Primary Retinal Ganglion Cell Cultures Research Articles

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Effect of papaverine on axonal outgrowth of primary retinal ganglion cells of Sprague Dawley rats

Increasing the level of cyclic adenosine 3, 5′-monophosphate is an important mechanism for axon outgrowth and recovery of central nervous system function. This study aimed to investigate the effects of papaverine, a non-specific phosphodiesterase inhibitor, on axon outgrowth of primary retinal ganglion cells from Sprague Dawley rats. Experiments were performed on primary retinal ganglion cells extracted from Sprague Dawley rat pups within 48–72 h of birth. At 24 h after seeding, immunofluorescence was used to identify and calculate the purity of retinal ganglion cells isolated by an improved two-step immunopanning method developed by author Sujia Ma. The effects of a range of papaverine concentrations on axon outgrowth of primary retinal ganglion cells cultures were observed by immunofluorescence and measured by ImageJ software at three different time points: 24, 48, and 72 h. The ability of papaverine to enable retinal ganglion cells to overcome the inhibitory effects of glial scar component chondroitin sulfate proteoglycans was examined using chondroitin sulfate proteoglycans-coated culture plates. Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt, a blocking agent of cyclic adenosine 3, 5′-monophosphate, and dibutyryl cyclic adenosine 3, 5′-monophosphate, an analogue of cyclic adenosine 3, 5′-monophosphate, were used to explore the mechanism of papaverine in promoting retinal ganglion cells axon outgrowth. Our study shows 2 μg/mL papaverine concentration significantly promoted axon outgrowth in primary retinal ganglion cells and restored axon outgrowth of these cells on chondroitin sulfate proteoglycans. Axon outgrowth was blocked by Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt and obviously promoted by dibutyryl cyclic adenosine 3, 5′-monophosphate. Our study is the first to describe the use of papaverine to promote axon outgrowth of retinal ganglion cells. These results may help to expand the application of papaverine, and they provide a cytological basis for papaverine in the treatment of optic nerve injury caused by glaucoma and other diseases.

Increased bioavailability of cyclic guanylate monophosphate prevents retinal ganglion cell degeneration

The nitric oxide – guanylyl cyclase-1 – cyclic guanylate monophosphate (NO-GC-1–cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma – primary open angle glaucoma (POAG; GC-1−/− mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.

Impact of Graphene on the Efficacy of Neuron Culture Substrates.

How graphene influences the behavior of living cells or tissues remains a critical issue for its application in biomedical studies, despite the general acceptance that graphene is biocompatible. While direct contact between cells and graphene is not a requirement for all biomedical applications, it is often mandatory for biosensing. Therefore, it is important to clarify whether graphene impedes the ability of cells to interact with biological elements in their environment. Here, a systematic study is reported to determine whether applying graphene on top of matrix substrates masks interactions between these substrates and retinal ganglion cells (RGCs). Six different platforms are tested for primary RGC cultures with three platforms comprised of matrix substrates compatible with these neurons, and another three having a layer of graphene placed on top of the matrix substrates. The results demonstrate that graphene does not impede interactions between RGCs and underlying substrate matrix, such that their positive or negative effects on neuron viability and vitality are retained. However, direct contact between RGCs and graphene reduces the number, but increases basal activity, of functional cation channels. The data indicate that, when proper baselines are established, graphene is a promising biosensing material for in vitro applications in neuroscience.

Müller cells increase survival of retinal ganglion cells – a coculture model of primary retinal ganglion cells and primary Müller cells

PurposeMüller cells are considered to be vital in the maintenance of retinal ganglion cells (RGCs), and since RGCs are essential to maintain the neuronal function of the retina, a functioning symbiotic partnership between Müller cells and RGCs is fundamental. The present study evaluates glia‐neuron interactions in a coculture model of primary Müller cells and primary RGCs.MethodsTo investigate the Müller cell‐RGC interaction we developed a coculture model, in which primary Müller cells from mice were grown in inserts on top of pure primary RGC cultures likewise from mice. The impact of 24 h of starvation on the ability of Müller cells to protect RGCs was evaluated. Moreover, changes in glutamate uptake were studied in Müller cells as well as cell viability in response to starvation.ResultsThe presence of Müller cells significantly increased the survival of RGCs during normal conditions as well as during 24 h of pre‐starvation. Glutamate uptake capacity was significantly increased during starvation along with an increased Vmax. Starvation induced significantly reduced cell viability both in Müller cells and RGCs with a significantly greater reduction of cell viability in Müller cells.ConclusionsThe present study reveals an increased survival of RGCs in presence of Müller cells and herewith we confirm Müller cells as being key players in RGC homeostasis. Moreover, we verify the coculture model as being a unique approach to study the glia‐neuron partnership in inner retinal diseases.

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.

Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

Glia-Neuron Interactions in the Retina Can Be Studied in Cocultures of Müller Cells and Retinal Ganglion Cells.

Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller cells on RGC survival. To investigate the Müller cell/RGC interactions we developed a coculture model, in which primary Müller cells were grown in inserts on top of pure primary RGC cultures. The impact of starvation and mitochondrial inhibition on the Müller cell ability to protect RGCs was studied. Moreover, the ability of Müller cells to remove glutamate from the extracellular space was investigated. RGC survival was evaluated by cell viability assays and glutamate uptake was assessed by kinetic uptake assays. We demonstrated a significantly increased RGC survival in presence of untreated and prestarved Müller cells. Additionally, prestarved Müller cells significantly increased RGC survival after mitochondrial inhibition. Finally, we revealed a significantly increased ability to take up glutamate in starved Müller cells. Overall, our study confirms essential roles of Müller cells in RGC survival. We suggest that targeting Müller cell function could have potential for future treatment strategies to prevent blinding neurodegenerative retinal diseases.

Energy supply is critical for Müller cells ability to transport glutamate

AbstractPurpose To study the effect of energy deprivation in Müller cells ability to maintain their function as well as their ability to protect retinal ganglion cells (RGC).Methods The human Müller cell line, MIO‐M1 and primary mouse Müller cells were used to study changes in glutamate uptake, glutamate release, excitatory amino acid transporter (EAAT) protein expression, ATP levels and glycogen content, when cells were compromised from energy. Moreover, a co‐culture system of primary RGC cultures and primary Müller cells, separated by an insert, was used to evaluate the role of Müller cells in RGC survival.Results EAAT1 and EAAT2 proteins were up‐regulated in energydeprived Müller cells and glutamate uptake was significantly increased in the absence of glucose, as opposed to conditions with sodium depletion or in the presence of the EAAT‐inhibitor tfb‐TBOA, in which glutamate uptake was decreased. The intracellular glycogen content decreased in a time‐dependent manner, whereas the ATP levels were sustained following energy deprivation. Co‐cultures of RGC and Müller cells revealed better survival of glutamate treated RGC in the presence of Müller cells compared to controls. Energy deprivation of Müller cells enhanced their ability to protect RGC.Conclusion The present findings revealed an up‐regulation of EAAT1 and EAAT2 in energy compromised Müller cells as well as an increased ability to remove glutamate from the extracellular space. Co‐cultures of RGC and Müller cells revealed a protective role of Müller cells when RGC were exposed to glutamate. Hence, energy failure may result in an increased ability to protect RGC from glutamate‐induced excitotoxicity, whereas malfunction of glutamate uptake in Müller cells may contribute to RGC death.

NF-κB-mediated nitric oxide production and activation of caspase-3 cause retinal ganglion cell death in the hypoxic neonatal retina.

Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells (RGCs) through production of inflammatory mediators, nitric oxide (NO), and free radicals. The present study was aimed at elucidating the pathway through which hypoxia results in overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs. Wistar rats (1 day old) were exposed to hypoxia and their retinas were studied at 3 hours to 14 days after exposure. The protein expression of nuclear factor-κB (NF-κB) and neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was analyzed using Western blotting and double-immunofluorescence, whereas the concentration of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was evaluated by caspase-3 immunolabeling. Following hypoxic exposure, NF-κB-mediated expression of nNOS, which was localized to the RGCs, and subsequent NO production was significantly increased in the developing retina. In primary cultures of RGCs subjected to hypoxia, the upregulation of nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), an nNOS inhibitor or BAY, an NF-κB inhibitor. Hypoxia-induced apoptosis of RGCs, which was evident with caspase-3 labeling, also was suppressed when these cells were treated with 7-NINA or BAY. Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by NF-κB and the resulting increased release of NO by RGCs causes their apoptosis through caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective strategy against hypoxia-induced RGCs death in the developing retina.

Hypoxia-induced retinal ganglion cell damage through activation of AMPA receptors and the neuroprotective effects of DNQX

Hypoxia-induced glutamate accumulation in neural tissues results in damage to neurons through excitotoxic mechanisms via activation of glutamate receptors (GluRs). Here we examine whether hypoxia in the developing retina would cause activation of the ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) GluRs and increase in Ca2+ influx into retinal ganglion cells (RGCs) that might ultimately lead to their death. Neonatal Wistar rats were subjected to hypoxia for 2h and then sacrificed at various time points after the exposure together with normal age matched control rats. Primary cultures of RGCs were also prepared and subjected to hypoxia. Expression of AMPA glutamate receptor (GluR) 1–4 was examined in the retina. Additionally, expression of GluRs, intracellular Ca2+ influx, reactive oxygen species (ROS) generation and cell death were investigated in cultured RGCs. GluR1-4 mRNA and protein expression showed a significant increase (P < 0.01) over control values after the hypoxic exposure both in vivo and in vitro. Cells expressing GluR1-4 in the retina were identified as RGCs by double immunofluorescence labeling with Thy1.1. Increased intracellular Ca2+ in cultured RGCs following hypoxic exposure was reduced (P < 0.01) by 10 μM AMPA antagonist 6, 7-dinitroquinoxaline-2,3-dione (DNQX). Our results suggest that following a hypoxic insult, an increased amount of glutamate accumulates in the neonatal retina. This would then activate AMPA receptors which may damage RGCs through increased Ca2+ accumulation and ROS generation. The involvement of AMPA receptors in damaging the RGCs is evidenced by suppression of intracellular Ca2+ influx by DNQX which also decreased ROS generation and cell death by 50%.

Diabetes and Overexpression of proNGF Cause Retinal Neurodegeneration via Activation of RhoA Pathway

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotosin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75NTR, cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75NTR and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.

SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells

Activation of SIRT1, an NAD+-dependent deacetylase, prevents retinal ganglion cell (RGC) loss in optic neuritis, an inflammatory demyelinating optic nerve disease. While SIRT1 deacetylates numerous protein targets, downstream mechanisms of SIRT1 activation mediating this neuroprotective effect are unknown. SIRT1 increases mitochondrial function and reduces oxidative stress in muscle and other cells, and oxidative stress occurs in neuronal degeneration. We examined whether SIRT1 activators reduce oxidative stress and promote mitochondrial function in neuronal cells. Oxidative stress, marked by reactive oxygen species (ROS) accumulation, was induced in RGC-5 cells by serum deprivation, or addition of doxorubicin or hydrogen peroxide, and resulted in significant cell loss. SIRT1 activators resveratrol (RSV) and SRTAW04 reduced ROS levels and promoted cell survival in RGC-5 cells as well as primary RGC cultures. Effects were blocked by SIRT1 siRNA. SIRT1 activators also increased expression of succinate dehydrogenase (SDH), a mitochondrial enzyme, and promoted deacetylation of PGC-1α, a co-enzyme involved in mitochondrial function. Results show SIRT1 activators prevent cell loss by reducing oxidative stress and promoting mitochondrial function in a neuronal cell line. Results suggest SIRT1 activators can mediate neuroprotective effects during optic neuritis by these mechanisms, and they have the potential to preserve neurons in other neurodegenerative diseases that involve oxidative stress.

Early Onset and Differential Temporospatial Expression of Melanopsin Isoforms in the Developing Chicken Retina

Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In this study, the presence of nonvisual phototransduction cascade components in the developing chicken retina and primary RGCs cultures was investigated, focusing on the two Opn4 genes: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. Retinas were dissected at different embryonic (E) and postnatal (P) days, and primary RGC cultures were obtained at E8 and kept for 1 hour to 5 days. Samples were processed for RT-PCR and immunochemistry. Embryonic retinas expressed the master eye gene Pax6, the prospective RGC specification gene Brn3, and components of the nonvisual phototransduction cascade, such as Opn4m and the G protein q (Gq) mRNAs at very early stages (E4-E5). By contrast, expression of photoreceptor cell markers (CRX, red-opsin, rhodopsin, and α-transducin) was observed from E7 to E12. Opn4m protein was visualized in the whole retina as early as E4 and remained elevated from E6 to the postnatal days, whereas Opn4x was weakly detected at E8 and highly expressed after E11. RGC cultures expressed Gq mRNA, as well as both Opn4 mRNAs and proteins. Opn4m was restricted exclusively to the GC layer at all ages, whereas Opn4x was limited to the forming GC layer and optic nerve at E8, but by E15, its expression was mostly in Prox1(+) horizontal cells. The early expression onset of nonvisual phototransduction molecules could confer premature photosensitivity to RGCs, while the appearance of Opn4x expression in horizontal cells suggests the identification of a novel type of photosensitive cell in birds.