Glutamate-induced Death Research Articles

Isoliquiritigenin Protects Neuronal Cells against Glutamate Excitotoxicity.

It is considered that glutamate excitotoxicity may be a major factor in the pathological death of neurons and mediate the development of neurodegenerative diseases in humans. Here, we show that isoliquiritigenin (ILG) at a concentration of 0.5-5 µM protects primary neuroglial cell culture from glutamate-induced death (glutamate 100 µM). ILG (1 µM) prevented a sharp increase in [Ca2+]i and a decrease in mitochondrial potential (ΔΨm). With the background action of ILG (1-5 µM), there was an increase in oxygen consumption rate (OCR) in response to glutamate, as well as in reserve respiration. The neuroprotective effect of ILG (5 µM) was accompanied by an increase in non-mitochondrial respiration. The results show that ILG can protect cortical neurons from death by preventing the development of calcium deregulation and limiting mitochondrial dysfunction caused by a high dose of glutamate. We hypothesize that ILG will be useful in drug development for the prevention or treatment of neurodegenerative diseases accompanied by glutamate excitotoxicity.

Prevention of Cell Death by Activation of Hydroxycarboxylic Acid Receptor 1 (GPR81) in Retinal Explants.

Background: Progressive retinal ganglion cell (RGC) dysfunction and death are common characteristics of retinal neurodegenerative diseases. Recently, hydroxycarboxylic acid receptor 1 (HCA1R, GPR81) was identified as a key modulator of mitochondrial function and cell survival. Thus, we aimed to test whether activation of HCA1R with 3,5-Dihydroxybenzoic acid (DHBA) also promotes RGC survival and improves energy metabolism in mouse retinas. Methods: Retinal explants were treated with 5 mM of the HCA1R agonist, 3,5-DHBA, for 2, 4, 24, and 72 h. Additionally, explants were also treated with 15 mM of L-glutamate to induce toxicity. Tissue survival was assessed through lactate dehydrogenase (LDH) viability assays. RGC survival was measured through immunohistochemical (IHC) staining. Total ATP levels were quantified through bioluminescence assays. Energy metabolism was investigated through stable isotope labeling and gas chromatography-mass spectrometry (GC-MS). Lactate and nitric oxide levels were measured through colorimetric assays. Results: HCA1R activation with 3,5-DHBAincreased retinal explant survival. During glutamate-induced death, 3,5-DHBA treatment also increased survival. IHC analysis revealed that 3,5-DHBA treatment promoted RGC survival in retinal wholemounts. 3,5-DHBA treatment also enhanced ATP levels in retinal explants, whereas lactate levels decreased. No effects on glucose metabolism were observed, but small changes in lactate metabolism were found. Nitric oxide levels remained unaltered in response to 3,5-DHBA treatment. Conclusion: The present study reveals that activation of HCA1R with 3,5-DHBA treatment has a neuroprotective effect specifically on RGCs and on glutamate-induced retinal degeneration. Hence, HCA1R agonist administration may be a potential new strategy for rescuing RGCs, ultimately preventing visual disability.

Neuroprotective Effects of Isolated Fraction from <I>Sapindus laurifolia</I> Extract in Hippocampal Neuronal HT22 Cells

Glutamate is a major endogenous excitatory neurotransmitter in the brain and contributes to the development of neurodegenerative diseases by excessive activation. The purpose of the present study was to determine the neuroprotective effect of Sapindus laurifolia (MESL) Fraction A against glutamate-induced oxidative stress and to assess the underlying mechanism. MESL Fraction A was subjected to a neuroprotective effect assay in HT22 mouse hippocampal cells. The mechanism underlying the neuroprotective effect of MESL Fraction A was evaluated by assaying Reactive Oxygen Species (ROS) levels, intracellular Ca2+ levels, mitochondrial membrane potential and glutathione level and antioxidant enzyme activity in HT22 cells. MESL Fraction A significantly decreased glutamate-induced death of HT22 cells (88.23 ± 1.65% relative neuroprotection). MESL Fraction A reduced the intracellular ROS and Ca2+ levels and increased the glutathione level and glutathione reductase and glutathione peroxide activities. Moreover, MESL Fraction A attenuated the mitochondrial membrane potential in HT22 cells. These results suggested that MESL Fraction A exerts a neuroprotective effect against oxidative stress HT22 cells, which was mediated by its antioxidant activity.

NRG1-ErbB4 signaling promotes functional recovery in a murine model of traumatic brain injury via regulation of GABA release.

Traumatic brain injury (TBI) is a serious health problem in the world. However, little is known about the pathogenesis and molecular mechanisms of TBI. Here, we show that TBI activates neuregulin 1 (NRG1)-ErbB4 signaling, with an increased expression of NRG1 and ErbB4 in the traumatic region. Specifically knocking out ErbB4 in parvalbumin-positive (PV+) interneurons exacerbates motor function deficits in mice after TBI. Consistently, PV-ErbB4-/- mice showed larger necrotic area and more edema when compared with PV-ErbB4+/+ mice. Replenishment of NRG1 through intranasal application of the recombinant protein in PV-ErbB4+/+ mice enhanced neurological function. Moreover, using an in vitro neuronal culture system, we found that NRG1-ErbB4 signaling protects neurons from glutamate-induced death, and such protective effects could be diminished by GABA receptor antagonist. These results indicate that NRG-ErbB4 signaling protects cortical neurons from TBI-induced damage, and such effect is probably mediated by promoting GABA activity. Taken together, these findings unveil a previously unappreciated role for NRG1-ErB4 signaling in preventing neuronal cell death during functional recovery after TBI.

Astrocyte-Conditioned Medium Protects Prefrontal Cortical Neurons from Glutamate-Induced Cell Death by Inhibiting TNF-α Expression

Objective: Both excitotoxicity and neurotrophin deficiency may contribute to the etiology of depression and neurodegeneration. Astrocytes not only regulate glutamate metabolism and clearance, they also produce neurotrophins in the brain. However, the direct interaction between neurons and astrocytes remains unknown. Methods: This study evaluated the cellular mechanisms by which astrocyte-conditioned medium (ACM) protects prefrontal cortical neurons from glutamate-induced death by measuring cell viability and morphology as well as mRNA and protein expression of brain-derived neurotrophic factor (BDNF), BDNF receptors, glial cell line-derived neurotrophic factor (GDNF), and the proinflammatory cytokine, tumor necrosis factor (TNF)-α. Neurons and astrocytes were purified from the brains of neonatal 1-day-old Sprague-Dawley rats. ACM was harvested after exposing astrocytes to culture medium containing 100 μM glutamate for 48 h. Results: Glutamate insult (100 μM for 6 h) significantly reduced neuronal cell viability and increased the mRNA expression of BDNF. Glutamate (24 h) decreased neuronal viability and the expression of BDNF, but increased mRNA expression of GFAP, p75 neurotrophin receptor (p75^NTR), and TNF-α. ACM pretreatment (2 h) reversed glutamate-decreased cell viability and increased BDNF, but reduced the expression of GDNF, P75^NTR, and TNF-α at the mRNA level. Western blotting generally confirmed the mRNA expression following 24 glutamate insults. Furthermore, the glutamate-induced decrease in the protein expression of BDNF and full-length TrkB receptor and increase in pro-BDNF, truncated TrkB isoform 1 receptor, p75^NTR, GDNF, and TNF-α were significantly attenuated by ACM pretreatment. Conclusions: The study demonstrates that ACM exerts neuroprotective effects on cell viability, and this effect is most likely mediated through the modulation of neurotrophin and TNF-α expression.

Bergenin Ameliorates Glutamate-Induced Death of Hippocampal Neuronal Cells through Modulation of the Antioxidant Activity and reducing the redox oxygen Species Creation

Oxytosis is a disproportion between the manufacturing and manifestation of reactive oxygen species and the ability of a biological system to restore the resulting injury. In mouse hippocampal cells, HT22, the neuroprotective impact of Bergenin against glutamate-induced oxidative stress was explored. The findings showed that bergenin protected cells against toxicity caused by glutamate at 10 μM and improved their impact in a dose-dependent way until peak protection at 30 μM was achieved. Pretreatment with bergenin, which was clarified using a Cell viability assay (WST-1Assay), considerably suppressed the elevation of glutamate-induced reactive oxygen species and consequently secure HT22 cells against death from glutamate. And this protection reduced by glutamate through restoration of glutathione concentrations. Besides upregulating glutathione activity, the antioxidant ability to scavenge ROS also activates PPARπ, which controls various pro-inflammatory cytokines.

Sanguiin H-11 from Sanguisorbae radix protects HT22 murine hippocampal cells against glutamate-induced death

Excessive glutamate level induces neuronal death in acute brain injuries and chronic neurodegenerative diseases. Natural compounds from medicinal and food plants have been attracting interest as a treatment for neurological disorders. Sanguiin H-11 (SH-11), a hydrolysable ellagitannin, inhibits neutrophil movement and nitric oxide -production. However, its neuroprotective effect has not been studied. Therefore, the present study examined the protective effect of SH-11 from Sanguisorbae radix and its mechanism against glutamate-induced death in HT22 cells. Our results showed that SH-11 possessed a strong antioxidant activity and prevented glutamate-induced death in HT22 cells. As a strong antioxidant, SH-11 significantly reduced glutamate-induced increases in intracellular reactive oxygen species accumulation and calcium ion influx. Western blotting analysis showed that glutamate-induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinases 1/2, c-Jun N-terminal kinase, and p38, was significantly decreased by SH-11. Furthermore, SH-11 significantly decreased the number of annexin V-positive HT22 cells, which is indicating apoptotic cell death. In conclusion, our results suggested that SH-11 exerted a potent neuroprotective activity against glutamate-mediated apoptotic cell death by inhibiting oxidative stress-mediated MAPK activation.

Neuroprotective effects of Magnoliae Flos extract in mouse hippocampal neuronal cells

Magnoliae Flos (MF) is a traditional medicinal herb used for managing rhinitis, sinusitis and headache. The purpose of the present study was to determine the neuroprotective effect of MF against glutamate-induced oxidative stress and to assess the underlying mechanism. Glutamate is a major endogenous excitatory neurotransmitter in the brain and contributes to the development of neurodegenerative diseases by excessive activation. MF extract was subjected to a neuroprotective effect assay in HT22 mouse hippocampal cells. The mechanism underlying the neuroprotective effect of MF extract was evaluated by assaying reactive oxygen species (ROS) levels, intracellular Ca2+ levels, mitochondrial membrane potential, glutathione level and antioxidant enzyme activity in HT22 cells. MF extract significantly decreased glutamate-induced death of HT22 cells (80.83 ± 7.34% relative neuroprotection). MF extract reduced the intracellular ROS and Ca2+ levels and increased the glutathione level and glutathione reductase and glutathione peroxide activities. Moreover, MF extract attenuated the mitochondrial membrane potential in HT22 cells. These results suggested that MF extract exerts a neuroprotective effect against oxidative stress HT22 cells, which was mediated by its antioxidant activity.

Inhibition of Bcl-xL prevents pro-death actions of \u0394N-Bcl-xL at the mitochondrial inner membrane during glutamate excitotoxicity

ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL. We now report that a high concentration of ABT-737 (1 μM), or a more selective Bcl-xL inhibitor WEHI-539 (5 μM) enhances glutamate-induced neurotoxicity while a low concentration of ABT-737 (10 nM) or WEHI-539 (10 nM) is neuroprotective. High ABT-737 markedly increased ΔN-Bcl-xL formation, aggravated glutamate-induced death and resulted in the loss of mitochondrial membrane potential and decline in ATP production. Although the usual cause of death by ABT-737 is thought to be related to activation of Bax at the outer mitochondrial membrane due to sequestration of Bcl-xL, we now find that low ABT-737 not only prevents Bax activation, but it also inhibits the decline in mitochondrial potential produced by glutamate toxicity or by direct application of ΔN-Bcl-xL to mitochondria. Loss of mitochondrial inner membrane potential is also prevented by cyclosporine A, implicating the mitochondrial permeability transition pore in death aggravated by ΔN-Bcl-xL. In keeping with this, we find that glutamate/ΔN-Bcl-xL-induced neuronal death is attenuated by depletion of the ATP synthase c-subunit. C-subunit depletion prevented depolarization of mitochondrial membranes in ΔN-Bcl-xL expressing cells and substantially prevented the morphological change in neurites associated with glutamate/ΔN-Bcl-xL insult. Our findings suggest that low ABT-737 or WEHI-539 promotes survival during glutamate toxicity by preventing the effect of ΔN-Bcl-xL on mitochondrial inner membrane depolarization, highlighting ΔN-Bcl-xL as an important therapeutic target in injured brain.

Neuroprotective effect of Aronia melanocarpa extract against glutamate-induced oxidative stress in HT22 cells

BackgroundGlutamate (an endogenous excitatory neurotransmitter) at high concentrations contributes to the development of neurodegenerative diseases. Aronia melanocarpa (A. melanocarpa) berries contain anthocyanins and have high antioxidant activities. In this study, we evaluated whether A. melanocarpa berries could protect neuronal cells against glutamate-induced oxidative stress.MethodA. melanocarpa berries exerted a protective effect against cytotoxicity in HT22 mouse hippocampal cells by MTT assay. We evaluated oxidative stress parameters including ROS level, intracellular Ca2+ level, glutathione level and antioxidant enzyme activity in HT22 cells to elucidate the mechanism of its neuroprotective effect.ResultsA. melanocarpa berries decreased glutamate-induced death of HT22 cells. In addition, A. melanocarpa berries reduced ROS and intracellular Ca2+ levels. Glutathione level, antioxidant enzymes, glutathione reductase and glutathione peroxide activities and mitochondrial membrane potential were also increased in HT22 cells.ConclusionThese results suggested that A. melanocarpa berries protected HT22 cells by exerting an antioxidant effect.

On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death.

Ferroptosis is a form of regulated necrosis associated with the iron-dependent accumulation of lipid hydroperoxides that may play a key role in the pathogenesis of degenerative diseases in which lipid peroxidation has been implicated. High-throughput screening efforts have identified ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent inhibitors of ferroptosis − an activity that has been ascribed to their ability to slow the accumulation of lipid hydroperoxides. Herein we demonstrate that this activity likely derives from their reactivity as radical-trapping antioxidants (RTAs) rather than their potency as inhibitors of lipoxygenases. Although inhibited autoxidations of styrene revealed that Fer-1 and Lip-1 react roughly 10-fold more slowly with peroxyl radicals than reactions of α-tocopherol (α-TOH), they were significantly more reactive than α-TOH in phosphatidylcholine lipid bilayers − consistent with the greater potency of Fer-1 and Lip-1 relative to α-TOH as inhibitors of ferroptosis. None of Fer-1, Lip-1, and α-TOH inhibited human 15-lipoxygenase-1 (15-LOX-1) overexpressed in HEK-293 cells when assayed at concentrations where they inhibited ferroptosis. These results stand in stark contrast to those obtained with a known 15-LOX-1 inhibitor (PD146176), which was able to inhibit the enzyme at concentrations where it was effective in inhibiting ferroptosis. Given the likelihood that Fer-1 and Lip-1 subvert ferroptosis by inhibiting lipid peroxidation as RTAs, we evaluated the antiferroptotic potential of 1,8-tetrahydronaphthyridinols (hereafter THNs): rationally designed radical-trapping antioxidants of unparalleled reactivity. We show for the first time that the inherent reactivity of the THNs translates to cell culture, where lipophilic THNs were similarly effective to Fer-1 and Lip-1 at subverting ferroptosis induced by either pharmacological or genetic inhibition of the hydroperoxide-detoxifying enzyme Gpx4 in mouse fibroblasts, and glutamate-induced death of mouse hippocampal cells. These results demonstrate that potent RTAs subvert ferroptosis and suggest that lipid peroxidation (autoxidation) may play a central role in the process.

Pinacidil and levamisole prevent glutamate-induced death of hippocampal neuronal cells through reducing ROS production

Activators of both adenosine 5′-triphosphate (ATP)-sensitive K+ (KATP) channel and cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel have significant in vivo and in vitro neuroprotection against glutamate-induced death of some neuronal cells. Here, the effect of the KATP channel activator, pinacidil, and the CFTR Cl− channel opener, levamisole, against glutamate-induced oxidative stress were investigated in mouse hippocampal cells, HT22. The results from cell viability assay (WST-1) showed that pinacidil and levamisole weakly protected cells against glutamate-induced toxicity at 10 μM and their effect increased in a dose-dependent manner till reach maximum protection at 300 μM. Pretreatment with pinacidil or levamisole significantly suppressed the elevation of reactive oxygen species (ROS) triggered by glutamate through stabilising mitochondrial membrane potential and subsequently protected HT22 cells against glutamate-induced death. HT22 cells viability was maintained by pinacidil and levamisole in presence of glutathione inhibitor, BSO. Also, pinacidil and levamisole pretreatment did not induce recovery of glutathione levels decreased by glutamate Expectedly, this protection was abolished by the KATP and CFTR Cl− channels blocker, glibenclamide. Thus, both pinacidil and levamisole protect HT22 cells against glutamate-induced cell death through stabilising mitochondrial membrane potential and subsequently decreasing ROS production.

Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc−, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc− subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.

TrkB is involved in the mechanism by which BDNF accelerates the glutamate-induced death of rat neuroblastoma B35 cells

Objective:Brain-derived neurotrophic factor (BDNF) binds to its high-affinity binding receptor, tropomyosin-related kinase (Trk) B, and can induce neuronal differentiation and survival. BDNF also accelerates neuronal cell death in a glutamate-induced model; however, it has been unknown whether the mechanism involves TrkB. In the current study, to determine the role of TrkB in neuronal cell death, we investigated TrkB involvement in BDNF acceleration of glutamate-induced neuronal death.Methods:A TrkB-stable transformant of rat neuroblastoma B35 (B35TrkB) cells was utilized to investigate whether TrkB is involved in BDNF acceleration of neuronal death. The cell viability of the B35TrkB cells was compared to that of mock vector-transgened B35 (B35mock) cells after treatment with/without BDNF and glutamate.Results:In both B35TrkB and B35mock cells, glutamate treatment decreased the cell viability. BDNF treatment further accelerated the decrease in the viability of B35TrkB cells, but not that in the viability of B35mock cells. At glutamate concentrations that did not significantly decrease cell viability, BDNF increased the cell viability of B35TrkB, but not that of B35mock. A mitogen-activated protein kinase (MAPK) inhibitor, U0126, suppressed BDNF’s accelerating effect on cell death. Although B35 parental cells endogenously express other neurotrophin receptors such as TrkA, nerve growth factor β (a ligand of TrkA and p75NTR) could not influence the viability of B35TrkB or B35mock cells.Conclusion:These results indicate that TrkB is an intermediator for the trophic and toxicity-exacerbating effects of BDNF against cell viabilities at non-cytotoxic and cytotoxic glutamate concentrations, respectively.

Tonicity-responsive enhancer binding protein haplodeficiency attenuates seizure severity and NF-κB-mediated neuroinflammation in kainic acid-induced seizures

Kainic acid (KA)-induced seizures followed by neuronal death are associated with neuroinflammation and blood-brain barrier (BBB) leakage. Tonicity-responsive enhancer binding protein (TonEBP) is known as a transcriptional factor activating osmoprotective genes, and in brain, it is expressed in neuronal nuclei. Thus dysregulation of TonEBP may be involved in the pathology of KA-induced seizures. Here we used TonEBP heterozygote (+/-) mice to study the roles of TonEBP. Electroencephalographic study showed that TonEBP (+/-) mice reduced seizure frequency and severity compared with wild type during KA-induced status epilepticus. Immunohistochemistry and western blotting analysis showed that KA-induced neuroinflammation and BBB leakage were dramatically reduced in TonEBP (+/-) mice. Similarly, TonEBP-specific siRNA reduced glutamate-induced death in HT22 hippocampal neuronal cells. TonEBP haplodeficiency prevented KA-induced nuclear translocation of NF-κB p65 and attenuated inflammation. Our findings identify TonEBP as a critical regulator of neuroinflammation and BBB leakage in KA-induced seizures, which suggests TonEBP as a good therapeutic target.

The antioxidant idebenone fails to prevent or attenuate chronic experimental autoimmune encephalomyelitis in the mouse

Oxidative stress and mitochondrial dysfunction appear to contribute to neurodegenerative processes during multiple sclerosis (MS). Thus, antioxidants may represent a therapeutic option for MS. The antioxidant idebenone was proven to be beneficial in Friedreich's ataxia and Leber's hereditary optic neuropathy, two disorders caused by mitochondrial alterations. Here we showed that idebenone protected neuronal HT22 cells from glutamate-induced death in vitro. However, in experimental autoimmune encephalomyelitis, idebenone failed to affect disease incidence or onset when applied preventively, or to reduce disease severity when applied therapeutically. Histopathological examination of CNS from idebenone treated mice showed no improvement in inflammation, demyelination, or axonal damage. Thus, we hypothesize that idebenone treatment will likely not benefit patients with MS.

Study of the protective effect of calcium channel blockers against neuronal damage induced by glutamate in cultured hippocampal neurons

BackgroundThe aim of this study was to examine the putative protective effect of calcium channel blockers on hippocampal neurons in the experimental model of excitotoxic damage. MethodsSeven-day old primary dissociated cultures of rat hippocampal neural cells containing one of the following calcium channel blockers: cinnarizine, flunarizine or nimodipine were exposed to glutamate-induced injury. Quantitative assessments of neuronal injury were accomplished by measuring lactate dehydrogenase (LDH) activity in the media 24h after exposure to glutamate and by counting and establishing the apoptotic and necrotic cells in flow cytometry with Annexin V-FITC/PI staining. ResultsIn our experiment, glutamate induced a 339% elevation of apoptotic cells and a 289% increase of necrotic cells in hippocampal neurons as compared to control cultures without drugs. In cultures containing flunarizine, glutamate-induced cell apoptosis was suppressed by 62% while necrosis showed no significant alternation. Cinnarizine exerted no anti-apoptotic effects on glutamate-injured cultured hippocampal neurons, while nimodipine intensified the apoptotic pathway of cell death and promoted an increase in the number of apoptotic neurons by 26%. When cinnarizine or nimodipine were used, the percentage of necrotic cells was significantly lower when compared with glutamate-injured cultures and it amounted to 44% and 24% for cinnarizine and nimodipine, respectively. ConclusionsThe obtained results suggest the beneficial anti-apoptotic potential of flunarizine and the anti-necrotic potential of cinnarizine against glutamate-induced death of cultured hippocampal neurons. Nimodipine can protect neurons against necrosis, but has an intensified adverse pro-apoptotic effect on cultured neurons in the experimental model of excitotoxic injury.

Hexane extract from Polygonum multiflorum attenuates glutamate-induced apoptosis in primary cultured cortical neurons

Ethnopharmacological relevancePolygonum multiflorum has traditionally had wide use as an anti-aging treatment in East Asian countries. We investigated the neuroprotective effects of Polygonum multiflorum against glutamate-induced neurotoxicity with a focus on the anti-apoptotic mechanism in primary cultured cortical neurons. Material and methodsCell viability, cytotoxicity, morphological, flow cytometry, Western blot, and caspase activity assays were performed for examination of the neuroprotective effects of active hexane extract from Polygonum multiflorum (HEPM). ResultsPretreatment with HEPM resulted in significantly decreased glutamate-induced neurotoxicity in a concentration-dependent manner and also resulted in drastically inhibited glutamate-induced apoptosis. Treatment with HEPM resulted in decreased expression of glutamate-induced death receptor (DR)4, and enhanced expression of glutamate-attenuated anti-apoptotic proteins, including Bcl-2, XIAP, and cIAP-1, and slightly reduced glutamate-induced cleavage of Bid. In addition, treatment with HEPM resulted in suppressed glutamate-induced activation of caspase-8, caspase-9, and caspase-3, and, subsequently, decreased degradation of poly(ADP-ribose) polymerase, β-catenin, and phospholipase Cγ1 protein, which are downstream targets of activated caspase-3. ConclusionsThe results of this study demonstrated that HEPM exerts a neuroprotective effect against glutamate-induced neurotoxicity via inhibition of apoptosis. This protection may be mediated through suppression of DR4 and up-regulation of Bcl-2, XIAP, and cIAP-1, as well as inhibition of caspase activation, resulting in prevention of apoptosis of cortical neurons.

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin-releasing factor (CRF). Adenosine A(2A) receptors contribute to glutamate excitoxicity and their blockade is effective in stress-induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A(2A) and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF(1)R and CRF(2)R were challenged with glutamate (20-1000 μM, 24 h). CRF(1)R was found to co-localize with neuronal markers and CRF(2)R to be present in both neuronal and glial cells. The effects of the CRF and A(2A) receptors ligands on cell viability were measured using propidium iodide and Syto-13 fluorescence staining. Glutamate decreased cell viability in a concentration-dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF(1)R or CRF(2)R with antalarmin (10 nM) or anti-Sauvagine-30 (10 nM), respectively. The blockade of A(2A) receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF(2)R, but not on CRF(1)R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate-induced death. The neuroprotection achieved by A(2A) receptors blockade requires CRF(2)R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A(2A) receptor antagonists against stress-induced noxious effects.