cFLIP in the molecular regulation of astroglia-driven neuroinflammation in experimental glaucoma.

BACKGROUND: Recent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL. METHODS: Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension in mouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western blot analysis of selected proteins in freshly isolated samples of astroglia. RESULTS: Immunoassays and immunolabeling of retina and optic nerve tissues presented reduced production of various proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks of ocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA and increased phospho-RelB in parallel with an increase in caspase-8 cleavage products. CONCLUSIONS: A prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion in astroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might reinforce cell survival in cFLIP-deleted astroglia.

Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype.

The microtubule-associated protein Tau is a key player in various neurodegenerative conditions, including Alzheimer's disease (AD) and Tauopathies, where its hyperphosphorylation disrupts neuronal microtubular lattice stability. Glaucoma, a neurodegenerative disorder affecting the retina, leads to irreversible vision loss by damaging retinal ganglion cells and the optic nerve, often associated with increased intraocular pressure. Prior studies have indicated Tau expression and phosphorylation alterations in the retina in both AD and glaucoma, yet the causative or downstream nature of Tau protein changes in these pathologies remains unclear. This study investigates the impact of Tau protein modulation on retinal neurons under normal and experimental glaucoma conditions. Employing AAV9-mediated gene therapy for Tau overexpression and knockdown, both manipulations were found to adversely affect retinal structural and functional measures as well as neuroprotective Akt/Erk survival signalling in healthy conditions. In the experimental glaucoma model, Tau overexpression intensified inner retinal degeneration, while Tau silencing provided significant protection against these degenerative changes. These findings underscore the critical role of endogenous Tau protein levels in preserving retinal integrity and emphasize the therapeutic potential of targeting Tau in glaucoma pathology.

Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma.

Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the retinal ganglion cells (RGCs), leading to irreversible vision loss. Nowadays, the traditional therapeutic approach to glaucoma consists of lowering the intraocular pressure (IOP), which does not address the neurodegenerative features of the disease. Besides animal models of glaucoma, there is a considerable need for in vitro experimental models to propose new therapeutic strategies for this ocular disease. In this study, we elucidated the pathological mechanisms leading to neuroretinal R28 cell death after exposure to glutamate and hydrogen peroxide (H2O2) in order to develop new therapeutic approaches for oxidative stress-induced retinal diseases, including glaucoma. We were able to show that glutamate and H2O2 can induce a decrease in R28 cell viability in a concentration-dependent manner. A cell viability of about 42% was found after exposure to 3 mM of glutamate and about 56% after exposure to 100 µM of H2O2 (n = 4). Label-free quantitative mass spectrometry analysis revealed differential alterations of 193 and 311 proteins in R28 cells exposed to 3 mM of glutamate and 100 µM of H2O2, respectively (FDR < 1%; p < 0.05). Bioinformatics analysis indicated that the protein changes were associated with the dysregulation of signaling pathways, which was similar to those observed in glaucoma. Thus, the proteomic alteration induced by glutamate was associated with the inhibition of the PI3K/AKT signaling pathway. On the other hand, H2O2-induced toxicity in R28 cells was linked to the activation of apoptosis signaling and the inhibition of the mTOR and ERK/MAPK signaling pathways. Furthermore, the data show a similarity in the inhibition of the EIF2 and AMPK signaling pathways and the activation of the sumoylation and WNT/ß-catenin signaling pathways in both groups. Our findings suggest that the exposure of R28 cells to glutamate and H2O2 could induce glaucoma-like neurodegenerative features and potentially provide a suitable tool for the development of new therapeutic strategies for retinal diseases.

POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells.

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON-OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2-/- mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

The neurostructural consequences of glaucoma and their overlap with disorders exhibiting emotional dysregulations: A voxel-based meta-analysis and tripartite system model.

BACKGROUND: Glaucoma, a progressive neurodegenerative disorder leading to irreversible blindness, is associated with heightened rates of generalized anxiety and depression. This study aims to comprehensively investigate brain morphological changes in glaucoma patients, extending beyond visual processing areas, and explores overlaps with morphological alterations observed in anxiety and depression. METHODS: A comparative meta-analysis was conducted, using case-control studies of brain structural integrity in glaucoma patients. We aimed to identify regions with gray matter volume (GMV) changes, examine their role within distinct large-scale networks, and assess overlap with alterations in generalized anxiety disorder (GAD) and major depressive disorder (MDD). RESULTS: Glaucoma patients exhibited significant GMV reductions in visual processing regions (lingual gyrus, thalamus). Notably, volumetric reductions extended beyond visual systems, encompassing the left putamen and insula. Behavioral and functional network decoding revealed distinct large-scale networks, implicating visual, motivational, and affective domains. The insular region, linked to pain and affective processes, displayed reductions overlapping with alterations observed in GAD. LIMITATIONS: While the study identified significant morphological alterations, the number of studies from both the glaucoma and GAD cohorts remains limited due to the lack of independent studies meeting our inclusion criteria. CONCLUSION: The study proposes a tripartite brain model for glaucoma, with visual processing changes related to the lingual gyrus and additional alterations in the putamen and insular regions tied to emotional or motivational functions. These neuroanatomical changes extend beyond the visual system, implying broader implications for brain structure and potential pathological developments, providing insights into the overall neurological consequences of glaucoma.

TGFß Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage.

Purpose: Mutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ocular anterior segment dysgenesis (ASD) and glaucoma. We previously showed that transforming growth factor beta (TGFß) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFß signaling ameliorated ASD, supporting a role for the TGFß pathway in disease pathogenesis. Here, we tested whether altered TGFß signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice. Methods: To test the role of TGFß signaling in glaucoma-relevant phenotypes, we genetically reduced TGFß signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFß ligands in Col4a1+/G1344D mice. We performed slit-lamp biomicroscopy and optical coherence tomography for qualitative and quantitative analyses of anterior and posterior ocular segments, histological analyses of ocular tissues and optic nerves, and intraocular pressure assessments using rebound tonometry. Results: Col4a1+/G1344D mice showed defects of the ocular drainage structures, including iridocorneal adhesions, and phenotypes consistent with glaucomatous neurodegeneration, including thinning of the nerve fiber layer, retinal ganglion cell loss, optic nerve head excavation, and optic nerve degeneration. We found that reducing TGFß receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice. Conclusions: Our results suggest that elevated TGFß signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

Lhx2 promotes axon regeneration of adult retinal ganglion cells and rescues neurodegeneration in mouse models of glaucoma.

The axons of retinal ganglion cells (RGCs) form the optic nerve, transmitting visual information from the eye to the brain. Damage or loss of RGCs and their axons is the leading cause of visual functional defects in traumatic injury and degenerative diseases such as glaucoma. However, there are no effective clinical treatments for nerve damage in these neurodegenerative diseases. Here, we report that LIM homeodomain transcription factor Lhx2 promotes RGC survival and axon regeneration in multiple animal models mimicking glaucoma disease. Furthermore, following N-methyl-D-aspartate (NMDA)-induced excitotoxicity damage of RGCs, Lhx2 mitigates the loss of visual signal transduction. Mechanistic analysis revealed that overexpression of Lhx2 supports axon regeneration by systematically regulating the transcription of regeneration-related genes and inhibiting transcription of Semaphorin 3C (Sema3C). Collectively, our studies identify a critical role of Lhx2 in promoting RGC survival and axon regeneration, providing a promising neural repair strategy for glaucomatous neurodegeneration.

NADPH and NAC synergistically inhibits chronic ocular hypertension-induced neurodegeneration and neuroinflammation through regulating p38/MAPK pathway and peroxidation.

Glaucoma, the leading cause of irreversible blindness worldwide, is characterized by neurodegeneration and neuroinflammation with retinal NAD/NADP and GSH decline. Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADP) and glutathione (GSH) are two redox reducers in neuronal and glial metabolism. However, therapeutic strategies targeting NAD/NADP or GSH do not exert ideal effects, and the underlying mechanisms are still poorly understood. We assessed morphological changes in retinal ganglion cells (RGCs), the affected neurons in glaucoma, and Müller cells, the major glial cells in the retina, as well as the levels of phosphorylated p38 (p-p38) and Caspase-3 in glaucoma patients. We constructed a modified chronic ocular hypertensive rat model and an oxygen-glucose deprivation (OGD) cell model. After applying NADPH and N-acetylcysteine (NAC), a precursor to cysteine, the rate-limiting substrate in GSH biosynthesis, to cells, apoptosis, axonal damage and peroxidation were reduced in the RGCs of the NAC group and p-p38 levels were decreased in the RGCs of the NADPH group, while in stimulated Müller cells cultured individually or cocultured with RGCs, gliosis and p38/MAPK, rather than JNK/MAPK, activation were inhibited. The results were more synergistic in the rat model, where either NADPH or NAC showed crossover effects on inhibiting peroxidation and p38/MAPK pathway activation. Moreover, the combination of NADPH and NAC ameliorated RGC electrophysiological function and prevented Müller cell gliosis to the greatest extent. These data illustrated conjoined mechanisms in glaucomatous RGC injury and Müller cell gliosis and suggested that NADPH and NAC collaborate as a neuroprotective and anti-inflammatory combination treatment for glaucoma and other underlying human neurodegenerative diseases.

CTRP 9 mitigates the apoptosis and unfolded protein response of OGD/R-induced retinal ganglion cells by regulating the AMPK pathway.

C1q/TNF-related protein-9 (CTRP9) has been reported to play roles in several types of retinal diseases. However, the role and the potential mechanism of CTRP9 in glaucoma are still incompletely understood. The expression of CTRP9 in OGD/R-induced retinal ganglion cells (RGCs) was detected by quantitative real-time polymerase chain reaction and western blot assay. Cell proliferation was identified by cell counting Kit-8 assay. Flow cytometry, enzyme-linked immunosorbent assay and western blot assay were performed to assess cell apoptosis. Unfolded protein response (UPR), endoplasmic reticulum (ER) stress and the AMPK pathway were evaluated by western blot assay. The data showed that the expression of CTRP9 was significantly downregulated in OGD/R-induced 661W cells. OGD/R treatment reduced cell viability, promoted cell apoptosis and activated the UPR and ER stress. The overexpression of CTRP9 reversed the effects of OGD/R on 661W cell viability, apoptosis, the UPR and ER stress, as well as the AMPK pathway. However, Compound C, an inhibitor of AMPK signaling, reversed the protection of CTRP9 overexpression against injury from OGD/R in 661W cells. In summary, the results revealed that CTRP9 abated the apoptosis and UPR of OGD/R-induced RGCs by regulating the AMPK pathway, which may provide a promising target for the treatment of glaucoma.

Chromatic vision and structural assessment in primary congenital glaucoma.

Primary congenital glaucoma is a rare disease that occurs in early birth and can lead to low vision. Evaluating affected children is challenging and there is a lack of studies regarding color vision in pediatric glaucoma patients. This cross-sectional study included 21 eyes of 13 children with primary congenital glaucoma who were assessed using the Farnsworth D-15 test to evaluate color vision discrimination and by spectral domain optical coherence tomography to measure retinal fiber layer thickness. Age, visual acuity, cup-to-disc ratio and spherical equivalent data were also collected. Global and sectional circumpapillary and macular retinal fiber layer thicknesses were measured and compared based on color vision test performance. Four eyes (19%) failed the color vision test with diffuse dyschromatopsia patterns. Only age showed statistical significance in color vision test performance. Global and sectional circumpapillary and macular retinal fiber layer thicknesses were similar between the color test outcomes dyschromatopsia and normal. While the color vision test could play a role in assessing children with primary congenital glaucoma, further studies are needed to correlate it with damage to retinal fiber layer thickness.

Microenvironment-regulated dual-hydrophilic coatings for glaucoma valve surface engineering.

Glaucoma valves (GVs) play an essential role in treating glaucoma. However, fibrosis after implantation has limited their long-term success in clinical applications. In this study, we aimed to develop a comprehensive surface-engineering strategy to improve the biocompatibility of GVs by constructing a microenvironment-regulated and dual-hydrophilic antifouling coating on a GV material (silicone rubber, SR). The coating was based on a superhydrophilic polydopamine (SPD) coating with good short-range superhydrophilicity and antifouling abilities. In addition, SPD coatings contain many phenolic hydroxyl groups that can effectively resist oxidative stress and the inflammatory microenvironment. Furthermore, based on its in situ photocatalytic free-radical polymerization properties, the SPD coating polymerized poly 2-methylacryloxyethylphosphocholine, providing an additional long-range hydrophilic and antifouling effect. The in vitro test results showed that the microenvironment-regulated and dual-hydrophilic coatings had anti-protein contamination, anti-oxidation, anti-inflammation, and anti-fiber proliferation capabilities. The in vivo test results indicated that this coating substantially reduced the fiber encapsulation formation of the SR material by inhibiting inflammation and fibrosis. This design strategy for dual hydrophilic coatings with microenvironmental regulation can provide a valuable reference for the surface engineering design of novel medical implantable devices. STATEMENT OF SIGNIFICANCE: Superhydrophilic polydopamine (SPD) coatings were prepared on silicone rubber (SR) by a two-electron oxidation method. Introduction of pMPC to SPD surface using photocatalytic radical polymerization to obtain a dual-hydrophilic coating. The dual-hydrophilic coating effectively modulates the oxidative and inflammatory microenvironment. This coating significantly reduced protein contamination and adhesion of inflammatory cells and fibroblasts in vitro. The coating-modified SR inhibits inflammatory and fibrosis responses in vivo, promising to serve the glaucoma valves.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

A novel HDAC8 inhibitor H7E exerts retinoprotective effects against glaucomatous injury via ameliorating aberrant Müller glia activation and oxidative stress.

Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma.

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.

Sirt6 protects retinal ganglion cells and optic nerve from degeneration during aging and glaucoma.

Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.

Longitudinal changes in optic disc cupping from the baseline in chiasmal lesion optic neuropathy and glaucoma.

We aimed to investigate the changes in cupping in chiasmal lesion optic neuropathy (chON) compared to baseline optic disc and glaucoma. We used a novel study design to enroll patients who had fundus photographs incidentally taken during routine health check-ups prior to the onset of optic neuropathy. In 31 eyes (21 patients) with chON and 33 eyes (30 patients) with glaucoma, we investigated the change in cup-to-disc (C/D) area from the baseline to overt cupping using flicker analysis. Compared to the baseline, 23 eyes (74.2%) had increased cup size and 3 (9.7%) had vascular configuration changes in the chONgroup; in contrast, all glaucoma eyes exhibited changes in cup size and vascular configuration. The increase in C/D area ratio was significantly smaller in chON (0.04 ± 0.04) compared to glaucoma (0.10 ± 0.04, P < 0.001); the minimum residual neuroretinal rim width showed a more pronounced difference (29.7 ± 8.2% vs 7.1 ± 3.9%, P < 0.001). The changes distributed predominantly towards the nasal direction in chON, contrasting the changes to the arcuate fibers in glaucoma. In conclusion, our results provide the first longitudinal evidence of true pathological cupping in chONcompared to photographically disease-free baseline. The marked difference in the residual minimum rim width reaffirms the importance of rim obliteration in the differential diagnosis between the two diseases.

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.