Glaucoma Model Research Articles

A Neuroprotective Peptide Modulates Retinal cAMP Response Element-Binding Protein (CREB), Synapsin I (SYN1), and Growth-Associated Protein 43 (GAP43) in Rats with Silicone Oil-Induced Ocular Hypertension

This study evaluated the neuroprotective potential of peptain-1 conjugated to a cell-penetrating peptide (CPP-P1) in an ocular hypertension model of glaucoma. Brown Norway (BN) rats were subjected to intraocular pressure (IOP) elevation via intracameral injection of silicone oil (SO), with concurrent intravitreal injections of either CPP-P1 or a vehicle. Retinal cross-sections were analyzed for markers of neuroprotection, including cAMP response element-binding protein (CREB), phosphorylated CREB (p-CREB), growth-associated protein-43 (GAP43), synapsin-1 (SYN1), and superoxide dismutase 2 (SOD2). Hematoxylin and eosin staining was used to assess retinal-layer thickness. SO-treated rats exhibited significant reductions in the thickness of the inner nuclear layer (INL, 41%, p = 0.016), inner plexiform layer (IPL, 52%, p = 0.0002), and ganglion cell layer (GCL, 57%, p = 0.001). CPP-P1 treatment mitigated these reductions, preserving INL thickness by 32% (p = 0.059), IPL by 19% (p = 0.119), and GCL by 31% (p = 0.057). Increased levels of CREB (p = 0.17) and p-CREB (p = 0.04) were observed in IOP-elevated, CPP-P1-treated retinas compared to IOP-elevated, vehicle-treated retinas. Although overall GAP43 levels were low, there was a modest increase in expression within the IPL and GCL in SO- and CPP-P1-treated retinas (p = 0.15 and p = 0.09, respectively) compared to SO- and vehicle-treated retinas. SO injection reduced SYN1 expression in both IPL and GCL (p = 0.01), whereas CPP-P1 treatment significantly increased SYN1 levels in the IPL (p = 0.03) and GCL (p = 0.002). While SOD2 expression in the GCL was minimal across all groups, a trend toward increased expression was observed in CPP-P1-treated animals (p = 0.16). The SO model was replicated with SO removal after 7 days and monitored for 21 days followed by retinal flat-mount preparation to assess retinal ganglion cell (RGC) survival. A 42% loss in RGCs (p = 0.009) was observed in SO-injected eyes, which were reduced by approximately 37% (p = 0.03) with CPP-P1 treatment. These findings suggest that CPP-P1 is a promising neuroprotective agent that promotes retinal ganglion cell survival and the preservation of other retinal neurons, potentially through enhanced CREB signaling in a rat model of SO-induced ocular hypertension.

The role of neurotrophic factors in retinal ganglion cell resiliency

Many retinal diseases are characterized by direct or indirect retinal ganglion cell (RGC) neurodegeneration. In glaucoma and optic nerve neuropathies, RGCs are the primary affected cells, whereas in photoreceptor dystrophies, RGC loss is secondary to the death of rods and cones. The death of RGCs in either case will irreversibly cause loss of vision, as RGCs are the sole output neurons of the retina. RGC neurodegeneration affects certain neurons preferentially, resulting in subpopulations of resilient and susceptible cells. Neurotrophins (NTs) are known to mediate neuronal survival through the downstream activation of various anti-apoptotic pathways. In this review, we summarize the current methods of RGC identification and quantification in animal models of direct or indirect neurodegeneration, and describe the advantages and disadvantages associated with these techniques. Using these techniques, multiple studies have uncovered the potential role of NTs in protecting RGCs during direct neurodegeneration, with BDNF and NGF delivery promoting RGC survival in models of experimental glaucoma. Many fewer studies have addressed similar questions in retinal diseases where RGC loss is secondary to photoreceptor degeneration, yielding conflicting results. Our analysis suggests that these seemingly contradictory results can be explained by the varying onset and geographic distribution of photoreceptor death.

BMP4-GPX4 can improve the ferroptosis phenotype of retinal ganglion cells and enhance their differentiation ability after retinal stem cell transplantation in glaucoma with high intraocular pressure.

Activation of bone morphogenetic protein (BMP) 4 signaling promotes the survival of retinal ganglion cell (RGC) after acute injury. In this study, we investigated the role of the BMP4 signaling pathway in regulating the degeneration of retinal ganglion cells (RGCs) in a mouse glaucoma model and its potential application in retinal stem cell. Our results demonstrate that BMP4-GPX4 not only reduces oxidative stress and iron accumulation but also promotes neuroprotective factors that support the survival of transplanted RSCs into the host retina. These findings suggest a novel therapeutic approach for glaucoma involving the modulation of the BMP4-GPX4 pathway to protect RGCs and improve visual function through enhanced RSC differentiation.

Pyruvate and Related Energetic Metabolites Modulate Resilience Against High Genetic Risk for Glaucoma.

Glaucoma polygenic risk scores (PRS) effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study in the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve=0.579), they offered modest prediction improvement in PRS-only-based models (P=0.004). We identified a metabolomic signature associated with resilience in the top PRS decile, with elevated glycolysis-related metabolites-lactate (P=8.8E-12), pyruvate (P=1.9E-10), and citrate (P=0.02)-linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (P interaction =0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratio highest vs. lowest total resilience metabolite quartile =0.71, 95% Confidence Interval [CI]=0.64-0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (P=0.002) and optic nerve damage (P<0.0003) in Lmx1b V265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.

Impaired axonal transport contributes to neurodegeneration in a Cre-inducible mouse model of myocilin-associated glaucoma.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that accurately replicates these human POAG features has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing the outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant loss of retinal ganglion cells (RGCs) and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.

FABP5 regulates ROS-NLRP3 inflammasome in glutamate-induced retinal excitotoxic glaucomatous model.

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.

Rapamycin protects glucocorticoid-induced glaucoma model mice against trabecular meshwork fibrosis by suppressing mTORC1/2 signaling.

Systemic or local use of glucocorticoids (GCs) can induce pathological elevation of intraocular pressure (IOP), potentially leading to permanent visual loss. Previous studies have demonstrated that rapamycin (Rapa) inhibits the activation of retinal glial cells and the production of neuroinflammation, achieving neuroprotective goals. However, there has been little research on the effect of Rapa on the trabecular meshwork (TM). This study aimed to investigate the protective effect and potential mechanism of Rapa in a glucocorticoid-induced glaucoma (GIG) model. Our findings indicate that Rapa significantly inhibited the IOP increase induced by dexamethasone acetate (Dex-Ac) and improved TM fibrosis and retinal ganglion cell (RGC) damage. In cultured human trabecular meshwork cells (HTMCs) treated with dexamethasone (Dex) and Rapa under different conditions revealed that Rapa inhibits Dex-induced HTMC fibrosis and cytoskeletal changes. This effect may result from the specific suppression of the mechanistic target of rapamycin complex 1 (mTORC1) pathway by Rapa, which reduces abnormal extracellular matrix (ECM) deposition. Alternatively, the improvement in cytoskeleton entanglement might be due to the inhibition of the mechanistic target of rapamycin complex2 (mTORC2) pathway. These two potential mechanisms may collectively contribute to the protective effects of Rapa in GIG. This study provides a new theoretical basis for using of Rapa in the treatment of GIG.

Self-generating electricity system driven by aqueous humor flow and trabecular meshwork contraction motion activated BCKa for glaucoma intraocular pressure treatment.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma and the leading cause of irreversible vision loss and blindness worldwide. Intraocular pressure (IOP) is the only modifiable risk factor, and prompt treatment to lower IOP can effectively slow the rate of vision loss due to glaucoma. Trabecular meshwork (TM) cells can maintain IOP homeostasis by correcting and adjusting the resistance to aqueous humor outflow in response to sustained pressure changes. TM cells' function is reduced, and membrane ion channels are impaired in POAG. The dysfunction of Large conductance Ca2+-activated K+ (BKCa) plays a central role in the pathogenesis of POAG. In this work, we targeted MXene nanoparticles (MXene-RGD) with piezoelectric response to TM cells in a 3D model of glaucoma in vitro as well as in the rabbit Transient Ocular Hypertension (OHT) Model in vivo. MXene-RGD gives the TM electromechanical transfer properties, while the self-enhancing and self-generated electricity properties of the TM are determined by the aqueous humor flow rate and the size of the deformation of the TM. MXene-RGD is nontoxic, as illustrated by a cell toxicity study and histological examination. In a 3D in vitro model of high-pressure glaucoma, whole-cell patch-clamp confirmed that piezoelectric stimulation turns on BKCa, which reduces the volume of the cell. MXene-RGD was injected into the anterior chamber with minimal trauma, i.e., anterior chamber injection, and specifically targeted to TM cells. The OHT model in vivo confirmed the potential IOP-lowering ability of MXene-RGD. We evaluated the ion channels involved in the reduction of IOP by MXene-RGD by pre-treatment with a BKCa channel blocker (iberiotoxin, IbTX) and a voltage-gated Ca2+channel blocker (nifedipine). Quantitative qPCR analysis showed that MXene-RGD inhibited the upregulation of mRNA expression levels of the myofibroblast marker α-smooth muscle actin (α-SMA) and the inflammatory response marker interleukin-6 (IL-6) induced by IOP. Histology confirmed that MXene-RGD attenuated IOP-induced proliferation and collagen production in the TM. Taken together, we present for the first time a minimally invasive surgical approach for targeting TM cells for POAG by utilizing piezoresponse nanomaterials to target BKCa to repair or awaken the ability of TM cells to regulate IOP homeostasis on their own.

Citicoline and coenzyme Q10 (COQUN Combo) reduce inflammation in an experimental glaucoma model

Aims/Purpose: To investigate the anti‐inflammatory effects of citicoline and coenzyme Q10 (COQUN Combo) in an experimental model of ocular hypertension (OHT).Methods: Swiss albino mice were used: vehicle control (Vehicle n = 6), COQUN Combo control (COQUN Combo n = 5), laser vehicle (LG+Veh, n = 6), and laser COQUN Combo (LG+COQUN Combo, n = 6). COQUN Combo was administered daily with gelatin 15 days prior to laser induction and 3 days post‐induction in COQUN Combo and LG+COQUN Combo groups. The Vehicle and LG+Veh groups received neutral gelatin. OHT was induced in the left eye by laser photocoagulation of the limbal and episcleral veins, analyzing hypertensive eyes and their contralateral eyes. Retinal whole‐mounts were processed with: 1) Anti Iba‐1 to quantify: Number of Iba‐1+ cells (Iba1‐cn) in outer segments (OS), outer plexiform layer (OPL) and inner plexiform layer (IPL); Retinal area occupied by Iba‐1+ cells (Iba1‐RA) in the nerve fiber layer‐ganglion cell layer (NFL‐GCL); Cell body area (Iba1‐cba) of Iba‐1+ cells in OPL, IPL and NFL‐GCL; and arbor area (Iba1‐aa) of Iba‐1+ cells in OPL and IPL. 2) Anti‐P2RY12 to differentiate activated microglia.Results: No significant differences were observed between the control groups. Both OHT eyes and their contralaterals of LG+Veh, as well as LG+COQUN Combo OHT eyes and their contralaterals, showed significant differences compared to controls in Iba1‐cn, Iba1‐ RA, Iba1‐cba, and Iba1‐aa in all layers analyzed. However, these changes were significantly smaller in LG+COQUN Combo OHT and their contralateral eyes compared to LG+Veh OHT and their contralateral eyes. In the Vehicle, Iba‐1+ cells were P2RY12+. P2RY12 expression was downregulated in both OHT eyes of LG+Veh and LG+ COQUN Combo; however, this downregulation was less pronounced in the OHT eyes of LG+COQUN Combo. In the contralateral eyes, P2RY12 expression remained similar to that of the controlsConclusions: COQUN Combo can attenuate the inflammatory process in glaucoma.

Effectiveness of intravitreal antitNF‐ALFA for neuroinflammation control and neuroprotection in experimental rabbit glaucoma model

Aims/Purpose: The aim of the study is to create a microbead induced ocular hypertension (OHT) model in rabbits, and to evaluate the effects of intravitreal and intraperitoneal injections of Adalimumab (ADA) on the number of retinal ganglion cells (RGC), retinal nerve fiber layer (RNFL) and ganglion cell complex (GCC) thickness.Methods: 15 rabbits of mixed strain (9–12 months old, 3.5‐4kg) were randomized into 4 groups. OHT was induced with microbead injection into the anterior chamber of right eyes. On 7th, 14th days of OHT, ADA was injected at one of two concentrations (2.5mg group 1: G1/5mg group 2:G2) into right eyes of two groups, intraperitoneally 5mg/kg into the third group (G3), and balanced salt solution into fourth group (sham). The left eyes were used as controls. At 40th day, the rabbits were euthanized. Retinal and optic nerve head (ONH) histology was studied with hematoxylin‐eosin and toluidine blue staining.Results: OHT was induced with microbeads in 13 eyes. 2 rabbits were excluded (endophthalmitis, total hyphema). The average pre‐OHT IOP from all eyes 9.7±09mmHg (8‐11). Statistically significant IOP increase was observed in all subject eyes on day 7 (14±1.4 (12‐18)) (p = 0.01) and maintained until ADA injection (day 21). RGCs in the eyes with elevated IOP were 7.2±1.2 and 7.6±1.5, 6.3+1.1 cells in ADA treated groups, G1, G2, G3, respectively. There was no statistically significant difference between treatment groups. The average RNFL was 125.9±10.9 μm in control, 60.2±9.4 μm in G1, 45.7±2.9 μm in G2, 22.8±2.9 μm in G3, 21.7±10 μm in sham. Significant elevation was observed in G1 compared to all treatment groups. (p = 0.01). The average GCC of control was 45.5±1.9 μm, G1 41.2±2.2 μm, G2 39.3±3.3 μm, G3 34.1±1.6 μm, sham was 38.3±1.1 μm. Significant increase was observed in G1 compared to G3. (p = 0.01).Conclusions: OHT triggers inflammatory response in which TNF‐α expression is increased around the ONH. Blocking TNF‐α might prevent axonal degeneration and RGC loss by inhibiting this response. These findings may further contribute to the literature for a new treatment strategy for glaucoma using TNF‐α antagonists or inflammation suppressors.

Impact of citicoline and coenzyme Q10 (COQUN Combo) on retinal ganglion cell survival in a experimental glaucoma model

Aims/Purpose: Treatments for glaucoma damage have focused on lowering intraocular pressure (IOP). However, despite controlled IOP, the neurodegenerative process continues. Therefore, it is necessary to study new molecules with neuroprotective properties for treatment. The aim of this study was to investigate the survival of retinal ganglion cells (RGCs) in an experimental model of ocular hypertension (OHT) following the administration of a combination of citicoline and coenzyme Q10 (COQUN Combo).Methods: Four groups of Swiss albino mice were used: vehicle control (Vehicle, n = 6), COQUN Combo control (COQUN Combo, n = 5), laser with vehicle (LG+Veh, n = 6), and laser with COQUN Combo (LG+COQUN Combo, n = 6). COQUN Combo was administered daily mixed with gelatin, 15 days prior to laser induction and 7 days post‐induction in COQUN Combo and LG+COQUN Combo groups. The Vehicle and LG+Veh groups received only neutral gelatin. OHT was induced in the left eye by laser photocoagulation of the limbal and episcleral veins, analyzing hypertensive eyes and their contralateral. IOP was measured different time points after‐laser (24h, 48h, 3, 5, and 7 days). Retinal whole‐mounts were processed with 1) Anti‐Brn3a to semi‐automatically quantify the number of Brn3a+ RGCs; and 2) Anti‐melanopsin to manually quantify the number of melanopsin+ RGCs.Results: At 24 hours post‐laser, both vehicle and COQUN Combo hypertensive eyes exhibited a significant increase in IOP. However, the increase was lower in the COQUN Combo OHT eyes (p value &lt; 0.0001). In vehicle OHT eyes, the number of Brn3a+ RGCs was significantly lower compared to the vehicle control. However, the COQUN Combo‐treated OHT eyes maintained similar values to vehicle control eyes, as did the contralateral vehicle and contralateral COQUN Combo eyes. The number of melanopsin+ RGCs was similar in all study groups.Conclusions: The COQUN Combo may be a neuroprotective strategy in glaucomatous pathology.

Artificial intelligence-enabled discovery of a RIPK3 inhibitor with neuroprotective effects in an acute glaucoma mouse model.

Retinal ganglion cell (RGC) death caused by acute ocular hypertension is an important characteristic of acute glaucoma. Receptor-interacting protein kinase 3 (RIPK3) that mediates necroptosis is a potential therapeutic target for RGC death. However, the current understanding of the targeting agents and mechanisms of RIPK3 in the treatment of glaucoma remains limited. Notably, artificial intelligence (AI) technologies have significantly advanced drug discovery. This study aimed to discover RIPK3 inhibitor with AI assistance. An acute ocular hypertension model was used to simulate pathological ocular hypertension in vivo . We employed a series of AI methods, including large language and graph neural network models, to identify the target compounds of RIPK3. Subsequently, these target candidates were validated using molecular simulations (molecular docking, absorption, distribution, metabolism, excretion, and toxicity [ADMET] prediction, and molecular dynamics simulations) and biological experiments (Western blotting and fluorescence staining) in vitro and in vivo . AI-driven drug screening techniques have the potential to greatly accelerate drug development. A compound called HG9-91-01, identified using AI methods, exerted neuroprotective effects in acute glaucoma. Our research indicates that all five candidates recommended by AI were able to protect the morphological integrity of RGC cells when exposed to hypoxia and glucose deficiency, and HG9-91-01showed a higher cell survival rate compared to the other candidates. Furthermore, HG9-91-01 was found to protect the retinal structure and reduce the loss of retinal layers in an acute glaucoma model. It was also observed that the neuroprotective effects of HG9-91-01 were highly correlated with the inhibition of PANoptosis (apoptosis, pyroptosis, and necroptosis). Finally, we found that HG9-91-01 can regulate key proteins related to PANoptosis, indicating that this compound exerts neuroprotective effects in the retina by inhibiting the expression of proteins related to apoptosis, pyroptosis, and necroptosis. AI-enabled drug discovery revealed that HG9-91-01 could serve as a potential treatment for acute glaucoma.

Unveiling Key Genes Modulating Retinal Cell Survival and Autophagy in Glaucoma.

Glaucoma is a leading cause of irreversible blindness, with rising incidence globally. Effective treatment is challenging due to limited understanding of the disease mechanisms. Growth factor activity is crucial in glaucoma, with potential to reduce retinal ganglion cell (RGC) apoptosis and slow disease progression. This study aims to identify and analyze differentially expressed genes (DEGs) involved in growth factor activity to uncover new therapeutic targets. We analyzed the GSE9944 dataset from the Gene Expression Omnibus (GEO) to identify DEGs associated with glaucoma, resulting in 94 DEGs, including 29 down-regulated and 65 up-regulated genes. Functional enrichment and protein-protein interaction (PPI) network analyses were conducted using bioinformatics tools, highlighting the roles of Bone Morphogenetic Protein 1 (BMP1), Pleiotrophin (PTN), and f fibroblast Growth Factor 7 (FGF7). Aberrant expression vectors for these genes were transfected into RGCs derived from a glaucoma model to evaluate their impact on cell viability, apoptosis, and autophagy. Bioinformatics analysis of the GSE9944 dataset identified 94 DEGs, with 29 down-regulated and 65 up-regulated genes. Functional enrichment analysis revealed that these DEGs were involved in pathways related to growth factor activity, apoptosis, and autophagy, processes highly relevant to glaucoma pathogenesis. PPI network analysis identified BMP1, PTN, and FGF7 as central hub genes involved in extracellular matrix organization and growth factor signaling. In experimental validation using RGCs, we found that up-regulation of BMP1 significantly enhanced RGC viability and reduced apoptosis. Conversely, silencing PTN and FGF7 provided protective effects, enhancing RGC survival. Silencing BMP1 and upregulating PTN and FGF7 led to increased RGC apoptosis. Additionally, BMP1 was found to inhibit autophagy in RGCs, whereas PTN and FGF7 promoted autophagic activity, suggesting differential regulatory roles in glaucoma pathogenesis. Overall, BMP1, PTN, and FGF7 play critical roles in the regulation of RGC activity and autophagy in glaucoma, making them promising molecular targets for future therapeutic interventions.

Autoencoder-based phenotyping of ophthalmic images highlights genetic loci influencing retinal morphology and provides informative biomarkers.

Genome-wide association studies (GWAS) have been remarkably successful in identifying associations between genetic variants and imaging-derived phenotypes. To date, the main focus of these analyses has been on established, clinically-used imaging features. We sought to investigate if deep learning approaches can detect more nuanced patterns of image variability. We used an autoencoder to represent retinal optical coherence tomography (OCT) images from 31,135 UK Biobank participants. For each subject, we obtained a 64-dimensional vector representing features of retinal structure. GWAS of these autoencoder-derived imaging parameters identified 118 statistically significant loci; 41 of these associations were also significant in a replication study. These loci encompassed variants previously linked with retinal thickness measurements, ophthalmic disorders and/or neurodegenerative conditions. Notably, the generated retinal phenotypes were found to contribute to predictive models for glaucoma and cardiovascular disorders. Overall, we demonstrate that self-supervised phenotyping of OCT images enhances the discoverability of genetic factors influencing retinal morphology and provides epidemiologically-informative biomarkers. Code and data links available at https://github.com/tf2/autoencoder-oct. Supplementary data are available at Bioinformatics online.

A Reduction in Mitophagy Is Associated with Glaucomatous Neurodegeneration in Rodent Models of Glaucoma.

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.

Oral supplementation with Nicotinamide Riboside treatment protects RGCs in DBA/2J mouse model.

The aim of this study was to test whether oral administration of nicotinamide riboside (NR), the nicotinamide adenine dinucleotide (NAD+) precursors, protect retina ganglion cells (RGCs) from neurodegeneration in DBA/2J (D2) mice, which is a widely used mouse model of age-related inherited glaucoma. Oral NR or NAM administration (NR low dose: 1150mg/kg; NR high dose: 4200mg/kg; NAM low dose group: 500mg/kg; NAM high dose: 2000mg/kg of body weight per day) essentially started when D2 mice were 4 or 9 months old and continued up to 12 months old. Control cohort identically received food/water without NAM or NR. Intraocular pressure (IOP) was measured every month until experiment completion. Pattern electroretinography (PERG) was recorded. Retinas were harvested for whole mount immunofluorescence staining with RGCs marker Brn3a and imaged by fluorescent confocal microscopy. Optic nerves were harvested for axon staining and quantification. Retinal NAD+ levels were enzymatically assayed. NR oral supplementary treatment started at 4 months old robustly increased retinal NAD+ levels in D2 mice (NRHigh vs. vehicle: 273.7±23.59% vs. 108.70±12.10%, p<0.001). In aged vehicle group (12 months old), there was significantly diminution of the P1 and N2 components of PERG response compare with naïve group (naïve vs. vehicle: P1: 7.82± 0.70uV vs 1.63± 0.17uV, p<0.0001; N2: -13.29± 0.83uV vs. -3.22± 0.27uV, p<0.0001; Kruskal-Wallis test with Dunn's multiple comparison test). NR treatment preserved aged D2 visual function when mice were 9 and 12 months old. In addition, long-term NR high dose treatment significantly protected against total RGCs loss and optic nerve atrophy (RGC: NRHigh vs. vehicle: 1412±62.00vs 475.2±94.68 cells/field, p<0.00001; axon numbers: NRHigh vs. vehicle: 23990±1159 vs 8573±1160, n=41-53, p<0.0001). Furthermore, long-term NR supplementation prevent iris depigmentation and delayed IOP elevation. NR oral supplementary treatment significantly preserved RGC and axon numbers, potentially preserves retinal function via elevated retinal NAD+ level in aged D2 mice. Interestingly, NR treatment also prevented iris atrophy, delayed IOP elevation associated with this glaucoma model. NR oral supplementation thus treated several aspects of murine pigment dispersion glaucoma. Given parallels between this model and glaucoma in human, out data indicate that NR is worth exploring as a therapeutic candidate in treatment of glaucoma.

Identifying Hub Genes for Glaucoma based on Bulk RNA Sequencing Data and Multi-machine Learning Models.

The aims of this study were to determine hub genes in glaucoma through multiple machine learning algorithms. Glaucoma has afflicted many patients for many years, with excessive pressure in the eye continuously damaging the nervous system and leading to severe blindness. An effective molecular diagnostic method is currently lacking. The present study attempted to reveal the molecular mechanism and gene regulatory network of hub genes in glaucoma, followed by an attempt to reveal the drug-gene-disease network regulated by hub genes. A microarray sequencing dataset (GSE9944) was obtained through the Gene Expression Omnibus database. The differentially expressed genes in Glaucoma were identified. Based on these genes, we constructed three machine learning models for feature training, Random Forest model (RF), Least absolute shrinkage and selection operator regression model (LASSO), and Support Vector Machines model (SVM). Meanwhile, Weighted Gene Co-Expression Network Analysis (WGCNA) was performed for GSE9944 expression profiles to identify Glaucoma-related genes. The overlapping genes in the four groups were considered as hub genes of Glaucoma. Based on these genes, we also constructed a molecular diagnostic model of Glaucoma. In this study, we also performed molecular docking analysis to explore the gene-drug network targeting hub genes. In addition, we evaluated the immune cell infiltration landscape in Glaucoma samples and normal samples by applying CIBERSORT method. 8 hub genes were determined: ATP6V0D1, PLEC, SLC25A1, HRSP12, PKN1, RHOD, TMEM158 and GSN. The diagnostic model showed excellent diagnostic performance (area under the curve=1). GSN might positively regulate T cell CD4 naïve as well as negatively regulate T cell regulation (Tregs). In addition, we constructed gene-drug networks in an attempt to explore novel therapeutic agents for Glaucoma. Our results systematically determined 8 hub genes and established a molecular diagnostic model that allowed the diagnosis of Glaucoma. Our study provided a basis for future systematic studies of Glaucoma pathogenesis.

Взаємозв’язок вмісту ендогенного сірководню в тканинах ока з внутрішньоочним тиском при експериментальній глаукомі

The modern scientific direction in the study of the pathogenesis of primary glaucoma is searching for specific biomarkers and regulatory mechanisms of this disease. The aim of the work was to study the level of hydrogen sulfide in eye tissues in animals with adrenaline-induced glaucoma (AIG) and its relationship with intraocular pressure (IOP). The glaucoma model (first group) was induced by injecting 0.1 ml of adrenaline solution into the ear vein for 3 months. In the second group, animals (during AIG simulation) received a hydrogen sulfide donor - daily instillations of 1% NaHS solution into the eye. The third (control) group consisted of intact animals. After 3 months, the level of endogenous hydrogen sulfide was determined in the tissues of the drainage zone of the eye, retina, optic nerve, and intraocular fluid. Glaucoma modeling in rabbits was characterized by a dynamic increase in IOP. A decrease of hydrogen sulfide level in all eye tissues in glaucoma was established. The maximum decrease was observed in the retina by 37.3% when compared with the control. In the second group, the level of endogenous hydrogen sulfide was higher than in the first group in the tissues of the drainage zone by 54%, the retina by 42%, the optic nerve by 37%, and the intraocular fluid by 60%. A decrease and gradual normalization of IOP were found in the second group. The presence of an inverse Spearman correlation between IOP indicators and the level of hydrogen sulfide in all experimental groups was established. We suggest that the established facts confirm the hypothesis regarding the role of a gas transmitter hydrogen sulfide in the pathogenesis of glaucoma.

Blind But Alive - Congenital Loss of atoh7 Disrupts the Visual System of Adult Zebrafish.

Vision is the predominant sense in most animal species. Loss of vision can be caused by a multitude of factors resulting in anatomic as well as behavioral changes. In mice and zebrafish, atoh7 mutants are completely blind as they fail to generate retinal ganglion cells (RGCs) during development. In contrast to mice, raising blind zebrafish to adulthood is challenging and this important model is currently missing in the field. Here, we report the phenotype of homozygous mutant adult zebrafish atoh7 mutants that have been raised using adjusted feeding and holding conditions. The phenotype of adult mutants was characterized using classical histology and immunohistochemistry as well as optical coherence tomography. In addition, the optokinetic response was characterized. Adult atoh7 mutants display dark body pigmentation and significantly reduced body length. They fail to form RGCs, the resulting nerve fiber layer as well as the optic nerve, and consequently behave completely blindly. In contrast, increased amounts of other retinal neurons and Müller glia are formed. In addition, the optic tectum is anatomically reduced in size, presumably due to the missing retinal input. Taken together, we provide a comprehensive characterization of a completely blind adult zebrafish mutant with focus on retinal and tectal morphology, as a useful model for glaucoma and optic nerve aplasia.

Exploring dysfunctional barrier phenotypes associated with glaucoma using a human pluripotent stem cell-based model of the neurovascular unit

Glaucoma is a neurodegenerative disease that results in the degeneration of retinal ganglion cells (RGCs) and subsequent loss of vision. While RGCs are the primary cell type affected in glaucoma, neighboring cell types selectively modulate RGCs to maintain overall homeostasis. Among these neighboring cell types, astrocytes, microvascular endothelial cells (MVECs), and pericytes coordinate with neurons to form the neurovascular unit that provides a physical barrier to limit the passage of toxic materials from the blood into neural tissue. Previous studies have demonstrated that these barrier properties may be compromised in the progression of glaucoma, yet mechanisms by which this happens have remained incompletely understood. Thus, the goals of this study were to adapt a human pluripotent stem cell (hPSC)-based model of the neurovascular unit to the study of barrier integrity relevant to glaucoma. To achieve this, hPSCs were differentiated into the cell types that contribute to this barrier, including RGCs, astrocytes, and MVECs, then assembled into an established Transwell®-insert model. The ability of these cell types to contribute to an in vitro barrier model was tested for their ability to recapitulate characteristic barrier properties. Results revealed that barrier properties of MVECs were enhanced when cultured in the presence of RGCs and astrocytes compared to MVECs cultured alone. Conversely, the versatility of this system to model aspects of barrier dysfunction relevant to glaucoma was tested using an hPSC line with a glaucoma-specific Optineurin (E50K) mutation as well as a paired isogenic control, where MVECs then exhibited reduced barrier integrity. To identify factors that could result in barrier dysfunction, results revealed an increased expression of TGFβ2 in glaucoma-associated OPTN(E50K) astrocytes, indicating a potential role for TGFβ2 in disease manifestation. To test this hypothesis, we explored the ability to modulate exogenous TGFβ2 in both isogenic control and OPTN(E50K) experimental conditions. Collectively, the results of this study indicated that the repurposing of this in vitro barrier model for glaucoma reliably mimicked some aspects of barrier dysfunction, and may serve as a platform for drug discovery, as well as a powerful in vitro model to test the consequences of barrier dysfunction upon RGCs in glaucoma.