Glaucoma Model Research Articles

Loss of neuroserpin exacerbates retinal ganglion cell dysfunction in neuroserpin knockout mice following chronic increase in intraocular pressure

AbstractPurpose: Recent studies have shown neuroserpin (NS) plays a critical role in several neurodegenerative diseases. The neuroprotective role of NS administration in the retina has been reported in an ischemic/reperfusion mouse model of glaucoma. Here, we studied the effect of neuroserpin loss on retinal integrity in normal and experimental glaucoma condition in NS knockout (NS−/−) mice.Methods: NS−/− and C57BL/6J wild type (WT) mice were subjected to weekly intraocular microbead injection for 8‐weeks (n = 40). Positive scotopic threshold response (pSTR) at −4.3log cd s/m2 and electroretinography (ERG) at 3.1 log cd s/m2 were recorded using Phoenix Ganzfeld at the 8‐week time point. Eye and optic nerve (ON) tissues were harvested after 8‐weeks for histological analysis (retina‐ haematoxylin and eosin and ON‐ toluidine blue staining) and biochemical evaluation.Results: NS−/− mice showed significant decline in pSTR amplitude (p < 0.0001) with loss of no. of cells in GCL (p < 0.0001) compared to WT in both normal and glaucoma (IOP: 28.5 ± 1.97 mmHg) conditions. However, no change in whole retinal scotopic ERG was observed in NS−/− and WT mice in both conditions. Plasmin inhibitory assay showed the loss of NS inhibitory activity in NS−/− retinas with or without glaucoma (p < 0.004). ON histology staining showed significant loss of axonal density (p < 0.0001) in NS−/− mice compared to WT in normal condition, however glaucoma exacerbated axonal loss in NS−/− compared to WT mice. Increased expression of autophagy markers, Beclin1 and LC3II/I were confirmed in NS−/− retinal lysates in both normal and glaucoma conditions. TUNEL positive cells were significantly higher in the GCL of retinal sections of NS−/− mice in glaucoma (p < 0.002).Conclusions: NS ablation worsens inner retinal structural and functional damage in glaucoma. This study establishes that absence of neuroserpin in the experimental glaucoma mice increases autophagic response in degenerating retinal cells. The exact molecular mechanism of neuroserpin on autophagy remains undefined. Future biochemical studies will explain the neuroserpin‐autophagy signalling network involvement in the retinal cells in glaucoma.

Characterization of retinal ganglion cells subtypes in the rat retina

AbstractPurpose: Retinal ganglion cells (RGCs) are a heterogeneous population of neurons that propagates visual information to the brain. Glaucoma is a disease characterized by the selective death of RGCs, which induce a progressive and irreversible loss of vision. In glaucoma, not all RGCs subtypes are affected equally; however, a rigorous characterization has not been fully described. Thus, the aim of this work is to fully characterize and quantify different RGCs subtypes at different localizations in rat retinas.Methods: RGCs were labelled by immunochemistry in control rat whole‐mount retinas. Images were captured using the optic disc as a reference for the different retinal zones. RGCs were labelled with an antibody against RBPMS that is considered the 100% of RGCs, and co‐labelled with subtype‐specific antibodies against: CART, Islet1/2, TBR2, SPP1. After quantification, the percentage of RGCs of each subtype were calculated in relation to the total RBPMS positive cells.Results: In the rat retina, the density of RGCs was 1853/mm2 in centre, 2041/mm2 in middle‐centre, 1760/mm2 in middle‐periphery and 916/mm2 in periphery. CART was expressed in 31.2% of the RGCs from the centre, in 32.1% from the middle‐centre, in 33.3% from the middle‐periphery and in 27.8% from the periphery of the retina. Islet1/2 was expressed in 72.4% of the central RGCs, in 79.3% from the middle‐central RGCs, in 77.8% from the middle‐peripheral RGCs and in 72.4% from the peripheral RGCs of the retina. TBR2 was expressed in 11.4% of the RGCs from the centre, in 13.3% from the middle‐centre, in 12.8% from the middle‐periphery and in 16.1% from the periphery of the retina. SPP1 slightly marked the soma of almost 100% of total RGCs.Conclusions: There is a characteristic pattern for each RGCs subtype in the rat retina. Therefore, we will compare these results with the same analysis in a rat glaucoma model allowing us to elucidate which RGCs subtypes are most susceptible.Supported by ELKARTEK (KK‐2019/00086), MINECO‐Retos (PID2019‐111139RB‐I00) and PIBA (2020_1_0026).

Saffron benefits for eye diseases

AbstractGlaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells (RGCs). An increase in the intraocular pressure is the principal risk factor but controlling this pressure does not always prevent glaucomatous damage, so other harmful factors would be implicated in this pathologic process. Several mechanisms have been proposed that leads to the RGC death in glaucoma, including glutamate excitotoxicity, aggregation of misfolded proteins, mitochondrial dysfunction, oxidative stress, neurotrophic deprivation, and neuroinflammation, among others. Therefore, early diagnosis of neurodegeneration, understanding of its pathogenic mechanisms, and the development of new neuroprotective therapies represent major challenges for treating this disease.During the inflammatory process that occurs in glaucoma, retinal glial cells, both microglia and macroglia (Müller cells and astrocytes), become activated and release factors that can be neuroprotective in some cases and neurodegenerative in others. Cytokine‐ and chemokine‐mediated signalling engages in the neuroinflammatory process that leads to retinal ganglion cell (RGC) damage in glaucoma. Thus, a substance with anti‐inflammatory activity may protect against RGC degeneration.Increasing evidence from both experimental models and clinical studies in patients supports the neuroprotective effect of saffron components in neurodegenerative conditions and in retinal neurodegenerative pathologies, due to its important anti‐inflammatory, anti‐apoptotic, and antioxidant properties.This study investigated the neuroprotective and anti‐inflammatory effects of a hydrophilic saffron extract standardized to 3% crocin content in a mouse model of unilateral, laser‐induced ocular hypertension (OHT). For this purpose, morphological signs of microglial activation and the expression of P2RY12, which demonstrates the increased microglial activation, were analysed. In addition, the possible regulation of cytokine and myokine production was also studied. Treatment with saffron extract decreased microglial signs of activation including reduction of microglial cell numbers and morphological features of its activation, including decrease soma size and process retraction, both in OHT and in contralateral eyes. In addition, also partially reversed OHT‐induced down‐regulation of P2RY12 and prevented retinal ganglion cell death in OHT eyes. Furthermore, most of the concentration of proinflammatory cytokines (IL‐1β, IFN‐γ, TNF‐α, and IL‐17), anti‐inflammatory cytokines (IL‐4 and IL‐10), Brain‐derived Neurotrophic Factor (BDNF), Vascular Endothelial Growth Factor (VEGF), and fractalkine were unaffected in response to laser‐induced OHT in both the OHT eye and its contralateral eye in animals treated with saffron extracts. Only IL‐6 levels were significantly increased in the OHT eye one day after laser induction compared with the control group.These results differed from those observed in this OHT glaucoma model in animals untreated with saffron, where microglial activation and down‐regulation of P2RY12 was more intense, and the significant changes in cytokine levels in both eyes in response to IOP increase were observed.All these findings indicate that oral administration of saffron extract could be beneficial in glaucoma by decreasing the neuroinflammation associated with increased intraocular pressure, reducing microglial activation, regulating the production of proinflammatory cytokines, VEGF, and fractalkine and preventing retinal ganglion cell death.

Macroglial activation over time in an experimental glaucoma model

AbstractGlaucoma is a neurodegenerative retinal disease characterized by irreversible loss of retinal ganglion cells (RGCs), leading to visual loss. Increased intraocular pressure (IOP) is the only risk factor for which treatment is available. However, other factors such as glial activation and dysfunction can also induce RGC death in glaucoma.Astrocytes and Müller glia (macroglial cells) play important roles in neuronal activity by providing physical and metabolic support to neurons. In glaucoma, when tissue damage occurs, macroglial cells undergo reactive gliosis, to defend the nerve tissue against damage and attempt to maintain its homeostasis. However, alterations in the function of these cells could cause damage and even neuronal death. In reactive macrogliosis, cells undergo complex biochemical and functional remodelling and exhibit morphological changes characterized by cell body thickening, increased number and length of cell processes, increased cell number and up‐regulation of cytoskeletal components such as gliofibrillary acidic protein (GFAP), which is considered one of the main markers of this process and may act as antigen‐presenting cells by expressing major histocompatibility complex class II (MHC‐II). Reactivation of macroglia can initially be beneficial, as it increases their metabolic activity, increases the expression of antioxidant defence, and restores ion, water and neurotransmitter balance. However, if macroglia become chronic, it is detrimental by directly or indirectly damaging the tissue and preventing its repair. Astrocytes may act in concert with microglia during the inflammatory process, so inflammatory mediators produced by astrocytes can chronically activate microglial cells, contributing to neuronal death, and similarly, inflammatory mediators released by microglia can chronically activate astrocytes.One of the most widely used models to elevate IOP is the unilateral laser‐induced ocular hypertension (OHT) model. Studies in this model have analysed the temporal pattern of RGC death and microglial cell activation at time points: 1, 3, 5, 8 and 15 days. Molecular changes in proinflammatory and anti‐inflammatory cytokines involved in the neuroinflammatory process, released by glial cells (microglia, astrocytes, and Müller cells) at the same time points mentioned above, have also been identified.Given the close relationship between the activation of macroglia and microglia and their involvement in the death of RGCs in this pathology, the present work analysed how the activation of retinal macroglial cells occurred at different time points (1, 3, 5, 8 and 15 days) after the induction of OHT in an experimental model of laser‐induced OHT in mice. For this purpose, we have analysed the GFAP‐labelled retinal area, the intensity of GFAP labelling, and the expression of MHC‐II in both OHT eyes and normotensive contralateral eyes compared to naïve eyes.In this study, in both OHT and contralateral eyes, macroglial cells showed morphological changes as well as in GFAP and MHC‐II expression at all time points analysed, being slightly more intense at 3–5 days, coinciding with the pattern of microglial activation previously studied, demonstrating this fact, a bilateral inflammatory process maintained over time in which microglia, astrocytes and Müller glia are involved.

Modulation of retinal cytokine expression by saffron in an experimental glaucoma model

AbstractPurpose: To analyse the anti‐inflammatory effect of saffron extract, by regulating the expression of cytokines and chemokines involved in the neuroinflammatory process leading to retinal ganglion cell (RGC) damage, in a mouse model of unilateral laser‐induced ocular hypertension (OHT).Methods: Three groups of Albino Swiss mice treated with saffron extract were used; saffron naïve group (SNG), saffron laser group 1 day (SLG1d) and saffron laser group 3 days (SLG3d). Both eyes with OHT (treated with laser photocoagulation) and their contralateral were analysed. Retinal samples were processed and multiarray kits (MILLIPLEX MAP Mouse Cytokine/Myokine Magnetic Bead Panel) were used to quantify the expression of: IL‐1β, IL‐4, IL‐6, IL‐10, IL‐17, IFN‐ϒ, TNF‐α, Brain‐derived Neurotrophic Factor (BDNF), Vascular Endothelial Growth Factor (VEGF) and Fractalkine. Immunohistochemical analysis was performed to locate cells expressing the factors and cytokines detected in the multiplex assay.Results: After saffron treatment, no significant differences were found, at 1 and 3 days after laser‐induced OHT, between the concentration of proinflammatory cytokines (IL‐1β, IFN‐γ, TNF‐α and IL‐17), anti‐inflammatory cytokines (IL‐4 and IL‐10), BDNF, VEGF, and fractalkine in both the OHT eye and its contralateral eye, compared to saffron naïve. IL‐6 levels increased significantly in the OHT eye in SLG1d, reaching normal values at SLG3d compared to SNG. These results are contrary to those found in OHT eyes and their contralateral not treated with saffron, in which changes in cytokine levels were observed.Conclusions: Saffron is effective in regulating the production of proinflammatory cytokines, VEGF, and fractalkine induced by increased IOP, thus protecting the retina from its related damage. Saffron could be beneficial as coadjutant therapies in the treatment of glaucoma, thus helping to decrease the inflammatory process that occurs in this pathology.

Retinal macroglial activation over time in an experimental glaucoma model

AbstractPurpose: The aim of this study was to analyse the effects of unilateral laser‐induced ocular hypertension (OHT) on macroglia in OHT and contralateral eyes at different time points after laser treatment (1, 3, 5, 8 and 15 days).Methods: Two groups of albino Swiss mice: naïve group (n = 6) and OHT group (n = 30, six animals for each time study group). In the OHT group, OHT was induced by laser photocoagulation of the limbal and episcleral veins of the left eyes. Retinal whole‐mounts were used to visualize the astroglial plexus throughout the retinal extension, analysing the GFAP‐labelled retinal area (GFAP‐RA), the intensity of GFAP labeling (GFAP‐IRI), and the intensity of MHC‐II expression (MHC‐II‐IRI) in both OHT eyes and normotensive contralateral eyes compared to naïve eyes.Results: In OHT and contralateral eyes, with respect to naïve eyes, at all the time points, we found the following: (i) astrocytes with thicker somas and more secondary processes, mainly in the intermediate (IR) and peripheral retina (PR); (ii) astrocytes with low GFAP‐IRI and only primary processes near the optic disc (OD); (iii) increased total GFAP‐RA, which was higher at 3 and 5 days, except for at 15 days; (iv) increased GFAP‐IRI in the IR and especially in the PR: (v) decreased GFAP‐IRI near the OD, mainly at 1 and 5 days; (vi) a significant increase in MHC‐II‐IRI, which was higher in the IR and PR; and (vii) GFAP+ and MHC‐II+ immunolabelling in the Müller glia.Conclusions: Increased IOP leads, both in OHT eyes and in their normotensive contralateral eye, to macroglial activation, demonstrated both by morphologic changes and by the overexpression of GFAP and MHC‐II. The main signs of activation were observed mainly in the IR and PR, being more intense at 3 and 5 days after induction. However, in the OD, astrocytes showed low expression of GFAP and MHC‐II. The bilateral macroglial activation observed in this study would be triggered by the immune response associated with neuroinflammation.

Tunable degrees of neurodegeneration in rats based on microsphere-induced models of chronic glaucoma

This study compares four different animal models of chronic glaucoma against normal aging over 6 months. Chronic glaucoma was induced in 138 Long–Evans rats and compared against 43 aged-matched healthy rats. Twenty-five rats received episcleral vein sclerosis injections (EPIm cohort) while the rest were injected in the eye anterior chamber with a suspension of biodegradable microspheres: 25 rats received non-loaded microspheres (N-L Ms cohort), 45 rats received microspheres loaded with dexamethasone (MsDexa cohort), and 43 rats received microspheres co-loaded with dexamethasone and fibronectin (MsDexaFibro cohort). Intraocular pressure, neuroretinal function, structure and vitreous interface were evaluated. Each model caused different trends in intraocular pressure, produced specific retinal damage and vitreous signals. The steepest and strongest increase in intraocular pressure was seen in the EPIm cohort and microspheres models were more progressive. The EPIm cohort presented the highest vitreous intensity and percentage loss in the ganglion cell layer, the MsDexa cohort presented the greatest loss in the retinal nerve fiber layer, and the MsDexaFibro cohort presented the greatest loss in total retinal thickness. Function decreased differently among cohorts. Using biodegradable microspheres models it is possible to generate tuned neurodegeneration. These results support the multifactorial nature of glaucoma based on several noxa.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-β1: Relevance to Pseudoexfoliation Glaucoma

Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, characterised by the production of exfoliation material and is estimated to affect 30 million people worldwide. Activation of the TGF-β pathway by TGF-β1 has been implicated in the pathogenesis of pseudoexfoliation glaucoma. To further investigate the role of TGF-β1 in glaucomatous changes in the trabecular meshwork (TM), we used RNA-Seq to determine TGF-β1 induced changes in the transcriptome of normal human trabecular meshwork (HTM) cells. The main purpose of this study was to perform a hypothesis-independent RNA sequencing analysis to investigate genome-wide alterations in the transcriptome of normal HTMs stimulated with TGF-β1 and investigate possible pathophysiological mechanisms driving XFG. Our results identified multiple differentially expressed genes including several genes known to be present in exfoliation material. Significantly altered pathways, biological processes and molecular functions included extracellular matrix remodelling, Hippo and Wnt pathways, the unfolded protein response, oxidative stress, and the antioxidant system. This cellular model of pseudoexfoliation glaucoma can provide insight into disease pathogenesis and support the development of novel therapeutic interventions.

Inhibition of ferroptosis promotes retina ganglion cell survival in experimental optic neuropathies

Retinal ganglion cell (RGC) death is a hallmark of traumatic optic neuropathy, glaucoma, and other optic neuropathies that result in irreversible vision loss. However, therapeutic strategies for rescuing RGC loss still remain challenging, and the molecular mechanism underlying RGC loss has not been fully elucidated. Here, we highlight the role of ferroptosis, a non-apoptotic form of programmed cell death characterized by iron-dependent lethal lipid peroxides accumulation, in RGC death using an experimental model of glaucoma and optic nerve crush (ONC). ONC treatment resulted in significant downregulation of glutathione peroxidase 4 (GPx4) and system xc(−) cystine/glutamate antiporter (xCT) in the rat retina, accompanied by increased lipid peroxide and iron levels. The reduction of GPx4 expression in RGCs after ONC was confirmed by laser-capture microdissection and PCR. Transmission electron microscopy (TEM) revealed alterations in mitochondrial morphology, including increased membrane density and reduced mitochondrial cristae in RGCs after ONC. Notably, the ferroptosis inhibitor ferrostatin-1 (Fer-1) significantly promoted RGC survival and preserved retinal function in ONC and microbead-induced glaucoma mouse models. In addition, compared to the apoptosis inhibitor Z-VAD-FMK, Fer-1 showed better effect in rescuing RGCs death in ONC retinas. Mechanistically, we found the downregulation of GPx4 mainly occurred in the mitochondrial compartment, accompanied by increased mitochondrial reactive oxygen species (ROS) and lipid peroxides. The mitochondria-selective antioxidant MitoTEMPO attenuated RGC loss after ONC, implicating mitochondrial ROS and lipid peroxides as major mechanisms in ferroptosis-induced RGC death in ONC retinas. Notably, administering Fer-1 effectively prevented the production of mitochondrial lipid peroxides, the impairment of mitochondrial adenosine 5′-triphosphate (ATP) production, and the downregulation of mitochondrial genes, such as mt-Cytb and MT-ATP6, in ONC retinas. Our findings suggest that ferroptosis is a major form of regulated cell death for RGCs in experimental glaucoma and ONC models and suggesting targeting mitochondria-dependent ferroptosis as a protective strategy for RGC injuries in optic neuropathies.

Sigma-1 receptor agonist, (+)-pentazocine, is neuroprotective in a Brown Norway rat microbead model of glaucoma

PurposeGlaucoma is a worldwide leading cause of irreversible blindness. Standard treatments lower intraocular pressure (IOP). Novel treatments to prevent optic nerve (ON) degeneration are needed. Here, we investigate the hypothesis that sigma-1 receptor (S1R) agonist (+)-pentazocine (PTZ) is neuroprotective in a Brown Norway (BN) rat, microbead model of glaucoma. MethodsBN rats (9–11 weeks, male and female) were treated by intraperitoneal injection, 3 times per week with (+)-PTZ (2 mg/kg) or vehicle (VEH) alone. Treatment started 1 week prior to intraocular injection of polystyrene microbeads to elevate IOP. IOP was measured 2–3 times per week. Five weeks post microbead injection, rats were euthanized. ONs were removed, then fixed and processed for 63x oil, light microscope imaging of toluidine blue stained ON cross sections. To facilitate comparison of ON morphology from VEH and (+)-PTZ treated rats with similar ocular hypertensive insults, rats were assigned to low (IOP ≤15.8 mmHg), moderate (15.8 < IOP <28.0 mmHg), and high (IOP ≥28.0 mmHg) groups based on average IOP in the microbead injected eye. Axon numbers, axon density, axonal and glial areas, axon loss, and axon size distributions of naïve, bead, and contralateral ONs were assessed using QuPath program for automated image analysis. Results(+)-PTZ treatment of BN rats protected ONs from damage caused by moderate IOP elevation. Treatment with (+)-PTZ significantly reduced axon loss and glial areas, and increased axon density and axonal areas compared to ONs from VEH treated rats with moderate IOP. (+)-PTZ-mediated neuroprotection was independent of IOP lowering effects. At average IOP ≥28.0 mmHg, (+)-PTZ treatment did not provide measurable neuroprotection. ONs from contralateral eyes exhibited subtle, complex changes in response to conditions in the bead eyes. ConclusionsS1R agonist (+)-PTZ shows promise as a neuroprotective treatment for glaucoma. Future studies to understand the complex molecular mechanisms by which (+)-PTZ provides this neuroprotection are needed.

Peptains block retinal ganglion cell death in animal models of ocular hypertension: implications for neuroprotection in glaucoma

Ocular hypertension is a significant risk factor for vision loss in glaucoma due to the death of retinal ganglion cells (RGCs). This study investigated the effects of the antiapoptotic peptides peptain-1 and peptain-3a on RGC death in vitro in rat primary RGCs and in mouse models of ocular hypertension. Apoptosis was induced in primary rat RGCs by trophic factor deprivation for 48 h in the presence or absence of peptains. The effects of intravitreally injected peptains on RGC death were investigated in mice subjected to retinal ischemic/reperfusion (I/R) injury and elevated intraocular pressure (IOP). I/R injury was induced in mice by elevating the IOP to 120 mm Hg for 1 h, followed by rapid reperfusion. Ocular hypertension was induced in mice by injecting microbeads (MB) or silicone oil (SO) into the anterior chamber of the eye. Retinal flatmounts were immunostained with RGC and activated glial markers. Effects on anterograde axonal transport were determined by intravitreal injection of cholera toxin-B. Peptain-1 and peptain-3a inhibited neurotrophic factor deprivation-mediated RGC apoptosis by 29% and 35%, respectively. I/R injury caused 52% RGC loss, but peptain-1 and peptain-3a restricted RGC loss to 13% and 16%, respectively. MB and SO injections resulted in 31% and 36% loss in RGCs following 6 weeks and 4 weeks of IOP elevation, respectively. Peptain-1 and peptain-3a inhibited RGC death; the loss was only 4% and 12% in MB-injected eyes and 16% and 15% in SO-injected eyes, respectively. Anterograde transport was defective in eyes with ocular hypertension, but this defect was substantially ameliorated in peptain-injected eyes. Peptains suppressed ocular hypertension-mediated retinal glial activation. In summary, our results showed that peptains block RGC somal and axonal damage and neuroinflammation in animal models of glaucoma. We propose that peptains have the potential to be developed as therapeutics against neurodegeneration in glaucoma.

Neuroprotection in Glaucoma: Basic Aspects and Clinical Relevance

Glaucoma is a neurodegenerative disease that affects primarily the retinal ganglion cells (RGCs). Increased intraocular pressure (IOP) is one of the major risk factors for glaucoma. The mainstay of current glaucoma therapy is limited to lowering IOP; however, controlling IOP in certain patients can be futile in slowing disease progression. The understanding of potential biomolecular processes that occur in glaucomatous degeneration allows for the development of glaucoma treatments that modulate the death of RGCs. Neuroprotection is the modification of RGCs and the microenvironment of neurons to promote neuron survival and function. Numerous studies have revealed effective neuroprotection modalities in animal models of glaucoma; nevertheless, clinical translation remains a major challenge. In this review, we select the most clinically relevant treatment strategies, summarize preclinical and clinical data as well as recent therapeutic advances in IOP-independent neuroprotection research, and discuss the feasibility and hurdles of each therapeutic approach based on possible pathogenic mechanisms. We also summarize the potential therapeutic mechanisms of various agents in neuroprotection related to glutamate excitotoxicity.

Loss of Retinogeniculate Synaptic Function in the DBA/2J Mouse Model of Glaucoma.

Retinal ganglion cell (RGC) axons comprise the optic nerve and carry information to the dorsolateral geniculate nucleus (dLGN), which is then relayed to the cortex for conscious vision. Glaucoma is a blinding neurodegenerative disease that commonly results from intraocular pressure (IOP)-associated injury leading to RGC axonal pathology, disruption of RGC outputs to the brain, and eventual apoptotic loss of RGC somata. The consequences of elevated IOP and glaucomatous pathology on RGC signaling to the dLGN are largely unknown yet are likely to contribute to vision loss. Here, we used anatomic and physiological approaches to study the structure and function of retinogeniculate (RG) synapses in male and female DBA/2J (D2) mice with inherited glaucoma before and after IOP elevation. D2 mice showed progressive loss of anterograde optic tract transport to the dLGN and vGlut2 labeling of RGC axon terminals while patch-clamp measurements of RG synaptic function showed that synaptic transmission was reduced in 9-month and 12-month D2 mice because of the loss of individual RGC axon inputs. TC neuron dendrites had reduced Sholl complexity at 12 months, suggestive of delayed reorganization following reduced synaptic input. There was no detectable change in RGC density in 11- to 12-month D2 retinas, quantified as the number of ganglion cell layer-residing somata immuno-positive for NeuN and immuno-negative for the amacrine marker choline acetyltransferase (ChAT). Thus, observed synaptic defects appear to precede RGC somatic loss. These findings identify glaucoma-associated and IOP-associated deficits in an important subcortical RGC projection target, shedding light on processes linking IOP to vision loss.

Neurodegeneration Markers Galectin-3 and Apolipoprotein E Are Elevated in the Aqueous Humor of Eyes With Glaucoma.

Galectin-3 (Gal-3) and apolipoprotein E (APOE) are markers of activated microglia in neurodegenerative diseases of the central nervous system, whose targeting is protective in mouse models of glaucoma. In this study, we examined levels of Gal-3 and APOE in human aqueous humor (AH) and defined their clinical associations with glaucoma. We collected AH from 59 glaucoma patients and 15 controls at the start of planned ophthalmic surgery. Gal-3 and APOE levels were quantified by enzyme-linked immunosorbent assay. Total protein in AH was quantified by bicinchoninic acid assay. Significant associations between Gal-3, APOE, and clinical covariates were defined using univariate and multivariate linear regression models. Gal-3 and APOE levels were significantly elevated in the AH of glaucoma patients compared to controls (P = 0.004 and P < 0.001, respectively). Gal-3 and APOE were positively correlated across the entire cohort (r = 0.65, P = 6.2E-9). No association was observed between Gal-3 and total protein or APOE and total protein (P = 0.35 and P = 0.50, respectively), indicating that their levels were not increased in glaucomatous AH due to nonspecific protein accumulation. Multivariate linear regression modeling revealed significant associations between Gal-3 and maximum recorded intraocular pressure (P = 0.009) and between APOE and number of past ophthalmic surgeries (P = 0.031). We demonstrate that Gal-3 and APOE are significantly elevated in the AH of eyes with glaucoma and are associated with a history of poorly controlled disease. Gal-3 and APOE in AH may inform clinical decision-making as quantifiable readouts of microglial activation in eyes with glaucoma.

GSK872 and necrostatin-1 protect retinal ganglion cells against necroptosis through inhibition of RIP1/RIP3/MLKL pathway in glutamate-induced retinal excitotoxic model of glaucoma

BackgroundGlaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-d-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear.MethodsThe glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting.ResultsWe found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1β (IL-1β), was markedly inhibited.ConclusionsOur findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.

Neuroprotective Effects of Transferrin in Experimental Glaucoma Models.

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

Role of CAST-Drp1 Pathway in Retinal Neuron-Regulated Necrosis in Experimental Glaucoma.

Numerous studies have indicated that excitatory amino acid toxicity, such as glutamate toxicity, is involved in glaucoma. In addition, excessive glutamate can lead to an intracellular calcium overload, resulting in regulated necrosis. Our previous studies have found that the calpastatin (CAST)-calpain pathway plays an important role in retinal neuron-regulated necrosis after glutamate injury. Although inhibition of the calpain pathway can decrease regulated necrosis, necrotic cells remain. It has been suggested that there are other molecules that participate in retinal neuron-regulated necrosis. CAST is an important regulator of dynamin-related protein 1 (Drp1)-mediated mitochondrial defects. Thus, the aim of this study was to determine whether the CAST-Drp1 pathway may be an underlying signaling axis in neuron-regulated necrosis. Using cultured retinal neurons and in an in-vivo glaucoma model induced by glutamate overload, members of the CAST-Drp1 pathway were assessed by immunofluorescence, Western blotting, Phos-tagTM SDS-PAGE, and co-immunoprecipitation assays. Moreover, the black and white box test was performed on the rats. We found that more retinal neuron-regulated necrosis and Drp1 activation as well as lower CAST levels were present in the glutamate-induced glaucoma model. Rats with glutamate-induced glaucoma exhibited impaired visual function. We also observed retinal neuron-regulated necrosis and Drp1 activity decreased, and impaired vision recovered after CAST active peptide application, indicating that the CAST-Drp1 pathway plays a critical role in retinal neuron-regulated necrosis and visual function. The results of this study indicate that the CAST-Drp1 pathway protects against retinal neuron-regulated necrosis, which may expand the therapeutic targets for the treatment of neurodegenerative disorders involving dysfunction of glutamate metabolism, such as glaucoma.

Neuroprotective Properties of Chlorogenic Acid and 4,5-Caffeoylquinic Acid from Brazilian arnica (Lychnophora ericoides) after Acute Retinal Ischemia.

Lychnophora is a genus of South American flowering plants in the daisy family, popularly known as "Brazilian arnica". It is used in traditional medicine as an anti-inflammatory and analgesic agent, whose active components are derived from chlorogenic acid (CGA) and C-flavonoids. Since the drugs currently used are ineffective to treat glaucoma, agents with antioxidant and anti-inflammatory properties may represent new alternatives in preventing cellular lesions in retinal ischemia. In this study, we report the neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic (CQA) acid, isolated from Lychnophora plants, in a rodent glaucoma model. Wistar rats were administered intravitreally with 10 µg CGA or CGA, and then subjected to acute retinal ischemia (ISC) by increasing intraocular pressure (IPO) for 45 minutes followed (or not) by 15 minutes of reperfusion (I/R). Qualitative and quantitative analyses of neurodegeneration were performed using hematoxylin-eosin or Fluoro-Jade C staining protocols. All retinas submitted to ISC or I/R exhibited matrix disorganization, pyknotic nuclei, and pronounced vacuolization of the cytoplasm in the ganglion cell layer (GCL) and inner nuclear layer (INL). Pretreatment with CGA or CQA resulted in the protection of the retinal layers against matrix disorganization and a reduction in the number of vacuolized cells and pyknotic nuclei. Also, pretreatment with CGA or CQA resulted in a significant reduction in neuronal death in the GCL, the INL, and the outer nuclear layer (ONL) after ischemic insult. Our study demonstrated that CGA and CQA exhibit neuroprotective activities in retinas subjected to ISC and I/R induced by IPO in Wistar rats.