Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy‐induced apoptosis via modulation of retinal glia in vivo

Adenoviral‐mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy‐induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRα) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral‐mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter‐1 (GLAST‐1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRα, pSTAT₃, GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-d-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p–c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.     

A role for polyamines in retinal ganglion cell excitotoxic death

Neuronal death due to excessive activation of N -methyl- d -aspartate (NMDA) receptors is a hallmark of neurodegenerative diseases. The polyamines: putrescine, spermine, and spermidine, bind to specific sites on the NMDA receptor and promote its activation, but their role in NMDA-induced neuronal death is ill defined. In this study, we characterized the role of polyamines in excitotoxic death of retinal ganglion cells (RGCs), a population of central neurons susceptible to NMDA-induced damage. Our data show that endogenous arginase I, the rate limiting enzyme for polyamine biosynthesis, is expressed in the intact, adult retina. Intraocular injection of NMDA visibly increased arginase I expression in Müller cells, the predominant glial cell-type in the mammalian retina. Inhibition of polyamine synthesis using di-fluoro-methyl-ornithine (DFMO) was markedly neuroprotective, while injection of exogenous polyamines in conjunction with NMDA exacerbated RGC death. Blockade of the polyamine binding sites on NMDA receptors using the non-competitive antagonist ifenprodil was neuroprotective, suggesting that polyamines contribute to excitotoxic death, at least partly, by binding to NMDA receptors. Importantly, we also demonstrate that NMDA leads to activation of both the Erk1/2 and PI3 K/Akt pathways, but only the PI3 K/Akt kinase was required for di-fluoro-methyl-ornithine-induced RGC survival. In summary, our study reveals that polyamines modulate neuronal death in the retina via different mechanisms that potentiate NMDA-triggered excitotoxicity.     

Inhibition of reactive gliosis attenuates excitotoxicity-mediated death of retinal ganglion cells

Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection.     

Effects of Dopamine D2-Like Receptor Antagonists on Light Responses of Ganglion Cells in Wild-Type and P23H Rat Retinas

In animal models of retinitis pigmentosa the dopaminergic system in the retina appears to be dysfunctional, which may contribute to the debilitated sight experienced by retinitis pigmentosa patients. Since dopamine D2-like receptors are known to modulate the activity of dopaminergic neurons, I examined the effects of dopamine D2-like receptor antagonists on the light responses of retinal ganglion cells (RGCs) in the P23H rat model of retinitis pigmentosa. Extracellular electrical recordings were made from RGCs in isolated transgenic P23H rat retinas and wild-type Sprague-Dawley rat retinas. Intensity-response curves to flashes of light were evaluated prior to and during bath application of a dopamine D2-like receptor antagonist. The dopamine D2/D3 receptor antagonists sulpiride and eticlopride and the D4 receptor antagonist L-745,870 increased light sensitivity of P23H rat RGCs but decreased light sensitivity in Sprague-Dawley rat RGCs. In addition, L-745,870, but not sulpiride or eticlopride, reduced the maximum peak responses of Sprague-Dawley rat RGCs. I describe for the first time ON-center RGCs in P23H rats that exhibit an abnormally long-latency (>200 ms) response to the onset of a small spot of light. Both sulpiride and eticlopride, but not L-745,870, reduced this ON response and brought out a short-latency OFF response, suggesting that these cells are in actuality OFF-center cells. Overall, the results show that the altered dopaminergic system in degenerate retinas contributes to the deteriorated light responses of RGCs.     

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.     

Neuroprotective effects of interleukin-6 on NMDA-induced rat retinal damage

This study shows that interleukin-6 (IL-6) combined with soluble interleukin-6 receptors (sIL-6R) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Eyes pretreated with a combined injection of IL-6 and sIL-6R had NMDA administered into the vitreous cavity. Morphometric analysis and retrograde labeling analysis found that pretreatment with either IL-6 or sIL-6R alone did not bring about any neuroprotective effect. However, pretreatment with a combined administration of IL-6 and sIL-6R induced a significant neuroprotective effect against NMDA-induced retinal damage. Apoptotic changes in the retina were assessed by the TUNEL method. The results indicated that pretreatment with IL-6 combined with sIL-6R prevents NMDA-induced apoptosis. Western blotting studies demonstrated upregulation of gp130 expression in the NMDA-injected retina. Present studies suggest that IL-6 combined with sIL-6R provides a neuroprotective effect on NMDA-induced retinal damage.     

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague-Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.     

Functional N-methyl-D-aspartate receptors are expressed in cone-driven horizontal cells in carp retina

Glutamate works as a major excitatory neurotransmitter in the vertebrate retina. Whole-cell recordings made from isolated carp cone horizontal cells (H1 cells) showed that N-methyl-D-aspartate (NMDA), co-applied with glycine, induced inward currents that were blocked by the NMDA receptor competitive antagonist D-2-amino-5-phosphonopentanoate (D-AP5) and 5,7-dichlorokynurenic acid (DCKA), a selective NMDA receptor antagonist acting at the glycine site on the NMDA receptor complex. Moreover, calcium imaging showed that NMDA caused a significant elevation of intracellular calcium levels ([Ca(2+)](i)) of H1 cells, which was also blocked by D-AP5. In contrast, neither inward currents nor changes in [Ca(2+)](i) could be induced by NMDA in rod horizontal cells (H4 cells). Intracellular recordings made from H1 cells in the isolated retina, superfused with Ringer's containing 1 mM Mg(2+), in the dark demonstrated that NMDA reduced the light-off overshoot of H1 cells. We therefore conclude that the functional NMDA receptor is expressed in carp H1 cells, from which this receptor has been thought to be absent, and this receptor may play a role in modulating cone-driven signal of horizontal cells in the dark.     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

Activated apoptotic and anti‐survival effects on rat hearts with fructose induced metabolic syndrome

Consumption of fructose has been linked to the development of metabolic syndrome, whereas the cardiomyopathic changes and cardiac apoptosis of dietary high‐fructose intake have not yet been clarified. The purpose of this study was to evaluate the effects of high‐fructose on cardiac apoptotic and survival pathways. Thirty‐two Wistar rats were randomly divided into a control group (CON), which received a standard chow diet, and a fructose‐induced metabolic syndrome group (FIMS), which received a 50% fructose‐content diet for 13 weeks. Histopathological analysis, TUNEL assays and Western blotting were performed on the excised hearts from both groups. The blood pressure, glucose, insulin, triglyceride and cholesterol levels were significantly increased in the FIMS group, compared with the CON group. The abnormal myocardial architecture, enlarged interstitial space and increased cardiac TUNEL‐positive apoptotic cells were observed in the FIMS group. The TNF‐α, TNF receptor 1, Fas ligand, Fas receptor, FADD, and activated caspase‐3 and 8 protein levels (Fas pathway) and the Bax, Bak, Bax/Bcl‐2, Bak/Bcl‐xL, cytosolic cytochrome c, and activated caspase‐3 and nine protein levels (mitochondria pathway) were increased in the FIMS group compared with those in the CON group. The IGFI, IGFI‐R, p‐PI3K, p‐Akt, Bcl‐2 and Bcl‐xL protein levels (survival pathway) were all significantly decreased in the FIMS group compared with those in the CON group. High‐fructose intake elevated blood pressure and glucose levels; moreover, high‐fructose diet activated cardiac Fas‐dependent and mitochondria‐dependent apoptotic pathways and suppressed the survival pathway, which might provide one possible mechanism for developing heart failure in patients with metabolic syndrome. Copyright © 2013 John Wiley & Sons, Ltd.     

Vision drives correlated activity without patterned spontaneous activity in developing Xenopus retina

Developing amphibians need vision to avoid predators and locate food before visual system circuits fully mature. Xenopus tadpoles can respond to visual stimuli as soon as retinal ganglion cells (RGCs) innervate the brain, however, in mammals, chicks and turtles, RGCs reach their central targets many days, or even weeks, before their retinas are capable of vision. In the absence of vision, activity-dependent refinement in these amniote species is mediated by waves of spontaneous activity that periodically spread across the retina, correlating the firing of action potentials in neighboring RGCs. Theory suggests that retinorecipient neurons in the brain use patterned RGC activity to sharpen the retinotopy first established by genetic cues. We find that in both wild type and albino Xenopus tadpoles, RGCs are spontaneously active at all stages of tadpole development studied, but their population activity never coalesces into waves. Even at the earliest stages recorded, visual stimulation dominates over spontaneous activity and can generate patterns of RGC activity similar to the locally correlated spontaneous activity observed in amniotes. In addition, we show that blocking AMPA and NMDA type glutamate receptors significantly decreases spontaneous activity in young Xenopus retina, but that blocking GABA(A) receptor blockers does not. Our findings indicate that vision drives correlated activity required for topographic map formation. They further suggest that developing retinal circuits in the two major subdivisions of tetrapods, amphibians and amniotes, evolved different strategies to supply appropriately patterned RGC activity to drive visual circuit refinement.     

Neuroprotectin D1 inhibits retinal ganglion cell death following axotomy

Neuroprotectin D1 (NPD1), a docosahexaenoic acid-derived autacoid, is an endogenous neuroprotective and anti-inflammatory mediator that is generated in the retina and brain. The effects of exogenous NPD1 on retinal ganglion cell (RGC) apoptosis and the role of 12/15-lipoxygenase (Alox15) in retina were evaluated after optic nerve transection (ONT). Treatment with NPD1 was associated with significant protection against RGC death. The percentage of RGC survival in NPD1-treated group was 30% at 2 weeks after ONT as compared with 12% of RGC survival in the ONT group without treatment. Endogenous NPD1 was a predominant lipid autocoid in uninjured and axotomized retinas. Alox15 mRNA expression was upregulated in retinas following ONT suggesting that amplification of 12/15-lipoxygenase (12/15-LOX) may represent a neuroprotective response in the rat retina. The density of RGCs was higher in the normal retina of 12/15-LOX-deficient mice as compared with congenic controls. Hence, the resident NPD1 has a potential role in the physiological and pathophysiological responses of the retina.     

Serine racemase expression and D-serine content are developmentally regulated in neuronal ganglion cells of the retina

d -Serine, the endogenous ligand for the glycine modulatory binding site of the NMDA receptor, and serine racemase, the enzyme that converts l -serine to d -serine, have been reported in vertebrate retina; initial reports suggested that localization was restricted to Müller glial cells. Recent reports, in which d -serine and serine racemase were detected in neurons of the brain, prompted the present investigation of neuronal expression of d -serine and serine racemase in retina and whether expression patterns were developmentally regulated. RT-PCR, in situ hybridization, western blotting, immunohistochemistry, and immunocytochemical methods were used to localize d -serine and serine racemase in intact retina obtained from 1 to 3 day, 3 week, and 18 week mouse retinas and in primary ganglion cells harvested by immunopanning from neonatal mouse retina. Results of these analyses revealed robust expression of d -serine and serine racemase in ganglion cells, both in intact retina and in cultured cells. The levels appear to be developmentally regulated with d -serine levels being quite high in ganglion cells of neonatal retinas and decreasing rapidly postnatally. Serine racemase levels are also developmentally regulated, with high levels detected during the early postnatal period, but diminishing considerably in the mature retina. This represents the first report of neuronal expression of d -serine and serine racemase in the vertebrate retina and suggests an important contribution of neuronal d -serine during retinal development.     

Modulation of microglia by Wolfberry on the survival of retinal ganglion cells in a rat ocular hypertension model

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of lipopolysaccharide decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.     

Neuroprotective Effects of NMDA Receptor Glycine Recognition Site Antagonism: Dependence on Glycine Concentration

Abstract: High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1–10 µ M ) before 500 µ M glutamate, 30 µ M NMDA, or 300 µ M kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 m M ) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 µ M –1 m M ) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 µ M ACEA 1021 was similar for glutamate and NMDA (18 ± 3 and 29 ± 9 µ M , respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

Modulation of microglia by Wolfberry on the survival of retinal ganglion cells in a rat ocular hypertension model

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of bacterial endotoxin lipopolysaccharide (LPS) decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.