2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and Necrosis

When excitotoxic mechanisms are blocked, severe or prolonged hypoxia and hypoxia-ischemia can still kill neurons, by a mechanism which is poorly understood. We studied this "non-excitotoxic hypoxic death" in primary cultures of rat dentate gyrus neurons. Many neurons subjected to hypoxia in the presence of blockers of ionotropic glutamate receptors developed the electron microscopic features of necrosis. They showed early mitochondrial swelling, loss of mitochondrial membrane potential and cytoplasmic release of cytochrome c, followed by activation of caspase-9, and by caspase-9-dependent activation of caspase-3. Caspase inhibitors were neuroprotective. These results suggest that "non-excitotoxic hypoxic neuronal death" requires the activation in many neurons of a cell death program originating in mitochondria and leading to necrosis.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Animals, Newborn; Caspases; Cell Survival; Cells, Cultured; Dentate Gyrus; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Activation; Hypoxia; L-Lactate Dehydrogenase; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; Mitochondria; Necrosis; Neurons; Neuroprotective Agents; Quinoxalines; Rats; Sodium Cyanide

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzylo

Topics: Amino Acid Transport System X-AG; Analysis of Variance; Animals; Animals, Newborn; Aspartic Acid; Cell Death; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Histocytochemistry; Hypoxia; Immunohistochemistry; In Vitro Techniques; Microtubule-Associated Proteins; Necrosis; Neurofilament Proteins; Neurons; Neuroprotective Agents; Propidium; Quinoxalines; Rats; Receptors, Glutamate; Time Factors

High extracellular potassium induces spreading depression-like depolarizations and elevations of extracellular glutamate. Both occur in the penumbra of a focal ischemic infarct, and may be responsible for the spread of cell death from the infarct core to the penumbra. We have modeled this situation with microdialysis of an isotonic high-potassium solution into the normal rat amygdala for 70 min. This elevates extracellular glutamate up to 8-fold or more and produces irreversibly damaged, acidophilic neurons. NMDA-receptor blockade protects neurons and reduces the elevation of extracellular glutamate. Here we investigated the effects of sodium channel blockade with the voltage-sensitive sodium channel blocker tetrodotoxin and the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide disodium (NBQX disodium) on high potassium-induced neuronal death and extracellular glutamate elevations. The acidophilic neurons produced are necrotic by ultrastructural examination. Tetrodotoxin, at dialysate concentrations of 33, 330 and 3300 microM (only a small fraction is extracted by tissue), markedly reduced the elevations of glutamate in rat amygdala at nearly all time points during high-potassium perfusion, but it reduced tissue edema only at the highest concentration, and it was neuroprotective only if dialyzed prior to high-potassium microdialysis (at 330 microM concentration). Although both 250 microM (6.2% is extracted by tissue) and 500 microM NBQX reduced elevations of glutamate, neither was neuroprotective, and neuropil edema was not reduced by either concentration. Our results suggest that in vivo, sodium influx through voltage-sensitive sodium channels but not through ligand-gated AMPA receptor channels contributes to high potassium-induced neuronal necrosis.

Topics: Amygdala; Animals; Cell Death; Dose-Response Relationship, Drug; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamic Acid; Ion Channel Gating; Male; Necrosis; Neurons; Potassium; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Isoflurane and pentobarbital can reduce alpha-amino-d-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated toxicity in vitro. However, their effect on AMPA toxicity in vivo is not known. The present study was undertaken to evaluate the effects of isoflurane and pentobarbital on the in vivo neurotoxicity produced by AMPA.. Wistar-Kyoto rats were allocated to one of seven groups (n = 8 per group): isoflurane 1 minimum alveolar concentration, isoflurane electroencephalogram burst suppression (EEG-BS), low-dose pentobarbital, pentobarbital EEG-BS, NBQX, conscious, and sham groups. AMPA 30 nm was injected into the cortex. An equivalent volume of cerebrospinal fluid was injected into the cortex in the sham group. In the NBQX group, 200 nm NBQX was injected into the cortex with the AMPA. In the isoflurane and pentobarbital groups, anesthesia was maintained for a period of 5 h. Animals in the conscious, NBQX, and sham groups were allowed to awaken immediately after the AMPA injection. Injury to the cortex was evaluated 48 h later.. Isoflurane reduced AMPA-induced cortical injury (4.5 +/- 1.9 mm3 and 1.7 +/- 0.8 mm3 in the 1 minimum alveolar concentration and EEG-BS groups, respectively) in comparison to the conscious group (7.2 +/- 0.8 mm3). Pentobarbital reduced cortical injury when administered in EEG-BS doses (2.2 +/- 0.7 mm3) but not when administered in sedative doses (8.6 +/- 0.9 mm3). NBQX reduced AMPA-induced cortical injury (1.2 +/- 0.5 mm3).. Isoflurane and pentobarbital reduced cortical AMPA excitotoxicity. The magnitude of injury reduction was similar to that produced by NBQX when the anesthetics were administered in EEG-BS doses. These results are consistent with the previously demonstrated ability of isoflurane and pentobarbital to inhibit AMPA receptor responses.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Anesthetics, Inhalation; Animals; Blood Pressure; Cerebral Cortex; Cerebral Infarction; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; GABA Modulators; Heart Rate; Image Processing, Computer-Assisted; Isoflurane; Necrosis; Pentobarbital; Quinoxalines; Rats; Rats, Inbred WKY; Receptors, AMPA

Ion-selective microelectrodes were used to study acute effects of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy- 5-methyl-4-isoxazole (AMPA) receptor blockade on posttraumatic calcium disturbances. An autoradiographic technique with 45 Ca2+ was used to study calcium disturbances at 8, 24, and 72 h. Compression contusion trauma of the cerebral cortex was produced by a 21-g weight dropped from a height of 35 cm onto a piston that compressed the brain 2 mm. Pre- and posttrauma interstitial [Ca2+] ([Ca2+]e) concentrations were measured in the perimeter, i.e., the shear stress zone (SSZ) and in the central region (CR) of the trauma site. For the [Ca2+]e studies the animals were divided into controls and groups pretreated with dizocilipine maleate (MK-801) or with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[F]quinoxaline (NBQX). In all groups, [Ca2+]e decreased from pretrauma values of approximately 1 mM to posttraumatic values of 0.1 mM in both the CR and the SSZ. This was followed by a slow restitution toward pretraumatic levels during the 2-h observation period. There was no significant difference in recovery pattern between controls and pretreated animals. Accumulation of 45Ca2+ and serum proteins was seen in the entire SSZ, while neuronal necrosis was confined to a narrow band within the SSZ. The CR was unaffected apart from occasional eosinophilic neurons and showed no accumulation of 45Ca2+. Posttraumatic treatment with MK-801 or NBQX had no obvious effect on neuronal injury in the SSZ. We conclude that (a) acute [Ca2+]e disturbances in compression contusion brain trauma are not affected by blockade of NMDA or AMPA receptors, (b) 45Ca2+ accumulation in the SSZ reflects mainly protein accumulation due to blood-brain barrier breakdown rather than cell death, and (c) acute cellular Ca2+ over-load per se does not seem to be a major determinant of cell death after cerebral trauma in our model.

Topics: Animals; Autoradiography; Brain Injuries; Calcium; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Ion Channels; Male; Necrosis; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

The effects of neurotrophins on several forms of neuronal degeneration in murine cortical cell cultures were examined. Consistent with other studies, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 all attenuated the apoptotic death induced by serum deprivation or exposure to the calcium channel antagonist nimodipine. Unexpectedly, however, 24-hour pretreatment with these same neurotrophins markedly potentiated the necrotic death induced by exposure to oxygen-glucose deprivation or N-methyl-D-aspartate. Thus, certain neurotrophins may have opposing effects on different types of death in the same neurons.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Calcium; Cell Death; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Mice; N-Methylaspartate; Necrosis; Nerve Degeneration; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; Neurotrophin 3; Quinoxalines; Receptors, AMPA

Pharmacologic inhibition of the N-methyl-D-aspartate (NMDA) glutamate receptor can reduce the neurologic injury associated with hypothermic circulatory arrest; however, other receptor subtypes, such as the alpha-amino-3-hydroxy-5-methylisoazole-4-propionic acid/kainate or AMPA/kainate subtype, may predominate in the adult brain. In this experiment, a selective AMPA antagonist, NBQX, was used in a canine survival model of hypothermic circulatory arrest. Twelve male dogs (20 to 25 kg) were placed on closed-chest cardiopulmonary bypass, subjected to 2 hours of hypothermic circulatory arrest at 18 degrees C, and rewarmed on cardiopulmonary bypass. All were mechanically ventilated and monitored for 20 hours before extubation and survived for 3 days. Six dogs received NBQX beginning 2 hours after arrest (3 mg/kg for 3 hours then 1.5 mg/kg for 2 hours). Control dogs received vehicle only. Neurologic recovery was assessed every 12 hours using a species-specific behavior scale that yielded a neurodeficit score ranging from 0 (normal) to 500 (brain dead). After sacrifice at 72 hours, brains were examined by receptor autoradiography and histologically for patterns of selective neuronal necrosis and scored blindly from 0 (normal) to 100 (severe injury). Dogs given NBQX had better neurologic function compared with controls (neurodeficit score, 58.6 +/- 15 versus 204 +/- 30; p < 0.004) and had less neuronal injury (18.2 +/- 3 versus 52.5 +/- 6; p < 0.004). Densitometric receptor autoradiography revealed preservation of neuronal NMDA receptor expression only in dogs given NBQX. These results suggest that antagonism of the non-NMDA glutamate receptor AMPA may be neuroprotective in adults after hypothermic circulatory arrest.

Topics: Animals; Autoradiography; Brain Diseases; Cardiopulmonary Bypass; Dogs; Electroencephalography; Heart Arrest, Induced; Hypothermia, Induced; Male; Necrosis; Neurons; Postoperative Care; Quinoxalines; Receptors, AMPA; Reperfusion Injury; Rewarming; Survival Rate; Time Factors

This study examines the relationship between the concentration of extracellular glutamate released during 30 min of forebrain ischemia, and the subsequent development of ischemic neural necrosis, in the presence of three agents which act at distinct sites on the glutamatergic synapse: a presynaptic inhibitor of glutamate release (5-(2,3,5-trichlorophenyl)-2,4-diamino-pyramidine ethane sulphonate (BW1003C87)); a competitive NMDA receptor antagonist (cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS19755)); and a competitive AMPA receptor antagonist (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX)). Pretreatment with either BW1003C87 or NBQX markedly attenuated the peak concentration of extracellular glutamate and offered protection from post-ischemic neuronal necrosis in the CA1 hippocampus. In contrast, pretreatment with CGS19755 had no effect on extracellular glutamate release and did not protect CA1 hippocampal neurons from ischemic injury.

Topics: Analysis of Variance; Animals; Binding, Competitive; Brain Ischemia; Disease Models, Animal; Glutamic Acid; Hippocampus; Injections, Intraperitoneal; Injections, Intravenous; Male; Microdialysis; N-Methylaspartate; Necrosis; Neurons; Observer Variation; Pipecolic Acids; Prosencephalon; Pyrimidines; Quinoxalines; Random Allocation; Rats; Rats, Wistar; Receptors, AMPA; Reperfusion Injury

Excitatory amino acids are implicated in the development of neuronal cell damage following periods of reversible cerebral ischemia or insulin-induced hypoglycemic coma. To explore the importance of glutamate receptor activation in the posthypoglycemic phase, we exposed rats to 20 min of insulin-induced severe hypoglycemia. The rats were treated immediately after the hypoglycemic insult with four regimes of glutamate receptor antagonists: (1) the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propriate)-receptor antagonist NBQX [2.3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline] given as a bolus dose of 30 mg.kg-1 i.p., followed by an i.v. infusion of 225 micrograms.kg-1.min-1 for 6 h; (2) the non-competitive NMDA-receptor antagonist, dizocilpine (MK-801) 1 mg.kg-1 given i.v.; (3) a combined NBQX treatment, (a bolus dose of 10 mg.kg-1 i.p., followed by an i.v. infusion of 225 micrograms.kg-1.min-1 for 6 h), with dizocilpine 0.33 mg.kg-1 given twice i.p. at 0 and 15 min after recovery and (4) the competitive NMDA-receptor blocker CGP 40,116 [D-(E)-2-amino-4-methyl-5-phosphono-3- pentenoic acid] 10 mg.kg-1 given i.p. In the striatum, all glutamate receptor blockers significantly decreased neuronal damage by approximately 30%. An approximately 50% decrease in neuronal damage was demonstrated in neocortex and hippocampus following the combined treatment with NBQX and dizocilpine, while protection was variable following the treatment with a single glutamate-receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; Animals; Blood Pressure; Brain Ischemia; Cell Death; Dizocilpine Maleate; Electroencephalography; Excitatory Amino Acid Antagonists; Insulin Coma; Male; Necrosis; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate