Interleukin-4 protects retinal ganglion cells and promotes axon regeneration.

BACKGROUND: The preservation of retinal ganglion cells (RGCs) and the facilitation of axon regeneration are crucial considerations in the management of various vision-threatening disorders. Therefore, we investigate the efficacy of interleukin-4 (IL-4), a potential therapeutic agent, in promoting neuroprotection and axon regeneration of retinal ganglion cells (RGCs) as identified through whole transcriptome sequencing in an in vitro axon growth model. METHODS: A low concentration of staurosporine (STS) was employed to induce in vitro axon growth. Whole transcriptome sequencing was utilized to identify key target factors involved in the molecular mechanism underlying axon growth. The efficacy of recombinant IL-4 protein on promoting RGC axon growth was validated through in vitro experiments. The protective effect of recombinant IL-4 protein on somas of RGCs was assessed using RBPMS-specific immunofluorescent staining in mouse models with optic nerve crush (ONC) and N-methyl-D-aspartic acid (NMDA) injury. The protective effect on RGC axons was evaluated by anterograde labeling of cholera toxin subunit B (CTB), while the promotion of RGC axon regeneration was assessed through both anterograde labeling of CTB and immunofluorescent staining for growth associated protein-43 (GAP43). RESULTS: Whole-transcriptome sequencing of staurosporine-treated 661 W cells revealed a significant upregulation in intracellular IL-4 transcription levels during the process of axon regeneration. In vitro experiments demonstrated that recombinant IL-4 protein effectively stimulated axon outgrowth. Subsequent immunostaining with RBPMS revealed a significantly higher survival rate of RGCs in the rIL-4 group compared to the vehicle group in both NMDA and ONC injury models. Axonal tracing with CTB confirmed that recombinant IL-4 protein preserved long-distance projection of RGC axons, and there was a notably higher number of surviving axons in the rIL-4 group compared to the vehicle group following NMDA-induced injury. Moreover, intravitreal delivery of recombinant IL-4 protein substantially facilitated RGC axon regeneration after ONC injury. CONCLUSION: The recombinant IL-4 protein exhibits the potential to enhance the survival rate of RGCs, protect RGC axons against NMDA-induced injury, and facilitate axon regeneration following ONC. This study provides an experimental foundation for further investigation and development of therapeutic agents aimed at protecting the optic nerve and promoting axon regeneration.

Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

Embolism, hyperglycemia, high intraocular pressure-induced increased reactive oxygen species (ROS) production, and microglial activation result in endothelial/retinal ganglion cell death. Here, we conducted in vitro and in vivo ischemia/reperfusion (I/R) efficacy studies of a hybrid antioxidant-nitric oxide donor small molecule, SA-10, to assess its therapeutic potential for ocular stroke. METHODS: To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1ß and IFN-γ for 1 h, followed by the addition of SA-10 (10 µM). Inhibition of microglial activation, ROS scavenging, cytoprotective and anti-inflammatory activities were measured. In vivo I/R-injured mouse retinas were treated with either PBS or SA-10 (2%) intravitreally, and pattern electroretinogram (ERG), spectral-domain optical coherence tomography, flash ERG and retinal immunocytochemistry were performed. RESULTS: SA-10 significantly inhibited microglial activation and inflammation in vitro. Compared to the control, the compound SA-10 significantly attenuated cell death in both microglia (43% vs. 13%) and R28 cells (52% vs. 17%), decreased ROS (38% vs. 68%) production in retinal microglia cells, preserved neural retinal function and increased SOD1 in mouse eyes. CONCLUSION: SA-10 is protective to retinal neurons by decreasing oxidative stress and inflammatory cytokines.

Ca2+/Calmodulin-Dependent Protein Kinase II Enhances Retinal Ganglion Cell Survival But Suppresses Axon Regeneration after Optic Nerve Injury.

Neuroprotection after injury or in neurodegenerative disease remains a major goal for basic and translational neuroscience. Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of the exogenous expression of various Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in mice. A dramatic increase in RGC survival following the optic nerve trauma was elicited by the expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival. Notably, in a dose-escalation study, AAV-expressed CaMKII was more potent for axon growth suppression than the promotion of survival. That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of the optic nerve axon regeneration demonstrates the need for understanding the intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.

Light affects the prefrontal cortex via intrinsically photosensitive retinal ganglion cells.

The ventromedial prefrontal cortex (vmPFC) is a part of the limbic system engaged in the regulation of social, emotional, and cognitive states, which are characteristically impaired in disorders of the brain such as schizophrenia and depression. Here, we show that intrinsically photosensitive retinal ganglion cells (ipRGCs) modulate, through light, the integrity, activity, and function of the vmPFC. This regulatory role, which is independent of circadian and mood alterations, is mediated by an ipRGC-thalamic-corticolimbic pathway. Lack of ipRGC signaling in mice causes dendritic degeneration, dysregulation of genes involved in synaptic plasticity, and depressed neuronal activity in the vmPFC. These alterations primarily undermine the ability of the vmPFC to regulate emotions. Our discovery provides a potential light-dependent mechanism for certain PFC-centric disorders in humans.

Human adipose tissue-derived stem cell extracellular vesicles attenuate ocular hypertension-induced retinal ganglion cell damage by inhibiting microglia- TLR4/MAPK/NF-κB proinflammatory cascade signaling.

Microglia-mediated neuroinflammatory responses are recognized as a predominant factor during high intraocular pressure (IOP)-induced retinal and optic nerve injury along with potential therapeutic targets for the disease. Our previous research indicated that mesenchymal stem cell (MSC) treatment could reduce high IOP-induced neuroinflammatory responses through the TLR4 pathway in a rat model without apparent cell replacement and differentiation, suggesting that the anti-neuroinflammatory properties of MSCs are potentially mediated by paracrine signaling. This study aimed to evaluate the anti-neuroinflammatory effect of human adipose tissue-derived extracellular vesicles (ADSC-EVs) in microbead-induced ocular hypertension (OHT) animals and to explore the underlying mechanism since extracellular vesicles (EVs) are the primary transporters for cell secretory action. The anti-neuroinflammatory effect of ADSC-EVs on LPS-stimulated BV-2 cells in vitro and OHT-induced retinal and optic nerve injury in vivo was investigated. According to the in vitro research, ADSC-EV treatment reduced LPS-induced microglial activation and the TLR4/NF-κB proinflammatory cascade response axis in BV-2 cells, such as CD68, iNOS, TNF-α, IL-6, and IL-1ß, TLR4, p-38 MAPK, NF-κB. According to the in vivo data, intravitreal injection of ADSC-EVs promoted RGC survival and function, reduced microglial activation, microglial-derived neuroinflammatory responses, and TLR4/MAPK/NF-κB proinflammatory cascade response axis in the OHT mice. Our findings provide preliminary evidence for the RGC protective and microglia-associated neuroinflammatory reduction effects of ADSC-EVs by inhibiting the TLR4/MAPK/NF-κB proinflammatory cascade response in OHT mice, indicating the therapeutic potential ADSC-EVs or adjunctive therapy for glaucoma.

Exploring Epigenetic Modifications as Potential Biomarkers and Therapeutic Targets in Glaucoma.

Glaucoma, a complex and multifactorial neurodegenerative disorder, is a leading cause of irreversible blindness worldwide. Despite significant advancements in our understanding of its pathogenesis and management, early diagnosis and effective treatment of glaucoma remain major clinical challenges. Epigenetic modifications, encompassing deoxyribonucleic acid (DNA) methylation, histone modifications, and non-coding RNAs, have emerged as critical regulators of gene expression and cellular processes. The aim of this comprehensive review focuses on the emerging field of epigenetics and its role in understanding the complex genetic and molecular mechanisms underlying glaucoma. The review will provide an overview of the pathophysiology of glaucoma, emphasizing the intricacies of intraocular pressure regulation, retinal ganglion cell dysfunction, and optic nerve damage. It explores how epigenetic modifications, such as DNA methylation and histone modifications, can influence gene expression, and how these mechanisms are implicated in glaucomatous neurodegeneration and contribute to glaucoma pathogenesis. The manuscript discusses evidence from both animal models and human studies, providing insights into the epigenetic alterations associated with glaucoma onset and progression. Additionally, it discusses the potential of using epigenetic modifications as diagnostic biomarkers and therapeutic targets for more personalized and targeted glaucoma treatment.

Role of PKN1 in Retinal Cell Type Formation.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1-/- retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

The Role of Complement Dysregulation in Glaucoma.

Glaucoma is a progressive neurodegenerative disease characterized by damage to the optic nerve that results in irreversible vision loss. While the exact pathology of glaucoma is not well understood, emerging evidence suggests that dysregulation of the complement system, a key component of innate immunity, plays a crucial role. In glaucoma, dysregulation of the complement cascade and impaired regulation of complement factors contribute to chronic inflammation and neurodegeneration. Complement components such as C1Q, C3, and the membrane attack complex have been implicated in glaucomatous neuroinflammation and retinal ganglion cell death. This review will provide a summary of human and experimental studies that document the dysregulation of the complement system observed in glaucoma patients and animal models of glaucoma driving chronic inflammation and neurodegeneration. Understanding how complement-mediated damage contributes to glaucoma will provide opportunities for new therapies.

Nfe2l3 promotes neuroprotection and long-distance axon regeneration after injury in vivo.

Nuclear factor erythroid 2 like (Nfe2l) gene family members 1-3 mediate cellular response to oxidative stress, including in the central nervous system (CNS). However, neuronal functions of Nfe2l3 are unknown. Here, we comparatively evaluated expression of Nfe2l1, Nfe2l2, and Nfe2l3 in singe cell RNA-seq (scRNA-seq)-profiled cortical and retinal ganglion cell (RGC) CNS projection neurons, investigated whether Nfe2l3 regulates neuroprotection and axon regeneration after CNS injury in vivo, and characterized a gene network associated with Nfe2l3 in neurons. We showed that, Nfe2l3 expression transiently peaks in developing immature cortical and RGC projection neurons, but is nearly abolished in adult neurons and is not upregulated after injury. Furthermore, within the retina, Nfe2l3 is enriched in RGCs, primarily neonatally, and not upregulated in injured RGCs, whereas Nfe2l1 and Nfe2l2 are expressed robustly in other retinal cell types as well and are upregulated in injured RGCs. We also found that, expressing Nfe2l3 in injured RGCs through localized intralocular viral vector delivery promotes neuroprotection and long-distance axon regeneration after optic nerve injury in vivo. Moreover, Nfe2l3 provided a similar extent of neuroprotection and axon regeneration as viral vector-targeting of Pten and Klf9, which are prominent regulators of neuroprotection and long-distance axon regeneration. Finally, we bioinformatically characterized a gene network associated with Nfe2l3 in neurons, which predicted the association of Nfe2l3 with established mechanisms of neuroprotection and axon regeneration. Thus, Nfe2l3 is a novel neuroprotection and axon regeneration-promoting factor with a therapeutic potential for treating CNS injury and disease.

SOX9 depletion attenuates retinal ganglion cell ferroptosis through blocking ERK/p38 signaling.

BACKGROUND: Retinal ischemia-refusion (I/R) is a leading cause of irreversible blindness worldwide. This study aims to explore the regulatory role of SOX9 in retinal I/R injury, and attempts to elucidate its potential regulatory mechanism. METHODS: Retinal I/R injury model was established in vivo, and the histological changes was examined by hematoxylin and eosin (H&E) staining and immunofluorescent assay was performed to examine SOX9 expression. Oxygenation-glucose deprivation/reoxygenation (OGD/R)-induced retinal ischemia/reperfusion (I/R) injury in 661 W cells was constructed as an in vitro cellular model of glaucoma. The production of cytokines, lactate dehydrogenase (LDH) and the antioxidant enzymes were assessed by their commercial kits. Cellular reactive oxygen species (ROS) and lipid ROS was detected using DCFH-DA and C11-BODIPY 581/591 staining, respectively. Lipid peroxidation and Fe2+ level were detected to assess the ferroptosis level. Protein expression was examined by western blot. LM22B-10, the agonist of ERK signaling, was used to pretreat 661 W cells for mechanism investigation. RESULTS: SOX9 was aberrantly upregulated following retinal I/R injury both in vivo and in vitro. SOX9 knockdown exerted a protective role against OGD/R-triggered oxidative stress, inflammatory response and ferroptosis in 661 W cells. Further, ERK/p38 signaling was activated in 661 W cells following OGD/R induction, which was repressed by SOX9 knockdown, and the ERK signaling agonist partially counteracted the protective role of SOX9 knockdown against oxidative stress, inflammatory response and ferroptosis in OGD/R-induced 661 W cells. CONCLUSION: Collectively, inhibiting SOX9 to block oxidative stress, inflammation and ferroptosis by inactivating ERK/p38 signaling might be effective to prevent retinal I/R injury, thereby alleviating glaucoma.

Loss of Sarm1 reduces retinal ganglion cell loss in chronic glaucoma.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

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-ß-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.

Retinal response to systemic inflammation differs between sexes and neurons.

Background: 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. Methods: 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. Results: 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-1ß. 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. Conclusions: 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.

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

OBJECTIVE: To 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. METHODS: A 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. RESULTS: OCT 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, IL1ß, PPARG, TGFß1, and NOTCH3. qRT-PCR analysis showed that AS-IV could upregulate the mRNA expression levels of SMAD3, TGF-ß1, and NOTCH3, and downregulate the mRNA expression levels of HIF1α, AKT1, CTNNB1, SMAD2, SMAD3, and IL-1ß in high glucose-induced RGC cells. CONCLUSION: The 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-1ß were identified as the crucial candidate molecules responsible for the protective effects of astragaloside IV on RGCs.

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 ß-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.

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.

Ciliary neurotrophic factor mediated growth of retinal ganglion cell axons on PGS/PCL scaffolds.

Ciliary neurotrophic factor (CNTF) promotes survival and/or differentiation of a variety of neuronal cells including retinal ganglion cells (RGCs). Delivery of CNTF requires a suitable medium capable of mediating diffusion and premature release of CNTF within the target tissue. Polymeric tissue-engineered scaffolds have been readily used as substrates for cell transplantation, expansion, and differentiation and, as carriers of cell growth factors. Their functions to CNTF release for RGC proliferation have remained so far unexplored, especially to CNTF affinity to the scaffold and subsequent RGC fate. Electrospunpoly(glycerol sebacate)/poly(ϵ-caprolactone) (PGS/PCL) biopolymer scaffolds have recently shown promising results in terms of supporting regeneration of RGC neurites. This work explores covalent immobilization of CNTF on PGS/PCL scaffold and the way immobilised CNTF mediates growth of RGC axons on the scaffold. Anex-vivothree-dimensional model of rodent optic nerve on PGS/PCL revealed that RGC explants cultured in CNTF mediated environment increased their neurite extensions after 20 d of cell culture employing neurite outgrowth measurements. The CNTF secretion on PGS/PCL scaffold was found bio-mimicking natural extracellular matrix of the cell target tissue and, consequently, has shown a potential to improve the overall efficacy of the RGC regeneration process.

Identification of circular RNA-Dcaf6 as a therapeutic target for optic nerve crush-induced RGC degeneration.

The death of retinal ganglion cells (RGCs) can cause irreversible injury in visual function. Clarifying the mechanism of RGC degeneration is critical for the development of therapeutic strategies. Circular RNAs (circRNAs) are important regulators in many biological and pathological processes. Herein, we performed circRNA microarrays to identify dysregulated circRNAs following optic nerve crush (ONC). The results showed that 221 circRNAs were differentially expressed between ONC retinas and normal retinas. Notably, the levels of circular RNA-Dcaf6 (cDcaf6) expression in aqueous humor of glaucoma patients were higher than that in cataract patients. cDcaf6 silencing could reduce oxidative stress-induced RGC apoptosis in vitro and alleviate retinal neurodegeneration in vivo as shown by increased neuronal nuclei antigen (NeuN, neuronal bodies) and beta-III-tubulin (TUBB3, neuronal filaments) staining and reduced glial fibrillary acidic protein (GFAP, activated glial cells) and vimentin (activated glial cells) staining. Collectively, this study identifies a promising target for treating retinal neurodegeneration.

A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

BAFF deficiency aggravated optic nerve crush-induced retinal ganglion cells damage by regulating apoptosis and neuroinflammation via NF-κB-IκBα signaling.

Loss of retinal ganglion cells (RGCs) is a primary cause of visual impairment in glaucoma, the pathological process is closely related to neuroinflammation and apoptosis. B-cell activating factor (BAFF) is a fundamental survival factor mainly expressed in the B cell lineage. Evidence suggests its neuroprotective effect, but the expression and role in the retina have not yet been investigated. In this study, we adopt optic nerve crush (ONC) as an in vivo model and oxygen-glucose deprivation/reoxygenation (OGD/R) of RGCs as an in vitro model to investigate the expression and function of BAFF. We found that BAFF and its receptors were abundantly expressed in the retina and BAFF inhibition exacerbated the caspase 3-mediated RGCs apoptosis, glial cell activation and pro-inflammatory cytokines expression, which may be caused by the activation of the NF-κB pathway in vivo. In addition, we found that BAFF treatment could alleviate RGCs apoptosis, pro-inflammatory cytokines expression and NF-κB pathway activation, which could be reversed the effect by blockade of the NF-κB pathway in vitro. Meanwhile, we found that microglia induced to overexpress BAFF in the inflammatory microenvironment in a time-dependent manner. Taken together, our results indicated that BAFF deficiency promoted RGCs apoptosis and neuroinflammation through activation of NF-κB pathway in ONC retinas, suggesting that BAFF may serve as a promising therapeutic target for the treatment of glaucoma.