Retinal Ganglion Cell Survival Research Articles

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.

Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma

Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.

Ochratoxin A induces mitochondrial dysfunction, oxidative stress, and apoptosis of retinal ganglion cells (RGCs), leading to retinal damage in mice

PurposeOchratoxin A (OTA) contamination of food and feed is a serious problem worldwide. OTA is considered a carcinogen and immunotoxic, nephrotoxic, and neurotoxic mycotoxin. The present study aims to determine the toxic effects of OTA on retinal ganglion cells (RGCs) and assess the resulting impairment of retinal function in mice.MethodsRGC-5 cells were exposed to OTA (100 and 200 μg/L) for 3 days, and the mice were fed OTA-contain (100 and 200 μg/kg) diets for 4 weeks. Antioxidant indices were detected by spectrophotometer. The apoptosis of RGC-5 cells was determined by flow cytometry. Mitochondrial morphology and mitochondrial membrane potential were detected by immunofluorescence. RGC survival was determined by immunofluorescence staining with Brn3a. Flash electroretinography (ERG) was conducted to assess visual function.ResultsThe oxidative-antioxidant balance suggested that OTA-induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the OTA-exposed RGC-5 cells, and the reduced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in the OTA exposed group. Furthermore, OTA exposure led to remarkable apoptosis in RGC-5 cells. The mitochondrial detection showed that OTA caused significant mitochondrial membrane potential reduction and mitochondrial fragmentation, which may be the cause of apoptosis of RGC-5 cells. Additionally, in vivo experiments demonstrated that OTA resulted in significant death of RGCs and subsequent retinal dysfunction in mice.ConclusionOchratoxin A induces mitochondrial dysfunction, oxidative stress, and RGCs death in mice.

Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF-macrophage pathway

Optic neuropathies, characterized by injury of retinal ganglion cell (RGC) axons of the optic nerve, cause incurable blindness worldwide. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) represent a promising "cell-free" therapy for regenerative medicine; however, the therapeutic effect on neural restoration fluctuates, and the underlying mechanism is poorly understood. Here, we illustrated that intraocular administration of MSC-sEVs promoted both RGC survival and axon regeneration in an optic nerve crush mouse model. Mechanistically, MSC-sEVs primarily targeted retinal mural cells to release high levels of colony-stimulating factor 3 (G-CSF) that recruited a neural restorative population of Ly6Clow monocytes/monocyte-derived macrophages (Mo/MΦ). Intravitreal administration of G-CSF, a clinically proven agent for treating neutropenia, or donor Ly6Clow Mo/MΦ markedly improved neurological outcomes in vivo. Together, our data define a unique mechanism of MSC-sEV-induced G-CSF-to-Ly6Clow Mo/MΦ signaling in repairing optic nerve injury and highlight local delivery of MSC-sEVs, G-CSF, and Ly6Clow Mo/MΦ as therapeutic paradigms for the treatment of optic neuropathies.

Synergistic Protection of Retinal Ganglion Cells (RGCs) by SARM1 Inactivation with CNTF in a Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy.

We evaluated whether inhibiting sterile alpha and (Toll/interleukin receptor (TIR)) motif-containing 1 (SARM1) activity protects retinal ganglion cells (RGCs) following ischemic axonopathy (rodent nonarteritic anterior ischemic optic neuropathy: rNAION) by itself and combined with ciliary neurotrophic factor (CNTF). Genetically modified SARM1(-) rats were rNAION-induced in one eye and compared against equivalently induced wild-type animals of the same background. Optic nerve (ON) diameters were quantified using optical coherence tomography (SD-OCT). RGCs were quantified 30 d post-induction using retinal stereology for Brn3a(+) nuclei. ON sections were analyzed by TEM and immunohistochemistry. SARM1(-)(-) and WT animals were then bilaterally sequentially rNAION-induced. One eye received intravitreal vehicle injection following induction; the contralateral side received CNTF and was analyzed 30 d post-induction. Inhibiting SARM1 activity suppressed axonal collapse following ischemic axonopathy. SARM1(-) animals significantly reduced RGC loss, compared with WT animals (49.4 ± 6.8% RGC loss in SARM1(-) vs. 63.6 ± 3.2% sem RGC loss in WT; Mann-Whitney one-tailed U-test, (p = 0.049)). IVT-CNTF treatment vs. IVT-vehicle in SARM1(-) animals further reduced RGC loss by 24% at 30 d post-induction, but CNTF did not, by itself, improve long-term RGC survival in WT animals compared with vehicle (Mann-Whitney one-tailed t-test; p = 0.033). While inhibiting SARM1 activity is itself neuroprotective, combining SARM1 inhibition and CNTF treatment generated a long-term, synergistic neuroprotective effect in ischemic neuropathy. Combinatorial treatments for NAION utilizing independent neuroprotective mechanisms may thus provide a greater effect than individual treatment modalities.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice.

To investigate the effects of Sonic hedgehog (Shh) gene-modified bone marrow mesenchymal stem cells (MSCs) on graft-induced retinal gliosis and retinal ganglion cells (RGCs) survival in diabetic mice. Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs (MSC-Shh). Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs (MSC-Gfp; control) were administered in diabetic mice. After 4wk, the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography, and markers of gliosis were examined by immunofluorescence and Western blotting. The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence. The mechanisms underlying the effects of MSC-Shh was investigated. A significant reduction of proliferative vitreoretinopathy (PVR) was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp. Significant downregulation of glial fibrillary acidic protein (GFAP) was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp. The extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (AKT) and phosphatidylin-ositol-3-kinase (PI3K) pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp. Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) levels were significantly increased in the host retina, and RGCs loss was significantly prevented after MSC-Shh administration. MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice. The ERK1/2, AKT and PI3K pathways are involved in this process. MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Combined treatment of human mesenchymal stem cells and green tea extract on retinal ganglion cell regeneration in rats after optic nerve injury

Retinal ganglion cell (RGC) death and axonal loss cause irreversible vision loss upon optic nerve (ON) injury. We have independently demonstrated that mesenchymal stem cells (MSCs) and green tea extract (GTE) promote RGC survival and axonal regeneration in rats with ON injury. Here we aimed to evaluate the combined treatment effect of human bone marrow-derived MSCs (hBM-MSCs) and GTE on RGC survival and axonal regeneration after ON injury. Combined treatment of hBM-MSCs and GTE promoted RGC survival and neurite outgrowth/axonal regeneration in ex vivo retinal explant culture and in rats after ON injury. GTE increased Stat3 activation in the retina after combined treatment, and enhanced brain-derived neurotrophic factor secretion from hBM-MSCs. Treatment of 10 μg/mL GTE would not induce hBM-MSC apoptosis, but inhibited their proliferation, migration, and adipogenic and osteogenic differentiation in vitro with reducing matrix metalloproteinase secretions. In summary, this study revealed that GTE can enhance RGC protective effect of hBM-MSCs, suggesting that stem cell priming could be a prospective strategy enhancing the properties of stem cells for ON injury treatment.

AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes.

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

Müller glial cells located in the peripheral retina are more susceptible to high pressure: implications for glaucoma

BackgroundGlaucoma, a progressive neurodegenerative disease, is a leading cause of irreversible vision loss worldwide. This study aims to elucidate the critical role of Müller glia (MG) in the context of retinal ganglion cell (RGC) death, particularly focusing on the influence of peripheral MG sensitivity to high pressure (HP).MethodsCo-cultures of porcine RGCs with MG were isolated from both the central and peripheral regions of pig retinas and subjected to both normal and HP conditions. Mass spectrometry analysis of the MG-conditioned medium was conducted to identify the proteins released by MG under all conditions.ResultsPeripheral MG were found to secrete a higher quantity of neuroprotective factors, effectively promoting RGC survival under normal physiological conditions. However, under HP conditions, co-cultures with peripheral MG exhibited impaired RGC survival. Moreover, under HP conditions, peripheral MG significantly upregulated the secretion of proteins associated with apoptosis, oxidative stress, and inflammation.ConclusionsThis study provides robust evidence suggesting the involvement of MG in RGC death in glaucoma, thus paving the way for future therapeutic investigations.

Metabolic effect of the retina in a murine model of AD

Aims/Purpose: The aim of the study is to evaluate energy metabolism and retinal tissue survival in retinal explants in a murine model of AD (TgSwDI) during aging.Methods: The 7‐, 12‐ and 24‐month‐old TgSwDI mouse model was compared with age‐matched wild‐type (WT) mice. Characterization of the mouse model was assessed by Aβ plaques staining. Cell viability was evaluated by lactate dehydrogenase (LDH) assay of retinal explants at 4 and 24 h of incubation with culture media. Retinal ganglion cell (RGC) survival was quantified by RBPMS staining in 7‐month‐old mice. Glucose metabolism was analysed by gas chromatography mass spectrometry (GC–MS) using uniformly labelled glucose by incubating whole retinas for 30 min with glucose in 7‐month‐old mice. And lactate, produced by glycolysis, was evaluated by the lactate assay in 7‐month‐old mice.Results: The presence of Aβ plaques and vascularization was detected in the retinas of TgSwDI mice, whereas they were not found in the retinas of control mice. At 7 months, the viability of retinal explants was reduced in the TgSwDI model compared to the age‐matched WT model using the LDH assay. Compared to controls, RGC survival in the TgSwDI model tended to decrease. We performed GC–MS to evaluate glucose metabolism and observed no difference between the TgSwDI and WT groups. Lactate production showed no significant differences between the groups.Conclusions: The presence of Aβ plaques was detected in the retinas of the TgSwDI model, indicating that it is a good model for studying the retina in AD. Retinal tissue viability was altered at different ages in the TgSwDI model, and RGC survival was also reduced in the AD model, substantiating that AD affect the retina. We did not detect any differences between WT and TgSwDI groups in energy metabolism, implying that alterations in retinal tissue and cell survival may be related to other pathways. Further studies are needed to explore this, ultimately providing novel insight to address visual function affected by AD.

Retinal response to systemic inflammation differs between sexes and neurons.

Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1β. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

Immunonutrition in glaucoma patients: A new microbiota‐based treatment

Glaucoma is one of the main causes of irreversible blindness, producing 12.3% of blindness worldwide. There are about 4.5 million people with glaucoma in the world. However, the real number should be much higher if we consider all people with undiagnosed glaucoma.Glaucoma is an age‐related multifactorial disease determined by both genetic and environmental factors. The cause remains unknown. So, efforts are focused on preventing blindness through early diagnosis and hypotensive treatment, which is not always effective. Currently, neuroprotective treatment is also considered.Among the most studied processes in relation to optic nerve degeneration in glaucoma are inflammation and apoptosis. Several studies show that genes associated with inflammation are upregulated in the trabecular meshwork, which could be related to the onset or progression of glaucoma. However, the most visible manifestations of inflammation in glaucoma occur on the ocular surface. This produces, for example, an infiltration of inflammatory cells and a reduction in corneal sensitivity. Like inflammatory processes, apoptosis has been associated with glaucomatous disease. In fact, vision loss is caused by the death of retinal ganglion cells (RGCs), whose axons form the fibres of the optic nerve. The survival of RGCs is key to the prevention of glaucomatous blindness.The ocular microbiota has recently been studied, analysing the possible association between the inflammatory molecules produced by the ocular microbiome and the etiopathogenic mechanisms of this optic neuropathy.The aim of these studies is the development of probiotics as a therapy for glaucoma. Probiotic strains allow a rapid and stable recovery of the normal ocular microbiota, providing faster anti‐inflammatory and antihistamine effects. In addition, they are antagonists of the pathogens without the risk of the appearance of resistance, which avoids the use of antibiotics. In this way, probiotics represent an alternative to the abusive use of antibiotics, thus avoiding the problem of resistance development. In this sense, polyphenols are very interesting since they are capable of exerting neuroprotective and immunomodulatory effects and also can remodel the microbiota.

RNA‐binding protein ELAVL1‐mediated neuroprotection of retinal ganglion cells

Aims/Purpose: To evaluate the impact of ELAVL1 overexpression on survival and function of retinal ganglion cells (RGC) in rat glaucoma model.Methods: The study included ex vivo and in vivo approaches. Ex vivo, we used rat retinal explants to investigate, whether AAV‐ELAVL1 delivery affects the survival of RGC in the degeneration model induced by axotomy. In vivo approach investigated histology and function of RGC in rat glaucoma model. Rats received intravitreal injection of AAV‐ELAVL1, AAV‐ELAVL1(S202A) or AAV‐control. To induce chronic glaucoma, unilateral episcleral vein cauterization was performed. Animals underwent electroretinography tests (ERG) during the experiment. Collected retinas and optic nerves were processed for immunostainings, western blots, and stereology.Results: In ex vivo retinal explants culture, the RGC count was 162 ± 35, 148 ± 22 and 149 ± 28 for AAV‐ELAVL1, AAV‐ELAVL1(S202A) and control, respectively (p < 0.03). The IOP values were similar in all experimental groups, and on average, they were significantly higher than in healthy eyes (p < 0.001). In retinal histology, clear neuroprotective effect in the AAV‐ELAVL1 group was observed. After AAV therapy, both constructs (ELAVL1 and ELAVL1(S202A) showed a significant increase in protein expression in RGC, which was also associated with a significant increase in the expression of BDNF in the retinas, especially after AAV‐ELAVL1 treatment. In functional tests, the PhNR response was significantly reduced in AAV‐control glaucoma (p < 0.001). The application of AAV‐ELAVL1 treatment resulted in the preservation of RGC function at a level very similar to the situation before the induction of glaucoma.Conclusions: The AAV‐ELAVL1 approach showed a very strong neuroprotective effect in a rat glaucoma model. The neuroprotective effect is associated with antioxidant and anti‐inflammatory activity with the increase in BDNF expression, which intensifies the neuroprotective effect.

Small extracellular vesicles derived from reactive microglia contribute to retinal neuroinflammation and neurodegeneration

Glaucoma is a leading cause of blindness worldwide and is characterized by progressive degeneration of retinal ganglion cells and damage to the optic nerve. Elevated intraocular pressure is a main risk factor. Moreover, chronic neuroinflammation plays an important role in glaucoma. Microglia become reactive when challenged with elevated pressure, releasing cytotoxic factors that contribute to retinal ganglion cell death. We have been reporting that controlling microglia‐mediated neuroinflammation in experimental glaucoma is sufficient to protect retinal ganglion cells from damage.Small extracellular vesicles are constitutively released by most cells and are important vehicles of intercellular communication, conveying lipids, proteins and genetic material. Small extracellular vesicles derived from microglia exposed to elevated pressure trigger a pro‐inflammatory response in microglia, as determined by increased production of proinflammatory cytokines, microglia motility, phagocytosis, and proliferation. These small extracellular vesicles also increase cell death and impact the survival of retinal ganglion cells. The depletion of retinal microglia halt neuroinflammation induced by small extracellular vesicles isolated from microglia at elevated pressure, suggesting that microglial small extracellular vesicles preferentially interact with microglia. Small extracellular vesicles isolated from microglia challenged with elevated pressure are also enriched in proteins associated with inflammatory signalling and RNA processing and in miRNAs that modulate the NF‐kappaB pathway.This route of communication between microglia involving small extracellular vesicles helps to clarify the pathological mechanisms in glaucoma and may open new perspectives for the development of therapies targeting microglia neuroinflammation.Funding: FCT, Portugal (UID/NEU/04539/2019; UIDB/04539/2020; UIDP/04539/2020), COMPETE‐FEDER (FCOMP‐01‐0124‐FEDER‐028417; POCI‐01‐0145‐FEDER‐007440), Centro 2020 Regional Operational Programme (CENTRO‐01‐0145‐FEDER‐000008: BRAINHEALTH2020).

Menopause‐induced neurodegeneration of retinal ganglion cells in a model of an acute retinal ischemia in rats

Aims/Purpose: To assess the influence of surgical menopause on the function and survival of retinal ganglion cells in a rat model of optic nerve crush.Methods: A total of 27 rats, aged 8 weeks, were divided into two groups based on the duration between ovariectomy (OVE) and euthanasia. Subgroups consisted of OVE, OVE combined with optic nerve crush (ONC), and ONC alone. All surgical procedures were conducted under general anaesthesia. Menopause was induced through OVE, which involved the surgical removal of ovaries. ONC was performed to induce acute retinal ischemia. Retinal function was evaluated using electroretinography (ERG). Retinas were stained for β3‐tubulin, NeuN, Erα, Erβ, and Iba1. Cell loss ratios were assessed through manual counting using ImageJ software.Results: After a two‐week menopause period, a reduction in retinal interneurons (RI) density was observed, but no significant decline was observed in retinal ganglion cell (RGC) count. The counts for NeuN were 373 ± 58 and 317 ± 55 (healthy vs. OVE, p < 0.02), and for β3‐tubulin were 197 ± 42 and 212 ± 38 (healthy vs. OVE, p > 0.05), respectively. Seven weeks after OVE, menopause affected RI but not RGC, with counts of 404 ± 49 and 378 ± 61 (healthy vs. OVE, p < 0.05), and 286 ± 32 and 287 ± 51 (healthy vs. OVE, p > 0.05) for NeuN and β3‐tubulin, respectively. Following the ONC procedure, the NeuN cell count was 345 ± 28 and 286 ± 40 (ONC and ONC + OVE, respectively, p < 0.0001), and for β3‐tubulin it was 221 ± 28 and 200 ± 37 (ONC and ONC + OVE, respectively, p < 0.05). In the preliminary analysis of ERG, a significant decrease in PhNR amplitudes was observed in the OVE group, while PhNR was severely diminished in the OVE + ONC group.Conclusions: Deprivation of estrogens through surgical menopause leads to accelerated retinal neurodegeneration, primarily affecting retinal interneurons. The absence of estrogens increases the vulnerability of RGCs to insults, resulting in expedited RGC loss during acute retinal ischemia.

Minocycline Administration Does Not Have an Effect on Retinal Ganglion Cell Survival in a Murine Model of Ocular Hypertension.

This study aims to investigate two key aspects in a mouse model of ocular hypertension (OHT): first, the time course of retinal ganglion cell (RGC) death and the parallel activation of caspase-3 (a-Casp3+ cells) to narrow the therapeutic window; and second, the effect of caspase-3 and microglia inhibition by minocycline on RGC rescue in this model. RGC loss after OHT induction was significant at day 7 and progressed to 30 days. However, anatomical RGC death was preceded by significant Casp3 activation on day 3. Microglial inhibition by minocycline did not alter the course of OHT or rescue RGCs but resulted in a decrease in a-Casp3+ cells and phagocytic and total microglia. Therefore, RGC death commitment occurs earlier than their loss of Brn3a expression, microglial cells do not exacerbate RGC loss, and while this death is primarily apoptotic, apoptosis inhibition does not rescue RGCs, suggesting that alternative death pathways play a role in glaucomatous injury.

Germacrone Protects against NF-κB-Mediated Inflammatory Signaling, Apoptosis, and Retinal Ganglion Cell Survival in a Rat Glaucoma Model.

Retinal ischemia-reperfusion (I/R) is a pathological phenomenon that causes cellular destruction in several ocular disorders, so there is a need for novel possible neuroprotective drugs. Researchers have reported numerous neuroprotective effects of Germacrone (GM). Therefore, this study aimed to elucidate the underlying processes of GM that may contribute to glaucoma development. 40 healthy rats underwent retinal ischemia-reperfusion (I/R) damage. The animals were divided into control, I/R-induced, GM-1d, and GM-7d. After 7 days of I/R, mice were sacrificed and retinal tissue removed. An enzyme-linked immunosorbent assay (ELISA) was used to assess retinal Malondialdehyde (MDA) and 8-OHdG levels after oxidative injury. The Fluro-Gold (FG) labelling assay counted retinal ganglion cells (RGC) before and after labelling. DNA from retinal tissue RNA was amplified. Western blotting and real-time qRT-PCR were utilised to assess Bax, Casapses-3, Bcl-2, retinal NF-kB, and COX-2 expression. Retinal cell apoptotic mediator expression was measured by a TUNEL assay. Retinal I/R damage increases ganglion cell death. Long-term GM treatment (GM-7d) reduced NF-κB activation and raised COX-2 expression, which suggests antioxidant potential. TUNEL-positive apoptotic cells were reduced in long-term GM-treated rats. In GM-treated retinas, the Bax-Bcl-2 ratio was identical to the control group and significantly different from the I/R group. GM reduces I/R-induced retinal cell damage by inhibiting RG cell death. Seven days after GM therapy, histology showed retinal tissue loss. NF-κB signaling and intrinsic mitochondrial apoptosis are possible mechanisms that may be attenuated by GM and are attributed to a retinal protective effect.

Chitosan-Rapamycin Carbon Dots Alleviate Glaucomatous Retinal Injury by Inducing Autophagy to Promote M2 Microglial Polarization.

Glaucoma is a prevalent cause of irreversible vision impairment, characterized by progressive retinal ganglion cells (RGCs) loss, with no currently available effective treatment. Rapamycin (RAPA), an autophagy inducer, has been reported to treat glaucoma in rodent models by promoting RGC survival, but its limited water solubility, systemic toxicity, and pre-treatment requirements hinder its potential clinical applications. Chitosan (CS)-RAPA carbon dot (CRCD) was synthesized via hydrothermal carbonization of CS and RAPA and characterized by transmission electron microscopy, Fourier transform infrared spectra, and proton nuclear magnetic resonance. In vitro assays on human umbilical cord vein endothelial and rat retinal cell line examined its biocompatibility and anti-oxidative capabilities, while lipopolysaccharide-stimulated murine microglia (BV2) assays measured its effects on microglial polarization. In vivo, using a mouse retinal ischemia/reperfusion (I/R) model by acute intraocular pressure elevation, the effects of CRCD on visual function, RGC apoptosis, oxidative stress, and M2 microglial polarization were examined. CRCD exhibited good water solubility and anti-oxidative capabilities, in the form of free radical scavenging. In vitro, CRCD was bio-compatible and lowered oxidative stress, which was also found in vivo in the retinal I/R model. Additionally, both in vitro with lipopolysaccharide-stimulated BV2 cells and in vivo with the I/R model, CRCD was able to promote M2 microglial polarization by activating autophagy, which, in turn, down-regulated pro-inflammatory cytokines, such as IL-1β and TNF-α, as well as up-regulated anti-inflammatory cytokines, such as IL-4 and TGF-β. All these anti-oxidative and anti-inflammatory effects ultimately aided in preserving RGCs, and subsequently, improved visual function. CRCD could serve as a potential novel treatment strategy for glaucoma, via incorporating RAPA into CDs, in turn not only mitigating its toxic side effects but also enhancing its therapeutic efficacy.

Monocytic involvement in the neuroinflammatory response after retinal injury

Aims/Purpose: Inflammation and disease lead to the recruitment of blood monocyte‐derived macrophages (MDM) to the central nervous system. The role of MDMs following a neuroinflammatory process after a lesion remains elusive because MDMs that enter the neural parenchyma are difficult to distinguish from microglia. The aim of this study is to elucidate the response of recruited MDMs after selective retinal ganglion cell (RGC) injury.Methods: Unilateral complete intra‐orbital optic nerve (ON) crush was performed in adult C57BL/6J LysM‐GFP mice carrying the selective marker green fluorescent protein (GFP) in myeloid MDM but not microglia, 1 week after retrograde labelling of RGCs with the neuronal tracer hydroxystilbamidine (OHSt) from the superior colliculi. This injury model allows to investigate how the retinal mononuclear phagocytic system responds to a neurodegenerative process via the phagocytic activity of its main cellular components, resident microglia and recruited MDM, both in the injured retina and its contralateral uninjured fellow retina, at 5‐, 7‐ or 10‐days post‐lesion.Results: At all‐time points after injury, microglial phagocytic activity was statistically significant in the injured retina compared to the uninjured fellow retina. In comparison to naïve retinas, the number of MDMs increases significantly after injury in both injured and uninjured retinas. In injured retinas the increase is statistically higher. However, only a subset of the recruited MDMs shows phagocytic activity in the injured retinas 10 days after the lesion but not before. In contrast, the population of MDMs in the contralateral uninjured retina shows no signs of phagocytic activity after the lesion.Conclusions: In the injured retina, recruited MDMs show an initial quiescent stage, with no phagocytic activity compatible with a neuroprotective state, followed by a delayed phase with detrimental behaviour for RGC survival. In the uninjured fellow retinas however, MDMs retain a quiescent stage. It is tempting to suggest that following ON injury, recruited MDMs adopt an initial neuroprotective stage followed by a detrimental phagocytic activity in the injured retina, whereas in the uninjured retina MDMs remain in the initial stage.

DJ-1 regulates mitochondrial function and promotes retinal ganglion cell survival under high glucose-induced oxidative stress.

This study aimed to investigate the antioxidative and neuroprotective effects of DJ-1 in mitigating retinal ganglion cell (RGC) damage induced by high glucose (HG). A diabetic mouse model and an HG-induced R28 cell model were employed for loss- and gain-of-function experiments. The expression levels of apoptosis and oxidative stress-related factors, including Bax, Bcl-2, caspase3, Catalase, MnSOD, GCLC, Cyto c, and GPx-1/2, were assessed in both animal and cell models using Western blotting. Retinal structure and function were evaluated through HE staining, electroretinogram, and RGC counting. Mitochondrial function and apoptosis were determined using JC-1 and TUNEL staining, and reactive oxygen species (ROS) measurement. In the mouse model, hyperglycemia resulted in reduced retinal DJ-1 expression, retinal structural and functional damage, disrupted redox protein profiles, and mitochondrial dysfunction. Elevated glucose levels induced mitochondrial impairment, ROS generation, abnormal protein expression, and apoptosis in R28 cells. Augmenting DJ-1 expression demonstrated a restoration of mitochondrial homeostasis and alleviated diabetes-induced morphological and functional impairments both in vivo and in vitro. This study provides novel insights into the regulatory role of DJ-1 in mitochondrial dynamics, suggesting a potential avenue for enhancing RGC survival in diabetic retinopathy.