Atorvastatin prevents cell damage via modulation of oxidative stress,glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation

Oxygen–glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.     

Atorvastatin Prevents Glutamate Uptake Reduction Induced by Quinolinic Acid Via MAPKs Signaling

Statins have been shown to promote neuroprotection in a wide range of neurological disorders. However, the mechanisms involved in such effects of statins are not fully understood. Quinolinic acid (QA) is a neurotoxin that induces seizures when infused in vivo and promotes glutamatergic excitotoxicity in the central nervous system. The aim of this study was to evaluate the putative glutamatergic mechanisms and the intracellular signaling pathways involved in the atorvastatin neuroprotective effects against QA toxicity. Atorvastatin (10 mg/kg) treatment for 7 days prevented the QA-induced decrease in glutamate uptake, but had no effect on increased glutamate release induced by QA. Moreover, atorvastatin treatment increased the phosphorylation of ERK1 and prevented the decrease in Akt phosphorylation induced by QA. Neither atorvastatin treatment nor QA infusion altered glutamine synthetase activity or the levels of phosphorylation of p38^MAPK or JNK1/2 during the evaluation. Inhibition of MEK/ERK signaling pathway, but not PI3K/Akt signaling, abolished the neuroprotective effect of atorvastatin against QA-induced decrease in glutamate uptake. Our data suggest that atorvastatin protective effects against QA toxicity are related to modulation of glutamate transporters via MAPK/ERK signaling pathway.     

Chronic Stress and Lithium Treatments Alter Hippocampal Glutamate Uptake and Release in the Rat and Potentiate Necrotic Cellular Death After Oxygen and Glucose Deprivation

This study was undertaken to evaluate the effects of chronic variate stress and lithium treatment on glutamatergic activity and neuronal vulnerability of rat hippocampus. Male Wistar rats were simultaneously treated with lithium and submitted to a chronic variate stress protocol during 40?days, and afterwards the hippocampal glutamatergic uptake and release, measured in slices and synaptosomes, were evaluated. We observed an increased synaptosomal [(3)H]glutamate uptake and an increase in [(3)H]glutamate stimulated release in hippocampus of lithium-treated rats. Chronic stress increased basal [(3)H]glutamate release by synaptosomes, and decreased [(3)H]glutamate uptake in hippocampal slices. When evaluating cellular vulnerability, both stress and lithium increased cellular death after oxygen and glucose deprivation (OGD). We suggest that the manipulation of glutamatergic activity induced by stress may be in part responsible for the neuroendangerment observed after stress exposure, and that, in spite of the described neuroprotective effects of lithium, it increased the neuronal vulnerability after OGD.     

Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 M) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.     

Guanosine promotes cytotoxicity via adenosine receptors and induces apoptosis in temozolomide-treated A172 glioma cells

Gliomas are a malignant tumor group whose patients have survival rates around 12 months. Among the treatments are the alkylating agents as temozolomide (TMZ), although gliomas have shown multiple resistance mechanisms for chemotherapy. Guanosine (GUO) is an endogenous nucleoside involved in extracellular signaling that presents neuroprotective effects and also shows the effect of inducing differentiation in cancer cells. The chemotherapy allied to adjuvant drugs are being suggested as a novel approach in gliomas treatment. In this way, this study evaluated whether GUO presented cytotoxic effects on human glioma cells as well as GUO effects in association with a classical chemotherapeutic compound, TMZ. Classical parameters of tumor aggressiveness, as alterations on cell viability, type of cell death, migration, and parameters of glutamatergic transmission, were evaluated. GUO (500 and 1000 μM) decreases the A172 glioma cell viability after 24, 48, or 72 h of treatment. TMZ alone or GUO plus TMZ also reduced glioma cell viability similarly. GUO combined with TMZ showed a potentiation effect of increasing apoptosis in A172 glioma cells, and a similar pattern was observed in reducing mitochondrial membrane potential. GUO per se did not elevate the acidic vesicular organelles occurrence, but TMZ or GUO plus TMZ increased this autophagy hallmark. GUO did not alter glutamate transport per se, but it prevented TMZ-induced glutamate release. GUO or TMZ did not alter glutamine synthetase activity. Pharmacological blockade of glutamate receptors did not change GUO effect on glioma viability. GUO cytotoxicity was partially prevented by adenosine receptor (A₁R and A_2AR) ligands. These results point to a cytotoxic effect of GUO on A172 glioma cells and suggest an anticancer effect of GUO as a putative adjuvant treatment, whose mechanism needs to be unraveled.     

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway

The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6 h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38^MAPK and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (−25%), diminished the phosphorylation of Akt and augmented p38^MAPK and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway.     

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.     

Neuroprotective effect of GMP in hippocampal slices submitted to an in vitro model of ischemia

1. Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage observed in rat hippocampal slices submitted to an in vitro model of ischemia with or without the presence of the ionotropic glutamate receptor agonist, Kainic acid (KA).2. Cellular injury was evaluated by MTT reduction, lactate dehydrogenase (LDH) release assay, and measurement of intracellular ATP levels.3. In slices submitted to ischemic conditions, 1 mM GMP partially prevented the decrease in cell viability induced by glucose and oxygen deprivation and the addition of KA.4. KA or N-methyl-D-aspartate (NMDA) receptor antagonists, -D-glutamylamino-methylsulfonate (GAMS) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, 20 M) also prevented toxicity in hippocampal slices under ischemic conditions, respectively.5. The association of GMP with GAMS or MK-801 did not induce additional protection than that observed with GMP or that classical glutamate receptor antagonists alone.6. GMP, probably by interacting with ionotropic glutamate receptors, attenuated the damage caused by glucose and oxygen deprivation in hippocampal slices. This neuroprotective action of GMP in this model of excitotoxicity is of outstanding interest in the search for effective therapies against ischemic injury.     

Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation

Guanosine, the endogenous guanine nucleoside, prevents cellular death induced by ischemic events and is a promising neuroprotective agent. During an ischemic event, nitric oxide has been reported to either cause or prevent cell death. Our aim was to evaluate the neuroprotective effects of guanosine against oxidative damage in hippocampal slices subjected to an in vitro ischemia model, the oxygen/glucose deprivation (OGD) protocol. We also assessed the participation of nitric oxide synthase (NOS) enzymes activity on the neuroprotection promoted by guanosine. Here, we showed that guanosine prevented the increase in ROS, nitric oxide, and peroxynitrite production induced by OGD. Moreover, guanosine prevented the loss of mitochondrial membrane potential in hippocampal slices subjected to OGD. Guanosine did not present an antioxidant effect per se. The protective effects of guanosine were mimicked by inhibition of neuronal NOS, but not of inducible NOS. The neuroprotective effect of guanosine may involve activation of cellular mechanisms that prevent the increase in nitric oxide production, possibly via neuronal NOS.     

Neuroprotective effects of arachidonic acid against oxidative stress on rat hippocampal slices

Arachidonic acid (AA), 5,8,11,14-eicosateraenoic acid is abundant, active and necessary in the human body. In the present study, we reported the neuroprotective effects and mechanism of arachidonic acid on hippocampal slices insulted by glutamate, NaN(3) or H(2)O(2)in vitro. Different types of models of brain injury in vitro were developed by 1mM glutamate, 10mM NaN(3) or 2mM H(2)O(2). After 30 min of preincubation with arachidonic acid or linoleic acid, hippocampal slices were subjected to glutamate, NaN(3) or H(2)O(2), then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride method. Endogenous antioxidant enzymes activities (SOD, GSH-PX and catalase) in hippocampal slices were evaluated during the course of incubation. MK886 (5 microM; a noncompetitive inhibitor of proliferator-activated receptor [PPAR]alpha), BADGE (bisphenol A diglycidyl ether; 100 microM; an antagonist of PPARgamma) and cycloheximide (CHX; 30 microM; an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by arachidonic acid. Population spikes were recorded in randomly selected hippocapal slices. Arachidonic acid (1-10 microM) dose dependently protected hippocampal slices from glutamate and H(2)O(2) injury (P<0.01), and arachidonic acid (10 microM) can significantly improve the activities of Cu/Zn-SOD in hippocampal slices after 1h incubation. In addition, 10 microM arachidonic acid significantly increased the activity of Mn-SOD and catalase, and decreased the activities of Cu/Zn-SOD to control value after 3h incubation. These secondary changes of SOD during incubation can be reversed by indomethacine (10 microM; a nonspecific cyclooxygenase inhibitor) or AA 861 (20 microM; a 5-lipoxygenase inhibitor). Its neuroprotective effect was completely abolished by BADGE and CHX. These observations reveal that arachidonic acid can defense against oxidative stress by boosting the internal antioxidant system of hippocampal slices. Its neuroprotective effect may be mainly mediated by the activation of PPARgamma and synthesis of new protein in tissue.     

Neuroprotective effects of Ptychopetalum olacoides Bentham (Olacaceae) on oxygen and glucose deprivation induced damage in rat hippocampal slices

Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae) are used in traditional medicine by patients presenting age associated symptoms and those recovering from stroke. The aim of this study is to evaluate the neuroprotective properties of PO ethanol extract (POEE) using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation). Mitochondrial activity, an index of cell viability, was assessed by the MTT assay; in addition, the free radicals content was estimated by the use of dichlorofluorescein diacetate as probe. The OGD ischemic condition significantly impaired cellular viability, and increased free radicals generation. In non-OGD slices, incubation with POEE (0.6 microg/ml) increased (approximately 40%) mitochondrial activity, without affecting free radicals levels. In comparison to OGD controls, slices incubated with POEE (0.6 microg/ml) during and after OGD exposure had significantly increased cellular viability. In addition, at this same concentration, POEE prevented the increase of free radicals content induced by OGD. In view of the fact that respiratory chain inhibition and increased generation of free radicals are major consequences of the ischemic injury, this study suggests that Ptychopetalum olacoides contains useful neuroprotective compounds and, therefore, deserves further scrutiny.     

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti‐inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A₁ receptor, phosphatidylinositol‐3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin‐sensitive G‐protein‐coupled signaling, blockade of adenosine A_2A receptors (A_2AR), but not via A₁ receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.     

Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons

Epidemiological data from retrospective and case-control studies have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer's disease. In addition, ERT has been found to promote cellular correlates of memory and to promote neuronal survival both in vivo and in vitro. Phytoestrogens have been proposed as potential alternatives to ERT. To determine whether phytoestrogens exert estrogen agonist effect in neural tissue, investigations of neuroprotective and neurotrophic efficacy of phytoestrogens were conducted. Six phytoestrogens, genistein, genistin, daidzein, daidzin, formononetin, and equol, were tested for their neuroprotective efficacy against two toxic insults, glutamate excitotoxicity and beta-amyloid(25-35). Neuronal membrane damage was quantitatively measured by lactate dehydrogenase (LDH) release, and neuronal mitochondrial viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid (MTT) assay. Results of these studies demonstrated that all phytoestrogens induced a modest but significant reduction in LDH release following exposure to glutamate and beta-amyloid(25-35). In contrast, none of phytoestrogens induced a significant increase in reduced MTT levels, which occurred in the presence of a full estrogen agonist, 17beta-estradiol. Analysis of the neurotrophic potential of genistein and daidzein, two phytoestrogens that exerted a significant reduction in LDH release, demonstrated that neither of these molecules promoted hippocampal neuron process outgrowth. Results of these analyses indicate that although phytoestrogens exert a neuroprotective effect at the plasma membrane, they do not sustain neuron mitochondrial viability nor do they induce cellular correlates of memory as neurite outgrowth and synaptogenesis are putative mechanisms of memory. Data derived from these investigations would predict that phytoestrogens could exert some neuroprotective effects analogous to that of antioxidants, but that these molecules are not functional equivalents to endogenously active 17beta-estradiol or to estrogen replacement formulations and, therefore, would raise the concern that they may not reduce the risk of Alzheimer's disease or sustain memory function in postmenopausal women.     

Protective Effects of Paeoniflorin Against Glutamate-Induced Neurotoxicity in PC12 Cells via Antioxidant Mechanisms and Ca<Superscript>2+</Superscript> Antagonism

Preclinical and clinical investigations have shown hippocampal neuronal atrophy and destruction were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Paeoniflorin, a monoterpene glycoside, has been reported to display antidepressant-like effects in animal models of behavioral despair. The present study aimed to examine the protective effect of paeoniflorin on glutamate-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that pretreatment with paeoniflorin elevated cell viability, inhibited apoptosis, decreased levels of intracellular reactive oxygen species and malondialdehyde, and enhanced activity of superoxide dismutase in glutamate-treated PC12 cells. Pretreatment with paeoniflorin also reversed the increased intracellular Ca²⁺ concentration and the reduced Calbindin-D28K mRNA level caused by glutamate in PC12 cells. The results suggest that paeoniflorin exerts a neuroprotective effect on glutamate-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative stress and Ca²⁺ overload. This neuroprotective effect may be one of the action pathways accounting for the in vivo antidepressant activity of paeoniflorin.     

Lithium and Valproate Protect Hippocampal Slices Against ATP-induced Cell Death

Lithium and valproate (VPA) are the most commonly prescribed mood-stabilizing drugs. Recently, several studies have reported their neuroprotective properties in several models of neural toxicity and, in some pathological conditions, large amounts of intracellular ATP can be released from damaged cells. In the present study, we investigate the potential neuroprotective effect of lithium and VPA against ATP-induced cell death in hippocampal slices of adult rats. Acute (in vitro) and chronic (in vivo) treatment at therapeutic doses with lithium or VPA significantly prevent the ATP-induced cell death. Lithium and VPA also exerted a synergic effect in the prevention of ATP-induced cell death. Moreover, hippocampal slices prepared from rats chronically treated with lithium or VPA presented a significant reduction in cell death in the presence of cytotoxic extracellular ATP. Although further investigations are necessary, our results show the neuroprotective effect of lithium and VPA against neuronal death induced by extracellular ATP, probably through a different pathway, and suggest novel uses of these drugs in neurogenerative diseases. L. C. Wilot and A. Bernardi equally contributed by this work.     

Neuroprotective Effects of Cactus Polysaccharide on Oxygen and Glucose Deprivation Induced Damage in Rat Brain Slices

1. The neuroprotective effect of cactus polysaccharide (CP) on oxygen and glucose deprivation (OGD) and reoxygenation (REO)-induced damage in the cortical and hippocampal slices of rat brain was investigated. 2. Cell viability was evaluated by using the 2, 3, 5-triphenyl tetrazolium chloride (TTC) method. The fluorescence of propidium iodide (PI) staining was used for quantification of cellular survival, and lactate dehydrogenase (LDH) activity in incubation medium was assessed by LDH assay to evaluate the degree of injury. 3. The OGD ischemic condition significantly decreased cellular viability and increased LDH release in the incubation medium. CP (0.2 mg/l∼2 mg/l) protected brain slices from OGD injury in a dosage dependent manner as demonstrated by increased A 490 value of TTC, decreased PI intensity and LDH release. At the above concentration, CP also prevented the increase of nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity induced by OGD. 4. CP can protect the brain slices (cortical and hippocampus) against injury induced by OGD. Its neuroprotective effect may be partly mediated by the NO/iNOS system induced by OGD insult. Xianju Huang and Qin Li have contributed equally to this article.     

Daphnetin, a Natural Coumarin Derivative, Provides the Neuroprotection Against Glutamate-Induced Toxicity in HT22 Cells and Ischemic Brain Injury

Daphnetin (DAP), a coumarin derivative, has been reported to have multiple pharmacological actions including analgesia, antimalarial, anti-arthritic, and anti-pyretic properties. It is unclear whether DAP has neuroprotective effects on ischemic brain injury. In this study, we found that DAP treatment (i.c.v.) reduced the infarct volume at 24 h after ischemia/reperfusion injury and improved neurological behaviors in a middle cerebral artery occlusion mouse model. Moreover, we provided evidences that DAP had protective effects on infarct volume in neonate rats even it was administrated at 4 h after cerebral hypoxia/ischemia injury. To explore its neuroprotective mechanisms of DAP, we examined the protection of DAP on glutamate toxicity-induced cell death in hippocampal HT-22 cells. Our results demonstrated that DAP protected against glutamate toxicity in HT-22 cells in a concentration-dependent manner. Further, we found that DAP maintained the cellular levels of glutathione and superoxide dismutase activity, suggesting the anti-oxidatant activity of DAP. Since DAP has been used for the treatment of coagulation disorder and rheumatoid arthritis for long time with a safety profile, DAP will be a promising agent for the treatment of stroke.     

Glutamate-induced Toxicity in Hippocampal Slices Involves Apoptotic Features and p38MAPK Signaling

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 μM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38^MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38^MAPK pathway.     

脑益康药物血清对谷氨酸诱导海马神经元损伤的保护作用

目的:探讨脑益康药物血清对谷氨酸(Glu)诱导的海马神经元损伤的保护作用。方法:大鼠海马神经元培养后,采用形态学观察、MTT法及DAPI染色法检测脑益康药物血清对Glu损伤细胞活力的影响,采用RT-PCR和免疫组化方法检测脑益康药物血清对Glu损伤细胞PTEN表达的影响。结果:脑益康药物血清可明显提高Glu损伤的海马神经元的细胞活力,减少PTEN的表达。结论:脑益康药物血清对Glu诱导的海马神经元损伤有保护作用,其机制可能与减少PTEN表达,抑制神经元凋亡有关。     

Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.