Degeneration Of Retinal Ganglion Cells Research Articles

Role of antioxidants in glaucoma medical treatment

AbstractGlaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells. Oxidative stress has been related to the cell death in this disease. Theoretically, this deleterious consequence can be reduced by antioxidants substances. This presentation intends to update the knowledge about oxidative stress mechanisms in relation with glaucoma and the effect of antioxidant coadjuvant therapy on this disease.

Correlations between visual function and inner retinal structure in OPA1 autosomal dominant optic atrophy

AbstractPurpose: To evaluate the influence of autosomal dominant optic atrophy (ADOA) on visual function and inner retinal layer thickness in patients harbouring a pathogenic variant in the OPA1 gene.Methods: We retrospectively investigated clinical data of 59 patients (118 eyes) with ADOA and genetically confirmed OPA1 variants. Visual function was evaluated by best corrected visual acuity (VA), and by mean deviation (MD) of automated static threshold perimetry. Macular ganglion cell layer (mGCL) volume and peripapillary retinal nerve fibre layer (pRNFL) thickness were measured by optical coherence tomography. The analysis was performed for all available data from both eyes in the whole sample. Furthermore, patients were stratified in 2 groups based on variant type (haploinsufficiency: 86 eyes, missense variants: 14 eyes).Results: The mean age at examination was 30.5 ± 16.1 years, VA was 0.55 ± 0.33 logMAR, MD was −4.73 ± 3.59 dB, mGCL volume was 0.60 ± 0.10 mm3, and pRNFL thickness was 63.8 ± 10.6 μm. VA correlated significantly with mGCL volume (p < 0.001) and with pRNFL thickness (p = 0.021). MD also correlated with pRNFL thickness (p = 0.017). Patient age was associated with mGCL volume (p < 0.001) and pRNFL thickness (p = 0.024), but not with VA or MD. MD was significantly lower in the missense group than in the haploinsufficiency group (−9.31 ± 4.07 dB vs. −4.03 ± 2.85 dB; p < 0.001), whereas VA did not differ between groups. Thickness of the pRNFL was also lower in the patients with missense variants, but only the nasal sector was significantly thinner (48.2 ± 12.8 μm vs. 58.2 ± 19.2 μm; p = 0.035).Conclusions: Visual function and inner retinal structure are closely associated in ADOA patients with OPA1 variants. Increasing age may be accompanied by a continuing degeneration of retinal ganglion cells, but seems to have less effect on further loss of visual function. Patients carrying missense variants show more pronounced optic atrophy and visual field defect.

Autophagy and retinal ganglion cell death in glaucoma: Friend or foe?

AbstractGlaucoma, a leading cause of irreversible blindness worldwide, is an age‐related neurodegenerative disease characterized by progressive alterations of the optic nerve head and degeneration of retinal ganglion cells (RGCs). Although elevated intraocular pressure (IOP) has been identified as the main and the only modifiable risk factor, the pathogenesis of the disease is still unclear. Indeed, lowering IOP is often insufficient to prevent the damage progression leaving unmet the therapeutic need of IOP‐independent pharmacological targets.Autophagy, and the more selective mitophagy, are responsible for breaking down cellular components, preserving cellular homeostasis and preventing the accumulation of altered and aggregated proteins and damaged organelles.Several evidence have shown that modulation of autophagy is a recurrent and functional response of RGCs to IOP‐dependent or independent glaucoma‐related insults. However, in RGCs, autophagy has been found to take part to either neuroprotection as well as neuronal death, depending on the experimental setting (i.e. animal model, timing of the experiments, drugs/doses used to modulate the pathway).Here we describe the dynamic changes of autophagy flux and the modulation of mitophagy related proteins in a model of acute glaucoma induced in C57BL/6J mice by transient elevation of intraocular pressure (IOP). We also report and discuss experimental evidence supporting the pro‐survival role of autophagy and mitophagy induction in mice subjected to ocular hypertension suggesting the possibility to targeting autophagy to achieve retinal neuroprotection in glaucoma.

ELAVL1/HuR regulates both endo‐ and exogenous neuroprotection in retinal ganglion cells

AbstractPurpose: To evaluate the impact of HuR gene silencing on the ratio of age‐related degeneration of Retinal Ganglion Cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of HuR‐ silenced RGC in rat glaucoma model treated with Metallothionein.Methods: Thirty‐five eight‐week‐old male Long Evans rats were divided into two groups: experimental and control. Animals from experimental group received intravitreal injection of AAV‐shRNA‐HuR. Control group received injection of AAV‐shRNA scramble control. Animals from both groups were sacrificed in 3 different time points: 2, 4 and 6 months after injection. Both healthy and treated retinas from each animal were collected and processed as whole mounts for immunostainings and RGC count. For the second trial, animals were divided into groups and transfected with virus particles as described above. To induce chronic glaucoma model, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Half of animals from each group received intravitreal injection of Metallothionein (MT), the other group received PBS injection. During the experiment IOP was monitored. Both healthy and treated retinas from each animal were collected and processed as whole mounts for immunostainings and RGC count.Results: RGC count (per visual field under 20× magnification) was 310 ± 31, 296 ± 25, 189 ± 41 in experimental group and 399 ± 51, 395 ± 49, 390 ± 23 in control respectively for 2, 4 and 6 months after injection (Kaplan‐Mayer trend rank p < 0.0001). Loss of RGC in central retina was 33.7% in animals from shRNA‐MT‐treated glaucoma and 11.4% in shRNA control‐MT‐treated glaucoma (p < 0.05). In peripheral part of the retina the loss was 37.4% in animals from shRNA‐MT‐treated glaucoma and 11.5% in shRNA control‐MT‐treated glaucoma (p < 0.01).Conclusions: Silencing of hur gene enhanced age‐related loss of RGC which translates into impaired endogenous neuroprotection pathways, moreover the absence of HuR critically limits neuroprotective activity of exogenously delivered Metallothionein.

Retrograde Optic Nerve Degeneration in Pituitary Adenoma: A Study with RE-PERG.

RE-PERG is altered in presence of primary neuronal degeneration of retinal ganglion cells, both in glaucoma and other diseases. A previous study showed that in a model of retrograde degeneration (vascular dementia) RE-PERG was normal. In this study, we enrolled patients with pituitary adenoma (PA) to evaluate RE-PERG findings in another model of retrograde degeneration compared with healthy controls (HC). Based on the outcome of the present and our previous studies with RE-PERG, and reviewing the literature, we discuss the physiopathology of glaucoma. Twelve PA patients and 14 age-matched HC were recruited. All participants performed visual field (VF) test, retinal nerve fiber layer (RNFL) and ganglion cells complex (GCC) thickness measurement by means of optical coherence tomography (OCT), visual evoked potentials (VEPs) and RE-PERG, a non-invasive, fast steady-state pattern electroretinogram (SS-PERG) sampled in five consecutive blocks of 130 events. VEPs amplitude was significantly lower in PA with respect to HC (6.8±0.6 vs 7.4±0.6 µV; p=0.045). VEPs latency was higher in PA (123.2±5.8 vs 103.6±4.1 msec; p<0.01). As for VF, mean defect (MD) and pattern standard deviation (PSD) were higher in PA (-6.6±2.6 vs -0.01±1.02 dB; p<0.01 and 8.5±3.1 vs 1.5±0.3; p<0.01, respectively). RNFL thickness was lower in PA (88±8.1 vs 97±9.3 µ; p=0.01). There was no statistically significant difference between PA and HC for RE-PERG. There was a significant correlation among MD, PSD, VEPs amplitude, PERG amplitude and RNFL thickness in the PA group, whereas no correlation was found with SDPh, which remains as normal as in the HC group. Our findings confirm that RE-PERG is not altered in retrograde degeneration. Based on the outcome of the present and our previous studies about RE-PERG and glaucoma, we assume that in glaucoma a double mechanism of retinal ganglion cells degeneration, both retrograde and primary, can coexist.

σ2R/TMEM97 in retinal ganglion cell degeneration

The sigma 2 receptor (σ2R) was recently identified as an endoplasmic reticulum (ER) membrane protein known as transmembrane protein 97 (TMEM97). Studies have shown that σ2R/TMEM97 binding compounds are neuroprotective, suggesting a role of σ2R/TMEM97 in neurodegenerative processes. To understand the function of σ2R/TMEM97 in neurodegeneration pathways, we characterized ischemia-induced retinal ganglion cell (RGC) degeneration in TMEM97−/− mice and found that RGCs in TMEM97−/− mice are resistant to degeneration. In addition, intravitreal injection of a selective σ2R/TMEM97 ligand DKR-1677 significantly protects RGCs from ischemia-induced degeneration in wildtype mice. Our results provide conclusive evidence that σ2R/TMEM97 plays a role to facilitate RGC death following ischemic injury and that inhibiting the function of σ2R/TMEM97 is neuroprotective. This work is a breakthrough toward elucidating the biology and function of σ2R/TMEM97 in RGCs and likely in other σ2R/TMEM97 expressing neurons. Moreover, these findings support future studies to develop new neuroprotective approaches for RGC degenerative diseases by inhibiting σ2R/TMEM97.

Adeno‐associated virus (AAV) knock‐down of amyloid precursor protein attenuates retinal ganglion cell degeneration in experimental glaucoma

AbstractPurpose: Retinal ganglion cell (RGC) degeneration and visual field loss are distinctive features of primary open angle glaucoma. Similar pathologies have been observed in Alzheimer's Disease patients, concomitant to amyloid‐β aggregation. Here, we investigated the effect of amyloid precursor protein (APP) modulation in the retina and examined the potential neuroprotective effects of APP knock‐down (APP‐KD) in experimental glaucoma.Methods: AAV‐PHP.eB constructs for APP‐KD (NM_000484) and positive scramble controls were transfected in SH‐SY5Y neuroblastoma cells to validate their expression. Wild‐type C57BL/6J mice (n = 30) were then transduced with APP‐KD (n = 10) or scramble (n = 10) vectors intravenously. Experimental chronic glaucoma was induced via weekly intraocular microbead injections for 8 weeks. Inner retinal function was assessed using positive scotopic threshold response (pSTR) amplitudes. Structural RGC changes were quantified using haematoxylin and eosin staining and manual cell count.Results: All microbead‐injected eyes showed significantly increased intraocular pressure (Mean ± SEM; Microbead vs. Control; 27.98 ± 0.78 mmHg vs. 10.01 ± 0.13 mmHg). In glaucomatous conditions, there was significant preservation of the RGC layer in APP‐KD treated eyes, compared to scramble and microbead‐only eyes (p &lt; 0.002) (Mean ± SEM; Microbead/APP‐KD vs. Microbead/Scramble vs. Microbead‐only; 41.57 ± 1.61 vs. 37.73 ± 1.50 vs. 35.43 ± 0.95 cells/300 μm). However, there was no significant changes in pSTR amplitudes of glaucomatous APP‐KD eyes, compared to scramble and microbead‐only eyes (p = 0.653).Conclusions: AAV‐PHP.eB mediated APP‐KD confers significant protection of the RGC layer in glaucomatous eyes, however this protection was not sustained functionally. The molecular mechanisms by which APP‐KD may impart neuroprotection and ameliorate APP neurotoxicity are unclear. Further studies will include biochemical evaluation of synaptic and cellular stress markers.

Retinal glial activation in the contralateral eyes in a mouse model of unilateral laser‐induced glaucoma

AbstractGlaucoma is a neurodegenerative chronic pathology, characterized by the loss of retinal ganglion cells (RGC), which leads to an irreversible vision field loss. The glaucomatous optic neuropathy is usually a bilateral disease, though asymmetric in time: this means that the damage initially present in one of the eyes, eventually also appears with a time delay in the contralateral eye. Notably, recent experimental models of unilateral ocular hypertension (OHT) have shown that the fellow normotensive contralateral eyes to hypertensive eyes exhibit changes in glial cells with little or no damage to RGCs.It has been reported that although increased intraocular pressure (IOP) may be the main trigger for this pathology, other pathogenic mechanisms such as neuroinflammation are involved in the RGCs death. Microglial cells constitute the population of immune resident cells, and they play an important role in the physiology and survival of RGC.One of the most widely used experimental models of glaucoma is the unilateral laser‐induced OHT model. In these animals, OHT induces an alteration of retrograde axonal transport in RGCs and a subsequent degeneration of these cells. In this model, at different time‐points after OHT induction (1, 3, 5, 8 and 15 days), the microglia exhibited different signs of activation, such as proliferation and migration to areas of damage, retraction, reorientation, and hyper‐ramification of their processes, soma thickening and the appearance of amoeboid‐ and rod‐like microglia with macrophagic capacity, related to RGC degeneration. All these changes were most intense at 3–5 days after OHT induction in the OHT eye. This was confirmed by the decreased expression of the purinergic receptor P2RY12, which would indicate higher microglial activation.In this model, at all time‐points after OHT induction, the activation of the macroglia (astrocytes and Müller cells) has also been demonstrated, showing morphological changes, as well as an overexpression of glial fibrillary acidic protein (GFAP).In this laser‐induced OHT model, an MHC‐II overexpression was observed in the microglia, astrocytes, and Müller cells, allowing these cells to induce a T‐cell mediated immune response.Significant changes in the expression of proinflammatory (IL‐1β, IL‐6, INF‐γ, IL‐17), anti‐inflammatory (IL‐4, IL‐10) and myokines (Fractalkine, BDNF, VEGF) cytokines were also observed.The same changes in glial cells (microglia, astrocytes and Müller cells) as well as in the expression of different cytokines and myokines were observed in the contralateral normotensive eyes at the different time points after OHT induction although with a lower intensity.Based on the alterations found in the contralateral eye, it can be concluded that this eye should not be used as a control eye in experimental models of unilateral glaucoma. Changes in contralateral normotensive eyes could exhibit the initial events of glaucomatous neurodegeneration, probably mediated by inflammatory mechanisms. Therefore, studies focusing on contralateral eyes could improve our understanding of glaucomatous pathophysiology. The study of the contralateral eye appears to have the potential for discovering points of intervention to which future neuroprotective therapeutics can be directed.

Microglia: Friends or Foes in Glaucoma? A Developmental Perspective.

Glaucoma is the most prevalent form of optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to irreversible loss of vision. The mechanism underlying glaucomatous degeneration remains poorly understood. However, evidence suggests that microglia, which regulate RGC numbers and synaptic integrity during development and provide homeostatic support in adults, may contribute to the disease process. Hence, microglia represent a valid cellular target for therapeutic approaches in glaucoma. Here, we provide an overview of the role of microglia in RGC development and degeneration in the backdrop of neurogenesis and neurodegeneration in the central nervous system and discuss how pathological recapitulation of microglia-mediated developmental mechanisms may help initiate or exacerbate glaucomatous degeneration.

Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.

The heterogeneity of astrocytes in glaucoma.

Glaucoma is a leading cause of blindness with progressive degeneration of retinal ganglion cells. Aging and increased intraocular pressure (IOP) are major risk factors. Lowering IOP does not always stop the disease progression. Alternative ways of protecting the optic nerve are intensively studied in glaucoma. Astrocytes are macroglia residing in the retina, optic nerve head (ONH), and visual brain, which keep neuronal homeostasis, regulate neuronal activities and are part of the immune responses to the retina and brain insults. In this brief review, we discuss the activation and heterogeneity of astrocytes in the retina, optic nerve head, and visual brain of glaucoma patients and animal models. We also discuss some recent transgenic and gene knockout studies using glaucoma mouse models to clarify the role of astrocytes in the pathogenesis of glaucoma. Astrocytes are heterogeneous and play crucial roles in the pathogenesis of glaucoma, especially in the process of neuroinflammation and mitochondrial dysfunction. In astrocytes, overexpression of Stat3 or knockdown of IκKβ/p65, caspase-8, and mitochondrial uncoupling proteins (Ucp2) can reduce ganglion cell loss in glaucoma mouse models. Based on these studies, therapeutic strategies targeting the heterogeneity of reactive astrocytes by enhancing their beneficial reactivity or suppressing their detrimental reactivity are alternative options for glaucoma treatment in the future.

Astrocytic dysfunction induced by ABCA1 deficiency causes optic neuropathy.

Astrocyte abnormalities have received great attention for their association with various diseases in the brain but not so much in the eye. Recent independent genome-wide association studies of glaucoma, optic neuropathy characterized by retinal ganglion cell (RGC) degeneration, and vision loss found that single-nucleotide polymorphisms near the ABCA1 locus were common risk factors. Here, we show that Abca1 loss in retinal astrocytes causes glaucoma-like optic neuropathy in aged mice. ABCA1 was highly expressed in retinal astrocytes in mice. Thus, we generated macroglia-specific Abca1-deficient mice (Glia-KO) and found that aged Glia-KO mice had RGC degeneration and ocular dysfunction without affected intraocular pressure, a conventional risk factor for glaucoma. Single-cell RNA sequencing revealed that Abca1 deficiency in aged Glia-KO mice caused astrocyte-triggered inflammation and increased the susceptibility of certain RGC clusters to excitotoxicity. Together, astrocytes play a pivotal role in eye diseases, and loss of ABCA1 in astrocytes causes glaucoma-like neuropathy.

Microglia activation in the hippocampus mediates retinal degeneration-induced depressive-like behaviors via the NLRP3/IL-1β pathway

Epidemiological studies have shown that patients with glaucoma are more prone to depression, but the mechanism of comorbid depression in patients with glaucoma remains unknown. Excessive neuroinflammation has been shown to participate in glaucoma-induced retinal degeneration and hippocampal neural apoptosis in depression. However, little research has been conducted to determine whether neuroinflammation contributes to glaucoma-induced depression. Since the degeneration of retinal ganglion cells is a hallmark of glaucoma, we investigated the role of microglia-induced neuroinflammation in retinal degeneration-induced depression and its potential mechanism. An N-methyl-D-aspartate (NMDA)-induced retinal degeneration model was established, and behavioral tests were conducted at 3, 7, 14, and 21 days after retinal degeneration. After tissue collection, we used immunohistochemistry to assess the activation of microglia and real-time polymerase chain reaction to measure the levels of pro-inflammatory cytokines and the NOD-, LRR-, and pyrin-domain containing protein 3 (NLRP3) inflammasome. The mice exhibited depressive-like behaviors 14 and 21 days after retinal degeneration, based on the open field test, tail suspension test, and forced swimming test. Mice also displayed a lower body weight gain than the control group. In addition, microglial activation was observed in the hippocampus. Microglial proliferation was first observed in the dentate gyrus on day 3, while the number of microglia in cornu ammonis 1 grew the most. Moreover, not only was the expression of pro-inflammatory cytokines, including interleukin-1β, interleukin-18, and interleukin-6 promoted, but the messenger ribonucleic acid levels of the NLRP3 inflammasome were also increased. In conclusion, our research shows that NMDA-induced retinal degeneration can induce depressive-like behaviors, which may be attributed to hippocampal neuroinflammation.

The top 10 most frequently involved genes in hereditary optic neuropathies in 2186 probands.

Hereditary optic neuropathies are caused by the degeneration of retinal ganglion cells whose axons form the optic nerves, with a consistent genetic heterogeneity. As part of our diagnostic activity, we retrospectively evaluated the combination of Leber hereditary optic neuropathy mutations testing with the exon sequencing of 87 nuclear genes on 2186 patients referred for suspected hereditary optic neuropathies. The positive diagnosis rate in individuals referred for Leber hereditary optic neuropathy testing was 18% (199/1126 index cases), with 92% (184/199) carrying one of the three main pathogenic variants of mitochondrial DNA (m.11778G>A, 66.5%; m.3460G>A, 15% and m.14484T>C, 11%). The positive diagnosis rate in individuals referred for autosomal dominant or recessive optic neuropathies was 27% (451/1680 index cases), with 10 genes accounting together for 96% of this cohort. This represents an overall positive diagnostic rate of 30%. The identified top 10 nuclear genes included OPA1, WFS1, ACO2, SPG7, MFN2, AFG3L2, RTN4IP1, TMEM126A, NR2F1 and FDXR. Eleven additional genes, each accounting for less than 1% of cases, were identified in 17 individuals. Our results show that 10 major genes account for more than 96% of the cases diagnosed with our nuclear gene panel.

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

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

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

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

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 μg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

The neuroprotective effect of melatonin in glutamate excitotoxicity of R28 cells and mouse retinal ganglion cells.

Glaucoma is the leading cause of irreversible blindness. The progressive degeneration of retinal ganglion cells (RGCs) is the major characteristic of glaucoma. Even though the control of intraocular pressure could delay the loss of RGCs, current clinical treatments cannot protect them directly. The overactivation of N-methyl-D-aspartic acid (NMDA) receptors by excess glutamate (Glu) is among the important mechanisms of RGC death in glaucoma progression. Melatonin (MT) is an indole neuroendocrine hormone mainly secreted by the pineal gland. This study aimed to investigate the therapeutic effect of MT on glutamate excitotoxicity of mouse RGCs and R28 cells. The Glu-induced R28 cell excitotoxicity model and NMDA-induced retinal injury model were established. MT was applied to R28 cells and the vitreous cavity of mice by intravitreal injection. Cell counting kit-8 assay and propidium iodide/Hoechst were performed to evaluate cell viability. Reactive oxygen species and glutathione synthesis assays were used to detect the oxidative stress state of R28 cells. Retina immunofluorescence and hematoxylin and eosin staining were applied to assess RGC counts and retinal structure. Flash visual-evoked potential was performed to evaluate visual function in mice. RNA sequencing of the retina was performed to explore the underlying mechanisms of MT protection. Our results found that MT treatment could successfully protect R28 cells from Glu excitotoxicity and decrease reactive oxygen species. Also, MT rescued RGCs from NMDA-induced injury and protected visual function in mice. This study enriches the indications of MT in the treatment of glaucoma, providing practical research ideas for its comprehensive prevention and treatment.

Hematogenous Macrophages Contribute to Fibrotic Scar Formation After Optic Nerve Crush.

Although glial scar formation has been extensively studied after optic nerve injury, the existence and characteristics of traumatic optic nerve fibrotic scar formation have not been previously characterized. Recent evidence suggests infiltrating macrophages are involved in pathological processes after optic nerve crush (ONC), but their role in fibrotic scar formation is unknown. Using wild-type and transgenic mouse models with optic nerve crush injury, we show that macrophages infiltrate and associate with fibroblasts in the traumatic optic nerve lesion fibrotic scar. We dissected the role of hematogenous and resident macrophages, labeled with Dil liposomes intravenously administered, and observed that hematogenous macrophages (Dil+ cells) specifically accumulate in the center of traumatic fibrotic scar while Iba-1+ cells reside predominantly at the margins of optic nerve fibrotic scar. Depletion of hematogenous macrophages results in reduced fibroblast density and decreased extracellular matrix deposition within the fibrotic scar area following ONC. However, retinal ganglion cell degeneration and function loss after optic nerve crush remain unaffected after hematogenous macrophage depletion. We present new and previously not characterized evidence that hematogenous macrophages are selectively recruited into the fibrotic core of the optic nerve crush site and critical for this fibrotic scar formation.

New strategies for neuro protection in glaucoma.

Glaucoma is a progressive, irreversible loss of retinal ganglion cells (RGCs) and axons that results in characteristic optic atrophy and corresponding progressive visual field defect. The exact mechanisms underlying glaucomatous neuron loss are not clear. The main risk factor for glaucoma onset and development is high intraocular pressure (IOP), however traditional IOP-lowering therapies are often not sufficient to prevent degeneration of RGCs and the vision loss may progress, indicating the need for complementary neuroprotective therapy. This review summarizes the progress for neuro protection in glaucoma in recent 5 years, including modulation of neuroinflammation, gene and cell therapy, dietary supplementation, and sustained-release system.