dizocilpine-maleate and thiazolyl-blue

Microglia represent the main cellular targets of HIV-1 in the brain. Infected and/or activated microglia play a pathogenic role in HIV-associated neurocognitive disorders (HAND) by instigating primary dysfunction and subsequent death of neurons. Although microglia are known to secrete neurotoxins when infected with HIV-1, the detailed mechanism of neurotoxicity remains unclear. Using a human microglia primary culture system and macrophage-tropic HIV-1 strains, we have now demonstrated that HIV-1 infection of microglia resulted in a significant increase in extracellular glutamate concentrations and elevated levels of neurotoxicity. RNA and protein analysis revealed upregulation of the glutamate-generating enzyme glutaminase isoform glutaminase C in HIV-1-infected microglia. The clinical relevance of these findings was further corroborated with investigation of postmortem brain tissues. The glutaminase C levels in the brain tissues of HIV dementia individuals were significantly higher than HIV serum-negative control and correlated with elevated concentrations of glutamate. When glutaminase was subsequently inhibited by siRNA or by a small molecular inhibitor, the HIV-induced glutamate production and the neuronal loss was diminished. In conclusion, these findings support glutaminase as a potential component of the HAND pathogenic process as well as a novel therapeutic target in their treatment.

Topics: Analysis of Variance; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Brain; Caspase 3; Cells, Cultured; Chromatography, High Pressure Liquid; Disintegrins; Dizocilpine Maleate; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; Fetus; Gene Expression Regulation, Viral; Glutamic Acid; Glutaminase; Glutamine; HIV Infections; HIV-1; Humans; Microglia; Microtubule-Associated Proteins; Receptors, Cell Surface; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Time Factors

We used human NT2-N neurons to investigate delayed effects of short-term exposure to unconjugated bilirubin (UCB). Cell viability was evaluated with MTT reduction assays and nuclear morphology. A 6-h exposure to 1, 5, or 25 microM UCB and serum deprivation (SED) significantly diminished MTT reduction. 96 h after rescue of neurons with removal of UCB and re-incubation in the original serum-containing medium, delayed effects were evident as recovery (1 microM UCB), intermediate cell death (5 microM UCB), or near complete cell death (25 microM UCB). The impact of 6 h of SED alone appeared to be modest in rescued neurons. In this model, co-treatment with the specific caspase-3 inhibitor, zDEVD.FMK (100 microM), or the pancaspase inhibitor zVAD.FMK (100 microM) did not improve viability in rescued neurons exposed to 5 microM UCB, while treatment with the NMDA receptor antagonist MK-801 (1 microM) enhanced the number of undamaged nuclei (86 +/- 14% versus 50 +/- 12%, P = 0.001). MK-801 had, however, no impact on MTT reduction. In a different model with a 102-h continuous exposure to UCB and SED, we found a significant additional toxic impact of serum deprivation. Separate experiments suggested that this was a result of late caspase-mediated toxicity. We conclude that UCB-mediated effects may be reversible in this model. Blockade of excitotoxic mechanisms, but not caspase activity may prevent delayed cell death.

Topics: Apoptosis; Bilirubin; Caspase Inhibitors; Cell Death; Cell Line; Cell Nucleus; Culture Media, Serum-Free; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Humans; Neurons; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Thiazoles

The neuroprotective effects of Delta(9)-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

Topics: alpha-Tocopherol; Animals; Benzimidazoles; Benzoxazines; Blotting, Northern; Blotting, Western; Calcium Channel Blockers; Calcium Channels; Capsaicin; Cell Count; Cell Death; Cell Lineage; Dizocilpine Maleate; DNA, Single-Stranded; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Excitatory Amino Acid Agonists; Immunohistochemistry; In Situ Nick-End Labeling; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Neuroprotective Agents; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Ruthenium; Tetrazolium Salts; Thiazoles; TRPC Cation Channels

The present study was performed to examine how the stimulation of gamma-aminobutyric acid (GABA) receptor affects amyloid beta protein (25-35) (Abeta (25-35)), a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. Abeta (25-35) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca(2+) channel blocker, and N(G)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor. Pretreatment with muscimol, a GABAA receptor agonist, over a concentration range of 0.1-10microM 24h before the treatment with 10microM Abeta (25-35) showed concentration-dependent inhibition on the Abeta (25-35)-induced neuronal apoptotic death. However, baclofen (1 and 10microM), a GABAB receptor agonist, failed to inhibit the Abeta (25-35)-induced neuronal death. In addition, pretreatment with muscimol (1microM) for 24h inhibited the Abeta (25-35) (10microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity in cultured neurons. These neuroprotective effects of muscimol (1microM) were completely blocked by the simultaneous treatment with 10microM bicuculline, a GABAA receptor antagonist, indicating that the protective effects of muscimol were due to GABAA receptor stimulation. When, however, treated just 15min before the treatment with Abeta (25-35), muscimol (1microM) did not show any protective effect against Abeta (25-35) (10microM)-induced neurotoxicity in cultured neurons. These results suggest that the chronic activation of GABAA receptor may ameliorate Abeta-induced neurotoxicity by interfering with the increase of [Ca(2+)]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Baclofen; Blotting, Western; Calcium; Calcium Channel Blockers; Caspase 3; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Female; GABA Agonists; Glutamic Acid; Muscimol; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Peptide Fragments; Pregnancy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, GABA-A; Tetrazolium Salts; Thiazoles; Time Factors; Verapamil

In search of novel therapeutic approaches for Alzheimer's disease (AD), we report herein the identification, design, synthesis, and pharmacological activity of (4-ethyl-piperaz-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity. (4-ethyl-piperaz-1-yl)-phenylmethanone is a common substructure shared by molecules isolated from plants of the Asteraceae genus, traditionally used as restorative of lost or declining mental functions. (4-Ethyl-piperaz-1-yl)-phenylmethanone displayed strong neuroprotective properties against Abeta1-42 and reversed Abeta1-42-induced ATP depletion on neuronal cells, suggesting a mitochondrial site of action. Abeta1-42 has been described to induce a hyperactivity of the glutamate network in neuronal cells. (4-Ethyl-piperaz-1-yl)-phenylmethanone also inhibited the neurotoxic effect that glutamate displayed on PC12 cells, suggesting that the reduction of glutamate-induced neurotoxicity may be one of the mechanisms by which this compound exerts its neuroprotective properties against the deleterious effects of the Abeta1-42. These data suggest that the identified (4-ethyl-piperaz-1-yl)-phenylmethanone chemical entity exerts neuroprotective properties and may serve as a lead compound for the development of novel therapies for AD.

Topics: Adenosine Triphosphate; Amyloid beta-Peptides; Analysis of Variance; Animals; Cell Survival; Chromatography, Thin Layer; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Free Radicals; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Phytoestrogens; Piperazines; Plant Preparations; Rats; Tetrazolium Salts; Thiazoles

The human immunodeficiency virus type 1 (HIV-1) glycoprotein gp120 causes neuronal cell death; however, the molecular mechanisms of the neurotoxic effect remain largely unresolved. It has been suggested that gp120 evokes cell death by inducing the release of neurotoxins, including glutamate. The objective of this work was to examine the role of glutamate in gp120-mediated neurotoxicity. We used as an experimental tool cerebellar granule cells prepared from 8-day-old rat cerebella, in which both glutamate and gp120 cause cell death. Cerebellar granule neurons were exposed to gp120 or glutamate alone or in combination with the glutamate receptor antagonist MK801 as well as other antiglutamatergic compounds. Cell viability was measured at various times by using several markers of cell death and apoptosis. MK801, at a concentration that blocked glutamate-induced neuronal cell death, failed to prevent gp120-mediated apoptotic cell death. Moreover, interleukin-10, which has previously been shown to block glutamate toxicity in these neurons, was not neuroprotective against gp120. Because gp120 toxicity is mediated by activation of the chemokine receptor CXCR4, neurons were incubated with the CXCR4 inhibitor AMD3100. This compound prevented gp120- but not glutamate-mediated cell death. These findings suggest that gp120 is toxic to neurons even in the absence of the virus and that the toxic mechanism involves primarily activation of CXCR4 receptor. Therefore, antagonists to the CXCR4 receptor may be more suitable compounds for inhibiting HIV-1 neurotoxicity.

Topics: Animals; Animals, Newborn; Anti-HIV Agents; Benzimidazoles; Benzylamines; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Cerebellum; Cyclams; Dizocilpine Maleate; Drug Interactions; Enzyme Activation; Glutamic Acid; Heterocyclic Compounds; HIV Envelope Protein gp120; Immunohistochemistry; Interleukin-10; Neurons; Neuroprotective Agents; Neurotoxins; Propidium; Rats; Rats, Sprague-Dawley; Receptors, CXCR4; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Thiazoles; Time Factors

Phospholipase C (PLC)-delta1 protein appears to accumulate aberrantly in Alzheimer's disease brains and its expression is reported to be induced by overstimulation of N-methyl-D-aspartate (NMDA) receptor, but there is little knowledge on its physiological role. To clarify this, we examined the expression profile of PLC-delta1 in primary cultured rat cortical neurons treated with NMDA or peroxynitrite, in comparison with those of PLC-beta1 and -gamma1, the overexpression of both of which protects cells from oxidative stress. Overstimulation of NMDA receptor decreased and increased the expression of PLC-beta1 and -delta1, respectively, but did not affect that of PLC-gamma1, in the neurons. The viability of neurons decreased depending on the period of treatment with S-nitroso-N-acetyl D,L-penicillamine (SNAP), there being a significant decrease on 9 h treatment. On examination of the expression profiles of PLC isozymes after treatment of neurons with SNAP, PLC-beta1 was found to be increased after 1h treatment and decreased after 9 h treatment, while PLC-delta1 was significantly increased, especially after 5 h treatment. Peroxynitrite treatment caused a dose-dependent decrease in the viability of neurons, and expression of PLC-beta1 was increased by a nontoxic level of peroxynitrite and decreased by a toxic level of it, while that of PLC-delta1 was increased by a sublethal level of it. These findings suggested that induction of PLC-beta1 might protect neurons from oxidative stress, but that of PLC-delta1 might have the opposite role, although both isozymes responded to oxidative stress.

Topics: Animals; Blotting, Western; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Isoenzymes; N-Methylaspartate; Neurons; Penicillamine; Peroxynitrous Acid; Rats; Tetrazolium Salts; Thiazoles; Time Factors; Type C Phospholipases

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.

Topics: Activating Transcription Factor 2; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Calcium; Cell Count; Cell Survival; Cells, Cultured; Cerebellum; Chromatin; Cyclic AMP Response Element-Binding Protein; Dizocilpine Maleate; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glutamic Acid; Humans; Membrane Potentials; Microscopy, Confocal; Minocycline; N-Methylaspartate; Neurons; Neuroprotective Agents; p38 Mitogen-Activated Protein Kinases; Patch-Clamp Techniques; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Serine; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factors

The haem precursor 5-aminolevulinic acid (ALA) has been proposed to be involved in the neurological dysfunctions presented by patients with acute porphyrias. The effects of ALA on the [3H]glutamate and [3H]MK-801 (dizocilpine) binding to rat cortical membranes and on [3H]glutamate uptake by rat astrocyte cultures were evaluated in the present study in order to elucidate the interaction of ALA with the glutamatergic system and its possible contribution to the in vivo excitatory properties of ALA. ALA (0-1mM) did not affect the binding of 100 nM [3H]glutamate, nor the equilibrium binding constants (K(d) and B(max)) of this neurotransmitter in rat or human cortical membranes. The binding of the NMDA-channel blocker, [3H]MK-801, was not affected by ALA (0-10mM) either. ALA (0-3mM) dose-dependently inhibited glutamate uptake by astrocyte cultures. ALA significantly reduced both the K(m) and V(max) of glutamate uptake indicating an uncompetitive inhibition. The inhibitory effect was irreversible and apparently related to the selective inhibition of the GLT-1 (EAAT2) subtype of glutamate transporter. The finding that ALA significantly increased astrocyte lipoperoxidation in astrocytes incubated under these conditions suggests that the inhibitory effect of ALA might be related to an oxidative damage of the transporter. We propose that the inhibition of glutamate uptake may underlie ALA-induced convulsions.

Topics: Aminolevulinic Acid; Animals; Astrocytes; Bucladesine; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Female; Glutamic Acid; Humans; Kinetics; Nerve Tissue Proteins; Rats; Rats, Wistar; Synaptic Transmission; Tetrazolium Salts; Thiazoles; Thiobarbituric Acid Reactive Substances

In this study, we demonstrated that a snake presynaptic toxin, beta-bungarotoxin (beta-BuTX), was capable of binding to NMDA receptors of the cultured primary neurons (cerebellar granule neurons, CGNs). We labeled beta-BuTX with fluorescent FITC (FITC-beta-BuTX) and showed that the binding of FITC-beta-BuTX was inhibited by unlabeled beta-BuTX and MK801 (an NMDA receptor antagonist). Meanwhile, the binding of [3H]-MK801 was also reduced by unlabeled MK801 and beta-BuTX. In addition, beta-BuTX produced a very potent neurotoxic effect on mature CGNs with the EC(50) of 3ng/ml (equivalent to 144pM), but was less effective in immature CGNs. We explored the signaling pathway of neuronal death and found that it was apparently due to the excessive production of reactive oxygen species (ROS) induced by beta-BuTX. MK801 and antioxidants (Vitamin C, N-acetylcysteine (NAC), melatonin, epigallocatechin gallate (EGCG), superoxide dismutase (SOD) and catalase) attenuated not only ROS production but also beta-BuTX-neurotoxicity. The downstream signaling of ROS was identified as the activation of caspase-3. Caspase inhibitor (z-DEVD-fmk) and antioxidants depressed both caspase-3 activation and neurotoxicity. Based on these findings and our previous reports, we conclude that the binding and activation of NMDA receptors by beta-BuTX was crucial step to produce the potent neurotoxic effect. The binding of NMDA receptors resulted in excessive Ca(2+) influx, followed by ROS production and activation of caspase-3. This snake toxin is considered not only to be a useful tool for exploring the death-signaling pathway of neurotoxicity, but also provides a model for searching neuroprotective agents.

Topics: Animals; Antioxidants; Bungarotoxins; Caspases; Cell Nucleus; Cell Survival; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Kinetics; Male; Neurites; Neurons; Neurotoxins; Rats; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Tetrazolium Salts; Thiazoles

During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures.. Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively.. At clinically relevant concentrations (0.05-10 microm), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol.. Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.

Topics: Amino Acid Transport System X-AG; Amino Acids; Anesthetics, Intravenous; Animals; Brain Ischemia; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Gangliosides; Glucose; Glutamic Acid; Immunohistochemistry; L-Lactate Dehydrogenase; Microscopy, Phase-Contrast; Neurons; Neuroprotective Agents; Oxygen; Pregnancy; Propofol; Rats; Rats, Wistar; Tetrazolium Salts; Thiazoles

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.

Topics: Agmatine; Animals; Botulinum Toxins; Cell Death; Cell Survival; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; L-Lactate Dehydrogenase; Neurons; Potassium; Quinoxalines; Rats; Tetrazolium Salts; Thiazoles

Hypoxia/reoxygenation (H/R) causes cell injury/death. We examined the protection by drugs intervening at various stages of the injury cascade in cultured neurons and glia. Primary cultures of rat cortical neurons and mixed glia were subjected to H/R. Measurements of cell death (by lactate dehydrogenase release into the medium) and viability (by MTT reduction) indicated that H/R led to time-dependent injury in both neuronal and mixed glial cultures. The extent of cell injury in neurons was significantly greater than in glia cells. Pretreatment with (+)-MK-801 hydrogen maleate (MK-801) (an N-methyl-D-aspartate antagonist), N(omega)-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of nitric oxide synthase) or free radical scavengers reduced the extent of the H/R-elicited neuronal damage. MK-801, in contrast, was without effect on glial cells while L-NAME was effective. Our results suggest differential mechanism(s) and susceptibility to injury caused by H/R for neurons and mixed glia.

Topics: Animals; Antioxidants; Astrocytes; Cell Death; Cells, Cultured; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Free Radicals; Hypoxia-Ischemia, Brain; L-Lactate Dehydrogenase; Neuroglia; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pregnancy; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tetrazolium Salts; Thiazoles

While seizure attack is one of the serious complications during the hyperbaric oxygen (HBO) therapy, there is still no direct evidence showing that HBO can induce neuronal damage in the brain. The objective of this study was first to investigate whether HBO would lead to neurotoxicity in the primary rat cortical culture. Second, since alterations in neurotransmitters have been suggested in the pathophysiology of central nervous system (CNS) oxygen toxicity, the protective effects of the N-methyl-D-aspartate (NMDA) receptor antagonism and nitric oxide (NO) synthase inhibition on the HBO-induced neuronal damage were examined. The results showed that HBO exposure to 6 atmosphere absolute pressure (ATA) for 30, 60, and 90 min increased the lactate dehydrogenase (LDH) activity in the culture medium in a time-dependent manner. Accordingly, the cell survival, measured by the 3,(4,5-dimethyl-2-thiazolyl)2, 5-diphenyl-tetrazolium bromide (MTT) assay, was decreased after HBO exposure. Pretreatment with the NMDA antagonist MK-801 protected the cells against the HBO-induced damage. The protective effect was also noted in the cells pretreated with L-N(G)-nitro-arginine methyl ester, an NO synthase inhibitor. Thus, our results suggest that activation of NMDA receptors and production of NO play a role in the neurotoxicity produced by hyperbaric oxygen exposure.

Topics: Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Culture Media, Conditioned; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hyperbaric Oxygenation; L-Lactate Dehydrogenase; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxygen; Pressure; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Thiazoles

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.

Topics: Analysis of Variance; Animals; Apoptosis; Benzothiadiazines; Binding, Competitive; Blotting, Western; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Diuretics; Dizocilpine Maleate; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Ethanol; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Intracellular Fluid; Long-Term Potentiation; N-Methylaspartate; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride Symporter Inhibitors; Tetrazolium Salts; Thiazoles

IgGs from sera containing antiphospholipid antibodies (aPL), detected as antibodies to cardiolipin, or control sera were incubated with rat cerebellar granule cells in primary culture. Using a mitochondrial dehydrogenase activity assay (MTT test), aPL IgGs were shown to decrease MTT metabolism after 24 h incubation with the cells, and to cause non-toxic amounts of glutamate to become neurotoxic when added to the cells for 45 min. Acute and chronic aPL toxicity were prevented by MK-801. Sera containing aPL bound to intact cerebellar neurons, as revealed by an immunofluorescent technique. These results suggest that antiphospholipid antibodies interfere with excitatory pathways in glutamatergic cerebellar granule cells by a mechanism involving overactivation of the NMDA glutamate receptor.

Topics: Animals; Antibodies, Antiphospholipid; Cerebellum; Coloring Agents; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Humans; Immunoglobulin G; Mitochondria; Neurons; Neurotoxins; Oxidation-Reduction; Oxidoreductases; Rats; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Thiazoles