injection fundus fluorescence imaging using a Spectralis imaging platform (Heidelberg Engineering) documented TcapQ488 activation in RGC cell bodies. Optical coherence tomography (OCT), slit-lamp examinations, intraocular pressure measurements, and visual electrophysiology testing were performed to monitor probe tolerability. For comparison, a negative control, non-cleavable, non-quenched probe (dTcap488, 6 nmol), was delivered intravitreally to a normal healthy eye. In normal healthy eyes, intravitreal injection of 3 nmol of TcapQ488 was well-tolerated, while 12 nmol of TcapQ488 to the healthy eye caused extensive probe activation in the ganglion cell layer (GCL) and eventual retinal nerve fiber layer thinning. In HEA eyes, the HEA procedure followed by intravitreal TcapQ488 (3 nmol) injection resulted in probe activation within cell bodies in the GCL, confined to the HEA-treated inferior retina, indicating cell injury and slow axonal transport in the GCL. However, in contrast to rodents, a vitreal haze that lasted 2-12 weeks obscured rapid high-resolution imaging of the fundus. By contrast, intravitreal TcapQ488 injection prior to the HEA procedure led to minimal probe labeling in the GCL. The results of the dTcap488 control experiments indicated that fast axonal transport carried the probe out of the retina after cell body uptake. No evidence of pan-retinal toxicity or loss of retino-cortical function was detected in any of the three NHPs tested. Overall, these data provide evidence of TcapQ488 activation, without toxicity, in NHP HEA eyes that had been intravitreally injected with 3 nmol of the probe. Compared to rodents, unexpectedly rapid axonal transport in the NHPs reduced the capacity to visualize RGC cell bodies and axons through the backdrop of an intravitreal haze. Nonetheless, although intravitreal clearance rates did not scale to NHPs, HEA-induced reductions in axonal transport enhanced probe visualization in the cell body., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2024 Qiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glaucoma is a heterogenous group of optic neuropathies characterized by the degeneration of optic nerve axons and the progressive loss of retinal ganglion cells (RGCs), which could ultimately lead to vision loss. Elevated intraocular pressure (IOP) is a major risk factor in the development of glaucoma, and reducing IOP remains the main therapeutic strategy. Endothelin-1 (ET-1), a potent vasoactive peptide, has been shown to produce neurodegenerative effects in animal models of glaucoma. However, the detailed mechanisms underlying ET-1-mediated neurodegeneration in glaucoma are not completely understood. In the current study, using a Seahorse Mitostress assay, we report that ET-1 treatment for 4 h and 24 h time points causes a significant decline in various parameters of mitochondrial function, including ATP production, maximal respiration, and spare respiratory capacity in cultured RGCs. This compromise in mitochondrial function could trigger activation of mitophagy as a quality control mechanism to restore RGC health. Contrary to our expectation, we observed a decrease in mitophagy following ET-1 treatment for 24 h in cultured RGCs. Using Morrison's model of ocular hypertension in rats, we investigated here, for the first time, changes in mitophagosome formation by analyzing the co-localization of LC-3B and TOM20 in RGCs. We also injected ET-1 (24 h) into transgenic GFP-LC3 mice to analyze the formation of mitophagosomes in vivo. In Morrison's model of ocular hypertension, as well as in ET-1 injected GFP-LC3 mice, we found a decrease in co-localization of LC3 and TOM20, indicating reduced mitophagy. Taken together, these results demonstrate that both ocular hypertension and ET-1 administration in rats and mice lead to reduced mitophagy, thus predisposing RGCs to neurodegeneration.

Ischemic-anoxic injury plays an important role in the pathophysiology of diabetes retinopathy, optic neuropathy, even glaucoma and other ocular diseases. It may ultimately cause damage to neuronal death like retinal ganglion cells (RGCs) and subsequent visual loss. RGCs are essential elements of the retina and optic nerve that are crucial to visual formation. Ischemic-anoxic injury, inflammation, and oxidative stress are vital causes of RGC death. Thus, neuroprotection is essential for the treatment of these ocular diseases. Recent studies have shown the neuroprotective property of apelin-13 in many disease models. In this study, we isolated RGCs and found that apelin-13 promoted the viability of RGCs and increased the phosphorylation of Protein kinase B (PKB, Akt) in an in vitro oxygen-glucose deprivation model. Moreover, apelin-13 increased the expressions of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADPH) and reduced the level of reactive oxygen species (ROS). And, we also found that apelin-13 could promote the expressions of glucose transporter-1 (GLUT1) and adenosine triphosphate (ATP). These results indicated that apelin-13 could delay or stop RGC death, which might be as potential therapeutic targets for treatment of diseases mediated by ischemic-anoxic damage like diabetes retinopathy, optic neuropathy, even glaucoma., Competing Interests: Declarations. Ethical approval: All animal protocols were approved by the Animal Welfare and Ethics Committee of the Third Military Medical University. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This review explores the intricate relationship between glaucoma and circadian rhythm disturbances. As a principal organ for photic signal reception and transduction, the eye plays a pivotal role in coordinating the body's circadian rhythms through specialized retinal ganglion cells (RGCs), particularly intrinsically photosensitive RGCs (ipRGCs). These cells are critical in transmitting light signals to the suprachiasmatic nucleus (SCN), the central circadian clock that synchronizes physiological processes to the 24-hour light-dark cycle. The review delves into the central circadian body clock, highlighting the importance of the retino-hypothalamic tract in conveying light information from the eyes to the SCN. It underscores the role of melanopsin in ipRGCs in absorbing light and initiating biochemical reactions that culminate in the synchronization of the SCN's firing patterns with the external environment. Furthermore, the review discusses local circadian rhythms within the eye, such as those affecting photoreceptor sensitivity, corneal thickness, and intraocular fluid outflow. It emphasizes the potential of optical coherence tomography (OCT) in studying structural losses of RGCs in glaucoma and the associated circadian rhythm disruption. Glaucomatous retinal damage is identified as a cause of circadian disruption, with mechanisms including oxidative stress, neuroinflammation, and direct damage to RGCs. The consequences of such disruption are complex, affecting systemic and local circadian rhythms, sleep patterns, mood, and metabolism. Countermeasures, with implications for glaucoma management, are proposed that focus on strategies to improve circadian health through balanced melatonin timing, daylight exposure, and potential chronotherapeutic approaches. The review calls for further research to elucidate the mechanisms linking glaucoma and circadian disruption and to develop effective interventions to address this critical aspect of the disease., (© 2024 The Author(s). Published by IMR Press.)

Optic neuropathy such as glaucoma, stemming from retinal ganglion cell (RGC) degeneration, is a leading cause of visual impairment. Given the substantial loss of RGCs preceding clinical detection of visual impairment, cell replacement therapy emerges as a compelling treatment strategy. Human-induced pluripotent stem cells (hiPSCs) serve as invaluable tools for exploring the developmental processes and pathological mechanisms associated with human RGCs. Utilizing a 3D stepwise differentiation protocol for retinal organoids, we successfully differentiated RGC precursors from hiPSCs harboring a BRN3B-GFP RGC reporter, verified by GFP expression. Intravitreal transplantation of enriched RGC precursors into healthy or N-methyl-D-aspartate (NMDA)-injured mice demonstrated their survival, migration, and integration into the proper retinal layer, the ganglion cell layer, after 3 weeks. Notably, these transplanted cells differentiated into marker-positive RGCs and extended neurites. Moreover, enhanced cell survival was observed with immunosuppressive and anti-inflammatory treatments of the host prior to transplantation. These data underscore the potential of transplanted RGC precursors as a promising therapeutic avenue for treating degenerative retinal diseases resulting from RGC dysfunction.

Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration., (© 2024 Masin et al.)

Objective: This investigation was to determine the relationship between changes in the expression levels of miR-134 and the E2F transcription factor 6 (E2F6) in mediating control of apoptosis in N-methyl-D-aspartate (NMDA)-induced glaucomatous mice., Methods: Morphological and structural changes were quantitatively analyzed along with apoptosis in the retinal ganglion cell (RGC) layer, internal plexiform layer and RGCs. Glaucomatous RGCs were transfected, and cell viability and apoptosis were examined. The targeting relationship between miR-134 and E2F6 was analyzed, as well as their expression pattern., Results: Intravitreal injection of NMDA induced a significant reduction in the number of RGCs and thinning of IPL thickness. miR-134 was highly expressed and E2F6 was lowly expressed in glaucoma mice. Suppression of miR-134 or E2F6 overexpression inhibited apoptosis in the glaucomatous RGCs and instead their proliferative activity. MiR-134 targeted inhibition of E2F6 expression. Suppressing rises in E2F6 expression reduced the interfering effect of miR-134 on glaucomatous RGC development., Conclusion: Depleting miR134 expression increases, in turn, E2F6 expression levels and in turn reduces glaucomatous RGC apoptosis expression.

Background and Purpose: Retinal ganglion cells (RGCs) are the output stage of retinal information processing, via their axons forming the optic nerve (ON). ON damage leads to axonal degeneration and death of RGCs, and results in vision impairment. Nerve growth factor (NGF) signalling is crucial for RGC operations and visual functions. Here, we investigate a new neuroprotective mechanism of a novel therapeutic candidate, a p75-less, TrkA-biased NGF agonist (hNGFp) in rat RGC degeneration, in comparison with wild type human NGF (hNGFwt)., Experimental Approach: Both neonate and adult rats, whether subjected or not to ON lesion, were treated with intravitreal injections or eye drops containing either hNGFp or hNGFwt. Different doses of the drugs were administered at days 1, 4 or 7 after injury for a maximum of 10 days, when immunofluorescence, electrophysiology, cellular morphology, cytokine array and behaviour studies were carried out. Pharmacokinetic evaluation was performed on rabbits treated with hNGFp ocular drops., Results: hNGFp exerted a potent RGC neuroprotection by acting on microglia cells, and outperformed hNGFwt in rescuing RGC degeneration and reducing inflammatory molecules. Delayed use of hNGFp after ON lesion resulted in better outcomes compared with treatment with hNGFwt. Moreover, hNGFp-based ocular drops were less algogenic than hNGFwt. Pharmacokinetic measurements revealed that biologically relevant quantities of hNGFp were found in the rabbit retina., Conclusions and Implications: Our data point to microglia as a new cell target through which NGF-induced TrkA signalling exerts neuroprotection of the RGC, emphasizing hNGFp as a powerful treatment to tackle retinal degeneration., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

This study aimed to explore the role of melatonin in oxidative stress-induced injury on retinal ganglion cells and the underlying mechanisms. The immortalized RGC-5 cells were treated with H 2 O 2 to induce oxidative injury. Cell viability was measured by Cell Counting Kit-8, and apoptosis was determined by flow cytometry and western blot assays. Reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels were examined to evaluate oxidative stress levels. In addition, Thioredoxin-1 (Trx1) was silenced in RGC-5 cells using small interfering RNA followed by signaling pathway examination to explore the underlying mechanisms of melatonin in alleviating oxidative injury. Melatonin pre-treatment significantly alleviated H 2 O 2 -induced apoptosis in RGC-5 cells. Melatonin also markedly reversed the upregulation of cleaved-caspase 3, cleaved-caspase 9, and Bax expression and downregulation of Bcl-2 expression induced by H 2 O 2 . Further analyses presented that melatonin significantly attenuated the increase of ROS, LDH, and MDA levels in RGC-5 cells after H 2 O 2 treatment. Melatonin also abolished the downregulated expression of Superoxide dismutase type 1, Trx1, and Thioredoxin reductase 1, and the reduced activity of thioredoxin reductase in RGC-5 cells after H 2 O 2 treatment. Notably, Trx1 knockdown significantly mitigated the protective effect of melatonin in alleviating H 2 O 2 -induced apoptosis and oxidative stress, while administration of compound C, a common inhibitor of c-Jun N-terminal kinase (JNK) signaling, partially reversed the effect of Trx1 silencing, thereby ameliorating the apoptosis and oxidative injury induced by H 2 O 2 in RGC-5 cells. Melatonin could significantly alleviate oxidative stress-induced injury of retinal ganglion cells via modulating Trx1-mediated JNK signaling pathway., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ocular neurodegenerative diseases, particularly glaucoma, represent a significant global cause of blindness, with current therapies inadequately addressing the degeneration of the retina and optic nerve. Recent research has identified the sigma-2 receptors as a potential druggable target to offer neuroprotection in managing ocular neurodegenerative disorders. This study investigates the neuroprotective potential of CT2074, a sigma-2 receptor modulator, in a mouse model of primary open-angle glaucoma. Male mice were subjected to unilateral magnetic bead-induced elevation of intraocular pressure (IOP) and received daily oral administration of CT2074, commencing three days prior to ocular hypertension (OHT) induction, and continuing for three weeks. Mice received bilateral intraocular injections of cholera toxin B-488 (CTB) to assess retinal ganglion cell (RGC) anterograde transport. Retina, optic nerve, and brain tissues were collected three weeks post OHT induction for quantification of RGC and axon number, with contralateral retinas and cerebelli preserved for assessment of drug exposure. CT2074 was observed in the retina at levels exceeding the 95% receptor occupancy concentration. RGC quantification showed a significant reduction in the Vehicle group compared to Naïve and CT2074 groups. Notably, the CT2074 treatment group exhibited significantly higher RGC density than the Vehicle (p < 0.0001) and was no different than Naïve. Analysis of RGC axons in optic nerve cross-sections revealed significant axonal loss in both the Vehicle and CT2074 groups compared to Naïve, though the CT2074-treated group had significantly higher axon number compared to the Vehicle. Anterograde transport in the Vehicle and CT2074 groups did not differ. This study underscores the potential of CT2074 administered orally to protect RGCs exposed to elevated IOP, as evidenced by substantial preservation of RGCs and their axons compared to Vehicle-treated mice. These findings signify a promising avenue for the development of sigma-2 receptor-targeted therapeutics in glaucoma and related neurodegenerative diseases, addressing a critical unmet need in the field of ocular neuroprotection., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Traumatic optic neuropathy refers to optic nerve (ON) injury by trauma, including explosion and traffic accident. Retinal ganglion cell (RGC) death is the critical pathological cause of irreversible visual impairment and blindness in ON injury. We previously investigated the patterns of 11 modes of cell death in mouse retina post-ON injury. Here we aimed to identify additional signalling pathways regulating RGC survival in rodents post-ON injury. RNA sequencing analysis identified the upregulation of inflammation and cellular senescence-related genes in retina post-ON injury, which were confirmed by immunoblotting and immunofluorescence analyses. Increased expression of senescence-associated β-galactosidase (SA-βgal) in RGCs and activation of microglia were also found. Transforming growth factor-β receptor type II inhibitor (LY2109761) treatment suppressed p15 Ink4b and p21 Cip1 protein and SA-βgal expression and promoted RGC survival post-ON injury with decreasing the expression of cell death markers in retina. Consistently, senolytics (dasatinib and quercetin) treatments can promote RGC survival and alleviate the reduction of ganglion cell complex thickness and pattern electroretinography activity post-ON injury with reducing SA-βgal, p15 Ink4b , p21 Cip1 , microglial activation and cell death marker expression. In summary, this study revealed the activation of cellular senescence in rodent retina post-ON injury and contribute to RGC survival regulation. Targeting cellular senescence can promote RGC survival after ON injury, suggesting a potential treatment strategy for traumatic optic neuropathy., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Rhythmic, daily fluctuations in minute ventilation are controlled by the endogenous circadian clock located in the suprachiasmatic nucleus (SCN). While light serves as a potent synchronizer for the SCN, it also influences physiology and behavior by activating Brn3b-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). It is currently unclear the extent to which the external light environment shapes daily ventilatory patterns independent of the SCN. To determine the relative influence of environmental light versus circadian timing on the organization of daily rhythms in minute ventilation, we used whole-body plethysmography to measure the breathing of mice housed on a non-entraining T28 cycle (14 h light:14 h dark). Using this protocol, we found that minute ventilation exhibits a ~28-h rhythm with a peak at dark onset that coincides with the light:dark cycle and the animals' locomotor activity. To determine if this 28-h rhythm in minute ventilation was mediated by Brn3b-expressing ipRGCs, we measured the breathing of Brn3bDTA mice housed under the T28 cycle. Brn3bDTA mice lack the Brn3b-expressing ipRGCs that project to many non-SCN brain regions. We found that despite rhythmic light cues occurring on a 28-h basis, Brn3bDTA mice exhibited 24-h rhythms in minute ventilation, locomotor activity, and core body temperature consistent with organization by the SCN. The 24-h minute ventilation rhythm of Brn3bDTA mice was found to be driven predominantly by tidal volume rather than respiratory rate. These data indicate that the external light:dark cycle can directly drive daily patterns in minute ventilation by way of Brn3b-expressing ipRGCs. In addition, these data strongly suggest that the activation of Brn3b-expressing ipRGCs principally organizes daily patterns in breathing and locomotor activity when light:dark cues are presented in opposition to endogenous clock timing., Competing Interests: Conflict of Interest StatementThe authors have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Being part of the mature mammalian central nervous system, impairments of the retina and optic nerves caused by trauma or diseases often cannot be restored. Progressive degeneration of retinal ganglion cells (RGCs) in glaucoma and other optic neuropathies gradually leads to permanent vision loss, which currently has no cure. The purpose of this study is to develop a biocompatible scaffold to support RGC survival and guide axon growth, facilitating optic nerve repair and regeneration. We here report that electrical stimulation (ES) significantly promoted neurite outgrowth and elongation from primary RGCs, mediated through glutamate receptor signaling. To mimic prolonged glutamate stimulation and facilitate sustained nerve growth, we fabricated biocompatible poly-γ-benzyl-L-glutamate (PBG) scaffolds for controlled glutamate release. These PBG scaffolds supported RGC survival and robust long-distance nerve growth in both retinal explants and isolated RGC cultures. In contrast, control polycaprolactone (PCL) scaffolds with similar physical structures showed little benefits on RGC survival or nerve growth. Moreover, PBG scaffolds promoted the differentiation and neurite outgrowth from embryonic stem cell-derived RGC progenitors. The aligned PBG scaffold drove directed nerve elongation along the fiber alignment. Transplantation of PBG-coated biocompatible conduits induced robust optic nerve regeneration in adult mice following nerve transection. Together, the findings present the exciting possibility of driving optic nerve regeneration and RGC progenitor cell differentiation by imitating ES or glutamate signaling. PBG presents a permissive biomaterial in supporting robust and directed axon growth with promising clinical applications in the future. STATEMENT OF SIGNIFICANCE: We here reported compelling findings that demonstrate the potent regenerative effects of a bioengineered scaffold incorporating poly-γ-benzyl-L-glutamate (PBG) on the optic nerve. Retinal ganglion cell (RGC) axons, which form the optic nerve, are incapable of regenerating in adulthood, posing a significant hurdle in restoring vision for patients with optic nerve diseases or injuries. Built upon the finding that electrical stimulation promotes RGC axonal growth through glutamate signaling, we developed PBG scaffolds to provide sustained glutamate stimulation and showed their exceptional effects on driving directed axonal elongation in cultured RGCs and neural progenitors, as well as supporting robust optic nerve regeneration after transection in vivo. The findings hold great promise for reversing vision loss in patients with optic nerve conditions., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests in financial or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Purpose: The objective of this study was to observe the macular pigment optical density (MPOD) and the relationship between MPOD and retinal thickness in Chinese primary angle-closure glaucoma (PACG) patients by the one-wavelength reflectometry method., Methods: This study was a prospective comparative observational study, including 39 eyes from 39 PACG patients (15 men and 24 women, mean age 61.89 ± 12.30) and 41 eyes from 41 controls (20 men and 21 women, mean age 63.24 ± 14.02). We measured the MPOD 7-degree area by the one-wavelength reflectometry method and analyzed both the max and mean optical density (OD). The central retinal thickness (CRT) and the total thickness of the macular ganglion cell layer (GCL), and inner plexiform layer (IPL)were measured by spectral-domain-optical coherence tomography (SD-OCT). Statistical methods such as Shapiro-Wilk test, Fisher's exact test, chi-square test, two independent samples test and Spearman's correlation coefficient were used to observe the differences in the MPOD between normal subjects and PACG patients and the correlation between the MPOD and retinal thickness., Results: The max optical density (Max OD) (PACG group: 0.302 ± 0.067d.u, control group: 0.372 ± 0.059d.u., p  < .001) and mean optical density (Mean OD) (PACG group: 0.124 ± 0.035d.u., control group: 0.141 ± 0.028d.u., p  < 0.05) were significantly reduced in PACG patients compared with control subjects. Significant decreases in GCL + IPL thickness (PACG group: 74.71 ± 39.56 μm, control group:113.61 ± 8.14 μm, p  < 0.001) and CRT (PACG group: 254.49 ± 41.47 μm, control group:329.10 ± 18.57 μm, p  < 0.001) were also observed in PACG eyes. There was no statistically significant correlation between the MPOD and GCL + IPL thickness ( p  = .639, p  = .828)., Conclusions: MPOD was significantly lower in Chinese PACG patients than in the control group, potentially due to thinning of the GCL + IPL thickness. This study provides insights for the pathophysiology, assessment of PACG and potential guidance for lifestyle modifications.

In vitro primary cell culture models of retinal ganglion cells (RGC) are widely used to study pathomechanisms of diseases such as glaucoma. The biomechanic interaction with the culture substrate is known to influence core cellular functions. RGC cultures, however, are usually grown on rigid plastic or glass substrates. We hypothesized that soft polyacrylamide gel substrates may alter survival and neurite outgrowth of primary cultured RGC. Primary retinal cultures from postnatal (day 1-6) Wistar rats were grown on glass coverslips or polyacrylamide (PA) gel substrate with different Young's elastic moduli (0.75, 10 or 30 kPa). Substrates were coated with Poly-l-lysine and/or laminin. RGC were immunostained with anti-beta-III-tubulin. Total neurite length, growth cone morphology, RGC density, mitochondrial morphology and transport as well as pro-survival pathways (Erk1/2, Akt, CREB) were assessed. PA gel substrates of E = 10 kPa significantly increased the total neurite length by factor 1.5 compared to glass (p = 0.02). The growth cone area was significantly larger by factor 5.3 on 30 kPa gels (p = 0.01). The presence of a substrate coating was more important for neurite outgrowth and RGC survival on PA gels (poly-l-lysine > laminin) than on glass. Neither mitochondrial morphology and motility nor the activation of pro-survival pathways significantly differed between the four substrates. PA gel substrates significantly enhanced RGC neurite outgrowth. The signaling cascades mediating this effect remain to be determined., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)