Cultured Rat Retinal Ganglion Cells Research Articles

In Silico Ocular Pharmacokinetic Modeling: Delivery of Topical FK962 to Retina.

To establish the in silico ocular pharmacokinetic modeling for eye drops, and to simulate the dose regimen for FK962 in human choroid/retinal diseases. Pharmacokinetics for FK962 in vivo was performed by a single instillation of drops containing 0.1% 14C-FK962 in rabbit eyes. Permeation of FK962 across the cornea, sclera, and choroid/retina was measured in vitro. Neurite elongation by FK962 was measured in cultured rat retinal ganglion cells. Parameters from the experimental data were used in an improved in silico model of ocular pharmacokinetics of FK962 in man. The mean concentration of FK962 in ocular tissues predicted by in silico modeling was consistent with in vivo results, validating the in silico model. FK962 rapidly penetrated into the anterior and posterior segments of the eye and then diffused into the vitreous body. The in silico pharmacokinetic modeling also predicted that a dose regimen of 0.0054% FK962 twice per day would produce biologically effective concentrations of FK962 in the choroid/retina, where FK962 facilitates rat neurite elongation. Our in silico model for ocular pharmacokinetics is useful (1) for predicting drug concentrations in specific ocular tissues after topical instillation, and (2) for suggesting the optimal dose regimens for eye drops. The pharmacodynamics for FK962 produced by this model may be useful for clinical trials against retinal neuropathy.

In vitro and in vivo neuroprotective effects of cJun N-terminal kinase inhibitors on retinal ganglion cells.

BackgroundThe c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied.ResultsJNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p < 0.05) protected against glutamate excitotoxicity and trophic factor withdrawal induced RGC death in culture. In the I/R model, phosphorylation of JNK (pJNK) in the retina was significantly (p < 0.05) increased after injury. I/R injury significantly (p < 0.05) decreased the thickness of retinal layers, including the whole retina, inner plexiform layer, and inner nuclear layer and cell numbers in the GCL. Administration of SP600125 for 28 days protected against all these degenerative morphological changes (p < 0.05). In addition, SP600125 significantly (p < 0.05) protected against I/R-induced reduction in scotopic ERG b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. SP600125 also protected against the I/R-induced losses in volume and levels of synaptic markers in the SC. Moreover, the protective effects of SP600125 in the retina and SC were also detected even with only 7 days (Days 1–7 after I/R) of SP600125 treatment.ConclusionsOur results demonstrate the important role the JNK pathway plays in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection of RGCs in the retina.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0093-4) contains supplementary material, which is available to authorized users.

Baicalein Inhibits Amadori-Glycated Albumin-Induced MCP-1 Expression in Retinal Ganglion Cells via a MicroRNA-124-Dependent Mechanism.

The purpose of this study was to characterize the inflammatory effect of amadori-glycated albumin (AGA) in cultured rat retinal ganglion cells (RGCs) and to further explore the potential mechanism of the anti-inflammatory effects of baicalein. Primary rat retinal neurons were separated and cultured. The levels of monocyte chemotactic protein-1 (MCP-1) mRNA and soluble MCP-1 produced by RGCs in response to AGA were measured with quantitative reverse transcription-PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the expression of microRNA-124 (miR-124) and histone deacetylases (HDACs) were detected in cultured rat RGCs by qRT-PCR in the presence or absence of baicalein. Luciferase reporter assays were used to validate the regulation of a putative target of miR-124. Amadori-glycated albumin stimulation increased the expression of MCP-1 and inhibited the expression of miR-124 in cultured rat RGCs. In addition, miR-124 directly controlled MCP-1 expression by binding directly to 3'-untranslated region (3'-UTR) of MCP-1. Next, we demonstrated that miR-124 expression was suppressed by HDACs and that treatment of RGCs with HDACs inhibitors increased miR-124 expression and decreased MCP-1 production. Furthermore, application of baicalein in RGCs attenuated AGA-induced MCP-1 expression and upregulated expression of miR-124 by controlling HDAC4 and HDAC5. Collectively, these data suggest that baicalein inhibits AGA-induced MCP-1 expression in retinal ganglion cells via a microRNA-124-dependent mechanism.

Age-related intraneuronal accumulation of αII-spectrin breakdown product SBDP120 in the human cerebrum is enhanced in Alzheimer's disease

Spectrins are a part of cytoskeletal platform that lines the intracellular side of plasma membrane, which can be proteolyzed by calcium-sensitive enzymes including calpains and caspases. Caspase-3 mediated αII-spectrin proteolysis results in the release of a 120kDa spectrin breakdown product (SBDP120), known to occur in conditions with cell death. In rodents, intraneuronal SBDP120 accumulation in the forebrain develops with age, which is enhanced in transgenic models of Alzheimer's disease (AD). The present study was set to explore age-related SBDP120 formation and its relevance to AD-type hallmark lesions in the human brains. SBDP120 immunoreactivity (IR) was detected in neuronal somata and dendrites in the cortex and hippocampal formation in postmortem brains from aged (n=10, mean age=84.2) and AD (n=10, mean age=84.8) subjects, but not mid-aged controls (n=10, mean age=58.2). The overall density of SBDP120 IR quantified in the temporal neocortex was increased in the aged and AD groups, more robust in the latter, relative to mid-aged control, while no regional, laminar or cellular association was found between SBDP120 accumulation and Aβ deposition or phosphorylated-tau aggregation. In cultured rat retinal ganglion cells (RGC-5), SBDP120 elevation occurred with caspase-3 activation following oxygen as well as serum deprivation, suggestive of SBDP120 formation in stressful conditions with and without apparent neuronal death. These results confirm an age-related intraneuronal SBDP120 accumulation in the human cerebrum that is enhanced in AD. This neuronal change appears to occur independent of amyloid deposition, tau pathology and overt neuronal death.

Antiproliferative and Cytotoxic Properties of Moxifloxacin on Rat Retinal Ganglion Cells

Purpose: To evaluate the antiproliferative and cytotoxic properties of moxifloxacin on cultured rat retinal ganglion cells (RGC5).Materials and Methods: Rat retinal ganglion cells were exposed to various concentration of moxifloxacin (5–1500 μg/mL). For antiproliferative properties, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test was performed. Cellular cytotoxicity was assessed by using the Live/Dead viability/cytotoxicity assay and analyzed by fluorescence microscopy after 24 and 72 h of incubation, respectively.Results: Neither cytotoxic nor antiproliferative effect of moxifloxacin was observed below 50 μg/mL on RGC5 cells after 24 and 72 h of incubation. At higher concentrations of moxifloxacin (150 μg/mL, 500 μg/mL, and 1500 μg/mL (p < 0.001)) the number of viable cells and the proliferation rate of RGC5 were significantly reduced.Conclusions: These results suggest a dose-dependent cytotoxic and antiproliferative effect of moxifloxacin on RGC5. Therefore, intracameral application of moxifloxacin should be used cautiously in patients with increased risk of retinal ganglion cells damage, particularly in glaucoma patients.

Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

Slow Excitation of Cultured Rat Retinal Ganglion Cells by Activating Group I Metabotropic Glutamate Receptors

As in many CNS neurons, retinal ganglion cells (RGCs) receive fast synaptic activation through postsynaptic ionotropic receptors. However, the potential role of postsynaptic group I metabotropic glutamate receptors (mGluRs) in these neurons is unknown. In this study we first demonstrated that the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased intracellular calcium concentration in neurons within the ganglion cell layer of the rat retina. This prompted us to use an immunopanned-RGC and cortical astroglia coculture preparation to explore the effect of group I mGluR activation on the electrophysiological properties of cultured RGCs. Using perforated patch-clamp recordings in current-clamp configuration, we found that application of DHPG increased spontaneous spiking and depolarized the resting membrane potential of RGCs. This boosting effect was attributed to an increase in membrane resistance due to blockade of a background K(+) conductance. Further experiments showed that the group I mGluR-sensitive K(+) conductance was not blocked by 3 mM Cs(+), but was sensitive to acidification. Pharmacological studies indicated that the effect of DHPG on RGCs was mediated by the mGluR1 rather than the mGluR5 receptor subtype. Our results suggest a facilitatory role for group I mGluR activation in modulating RGC excitability in the mammalian inner retina.

Serum Deprivation Induces Apoptotic Cell Death of Transformed Rat Retinal Ganglion Cells via Mitochondrial Signaling Pathways

Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappaB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappaB (NF-kappaB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

Neuroprotective effects of B vitamins on cultured rat retinal neurons and ganglion cells

目的 评价维生素B1、B6、B12及其衍生物甲钴胺对视网膜神经细胞及神经节细胞(RGCs)的生存和轴突再生伸长的影响.方法采用新生大鼠视网膜神经细胞体外原代培养技术,与不同浓度的B族维生素共同培养,MTT比色法检测细胞活力,进行HE染色和抗Thy1免疫细胞化学染色,测量视网膜神经细胞和RGCs轴突长度,比较各种维生素对细胞轴突伸长的影响.结果维生素B1、B1、B12和甲钴胺的最有效作用浓度分别为100、100、1.0、1.0μmol/L;高密度培养该营养作用更明显.用该浓度作用于细胞,维生素B6、B12和甲钴胺可促进视网膜神经细胞和RGCs轴突伸长.结论B族维生素在体外短期内能够显著提高视网膜神经细胞和RGCs的活力,促进上述细胞轴突再生伸长。

Pathophysiology of the optic neuropathy associated with Friedreich ataxia.

To describe the optic neuropathy associated with the genetic defect in Friedreich ataxia and suggest a pathophysiologic mechanism. An experimental model of retinal ganglion cell death in the presence of metal chelation was used to test a hypothetical mechanism for the optic neuropathy of Friedreich ataxia. Study of cultured rat retinal ganglion cells suggests that abnormal regulation of intracellular iron levels could increase sensitivity to reactive oxygen species and lead to cell death in these metabolically active tissues. We hypothesize that decreased expression of frataxin, the mutated gene in Friedreich ataxia, could cause an optic neuropathy by increasing the sensitivity of retinal ganglion cells to oxidative stress.

Enhanced metabolic activity coincides with survival and differentiation of cultured rat retinal ganglion cells exposed to glutamate

Neurotransmitters are prominent candidates for trans-cellular signals that influence the development of the CNS. The present study has examined the effect of glutamate on survival, differentiation and metabolic activity of cultured rat retinal ganglion cells at 3 days in vitro. Retinal cultures from neonatal Wistar rats were treated with glutamate for 48 h. The metabolic activity was markedly increased in the retinal ganglion cells exposed to 20 μM glutamate. This was accompanied by an enhanced survival of these neurons. The number of differentiated retinal ganglion cells as determined by microtubule-associated protein-2 labeling was significantly increased following exposure to low but not higher doses of glutamate. The effect of glutamate on the metabolic activity and differentiation was blocked by tetrodotoxin. The results of the present study shows that glutamate has a significant effect on survival, differentiation and metabolic activity. An increase in the metabolic activity indicates an enhancement in the electrical activity. Thus, our results are consistent with the hypothesis that glutamate is critically involved in the regulation of electrical activity in developing rat retinal ganglion cells.

Existence of Ionotropic Glutamate Receptor Subtypes in Cultured Rat Retinal Ganglion Cells Obtained by the Magnetic Cell Sorter Method and Inhibitory Effects of 20-Hydroxyecdysone, a Neurosteroid, on the Glutamate Response

Glutamate and neurosteroids are known to exist in retinal ganglion cells (RGC). Therefore, patch clamp studies using the whole-cell recording method were performed to determine whether or not ionotropic glutamate receptor subtypes, i.e., N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors, were present on RGC obtained by the magnetic cell sorter (MACS) method and cultures. In addition, the effects of 20-hydroxyecdysone (20-HE), a neuro-steroid, on inward currents induced by NMDA, AMPA and kainate were examined at a holding potential of -60 mV. The current-voltage relationship for NMDA in the presence of glycine and Mg2+-free, as well as those for AMPA and kainate were linear, with a reversal potential of around 0 mV. NMDA-induced currents were blocked by MK-801, while both AMPA- and kainate-induced currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Application of 20-HE in the bath resulted in significant inhibitions on NMDA-, AMPA- and kainate-induced currents. Thus, NMDA, AMPA and kainate receptors were confirmed to exist on MACS-separated cultured RGC. Moreover, 20-HE inhibited NMDA receptor-mediated currents most prominently and AMPA- and kainate-mediated currents moderately, suggesting that neurosteroids may be playing a role in modulating glutamate-mediated transmission in RGC, and 20-HE might be useful for preventing glutamate neurotoxicity.

Pharmacological characteristics of ionotropic glutamate receptor subtypes in cultured rat retinal ganglion cells — a whole-cell patch clamp study

Pharmacological characteristics of ionotropic glutamate receptor subtypes in cultured rat retinal ganglion cells — a whole-cell patch clamp study

Ca2+-Permeable P2X Receptor Channels in Cultured Rat Retinal Ganglion Cells

ATP has been identified as an excitatory neurotransmitter in both the CNS and peripheral nervous system; however, little is known about the functional properties of ATP-gated channels in central neurons. Here we used a culture preparation of the postnatal rat retina to test the responsiveness of identified retinal ganglion cells (RGCs) and putative amacrines to exogenous ATP and other purinoceptor agonists. Rapidly activating ATP-induced currents (IATP) were exclusively generated in a subpopulation (approximately 65%) of RGCs. The latter were identified by Thy1.1 immunostaining, repetitive firing patterns, and activation of glutamatergic autaptic currents. None of the putative amacrine cells was ATP-sensitive. IATP could be induced with ATP, ADP, and alpha,beta-mATP but not with adenosine. It was antagonized by suramin. The current-voltage relationship of IATP showed marked inward rectification. Dose-response analysis yielded an EC50 of 14.5 microM, with a Hill coefficient of 0.9. Noise analysis of IATP suggested a mean single channel conductance of 2.3 pS. Retinal P2X purinoceptor channels exhibited a high permeability for Ca2+. PCa/PCs obtained from reversal potentials of IATP under bi-ionic conditions amounted to 2. 2 +/- 0.7. In the majority of cells, the decay of IATP was biphasic. The degree of current inactivation during the first 2 sec of agonist application was highly variable. Heterogeneity was also found with respect to the sensitivity to ADP and alpha,beta-mATP and the blocking action of suramin, suggesting expression of multiple P2X receptor subtypes. Our results indicate that activation of P2X receptor channels represents an important pathway for Ca2+ influx in postnatal RGCs.

P–85 - Presence of ionotropic glutamate receptor subtypes in cultured rat retinal ganglion cells: a whole cell patch clamp study.

P–85 - Presence of ionotropic glutamate receptor subtypes in cultured rat retinal ganglion cells: a whole cell patch clamp study.

P–87 - Non-N-methyl-D-aspartate receptors-mediated influx of Ca2+ in cultured rat retinal ganglion cells

P–87 - Non-N-methyl-D-aspartate receptors-mediated influx of Ca2+ in cultured rat retinal ganglion cells

P–71 - Maintenance of glutamate receptor functions in cultured rat retinal ganglion cells separated with the magnetic cell sorter.

P–71 - Maintenance of glutamate receptor functions in cultured rat retinal ganglion cells separated with the magnetic cell sorter.

Hypoxic and Excitotoxic Damage to Cultured Rat Retinal Ganglion Cells

The cytotoxic effects of hypoxia and excitatory amino acids on cultured retinal ganglion cells were studied. The influence of coculture with retinal Müller glia and cortical astrocytes on cell survival was evaluated, as were the effects of the N-methyl- dl-aspartate inhibitor, MK-801. Dissociated retinal neurons from 7-day-old Sprague–Dawley rats were plated on a laminin substrate, neocortex-derived glial monolayers, or Müller cell monolayers. Ganglion cells were labeled by injection of DiI into the superior colliculus 2 days prior to dissociation. Exposure of cultured ganglion cells to glutamate and N-methyl- dl-aspartate showed a time- and concentration-dependent survival rate. Exposure of cells to hypoxia demonstrated a survival rate that was dependent on time and O 2concentration. Excitotoxic and hypoxic damage was entirely blocked by the specific non-competitive inhibitor of N-methyl- dl-aspartate, MK-801. Retinal ganglion cells cultured on cortical astrocytes and retina-derived Müller glia showed significantly better survival rates ( P<0.001) than cells cultured on laminin-coated dishes under control conditions, in hypoxia (9% to 15% O 2), and after exposure to 200μ mglutamate. Retinal ganglion cells cultured on Müller glia showed significantly better survival rates ( P<0.01) than those cultured on cortical astrocytes under conditions of hypoxia (9% to 15% O 2) and exposure to 200μ mglutamate. The results demonstrate that excitotoxic and hypoxic damage to cultured retinal ganglion cells is moderated by NMDA receptor blockade and by the presence of glial cells, especially retinal Müller cells. This system may provide a useful model for studying the pathophysiology of excitotoxicity and hypoxia on cultured retinal ganglion cells, and may be used to help identify potentially clinically useful therapeutic agents.

Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture

The signaling mechanisms that control the survival of CNS neurons are poorly understood. Here we show that, in contrast to PNS neurons, the survival of purified postnatal rat retinal ganglion cells (RGCs) in vitro is not promoted by peptide trophic factors unless their intracellular cAMP is increased pharmacologically or they are depolarized by K + or glutamate agonists. Long-term survival of most RGCs in culture can be promoted by a combination of trophic factors normally produced along the visual pathway, including BDNF, CNTF, IGF1, an oligodendrocyte-derived protein, and forskolin. These results suggest that neurotransmitter stimulation and electrical activity enhance the survival of developing RGCs and raise the question of whether the survival control mechanisms of PNS and CNS neurons are different.

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.