6-cyano-7-nitroquinoxaline-2-3-dione and Necrosis

In term and preterm neonates, massive glutamate release can lead to excitotoxic white-matter and cortical lesions. Because of its high permeability toward calcium, the N-methyl-D-aspartic acid (NMDA) receptor is thought to play an important role in excitotoxic lesions and NMDA antagonists therefore hold promise for neuroprotection. We found that, in neonatal mouse cortex, a given NMDA concentration exerted either excitotoxic or antiapoptotic effects depending on the cortical layers. In layer VI, NMDA led to excitotoxicity, sustained calcium mobilization, and necrosis of Gad67GFP neurons. In the immature layers II-IV, NMDA decreased apoptosis and induced transient calcium mobilization. The NMDA antagonist MK801 acted as a potent caspase-3 activator in immature layers II-IV and affected gamma aminobutyric acid (GABA)ergic interneurons. The apoptotic effect of MK801-induced BAX expression, mitochondrial potential collapse and caspase-9 activation. In vivo Bax small interfering ribonucleic acid and a caspase-9 inhibitor abrogated MK801-induced apoptosis and pyknotic nucleus formation. Ketamine, an anesthetic with NMDA antagonist properties, mimicked the apoptotic effects of MK801. These data indicate a dual effect of glutamate on survival of immature and mature GABAergic neurons and suggest that ketamine may induce apoptosis of immature GABAergic neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Glutamate Decarboxylase; Glutamic Acid; Green Fluorescent Proteins; In Vitro Techniques; Interneurons; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mice; Mice, Transgenic; N-Methylaspartate; Necrosis; RNA, Small Interfering

The ratio of necrosis to apoptosis and the mechanisms of apoptosis were studied during neurodegeneration induced by glutamate and selective agonists of glutamate receptors - N-methyl-D-aspartate (NMDA) and kainate. Experiments were performed on primary cultures (seven days in vitro) of rat cerebral cortex neurons. Apoptosis and necrosis were identified using a vital fluorescence rapid test with staining with acridine orange and ethidium bromide. Immunocytochemistry in combination with confocal microscopy was used to visualize apoptotic proteins. Agonists (240 min) caused neuron death via both processes, though the proportion of necrotic cells when neurodegeneration was induced by NMDA and kainate was significantly less than when neurodegeneration was induced with glutamate. The neurotoxic effect of 3 mM glutamate was mediated via alpha-amino-3-(3-hydroxy-5-methylisoxazole-4-yl)propionate (AMPA) and kainate receptors, as it was blocked by 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX). Activation of NMDA receptors led to the development of apoptosis without involvement of caspases, due to the direct action of apoptosis-inducing factor (AIF) on neuron nuclei. Activation of AMPA-kainate receptors was accompanied by the development of apoptosis via the caspase-dependent pathway. Thus, these data identified the receptor dependence of the mechanisms of apoptosis during the neurotoxic action of glutamate.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Active Transport, Cell Nucleus; Animals; Apoptosis; Apoptosis Inducing Factor; Caspase 3; Cell Nucleus; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Kainic Acid; N-Methylaspartate; Necrosis; Neurons; Rats; Rats, Wistar; Receptors, Glutamate; Tumor Suppressor Protein p53

A contribution of necrosis and apoptotis as well as the particular apoptosis pathways in neuro-degeneration induced by glutamate and selective glutamate receptor agonists, NMDA and kainate, were studied. In experiments on primary neuron cultures of 7 days in vitro from embryonic rat cortex, the necrosis and apoptosis were recognized using vital fluorescence acridine orange and ethidium bromide staining. Immunostaining was used to visualize apoptotic peptides such as P53, Cas-3 and AIF. Death of neurons occurred by both necrosis and apoptosis following 240 min 3 mM glutamate, 30 microM NMDA and 30 microM kainate exposure. Quantities of necrotic neurons in the presence of NMDA and kainate were substantially reduced when compared to the glutamate action. The glutamate effects were realized through predominant activation of AMPA- and kainate receptors, since it could be greatly suppressed by 30 microM CNQX. AIF but not Cas-3, was found in a large amount of neurons when apoptosis was evoked by the selective NMDA receptor activation. On the contrary, during apoptosis induced by glutamate and kainate, many cells contained Cas-3 in nuclei rather than the AIF. The data suggest that apoptosis induced by the NMDA receptor activation develops through the caspase-3-independent pathway that involves direct AIF accumulation in nuclei. The AMPA/kainate receptor mediated apoptosis includes the caspase-3-dependent mechanism.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Active Transport, Cell Nucleus; Animals; Apoptosis; Apoptosis Inducing Factor; Caspase 3; Cell Nucleus; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Kainic Acid; N-Methylaspartate; Necrosis; Neurons; Rats; Rats, Wistar; Receptors, Glutamate; Tumor Suppressor Protein p53

Sustained alteration in [Ca(2+)]i triggers neuronal death. We examined morphological and signaling events of Ca(2+)-deficiency-induced neuronal death. Cortical cell cultures exposed to 20 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 microg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4-12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons deficient in [Ca(2+)]i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Apoptosis; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Chelating Agents; Chromans; Cycloheximide; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Egtazic Acid; Fetus; Kinetics; Mice; Mice, Inbred ICR; Necrosis; Neocortex; Neuroglia; Neurons; Neuroprotective Agents; Reactive Oxygen Species; Time Factors

Ischemic injury of immature oligodendrocytes is a major component of the brain injury associated with cerebral palsy, the most common human birth disorder. We now report that cultured immature oligodendrocytes [O4(+)/galactoceramide (GC)(-)] are exquisitely sensitive to ischemic injury (80% of cells were dead after 25.5 min of oxygen and glucose withdrawal). This rapid ischemic cell death was mediated by Ca(2+) influx via non-NMDA glutamate receptors. The receptors were gated by the release of glutamate from the immature oligodendrocytes themselves via reverse glutamate transport and included a significant element of autologous feedback of glutamate from cells onto their own receptors. High (> or = 100 microM) extracellular glutamate was protective against ischemic injury as a result of non-NMDA glutamate receptor desensitization. Other potential pathways of Ca(2+) influx, such as voltage-gated Ca(2+) channels, NMDA receptors, or the Na(+)-Ca(2+) exchanger, did not significantly contribute to ischemic Ca(2+) influx or cell injury. Release of Ca(2+) from intracellular stores was also not an important factor. In agreement with previous studies, more mature oligodendrocytes (O4(-)/GC(+)) were found to be less sensitive to ischemic injury than were the immature cells studied here.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Brain Ischemia; Calcium; Cell Death; Cell Size; Cells, Cultured; Cellular Senescence; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Extracellular Space; Feedback; Glutamic Acid; Necrosis; Oligodendroglia; Rats; Rats, Long-Evans; Receptors, Glutamate

Evidence has accumulated that Zn2+ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn2+ neurotoxicity. Inclusion of 30-300 microM Zn2+ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn2+ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn2+ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn(2+)-induced neuronal necrosis. Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn(2+)-induced free radical injury. Kainate or AMPA facilitated Zn2+ entry and potentiated Zn2+ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn2+ neurotoxicity. These findings indicate that entry and accumulation of Zn2+ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Brain-Derived Neurotrophic Factor; Cells, Cultured; Cerebral Cortex; Chromans; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Dopamine Agonists; Drug Synergism; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Free Radicals; Hypoglycemic Agents; Insulin; Kainic Acid; Lipid Peroxidation; Mice; Microscopy, Electron; Mitochondrial Swelling; Necrosis; Nerve Degeneration; Neurons; Neurotoxins; Oligopeptides; Oxidative Stress; Pregnancy; Zinc

Indirect glutamate toxicity can be demonstrated by exposing dissociated rat hippocampal cultures to the media of the same culture transiently exposed (1 min) to glutamate (0.5 mM). The toxicity was maximum when the media was collected 5 min after the glutamate exposure. While the primary glutamate toxicity was attenuated by ionotropic glutamate receptor antagonists, the transferred, indirect toxicity was unaffected by the same antagonists. The changes in nuclear morphology indicated chromatin condensation and nuclear fragmentation in both primary and transferred toxicity. The stain for DNA damage by TUNEL method also revealed cells staining positive in both primary and transferred glutamate toxicity. These observations demonstrate that glutamate-induced neurotoxicity can be propagated to the uninjured cells by an unknown toxin released into the extracellular space. This neurotoxin induced both apoptosis and necrosis in cultured rat hippocampal cells.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Apoptosis; Cell Death; Culture Techniques; Dizocilpine Maleate; DNA Fragmentation; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Necrosis; Neuroglia; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

We examined the effects of nerve growth factor (NGF) on free radical neurotoxicity in striatal cell cultures. Following exposure to 30 microM Fe2+ or 1 mM L-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, striatal neurons underwent cell body swelling and then widespread death over the next 24 h. The degeneration was prevented by addition of 100 microM trolox, an antioxidant. Addition of 100 ng/ml BDNF beginning 12 h before Fe2+ or BSO potentiated necrosis of most striatal neurons after exposure to 10 microM Fe2+ or 1 mM BSO. In contrast, treatment with 100 ng/ml NGF selectively potentiated the oxidative degeneration of striatal cholinergic neurons. The present findings provide additional evidence that NGF, like other neurotrophins, can potentiate oxidative neuronal cell necrosis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholinesterase; Animals; Antioxidants; Brain-Derived Neurotrophic Factor; Buthionine Sulfoximine; Cells, Cultured; Chromans; Corpus Striatum; Cycloheximide; Dizocilpine Maleate; Drug Synergism; Fetus; Free Radicals; Iron; Necrosis; Nerve Degeneration; Nerve Growth Factors; Neuroglia; Neurons; Neurotoxins; Rats; Rats, Sprague-Dawley