Loss Of Retinal Ganglion Cells Research Articles

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.

Visual Impairment in Pre-Clinical Models of Mild Traumatic Brain Injury.

Impairment in visual function is common after traumatic brain injury (TBI) in the clinical setting, a phenomenon that translates to pre-clinical animal models as well. In Morris et al. (2021), we reported histological changes following weight-drop-induced TBI in a rodent model including retinal ganglion cell (RGC) loss, decreased electroretinogram (ERG) evoked potential, optic nerve diameter reduction, induced inflammation and gliosis, and loss of myelin accompanied by markedly impaired visual acuity. In this review, we will describe several pre-clinical TBI models that result in injuries to the visual system, indicating that visual function may be impaired following brain injury induced by a number of different injury modalities. This underscores the importance of understanding the role of the visual system and the potential detrimental sequelae to this sensory modality post-TBI. Given that most commonly employed behavioral tests such as the Elevated Plus Maze and Morris Water Maze rely on an intact visual system, interpretation of functional deficits in diffuse models may be confounded by off- target effects on the visual system.

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

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 (TGFβ) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFβ signaling ameliorated ASD, supporting a role for the TGFβ pathway in disease pathogenesis. Here, we tested whether altered TGFβ signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice. To test the role of TGFβ signaling in glaucoma-relevant phenotypes, we genetically reduced TGFβ signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFβ 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. 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 TGFβ receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice. Our results suggest that elevated TGFβ signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

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

BackgroundNADPH 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.MethodsEx 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.ResultsWe 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.ConclusionsCollectively, 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.

Protection against Oxidative Stress by Coenzyme Q10 in a Porcine Retinal Degeneration Model.

Oxidative stress plays an important role in neurodegenerative diseases, including glaucoma. Therefore, we analyzed if the antioxidant coenzyme Q10 (CoQ10), which is also commercially available, can prevent retinal degeneration induced by hydrogen peroxide (H2O2) in a porcine organ culture model. Retinal explants were cultivated for eight days, and H2O2 (500 µM, 3 h) induced the oxidative damage. CoQ10 therapy was applied (700 µM, 48 h). Retinal ganglion cells (RGCs) and microglia were examined immunohistologically in all groups (control, H2O2, H2O2 + CoQ10). Cellular, oxidative, and inflammatory genes were quantified via RT-qPCR. Strong RGC loss was observed with H2O2 (p ≤ 0.001). CoQ10 elicited RGC protection compared to the damaged group at a histological (p ≤ 0.001) and mRNA level. We detected more microglia cells with H2O2, but CoQ10 reduced this effect (p = 0.004). Cellular protection genes (NRF2) against oxidative stress were stimulated by CoQ10 (p ≤ 0.001). Furthermore, mitochondrial oxidative stress (SOD2) increased through H2O2 (p = 0.038), and CoQ10 reduced it to control level. Our novel results indicate neuroprotection via CoQ10 in porcine retina organ cultures. In particular, CoQ10 appears to protect RGCs by potentially inhibiting apoptosis-related pathways, activating intracellular protection and reducing mitochondrial stress.

Mesenchymal stem cells for repairing glaucomatous optic nerve.

Glaucoma is a common and complex neurodegenerative disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Currently, there is no effective method to address the cause of RGCs degeneration. However, studies on neuroprotective strategies for optic neuropathy have increased in recent years. Cell replacement and neuroprotection are major strategies for treating glaucoma and optic neuropathy. Regenerative medicine research into the repair of optic nerve damage using stem cells has received considerable attention. Stem cells possess the potential for multidirectional differentiation abilities and are capable of producing RGC-friendly microenvironments through paracrine effects. This article reviews a thorough researches of recent advances and approaches in stem cell repair of optic nerve injury, raising the controversies and unresolved issues surrounding the future of stem cells.

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

Expression of IL-4 and IL-12 in the aqueous humor of patients with chronic primary angle-closure glaucoma.

Angle-closure glaucoma is a common type of glaucoma in Asian populations. However, the role of inflammatory cytokines in angle-closure glaucoma is yet to be elucidated. Therefore, this study aimed to examine the expression of interleukin-4 (IL-4) and IL-12 in the aqueous humor of patients with chronic primary angle-closure glaucoma (CPACG) and elucidate the correlations between IL-4 and IL-12 concentrations in the aqueous humor, the degree of visual field defects, and retinal nerve fiber layer (RNFL) thickness in patients with CPACG. Aqueous humor samples were obtained from 31 patients diagnosed with CPACG at the Shaoxing People's Hospital between April 2022 and March 2023 and from 30 individuals with cataract (control). Based on the degree of the mean defect (MD), patients were divided into three groups: group A (MD ≤ -6dB, n= 10), group B (-6dB< MD< -12dB, n= 9), and group C (MD ≥ - 12dB, n= 12). RNFL thickness was measured using an optical coherence tomograph, and the concentrations of IL-4 and IL-12 in the aqueous humor were measured using Luminex technology. Aqueous humor concentration of IL-4 was significantly higher (p = 0.036) in the CPACG group than in the cataract group. However, there was no significant difference (p > 0.05) in IL- 12 concentration between the two groups. Additionally, there were no significant differences (p > 0.05) in IL-4 and IL-12 levels among patients with varying degrees of visual field defects (groups A, B, and C). Spearman's correlation analysis showed that IL-4 and IL-12 concentrations were not correlated (p > 0.05) with RNFL thickness around the optic disc and the degree of visual field defects. Conclusively, IL-4 may play an important role in the pathogenesis of CPACG. Given that IL-4 and IL-12 concentrations in the aqueous humor were not significantly correlated with RNFL thickness and the degree of visual field defects, the increase in IL-4 and IL-12 expression may not induce apoptosis and loss of retinal ganglion cells or affect RNFL thickness as well as the degree of visual field defects.

Intranasal Resveratrol Nanoparticles Enhance Neuroprotection in a Model of Multiple Sclerosis.

Resveratrol is a natural polyphenol which has a very low bioavailability but whose antioxidant, anti-inflammatory and anti-apoptotic properties may have therapeutic potential for the treatment of neurodegenerative diseases such as multiple sclerosis (MS). Previously, we reported the oral administration of resveratrol nanoparticles (RNs) elicited a neuroprotective effect in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, at significantly lower doses than unconjugated resveratrol (RSV) due to enhanced bioavailability. Furthermore, we demonstrated that the intranasal administration of a cell-derived secretome-based therapy at low concentrations leads to the selective neuroprotection of the optic nerve in EAE mice. The current study sought to assess the potential selective efficacy of lower concentrations of intranasal RNs for attenuating optic nerve damage in EAE mice. EAE mice received either a daily intranasal vehicle, RNs or unconjugated resveratrol (RSV) for a period of thirty days beginning on the day of EAE induction. Mice were assessed daily for limb paralysis and weekly for visual function using the optokinetic response (OKR) by observers masked to treatment regimes. After sacrifice at day 30, spinal cords and optic nerves were stained to assess inflammation and demyelination, and retinas were immunostained to quantify retinal ganglion cell (RGC) survival. Intranasal RNs significantly increased RGC survival at half the dose previously shown to be required when given orally, reducing the risk of systemic side effects associated with prolonged use. Both intranasal RSV and RN therapies enhanced RGC survival trends, however, only the effects of intranasal RNs were significant. RGC loss was prevented even in the presence of inflammatory and demyelinating changes induced by EAE in optic nerves. The intranasal administration of RNs is able to reduce RGC loss independent of the inflammatory and demyelinating effects on the optic nerve and the spinal cord. The concentration of RNs needed to achieve neuroprotection is lower than previously demonstrated with oral administration, suggesting intranasal drug delivery combined with nanoparticle conjugation warrants further exploration as a potential neuroprotective strategy for the treatment of optic neuritis, alone as well as in combination with glucocorticoids.

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7days or 1month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

Effect of astrocyte GPER on the optic nerve inflammatory response following optic nerve injury in mice

Activated astrocytes are a primary source of inflammatory factors following traumatic optic neuropathy (TON). Accumulation of inflammatory factors in this context leads to increased axonal damage and loss of retinal ganglion cells (RGCs). Therefore, in the present study, we explored the role of the astrocyte G protein-coupled estrogen receptor (GPER) in regulating inflammatory factors following optic nerve crush (ONC), and analyzed its potential regulatory mechanisms. Overall, our results showed that GPER was abundantly expressed in the optic nerve, and co-localized with glial fibrillary acidic proteins (GFAP). Exogenous administration of G-1 led to a significant reduction in astrocyte activation and expression of inflammation-related factors (including IL-1β, TNF-α, NFκB, and p-NFκB). Additionally, it dramatically increased the survival of RGCs. In contrast, astrocytes were activated to a greater extent by exogenous G15 administration; however, RGCs survival was significantly reduced. In vitro, GPER activation significantly reduced astrocyte activation and the release of inflammation-related factors. In conclusion, activation of astrocyte GPER significantly reduced ONC inflammation levels, and should be explored as a potential target pathway for protecting the optic nerve and RGCs after TON.

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.

Resveratrol Ameliorates Retinal Ischemia-Reperfusion Injury by Modulating the NLRP3 Inflammasome and Keap1/Nrf2/HO-1 Signaling Pathway.

Glaucoma, as an ischemia-reperfusion (I/R) injury disease, leading irreversible blindness through the loss of retinal ganglion cells (RGCs), mediated by various pathways. Resveratrol (Res) is a polyphenolic compound that exerts protective effects against I/R injury in many tissues. This article aimed to expound the underlying mechanisms through which Res protects RGCs and reduces visual dysfunction in vivo. An experimental glaucoma model was created using 6-8-week wild-type male C57BL/6J mice. Res was injected intraperitoneally for 5 days. The mice were then grouped according to the number of days after surgery and whether Res treatment was administered. We applied the Brn3a-labeled immunofluorescence staining and flash electroretinography (ERG) to assess the survival of RGCs and visual function. The expression of components of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, the interleukin-1-beta (IL-1β), and vital indicators of kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme-oxygenase 1 (HO-1) pathway at the protein and RNA levels were detected respectively. The survival of RGCs was reduced after surgery compared to controls, whereas Res application rescued RGCs and improved visual dysfunction. In conclusion, our results discovered that Res administration showed neuroprotective effects through inhibition of the NLRP3 inflammasome pathway and activation of Keap1/Nrf2/HO-1 pathway. Thus, we further elucidated the potential of Res in glaucoma therapy.

Impact of Acute Ocular Hypertension on Retinal Ganglion Cell Loss in Mice.

To assess the correlation between intraocular pressure (IOP) levels and retinal ganglion cell (RGC) loss across different fixed-duration episodes of acute ocular hypertension (AOH). AOH was induced in Thy1-YFP-H transgenic mice by inserting a needle connected to a saline solution container into the anterior chamber. Thirty-one groups were tested, each comprising three to five mice exposed to IOP levels ranging from 50 to 110mm Hg in 5/10mm Hg increments for 60/90/120minutes and a sham control group. The YFP-expressing RGCs were quantified by confocal scanning laser ophthalmoscopy, whereas peripapillary ganglion cell complex thickness was measured using spectral-domain optical coherence tomography. Changes in RGC count and GCCT were determined from values measured 30days after AOH relative to baseline (before AOH). In the 60-minute AOH groups, RGC loss varied even when IOP was increased up to 110mm Hg (36.8%-68.2%). However, for longer durations (90 and 120minutes), a narrow range of IOP levels (60-70mm Hg for 90-minute duration; 55-65mm Hg for 120-minute duration) produced a significant difference in RGC loss, ranging from <25% to >90%. Additionally, loss of YFP-expressing RGCs was comparable to that of total RGCs in the same retinas. Reproducible RGC loss during AOH depends on precise durations and IOP thresholds. In the current study, the optimal choice is an AOH protocol set at 70mm Hg for a duration of 90minutes. This study can assist in determining the optimal duration and intensity of IOP for the effective utilization of AOH models.

Mesenchymal Stromal Cells from Human Wharton's Jelly Modulate the Intraocular Immune Response in a Glucocorticoid Hypertension Model: An Exploratory Analysis.

Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells. Recent research suggests immunological changes such as cytokine imbalance may affect its pathophysiology. This implies that immunomodulation, like that of mesenchymal cells, could be a potential therapeutic avenue for this disease. However, the effects of intravitreal injections of human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) on intraocular immune response have not been assessed in ocular hypertension (OH) models. We explored this by measuring cytokine levels and expression of other markers, such as glial fibrillary acidic protein (GFAP) and T cells, in 15 randomly divided New Zealand rabbits: G1: OH, G2: hWJ-MSCs, and G3: OH+hWJ-MSCs. We analyzed the aqueous humor (IL-6, IL-8, and TNF-α) and vitreous humor (IFN-γ, IL-10, and TGF-β) using ELISA and flow cytometry (cell populations), as well as TCD3+, TCD3+/TCD4+, and TCD3+/TCD8+ lymphocytes, and GFAP in the retina and optic nerve through immunohistochemistry. We found a decrease in TNF-α, IL-6, IFN-γ, IL-10, and IL-8 in G3 compared to G1 and an increase in TGF-β in both G2 and G3. TCD3+ retinal infiltration in all groups was primarily TCD8+ rather than TCD4+ cells, and strong GFAP expression was observed in both the retina and optic nerves in all groups. Our results suggest that cellular and humoral immune responses may play a role in glaucomatous optic neuropathy and that intravitreal hWJ-MSCs can induce an immunosuppressive environment by inhibiting proinflammatory cytokines and enhancing regulatory cytokines.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Six-week-old wildtype (WT+ONA) and βB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Cilastatin as a Potential Anti-Inflammatory and Neuroprotective Treatment in the Management of Glaucoma.

Glaucoma is a neurodegenerative disease that causes blindness. In this study, we aimed to evaluate the protective role of cilastatin (CIL), generally used in the treatment of nephropathologies associated with inflammation, in an experimental mouse model based on unilateral (left) laser-induced ocular hypertension (OHT). Male Swiss mice were administered CIL daily (300 mg/kg, i.p.) two days before OHT surgery until sacrifice 3 or 7 days later. Intraocular Pressure (IOP), as well as retinal ganglion cell (RGC) survival, was registered, and the inflammatory responses of macroglial and microglial cells were studied via immunohistochemical techniques. Results from OHT eyes were compared to normotensive contralateral (CONTRA) and naïve control eyes considering nine retinal areas and all retinal layers. OHT successfully increased IOP values in OHT eyes but not in CONTRA eyes; CIL did not affect IOP values. Surgery induced a higher loss of RGCs in OHT eyes than in CONTRA eyes, while CIL attenuated this loss. Similarly, surgery increased macroglial and microglial activation in OHT eyes and to a lesser extent in CONTRA eyes; CIL prevented both macroglial and microglial activation in OHT and CONTRA eyes. Therefore, CIL arises as a potential effective strategy to reduce OHT-associated damage in the retina of experimental mice.

Ameliorative effect of resveratrol on acute ocular hypertension induced retinal injury through the SIRT1/NF-κB pathway

Glaucoma is a kind of neurodegenerative disorder characterized by irreversible loss of retinal ganglion cells (RGCs) and permanent visual impairment. It is reported that resveratrol (RES) is a promising drug for neurodegenerative diseases. However, the detailed molecular mechanisms underlying its protective potential have not yet been fully elucidated. The present study sought to investigate whether resveratrol could protect RGCs and retinal function triggered by acute ocular hypertension injury through the SIRT1/NF-κB pathway. An experimental glaucoma model was generated in C57BL/6J mice. Resveratrol was intraperitoneally injected for 5 days. Sirtinol was injected intravitreally on the day of retinal AOH injury. RGC survival was determined using immunostaining. TUNEL staining was conducted to evaluate retinal cell apoptosis. ERG was used to evaluate visual function. The proteins Brn3a, SIRT1, NF-κB, IL-6, Bax, Bcl2, and Cleaved Caspase3 were determined using western blot. The expression and localisation of SIRT1 and NF-κB in the retina were detected by immunofluorescence. Our data indicated that resveratrol treatment significantly increased Brn3a-labelled RGCs and reduced RGC apoptosis caused by AOH injury. Resveratrol administration also remarkably decreased NF-κB, IL-6, Bax, and Cleaved Caspase3 proteins and increased SIRT1 and Bcl2 proteins. Furthermore, resveratrol treatment obviously inhibited the reduction in ERG caused by AOH injury. Importantly, simultaneous administration of resveratrol and sirtinol abrogated the protective effect of resveratrol, decreased NF-κB protein expression, and increased SIRT1 protein levels. These results suggest that resveratrol administration significantly mitigates retinal AOH-induced RGCs loss and retinal dysfunction, and that this neuroprotective effect is partially regulated through the SIRT1/NF-κB pathway.

TRPV4 antagonist suppresses retinal ganglion cell apoptosis by regulating the activation of CaMKII and TNF-α expression in a chronic ocular hypertension rat model

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs), leading to irreversible visual function impairment. Sustained increase in intraocular pressure represents a major risk factor for glaucoma, yet the underlying mechanisms of RGC apoptosis induced by intraocular pressure remains unclear. This study aims to investigate the role of TRPV4 in RGC apoptosis in a rat model of chronic ocular hypertension (COH) and the underlying molecular mechanism. In the COH rat models, we evaluated the visual function, retinal pathological changes and RGC apoptosis. TRPV4 expression and downstream signaling molecules were also detected. We found that RGC density decreased and RGC apoptosis was induced in COH eyes compared with control eyes. TRPV4 expression increased significantly in response to elevated IOP. TRPV4 inhibition by the TRPV4 antagonist HC-067047 (HC-067) suppressed RGC apoptosis and protected visual function. HC-067 treatment upregulated the phosphorylation of CaMKII in both control and COH eyes. Finally, HC-067 treatment suppressed the production of TNF-α induced by ocular hypertension. The TRPV4 antagonist HC-067 might suppress RGC apoptosis by regulating the activation of CaMKII and inhibiting the production of TNF-α in the COH model. This indicated that TRPV4 antagonists may be a potential and novel therapeutic strategy for glaucoma.

Pharmacologic activity of mGLUR4 agonist in a model of primary open-angle glaucoma: A preclinical experimental study

Background. Worldwide, more than 67 million people suffer from glaucoma. In Russia, this number exceeds 1.08 million people. Annually, the number of primary cases increases by 3–4%. The increasing prevalence of glaucoma intensifies the search for neuroprotectants that can reduce the loss of retinal ganglion cells, thereby impeding the progression of the disease. Objective. To study of the pharmacological activity of mGLUR4 agonist, ZC64-0001 substance, on a model of primary open-angle glaucoma. Methods. The conducted preclinical study involved 60 sexually mature male Wistar rats, weighing 180–220 g. The observation period was 73 days. The animals were divided into 6 groups with 10 animals in each. Group 1 included intact animals (without any manipulations); Group 2 included negative control animals with the water injected in the anterior chamber of the eye; in Group 3–6, primary open-angle glaucoma was modelled by injecting 1% hyaluronic acid solution into the anterior chamber of the eye once every 7 days from day 1 to day 62 of the study. Animals in Group 4 were treated intragastrically with H-[(4-chlorophenyl)methyl]-1,6-dihydro-4-methoxy-1-(2-methylphenyl)-6-oxo-3-pyridazinecarboxamide under the laboratory code of ZC64-0001 at a dose of 10 mg/kg. Animals in Group 5 received Mexidol as a comparison drug intramuscularly at a dose of 25.7 mg/kg. Animals in Group 6 were treated with a Timolol instillation at a dose of 0.009 ml/kg. The studied compounds were administered from day 63 of the study once a day for 10 days. The evaluated indicators included the level of microcirculation in the retina, the amplitude of a-wave and b-wave of the electroretinogram, and the number of retinal ganglionic layer nuclei in the setting of the conducted treatment. Statistical processing of the data was performed using the Statistica 10.0 software (StatSoft, USA). Differences were determined at the significance level of p ˂ 0.05. Results. ZC64-0001 increased the level of microcirculation relative to the group with modelled glaucoma by 11.5%, with this indicator being statistically significantly different from that both in the group with modelled glaucoma and the intact group ( р &lt; 0.05). In the group of animals receiving ZC64-0001, the amplitude of a-wave increased relative to the primary glaucoma group by 17.7%, with this index being statistically different from the groups of intact animals and negative control (р &lt; 0.05). The b-wave amplitude increased by 34.4% relative to the group with modelled glaucoma, being statistically different from the intact group, negative control group, pathology modelled group, and comparison drug groups ( р &lt; 0.05). Administration of ZC64-0001 increased the number of retinal ganglion cell nuclei relative to the group with modelled glaucoma by 41.0%, which had a statistically significant difference from all the studied groups ( р &lt; 0.05). Conclusion. The ZC64-0001 compound demonstrated high neuroprotective properties in a model of primary open-angle glaucoma, leading to an improvement in retinal microcirculation, an increase in the wave amplitude according to the conducted electrophysiological study, and an increase in the number of ganglion cell nuclei.