Apoptosis Of Retinal Ganglion Cells Research Articles

Neuroprotective effects and mechanisms of action of artemisinin in retinal ganglion cells in a mouse model of traumatic optic neuropathy

IntroductionTraumatic optic neuropathy is known to be a critical condition that can cause blindness; however, the specific mechanism underlying optic nerve injury is unclear. Recent studies have reported that artemisinin, considered vital in malaria treatment, can also be used to treat neurodegenerative diseases; however, its precise role and mechanism of action remain unknown. Therefore, in this study, we aimed to investigate the impact and probable mechanism of action of artemisinin in retinal ganglion cells (RGCs) in a mouse model of traumatic optic neuropathy induced by optic nerve crush (ONC). MethodsONC was induced in the left eye of mice by short-term clamping of the optic nerve; oral artemisinin was administered daily. The neuroprotective effect of the drug was assessed using Tuj-1 staining in RGCs. In addition, the inflammatory response and the expression levels of phosphorylated tau protein and tau oligomers were observed using RT-qPCR, TUNEL assay, and fluorescence staining to investigate the underlying mechanisms. ResultsArtemisinin increased the survival rate of RGCs 14 days after ONC. Artemisinin significantly reduced the levels of inflammatory factors such as CXCL10, CXCR3, and IL-1β in the retina and decreased the apoptosis of RGCs. Moreover, downregulation of the phosphorylation of tau proteins and the expression of tau oligomers were observed after artemisinin treatment. ConclusionOur results suggest that artemisinin can increase the survival rate of RGCs after ONC and reduce their apoptosis. This effect may be achieved by inhibiting the inflammatory response it triggers and downregulating tau protein phosphorylation and tau oligomer expression. These findings suggest the potential application of artemisinin as a therapeutic agent for neuropathy.

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models

BackgroundRetinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR).MethodsAn in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted.ResultsAPI treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission.ConclusionAPI protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

SiRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells.

Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications. RGCs were treated with siRhoA for 24 hours in vitro. Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of H2O2 for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay in situ, and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against H2O2-induced injury in RGCs in vitro. Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM). With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented H2O2-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway. The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.

A novel function and mechanism of ischemia-induced retinal astrocyte-derived exosomes for RGC apoptosis of ischemic retinopathy

Retinal ischemia is a common clinical event leading to retinal ganglion cell (RGC) death, resulting in irreversible vision loss. In the retina, glia-neuron communication is crucial for multiple functions and homeostasis. Extracellular vesicles, notably exosomes, play a critical role. The functions and mechanisms of retinal astrocyte-secreted exosomes remain unclear. Here, we isolated astrocyte-derived exosomes under hypoxia or normoxia and explored their role in an in vivo retinal ischemia-reperfusion (RIR) model. We found that hypoxia triggered astrocytes to produce a significantly increased number of exosomes, which could be internalized by RGCs in vivo or in vitro. Also, in the RIR model, the hypoxia-induced exosomes ameliorated the RIR injury and suppressed the RGC apoptosis. Furthermore, microRNA sequencing of retinal astrocyte-secreted exosomes revealed different patterns of exosomal miRNAs under hypoxia, particularly enriched with miR-329-5p. We verified that miR-329-5p was specifically bound to MAPK8 mRNA, and subsequent JNK-pathway molecules were downregulated. We anticipated that the miR-329-5p/JNK pathway is a key to suppressing RGC apoptosis and preventing RIR injury. Such findings provided insights into the therapeutic potential of hypoxia-induced astrocyte-secreted exosomes and the miR-329-5p for treating retina ischemic diseases.

Resveratrol protects against diabetic retinal ganglion cell damage by activating the Nrf2 signaling pathway

ObjectiveOxidative stress-induced retinal neurodegenerative changes are among the pathological alterations observed in diabetic retinopathy. Resveratrol (RSV), a polyphenolic compound with diverse pharmacological effects, has shown preventive qualities in several neurodegenerative illnesses, including anti-inflammatory, anti-aging, and antioxidant benefits. However, its therapeutic efficacy in diabetic retinal neurodegeneration has not yet been thoroughly elucidated. Our study aimed to explore the protective mechanisms and therapeutic benefits of RSV on diabetic retinal neurodegeneration alterations. Materials and methodsUsing streptozotocin, we created a diabetic mouse model and conducted visual electrophysiological examinations on mice from the normal group, diabetic group, and diabetic group treated with RSV. Retinas were harvested for histological staining. Additionally, primary retinal ganglion cells cultured in high glucose conditions were used to assess malondialdehyde (MDA) levels and superoxide dismutase (SOD) levels upon siRNA-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) interference. Protein levels of Nrf-2, heme oxygenase-1 (HO-1), and transcriptional levels of them were also measured. ResultsWe demonstrated that RSV significantly improved the retinal morphology and function in the diabetic retinopathy model mice. The treated mice exhibited notable improvements in visual electrophysiology, with a significant reduction in retinal ganglion cell apoptosis. Following RSV treatment, the high glucose-cultured ganglion cells demonstrated a considerable rise in SOD levels and a substantial drop in MOD. Moreover, the protein expression of solute carrier family 7 member 11 (SLC7A11) and Nrf2 significantly increased. RT-PCR and Western blot results indicated a significant attenuation of RSV's therapeutic effects upon Nrf2 inhibition. ConclusionOur findings suggest that RSV may reduce oxidative stress levels in the retina and inhibit retinal ganglion cell apoptosis via reducing the Nrf2/HO-1 pathway, which lessens the harm that excessive glucose causes to the retina.

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.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

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.

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.

Müller cells under hydrostatic pressure modulate retinal cell survival via TRPV1/PLCγ1 complex-mediated calcium influx in experimental glaucoma.

Glaucoma, an irreversible blinding eye disease, is currently unclear whose pathological mechanism is. This study investigated how transient receptor potential cation channel subfamily V member 1 (TRPV1), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (PLCγ1), and P2X purinoceptor 7 (P2X7) modulate the levels of intracellular calcium ions (Ca2+ ) and adenosine triphosphate (ATP) in Müller cells and retinal ganglion cells (RGCs) under conditions of elevated intraocular pressure (IOP). Müller cells were maintained at hydrostatic pressure (HP). TRPV1- and PLCG1-silenced Müller cells and P2X7-silenced RGCs were constructed by transfection with short interfering RNA (siRNAs). RGCs were cultured with the conditioned media of Müller cells under HP. A mouse model of chronic ocular hypertension (COH) was established and used to investigate the role of TRPV1 in RGCs in vivo. Müller cells and RGCs were analyzed by ATP release assays, intracellular calcium assays, CCK-8 assays, EdU (5-ethynyl-2'-deoxyuridine) staining, TUNEL staining, flow cytometry, and transmission electron microscopy. In vivo changes in inner retinal function were evaluated by hematoxylin and eosin (H&E) staining and TUNEL staining. Western blot analyses were performed to measure the levels of related proteins. Our data showed that HP increased the levels of ATP and Ca2+ influx in Müller cells, and those increases were accompanied by the upregulation of TRPV1 and p-PLCγ1 expression. Suppression of TRPV1 or PLCG1 expression in Müller cells significantly decreased the ATP levels and intracellular Ca2+ accumulation induced by HP. Knockdown of TRPV1, PLCG1, or P2X7 significantly decreased apoptosis and autophagy in RGCs cultured in the conditioned media of HP-treated Müller cells. Moreover, TRPV1 silencing decreased RGC apoptosis and autophagy in the in vivo model of COH. Collectively, inhibition of TRPV1/PLCγ1 and P2X7 expression may be a useful therapeutic strategy for managing RGC death in glaucoma.

Melatonin antagonizes oxidative stress-induced apoptosis in retinal ganglion cells through activating the thioredoxin-1 pathway.

This study aimed to explore the role of melatonin in oxidative stress-induced injury on retinal ganglion cells and the underlying mechanisms. The immortalized RGC-5 cells were treated with H2O2 to induce oxidative injury. Cell viability was measured by Cell Counting Kit-8, and apoptosis was determined by flow cytometry and western blot assays. Reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels were examined to evaluate oxidative stress levels. In addition, Thioredoxin-1 (Trx1) was silenced in RGC-5 cells using small interfering RNA followed by signaling pathway examination to explore the underlying mechanisms of melatonin in alleviating oxidative injury. Melatonin pre-treatment significantly alleviated H2O2-induced apoptosis in RGC-5 cells. Melatonin also markedly reversed the upregulation of cleaved-caspase 3, cleaved-caspase 9, and Bax expression and downregulation of Bcl-2 expression induced by H2O2. Further analyses presented that melatonin significantly attenuated the increase of ROS, LDH, and MDA levels in RGC-5 cells after H2O2 treatment. Melatonin also abolished the downregulated expression of Superoxide dismutase type 1, Trx1, and Thioredoxin reductase 1, and the reduced activity of thioredoxin reductase in RGC-5 cells after H2O2 treatment. Notably, Trx1 knockdown significantly mitigated the protective effect of melatonin in alleviating H2O2-induced apoptosis and oxidative stress, while administration of compound C, a common inhibitor of c-Jun N-terminal kinase (JNK) signaling, partially reversed the effect of Trx1 silencing, thereby ameliorating the apoptosis and oxidative injury induced by H2O2 in RGC-5 cells. Melatonin could significantly alleviate oxidative stress-induced injury of retinal ganglion cells via modulating Trx1-mediated JNK signaling pathway.

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.

DERAJAT APOPTOSIS SEL GANGLION RETINA PASCA PEMBERIAN TAMBAHAN EKSTRAK MORINGA OLEIFERA ORAL DIBANDINGKAN DENGAN TERAPI STANDAR Studi Eksperimental pada Tikus Wistar Model Methanol Toxic Optic Neuropathy

Abstract&#x0D; Introduction &amp; Objectives : Methanol toxic optic neuropathy can lead to retinal ganglion cell apoptosis which causes visual disturbance. Moringa oleifera has been reported to have an anti-inflamatorry effect that can prevent inflammatory mediators from being relased in methanol toxic optic neuropathy&#x0D; Methods : Twenty-one Wistar methanol toxic optic neuropathy were divided into 3 groups, namely base line group, control group that given intravenous methylprednisolone 4.5mg/6 hours for 3 days, and given standard theraphy and additional administration of oral Moringa oleifera 300mg/kgbodyweight/day for 4 weeks. Assessment of the degree of apoptosis by Anatomical Pathology with histopatolocigal scaoring. The study used Mann Whitney comparative hypothesis test (Significant p&lt;0.05)&#x0D; Results : The degree of retinal ganglion cell apoptosis was significantly lower in the treatment and standard therapy groups compared to the base line group. The treatment group compared to the standard therapy group was not significantly different (p = 1,000)&#x0D; Conclusion : The degree of retinal ganglion cell apoptosis in rat model methanol toxic optic neuropathy was not significantly different between the group that was given additional oral Moringa oleifera extract compared to the standard theraphy group

EFFECT OF TOPICAL BRIMONIDINE ON NMDA RECEPTOR EXPRESSION AND RETINA GANGLION CELL DENSITY IN TRAUMATIC OPTIC NEUROPATHY RATS MODEL

Abstract&#x0D; Introduction &amp; Objectives : Traumatic optic neuropathy is a visual disturbance caused by acute injury to the optic nerve which results in retinal ganglion cell apoptosis through the glutamate excitotoxicity pathway. Brimonidine is an alpha 2-receptor agonist that acts as a neuroprotector in the glutamate excitotoxicity pathway and causes modulation of NMDA receptors associated with glutamate. Objective of this study is to prove the effect of topical brimonidine on NMDA receptor expression and retinal ganglion cell density in a rat model of traumatic optic neuropathy&#x0D; Methods : Rat models of traumatic optic neuropathy was carried out using the optic nerve crush method. The treatment group was given topical brimonidine 0.15% 1 drop/12 hours for 14 days. NMDA receptor expression was assessed by immunohistochemical staining and retinal ganglion cell density was assessed by Hematoxylin-eosin staining. Statistical analysis was performed to assess the correlation between NMDA receptor expression and retinal ganglion cell density&#x0D; Results : The Allred score of NMDA receptors expression in the treatment group was lower than control group, with significant difference (p=0.002). The mean density of retinal ganglion cells in the treatment and control group was 20,99  7,76 and 12,51  3,10, respectively with a significant difference (p=0.032). There was a significant correlation between NMDA receptor expression and retinal ganglion cell density in the treatment group (p=0.035) with strong negative correlation (r= -0.611)&#x0D; Conclusion : Topical administration of brimonidine to the Wistar rat model of traumatic optic neuropathy can suppress NMDA receptor expression and maintain retinal cell ganglion density

Inhibition of Caspase-3 Provides Neuroprotection for Retina Ganglion Cells againts NMDA-induced Exitotoxicity in Wistar Rats

Introduction &amp; ObjectivesGlaucoma is a disorder characterized by the presence of optic neuropathy. Visual impairment inglaucoma is caused by retinal ganglion cell (RGC) apoptosis associated with N-methyl-D-aspartate(NMDA)-mediated excitotoxicity. Excitotoxicity causes an increase in calcium in cells as well asactivation of caspase-3. Glaucoma therapy is currently given to reduce intraocular pressure (IOP),but the damage is still progressing progressively. Hesperidin is known as a neuroprotector that cansuppress calpain overactivation in NMDA-treated retina, thereby reducing the upregulation of TNF-,an inflammatory cytokine, inhibiting calpain activation by suppressing oxidative stress, andattenuating caspase-3 activation. The aim of this study to proving the effect of oral Hesperidin on theexpression of caspase-3 Retinal Ganglion Cell in Wistar rats model of NMDA-induced glaucoma.&#x0D; MethodsExperimental research with post test design only randomized control trial Wistar rats with NMDAinduction were divided into 2 treatment and control groups. The treatment group was given oralHesperidin at a dose of 100mg/kg/day while the control group was given Na-CMC 182 mg/kg/day for3 weeks. Retinal caspase-3 expression was examined by immunohistochemical staining. Data werecollected and processed and then the Mann- Whitney test was performed. (significant p&lt;0.05)&#x0D; ResultsThe mean percentage and intensity of RGC caspase-3 expression in the treatment group were 4.29 ±0.49 it was significantly lower than the control group 6.29 ± 0.95 (p value = 0.003).&#x0D; ConclusionHesperidin provides neuroprotection for RGC againts NMDA- induced exitotoxicity by inhibition ofcaspase-3.

EFFECT OF MEMANTINE AS N-METHYL D-ASPARTATE RECEPTOR ANTAGONIST ON CASPASE-9 LEVEL OF RETINAL GANGLION CELL IN METHANOL TOXIC OPTIC NEUROPATHY RAT MODEL

Abstract&#x0D; Introduction &amp; Objectives : Methanol toxic optic neuropathy (MTON) remains as a health issue in developing countries and there are no current definitive treatments. Accumulation of formic acid as metabolite of methanol causes mitochondrial dysfunction that can reduce ATP synthesis rate, disrupt axoplasmic transport, and elevate the reactive oxygen species (ROS). All of these mechanisms lead to the death of retinal ganglion cell (RGC) and optic nerve. RGC destruction in MTON has similar patterns with those that have been observed in glaucoma, ischemia, and trauma cases, that is through excessive activation of glutamate neurotransmitter via N-Methyl D-Aspartate receptors (NMDAR). Memantine is a NMDAR antagonist that inhibit the ion channel of NMDAR. This inhibitory effect of NMDAR by memantine is expected to reduce intracellular calcium level, prevent apoptosis of RGC, and prevent further mitochondrial dysfunction. Caspase-9 is essential to eliminate cells by executing apoptotic Objective: To observe the effect of Memantine on Caspase-9 level of RGC in Methanol-Intoxicated Rats.&#x0D; Methods : This experimental study was performed on 28 Methanol-Intoxicated Rats that are assigned into four treatment groups: K1 (MTON;enucleated day-3), K2 (MTON+Memantine; enucleated day-3), K3 (MTON; enucleated day-7), and K4 (MTON+Memantine; enucleated day-7). During the experiment, all groups were exposed to N2O:O2 gas and were given methanol orally. Caspase-9 level of RGC were measured immediately after enucleation.&#x0D; Results : The average of Caspase-9 level (ng/ml): K1=197,88±33,98; K2=205,64±63,66; K3=243,15±40,13; K4=150,08±119,60. The Caspase-9 level was significantly lower in K4 compared to K3 (p value =0.038).&#x0D; Conclusion : Memantine decrease the level of Caspase-9 of RGC in Methanol Toxic Optic Neuropathy rat model.

COENZYME Q10 EFFECT ON NMDA RECEPTOR EXPRESSION AND RETINAL GANGLION CELL DENSITY (Experimental Study in Traumatic Optic Neuropathy Model of Wistar Rat)

Abstract&#x0D; Introduction &amp; Objectives : Traumatic optic neuropathy is a visual disturbance condition after acute injury to optic nerve. This injury results in retinal ganglion cells ischemic leading to excitotoxicity and apoptosis. Coenzyme Q10 (CoQ10) is an antioxidant and cofactor in electron transport within mitochondria that inhibits glutamate excitotoxicity and thereby preventing retinal ganglion cell apoptosis. This research aims to prove the effect of coenzyme Q10 on NMDA receptor expression and retinal ganglion cell density in rat model of traumatic optic neuropathy.&#x0D; Methods : This study use post-test only randomized controlled groups design. Traumatic optic neuropathy wistar rats model was made by optic nerve crushed method, then given oral CoQ10 100mg/kgBW after 1 hour for 14 days in treatment group. Retinal histopathological specimen was made with immunohistochemistry and Haematoxyllin-eosin staining and examined under microscope with 400x magnification. Data was verified and statistically analyzed.&#x0D; Results : The NMDA receptor expression in the treatment group was significantly lower than control (p=0.009). The retinal ganglion cell density in the treatment group was significantly higher than control (p=0.009). There was a significant correlation between NMDA receptor expression and retinal ganglion cell density in the treatment group (p = 0.012) with a strong negative correlation direction (r = - 0.652).&#x0D; Conclusion : Administration of CoQ10 in traumatic optic neuropathy model rats affects NMDA receptor expression and retinal ganglion cell density. NMDA receptor expression was lower with higher retinal ganglion cell density in the treatment group. NMDA receptor expression was inversely proportional to retinal ganglion cell density in the treatment group.

Melatonin ameliorates retinal ganglion cell senescence and apoptosis in a SIRT1-dependent manner in an optic nerve injury model

Melatonin is involved in exerting protective effects in aged-related and neurodegenerative diseases through a silent information regulator type 1 (SIRT1)-dependent pathway. However, little was known about the impact of melatonin on retinal ganglion cell (RGC) senescence and apoptosis following optic nerve crush (ONC). Thus, this study aimed to examine the effects of melatonin on RGC senescence and apoptosis after ONC and investigate the involvement of SIRT1 in this process. To study this, an ONC model was established. EX-527, an inhibitor of SIRT1, was injected intraperitoneally into mice. And melatonin was administrated abdominally into mice after ONC every day. Hematoxylin & eosin staining, retina flat-mounts and optical coherence tomography were used to evaluate the loss of retina cells/neurons. Pattern electroretinogram (p-ERG) was performed to evaluate the function of RGCs. Immunofluorescence and western blot were used to evaluate protein expression. SA-β-gal staining was employed to detect senescent cells. The results demonstrated that melatonin partially rescued the expression of SIRT1 in RGC 3 days after ONC. Additionally, melatonin administration partly rescued the decreased RGC number and ganglion cell complex thickness observed 14 days after ONC. Melatonin also suppressed ONC-induced senescence and apoptosis index. Furthermore, p-ERG showed that melatonin improved the amplitude of P50, N95 and N95/P50 following ONC. Importantly, the protective effects of melatonin were reversed when EX-527 was administered. In summary, this study revealed that melatonin attenuated RGC senescence and apoptosis through a SIRT1-dependent pathway after ONC. These findings provide valuable insights for the treatment of RGC senescence and apoptosis.

Dysregulation of the circRNA_0087207/miR-548c-3p/PLSR1-TGFB2 axis in Leber hereditary optic neuropathy in vitro.

Leber hereditary optic neuropathy (LHON) is mainly the degeneration of retinal ganglion cells (RGCs) associated with high apoptosis and reactive oxygen species (ROS) levels, which is accepted to be caused by the mutations in the subunits of complex I of the mitochondrial electron transport chain. The treatment is still infant while efforts of correcting genes or using antioxidants do not bring good and consistent results. Unaffected carrier carries LHON mutation but shows normal phenotype, suggesting that the disease's pathogenesis is complex, in which secondary factors exist and cooperate with the primary complex I dysfunction. Using LHON patient-specific induced pluripotent stem cells (iPSCs) as the in vitro disease model, we previously demonstrated that circRNA_0087207 had the most significantly higher expression level in the LHON patient-iPSC-derived RGCs compared with the unaffected carrier-iPSC-derived RGCs. To elaborate the underlying pathologies regulated by circRNA_008720 mechanistically, bioinformatics analysis was conducted and elucidated that circRNA_0087207 could act as a sponge of miR-548c-3p and modulate PLSCR1/TGFB2 levels in ND4 mutation-carrying LHON patient-iPSC-derived RGCs. Using LHON iPSC-derived RGCs as the disease-based platform, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on targeted mRNA of miR-548c-3p showed the connection with apoptosis, suggesting downregulation of miR548c-3p contributes to the apoptosis of LHON patient RGCs. We showed that the downregulation of miR548c-3p plays a critical role in modulating cellular dysfunction and the apoptotic program of RGCs in LHON.

Discovery of astragaloside IV against high glucose-induced apoptosis in retinal ganglion cells: Bioinformatics and in vitro studies

ObjectiveTo examine the therapeutic mechanism of astragaloside IV (AS-IV) in the management of retinal ganglion cell (RGC) injury induced by high glucose (HG), a comprehensive approach involving the integration of network pharmacology and conducting in vitro and in vivo experiments was utilized. MethodsA rat model of diabetic retinopathy (DR) injury was created by administering streptozotocin through intraperitoneal injection. Additionally, a model of RGC injury induced by HG was established using a glucose concentration of 0.3 mmol/mL. Optical coherence tomography (OCT) images were captured 8 weeks after the injection of AS-IV. AS-IV and FBS were added to the culture medium and incubated for 48 h. The viability of cells was assessed using a CCK-8 assay, while the content of reactive oxygen species (ROS) was measured using DCFH-DA. Apoptosis was evaluated using Annexin V-PI. To identify the targets of AS-IV, hyperglycemia, and RGC, publicly available databases were utilized. The Metascape platform was employed for conducting GO and KEGG enrichment analyses. The STRING database in conjunction with Cytoscape 3.7.2 was used to determine common targets of protein–protein interactions (PPIs) and to identify the top 10 core target proteins in the RGC based on the MCC algorithm. qRT-PCR was used to measure the mRNA expression levels of the top10 core target proteins in RGCs. ResultsOCT detection indicated that the thickness of the outer nucleus, and inner and outer accessory layers of the retina increased in the AS-IV treated retina compared to that in the DM group but decreased compared to that in the CON group. Coculturing RGC cells with AS-IV after HG induction resulted in a significant increase in cell viability and a decrease in ROS and apoptosis, suggesting that AS-IV can reduce damage to RGC cells caused by high glucose levels by inhibiting oxidative stress. There were 14 potential targets of AS-IV in the treatment of RGC damage induced by high glucose levels. The top 10 core target proteins identified by the MCC algorithm were HIF1α, AKT1, CTNNB1, SMAD2, IL6, SMAD3, IL1β, PPARG, TGFβ1, and NOTCH3. qRT-PCR analysis showed that AS-IV could upregulate the mRNA expression levels of SMAD3, TGF-β1, and NOTCH3, and downregulate the mRNA expression levels of HIF1α, AKT1, CTNNB1, SMAD2, SMAD3, and IL-1β in high glucose-induced RGC cells. ConclusionThe findings of this study validate the efficacy of astragaloside IV in the treatment of DR and shed light on the molecular network involved. Specifically, HIF1α, AKT1, CTNNB1, SMAD2, SMAD3, and IL-1β were identified as the crucial candidate molecules responsible for the protective effects of astragaloside IV on RGCs.