Chemokine CX3CL1 protects rat hippocampal neurons against glutamate-mediated excitotoxicity

Excitotoxicity is a cell death caused by excessive exposure to glutamate (Glu), contributing to neuronal degeneration in many acute and chronic CNS diseases. We explored the role of fractalkine/CX3CL1 on survival of hippocampal neurons exposed to excitotoxic doses of Glu. We found that: CX3CL1 reduces excitotoxicity when co-applied with Glu, through the activation of the ERK1/2 and PI3K/Akt pathways, or administered up to 8 h after Glu insult; CX3CL1 reduces the Glu-activated whole-cell current through mechanisms dependent on intracellular Ca2+; CX3CL1 is released from hippocampal cells after excitotoxic insult, likely providing an endogenous protective mechanism against excitotoxic cell death.     

Adiponectin protects hippocampal neurons against kainic acid-induced excitotoxicity

Neuronal damage after seizure is correlated with blood–brain barrier (BBB) leakage. Adiponectin (Ad) has shown protective effects on endothelial function. In this study, we investigated the effects of Ad on cell survival and BBB integrity in the mouse hippocampus after kainic acid (KA) treatment. Twenty-four hours after intracerebroventricular injection of recombinant Ad, mice were treated with KA, and then sacrificed 48 h later. Decreased serum Ad and increased hippocampal Ad receptor 1 in the hippocampus of KA-treated mice were prevented by Ad pretreatment. Using cresyl violet staining, TUNEL analysis, and immunostaining for caspase-3, histological evaluation revealed that the marked cell death noted in the hippocampus of KA-treated mice was not observed in KA-treated mice pretreated with Ad. Impairment of the BBB, which was demonstrated by the presence of IgG, was inhibited by Ad pretreatment. Immunohistochemical analysis indicated that KA caused up-regulation of hippocampal VEGF, eNOS, and NF-κB levels, all of which were reduced in animals that received Ad pretreatment. These data indicate that Ad preserves the integrity of the BBB and has neuroprotective effects in an animal model of seizures.     

Neuroprotection of adenoviral-vector-mediated GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. In the present study, we examined the protective roles of adenoviral-vector-delivered GDNF (Ad-GDNF) in the hippocampus damaged by kainic-acid (KA)-induced excitotoxicity using GAD-67 immunoreactivity, immunoblot analysis, behavioral test, 5-bromo-2-deoxyuridine (BrdU) and TUNEL assay. Ad-GDNF was pre-inoculated into the KA-treated rat hippocampus 7 days before KA injection. Ad-GDNF resulted in the suppression of KA-induced tonic-clonic convulsions. In situ apoptosis assay demonstrated a significant reduction in apoptotic cells in the CA3 and dentate hilus regions of the Ad-GDNF-pre-inoculated rats (Ad-GDNF-KA), compared to the KA rats. Striking reductions in the density of GAD-67 neurons were also observed in the CA3 and dentate hilus regions of the KA rats. On the other hand, the number of GAD-67-positive cells was recovered to the control levels in the Ad-GDNF-KA rats. Immunoblot analysis further confirmed that GAD-67 and Bcl-2 expression increased in the Ad-GDNF-KA rats compared to KA rats. Taken together, these results suggest that Ad-GDNF may serve to control KA-induced hippocampal cell loss and behavioral seizure.     

Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons.

Pigment epithelial-derived factor (PEDF) has been shown to be a survival factor for cerebellar granule neurons. Here we investigated the ability of PEDF to enhance the survival of hippocampal neurons in culture, and to protect these neurons against acute glutamate toxicity. Hippocampal neurons prepared from 1- to 3-day postnatal rat brain were cultured for either 7 or 14 days in vitro (DIV). At 14 DIV, neurons were only slightly protected (13% +/- 4%) against 50 microM glutamate toxicity when treated with 1 microg/ml of PEDF for 3 successive days before glutamate exposure as measured by lactate dehydrogenase (LDH) release. In comparison, basic fibroblast growth factor (bFGF) at 10 ng/ml for the same treatment period protected 58% +/- 8% of the neurons against glutamate. Using quantitative image analysis of digitized micrographs, we found that the average size of neurons in young, developing hippocampal cultures (7 DIV), was greatly decreased by treatment with 50 microM glutamate. Treatment for up to 5 successive days with 1 microg/ml of PEDF before glutamate addition dramatically increased the average hippocampal neuron soma size, compared to cells treated with glutamate alone. Thus, PEDF may promote the growth of hippocampal neurons, and, if added to developing hippocampal neurons, can also protect these cells from subsequent injury, such as the excitotoxicity of glutamate.     

Phenformin suppresses calcium responses to glutamate and protects hippocampal neurons against excitotoxicity

Phenformin is a biguanide compound that can modulate glucose metabolism and promote weight loss and is therefore used to treat patients with type-2 diabetes. While phenformin may indirectly affect neurons by changing peripheral energy metabolism, the possibility that it directly affects neurons has not been examined. We now report that phenformin suppresses responses of hippocampal neurons to glutamate and decreases their vulnerability to excitotoxicity. Pretreatment of embryonic rat hippocampal cell cultures with phenformin protected neurons against glutamate-induced death, which was correlated with reduced calcium responses to glutamate. Immunoblot analyses showed that levels of the N-methyl-d-aspartate (NMDA) subunits NR1 and NR2A were significantly decreased in neurons exposed to phenformin, whereas levels of the AMPA receptor subunit GluR1 were unchanged. Whole-cell patch clamp analyses revealed that NMDA-induced currents were decreased, and AMPA-induced currents were unchanged in neurons pretreated with phenformin. Our data demonstrate that phenformin can protect neurons against excitotoxicity by differentially modulating levels of NMDA receptor subunits in a manner that decreases glutamate-induced calcium influx. These findings show that phenformin can modulate neuronal responses to glutamate, and suggest possible use of phenformin and related compounds in the prevention and/or treatment of neurodegenerative conditions.     

Development of N-methyl-D-aspartate excitotoxicity in cultured hippocampal neurons

Immature hippocampal neurons (E-18) were maintained in defined medium for up to 3 weeks and their susceptibility to N-methyl-D-aspartic acid (NMDA)-induced cell death was studied at various days in vitro. Upon acute exposure to NMDA (5 min), hippocampal neurons in vitro (8-12 days after plating) showed cell body swelling and dendritic degeneration that preceded cell death 24 h later. NMDA-induced neurodegeneration could be prevented by MK-801 treatment but not by tetrodotoxin. In contrast, immature (5-7 days old) neurons were unaltered by exposure to 500 microM NMDA for either 5 min or 24 h. One explanation for the resistance of immature neurons to glutamate neurotoxicity may be related to maturation of the NMDA receptor complex. Glutamate binding to the NMDA receptor in vivo increased from 14.6 +/- 1.6% (0 day) to 55.2 +/- 4.5% (day 7), 79 +/- 4.9% (day 14), 93.8 +/- 2.8% (day 21) until it reached the adult Sprague-Dawley value of 100 +/- 0.8% (day 90).     

Delta9-tetrahydrocannabinol protects hippocampal neurons from excitotoxicity

Excitotoxic neuronal death underlies many neurodegenerative disorders. Because cannabinoid receptor agonists act presynaptically to inhibit glutamate release, we examined the effects of Win 55212-2, a full agonist at CB(1) receptors, and Delta(9)-tetrahydrocannabinol (THC), a partial agonist, on the survival of neurons exposed to an excitotoxic pattern of synaptic activity. Reducing the extracellular Mg(2+) concentration ([Mg(2+)](o)) to 0.1 mM evoked an aberrant pattern of glutamatergic activity that produced synaptically mediated death of rat hippocampal neurons in culture. Neuronal viability was quantified with a multiwell fluorescence plate scanner equipped to detect propidium iodide fluorescence. Win 55212-2 (100 nM) and THC (100 nM) significantly reduced 0.1 mM [Mg(2+)](o)-induced cell death by 77 +/- 11% and 84 +/- 8%, respectively. Interestingly, the protection afforded by THC was not significantly different from that produced by Win 55212-2, suggesting that attenuation without a complete block of excitatory activity is sufficient for neuroprotection. The effect of prolonged drug exposure on the neuroprotection afforded by cannabinoid receptor agonists was also studied. When cultures were pretreated for 24 h with Win 55212-2 (100 nM) or THC (100 nM), inhibition of 0.1 mM [Mg(2+)](o)-induced toxicity was significantly reduced to 39 +/- 19% and 45 +/- 13%, respectively. Thus, desensitization of CB(1) receptors diminishes the neuroprotective effects of cannabinoids. This study demonstrates the importance of agonist efficacy and the duration of treatment on the neuroprotective effects of cannabinoids. It will be important to consider these effects on neuronal survival when evaluating pharmacologic treatments that modulate the endocannabinoid system.     

Taxol stabilizes [Ca]i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca²⁺]_i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca²⁺ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca²⁺]_i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca²⁺]_i was monitored using the Ca²⁺ indicator dye fura-2 showed that the elevation of [Ca²⁺]_i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca²⁺ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca²⁺ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.     

Psychosine blocks quisqualate-induced glutamate excitotoxicity in hippocampal CA sector neurons

Quisqualate is a potent specific agonist for Group 1 metabotropic glutamate receptors (mGluR's), that activate G protein-coupled phospholipase C (PLC) in a molecular signal-transduction mechanism that raises cytoplasmic Ca²⁺ and, when excessive, damages hippocampal neurons. Psychosine (β-galactosylsphingosine), a cationic lysosphingolipid occurring naturally in nervous tissues, dose-dependently inhibited PLC activation induced by metabotropic α₁-adrenergic receptor signaling in cultured rat brain astrocytes in vitro. In the present study, we have tested neuroprotective efficacy of psychosine in vivo, in a rat model of glutamate excitotoxicity induced by intracerebroventricular (i.c.v.) administration of quisqualate. A sublethal i.c.v. dose of quisqualate caused episodes of prolonged akinesia and convulsions, and major damage to pyramidal neurons of the hippocampal CA1 and CA3 sector, but not to granule cell neurons of the dentate gyrus. Prior infusion of psychosine greatly attenuated quisqualate-induced behaviors, and fully prevented destruction by quisqualate of vulnerable hippocampal neurons. Psychosine may prove useful in prophylaxis of neurodegenerative disorders that arise from intensive hippocampal Group 1 mGluR stimulation.     

Interferon-gamma is up-regulated in the hippocampus in response to intermittent fasting and protects hippocampal neurons against excitotoxicity

Dietary restriction (DR) increases the life span of many different organisms, and recent findings have demonstrated neuroprotective effects of DR in rodent and nonhuman primate models of neurodegenerative disorders. The neuroprotective mechanism of action of DR is unknown, but it may result from a mild cellular stress response involving increased production of neurotrophic factors. Because several different cytokines are known to be up-regulated in brain cells in response to stress, we determined whether DR affected cytokine expression in the rat brain. Levels of expression of interferon-gamma (IFN-gamma) and its receptor were significantly increased in the hippocampus of rats that had been maintained on an intermittent fasting DR regimen compared with rats on the ad libitum control diet. Pretreatment of embryonic rat hippocampal cell cultures with IFN-gamma protected neurons against glutamate-induced death. IFN-gamma-mediated neuroprotection was associated with an enhanced recovery of intracellular Ca(2+) concentrations following exposure to glutamate. Our data show that intermittent fasting DR stimulates IFN-gamma-mediated neuroprotective signaling in the hippocampus, suggesting a role for this cytokine in the previously reported ability of DR to protect neurons in animal models of severe epileptic seizures, stroke, and neurodegenerative disorders.     

Damage and plasticity in adult rat hippocampal trisynaptic circuit neurons after neonatal exposure to glutamate excitotoxicity

Hippocampal vulnerability to excitotoxicity has been widely studied along with its implication to learning and memory. Neonatal glutamate excitotoxicity induces loss of CA1 pyramidal neurons in adult rats concomitantly with some plastic changes in the dendritic spines of surviving neurons. At least in part, these may underlie the place learning impairments seen in previous studies based on a similar excitotoxicity-inducing model. In the present study, cytoarchitecture of dentate gyrus, CA3 and CA1 fields were evaluated in 120-day-old rats, after they had been neonatally treated with glutamate as monosodium salt. Dentate granule cells and CA1 pyramidal neurons were less than those counted in NaCl-treated control animals. In addition, dentate granule cells had more dendrites as well as more branched spines. Spine density in CA1 pyramidal neurons was greater than in the controls. Additionally, thin and mushroom spines were proportionally more abundant in monosodium glutamate-treated animals. No effects were seen in the hippocampal CA3 field. Our results strongly suggest a long-term induction of plastic changes in the cytoarchitecture of the hippocampal trisynaptic circuit neurons after cell death provoked by the monosodium glutamate-induced excitotoxicity. These plastic events as well as the aberrant expression of the glutamate NMDA receptors resulting from monosodium glutamate neonatal treatment could be strongly associated with the place learning impairments previously reported.     

Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia

Brain ischemia induces neuronal loss which is caused in part by excitotoxicity and free radical formation. Here, we report that mangiferin and morin, two antioxidant polyphenols, are neuroprotective in both in vitro and in vivo models of ischemia. Cell death caused by glutamate in neuronal cultures was decreased in the presence of submicromolar concentrations of mangiferin or morin which in turn attenuated receptor-mediated calcium influx, oxidative stress as well as apoptosis. In addition, both antioxidants diminished the generation of free radicals and neuronal loss in the hippocampal CA1 region due to transient forebrain ischemia in rats when administered after the insult. Importantly, neuroprotection by these antioxidants was functionally relevant since treated-ischemic rats performed significantly better in three hippocampal-dependent behavioral tests. Together, these results indicate that mangiferin and morin have potent neuroprotectant activity which may be of therapeutic value for the treatment of acute neuronal damage and disability.     

Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro

Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor (EGF and bFGF respectively), and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid (1 mM) led to specific, large decreases (75% reduction) in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF (500 pM each) prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10⁻⁵ M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm.     

Estrogen protects against beta-amyloid-induced neurotoxicity in rat hippocampal neurons by activation of Akt.

The cellular mechanisms underlying the neuroprotective effects of estrogen are only beginning to be elucidated. Here we examined the role of protein kinase B (Akt) activation in 17beta-estradiol (E2) inhibition of beta-amyloid peptide (31-35) (Abeta31-35)-induced neurotoxicity in cultured rat hippocampal neurons. Abeta31-35 (25-30 betaM) significantly decreased the total number of microtubule associated protein-2 positive cells (MAP2+). This decrease was significantly reversed by pre-treatment with 100 nM E2. Further, 100 nM E2 alone significantly increased the total number of protein kinase B and microtubule associated protein-2 positive cells compared with controls. Such E2-induced increases were inhibited by LY294002 (20 microM), a specific PI3-K inhibitor, as well as by tamoxifen, an estrogen receptor antagonist/selective estrogen receptor modulator. These results indicate that the neuroprotective effects of E2 may be mediated at least in part via estrogen receptor-mediated protein kinase B activation.     

Preconditioning by motor activity protects rat hippocampal CA1 neurons against prolonged ischemia

Recovery of orthodromic and antidromic population spikes in CA1 hippocampal slices of 30-day-old Wistar rats has been studied in the reperfusion period after prolonged (90 min) decapitation ischemia with and without preceding 15 min long non-voluntary motor activity of intact animals. The preconditioning motor activity significantly enhances the resistance of pyramidal neurons to ischemia at a temperature of 30 degrees C. The period of protection lasts for up to 40 min after the end of motor activity. In case the ischemia was started within 5-10 min after the preconditioning, complete restoration of the field potentials to preischemic control level could be achieved. These data are the first indication of the neuroprotective effect of preconditioning motor activity in CA1 damage after prolonged global ischemia.     

Antidepressants are neuroprotective against nutrient deprivation stress in rat hippocampal neurons

There is ample evidence that depression and stress can be ameliorated through the use of physical exercise and/or antidepressant drugs. Both have been shown to promote neuroprotection against atrophy of dendrites and neuronal death through the activation of pro-survival signaling pathways, such as that of phosphatidyl inositol 3' kinase (PI-3K) and mitogen-activated protein kinase (MAPK). Depriving neurons in culture of several vital nutrients provides a viable model of neuronal stress, trauma or insult that occurs in vivo. Therefore, we sought to evaluate if various antidepressants are indeed neuroprotective in this model of nutrient deprivation stress. In addition, we evaluated if three key pro-survival pathways (PI-3K, MAPK, protein kinase A) are necessary for such neuroprotection. We used quantitative Western blotting to evaluate the immunoreactivity levels of brain-derived neurotrophic factor, PI-3K, phospho-protein kinase B (P-Akt), phospho-MAPK and phospho-cyclic AMP response element-binding protein, and live/dead cytotoxicity assay to evaluate cell survival. We demonstrate that in the ideal conditions of nutrient supplement, norepinephrine, serotonin and three antidepressants increased all six outcome measures; however, in the absence of such nutrients, only P-Akt levels showed signs of decreasing. In the presence of pro-survival pathway inhibitor, however, five out of the six outcome measures decreased (not P-Akt), relative to those of the ideal conditions of nutrient supplement. Thus, pro-survival pathway integrity, which more directly affects gene expression, is more important than the presence of externally placed nutrients for cell survival. We discuss our results in the context of receptor and pathway cross-talk, indicating that pharmacological rescue of neuronal atrophy/death in the face of mood disorders requires that pro-survival pathways remain intact.     

洛伐他汀减轻NMDA诱导的兴奋性毒性损害

目的 探讨洛伐他汀(Lovastatin,LOV)对NMDA诱导的大鼠皮质神经元兴奋性毒性损害的神经保护作用.方法 选用胚胎17d的SD大鼠,取皮质神经元接种培养.通过台盼蓝排除实验评估细胞活力、TUNEL染色检测凋亡细胞及免疫荧光细胞化学技术测定神经元形态.结果 台盼蓝排除实验显示500nmol/L洛伐他汀预处理3d显著减轻NMDA诱导的大鼠皮质神经元兴奋性毒性损害,而不能减少蛋白激酶C抑制剂星形孢菌素诱导的细胞凋亡.LOV和L-甲羟戊酸(MVA)共同预处理不能减轻NMDA诱导的兴奋性毒性损害,提示其保护作用依赖于降低胆固醇水平.洛伐他汀的神经保护作用呈剂量和时程依赖性.TUNEL染色显示洛伐他汀预处理能减少NMDA诱导的细胞凋亡.免疫荧光细胞化学结果显示洛伐他汀能改善NMDA诱导的MAP-2神经元形态损害.结论 洛伐他汀选择性地减轻NMDA诱导的大鼠皮质神经元兴奋性毒性损害及形态损害,提示洛伐他汀对兴奋性毒性损害相关神经病理有潜在的神经保护作用.     

GDNF and neublastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures

The potential neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) and neublastin (NBN) against NMDA-induced excitotoxicity were examined in hippocampal brain slice cultures. Recombinant human GDNF (25-100 ng/ ml) or NBN, in medium conditioned by growth of transfected, NBN-producing HiB5 cells, were added to slice cultures I h before exposure to 10 microM NMDA for 48h. Neuronal cell death was monitored, before and during the NMDA exposure, by densitometric measurements of propidium iodide (PI) uptake and loss of Nissl staining. Both the addition of rhGDNF and NBN-containing medium significantly reduced the NMDA-induced PI uptake in the CA1 (p < 0.01), suggesting neuroprotective effects of these factors, beyond their well-known trophic effects on dopaminergic neurons.     

海马神经元兴奋性毒性模型的建立及其意义

目的探讨不同浓度谷氨酸对海马神经元的损伤作用,流式细胞仪在建立理想的兴奋性毒性模型中的应用。方法在体外原代培养10d的海马神经元中分别加入不同浓度的谷氨酸(100、200、400、600、800μmol/L),24h后相差显微镜下观察细胞形态变化,用MTT法检测存活率和流式细胞仪检测凋亡率以评定谷氨酸对海马神经元的损伤程度。结果不同浓度的谷氨酸组与对照组的细胞存活率差异有显著性(P<0.01),并呈浓度依赖性,随着谷氨酸浓度的升高,神经元的存活率降低;谷氨酸终浓度(100、200、400μmol/L)组的细胞凋亡率与对照组比较差异有显著性(P<0.01);600和800μmol/L谷氨酸组的细胞凋亡率与对照组比较差异无显著性(P>0.05),但细胞凋亡率随着谷氨酸浓度的增加而降低。结论过高浓度的谷氨酸导致细胞急性坏死而非迟发性凋亡,运用流式细胞仪检测体外培养海马神经元凋亡率是一种特异性高的检测方法,值得推广。