dizocilpine-maleate and Hypoxia

Hypoxia is the most common cause of neonatal seizures and encephalopathy. We have previously developed an in vivo experimental model of perinatal hypoxia which exhibits age-dependent acute and chronic epileptogenic effects. Between postnatal day (P) 10-12, the rat exhibits acute seizure activity during global hypoxia, while no seizures are induced at earlier (P5) or older (P60) ages. Rats exposed to hypoxia between P10-12 have reduced seizure thresholds to chemical convulsants in adulthood. The nonNMDA antagonists NBQX appears to suppress both the acute and long term epileptogenic effects of hypoxia. The age-dependency of the hyperexcitable response to hypoxia in vivo can be reproduced in vitro using hippocampal slices. In Mg(2+)-free media, hypoxia induced ictal discharges within 60 s of onset in 79% of slices from normal P10 rat pups compared to 11% of adult slices (p < 0.001). Model systems such as that described here allow for correlation of in vitro and in vivo electrophysiology and should provide data regarding the pharmacological and physiological characteristics of hypoxia-induced seizure activity in the immature brain which could ultimately be applied to therapeutic strategies.

Topics: Age Factors; Animals; Disease Models, Animal; Dizocilpine Maleate; Electroencephalography; Electrophysiology; Epilepsy, Temporal Lobe; Hippocampus; Hypoxia; In Vitro Techniques; Lorazepam; Quinoxalines; Rats

Chronic intermittent hypoxia (CIH), a component of sleep apnea-hypopnea syndrome, is suggested to cause damage to lung tissue, and the role of glutamate is not well studied. We used a chronic long-term intermittent hypobaric hypoxia (CLTIHH) model of rats to find out if such procedure causes lung injury and the potential effect of N-methyl-D-aspartate receptors (NMDARs) by using receptor antagonist MK-801 (dizocilpine). Thirty-two rats were placed into four groups; a control and three CLTIHH groups where rats were placed into a low-pressure chamber set to 430 mmHg for 5 h/day, 5 days/week, for 5 weeks. Only one group received MK-801 (0.3 mg/kg, ip) daily. We evaluated tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and nuclear factor (NF)-kB for the inflammatory process, superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPX), total antioxidant status (TAS), and total oxidant status (TOS) for oxidative stress, and caspase-9 levels. Blood plasma, bronchoalveolar fluid (BALF), and lung tissue extracts were evaluated. Both oxidant and inflammatory parameters were significantly increased in all the mediums of the CLTIHH groups except the group that received MK-801. Significant evidence was collected on MK-801 alleviating the effect of CLTIHH. Histological evaluations revealed lung damage and fibrotic changes in the CLTIHH groups. It was first shown that the CLTIHH procedure caused chronic lung injury, and that inflammation and oxidant stress were influential in the formation of lung injury. Secondly, NMDAR antagonist MK-801 effectively inhibited the development of lung injury and fibrosis.

Topics: Animals; Dizocilpine Maleate; Glutamic Acid; Hypoxia; Interleukin-6; Lung Injury; N-Methylaspartate; Oxidants; Oxidative Stress; Rats; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Tumor Necrosis Factor-alpha

We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms.. Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect.. Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect.. Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

Topics: Animals; Corpus Striatum; Dizocilpine Maleate; Dopamine; Dopamine Antagonists; Dopamine Plasma Membrane Transport Proteins; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Haloperidol; Hypoxia; Ligands; Nomifensine; Ouabain; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, sigma

We previously generated an ischemic stroke in a zebrafish model using N

Topics: Animals; Brain; Brain Ischemia; Cell Death; Disease Models, Animal; Dizocilpine Maleate; Drug Evaluation, Preclinical; Edaravone; Free Radical Scavengers; Gases; Hypoxia; Larva; Neurons; Nitrogen; Receptors, N-Methyl-D-Aspartate; Zebrafish

Harmful algal blooms are increasing in frequency and extent worldwide and occur nearly annually off the west coast of Florida where they affect both humans and wildlife. The dinoflagellate Karenia brevis is a key organism in Florida red tides that produces a suite of potent neurotoxins collectively referred to as the brevetoxins (PbTx). Brevetoxins bind to and open voltage gated sodium channels (VGSC), increasing cell permeability in excitable cells and depolarizing nerve and muscle tissue. Exposed animals may thus show muscular and neurological symptoms including head bobbing, muscle twitching, paralysis, and coma; large HABs can result in significant morbidity and mortality of marine life, including fish, birds, marine mammals, and sea turtles. Brevetoxicosis however is difficult to treat in endangered sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. In this study we used the freshwater turtle Trachemys scripta as a model organism to investigate the effects of the specific brevetoxin PbTx-3 in the turtle brain. Primary turtle neuronal cell cultures were exposed to a range of PbTx-3 concentrations to determine excitotoxicity. Agonists and antagonists of voltage-gated sodium channels and downstream targets were utilized to confirm the toxin's mode of action. We found that turtle neurons are highly resistant to PbTx-3; while cell viability decreased in a dose dependent manner across PbTx-3 concentrations of 100-2000nM, the EC

Topics: Animals; Calcium; Cell Survival; Cells, Cultured; Dizocilpine Maleate; Exocytosis; Female; Florida; Harmful Algal Bloom; Humans; Hypoxia; Marine Toxins; Neurons; Oxocins; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Tetrodotoxin; Turtles; Voltage-Gated Sodium Channels; Water Pollutants

The present study aims to investigate the effect of noscapine (0.5-2.5 μM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia.. Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively.. The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 μM noscapine. Increasing noscapine concentration up to 2.5 μM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 μM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 μM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD.. We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.

Topics: Animals; Brain Ischemia; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glucose; Hypoxia; Mice; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nimodipine; Nitric Oxide; Noscapine; Primary Cell Culture; Quinoxalines; Thapsigargin

Cerebral ischemia-reperfusion is associated with NMDA receptor-mediated calcium influx which activates neuronal nitric oxide synthase (nNOS) and consequently induces NO production. NO S-nitrosylates cellular protein and aggravates neuronal injury. Receptor-interacting protein 3 (RIP3) is a sensor molecule regulating cell apoptosis and necrosis. However, the roles of RIP3 in cerebral ischemic injury remain elusive. In this study, we reported that RIP3 could be S-nitrosylated by the exogenous NO donor GSNO in HEK293 cells and the Cys(119) residue was the key nitrosylation site. In addition, we found that cerebral ischemia induced RIP3 S-nitrosylation at different time points of reperfusion, which was coupling with RIP3 phosphorylation (which is associated with its activation) and its interaction with receptor-interacting protein 1 (RIP1), and this process facilitated cerebral ischemic injury. Treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7NI, diminished RIP3 S-nitrosylation and reduced neuronal damage. Taken together, these data demonstrated that NMDAR-dependent RIP3 S-nitrosylation induced by ischemia facilitated its activation in the early stages of ischemia, blocking this process could reduce the ischemia neuronal injury.

Topics: Aldehyde Oxidoreductases; Animals; Apoptosis; Brain Ischemia; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glucose; HEK293 Cells; Humans; Hypoxia; Male; Neurons; Nitric Oxide Synthase Type I; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Time Factors

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, Control rats. Given the similarity between the sustained HVR obtained after chronic exposure to developmental hyperoxia and the mature HVR, it was hypothesized that hyperoxia-induced plasticity and normal maturation share common mechanisms such as enhanced glutamate and nitric oxide signaling and diminished platelet-derived growth factor (PDGF) signaling. Rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth until 4-5 days of age were studied after intraperitoneal injection of drugs targeting these pathways. Hyperoxia rats receiving saline showed a sustained HVR to 12% O2, but blockade of NMDA glutamate receptors (MK-801) restored the biphasic HVR typical of newborn rats. Blockade of PDGF-β receptors (imatinib) had no effect on the pattern of the HVR in Hyperoxia rats, although it attenuated ventilatory depression during the late phase of the HVR in Control rats. Neither nitric oxide synthase inhibitor used in this study (nNOS inhibitor I and l-NAME) altered the pattern of the HVR in Control or Hyperoxia rats. Drug-induced changes in the biphasic HVR were not correlated with changes in metabolic rate. Collectively, these results suggest that developmental hyperoxia hastens the transition from a biphasic to sustained HVR by upregulating glutamate-dependent mechanisms and downregulating PDGF-dependent mechanisms, similar to the changes underlying normal postnatal maturation of the biphasic HVR.

Topics: Analysis of Variance; Animals; Animals, Newborn; Benzamides; Disease Models, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamic Acid; Hyperoxia; Hypoxia; Imatinib Mesylate; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Piperazines; Platelet-Derived Growth Factor; Pulmonary Ventilation; Pyrimidines; Rats; Rats, Sprague-Dawley

Recent studies have indicated that dynamic alterations in the structure of postsynaptic density (PSD) are involved in the pathogenesis of many central nervous system disorders, including ischemic stroke. Homer is the newly identified scaffolding protein located at PSD and regulates synaptic function. Homer1a, an immediate early gene, has been shown to be induced by several stimulations, such as glutamate, brain-derived neurotrophic factor, and trauma. However, whether acidosis mediated by acid-sensing ion channels (ASICs) and hypoxia during cerebral ischemia can change Homer1a expression remains to be determined.. We investigated that acidosis and hypoxia selectively and rapidly upregulated Homer1a expression, but not Homer1b/c in cultured cortical neurons. We also found that Homer1a exhibited induction expression in brain cortex of the middle cerebral artery occlusion (MCAO) rats. Additionally, acid-evoked Homer1a mRNA induction depended on extracellular signal-regulated kinase1/2 (ERK1/2) and Akt activity, and ASIC1a-mediated calcium influx whereas hypoxia depended only on ERK1/2 activity. Also, we demonstrated that continuous acidosis and hypoxia resulted in pronounced cell injury and Homer1a knockdown with small interfering RNA aggravated this damage induced by 3 h acid and hypoxia incubation in neuro-2a cells.. Homer1a might act as an activity-dependent regulator responding to extracellular stimuli during cerebral ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Amiloride; Animals; Carrier Proteins; Cell Survival; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Homer Scaffolding Proteins; Hypoxia; Infarction, Middle Cerebral Artery; Neurons; Peptides; Rats; Rats, Sprague-Dawley; Signal Transduction; Spider Venoms; Time Factors; Up-Regulation

When exposed to a hypoxic environment the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR. This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. Currently these mechanisms of HVR plasticity are unknown and we hypothesized that they involve glutamatergic synapses in the nucleus tractus solitarius (NTS), where afferent endings from arterial chemoreceptors terminate. To test this, we treated rats held in normoxia (CON) or 10% O2 (CSH) for 7 days and measured ventilation in conscious, unrestrained animals before and after microinjecting glutamate receptor agonists and antagonists into the NTS. In normoxia, AMPA increased ventilation 25% and 50% in CON and CSH, respectively, while NMDA doubled ventilation in both groups (P < 0.05). Specific AMPA and NMDA receptor antagonists (NBQX and MK801, respectively) abolished these effects. MK801 significantly decreased the HVR in CON rats, and completely blocked the acute HVR in CSH rats but had no effect on ventilation in normoxia. NBQX decreased ventilation whenever it was increased relative to normoxic controls; i.e. acute hypoxia in CON and CSH, and normoxia in CSH. These results support our hypothesis that glutamate receptors in the NTS contribute to plasticity in the HVR with CSH. The mechanism underlying this synaptic plasticity is probably glutamate receptor modification, as in CSH rats the expression of phosphorylated NR1 and GluR1 proteins in the NTS increased 35% and 70%, respectively, relative to that in CON rats.

Topics: Acclimatization; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Chemoreceptor Cells; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hypoxia; Male; N-Methylaspartate; Pulmonary Ventilation; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Reflex; Solitary Nucleus

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis, a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally, we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels, with a concomitant increase of the RIP1 protein. Together, these two related proteins form the necrosome, the complex responsible for induction of necroptotic cell death. Interestingly, we found that caspase-8 mRNA, a known negative regulator of necroptosis, was transiently decreased following OGD. Importantly, we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia, specifically in the CA1 area of the hippocampus. Moreover, we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together, our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury.

Topics: Animals; Antibodies; Apoptosis; Brain Ischemia; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Embryo, Mammalian; Glucose; Hippocampus; Hypoxia; Imidazoles; Indoles; L-Lactate Dehydrogenase; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Up-Regulation

Preconditioning is an experimental strategy for reducing ischemic brain damage. There are reports that brief exposure of neurons to NMDA-receptor antagonists may be an adequate preconditioning stressor. We studied effects of preconditioning of the cerebellar granule cells (CGC) in primary culture by 30-minute exposure to NMDA receptor antagonists 0.5 μM MK-801 or 5 μM memantine. CGC were challenged with oxygen and glucose deprivation (OGD) or excitotoxic glutamate and cell viability was tested 24 h later using calcein/ethidium homodimer-1 staining. We studied glutamate-induced increases in 45Ca uptake and in the intracellular Ca2+ level assessed with the fluorescent probe fluo-3. The number of living cells in OGD-treated cultures decreased by 42%. Preconditioning with MK-801 or memantine 24 h earlier reduced cell death to 8% and 30% and 48 h earlier to 27% and 33%, respectively. Pretreatment with MK-801 followed by the standard MK-801 wash out was slightly cytoprotective in a glutamate excitotoxicity test performed immediately; the protection increased significantly 24 h after preconditioning. In both cases the extensive wash out of MK-801 after preconditioning resulted in loss of cytoprotection. The increase in the intracellular Ca2+ level evoked by glutamate was decreased 24 h after preconditioning and even halved in the neuronal cultures 48 h after preconditioning with MK-801 and memantine. Glutamate-induced 45Ca uptake in these cells was decreased by 18%, irrespective of the time laps after preconditioning. These results demonstrate that preconditioning of CGC with NMDA receptor antagonists induces prolonged tolerance to OGD, which is accompanied by the reduction of glutamate-evoked calcium fluxes. The causal relationship between these effects may be suggested.

Topics: Analysis of Variance; Animals; Animals, Newborn; Cell Count; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; Memantine; Neurons; Rats; Rats, Wistar; Statistics, Nonparametric; Time Factors

During stroke the blood-brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7-fold after 6h OGD, which was significantly reduced by 10μM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after 6h OGD and was still higher (210%) after the 20h reoxygenation phase compared to normoxia. Ten micromolar MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.

Topics: Animals; Brain; Dizocilpine Maleate; Dose-Response Relationship, Drug; Endothelial Cells; Excitatory Amino Acid Antagonists; Glucose; Hypoxia; Mice; Oxygen; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Time Factors; Tritium; Up-Regulation

It is well established that hypoxia causes excess accumulation of glutamate in developing neural tissues. This study aimed to elucidate the mechanism by which glutamate can cause retinal ganglion cell (RGC) death through the N-methyl-D-aspartate (NMDA) receptors (NR) in the developing retina. One-day-old Wistar rats were exposed to hypoxia for 2 hours and then killed at different time points. Normal age-matched rats were used as controls. NR1, NR2A-D, and NR3A messenger RNA and protein expression showed significant increases over control values, notably at early time points (3 hours to 7 days) after the hypoxic exposure, and immunoexpression of NR1, NR2A-D and NR3A on retinal ganglion cells (RGCs) was enhanced in hypoxic rats and this was confirmed in cultured hypoxic RGCs. Ca(2+) influx in cultured RGCs was increased after hypoxic exposure, and the intracellular Ca(2+) concentration was suppressed by MK-801. Mitochondrial permeability transition pore opening, mitochondrial/cytosolic cytochrome c, and cytosolic caspase-3 expression levels were significantly increased in the hypoxic RGCs. These increases were reversed by MK-801, suggesting that the NMDA receptor subunits in the retina respond rapidly to the hypoxia-induced glutamate overload that leads to the cascade of events that result in RGC death.

Topics: Animals; Animals, Newborn; Cell Death; Cells, Cultured; Dizocilpine Maleate; Hypoxia; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Retina; Retinal Ganglion Cells

Developing central white matter is subject to ischemic-type injury during the period that precedes myelination. At this stage in maturation, central axons initiate a program of radial expansion and ion channel redistribution. Here we test the hypothesis that during radial expansion axons display heightened ischemic sensitivity, when clusters of Ca(2+) channels decorate future node of Ranvier sites.. Functionality and morphology of central axons and glia were examined during and after a period of modeled ischemia. Pathological changes in axons undergoing radial expansion were probed using electrophysiological, quantitative ultrastructural, and morphometric analysis in neonatal rodent optic nerve and periventricular white matter axons studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo.. Acute ischemic injury of central axons undergoing initial radial expansion was mediated by Ca(2+) influx through Ca(2+) channels expressed in axolemma clusters. This form of injury operated only in this axon population, which was more sensitive to injury than neighboring myelinated axons, smaller axons yet to initiate radial expansion, astrocytes, or oligodendroglia. A pharmacological strategy designed to protect both small and large diameter premyelinated axons proved 100% protective against acute ischemia studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo.. Recent clinical data highlight the importance of axon pathology in developing white matter injury. The elevated susceptibility of early maturing axons to ischemic injury described here may significantly contribute to selective white matter pathology and places these axons alongside preoligodendrocytes as a potential primary target of both injury and therapeutics.

Topics: Age Factors; Animals; Animals, Newborn; Apoptosis; Astrocytes; Axons; Disease Models, Animal; Dizocilpine Maleate; Glucose; Green Fluorescent Proteins; Hypoxia; Hypoxia-Ischemia, Brain; Mice; Mice, Transgenic; Myelin Sheath; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroprotective Agents; Oligodendroglia; omega-Agatoxin IVA; Optic Nerve; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Recovery of Function; Thy-1 Antigens

The present study was focused on identifying the expression of N-methyl D-aspartate receptor (NMDAR) subunits on activated microglia and to determine their role in the pathogenesis of periventricular white matter damage (PWMD) in neonatal rats following hypoxia. One day old wistar rats were subjected to hypoxia (5% O(2) ; 95% N(2) ) and the mRNA and protein expression of NMDAR subunits (NR1, NR2A-D, and NR3A) in the periventricular white matter (PWM) was determined at different time points (3,24 h, 3, 7, and 14 days) following hypoxic exposure. Immunoexpression of NR1 and NR2A-D was localized in amoeboid microglial cells (AMC) suggesting the presence of functional NMDARs in them. The expression of NMDAR in primary microglial cultures was ascertained by RT-PCR analysis and double immunofluorescence studies. The functionality of the microglial NMDAR in cultured microglial cells was examined by monitoring calcium movements in cells with fura-2. In primary microglial cultures, hypoxia induced the nuclear translocation of NF-κB which was suppressed by administration of MK801, an NMDAR antagonist. MK801 also down regulated the hypoxia-induced expression of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) production by microglia which may be mediated by the NF-κB signaling pathway. NO produced by microglia is known to cause death of oligodendrocytes in the developing PWM. In this connection, pharmacological agents such as MK801, BAY (NF-κB inhibitor), and 1400w (iNOS inhibitor) proved to be beneficial since they reduced the hypoxia-induced iNOS expression, NO production, and a corresponding reduction in the death of oligodendrocytes following hypoxia.

Topics: Amidines; Animals; Animals, Newborn; Benzylamines; Blotting, Western; Calcium; Cell Death; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Oligodendroglia; Protein Subunits; Protein Transport; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

This study addressed the hypotheses that exposure to chronic hypoxia (CH) and chronic hypercapnia (CHC) would modify the acute hypercapnic ventilatory response in the cane toad (Rhinella marina; formerly Bufo marinus or Chaunus marinus) and its regulation by NMDA-mediated processes. Cane toads were exposed to 10 days of CH (10% O(2)) or CHC (3.5% CO(2)) followed by acute in vivo hypercapnic breathing trials, conducted before and after an injection of the NMDA-receptor channel blocker, MK801 into the dorsal lymph sac. CH, CHC and MK801 did not alter ventilation under acute normoxic normocapnic conditions. CH blunted the increase in breathing frequency during acute hypercapnia while CHC had no effect. The effect of CH on breathing frequency was mediated by a decrease in the number of breaths per breathing episode. Neither CH nor CHC altered breath area (volume). MK801 augmented breathing frequency (via an increase in breaths per episode) and total ventilation during acute hypercapnia in control toads and toads exposed to CH; there was no effect of MK801 on the increase in breathing frequency or total ventilation, during acute hypercapnia in toads exposed to CHC. The results indicate that CH and CHC differentially alter breathing pattern. Furthermore, they indicate an absence of NMDA-mediated glutamatergic tone during normoxic normocapnia but that NMDA-mediated processes attenuate the increase in breathing frequency during acute hypercapnia under control conditions and following CH but not following CHC. Given that MK801 was administered systemically, the effects could be acting anywhere in the reflex pathway from CO(2)-sensing to respiratory motor output.

Topics: Animals; Bufo marinus; Dizocilpine Maleate; Hypercapnia; Hypoxia; N-Methylaspartate; Respiration; Respiratory Mechanics

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC(50)) values of 199 μmol l(-1) and 4132 μmol l(-1) for NH(3) and total ammonia ([T(Amm)]=[NH(3)]+[NH(4)(+)]), respectively. These values were ~5-6 times greater than corresponding NH(3) and T(Amm) LC(50) values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH(4)Cl (3-5 mmol l(-1)) for 5 days, despite 6-fold increases in plasma [T] to ~1300 μmol l(-1) and 3-fold increases in brain [T(Amm)] to 6700 μmol l(-1). Muscle [T(Amm)] increased by almost 8-fold from ~900 μmol kg(-1) wet mass (WM) to greater than 7000 μmol kg(-1) WM by 48 h, and stabilized. Although urea excretion rates (J(Urea)) increased by 2-3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000 μmol kg(-1) WM to 15,000 μmol kg(-1) WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8 mg kg(-1)) or ethanol (1-8 mg kg(-1)) increased trout survival time by 1.5-2.0-fold during exposure to 2 mmol l(-1) ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8-9.5 mmol l(-1) T(Amm)) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro expo

Topics: Ammonia; Animals; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamine; Goldfish; Hypoxia; Nitrogen; Oncorhynchus mykiss; Patch-Clamp Techniques; Receptors, N-Methyl-D-Aspartate; Toxicity Tests, Acute; Urea; Water Pollutants, Chemical

Although the glossopharyngeal nerve (IX) is mainly a sensory nerve, it innervates stylopharyngeus and some other pharyngeal muscles, whose excitations would likely improve upper airway patency since electrical IX stimulation increases pharyngeal airway size. As acute intermittent hypoxia (AIH) induces hypoglossal and genioglossal long-term facilitation (LTF), we hypothesized that AIH induces glossopharyngeal LTF, which requires serotonin 5-HT(2) and NMDA receptors. Integrated IX activity was recorded in anesthetized, vagotomized, paralyzed and ventilated rats before, during and after 5 episodes of 3-min isocapnic 12% O(2) with 3-min intervals of 50% O(2). Either saline, ketanserin (5-HT(2) antagonist, 2mg/kg) or MK-801 (NMDA antagonist, 0.2mg/kg) was (i.v.) injected 30-60 min before AIH. Both phasic and tonic IX activities were persistently increased (both P<0.05) after AIH in vehicle, but not ketanserin or MK-801, rats. Hypoxic glossopharyngeal responses were minimally changed after either drug. These data suggest that AIH induces both phasic and tonic glossopharyngeal LTF, which requires activation of 5-HT(2) and NMDA receptors.

Topics: Anesthesia; Animals; Blood Gas Analysis; Chronic Disease; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glossopharyngeal Nerve; Hypoxia; Injections, Intravenous; Ketanserin; Long-Term Potentiation; Male; Oxygen; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin, 5-HT2; Serotonin Antagonists; Time Factors; Vagotomy

These experiments were designed to determine the effects of EEG state on the response of rats to intermittent hypoxia and to test the hypotheses that short-term potentiation (STP) and ventilatory long term facilitation (vLTF) are state dependent; and that neurons with NMDA receptors in the dorso-ventral pontine respiratory group (dvPRG) modulate the development of STP and vLTF in rats. Low-doses of urethane anaesthesia (<1.3g/kg) that do not cause significant respiratory depression or reductions in sensitivity to hypoxia result in cycling between EEG states that superficially resemble wake and slow wave sleep in rats and are accompanied by changes in breathing pattern that closely resemble those seen when unanaesthetized rats cycle between wake and SWS. When changes between these states were accounted for, intermittent, poikilocapnic hypoxia did not produce a significant vLTF. However, there was a persistent STP of tidal volume and vLTF did develop after blockade of NMDAr in the region of the PBrKF complex by microinjection of MK-801. Blockade of NMDA-type glutamate receptor-mediated processes in the dorsal pons also caused animals to cycle into State III, but did not alter the response to either continuous or intermittent hypoxia indicating that the response to hypoxia was not state dependent. This shows that neurons in the region of the PRG inhibit STP and vLTF, but no longer do so if PRG NMDA receptor activation is blocked.

Topics: Anesthetics, Intravenous; Animals; Dizocilpine Maleate; Electroencephalography; Excitatory Amino Acid Antagonists; Hypoxia; Male; Microinjections; Neurons; Pons; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics; Sleep; Tidal Volume; Urethane; Wakefulness

NMDA receptors mediate hypoxia-induced ventilatory frequency and blood pressure increases in fish. Here we continue to resolve whether non-NMDA receptors participate in chemoreflexes. Shorthorn sculpins, instrumented for cardiorespiratory measurements, were kept unrestrained or positioned in a stereotaxic frame. Chemoreflexes were elicited (hypoxia/NaCN-induced) before/after administration of either the specific AMPA receptor antagonist, GYKI52466 (systemically), or the specific kainate receptor antagonist, UBP293 (microinjections into fourth ventricle). Immunohistochemistry was performed on medullary cross-sections to identify non-NMDA receptor subunits in the chemoreflex-pathway. Kainate receptors mediate the chemoreflex-mediated increase in ventilation amplitude, since the response was abolished by UBP293. GYKI52466 attenuated the ventilatory frequency increase, and induced more regular breathing patterns and higher heart rate in both normoxic and hypoxic conditions, suggesting that AMPA receptors also partake in cardiorespiratory control. This together with immunohistochemical findings of both AMPA and kainate receptor subunits in the chemoreflex-pathway, show that non-NMDA receptors play a role in both chemoreflex-activation and normoxic cardiorespiratory regulation in fish.

Topics: Alanine; Animals; Benzodiazepines; Blood Pressure; Chemoreceptor Cells; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fishes; Glutamic Acid; Heart Rate; Hypoxia; Immunohistochemistry; Medulla Oblongata; Microinjections; Oxygen; Poisons; Receptors, AMPA; Receptors, Kainic Acid; Reflex; Respiratory Mechanics; Seasons; Sodium Cyanide

Although several studies have provided evidence for the therapeutic potential of bone marrow-derived mononuclear cells (MNCs) in animal models of stroke, the mechanisms underlying their benefits remain largely unknown. We have determined the neuroprotective potential of MNCs in primary neuronal cultures exposed to various injuries in vitro. Cortical neurons in culture were exposed to oxygen-glucose deprivation, hypoxia, or hydrogen peroxide, and cell death was assayed by MTT, caspase-3 activation or TUNEL labelling at 24 hrs. Cultures were randomized to cotreatment with MNC-derived supernatants or media before injury exposure. In separate experiments, macrophage or microglial cultures were exposed to lipopolypolysacharide (LPS) in the presence and absence of MNC-derived supernatants. Neuronal cultures were then exposed to conditioned media derived from activated macrophages or microglia. Cytokines from the supernantants of MNC cultures exposed to normoxia or hypoxia were also estimated by enzyme-linked immunosorbant assay (ELISA). MNC-derived supernatants attenuated neuronal death induced by OGD, hypoxia, hydrogen peroxide, and conditioned macrophage/microglial media and contain a number of trophic factors, including interleukin-10, insulin-like growth factor-1, vascular endothelial growth factor, and stromal cell-derived factor-1. MNCs provide broad neuroprotection against a variety of injuries relevant to stroke.

Topics: Analysis of Variance; Animals; Bone Marrow Cells; Caspase 3; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Cytokines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Embryo, Mammalian; Glucose; Hypoxia; In Situ Nick-End Labeling; Microglia; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Sprague-Dawley

Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.

Topics: Analysis of Variance; Animals; Arginine; Blood Gas Analysis; Blood Pressure; Cyclic N-Oxides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Free Radical Scavengers; Hydrogen-Ion Concentration; Hypoxia; Imidazoles; In Vitro Techniques; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; N-Methylaspartate; Neuronal Plasticity; Neurons; Nitric Oxide; Nitric Oxide Synthase Type I; Paraventricular Hypothalamic Nucleus; Receptors, N-Methyl-D-Aspartate; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Time Factors; Vasopressins

In the present study, we investigated the effects of mebudipine and dibudipine, two new Ca(2+) channel blockers, on primary murine cortical neurons exposed to oxygen-glucose deprivation/reperfusion. The experiments were performed on cells after 11-16 days of culture. To initiate oxygen-glucose deprivation /reperfusion, the culture medium was replaced by glucose-free medium, and the cells were transferred to a humidified incubation chamber in a mixture of 95% N(2) and 5% CO(2) at 37 degrees C for 30 min. The cultures were pretreated with mebudipine and dibudipine 3 h prior to oxygen-glucose deprivation/reperfusion, in order to explore their effects on neurons under oxygen-glucose deprivation conditions. Cell viability and nitric oxide (NO) production were assessed by MTT assay and the modified Griess method, respectively. Exposure of murine cortical neuronal cells to 30 min oxygen-glucose deprivation significantly decreased cell viability and increased NO production. Pretreatment of the cultures with mebudipine and dibudipine significantly increased cell viability and decreased NO generation in a dose-dependent manner. However, the drugs had no protective effect in cells subjected to oxygen-glucose deprivation for 60 min. Pretreatment of cultures with MK-801 (10 microM), a non-competitive NMDA antagonist, decreased neuronal death after 30-min oxygen-glucose deprivation, while application of NBQX (30 microM), a selective AMPA-kainate receptor antagonist, partially attenuated the cell injury. oxygen-glucose deprivation -induced cytotoxicity and NO production were also inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and MK-801. We conclude that mebudipine and dibudipine could protect cortical neurons against oxygen-glucose deprivation /reperfusion-induced cell injury in a dose-dependent manner, and that this could be mediated partially by decreased NO production.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Formazans; Glucose; Hypoxia; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nifedipine; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Quinoxalines; Receptors, Kainic Acid; Reperfusion Injury; Tetrazolium Salts; Time Factors

The present study investigated whether the platelet-derived growth factor receptor (PDGFR)-beta-mediated mechanisms are involved in the hypoxic ventilatory response through modulating the N-methyl-D-aspartate (NMDA) function. The ventilatory changes during hypoxic challenge (10% O(2), 30 min) were measured plethysmographically in mice selectively lacking the PDGFR-beta in neurons (KO mice) and in control wild-type mice (WT mice) before and after blockade of NMDA receptors. In baseline breathing at rest, respiratory rate, tidal volume, and minute ventilation were similar between WT and KO mice. Hypoxia caused an increase of ventilation during the early period of exposure (an initial excitation), followed by a progressive decrease along with the exposure period (a late decline). The initial excitation occurred similarly in KO and WT mice, while the late decline was markedly attenuated in KO mice. Administration of an antagonist of NMDA receptors, dizocilpine (0.3 mg/kg, i.p.) decreased the initial excitation and hastened the late decline of hypoxic ventilatory response. Furthermore, the hypoxic ventilatory response in KO mice was indistinguishable from that in WT mice after blockade of NMDA receptors. The present study suggests that the PDGF-BB/PDGFR-beta signal axis contributes to the hypoxic ventilatory response by its inhibitory effect on the NMDA receptor-mediated function.

Topics: Animals; Dizocilpine Maleate; Humans; Hypoxia; Mice; Mice, Knockout; Pulmonary Ventilation; Receptor, Platelet-Derived Growth Factor beta; Receptors, N-Methyl-D-Aspartate

Global birth hypoxia in rats has been shown to produce long-term changes in central nervous system functions, known to be influenced by neonatal testosterone secretion. Birth hypoxia alters levels of several circulating hormones, but it is unknown if it affects neonatal testosterone.. Using a rat model of acute global hypoxia during cesarean section (C-section) birth, this study tested whether birth hypoxia affects neonatal testosterone. We then evaluated whether the observed hypoxia-induced changes in neonatal testosterone may be mediated via N-methyl-D-aspartate (NMDA) receptor activation and/or altered luteinizing hormone (LH), adrenocorticotropic hormone (ACTH) or corticosterone levels. Longer-term effects of birth hypoxia on testosterone-related function were also assessed.. Rats born by C-section + 15 min of anoxia had significantly higher plasma testosterone at 2 and 3 h after birth compared to controls born either vaginally or by C-section. Administration of an NMDA receptor antagonist at birth increased neonatal testosterone in both anoxic pups and controls. Pups exposed to birth anoxia under hypothermic conditions also showed increased neonatal testosterone. Circulating LH, follicle-stimulating hormone and corticosterone in neonates, and testosterone at adulthood were unaffected by birth hypoxia. However, plasma ACTH was significantly increased in anoxic neonates at 2 h after birth. Birth condition had no effect on anogenital distance or juvenile play behavior.. It is concluded that birth hypoxia augments plasma testosterone during the critical period of the neonatal testosterone surge, by a mechanism that is independent of NMDA-mediated LH secretion, but may involve increased circulating ACTH.

Topics: Adrenocorticotropic Hormone; Animals; Animals, Newborn; Behavior, Animal; Cesarean Section; Corticosterone; Disease Models, Animal; Dizocilpine Maleate; Female; Hypothermia; Hypoxia; Luteinizing Hormone; Parturition; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Testosterone

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Topics: Adaptation, Physiological; Adenosine Triphosphate; Animals; Dizocilpine Maleate; Down-Regulation; Energy Metabolism; Ethanol; Excitatory Amino Acid Antagonists; Gills; Goldfish; Hypoxia; Membrane Potentials; Oxygen; Patch-Clamp Techniques; Receptors, N-Methyl-D-Aspartate; Sodium-Potassium-Exchanging ATPase; Telencephalon; Time Factors

Hypoxic exposure produces a consistent decrease of rectal temperature (Tb), which is recognized as a potent protective response. While some of the neural mechanisms underlying this response have recently been described, it remains poorly known how these mechanisms evolve during post-natal development. We recently reported that in rat pups NMDA glutamate receptor limits Tb drop upon hypoxic exposure, an effect that has not been reported by others in adult rats. Accordingly, we tested the hypothesis that the implication of NMDA receptors on temperature control during hypoxic exposure evolves during development. To this aim, we evaluated the hypoxic (30 min - 12% O(2)) responses of Tb, metabolic rate, and ventilation in rats after injection of vehicle, or the NMDA receptor antagonist MK-801, at different ages (post-natal days 4, 10, 20 and 2-3 month-old - P4, P10, P20 and P60). MK-801 amplified the magnitude of the hypoxic-induced Tb drop in P4, P10 and P20 rats, but this effect was not apparent in adults. In P20 rats MK-801 tripled the hypoxic induced Tb drop, which was 0.5 degrees C in control and 1.4 degrees C in treated rats (p<0.0001). This effect was specific to temperature regulation, and was not accompanied by similar changes of other recorded parameters. MK-801 induced a significant decrease of the hypoxic ventilatory response in adults only. We conclude that NMDA glutamate receptor acts as a counter-regulatory factor that limits the hypoxic-induced drop of rectal temperature during post-natal development in rats.

Topics: Adaptation, Physiological; Aging; Analysis of Variance; Animals; Body Temperature Regulation; Dizocilpine Maleate; Energy Metabolism; Excitatory Amino Acid Antagonists; Female; Hypoxia; Male; Pulmonary Ventilation; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

We tested the hypothesis that glutamate, acting via NMDA-type receptors (NMDAr) in the Parabrachial/Kölliker fuse (PBrKF) nucleus of the pons, is involved both directly and indirectly (via changes in cortical activation state) in modulating breathing and ventilatory responses to hypoxia. To this end we examined the effects of MK-801, injected either systemically or directly into the PBrKF, on the breathing patterns of urethane-anaesthetized rats breathing air or an hypoxic gas mixture as electroencephalographic (EEG) activity alternated between State I (awake-like) and State III (NREM sleep-like) EEG patterns. Regardless of EEG state, systemic MK-801 reduced ventilation primarily by reducing tidal volume while microinjection of MK-801 into the PBrKF reduced ventilation by reducing breathing frequency. With both injections, EEG pattern changed from State I to III mimicking the change from wakefulness to NREM sleep that occurs in unanaesthetized rats given MK-801 systemically. Systemic injection of MK-801 delayed and reduced the response to hypoxia while microinjection of MK-801 into the PBrKF did not reduce the HVR but sustained the hypoxic increase in tidal volume well into the post-hypoxic recovery period. Thus, while NMDAr in the PBrKF complex of the pons play a role in modulating sleep/wake-like states as well as changes in breathing pattern associated with changes in cortical activation state, they are neither involved in the hypoxic ventilatory response nor in the change in hypoxic sensitivity associated with the changes in cortical activation state.

Topics: Analysis of Variance; Animals; Brain Mapping; Cerebral Cortex; Dizocilpine Maleate; Electroencephalography; Excitatory Amino Acid Antagonists; Hypoxia; Male; Microinjections; Pons; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiration; Respiratory System; Tidal Volume; Wakefulness

This study was designed to examine how systemic administration of an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, altered respiratory timing in unanesthetized rats under normoxia and hypoxia. To detect fine changes in inspiratory time (TI) and expiratory time (TE), and cycle duration (TTOT), we prepared a diaphragmatic electromyogram (EMGdia). Diaphragm electrodes and arterial and venous catheters were inserted into Wistar rats (n = 8) under pentobarbital anesthesia. The next day, EMGdia was recorded before and after intravenous administration of MK-801 (3 mg/kg) under normoxia and hypoxia (12% O2) without anesthesia, and the respiratory timing (TI, TE, TTOT), respiratory frequency (fR), and amplitude of the integrated EMGdia were measured. Arterial blood gases (ABGs), mean arterial pressure (MAP), and heart rate (fH) were also measured with the EMGdia. Under normoxia, MK-801 increased fR owing to a significant decrease in TE, and elevated both MAP and fH. Under hypoxia, MK-801 suppressed an increase in fR owing to a significant increase in TI, and did not accelerate fH. In both gaseous conditions, on ABGs, MK-801 did not alter partial pressure of O2 (PaO2) or CO2 (PaCO2), and slightly decreased pH (but not less than 7.4). MK-801 significantly decreased hypoxic response (%change from normoxia) in fR, and increased that in EMGdia amplitude, and did not alter a total ventilatory index (fRxEMGdia amplitude). The results suggest that an NMDA receptor-mediated mechanism partially determines fR through significant alterations in respiratory timing, particularly in which the hypoxic ventilatory response was obtained in unanesthetized rats.

Topics: Animals; Blood Gas Analysis; Blood Pressure; Diaphragm; Dizocilpine Maleate; Electromyography; Excitatory Amino Acid Antagonists; Heart Rate; Hypoxia; Injections, Intravenous; Male; Models, Animal; Pulmonary Ventilation; Rats; Rats, Wistar

Astrocytic glutamate transporters are considered an important target for neuroprotective therapies as the function of these transporters is abnormal in stroke and other neurological disorders associated with excitotoxicity. Recently, Rothstein et al., [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73-77] reported that beta-lactam antibiotics (including ceftriaxone, which easily crosses the blood-brain barrier) increase glutamate transporter 1 (GLT-1) expression and reduce cell death resulting from oxygen-glucose deprivation (OGD) in dissociated embryonic cortical cultures. To determine whether a similar neuroprotective mechanism operates in more mature neurons, which show a different pattern of response to ischemia than primary cultures, we exposed acute hippocampal slices obtained from rats treated with ceftriaxone for 5 days (200 mg/kg; i.p.) to OGD. Whole-cell patch clamp recording of glutamate-induced N-methyl-d-aspartate (NMDA) currents from CA1 pyramidal neurons showed a larger potentiation of these currents after application of 15 microM dl-threo-beta-benzyloxyaspartic acid (TBOA; a potent blocker of glutamate transporters) in ceftriaxone-injected animals than in untreated animals, indicating increased glutamate transporter activity. Western blot analysis did not reveal GLT-1 upregulation in the hippocampus. Delay to OGD-induced hypoxic spreading depression (HSD) recorded in slices obtained from ceftriaxone-treated rats was longer (6.3+/-0.2 vs. 5.2+/-0.2 min; P<0.001) than that in the control group, demonstrating a neuroprotective action of the antibiotic in this model. The effect of ceftriaxone was also tested in organotypic hippocampal slices obtained from P7-9 rats (>14 days in vitro). OGD or glutamate (3.5-5.0 mM) damaged CA1 pyramidal neurons as assessed by propidium iodide (PI) fluorescence. Similar damage was observed after pre-treatment with ceftriaxone (10-200 microM; 5 days) and ceftriaxone exposure did not result in GLT-1 upregulation as assayed by Western blot. Treatment of slice cultures with dibutyryl cAMP (100-250 microM; 5 days) increased GLT-1 expression but did not reduce cell damage induced by OGD or glutamate. Thus we confirm the neuroprotective effect of antibiotic exposure on OGD-induced injury, but sugge

Topics: Analysis of Variance; Animals; Animals, Newborn; Anti-Bacterial Agents; Aspartic Acid; Ceftriaxone; Cell Death; Disease Models, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Glutamic Acid; Hippocampus; Hypoxia; Membrane Potentials; Neurons; Neuroprotective Agents; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Stroke; Time Factors

Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity in ischemic stroke. We recently showed that NRG-1 reduced neuronal death following transient middle cerebral artery occlusion (tMCAO) by up to 90% with an extended therapeutic window. Here, we examined the neuroprotective potential of NRG-1 using a permanent MCAO ischemia (pMCAO) rat model. NRG-1 reduced infarction in pMCAO by 50% when administered prior to ischemia. We previously demonstrated using gene expression profiling that pMCAO was associated with an exaggerated excitotoxicity response compared to tMCAO. Therefore, we examined whether co-treatment with an inhibitor of excitotoxicity would augment the effect of NRG-1 following pMCAO. Both NRG-1 and the N-methyl-D-aspartate (NMDA) antagonist MK-801 similarly reduced infarct size following pMCAO. However, combination treatment with both NRG-1 and MK-801 resulted in greater neuroprotection than either compound alone, including a 75% reduction in cortical infarction compared to control. Consistent with these findings, NRG-1 reduced neuronal death using an in vitro ischemia model and this effect was augmented by MK-801. These results demonstrate the efficacy of NRG-1 in pMCAO rat focal ischemia model. Our findings further indicate the potential clinically relevance of NRG-1 alone or as a combination strategy for treating ischemic stroke.

Topics: Animals; Brain Infarction; Brain Ischemia; Cell Death; Cell Line, Tumor; Disease Models, Animal; Dizocilpine Maleate; Glucose; Hypoxia; Male; Neuregulin-1; Neuroblastoma; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tetrazolium Salts

We tested the hypothesis that glutamate NMDA receptors may help maintain metabolic rate and body temperature during acute or chronic hypoxic exposure in newborn rats. We recorded ventilation, metabolism ((.)V(O(2)) -- ((.)V(CO(2)) and rectal temperature, under normoxia, acute hypoxia (30 min -- 12% O(2)), or following 10 days of chronic hypoxia, in 10 days old male and female rats, receiving saline i.p. injection or the NMDA receptor antagonist MK-801. Acute hypoxia decreased rectal temperature and metabolism, and increased ventilation, and (.)V(E)/((.)V(O(2) and (.)V(E)/((.)V(CO(2) to the same extent in males and females. MK-801 injection amplified the metabolic decrease under acute (in males and females) and chronic (in males) hypoxia, prevented the increase of minute ventilation, while (.)V(E)/((.)V(O(2) or (.)V(E)/((.)V(CO(2)remained constant. Hence, NMDA glutamate receptors help to maintain metabolic rate, minute ventilation and body temperature at a determined level in acute (males and females) and chronic hypoxia (males only).

Topics: Analysis of Variance; Animals; Animals, Newborn; Body Temperature; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Hypoxia; Male; Metabolism; Pregnancy; Pulmonary Gas Exchange; Pulmonary Ventilation; Random Allocation; Rats; Receptors, N-Methyl-D-Aspartate; Sex Factors; Time Factors

This study examined whether a hypoxia-tolerant amphibian, the Cane toad, undergoes mammalian-like ventilatory acclimatisation to hypoxia (VAH) and whether chronic hypoxia (CH) alters NMDA-mediated regulation of the acute hypoxic ventilatory response (HVR). Toads were exposed to 10 days of CH (10% O2) followed by acute hypoxic breathing trials or an intra-arterial injection of NaCN. Trials were conducted before and after i.p. treatment with an NMDA-receptor channel blocker (MK801). CH blunted the acute HVR but did not alter resting breathing. MK801 did not alter resting ventilation. In control animals, MK801 augmented breathing frequency (fR) during acute hypoxia by increasing the number of breaths per episode. This effect was attenuated following CH although MK801 did enhance the number of episodes per minute during acute hypoxia. MK801 enhanced the fR response to NaCN in both groups. The results indicate that CH did not produce mammalian-like VAH (i.e. increased resting ventilation and an augmented acute HVR) but did alter MK801-sensitive regulation of breathing pattern and the acute HVR.

Topics: Acclimatization; Analysis of Variance; Animals; Bufo marinus; Cyanates; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; N-Methylaspartate; Pulmonary Ventilation; Respiration; Time Factors

In vitro studies suggest that glutamate receptor activation is important in the genesis of post-hypoxic preterm brain injury, but there are limited data on post-hypoxic N-methyl-D-aspartate (NMDA) receptor activation. We therefore examined an infusion of the specific, non-competitive NMDA receptor antagonist dizocilpine (2 mg kg(-1) bolus plus 0.07 mg kg(-1) h(-1) i.v.) from 15 min to 4 h after severe hypoxia-ischemia induced by umbilical cord occlusion for 25 min in fetal sheep at 70% of gestation. Dizocilpine suppressed evolving epileptiform transient activity in the first 6 h after reperfusion (2.3 +/- 0.9 versus 9.3 +/- 2.3 maximal counts min(-1), P < 0.05) and mean EEG intensity up to 11 h after occlusion (P < 0.05). Fetal extradural temperature transiently increased during the dizocilpine infusion (40.1 +/- 0.2 versus 39.3 +/- 0.1 degrees C, P < 0.05). After 3 days recovery, treatment was associated with a significant reduction in neuronal loss in the striatum (31 +/- 7 versus 58 +/- 2%, P < 0.05), expression of cleaved caspase-3 (111+/-7 versus 159 +/- 10 counts area(-1), P < 0.05) and numbers of activated microglia (57 +/- 9 versus 92 +/- 16 counts area(-1), P < 0.05); there was no significant effect in other regions or on loss of immature O4-positive oligodendrocytes. In conclusion, abnormal NMDA receptor activation in the first few hours of recovery from hypoxia-ischemia seems to contribute to post-hypoxic striatal damage in the very immature brain.

Topics: Animals; Brain Ischemia; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Electroencephalography; Female; Gestational Age; Hypoxia; Microglia; Neuroprotective Agents; Pregnancy; Pyramidal Cells; Sheep

Increased ventilation frequency (fV) in response to hypoxia in adult fish depends on ionotropic N-methyl-D-aspartate (NMDA) receptors. Nonetheless, the ontogeny of central control mechanisms mediating hypoxic ventilatory chemoreflexes in lower vertebrates has not been studied. Therefore, the aim of this study was to determine when the hypoxic ventilatory response during zebrafish (Danio rerio) development is mediated via NMDA receptors, by performing physiological experiments and western blot analysis of NMDA receptor subunits. Zebrafish larvae at stages 4-16 days post-fertilisation (dpf) were exposed to an hypoxic pulse in control groups and in groups treated with MK801 (NMDA receptor antagonist). The hypoxic increase in fV was present at all larval stages, and it matured during development. The reflex became MK801 sensitive at 8 dpf, but did not completely rely on a glutamatergic transmission until 13 dpf. This, together with changing subunit composition during the different stages (increasing amounts of NMDAR1 subunits and appearance of NMDAR2A subunits in adults), suggests that the amount of functional NMDA receptors needed to achieve a fully developed reflex is not attained until later stages. Furthermore, our results suggest that other non-NMDA receptor mechanisms are responsible for the hypoxia-induced increase in fV during the earlier developmental stages.

Topics: Animals; Dizocilpine Maleate; Hypoxia; Pulmonary Ventilation; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics; Zebrafish

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

Fetal alcohol exposure results in cognitive and neurobehavioral deficits, but the effects of modifying genetic loci on the severity of these sequelas have not been well characterized. Although the cAMP signaling pathway has been shown to be an important modulator of ethanol sensitivity in adult mice, its potential role in modulating ethanol-induced neurodegeneration has not been examined. Adenylyl cyclases (ACs) 1 and 8 produce cAMP in response to intracellular calcium elevation and modulate several aspects of neuronal function, including ethanol sensitivity. AC1 and AC8 are expressed widely throughout the brain of neonatal mice, and genetic deletion of both AC1 and AC8 in double-knock-out (DKO) mice enhances ethanol-induced neurodegeneration in the brains of neonatal mice. In addition, ethanol treatment induces significantly greater levels of caspase-3 activation in the brains of DKO mice compared with wild-type (WT) mice, reflecting higher numbers of apoptotic neurons. Administration of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate] or the GABA(A) receptor potentiator phenobarbital, which mimics components of the effects of ethanol on neurons, results in significantly greater neurodegeneration in the brains of neonatal DKO mice than WT mice. Furthermore, loss of a single calcium-stimulated AC isoform potentiates neurodegeneration after administration of ethanol, MK801, or phenobarbital. In contrast, the levels of physiological cell death, death after hypoxia/ischemia, and excitotoxic cell death are not increased in the brains of DKO mice. Thus, AC1 and AC8 are critical modulators of neurodegeneration induced by activity blockade in the neonatal brain and represent genetic loci that may potentially modify the severity of fetal alcohol syndrome.

Topics: Adenylyl Cyclases; Anilides; Animals; Animals, Newborn; Behavior, Animal; Blotting, Western; Brain; Calcium; Caspase 3; Caspases; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Ethanol; GABA Modulators; Hippocampus; Hypoxia; In Situ Hybridization; In Vitro Techniques; Mice; Mice, Inbred C57BL; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Oligopeptides; Phenobarbital; Silver Staining; Time Factors

Medical measures that bear no known danger for the adult brain may trigger active neuronal death in the developing brain. Pharmacological blockade of N-methyl-D-aspartate or activation of GABA(A) receptors, blockade of voltage-dependent sodium channels, and oxygen induce widespread apoptotic neurodegeneration during the period of rapid brain growth in rodents. Because such measures are often necessary in critically ill infants and toddlers, search for adjunctive neuroprotective strategies is warranted. We report that 17beta-estradiol ameliorates neurotoxicity of drugs that block N-methyl-D-aspartate receptors, activate GABA(A) receptors, or block voltage-gated sodium channels and reduces neurotoxicity of oxygen in the infant rat brain. This neuroprotective effect is reversed by tamoxifen and cannot be reproduced by 17alpha-estradiol. 17Beta-estradiol did not affect GABA(A) or N-methyl-D-aspartate currents in hippocampal neuronal cultures, indicating that direct modulation of neurotransmitter receptor/channel properties by this compound cannot explain neuroprotective effect. 17beta-Estradiol did, however, increase levels of phosphorylated extracellular signal-regulated kinase 1/2 and AKT, suggesting that activation of these prosurvival proteins may represent one mechanism for its neuroprotective action. 17Beta-estradiol and related compounds may be neuroprotective agents suitable for use in critically ill infants and toddlers. Its supplementation may particularly help to improve neurocognitive outcome in preterm infants who are prematurely deprived of maternal estrogen.

Topics: Animals; Animals, Newborn; Apoptosis; Bicuculline; Blotting, Western; Brain; Caenorhabditis elegans Proteins; Cell Count; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrogen Antagonists; Extracellular Signal-Regulated MAP Kinases; GABA Antagonists; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Membrane Potentials; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Phenobarbital; Phenytoin; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Receptors, GABA-A; Silver Staining; Tamoxifen

Chronic hypoxia increases the sensitivity of the central nervous system to afferent input from carotid body chemoreceptors. We hypothesized that this process involves N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms and predicted that chronic hypoxia would change the effect of the NMDA receptor blocker dizocilpine (MK-801) on the poikilocapnic hypoxic ventilatory response (HVR). Male Sprague-Dawley rats were studied before and after acclimatization to hypoxia (70 Torr inspiratory Po(2) for 9 days). We measured ventilation (VI) and the HVR before and after systemic MK-801 treatment (3 mg/kg ip). MK-801 resulted in a constant respiratory frequency (approximately 175 min(-1)) during acute exposure to 10% and 30% O(2) before and after acclimatization. MK-801 had no effect on tidal volume (VT) before acclimatization, but it significantly decreased Vt when the animals were breathing 10% O(2) after acclimatization. The net effect of MK-801 was to eliminate the O(2) sensitivity of Vi before (via changes in respiratory frequency) and after (via changes in VT) acclimatization. Hence, chronic hypoxia altered the effect of MK-801 on the acute HVR, primarily because of increased effects on Vt. This indicates that changes in NMDA receptor-mediated neurotransmission may be involved in ventilatory acclimatization to hypoxia. However, further experiments are necessary to determine the precise location of such plasticity in the central nervous system.

Topics: Acetamides; Adaptation, Physiological; Animals; Chronic Disease; Dizocilpine Maleate; Glycine; Hypoxia; Male; Pulmonary Gas Exchange; Pulmonary Ventilation; Rats; Rats, Sprague-Dawley; Respiration; Tidal Volume

The mechanisms and functional consequences of ischemia-induced injury during perinatal development are poorly understood. Subplate neurons (SPn) play a central role in early cortical development and a pathophysiological impairment of these neurons may have long-term detrimental effects on cortical function. The acute and long-term consequences of combined oxygen and glucose deprivation (OGD) were investigated in SPn and compared with OGD-induced dysfunction of immature layer V pyramidal cortical neurons (PCn) in somatosensory cortical slices from postnatal day (P)0-4 rats. OGD for 50 min followed by a 10-24-h period of normal oxygenation and glucose supply in vitro or in culture led to pronounced caspase-3-dependent apoptotic cell death in all cortical layers. Whole-cell patch-clamp recordings revealed that the majority of SPn and PCn responded to OGD with an initial long-lasting ischemic hyperpolarization accompanied by a decrease in input resistance (R(in)), followed by an ischemic depolarization (ID). Upon reoxygenation and glucose supply, the recovery of the membrane potential and R(in) was followed by a Na+/K+-ATPase-dependent postischemic hyperpolarization, and in almost half of the investigated SPn and PCn by a postischemic depolarization. Whereas neither a moderate (2.5 mm) nor a high (4.8 mm) increase in extracellular magnesium concentration protected the SPn from OGD-induced dysfunction, blockade of NMDA receptors with MK-801 led to a significant delay and decrease of the ID. Our data demonstrate that OGD induces apoptosis and a profound dysfunction in SPn and PCn, and underline the critical role of NMDA receptors in early ischemia-induced neuronal damage.

Topics: Animals; Animals, Newborn; Caspase 3; Caspases; Dizocilpine Maleate; Dose-Response Relationship, Radiation; Electric Stimulation; Enzyme Inhibitors; Glucose; Hypoglycemic Agents; Hypoxia; Immunohistochemistry; In Vitro Techniques; Magnesium; Membrane Potentials; Neurons; Neuroprotective Agents; Ouabain; Patch-Clamp Techniques; Rats; Somatosensory Cortex; Tolbutamide

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

Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the downstream pathways activated by intracellular calcium overload.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Death; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glucose; Hippocampus; Hypoxia; Male; Microscopy, Immunoelectron; Mitogen-Activated Protein Kinases; Neurons; Neuroprotective Agents; Organ Culture Techniques; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Time Factors

To determine whether hippocampal pyramidal neurons retain authentic functional properties in mature organotypic culture, hippocampal slice cultures were established from young adult rats (P20-21). Cultures maintained 7 days in vitro retained tight organization of neuronal layers, as opposed to the widening restructure of pyramidal neurons often observed in perinatal slices. CA3 and CA1 pyramidal neurons fired action potentials in response to current injection and exhibited spontaneous and evoked synaptic currents, indicating intact neuronal function and normal hippocampal neural circuitry. We also tested neuronal sensitivity of slice cultures to ischemic injury. Acute ischemic paradigm resulted in selective death of pyramidal neurons in the CA1 region, which was prevented by treatment with an NMDA-antagonist, MK-801. Robust efflux of excitatory and inhibitory amino acid neurotransmitters was detected during ischemia, consistent with changes shown in acute slices. In summary, hippocampal organotypic cultures prepared from young adult rats maintained neuronal architecture and synaptic activity in vitro and can be used in parallel with an acute slice system to model mature brain tissue to examine ischemic pathophysiology and neuroprotective treatment.

Topics: Action Potentials; Animals; Animals, Newborn; Azides; Brain Injuries; Cell Death; Disease Models, Animal; Dizocilpine Maleate; Electric Stimulation; Evaluation Studies as Topic; Excitatory Amino Acid Antagonists; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Hypoxia; Immunohistochemistry; Male; Models, Biological; Neurons; Neurotransmitter Agents; Organ Culture Techniques; Oxygen; Patch-Clamp Techniques; Phenothiazines; Phosphopyruvate Hydratase; Rats; Rats, Wistar; Synaptic Transmission

Postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line in a novel rapid differentiation procedure. These neurons were used to establish an in vitro assay system that allows the investigation of hypoxic/ischaemic cell damage and the development of neuroprotective strategies. In experiments of simulated ischaemia, the neurons were subjected to anoxia and hypoglycaemia. The viability of NT-2 neuronal cells was significantly reduced by anoxia especially in the presence of glutamate, reflecting the cellular vulnerability to excitotoxic conditions. The addition of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduced glutamate-induced neuronal damage. Calcium imaging showed that NT-2 neurons increased cytosolic calcium levels in response to stimulation with glutamate or NMDA, an effect that was abolished in calcium free medium and at low pH values. The NMDA receptor antagonists MK-801, AP 5 and ketamine reduced the NMDA-induced response, suggesting the presence of functional NMDA receptors in the human neuronal cells. The mitochondrial potential of neurons was estimated using the fluorescent dye rhodamine 123 (R123). The fluorescence imaging experiments indicated an energetic collapse of mitochondrial functions during anoxia, suggesting that the human NT-2 neurons can be used to investigate subcellular processes during the excitotoxic cascade.

Topics: Aging; Calcimycin; Calcium; Cell Death; Cell Line, Tumor; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescein; Glutamic Acid; Humans; Hypoxia; Ionophores; Mitochondria; N-Methylaspartate; Neurons; Teratocarcinoma; Time Factors

Acute hypoxia produces an increase in ventilation. When the hypoxia is sustained, the initial increase in ventilation is followed by a decrease in ventilation. Hypoxia causes changes in brain neurotransmitters depending on its severity and durations. The purpose of this study was to investigate the role of gamma-aminobutyric acid (GABA) and glutamate for hypoxic ventilatory response in rabbits. The experiments were performed in peripheral chemoreceptors intact and denervated rabbits anesthetized with Na-pentobarbitate. For intracerebroventricular (ICV) injections of reagents in each animal, cannula was placed in left lateral cerebral ventricle by stereotaxic method. After ICV injection of GABA (0.48 mg/kg), air breathing in both groups caused a depression of respiratory activity. On the other hand, after ICV injection of GABA, breathing of hypoxic gas mixture (8% O(2)-92% N(2)) in both groups produced the hypoxic hyperventilation. After ICV injection of GABA, blockade of GABA(A) receptors with bicuculline (0.2 mg/kg) did not prevent the hypoxic hyperventilation. In contrast, after ICV GABA injection, blockade of glutamate NMDA receptors with MK-801 (0.09 mg/kg) completely abolished the hypoxic hyperventilation observed while the animals were breathing hypoxic gas mixture. Our findings suggest that ICV injection of GABA causes respiratory depression in normoxic conditions, and that it increases ventilation in hypoxic conditions with or without peripheral chemoreceptor impulses by increasing glutamate.

Topics: Adjuvants, Anesthesia; Anesthesia; Animals; Aorta; Carotid Arteries; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; GABA Modulators; gamma-Aminobutyric Acid; Glutamic Acid; Hypoxia; Oxygen; Pentobarbital; Phrenic Nerve; Rabbits; Receptors, N-Methyl-D-Aspartate; Respiration

Birth complications involving fetal hypoxia and stress at adulthood, which are risk factors for schizophrenia, can produce alterations in subcortical dopamine (DA) function in rat models. As adults, rats born either by cesarean section (C-section) or by C-section with added global anoxia show increased stress-induced DA release from nucleus accumbens and increased amphetamine-induced locomotion, compared to vaginally born controls. Moreover, stress at adulthood interacts with these birth insults to modulate DA receptor and transporter levels. Glutamatergic transmission at the level of the nucleus accumbens, prefrontal cortex, and hippocampus are known to modulate subcortical DA activity. Thus, altered excitatory amino acid (EAA) function might contribute to the dopaminergic changes observed in rats after birth insult and/or stress at adulthood. To test this possibility, rats born vaginally, by C-section, or by C-section with 15 min of anoxia, were either repeatedly stressed (15 min of tail pinch daily for 5 days) at adulthood or received no stress, and levels of EAA receptor binding were measured by ligand autoradiography in limbic brain regions. As adults, rats born by C-section showed increases in AMPA receptor binding in nucleus accumbens shell, NMDA receptor binding in cingulate cortex, and kainate receptor binding in the hippocampal CA1 region. Anoxic rats showed increases in CA1 kainate receptor and anterior olfactory NMDA receptor binding. Stress at adulthood increased AMPA receptor binding in several regions of prefrontal cortex and reduced NMDA receptor binding in infralimbic cortex and dentate gyrus, across all birth groups. Two instances of interactions between birth insult and stress at adulthood were observed. Stress reduced cingulate cortex NMDA receptor binding and increased olfactory tubercle kainate receptor binding only in C-sectioned animals, but not in controls. The possibility that the observed EAA receptor changes contribute to dopaminergic dysfunction in these animal models is discussed, in light of known glutamate-DA interactions.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Autoradiography; Binding Sites; Birth Injuries; Brain; Cesarean Section; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Hypoxia; Iodine Isotopes; Male; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Stress, Physiological; Tritium

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Boron Compounds; Calcium; Calcium Channel Blockers; Cell Death; Clonazepam; Dantrolene; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fura-2; Glucose; Hippocampus; Hypoxia; In Situ Nick-End Labeling; Indoles; Intracellular Space; Ion Exchange; Lidocaine; Mibefradil; Nimodipine; Organ Culture Techniques; Quinoxalines; Rats; Rats, Wistar; Sodium; Sodium Channel Blockers; Thiazepines; Thiourea; Time Factors

This study seeks to discern the influence of the NMDA glutamate-mediated pathway in the early stimulatory and late depressant phases of the hypoxic ventilatory response. We addressed this issue by recording ventilation before and after intravenous administration of the NMDA receptor antagonist MK-801 during acute, steady-state hypoxic challenges in the anesthetized, spontaneously breathing rats. Minute ventilation and its volume and frequency components were calculated and compared at the peak and nadir of the hypoxic response. We found that NMDA receptor antagonism appreciably affected both ventilatory phases of hypoxia. The early stimulation of ventilation was attenuated and the late depression was accentuated. The latter consisted of abolishment of the characteristic sustenance of hypoxic ventilation above the baseline level in the depressant phase, so that ventilation declined down to the baseline after NMDA receptor blockade. The inability to uphold ventilation in the depressant phase suggests that the NMDA glutamate-mediated pathway is operative in shaping the late hypoxic ventilatory response. The role of the glutamatergic pathway may thus be extended beyond the hitherto recognized early ventilatory stimulation of hypoxia.

Topics: Anesthesia; Animals; Blood Pressure; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; Male; Rats; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics

The digastric muscle acts for both feeding (including mastication and swallowing) and respiration. In this study, we examined whether or not the muscle activity is detectable during anoxia in developing rats. Rats at 4 different ages, days 5, 10, 16, and 24, were exposed to 100% N2 under pentobarbital or ketamine-xylazine anesthesia, and the electromyograms of digastric muscles (dEMG) and the diaphragm (diaEMG) were examined simultaneously. Prior to the anoxic exposure, at all ages, the dEMG was similar to or less apparent than the diaEMG, which was detected at each inspiratory movement. In anoxia, we first observed dEMG activity, mostly sporadic (days 5 and 10) or mostly tonic (days 16 and 24), when diaEMG activity was temporarily suppressed (we termed it Phase 1). Second, synchronous phasic or tonic dEMG and phasic diaEMG were recorded temporarily before terminal apnea (we termed it Phase 2). These phenomena were also obtained in vagotomized rats (all ages) or in rats injected with the N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (days 16 and 24). In conclusion, our results suggest that in anoxia, dEMG activity is detectable during diaEMG suppression in early anoxia, irrespective of the developmental age, the anesthetic (pentobarbital or ketamine-xylazine), vagotomy, or MK-801 injections.

Topics: Animals; Animals, Newborn; Dizocilpine Maleate; Electromyography; Hypoxia; Masticatory Muscles; Neck Muscles; Rats; Rats, Wistar; Respiratory Mechanics; Trigeminal Nerve

We studied changes in organism's sensitivity and resistance to hypoxic hypoxia under conditions of NMDA receptor blockade with MK-801. Breathing hypoxic gas mixture after administration of MK-801 sharply decreased the mean blood pressure in the systemic circulation and slightly improved lung ventilation in animals. Respiratory arrest was observed in 40% animals. NMDA receptor blockade decreased organism's sensitivity and resistance to hypoxic hypoxia.

Topics: Animals; Blood Pressure; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; Male; Rats; Receptors, N-Methyl-D-Aspartate; Respiratory System

Hypoxia of the brain may alter further seizure susceptibility in a different way. In this study, we tried to answer the question how episode of convulsion induced by hypoxia (HS) changes further seizure susceptibility, and how N-methyl-D-aspartic acid (NMDA) and AMPA/KA receptor antagonists influence this process. Adult Albino Swiss mice exposed to hypoxia (5% O(2)) developed clonic/tonic convulsions after about 340 s. Mice which underwent 10 s but not 5 s seizures episode subsequently exhibited significantly increased seizure susceptibility to low doses (equal ED(16)) of bicuculline (BCC) and NMDA during a 3-week observation period. No morphological signs of brain tissue damage were seen in light microscope on the third day after a hypoxia-induced seizure (HS). Learning abilities assessed in passive avoidance test as well as spontaneous alternation were not disturbed after an HS episode. Pretreatment with AMPA/KA receptor antagonist NBQX effectively prolonged latency to HS and given immediately after seizure episode also attenuated subsequent convulsive susceptibility rise, however, NMDA receptor antagonist, MK-801, appeared to be ineffective. These results suggest that a seizure episode induced by hypoxia, depending on its duration, may play an epileptogenic role. The AMPA/KA receptor antagonist prolongs the latency to HS, and given after this episode, prevents the long-term epileptogenic effect.

Topics: Animals; Avoidance Learning; Bicuculline; Brain Chemistry; Dizocilpine Maleate; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; gamma-Aminobutyric Acid; Hypoxia; Mice; N-Methylaspartate; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Seizures

Oxygen and glucose deprivation (OGD) in cell cultures is generally studied in a medium, such as artificial cerebrospinal fluid (CSF), with an ion composition similar to that of the extracellular fluid of the normal brain (2 to 4 mmol/L K+, 2 to 3 mmol/L Ca2+; pH 7.4). Because the distribution of ions across cell membranes dramatically shifts during ischemia, the authors exposed mouse organotypic hippocampal tissue cultures to OGD in a medium, an ischemic cerebrospinal fluid, with an ion composition similar to the extracellular fluid of the brain during ischemia (70 mmol/L K+, 0.3 mmol/L Ca2+; pH 6.8). In ischemic CSF, OGD induced a selective and delayed cell death in the CA1 region, as assessed by propidium iodide uptake. Cell death was glutamate receptor dependent since blockade of the N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mitigated cell damage. Hyperglycemia aggravates ischemic brain damage whereas glucose in artificial CSF prevents oxygen deprivation-induced damage. The authors demonstrate that glucose in ischemic CSF significantly exacerbates cell damage after oxygen deprivation. This new model of "ischemia" can be useful in future studies of the mechanisms and treatment of ischemic cell death, including studies using genetically modified mice.

Topics: Animals; Brain Ischemia; Calcium; Cerebrospinal Fluid; Culture Techniques; Dizocilpine Maleate; Electrophysiology; Excitatory Amino Acid Antagonists; Glucose; Hippocampus; Hydrogen; Hypoxia; Ions; Mice; Mice, Inbred BALB C; Potassium; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

The influences of N-methyl-D-aspartate (NMDA) type glutamate receptor antagonism, by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate (MK-801), on breathing pattern and ventilatory chemoresponses, were assessed in anaesthetized vagotomized spontaneously breathing golden-mantled ground squirrels, Spermophilus lateralis. MK-801 was administered by either bilateral pressure micro-injection into a region of the rostral dorsolateral pons, containing the medial and lateral Parabrachial and Kölliker-Fuse nuclei (the Parabrachial complex, PbC), or by systemic injection. Both treatments induced apneusis. These data indicate that functional NMDA receptor-mediated processes located within the PbC terminate inspiration and actively prevent apneusis in vagotomized ground squirrels. Although both hypercapnia and hypoxia stimulated breathing during the apneusis, the responses were generally slight. The breathing frequency component of the hypercapnic ventilatory response was completely eliminated supporting the hypothesis that the PbC is an integral component of the control network for CO(2) chemoreflex responses. Differences in the results of systemic versus PbC MK-801 illustrate that NMDA receptor-mediated processes outside the PbC do influence ventilation. Our data also show that such processes outside the PbC lengthen both inspiration and expiration in this species, slowing ventilation, and are necessary for the expression of the hypoxic ventilatory response.

Topics: Animals; Apnea; Dizocilpine Maleate; Drug Administration Routes; Excitatory Amino Acid Antagonists; Female; Hypercapnia; Hypoxia; Injections, Intraperitoneal; Male; Microinjections; Pons; Receptors, N-Methyl-D-Aspartate; Sciuridae; Tidal Volume; Vagotomy; Ventilation; Work of Breathing

Tianeptine is an antidepressant with proven clinical efficacy and effects on hippocampal plasticity. Hypoxia increased lactate dehydrogenase (LDH) release from cortical neuronal cultures, and tianeptine (1, 10 and 100 microM) inhibited LDH release as efficiently as the N-methyl-D-aspartate (NMDA) antagonist, MK-801. However, tianeptine did not block apoptosis in cultured cortical neurones caused by NMDA, but reduced apoptosis when interleukin-1beta (IL-1beta) was included with NMDA. In 5-day old mice, intracerebral injection of ibotenate induced reproducible lesions in cortex and white matter. Lesion size was markedly reduced by co-administration of MK-801 (1 mg/kg i.p.) but neither by tianeptine or its enantiomers administered acutely (1, 3 or 10 mg/kg i.p.) nor by tianeptine administered chronically (10 mg/kg i.p. for 5 days). Chronic administration of IL-1beta (10 ng/kg i.p. for 5 days) prior to ibotenate injection exacerbated lesion size in cortex and white matter, and this exacerbation was prevented by chronic pre-treatment with tianeptine (10 mg/kg i.p.) or by acute administration of tianeptine (10 mg/kg i.p.) concomitantly with ibotenate. Thus tianeptine has neuroprotective effects against hypoxia in tissue culture and against the deleterious effects of cytokines in cortex and white matter, but not against NMDA receptor-mediated excitotoxicity.

Topics: Animals; Animals, Newborn; Antidepressive Agents, Tricyclic; Brain; Cell Hypoxia; Cells, Cultured; Cytokines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Hypoxia; Ibotenic Acid; Interleukin-1; L-Lactate Dehydrogenase; Mice; Neuronal Plasticity; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Stereoisomerism; Thiazepines

MAP2 (microtubule-associated protein 2) is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites, particularly near spines. MAP2 is degraded after ischemia and other metabolic insults, but the time course and initial triggers of that breakdown are not fully understood. This study determined that MAP2 resides in a complex with the NMDA receptor, suggesting that spatially localized changes may be important in the mechanism of MAP2 redistribution and breakdown after oxygen-glucose deprivation (OGD). Using OGD in the adult rat hippocampal slice as a model system, this study demonstrated that MAP2 breakdown occurs very early after OGD, with the first statistical decrease in MAP2 levels within the first 30 min after the insult. There is a dramatic redistribution of MAP2 to the somata of pyramidal neurons, particularly neurons at the CA1-subiculum border. Free radicals and nitric oxide are not involved in the damage to MAP2. NMDA-receptor activation plays a prominent role in the MAP2 breakdown. In direct response to NMDA receptor activation, calcium influx, likely through the receptor ion channel complex, as well as release of calcium from the mitochondria through activation of the 2Na(+)-Ca(2+) exchanger of mitochondria, triggers MAP2 degradation. The proteolysis of MAP2 is limited by endogenous calpain activity, likely via the spatial access of calpain to MAP2.

Topics: Animals; Calcium; Calcium Channel Blockers; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glucose; Glycoproteins; Hippocampus; Hypoxia; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Male; Microtubule-Associated Proteins; N-Methylaspartate; Neurons; Oxygen; Precipitin Tests; Protein Subunits; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors

Hypoxia is a pathogenetic factor in various inner ear diseases, and increasing importance is attached to the protection of the cochlea from traumatic influences. It was recently demonstrated in guinea pigs that magnesium can significantly reduce ischemia- and impulse noise-induced hearing loss. The aim of this study was to evaluate if magnesium has a protective effect on hypoxia-induced hair cell loss using an in vitro model of the new-born rat cochlea In view of the NMDA receptor-antagonistic action of magnesium, we tested MK 801, a highly potent and selective non-competitive NMDA receptor antagonist. Organotypic cochlea cultures were exposed to hypoxia (pO2 = 10-20 mm Hg at 37 degrees C) in DMEM medium containing magnesium (0.75 or 3.0 mmol/l) or MK801 (1 or 10 micromol/l) for 24 or 36 h. The cultures were phalloidin-labeled for counting the number of outer and inner hair cells (OHC/IHC). The mean damage in normoxic controls was 1-4%. IHC revealed a significantly higher susceptibility to hypoxia than OHC. In the normal magnesium group (0.75 mmol/l), 36-hour exposure to hypoxia caused a mean loss of about 25% OHC and 60% IHC. In the groups treated with either 3.0 mmol magnesium or 10 microm MK 801, the damage was significantly reduced to about 10% in OHC and 35% in IHC. This study supports previous in vivo observations in the guinea pig demonstrating the protective effects of magnesium on noise-induced impairment of inner ear oxygenation.

Topics: Animals; Animals, Newborn; Cochlea; Dizocilpine Maleate; Hair Cells, Auditory, Inner; Hair Cells, Auditory, Outer; Hypoxia; Magnesium; Neuroprotective Agents; Oxygen; Phalloidine; Rats; Receptors, N-Methyl-D-Aspartate; Time Factors

We investigated the effect of betaxolol on the decrease of mitochondrial aspartate aminotransferase (mAAT) activity in rat retinas induced by hypoxia in vitro. It is reported that mAAT decreases in ischemic or hypoxic retina and that the decrease is caused by Ca(2+)-dependent proteases such as calpain. Betaxolol is a compound that has beta(1)-adrenergic receptor blocking and voltage-dependent calcium channel blocking properties. The rat eye cups were maintained with Locke's solution saturated with 95% air - 5% CO(2). The eye cups were immersed in glucose-free Locke's solution saturated with 95% N(2) / 5% CO(2) (hypoxic solution). Ninety minutes of hypoxia caused a 20% decrease in mAAT activity. The eye cups incubated with the hypoxic solution containing 1 mM EGTA, 10 micro M MK-801 or 100 micro M betaxolol were protected from the decrease in mAAT activity, so that the residual mAAT activity was 50%, 45% or 40%, respectively, compared to the eye cups incubated in hypoxic solution alone, which had a 100% decrease in mAAT activity. In addition, co-incubation with EGTA and betaxolol had a greater protective effect against the mAAT decrease than a single application. This additive effect of betaxolol was dose-dependent. These results suggested that betaxolol had a protective effect against the decrease of mAAT caused by hypoxia and indicated that betaxolol might inhibit the Ca(2+) release from intracellular Ca(2+) stores.

Topics: Animals; Aspartate Aminotransferase, Mitochondrial; Betaxolol; Calcium; Dizocilpine Maleate; Dose-Response Relationship, Drug; Egtazic Acid; Excitatory Amino Acid Antagonists; Hypoxia; In Vitro Techniques; Neuroprotective Agents; Rats; Retina

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.

Topics: Animals; Apoptosis; Base Sequence; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; DNA Primers; Hypoxia; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Gasping is an important mechanism for survival that appears to be developmentally modulated by the glutamate-nitric oxide (NO) pathway. However, the temporal characteristics of NO brain tissue levels during gasping are unknown. We hypothesized that during anoxia-induced gasping, the gasping frequency would be closely correlated with caudal brainstem tissue NO concentrations in developing rats. Brainstem and cortical tissue NO levels were measured during anoxia using a voltammetric electrode in adult rats and 5-day-old pups during control conditions and following pretreatment with the NMDA receptor antagonist MK-801 (1 mg/kg) or the neuronal NO synthase inhibitor 7-nitro-indazole (7-NI; 100 mg/kg). In young animals, NO tissue levels followed a triphasic trajectory coincident with gasp frequency which was markedly altered by MK-801 and 7-NI, albeit with preservation of gasp frequency-NO tissue level relationships. In adult rats, 40-fold higher NO tissue levels occurred and followed a monophasic trajectory coincident with gasp patterning. In the cortex, monophasic increases in NO levels occurred at all ages. We conclude that anoxia-induced gasping neurogenesis is modulated via NMDA-NO mechanisms in the developing rat. We postulate that higher NO brainstem concentrations may favor early autoresuscitation, but limit anoxic tolerance.

Topics: Aging; Animals; Animals, Newborn; Brain Stem; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hypoxia; Indazoles; Neocortex; Nitric Oxide; Nitric Oxide Synthase; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiration; Respiration Disorders

The effects of blockade of N-methyl-D-aspartate (NMDA) type glutamate receptors by a non-competitive antagonist (MK-801) on cortical arousal, breathing pattern and ventilatory responses to hypoxia (10% O2 in N2) and hypercapnia (5% CO2 in air) were assessed in anesthetized (urethane) and unanesthetized golden-mantled ground squirrels (Spermophilus lateralis). Intra-cerebroventricular administration of MK-801 did not alter ventilation during wakefulness, although it did alter the pattern (breathing frequency and tidal volume components) of the hypercapnic ventilatory response, and suppressed the ventilatory response to hypoxia. Animals did not sleep following treatment with MK-801, and intravenous administration of MK-801 prevented expression of the sleep-like state normally observed in anesthetized animals. In anesthetized animals MK-801 elevated breathing frequency to levels observed without anesthesia, and suppressed the hypoxic ventilatory response. These data suggest that NMDA-type glutamatergic receptor-mediated processes influence cortical arousal and facilitate depression of breathing frequency during anesthesia and the hypoxic ventilatory response. Such processes are not essential for the hypercapnic ventilatory response.

Topics: Acute Disease; Animals; Arousal; Chronic Disease; Dizocilpine Maleate; Electroencephalography; Excitatory Amino Acid Antagonists; Female; Hypercapnia; Hypoxia; Male; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics; Sciuridae

Effects of intrauterine hypoxia-ischemia (HI) on expression of the NMDA receptor subunits as well as on [3H]MK-801 binding of the NMDA receptor were studied in 1-day to 30-day old rat brain. Intrauterine HI conditions were achieved on gestation day 17 by clamping the uterine vasculature for 30 min followed by removal of the clamps to permit reperfusion. As determined by reverse-transcriptase polymerase chain reaction, prenatal HI significantly reduced mRNA expression of the NRI subunit of the NMDA receptor in the hippocampus of 4, 8, and 30-day old rat brains. NR2A and NR2B subunit mRNAs were expressed in the hippocampus and the cortex of both the control and the prenatal HI rat brains. Intrauterine HI did not significantly affect expression of either the NR2A or NR2B subunit mRNA. Consistent with the RT-PCR data, protein expression of the NRI subunit in the hippocampus, but not the cortex, of 21-day old prenatal HI rat brains was significantly decreased as compared to the control rat brain. Intrauterine HI also significantly reduced binding affinity, but not the number of binding sites, of the NMDA receptor to [3H]MK-801, a noncompetitive antagonist of the NMDA receptor, in the hippocampus of 21-day old rat brain. The overall results suggest that prenatal HI-induced reduction of NRI expression and the altered binding ability of the NMDA receptor in the young rat brain may contribute to other long-lasting effects of intrauterine HI that we reported previously.

Topics: Aging; Animals; Animals, Newborn; Brain; Brain Ischemia; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Fetal Diseases; Hypoxia; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Receptors, N-Methyl-D-Aspartate; RNA, Messenger

The effects of citicoline and/or low dose of MK-801 (sufficient to prevent the development of seizures) on survival, neurological and behavioral recovery following transient hyperglycemic-oligemic-hypoxic insult have been evaluated in mice. Neurological recovery was assessed semi-quantitatively on the third and the 10th day after the insult, and behavioral tests evaluating spontaneous locomotor activity, motor coordination and spontaneous alternation performance were performed on day 10. Neither drug given alone did influence survival rate, but the combination of MK-801 and higher citicoline dose decreased mortality on day 10. Behavioral performance was markedly compromised by the insult. Citicoline, but not MK-801, slightly but significantly improved behavioral outcome in all three tests.. when brain ischemic insult is complicated with acute hyperglycemia, post-treatment with citicoline combined with MK-801 in low anti-convulsive dose improves survival and neurological recovery, and citicoline but not MK-801 enhances behavioral recovery.

Topics: Animals; Behavior, Animal; Brain Ischemia; Cytidine Diphosphate Choline; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hyperglycemia; Hypoxia; Male; Mice; Motor Activity; Nervous System

N-methyl-D-aspartate (NMDA) receptors play important roles in the neural control of respiration. We hypothesized that the brainstem circuit for respiratory control is modulated in response to chronic hypoxia during postnatal maturation, and the modulation may involve changes in the neurotransmission mediated by the NMDA receptors for inspiratory termination. Electrophysiological studies were performed on anesthetized, vagotomized, paralyzed and ventilated rats. Phrenic nerve activity was recorded in normoxic control and chronically hypoxic (CH) rats maintained in normobaric hypoxia (10% O2) for 4-5 weeks from birth. In normoxic rats, the NMDA receptor antagonist, dizocilpine (MK801, i.p.) irreversibly increased inspiratory time (Ti) by 53% and decreased expiratory time (Te) by 29%. However, MK801 did not change the Ti, Te, respiratory rate and peak phrenic nerve activity in CH rats. Results suggest that brainstem mechanisms underlying inspiratory termination mediated by NMDA receptors are modulated by early chronic hypoxia.

Topics: Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; Rats; Receptors, N-Methyl-D-Aspartate; Respiration

Topics: Anesthesia; Anesthetics, Inhalation; Anesthetics, Intravenous; Animals; Blood Gas Analysis; Carbon Dioxide; Dizocilpine Maleate; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Hypoglossal Nerve; Hypoxia; Isoflurane; Male; Oxygen; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiratory Mechanics; Urethane

To study in vivo phosphorylation of N-methyl-D-aspartate (NMDA) glutamate receptors and the recruitment of protein kinase C isoforms during acute hypoxia, dorsocaudal brainstem lysates were harvested from conscious rats exposed to either room air or hypoxia (10% O2 for 5 and 15 min). Increased phosphorylation of the NR-1 subunit at serine residue 896 occurred during hypoxia and was blocked by pre-treatment with MK-801. Immunoblots of soluble and particulate fractions revealed subcellular translocation for PKC-beta, -gamma, -delta, -epsilon, and -iota during hypoxia with no changes in PKC-alpha, -mu, and -zeta. Translocation of PKC-beta, -delta and -epsilon was selectively attenuated following MK-801. We demonstrate that hypoxia leads to PKC-mediated activation of NMDA receptors in the brainstem, and that PKC-beta, -delta and -epsilon are the most likely candidates for NR-1 phosphorylation.

Topics: Animals; Biological Transport; Brain Stem; Consciousness; Dizocilpine Maleate; Enzyme Activation; Excitatory Amino Acid Antagonists; Hypoxia; Isoenzymes; Nerve Tissue Proteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Signal Transduction

Neuronal protein synthesis is inhibited in CA1 pyramidal neurons for many hours after ischemia, hypoxia or hypoglycemia. This inhibition precedes cell death, is a hallmark characteristic of necrotic damage and may play a key role in the death of vulnerable neurons after these insults. The sequence of events leading to this inhibition remains to be fully elucidated. The protein synthesis failure after 7.5 min anoxia/aglycemia in the rat hippocampal slice can be prevented by blocking N-methyl-D-aspartate receptors in a reduced calcium environment during the insult. In this study, we demonstrate that N-methyl-D-aspartate exposure directly causes a dose-dependent, receptor-mediated and prolonged protein synthesis inhibition in CA1 pyramidal neurons. The free radical scavenger Vitamin E significantly attenuates this damage due to low concentrations of N-methyl-D-aspartate (10 microM). Free radical generation by xanthine/xanthine oxidase (XOD) can directly damage protein synthesis in neurons of the slice. Vitamin E, ascorbic acid and N-acetylcysteine can each prevent the damage due to anoxia/aglycemia and to higher concentrations of N-methyl-D-aspartate (50 microM), provided calcium levels are reduced concomitantly. These findings indicate that both free radicals and calcium play a role in the sequence of events leading to protein synthesis failure after energetic stress like anoxia/aglycemia. They further suggest that the mechanism by which N-methyl-D-aspartate receptor activation damages protein synthesis involves free radical generation.

Topics: Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Free Radical Scavengers; Free Radicals; Hippocampus; Hypoglycemia; Hypoxia; Male; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Vitamin E

The impact of hypoxia on somatostatin (SS) secretion from the median eminence (ME) of the hypothalamus and the possible glucocorticosteroid involvement in modulating secretion, were investigated in adult male rats exposed to hypoxia. SS levels were measured by specific radioimmunoassay during acute and prolonged hypoxia as well as after bilateral adrenalectomy (ADX) with or without a replacement with dexamethasone (DEX). The results were as follows: (a) acute hypoxia (5 km altitude, 10.8% O(2)) for 2 and 24 h markedly increased SS content in ME, but acute severe hypoxia (7 km, 8.2% O(2) for 24 h) markedly decreased SS level in ME. (b) Chronic hypoxia (10.8% O(2)) from 5 to 25 days exposure did not significantly affect SS content of ME. (c) ADX alone increased SS content of ME and this increase was further enhanced after 2 h exposure to hypoxia. (d) The increased SS in ME of ADX rats was blocked by replacement with DEX (500 microg/rat i.p.). The data presented suggest that acute hypoxia stress may increase or decrease SS content of ME in rats, depending on the severity and duration of the hypoxia and that the stimulatory action of hypoxia on SS content of ME be may in part mediated by the increased corticosterone levels during hypoxia.

Topics: Acute Disease; Adrenal Glands; Adrenalectomy; Altitude; Animals; Chronic Disease; Dexamethasone; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; Male; Median Eminence; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Somatostatin; Stress, Physiological; Time Factors

This study tests the hypothesis that brain tissue hypoxia results in modification of spermine-dependent activation of the cerebral N-methyl-D-aspartate (NMDA) receptor ion-channel in newborn piglet brains and that pretreatment with N(omega)-nitro-L-arginine (NNLA), an inhibitor of nitric oxide synthase, will reduce the hypoxia-induced modification of the spermine-dependent activation of the receptor. Piglets were assigned to one of four groups; normoxia or hypoxia with or without NNLA. The infusion of NNLA or vehicle lasted for 60 min while the animals were ventilated under either hypoxic or normoxic conditions. Cerebral tissue hypoxia was confirmed by measuring ATP and phosphocreatine (PCr) levels. P2 membranes were isolated and 3H-MK-801 binding was measured in the presence of spermine. Steady state 3H-MK-801 binding in the presence of spermine, showed an increase in receptor affinity in both normoxic (47% of control) and hypoxic (42% of control) animals without change in receptor density. During hypoxia, the spermine-dependent increase in the maximal response of the 3H-MK-801 binding correlated inversely with the ATP concentrations. NNLA pretreatment prior to hypoxia, resulted in a decrease in the slope of the regression line describing the relationship between cellular energy state (ATP) and percent change in maximal response to spermine compared with vehicle treated animals indicating attenuation of the response to hypoxia. We conclude that the spermine-dependent modification of the affinity of the NMDA receptor ion-channel as assessed by 3H-MK-801 binding is similar in hypoxic and normoxic cortical tissue. NNLA administration reduces the hypoxia-induced spermine-dependent activation of the receptor indicating that nitric oxide mediates modification of the spermine site activation of the NMDA receptor ion-channel complex.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Cerebral Cortex; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hypoxia; Nitric Oxide Synthase; Nitroarginine; Receptors, N-Methyl-D-Aspartate; Reference Values; Spermine; Swine

Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia-reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK-801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca(2+)-independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate-dependent iNOS expression was concentration-dependent and was blocked by EGTA and by the inhibitors of nuclear factor kappaB (NF-kappaB) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF-kappaB translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca(2+)-dependent activation of the transcription factor NF-kappaB. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia.

Topics: Animals; Chelating Agents; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Glucose; Glutamic Acid; Hypoxia; In Vitro Techniques; L-Lactate Dehydrogenase; Male; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prosencephalon; Pyrrolidines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thiocarbamates

Severe anoxia induces destabilisation of intracellular calcium homeostasis in neurones. The mechanism of this effect, and particularly the interrelationship between changes in intracellular concentration of free Ca(2+) ions and the content of the intracellular Ca(2+) stores, during and after anoxia, is not clear. We used a superfusion system of rat olfactory cortical slices for the fluorimetric estimation of changes in the intracellular concentration of free Ca(2+) ions and in the level of bound Ca(2+), utilising the fluorescent indicators Fura-2 and chlortetracycline, respectively. It was found that 10-min normoglycaemic anoxia results in simultaneous decrease in bound and increase in free Ca(2+) levels, whereas during 60-min reoxygenation, we detected an increase in both indices. The NMDA receptor antagonists MK-801 and APV attenuated changes in free Ca(2+) level during anoxia and reoxygenation and intensified anoxia-evoked decrease in bound Ca(2+) content, whereas a late post-anoxic increase in bound Ca(2+) was abolished. These data suggest that the influx of extracellular Ca(2+) to neurones via NMDA receptors, plays a critical role in the rise of intracellular free Ca(2+) concentration during and after anoxia. Biphasic changes in bound Ca(2+) content during anoxia and reoxygenation may reflect an anoxia-induced release of Ca(2+) from intracellular stores, followed later by a neuronal calcium overload and refilling of intracellular Ca(2+) binding sites.

Topics: 2-Amino-5-phosphonovalerate; Animals; Calcium; Chlortetracycline; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Fura-2; Hypoxia; In Vitro Techniques; Intracellular Membranes; Olfactory Pathways; Oxygen; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Time Factors

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+), which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca(2+) concentration [Ca(2+)](i), whereas the elevation of intracellular Na(+) has received less attention. In the present study we investigated the effects of inhibitors of different Na(+) channels and of the Na(+)/Ca(2+) exchanger, which couples the Na(+) to the Ca(2+) gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na(+) channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca(2+) accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca(2+) extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca(2+)](i) increase caused by the exchanger.

Topics: Animals; Brain Ischemia; Cell Death; Culture Techniques; Dizocilpine Maleate; Electrophysiology; Hippocampus; Homeostasis; Hypoglycemia; Hypoxia; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Tetrodotoxin; Thiourea

We, and others, have previously demonstrated that N-methyl-D-aspartate (NMDA) receptor is involved in hypoxia or ischemia-mediated responses. We found that the NMDA antagonist ketamine attenuates cortical nitric oxide release during cerebroischemia. It has been reported that ethanol (EtOH) antagonizes NMDA-induced responses in various systems. In the present study, the interaction of EtOH and KCl-evoked striatal dopamine release in vivo during acute hypoxia was examined. High-speed chronoamperometric recording techniques, using Nafion-coated carbon fiber electrodes, were used to evaluate extracellular dopamine (DA) concentration in the striatum of urethane-anesthetized Sprague-Dawley rats. KCl was directly applied to the striatum to evoke release of DA. These anesthetized animals were paralyzed with d-tubocurarine and connected to a respirator to allow controlled respiration. Systemic concentrations of oxygen were altered by changing the rate of the respirator. We previously reported that lowering the respiratory rates from 90 to 20 times/min for 5 min decreased arterial PO(2) and facilitated KCl-induced DA release in the striatum. In this study, we found that application of NMDA antagonist MK801 attenuates hypoxic DA release, suggesting that NMDA receptor is involved in this hypoxic reaction. In contrast, EtOH dose dependently enhanced KCl-evoked DA release during hypoxia. To further examine the interactions of excitatory amino acid and EtOH on DA release, glutamate was locally applied to the striatum. Glutamate-induced DA release was not affected by the systemic application of EtOH. Taken together, these data suggest that EtOH enhances DA release in vivo during short-term hypoxia, possibly through mechanisms other than excitatory amino acid pathways.

Topics: Animals; Blood Gas Analysis; Central Nervous System Depressants; Dizocilpine Maleate; Dopamine; Dose-Response Relationship, Drug; Electrochemistry; Ethanol; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoxia; Neostriatum; Potassium Chloride; Rats; Rats, Sprague-Dawley

The present study tests the hypothesis that nitration is a potential mechanism of N-methyl-D-aspartate (NMDA) receptor modification, by assessing the effect of peroxynitrite in vitro on the glutamate and ion-channel sites of the NMDA receptor in the fetal guinea pig. Nitration of NMDA receptor subunits was confirmed by Western blot. Following peroxynitrite exposure, (3)H-MK-801 bindings show an increase in the B(max) and a decrease in the K(d), while (3)H-glutamate bindings show a decrease in the K(d) with no change in the B(max). We conclude that peroxynitrite regulates the NMDA receptor function by increasing the affinity of the ion-channel and glutamate sites, and by exposing additional ion-channel sites. We propose that nitration of the NMDA receptor is a potential mechanism for the regulation of the receptor during hypoxia.

Topics: Animals; Cerebral Cortex; Dizocilpine Maleate; Fetus; Glutamic Acid; Guinea Pigs; Hypoxia; Ion Channels; Kinetics; Nitrates; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

An in vitro model of ischemia was developed and characterized using the acute rat hippocampal slice preparation. Neuroprotective concentrations of several competitive and noncompetitive glutamate subtype-selective antagonists (CGS-19755, MK-801, YM90K and GYKI-52466) were initially determined in anoxia-enhanced agonist-induced excitotoxicity experiments. Concentrations which proved to be effective in these studies were subsequently tested for their effectiveness against an ischemic episode. Ischemia was defined as a 30-min exposure to aglycemic media ending in 5 min of concurrent anoxia, a protocol which was arrived at by empirically determining the effect of various hypoglycemic and anoxic insults on the ability of hippocampal slices to retain their electrophysiological viability. Exposure to such an ischemic episode resulted in a loss of viability by most slices, an effect which was strongly dependent on extracellular calcium. AMPA antagonists applied alone produced no neuroprotective effect in the present model of in vitro ischemia, while NMDA antagonists applied alone had a modest neuroprotective effect. In contrast, the coapplication of 10 microM MK-801 and 300 microM GYKI-52466, noncompetitive NMDA and AMPA receptor antagonists, respectively, resulted in almost complete neuroprotection. This protection was comparable to that obtained by withholding extracellular calcium, indicating that the toxic effects of glutamate receptor overstimulation can be accounted for solely by calcium influx. The effect of this combination treatment on the survival rate of hippocampal slices was synergistic, that is greater than the sum of the effects of the individual compounds. The results indicate that neuroprotection against acute ischemic insults may require a combination therapy approach.

Topics: Animals; Anti-Anxiety Agents; Benzodiazepines; Brain Ischemia; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hypoxia; Male; Neuroprotective Agents; Pipecolic Acids; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.

Topics: Animals; Animals, Newborn; Brain Ischemia; Calcium; Dizocilpine Maleate; Enzyme Inhibitors; Guanidines; Hypoxia; Indazoles; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Oxygen; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

Topics: Animals; Benzodiazepines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; In Vitro Techniques; Ischemia; Male; Norepinephrine; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Stereoisomerism; Tritium

Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4-dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia-induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia-induced DA depletion, aglycemia- and ischemia-induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK-801 and also by comparing the glutamate-induced DA release with that during anoxia, aglycemia, or ischemia. MK-801 decreased the anoxia-induced DA depletion in a dose-dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia-induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK-801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Biological Transport; Brain Ischemia; Corpus Striatum; Dizocilpine Maleate; Dopamine; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Glucose; Glutamic Acid; Hypothermia; Hypoxia; Male; Neurons; Nomifensine; Organ Culture Techniques; Ouabain; Oxygen; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Tritium

We have recently demonstrated that inducible nitric oxide synthase (iNOS) is expressed in rat forebrain slices exposed to oxygen and glucose deprivation (OGD). Now, we have found that the expression of iNOS after OGD is time-dependent since 20 min of OGD produces the appearance of iNOS at earlier times than 10 min of OGD. OGD also causes neurotoxicity in this model, as revealed by the increase in excitatory amino acid, neuron specific enolase and lactate dehydrogenase (LDH) efflux to the incubation solution. Finally, the administration of the NMDA receptor antagonist MK-801 (100 nM) inhibits both the expression of iNOS and the release of LDH. Our findings demonstrate that this method may be considered an useful in vitro model of ischemia-reperfusion to determine the therapeutic role of neuroprotective tools.

Topics: Animals; Brain Ischemia; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Glucose; Hypoxia; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphopyruvate Hydratase; Prosencephalon; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The present study tests the hypothesis that Mg2+ modification of N-methyl-D-aspartate receptor ion channel opening is altered during hypoxia and correlates with the progressive decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in five normoxic and nine hypoxic ventilated piglets. In the hypoxic group, varying degrees of cerebral energy metabolism were achieved by administration of different fractions of inspired oxygen (FiO2) (5-9%) for varying durations of time and were documented by cortical tissue phosphocreatine levels. [3H]Dizocilpine maleate binding was performed with increasing concentrations of MgSO4 from 0.01 to 15 mM in cortical P2 membrane fractions. Mg2+ percentage activation and Mg2+ 50% inhibitory concentrations (IC50) were determined. The mean +/- S.D. phosphocreatine value was 3.0 +/- 1.3 micromol/g brain in the normoxic group and 1.4 +/- 1.0 micromol/g brain in the hypoxic group (P < 0.01). Low concentrations of Mg2+ (0.01-1 mM) increased [3H]dizocilpine maleate binding in the normoxic group (to 137 +/- 26% of baseline), significantly greater than in the hypoxic group (109 +/- 13%, P < 0.05). Receptor activation correlated with brain tissue levels of phosphocreatine, with percentage maximal activation decreasing linearly as phosphocreatine levels decreased (r=0.7). Higher levels of Mg2+ (1.5-15 mM) caused inhibition of [3H]dizocilpine maleate binding, with IC50 levels significantly higher in the normoxic group (3.2 +/- 1.1 mM) than in the hypoxic group (1.9 +/- 0.4 mM). Mg2+ IC50 values decreased in a linear fashion as phosphocreatine values decreased (r=0.9). The data demonstrate that, as brain cell energy metabolism decreases during hypoxia, maximal receptor activation by low levels of Mg2+ decreases and receptor inhibition by high levels of Mg2+ increases in a linear fashion. We speculate that, during hypoxia, dephosphorylation of the ion channel of the N-methyl-D-aspartate receptor increases Mg2+ blockade of the receptor by increasing Mg2+ accessibility to its binding site and that receptor modification may be initiated by subtle decreases in cortical oxygenation in the newborn brain.

Topics: Animals; Animals, Newborn; Cerebral Cortex; Dizocilpine Maleate; Energy Metabolism; Excitatory Amino Acid Antagonists; Hypoxia; Ion Channel Gating; Magnesium; Phosphocreatine; Receptors, N-Methyl-D-Aspartate; Swine

Effect of dizocilpine (0.5 mg/kg i.p.) on epileptic afterdischarges elicited by low-frequency electrical stimulation of the dorsal hippocampus was studied in rat pups aged 12 and 18 days. Repeated elicitation of afterdischarges (ADs) in control animals resulted in a progressive increase of the duration of ADs in both age groups. Dizocilpine (MK-801) injected after the first afterdischarge suppressed this prolongation in 12-day-old rats only. Hypobaric hypoxia (simulated altitude of 9000 m for one hour) led to a marked prolongation of the first afterdischarge in both age groups with a tendency to shorter ADs after repeated stimulations. Dizocilpine potentiated this tendency in 12-day-old rat pups so that it became statistically significant. Administration of dizocilpine before hypoxia prevented the increase in duration of the first afterdischarge in both age groups.

Topics: Animals; Dizocilpine Maleate; Electric Stimulation; Electroencephalography; Epilepsy; Hippocampus; Hypoxia; Male; Neuroprotective Agents; Rats; Rats, Wistar

The experimental evidence linking glutamate to ischemic neuronal injury is derived from in vitro or in vivo animal stroke models. We, therefore, developed an in vitro preparation to determine whether glutamate contributes to early neuronal swelling in oxygen and glucose deprived (OGD) human neocortical slices. In order to monitor neuronal swelling, we measured extracellular tissue resistance in brain slices by passing constant current pulses through two electrodes and recording the voltage drop between them. We verified that NMDA (30 microM) or OGD induced a rise in tissue resistance in rat neocortical slices. We then examined human neocortical slices from 11 patients undergoing resections for intractable epilepsy. Both the rodent and human neocortical slices swelled within 10 min of OGD. In both, the glutamate antagonist dizocilpine (MK-801) reduced the swelling. In the rats, MK-801 (5 microM) prolonged the latency to onset of neuronal swelling following OGD from 7.6 +/- 0.6 min (mean +/- S.E.M., n = 16) to 17.4 +/- 2.6 min (n = 6; p < 0.01). Other putative neuroprotective agents were much less effective in this paradigm. In the human slices, MK-801 again prolonged the latency to resistance increase from 8.6 +/- 0.4 min (n = 8) to 17.2 +/- 1.7 min (n = 9, p < 0.01). This is the direct demonstration that glutamate receptor activation leads to neuronal swelling in substrate deficient human brain. These results, which are similar to those obtained in the rodent brain slices, help validate the animal slices as appropriate models for the study of OGD in human brain.

Topics: Animals; Brain Edema; Cerebral Cortex; Dizocilpine Maleate; Electric Impedance; Excitatory Amino Acid Antagonists; Female; Glucose; Humans; Hypoxia; In Vitro Techniques; Male; Neuroprotective Agents; Neurotoxins; Rats

Glutamate significantly increased levels of spontaneous chemiluminescence (CL) in rat hippocampal slices incubated under hypoxic conditions. Although it has been previously shown that guanine nucleotides (GN) displace glutamate from several of its receptors, in our study only GMP, as well as the glutamate antagonist MK-801, was able to reverse the increase in CL provoked by glutamate. On the other hand, not only GTP or Gpp(NH)p failed to reverse the action of glutamate, but they increased CL production like glutamate. This effect of GTP/Gpp(NH)p was also reversed by GMP. We concluded that, under neurotoxic conditions, GMP acted as an antagonist and GTP or Gpp(NH)p acted as agonists of glutamate. These results reinforced the evidence of the existence of extracellular site(s) for GN and indicated a possible role for GN in excitotoxicity.

Topics: Animals; Cyclic AMP; Dizocilpine Maleate; Female; Glutamic Acid; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hippocampus; Hypoxia; In Vitro Techniques; Luminescent Measurements; Rats; Rats, Wistar

The present study tested the hypothesis that the increase in extracellular striatal dopamine during hypoxia is least partly associated with activation of N-methyl-D-aspartate (NMDA) and/or non-NMDA excitatory amino acid receptors. Studies were performed in anesthetized and mechanically ventilated 2-3 days old piglets. Hypoxic insult was induced by decreasing the oxygen fraction in inspired gas (FiO2) from 22 to 7% for 1 h, followed by 1 h reoxygenation at 22%. Cortical oxygen pressure was measured optically by oxygen dependent quenching of phosphorescence, and extracellular striatal dopamine was measured using in vivo microdialysis. The microdialysis probes were perfused with Ringer solution +/- 50 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) or 50 microM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). One hour of hypoxia decreased the cortical oxygen pressure from 46 +/- 3 Torr to 10 +/- 1.8 Torr. In striatum perfused with Ringer, statistically significant increase in extracellular dopamine, to 1050 +/- 310% of control, was observed after 20 min of hypoxia. By 40 min of hypoxia the extracellular level of dopamine increased to 4730 +/- 900% of control; by the end of the hypoxic period the values increased to 18,451 +/- 1670% of control. The presence of MK-801 in the perfusate significantly decreased the levels of extracellular dopamine during hypoxia. At 20, 40 and 60 min of hypoxia extracellular level of dopamine increased to 278 +/- 94% of control, 1530 +/- 339% of control and 14,709 +/- 1095 of control, respectively. The presence of NBQX caused a statistically significant decrease, by about 30%, in the extracellular dopamine compared to control, only at the end of the hypoxic period. It can be concluded that in striatum of newborn piglets, the excitatory NMDA receptors but not the non-NMDA receptors may be modulating the changes in extracellular levels of dopamine. The NMDA receptor antagonist, MK-801, may exert part of its reported neuroprotective effect to hypoxic stress in striatum by decreasing the levels of extracellular dopamine.

Topics: Animals; Animals, Newborn; Corpus Striatum; Dizocilpine Maleate; Dopamine; Excitatory Amino Acid Antagonists; Extracellular Space; Hypoxia; Oxygen; Oxygen Consumption; Pressure; Quinoxalines; Receptors, Amino Acid; Swine

Posthypoxic frequency decline (PHFD) refers to the undershoot in respiratory frequency that follows brief hypoxic exposures. Lateral pontine neurons are required for PHFD. The neurotransmitters involved in the circuit that activate and/or are released by these pontine neurons regulating PHFD are unknown. We hypothesized that N-methyl-D-aspartate (NMDA) receptors are required for PHFD, because of the similarity in respiratory pattern after blocking lateral pontine activity or NMDA receptors. Furthermore, we hypothesized that the location of these NMDA receptors could be visualized by optimizing binding affinity with spermidine. In vagotomized, anesthetized rats (n = 16), cardiorespiratory responses to hypoxia (8% O2, 30-90 s) were recorded before and after dizocilpine (10 microg-1 mg/kg iv), and NMDA receptors were mapped with [3H]dizocilpine (n = 6). Dizocilpine elicited a dose-related effect on PHFD, blocking PHFD at high doses. Resting arterial blood pressure and breathing frequency decreased with high doses of dizocilpine, but the respiratory response to hypoxia remained intact. Our novel anatomical data indicate that NMDA receptors were widespread but distributed differentially in the brain stem. We conclude that NMDA receptors are located in pontine and medullary respiratory-related regions and that PHFD requires NMDA-receptor activation.

Topics: Animals; Brain Stem; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Hypoxia; Male; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiration; Tissue Distribution

In the nucleus of the solitary tract, NMDA receptors are critical for the hypoxic ventilatory response while neuronal nitric oxide synthase (NOS) modulates the late component of this response. Nuclear factor (NF)-kappaB is a ubiquitous transcription factor that increases the expression of multiple stress-activated genes. We sought to examine temporal changes in expression of NF-kappaB within the dorsocaudal brain stem of conscious rats after exposures to 10% O2. Time-dependent increases in NF-kappaB occurred with hypoxia and peaked at 60 min. Pretreatment with the N-methyl-D-aspartate (NMDA)-receptor channel antagonist dizocilpine maleate (MK-801) markedly attenuated NF-kappaB complexes during hypoxia. In contrast, after NOS inhibition with NG-nitro-L-arginine methyl ester (L-NAME), although NF-kappaB was diminished in normoxia, increased NF-kappaB expression still occurred with hypoxia. Increased phosphorylation of the NF-kappaB regulatory unit [inhibitory (I)kappaB] was detected by immunoblotting and also peaked at 60 min. Phosphorylation of Ikappa-B during hypoxia was attenuated by MK-801 but not by L-NAME. Thus NMDA-receptor activation in the dorsocaudal brain stem during hypoxia elicits in NF-kappaB activity marked enhancements that are unaffected after NOS blockade.

Topics: Animals; Blotting, Western; Brain Stem; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hypoxia; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphorylation; Rats; Rats, Sprague-Dawley; Time Factors

Using dizocilpine (MK-801), we tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors are important controllers of cerebral O2 supply/consumption balance in newborn piglets both during normoxia and hypoxia. Twenty-five 2 to 7-day-old piglets were anesthetized and divided into four groups: (1) Normoxia (n = 6), (2) Normoxia + MK-801 (n = 6), (3) Hypoxia (n = 6), and (4) Hypoxia + MK-801 (n = 7). Regional cerebral blood flow (rCBF) in ml/min/100 g was measured using 14C-iodoantipyrine, and we determined arterial and venous O2 saturations by microspectrophotometry, calculating cerebral O2 consumption (VO2) in ml O2/min/100 g in the cortex, hypothalamus and pons. MK-801 did not significantly affect regional VO2 or rCBF in normoxic piglets. Hypoxia resulted in an increase in local rCBF compared to controls: from 41 +/- 6 to 103 +/- 18 in the cortex; 34 +/- 7 to 101 +/- 20 in the hypothalamus; and 45 +/- 10 to 95 +/- 11 in the pons. Pretreatment with MK-801 abolished this hypoxic flow effect in the cortex (51 +/- 2) and hypothalamus (49 +/- 5), but not in the pons (91 +/- 17). Similar results were observed for VO2 with control values of 1.9 +/- 0.3, 1.6 +/- 0.2 and 2.1 +/- 0.3 for the cortex, hypothalamus and pons respectively. Hypoxia resulted in an increase in the VO2 to 3.9 +/- 0.4 (cortex), 3.8 +/- 0.6 (hypothalamus) and 3.9 +/- 0.8 (pons). Pretreatment with MK-801 prior to hypoxia abolished these effects in the cortex (2.1 +/- 0.2) and hypothalamus (2.1 +/- 0.2), but not in the pons (2.9 +/- 0.2). These findings suggest that NMDA receptors may play a role in the control of cerebral metabolism during hypoxia in this immature porcine model.

Topics: Animals; Animals, Newborn; Blood Pressure; Brain; Carbon Dioxide; Cerebrovascular Circulation; Dizocilpine Maleate; Female; Heart Rate; Hemodynamics; Hypoxia; Male; Oxygen; Oxygen Consumption; Partial Pressure; Receptors, N-Methyl-D-Aspartate; Reference Values; Regional Blood Flow; Swine

1. The influence of voltage dependent calcium channel blocker (VDCC), nimodipine and N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 on the brain free arachidonic acid (FAA) level and on the learning ability in hypoxia-exposed rats was examined. 2. Some animals were decapitated after cerebral hypoxia had been obtained and the brain FAA level was determined by gas chromatography. The other animals were trained in a passive avoidance procedure and were exposed to hypoxic conditions immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 hours later. 3. Various doses of nimodipine (0.03; 0.1; 0.3 and 1.0 mg/kg) and MK-801 (0.03; 0.1 and 0.3 mg/kg) had been injected 30 minutes before biochemical or behavioral procedures started. 4. It was found that hypoxia strongly increased the brain FAA level and impaired the retention of the passive avoidance response. 5. Pretreatment with 0.3 mg/kg and 1.0 mg/kg of nimodipine prevented the brain FAA accumulation. It has also been shown that all tested doses of nimodipine significantly improved the retention deficit in the animals exposed to hypoxia. 6. Neither the one of tested doses of MK-801 influenced significantly the increase of the brain FAA level and/or passive avoidance behavior in hypoxic animals. 7. These results confirm the hypothesis that the brain FAA accumulation and cognitive impairment, caused by hypoxia, are maybe associated with disturbances in calcium homeostasis and that nimodipine may be useful in ameliorating the hypoxia-induced brain tissue damage. Blocade of NMDA receptor-channel complex by MK-801 was not sufficient to prevent hypoxia-induced neuronal damage.

Topics: Animals; Arachidonic Acid; Brain; Dizocilpine Maleate; Female; Hypoxia; Learning; Nimodipine; Rats; Rats, Wistar

Cerebral ischemia is known to induce endogenous adaptive mechanisms such as the activation of ATP-sensitive potassium channels that can prevent or delay neuronal injury. This process can be therapeutically mimicked by treatment with potassium channel openers. Primary neuronal cell cultures were derived from embryonic chick telencephalon and were exposed to chemical hypoxia (1 mM cyanide) or excitotoxic injury (1 mM L-glutamate). While treatments with the potassium channel openers bimakalim (1-10 microM) and EMD 57283 (0.1-10 microM) were clearly able to maintain neuronal viability after chemical hypoxia, similar concentrations of the drugs had negligible effects on glutamate-induced neurotoxicity. In contrast, both types of neuronal injury were sensitive to the protective action of the glutamate receptor antagonist dizocilpine (MK-801; 0.1-1 microM). The neuroprotective effect of bimakalim against chemically induced hypoxic injury was reversed by tolbutamide (1 microM), an ATP-sensitive potassium channel blocker. These experiments demonstrate neuroprotective effects of potassium channel openers that could be related to inhibition of neurotransmitter release.

Topics: Adenosine Triphosphate; Animals; Benzopyrans; Cell Survival; Chick Embryo; Dihydropyridines; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoxia; Neurons; Neuroprotective Agents; Potassium Channels; Pyridazines; Sodium Cyanide

Increased neuronal vulnerability to ischemia or hypoxia has been demonstrated following traumatic brain injury but not explained. Animal data suggest that neuronal damage after traumatic brain injury is caused mainly by massive glutamate release that activates N-methyl-D-aspartate (NMDA) receptors. Using rat models with controlled closed head injury (CHI) followed by hypoxia, we investigated extracellular concentrations of neuroactive amino acids in the hippocampus by in vivo microdialysis. CHI alone produced an immediate increase of glutamate and taurine; hypoxia alone did not alter amino acid concentrations. CHI followed by hypoxia produced a biphasic increase in extracellular glutamate and taurine, with an immediate peak after CHI and a prolonged plateau after hypoxia. Though changes in gamma-aminobutylic acid (GABA) concentration is also prolonged by combined traumatic and hypoxic insults, it showed less alteration than glutamate. Pre-treatment with dizocilpine maleate (MK-801), a non-competitive NMDA antagonist, did not affect the immediate peak of glutamate after CHI but significantly diminished the prolonged plateau after hypoxia. These findings suggest that traumatic brain injury may increase hypoxic release of glutamate, contributing to increased vulnerability to hypoxia. Our data suggest that MK-801 may be beneficial in preventing secondary neuronal damages by hypoxia.

Topics: Amino Acids; Animals; Brain Injuries; Dizocilpine Maleate; Hippocampus; Hypoxia; Male; Microdialysis; Rats; Rats, Sprague-Dawley

The NMDA receptor subtype is the major excitatory mediator for glutamate neurotoxicity. To assess its participation in the noxious effects of postnatal hypoxia, we have characterized the binding of the ionophoric marker of NMDA receptor, dizocilpine (MK-801). Binding of (+)-3-[125I]MK-801 to NMDA brain receptors under nonequilibrium conditions was quantified by in vitro autoradiography in rats exposed to hypoxia induced by 93% N2/6.5% O2 exposure for 70 min on Postnatal Day 4. Acute and long-lasting effects were investigated at 4 h after injury and on Postnatal Day 40. At the acute stage, a transient decrease in binding was found in several specific brain areas, hypothalamus, amygdaloid nuclei, entorhinal cortex, perirhinal cortex, and hippocampus, and no differences were found in temporal cortex, thalamus, and geniculate nucleus, when compared to sham-treated animals. At this early age, there was no increase of binding when slices from both groups were incubated in the presence of glutamate and glycine (Glu/Gly), positive allosteric modulators of MK-801 binding. In the 40-day-old brains, the binding to the NMDA receptors of hypoxiatreated animals was not different with respect to controls in most of the areas studied, but the Glu/Gly stimulation of binding in hypoxic rats showed a reduced, or absent, response to the allosteric modulators. In contrast, control rats showed a remarkable increase of the specific binding induced by the presence of the modulators in the incubation buffer. Binding of (+)-3-[125I]MK-801 was also performed at a higher concentration to clarify whether the altered response to Glu/Gly may be due to differences in the number of channels; however, the density of NMDA receptors at this concentration was similar in both control and hypoxia-treated rats. We conclude that the effect of exposure of newborn rats to hypoxia can generate acute and long-lasting effects on the NMDA receptor. The deleterious action of this kind of noxa on the CNS could be exerted by interference with normal glutamatergic transmission and hence over normal growth and development.

Topics: Acute Disease; Animals; Animals, Newborn; Brain Chemistry; Brain Damage, Chronic; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Hypoxia; Hypoxia, Brain; Organ Specificity; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors; Up-Regulation

We investigated the functional characteristics of the NMDA receptor that modulates hypoxia/hypoglycaemia-induced striatal dopamine release. Dopamine release was detected by fast cyclic voltammetry in rat neostriatal slices. Four variables were measured: T(on) -- time from initiation of hypoxia/hypoglycaemia to the onset of dopamine release, Tpk -- time from onset to maximum, deltaDA/delta(t) -- rate of dopamine release and DAmax -- maximum extracellular dopamine concentration. In controls, T(on) = 164.9 +/- 1.7 s, Tpk = 20.9 +/- 0.9 s, deltaDA/delta(t) = 5.31 +/- 0.44 microM/s and DAmax = 79.1 +/- 2.5 microM (means +/- S.E.M., n = 203). Cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755, 20 microM) lengthened, while N-methyl-D-aspartate (NMDA) (100 microM) shortened T(on). (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK 801, 1 and 10 microM) and dextromethorphan (10 and 100 microM) increased Tpk and decreased DAmax. Neither glycine (100 microM), 7-chlorokynurenic acid (50 microM) nor 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione (ACEA 1021, 100 microM) had any effect although 7-chlorokynurenic acid blocked the effect of NMDA. Increasing [Mg2+] from 1.3 to 3.7 mM, increased Tpk and decreased deltaDA/delta(t). Dithiothreitol (1 mM) accelerated T(on) while 5.5-dithio-bis-(2-nitrobenzoic acid) (1 mM) delayed T(on). Neither drug affected Tpk, DAmax or deltaDA/delta(t). Neither spermidine (100 microM) nor arcaine (100 microM) affected T(on), Tpk or deltaDA/delta(t) although arcaine decreased DAmax. In conclusion, hypoxia/hypoglycaemia-induced dopamine release was influenced by an NMDA receptor although modulation of the glycine recognition site of the receptor was ineffective, as were agents acting at polyamine modulatory zones. These findings highlight differences between recombinant and native NMDA receptors and suggest caution in extrapolating molecular biology to functional studies.

Topics: Animals; Biogenic Polyamines; Dextromethorphan; Dizocilpine Maleate; Dopamine; Excitatory Amino Acid Antagonists; Hypoglycemia; Hypoxia; In Vitro Techniques; Magnesium; Male; Neostriatum; Rats; Rats, Wistar; Receptors, Glutamate; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Phencyclidine

A series of 1,2,3,4-tetrahydroisoquinoline derivatives were synthesized and evaluated for anticonvulsant activity against intracerebro-ventriculas (i.c.v.) N-methyl-D-aspartate (NMDA)-induced seizures in mice. Among these compounds, (+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride ((+)-1a, FR115427) was the most effective anticonvulsant, and also protected CA1 hippocampal neurons from ischemia-induced neuronal degeneration in rats at 32 mg/kg i.p. In addition, (+)-1a showed anti-hypoxic activity in mice at 3.2-32 mg/kg i.p. The absolute configuration at the C-1 position of the isoquinoline ring was determined to be S by a single-crystal X-ray analysis of (+)-1a (+)-di-p-toluoyl-D-tartrate. Structure-activity relationships with regard to the anticonvulsant activity of this series of compounds are discussed, and the three-dimensional structures of (S)-(+)-1a and MK801 are compared.

Topics: Animals; Anticonvulsants; Brain Ischemia; Crystallography, X-Ray; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Hypoxia; Isoquinolines; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred ICR; Molecular Structure; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Seizures; Stereoisomerism; Structure-Activity Relationship; Tetrahydroisoquinolines

Induction of chemical anoxia, using sodium azide in cerebellar granule cells maintained in primary culture, was evaluated as an in vitro assay for screening of potential neuroprotective compounds. The purpose of this study was to evaluate sodium azide as an alternative to cyanide salts, compounds which, despite their unfavorable characteristics, are often used in assays for chemical anoxia. The viability of neuronal cultures after treatment with azide, with or without preincubation with calcium channel blockers, tetrodotoxin (TTX), or glutamate receptor antagonists, was monitored by subsequent incubation with the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), followed by isopropanol extraction and spectrophotometric quantification of cellularly reduced MTT. The azide-induced degeneration of neurons was shown to be dependent on the concentration as well as on the duration of incubation with submaximal concentrations of azide. Incubation of the neurons with nifedipine, a blocker of L-type voltage-sensitive calcium channels (L-VSCC), or with the noncompetitive N-methyl-D-aspartate (NMDA) subtype glutamate receptor antagonist MK-801, prior to addition of submaximal concentrations of azide, significantly attenuated azide-induced neuronal death. Blockers of N-type and Q-type VSCC (omega-conotoxin MVIIA and MVIIC, respectively) and the P-type VSCC blocker omega-agatoxin IVA had no effect in this assay. The sodium channel blocker TTX was without effect when added to neurons under depolarizing conditions, but potently and effectively protected cells when experiments were performed in a nondepolarizing buffer. The results show that chemical anoxia induced by incubation of cultured neurons with azide leads to detrimental effects, which may be quantitatively monitored by the capability of the cells to reduce MTT. This procedure is a suitable method for screening of compounds for possible protective effects against neuronal death induced by energy depletion. In addition, the results suggest involvement of L-type VSCC as well as of glutamate receptors in the pathways leading to neuronal degradation induced by energy depletion in cerebellar granule neurons. This would further support the notion that these pathways might be important in neurodegeneration induced by cerebral ischemia or anoxia.

Topics: Animals; Azides; Brain Ischemia; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Dose-Response Relationship, Drug; Female; Hypoxia; Mice; Mice, Inbred Strains; Mutagens; Nifedipine; Pregnancy; Sodium Azide

This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.

Topics: Animals; Binding, Competitive; Brain; Cerebral Cortex; Dizocilpine Maleate; Hippocampus; Hypoxia; Rats; Receptors, N-Methyl-D-Aspartate; Time Factors

The cytotoxic effects of hypoxia and excitatory amino acids on cultured retinal ganglion cells were studied. The influence of coculture with retinal Müller glia and cortical astrocytes on cell survival was evaluated, as were the effects of the N-methyl-DL-aspartate inhibitor, MK-801. Dissociated retinal neurons from 7-day-old Sprague-Dawley rats were plated on a laminin substrate, neocortex-derived glial monolayers, or Müller cell monolayers. Ganglion cells were labeled by injection of DiI into the superior colliculus 2 days prior to dissociation. Exposure of cultured ganglion cells to glutamate and N-methyl-DL-aspartate showed a time- and concentration-dependent survival rate. Exposure of cells to hypoxia demonstrated a survival rate that was dependent on time and O2 concentration. Excitotoxic and hypoxic damage was entirely blocked by the specific non-competitive inhibitor of N-methyl-DL-aspartate, MK-801. Retinal ganglion cells cultured on cortical astrocytes and retina-derived Müller glia showed significantly better survival rates (P < 0.001) than cells cultured on laminin-coated dishes under control conditions, in hypoxia (9% to 15% O2), and after exposure to 200 microM glutamate. Retinal ganglion cells cultured on Müller glia showed significantly better survival rates (P < 0.01) than those cultured on cortical astrocytes under conditions of hypoxia (9% to 15% O2) and exposure to 200 microM glutamate. The results demonstrate that excitotoxic and hypoxic damage to cultured retinal ganglion cells is moderated by NMDA receptor blockade and by the presence of glial cells, especially retinal Müller cells. This system may provide a useful model for studying the pathophysiology of excitotoxicity and hypoxia on cultured retinal ganglion cells, and may be used to help identify potentially clinically useful therapeutic agents.

Topics: Animals; Astrocytes; Cell Culture Techniques; Cell Survival; Cells, Cultured; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glutamic Acid; Hypoxia; N-Methylaspartate; Neuroglia; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Time Factors

1. We examined the role of nitric oxide (NO) in respiratory regulation in the nucleus tractus solitarii (NTS), where L-glutamate release associated with peripheral chemoreceptor activation modulates the hypoxic ventilatory response. 2. Experiments were performed in unanaesthetized freely moving rats. First, the effects on the hypoxic ventilatory response of sodium nitroprusside (SNP, a NO donor) or NG-monomethyl-L-arginine (L-NMMA, a NO synthase inhibitor), microinjected into the NTS, were investigated. Second, using in vivo microdialysis, changes in extracellular L-glutamate during hypoxia were examined in the presence of L-NMMA. Third, the effect of L-NMMA on ventilatory augmentation by exogenous L-glutamate was examined. Furthermore, we measured extracellular L-citrulline concentration changes during hypoxia in the NTS to assess NO formation indirectly and also examined the effect of MK-801 (an NMDA receptor antagonist) on L-citrulline levels during hypoxia. 3. SNP increased ventilation during both normoxia and hypoxia. L-NMMA did not alter ventilation or L-glutamate levels during normoxia but significantly attenuated the hypoxic ventilatory response and the increase in L-glutamate during hypoxia. The inhibition by L-NMMA was blocked by L-arginine. The ventilatory augmentation by exogenous L-glutamate was attenuated by L-NMMA. L-Citrulline increased during hypoxia, and this increase was inhibited by MK-801. 4. We provide the first in vivo evidence that, in the NTS, NO works as a retrograde messenger in an L-glutamate-releasing positive feedback system contributing to the augmentation of ventilation during hypoxia.

Topics: Animals; Arginine; Citrulline; Dizocilpine Maleate; Drug Combinations; Enzyme Inhibitors; Glutamic Acid; Hypercapnia; Hypoxia; Male; Microinjections; Nitric Oxide; Nitroprusside; omega-N-Methylarginine; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Respiration; Solitary Nucleus

Anoxic depolarization (AD) and failure of ion homeostasis play an important role in ischemia-induced neuronal injury. In the present study, different drugs with known ion-channel-modulating properties were examined for their ability to interfere with cardiac-arrest-elicited AD and with the changes in the extracellular ion activity in rat brain. Our results indicate that only drugs primarily blocking membrane Na+ permeability (NBQX, R56865, and flunarizine) delayed the occurrence of AD, while compounds affecting cellular Ca2+ load (MK-801 and nimodipine) did not influence the latency time. The ischemia-induced [Na+]e reduction was attenuated by R56865. Blockade of the ATP-sensitive K+ channels with glibenclamide reduced the [K+]e increase upon ischemia, indicating an involvement of the KATP channels in ischemia-induced K+ efflux. The KATP channel opener cromakalim did not affect the AD or the [K+]e concentration. The ischemia-induced rapid decline of extracellular calcium was attenuated by receptor-operated Ca2+ channel blockers MK-801 and NBQX, but not by the voltage-operated Ca2+ channel blocker nimodipine, R56865, and flunarizine.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Calcium; Dizocilpine Maleate; Flunarizine; Glyburide; Heart Arrest; Hypoxia; Ion Channels; Male; Nimodipine; Piperidines; Potassium; Potassium Channels; Quinoxalines; Rats; Rats, Wistar; Sodium; Thiazoles

In the present study, we investigated the possibility that MK-801 (dizocilpine), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, owes its potent neuroprotective properties to calcium channel blocking ability rather than to its NMDA receptor antagonism. Rat hippocampal slices were exposed to a long hypoxic period (20 min) from which only 13.8% recovered their neuronal function after 30 min of reoxygenation. The recovery rate of neuronal function from 20-min hypoxia was increased to 100% when slices were pretreated with 5 microM MK-801. DL-2-amino-5-phosphonovalerate (APV), a competitive NMDA receptor antagonist, even at relatively high concentration (100 microM), provided only marginal protection against such severe hypoxic insult. The L-type calcium channel blocker diltiazem (DILT) was more effective than APV in protecting hypoxic slices against neuronal damage. Combining suboptimal concentrations of DILT and MK-801 produced a neuroprotective effect with significantly exceeded the calculated additive effect of the two drugs. Such synergism could not be demonstrated between DILT and APV, a combination that produced only the expected additive neuroprotective effect. The observed synergy between the calcium channel blocker (DILT) and MK-801, along with other studies that demonstrated interaction between these two drugs, led us to postulate that MK-801 possesses calcium channel blocking properties through which its neuroprotective effect is exerted. These calcium channels could either be of the L-type or otherwise, channels which are being activated only under stressful conditions, such as hypoxia or ischemia.

Topics: Animals; Diltiazem; Dizocilpine Maleate; Dose-Response Relationship, Drug; Hippocampus; Hypoxia; Kinetics; Male; Membrane Potentials; Rats; Rats, Sprague-Dawley

The aim of the present study was to determine whether calcium channel antagonists attenuated hypoxia/hypoglycemia- or glutamate-induced reduction in 2-deoxyglucose (2-DG) uptake of hippocampal slices obtained from ethanol withdrawal rats. Ethanol withdrawal significantly potentiated the hypoxia/hypoglycemia- and glutamate-induced reductions in 2-DG uptake of hippocampal slices. Both nifedipine and flunarizine exhibited attenuating effects on ethanol withdrawal-induced potentiation of impairment of 2-DG uptake caused by hypoxia/hypoglycemia or glutamate. Hypoxia/hypoglycemia-induced deficit of 2-DG uptake was prevented by ethanol, but chronic consumption of ethanol resulted in the development of tolerance to neuroprotective effect. These findings suggest that the increased sensitivity of neurons to ischemic damage by ischemia may involve in the increased activity of calcium channels in the hippocampus.

Topics: Animals; Calcium Channel Blockers; Deoxyglucose; Dizocilpine Maleate; Dose-Response Relationship, Drug; Ethanol; Flunarizine; Glutamic Acid; Hippocampus; Hypoglycemia; Hypoxia; Male; Nifedipine; Rats; Rats, Wistar; Substance Withdrawal Syndrome

Mice were exposed to an atmosphere consisting of 7% O2 and 93% N2 or 5.5% O2 and 94.5% N2 for 60 min. The susceptibility of the mice to the convulsive effect of pentylenetetrazol (PTZ) was decreased, in comparison to that of naive or sham-exposed controls, 1 and 7 days after exposure to 7% O2. A significant protective effect against PTZ-induced seizures was not observed in mice exposed to 5.5% O2. N-methyl-D-aspartate (NMDA) administrated immediately after exposure to the hypoxic atmosphere, had no significant influence on the protective effect of hypoxia. Treatment of naive or sham-exposed mice with NMDA resulted in protection against PTZ-induced seizures when they were tested 7 days later. Dizolcipine (MK 801), at a dose of 0.01 mg/kg injected i.p. 10 min before hypoxia, abolished the protective effect of hypoxia; higher doses (0.1 or 0.3 mg/kg) of MK 801 were not effective. The adenosine A1 receptor antagonist 1,3-diethyl-8-phenylxanthine (DPX), administered at a dose of 0.1 mg/kg s.c. before hypoxia, blocked the decrease in the susceptibility to the convulsive effect of PTZ. DPX also blocked the protective effect, seen after 7 days, of NMDA given to control mice. These results suggest that both NMDA and adenosine A1 receptor-mediated processes were involved in the protective effect of moderate hypoxia against PTZ-evoked seizure.

Topics: Animals; Dizocilpine Maleate; Excitatory Amino Acid Agents; Hypoxia; Male; Mice; Mice, Inbred Strains; N-Methylaspartate; Pentylenetetrazole; Receptors, Purinergic; Seizures; Time Factors; Xanthines

The present study investigated the influence of MK-801 (N-methyl-D-aspartate receptor antagonist) and U83836E (antioxidative aminosteroid) on the permeability of sodium fluorescein through a cell barrier during hypoxia (2 h 95% N2/5% CO2). The barrier consisted of porcine brain capillary endothelial cells and of cerebral rat astrocytes cultivated on two sides of a filter. After hypoxia, the permeation of fluorescein was significantly increased (10.2 +/- 1.5 x 10(-3) cm/min, P < 0.001) compared to the normoxic control (2 h 95% O2/5% CO2, 1.8 +/- 0.6 x 10(-3) cm/min). The hypoxia-enhanced permeation was significantly (P < 0.05) reduced by 10 microM MK-801 (2.0 +/- 0.5 x 10(-3) cm/min) and 10 microM U83836E (3.1 +/- 1.3 x 10(-3) cm/min). The results demonstrate, for the first time in a cell culture system, that hypoxia impairs brain endothelial barrier function, and that this enhanced permeability can be influenced pharmacologically. It is concluded that two distinct pathogenic mechanisms are involved in hypoxic cerebral endothelial cell injury, and that cerebroprotection afforded by these agents may result, in part, from reductions in edema secondary to improved blood-brain barrier function.

Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain; Capillaries; Cells, Cultured; Cerebrovascular Circulation; Chromans; Dizocilpine Maleate; Endothelium, Vascular; Fluorescein; Fluoresceins; Free Radical Scavengers; Hypoxia; Piperazines; Rats; Rats, Wistar; Swine

The CA1 region of hippocampus is selectively vulnerable to a variety of insults, including hypoxia-ischemia and Alzheimer's disease, but the basis of this regional susceptibility is poorly understood. We examined the regional hippocampal sensitivity to mitochondrial metabolic disruption induced by malonate, an inhibitor of succinate dehydrogenase. The CA1 region was exquisitely sensitive to malonate and the dentate gyrus was extremely resistant; the CA3 region had intermediate sensitivity. This pattern of vulnerability is reminiscent of hypoxic-ischemic damage. Malonate damage was blocked by the N-methyl-D-aspartic acid (NMDA) antagonist, MK-801, but regional susceptibility to malonate did not correlate with the density of NMDA receptors. Instead, malonate toxicity was inversely correlated with activity of succinate dehydrogenase. Our results suggest that regional metabolic capacity may help to determine sensitivity to metabolic/excitotoxic insults such as hypoxia-ischemia.

Topics: Animals; Autoradiography; Brain Ischemia; Dizocilpine Maleate; Hippocampus; Hypoxia; Hypoxia, Brain; Male; Malonates; N-Methylaspartate; Rats; Rats, Sprague-Dawley

Substantiating evidence has raised the possibility that sigma ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of sigma ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the sigma binding site. However, sigma specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of sigma ligands, with either significant (sigma/PCP) or negligible (sigma) affinity for the PCP site of the NMDA receptor, in order to delineate a selective sigma site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only sigma ligands with affinity for the NMDA receptor [(+) and (-) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the sigma [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and kappa-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC50) value, of sigma/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the sigma site. In addition sigma/PCP, sigma or kappa-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, sigma/PCP, sigma and kappa-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the sigma or PCP binding sites. In conclusion, the ability of sigma and kappa-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Anti-Arrhythmia Agents; Antihypertensive Agents; Benzofurans; Brain Ischemia; Cell Death; Cells, Cultured; Dizocilpine Maleate; Hypoxia; Kainic Acid; Ligands; N-Methylaspartate; Neurons; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Phencyclidine; Receptors, sigma

1. We have studied three hypoxia-induced phenomena in the CA1 stratum pyramidale of the rat hippocampal slice: (a) the increase in extracellular potassium ion concentration ([K+]e) measured with ion-sensitive microelectrodes, (b) the intracellularly-recorded pyramidal cell hyperpolarization and (c) the extracellularly-recorded depression of the synaptically-evoked field potential recorded in stratum pyramidale. 2. The extracellular potassium ion concentration ([K+]e) rose from 3 mM to 4.1-4.4 mM at a time when the pyramidal cells hyperpolarized by about 6 mV and neurotransmission was virtually abolished. 3. Presumed glial cells depolarized in response to hypoxia. The shape and time course of this response was remarkably similar to the rise in [K+]e so induced. This is consistent with findings that glial cell membrane potential is dependent on transmembrane K+ gradient. 4. We investigated the effects of theophylline (100 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM) on these effects. We have found that these compounds attenuated by about half the hypoxia-induced increase in [K+]e; however, they did not reduce the hypoxia-induced hyperpolarization. We have confirmed that they dramatically reduced the suppression of excitatory transmission caused by the hypoxia. We conclude that adenosine A1 receptors may be involved in the alteration of K+ homeostasis in the hippocampal slice during hypoxia.

Topics: Adenosine; Animals; Corpus Striatum; Dizocilpine Maleate; Hypoxia; Male; Membrane Potentials; Microelectrodes; Neuroglia; Neuroprotective Agents; Phosphodiesterase Inhibitors; Potassium; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Theophylline; Xanthines

Changes in the activity of the NMDA receptor-gated ionic channels induced by potassium cyanide were studied in rat hippocampal slices utilizing a [3H]MK-801 binding technique. A 30-min exposure of slices to potassium cyanide (KCN) increased MK-801 binding by 252%. Co-application of N omega-nitro-L-arginine (NNLA), a competitive antagonist of nitric oxide (NO) synthase, reduced this increase by 72%. This inhibition by NNLA was completely reversed by an excess of L-arginine, a substrate for NO synthase, suggesting that the KCN-induced increase in MK-801 binding is mediated by NO synthase activity. KCN had no effect on MK-801 binding in synaptic membranes. In Ca(2+)-containing medium, KCN increased the release of glutamate, aspartate and glycine by 4- to 5-fold, and this was blocked by application of NNLA. NNLA inhibition was reversed by an excess of L-arginine, indicating that KCN-stimulated release of these amino acids is mediated by NO synthase activity. In Ca(2+)-free medium, a KCN-induced increase in MK-801 binding and in excitatory amino acid release was also observed, however, this increase was not influenced by NO-related agents, suggesting that these changes were not mediated by NO synthase activation. NNLA given after the end of exposure to KCN did not reverse the increase in MK-801 binding. These findings suggest that NO is involved in the initial activation of NMDA receptor-gated ionic channels and in the enhanced amino acid transmitter release induced by KCN, but that KCN can also induce some of these effects by a Ca(2+)- and NO-independent mechanism.

Topics: Amino Acids; Animals; Calcium; Culture Media; Dizocilpine Maleate; Hypoxia; In Vitro Techniques; Ion Channel Gating; Ion Channels; Neurotoxins; Nitric Oxide; Potassium Cyanide; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

Topics: Animals; Dizocilpine Maleate; Glutamic Acid; Hypoxia; Male; Medulla Oblongata; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Respiration; Respiration, Artificial; Substance P

Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calbindin 1; Calbindin 2; Calbindins; Calcimycin; Cell Death; Cells, Cultured; Dizocilpine Maleate; Glucose; Glutamic Acid; Hippocampus; Hypoxia; Immunohistochemistry; Kainic Acid; Mice; Nerve Tissue Proteins; Neurons; Oxygen Consumption; Rats; S100 Calcium Binding Protein G

1. An attempt has been made to test the hypothesis that, in the caudal part of nucleus tractus solitarii (NTS) where carotid sinus nerve (CSN) afferents project, L-glutamate (Glut) modulates the hypoxic ventilatory response. 2. Unanaesthetized, peripherally chemodenervated (carotid body denervated; CBD) and sham-operated, freely moving rats were used. During peripheral chemoreceptor stimulation by hypoxia (10% O2 for 30 min) or doxapram (Dox) infusion (2 mg kg-1 (30 min)-1), ventilation was recorded and successively, under the same conditions, the extracellular Glut concentration ([Glut]o) in the caudal NTS was measured by in vivo microdialysis. [Glut]o was also measured during hyperoxic hypercapnia (10% CO2-30% O2 for 30 min). 3. Furthermore, the effects on ventilation of exogenous Glut, the NMDA (N-methyl-D-aspartate) receptor antagonist MK-801 or the ionotropic receptor antagonist kynurenate microinjected into the caudal NTS were investigated in sham-operated rats. 4. In sham-operated rats, both ventilation and [Glut]o in NTS were increased during peripheral chemoreceptor stimulation. On the other hand, no increases in either ventilation or Glut release were observed in CBD rats. In spite of ventilatory augmentation during hypercapnia, no response of [Glut]o to hypercapnia was observed in either group. 5. Local Glut application into NTS increased ventilation. Pretreatment with MK-801 or kynurenate reduced the hypoxic ventilatory response. This reduction in ventilation was mainly due to the decrease in tidal volume. 6. These results suggest that hypoxia induced the release of Glut in NTS and that this effect was mediated by arterial chemosensory input.

Topics: Animals; Blood Pressure; Carotid Body; Chemoreceptor Cells; Denervation; Dizocilpine Maleate; Doxapram; Glutamic Acid; Heart Rate; Hypoxia; Infusions, Intravenous; Kynurenic Acid; Male; Microinjections; Rats; Rats, Sprague-Dawley; Respiration; Solitary Nucleus

Hippocampal slices of rats at postnatal day 7 were submitted to superfusion with Ca(2+)- and Mg(2+)-free, bicarbonate buffered ion balanced medium, and perfusate concentrations of eicosanoids: thromboxane B2 and 6-keto prostaglandin F1 alpha were determined by the radioimmunoassay. It was noted that the permanent presence of Ca2+ increased the basal eicosanoid level, and in these conditions modulation of eicosanoid production was lost, whereas temporary, a 20 min application of 1.3 mM Ca2+ did not influence significantly eicosanoid release. A 20 min application of the anoxic/aglycemic medium containing calcium did not change the content of eicosanoids in superfusates. A significant stimulation of the thromboxane B2 and 6-keto prostaglandin F1 alpha release was noted provided the application of the experimental medium was accompanied by a 10 min arrest of superfusion. This effect was inhibited by MK-801 and quinacrine, suggesting an involvement of NMDA receptors and phospholipase A2. We propose that a model of anoxic/aglycemic superfusion with a stop flow period allows retention of endogenous glutamate in the extracellular fluid, resembling a similar effect during in vivo ischemia, whereas during a continuous superfusion glutamate is immediately washed out. Consequently, an application of the anoxic/aglycemic medium accompanied by a temporary arrest of superfusion represents more adequate in vitro model of ischemia than a constant superfusion with this medium. In these conditions NMDA receptors mediate eicosanoid release.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Brain Ischemia; Calcium; Culture Techniques; Dizocilpine Maleate; Eicosanoids; Hippocampus; Hypoxia; N-Methylaspartate; Quinacrine; Radioimmunoassay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Thromboxane B2

The extracellular calcium concentration ([Ca2+]ec) was recorded by calcium-sensitive microelectrodes in the parietal cortex of 9-11 day old rats during anoxia. During the first 10 min of anoxia, [Ca2+]ec increased from 1.1 mM to 1.5 +/- 0.23 mM, and thereafter it started to decrease reaching below basal level after around 13 min. The [Ca2+]ec decrease was either slow and continuous, or biphased with a rapid initial decrease followed by a continuous slow decrease. After 60 min of anoxia, the [Ca2+]ec had reached 0.2-0.3 mM. Changes in [Ca2+]ec in animals treated with the NMDA receptor antagonist MK-801 (0.3 mg/kg i.p.) did not display any significant differences compared to controls. Thus, the strong neuroprotective effect of MK-801 in ischemic situations in the immature brain can not be explained by a prevention of calcium entry during anoxic depolarization.

Topics: Animals; Animals, Newborn; Calcium; Cerebral Cortex; Dizocilpine Maleate; Extracellular Space; Hypoxia; Osmolar Concentration; Rats; Rats, Sprague-Dawley

The effect of hypoxia on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex in the brain cell membrane of the newborn piglet was studied. Experiments were conducted on newborn piglets, 2-4 days of age, that were anesthetized and mechanically ventilated. Hypoxic hypoxia was induced in the experimental group by lowering the FiO2 to 5-7%. The control group was ventilated under normoxic conditions. Tissue hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine (PCr) in the hypoxic group (52% and 81% lower than the normoxic group, respectively). [3H]MK-801 binding characteristics (Bmax = number of receptors, Kd = dissociation constant) were used as an index of NMDA receptor modification. In hypoxic brains, Bmax decreased from the control level of 1.13 +/- 0.15 pmol/mg protein to 0.68 +/- 0.23 pmol/mg protein (P < 0.01) and the Kd value decreased (reflecting increased affinity) from 9.46 +/- 1.68 nM in the control brains to 4.87 +/- 1.42 nM (P < 0.01) in the hypoxic brains. The Na+,K(+)-ATPase activity, an index of brain cell membrane function, decreased from a control value of 46.5 +/- 0.4 to 40.5 +/- 2.3 mumol inorganic phosphate (Pi) mg protein/h (P < 0.005) during hypoxia. The results of this study indicate that hypoxia in newborn piglets modifies the NMDA receptor in the cerebral cortex, resulting in an increased affinity of the receptor channel. Hypoxia-induced modification of the NMDA ion/receptor complex may be a potential mechanism of cerebral excitotoxicity.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Cerebral Cortex; Dizocilpine Maleate; Hypoxia; Phosphocreatine; Receptors, N-Methyl-D-Aspartate; Sodium-Potassium-Exchanging ATPase; Swine

Using guinea-pig hippocampal slices, we determined the amount of various amino acids released into the medium during deprivation of oxygen and glucose. Within 10 min of slices being deprived of O2 and glucose, the amounts of serine, aspartate, alanine, glycine, GABA, taurine and threonine released into the medium increased up to 1.7 (serine), 1.6 (aspartate), 1.6 (alanine), 1.9 (glycine), 2.0 (GABA), 1.4 (taurine) and 1.8 (threonine) times the control levels, respectively. The amount of serine released 10 min after O2 and glucose deprivation was four times as great as that of glutamate. The dose-response effects of glutamate and serine were studied on the population spikes evoked in the granular cell layer. Bath application of 100 microM serine elevated the amplitude of the population spike to 117% and at 10 mM depressed it completely. The dose-response curve for glutamate displayed a similar pattern but the effectiveness was 10 times higher than that of serine. The combined application of glutamate (300 microM) and serine (2 mM) produced a dramatic reduction in and depression of the amplitude of the population spike, although 300 microM glutamate and 2 mM serine individually failed to show a significant effect. The population spike was depressed by the addition of 1 mM glutamate but, after washing, it recovered completely. On the other hand treatment with 1 mM glutamate together with 5 mM serine caused no recovery of the population spike even after removal of the agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Amino Acids; Animals; Dizocilpine Maleate; Glucose; Glutamates; Glutamic Acid; Guinea Pigs; Hippocampus; Hypoxia; In Vitro Techniques; Kynurenic Acid; Neurons; Phosphocreatine; Receptors, N-Methyl-D-Aspartate; Serine; Synaptic Transmission

Percent recovery of CA1 field EPSP amplitude following various anoxic aglycemic (AA) periods was examined in rat hippocampal slices superfused with MK-801 (0.1 microM, 1 microM, 10 microM) or Mg(2+)-free artificial cerebrospinal fluid. Slices treated with 0.1 microM MK-801 showed greater percent recuperation of EPSP amplitude following 3 min 30 s of AA (36 +/- 12% vs 6 +/- 4% in controls). Higher concentrations of MK-801 resulted in a greater recovery of EPSP amplitudes in more than one time period of AA, with 10 microM MK-801 providing protection in up to 4 min 30 s AA. Percent recuperation of EPSP amplitude was smaller in Mg(2+)-free slices following 2 min (34 +/- 15% vs 81 +/- 11% in controls) and 2 min 30 (25 +/- 14% vs 77 +/- 10% in controls) of AA. These results suggest that the activation of the N-methyl-D-aspartate (NMDA) receptor channel may contribute to irreversible AA induced synaptic failure in CA1.

Topics: Animals; Blood Glucose; Dizocilpine Maleate; Electrophysiology; Evoked Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Magnesium; Male; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission

The effects of the 21-aminosteroids, U-74500A and U-78517F (drugs endowed with lipid peroxidation inhibitor properties) were tested on hypoxia-induced functional failure in rat hippocampal slices. For comparison, the effects of the non-competitive N-methyl-D-aspartate antagonist, dizocilpine (MK-801) were studied. Perfusion of slices with 50 microM of MK-801 or with 50-100 microM of U-78517F, but not with 100-200 microM of U-74500A, significantly (P < 0.01) increased the incidence of reappearance of the CA1 population spikes after reoxygenation in rat hippocampal slices subjected to a 45-min hypoxic period followed by a 45-min reoxygenation period. Perfusion of slices with 12.5 microM of MK-801 plus 12.5 microM of U-78517F significantly (P < 0.05) increased the incidence of reappearance of the CA1 population spikes after reoxygenation with respect to perfusion of slices with 12.5 microM of U-78517F alone or with 12.5 microM of MK-801 alone. The results show that 21-aminosteroids have protective effects against hypoxia-induced functional failure in rat hippocampal slices. In addition, the data show that, under the same experimental conditions, the NMDA receptor antagonist, MK-801, was also able to improve hypoxia-induced functional failure. On the whole, the results suggest that the hypoxia-induced functional electrical failure might depend on both release of excitatory amino acids and oxygen free-radical-mediated membrane lipid peroxidation.

Topics: Animals; Chromans; Dizocilpine Maleate; Electrophysiology; Hippocampus; Hypoxia; Lipid Peroxides; Male; Piperazines; Pregnatrienes; Rats; Rats, Wistar

Because hypoxia increases brain extracellular glutamate levels, we hypothesized that gasping and increased sympathetic activity during severe hypoxia result from glutamergic excitation. To test this hypothesis, we exposed anesthetized paralyzed vagotomized glomectomized cats to hypoxia before and after N-methyl-D-aspartate (NMDA) glutamergic blockade (MK-801, 1 mg/kg iv) or non-NMDA blockade (NBQX, 3 mg/kg iv) while monitoring phrenic neurogram (PN) and inspiratory-synchronous (ISSN) and tonic (TSN) activity in cervical sympathetic neurogram (SN). Before hypoxia, MK-801 caused apneusis and reduced PN and ISSN amplitude by 38 and 84%, respectively, but TSN activity was unaffected. During hypoxia, MK-801 had no effect on PN gasping or TSN activity but reduced ISSN amplitude during gasping. Before hypoxia, NBQX reduced PN and ISSN amplitude by 54 and 60%, respectively but did not affect inspiratory timing or TSN activity. Gasping activity in PN and ISSN and TSN activity during hypoxia were unaffected by NBQX. We conclude that 1) ionotropic glutamergic receptor activation is important for eupneic phrenic patterning but is not involved in genesis of gasping, 2) NMDA receptor activation is involved in integration of respiratory and sympathetic activity, and 3) changes in TSN activity are independent of ionotropic glutamergic receptor activation.

Topics: Animals; Cats; Dizocilpine Maleate; Electrophysiology; Excitatory Amino Acid Antagonists; Female; Hypercapnia; Hypoxia; Male; Phrenic Nerve; Quinoxalines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Sympathetic Nervous System

Recent data suggest that the increase in ventilation during hypoxia may be related to the release of the excitatory amino acid neurotransmitter glutamate centrally. To further investigate this, we studied the effects of MK-801, a selective noncompetitive N-methyl-D-aspartate receptor antagonist, on the hypoxic ventilatory response in lightly anesthetized spontaneously breathing intact dogs. The cardiopulmonary effects of sequential ventriculocisternal perfusion (VCP) at the rate of 1 ml/min with mock cerebrospinal fluid (CSF, control) and MK-801 (2 mM) were compared during normoxia and 8 min of hypoxic challenge with 12% O2. Minute ventilation (VE), tidal volume (VT), and respiratory frequency (f) were recorded continuously, and hemodynamic parameters [heart rate (HR), blood pressure (MAP), cardiac output (CO), pulmonary arterial pressure, and pulmonary capillary wedge pressure] were measured periodically. Each dog served as its own baseline control before and after each period of sequential VCP under the two different O2 conditions. During 15 min of normoxia, there were no significant changes in the cardiopulmonary parameters with mock CSF VCP, whereas with MK-801 VCP for 15 min, VE decreased by approximately 27%, both by reductions in VT and f (17 and 9.5%, respectively). HR, MAP, and CO were unchanged. During 8 min of hypoxia with mock CSF VCP, VE increased by 171% associated with increased VT and f (25 and 125%, respectively). HR, MAP, and CO were likewise augmented. In contrast, the hypoxic response during MK-801 VCP was characterized by an increased VE of 84%, mainly by a rise in f by 83%, whereas the VT response was abolished. The cardiovascular excitation was also inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dizocilpine Maleate; Dogs; Glutamates; Glutamic Acid; Hemodynamics; Hypoxia; Neurotransmitter Agents; Receptors, N-Methyl-D-Aspartate; Respiration; Synaptic Transmission

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Chickens; Dizocilpine Maleate; Edema; Electron Transport; Glycolysis; Hypoglycemia; Hypoxia; Iodoacetates; Iodoacetic Acid; Ischemia; N-Methylaspartate; Potassium Cyanide; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Retina; Tetrodotoxin

The protective effect of the anticonvulsant MK-801 and the antitussive dextromethorphan, which are both N-methyl-D-aspartate receptor antagonists, and kynurenic acid, a broad-spectrum excitotoxin antagonist, was tested in cultured rat retinal cells in an hypoxic environment. The protective effect of these antagonists also was tested in cultured retinal cells and in intact adult rat retinas exposed to the exogenous excitotoxins L-glutamic acid and N-methyl-D-aspartic acid. MK-801 and kynurenic acid protected retinal neurons from hypoxic damage and from the toxicity of exogenous L-glutamic acid and N-methyl-D-aspartic acid. Dextromethorphan, a less potent antagonist, did not protect the retinal neurons from hypoxic damage or the toxicity of exogenous L-glutamic acid, but did attenuate N-methyl-D-aspartate toxicity. These results provide evidence that the synaptic release of excitatory transmitters, most likely glutamate and aspartate, mediate the death of hypoxic retinal neurons. Compounds related to MK-801 may have possible therapeutic applications in the management of retinal ischemia.

Topics: Animals; Culture Techniques; Dextromethorphan; Dizocilpine Maleate; Glutamates; Glutamic Acid; Hypoxia; Kynurenic Acid; N-Methylaspartate; Neurons; Rats; Rats, Inbred Strains; Retina

The Protective effects of dizocilpine maleate (DM) against anoxia in mice and ischemic damage in rats of 4-vessel occlusion (4-VO) were studied. DM 0.5 or 1.0 mg.kg-1 ip significantly prolonged the survival time of mice in closed containers. DM 0.5 and 1.0 mg.kg-1 ip 30 min prior to 4-VO obviously accelerated the electroencephalographic recovery, reduced the neuronal loss in the hippocampus, and increased the survival rate after 72-h reperfusion. These effects followed a dose-dependent manner. Our results indicate that selective non-competitive N-methyl-D-aspartate receptor blocker DM protects against anoxic and ischemic cerebral damage.

Topics: Animals; Brain Ischemia; Dizocilpine Maleate; Dose-Response Relationship, Drug; Electroencephalography; Hippocampus; Hypoxia; Male; Mice; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The effects of adenosinergic antagonists caffeine and DPCPX, and of the adenosinergic agonists L-PIA, CPA and CGS 21680 were investigated on fully and partially reversible hypoxia-induced electrophysiological changes in rat hippocampal slices. The influence of a high potassium solution and of the N-methyl-D-aspartate antagonist dizocilpine (MK 801) was also tested. The latency to obtain a 50% decrease in the amplitude of the CA1 population spike (CA1 PS) during a short- (5-10 min) lasting hypoxic period was significantly increased (P less than 0.01) by slice perfusion with caffeine (50 microM), DPCPX (0.2 microM), and by increasing (from 3 to 4 mM) the potassium concentration in the medium bathing the hippocampal slices. The latency was significantly decreased (P less than 0.01) by slice perfusion with L-PIA (0.2 microM) and CPA (0.05 microM). It was not significantly modified by CGS 21680 (5 microM). The incidence of reappearance of the CA1 PS during reoxygenation after long- (45 min) lasting hypoxia was significantly increased (P less than 0.05) by slice perfusion with MK 801 (50 microM), while it was not significantly affected by slice perfusion with caffeine (50 microM) or DPCPX (0.2 microM) or L-PIA (0.2 microM) or CPA (0.05 microM) or CGS 21680 (5 microM). The results indicate a prevalent involvement of the A1 adenosine receptors in the early mechanisms underlying hypoxia-induced reversible changes. Adenosine seems to have a limited role in the late mechanisms occurring after a long-lasting hypoxic period.

Topics: Adenosine; Animals; Caffeine; Dizocilpine Maleate; Electrophysiology; Hippocampus; Hypoxia; In Vitro Techniques; Male; Membrane Potentials; Phenethylamines; Phenylisopropyladenosine; Potassium; Purinergic Antagonists; Rats; Rats, Inbred Strains; Synapses; Xanthines

Neurotoxicity of glutamate in conjunction with subcritical hypoxia was determined in vitro using hippocampal neurons obtained from 18-day-old rat fetuses. Neurons were plated at a low density and maintained for 3 days in a chemically defined medium without glutamate. When glutamate + was added after subcritical hypoxic stress, a low dose of glutamate, even at 10 microM, could cause significant neuronal loss in the following 24 h. The observed neurotoxicity to low glutamate dose (10-100 microM) could completely be prevented by 5 microM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This protective effect of CNQX was more potent than that of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). The mechanism by which glutamate is transformed from a neurotransmitter to a neurotoxin is discussed.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Glutamates; Glutamic Acid; Hippocampus; Hypoxia; Nervous System Diseases; Neurons; Pregnancy; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Kainic Acid; Receptors, Neurotransmitter

We have investigated the contribution of excitatory amino acid receptor activation to the inhibition of protein synthesis observed after anoxia in rat hippocampal slices. Protein synthesis was assessed in normoxic medium by measuring the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. Protein synthesis was impaired after anoxia; the extent of inhibition was dependent on the duration of anoxia and on the time allowed for postanoxic recovery. There was a similar impairment under normoxic conditions when the N-methyl-D-aspartate (NMDA) receptor channel was activated by removing Mg2+ and adding NMDA. This was prevented by noncompetitive antagonists of the NMDA receptor channel (MK-801, phencyclidine, and N-allylnormetazocine). In contrast, incubation with the NMDA antagonists failed to prevent the protein synthesis inhibition caused by anoxia, although it moderately facilitated the postanoxic recovery. Protein synthesis was also impaired under normoxic conditions after incubation with quisqualate and kainate, agonists of non-NMDA glutamate receptors. This impairment was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of these receptors. Although 6-cyano-7-nitroquinoxaline-2,3-dione alone failed to prevent anoxic damage, when used in combination with an NMDA antagonist it did partially enhance the later recovery of protein synthesis. These results indicate that the activation of excitatory amino acid receptors cannot alone account for anoxia-induced impairment of protein synthesis in rat hippocampal slices.

Topics: Animals; Aspartic Acid; Carbon Radioisotopes; Dizocilpine Maleate; Glutamates; Glutamic Acid; Hippocampus; Hypoxia; Kainic Acid; Lysine; Male; N-Methylaspartate; Nerve Tissue Proteins; Phenazocine; Phencyclidine; Quisqualic Acid; Rats; Receptors, Amino Acid; Receptors, Cell Surface; Receptors, N-Methyl-D-Aspartate

Using positron emission tomography (PET), the potential of 18F-labelled fluoro-methyl-MK-801([18F]FMM) as a radioligand for in vivo studies of the NMDA receptor complex was investigated in baboons. In baseline conditions, there was a slight differential retention of [18F]FMM in cerebral cortex and striatum relative to cerebellum, compatible with specific binding. However, neither pretreatment with pharmacological doses of MK-801 or phencyclidine, nor severe, transient brain hypoxia, were able to clearly alter [18]FMM brain regional kinetics, indicating limited usefulness of this radioligand for in vivo PET investigations of the NMDA receptor.

Topics: Animals; Brain; Brain Chemistry; Dizocilpine Maleate; Hypoxia; Male; Papio; Phencyclidine; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Tomography, Emission-Computed

The purpose of this study was to determine whether prior transient cerebral ischemia, in conscious mice, would alter the biological responses resulting from excessive activation of N-methyl-D-aspartate (NMDA) receptors, in an early stage. The responses to the activation of NMDA receptors by an intracerebroventricular injection of NMDA, such as wild running, tonic and clonic convulsions, absence of the visual placing reflex, loss of the righting reflex, impaired motor function and a high mortality rate, were to a large extent prevented if 30 min before treatment, either a 10-min period of global cerebral ischemia was induced or a 1 nmol intraventricular injection of NMDA was given but not if either of the above procedures was done one day before the test dose of NMDA. In contrast, behavioral symptoms, in response to activation of non-NMDA-type glutamate receptors elicited by intraventricular injection of either kainic acid or AMPA, were not clearly affected. Transient systemic hypercapnic anoxia (22-sec exposure to 100% CO2 gas), before treatment with NMDA did not significantly reduce the NMDA-induced behavior. The severity of these behavioral responses and high mortality rate observed after intraventricular injection of pentylenetetrazole (PTZ, 30 mumol) were not altered by either prior global ischemic insult or by a preexposure to NMDA given intraventricularly. The NMDA antagonist, MK801 (0.1 and 0.3 mg/kg i.p.) greatly reduced the behavioral effects and mortality rate, resulting from the intraventricular injection of NMDA and somewhat reduced the effects of the intraventricular injection of PTZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Behavior, Animal; Carbon Dioxide; Dizocilpine Maleate; Female; Hypoxia; Injections, Intraventricular; Ischemic Attack, Transient; Mice; Mice, Inbred Strains; N-Methylaspartate; Nerve Degeneration; Pentylenetetrazole; Receptors, N-Methyl-D-Aspartate; Survival Rate

The effects of the non-competitive N-methyl-D-aspartate (NMDA) antagonists, (+)-MK-801 hydrogen maleate (MK-801) and phencyclidine hydrochloride (PCP), were tested against hypoxia-induced tonic convulsions and hippocampal epileptiform discharges. Systemic administration of MK-801 and PCP suppressed the hypoxia-induced tonic convulsions in dietary Mg(2+)-deficient mice in a dose-dependent manner. The ED50 values (and their 95% confidence limits) of MK-801 and PCP were 0.19 (0.14-0.26) and 1.14 (0.32-4.11) mumol/kg, respectively. These values were lower than those of the conventional anticonvulsants tested. Induction of epileptiform discharges following hypoxia was also prevented by the presence of MK-801 and PCP at concentrations of 5-30 microM. The hypoxia-induced epileptiform discharges, however, were reduced, but not blocked completely, by the application of MK-801 and PCP. These results strongly suggest that activation of NMDA receptors in the hippocampus plays a pivotal role in the induction of hypoxia-induced tonic convulsions in dietary Mg(2+)-deficient mice.

Topics: Animals; Anticonvulsants; Dizocilpine Maleate; Hippocampus; Hypoxia; Magnesium Deficiency; Male; Membrane Potentials; Mice; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Seizures

Topics: Animals; Animals, Newborn; Brain Diseases; Dizocilpine Maleate; Hypothermia, Induced; Hypoxia; Ischemia; Nervous System; Rats; Rats, Inbred Strains

Rats exposed on their first postnatal day to 100% nitrogen for 25 min developed hyperactivity and lower performance in passive avoidance task during development. Administration of MK-801 (0.5 mg/kg i.p.) 1 h before anoxia or (0.25 and 0.5 mg/kg) 1 h after completely reversed this behavioral impairment. Senescent rats (24-26 months) exposed to hypoxia (92% N2 + 8% O2) for 5 h failed in their performance in C.A.R., 30 days later. Pretreatment with MK-801 (1 mg/kg i.p.) completely reversed this impairment. These data suggest that activation of endogenous NMDA receptors produces different behavioral consequences in neonatal and senescent rats and that MK-801 administration close to exposure of animals to anoxia or hypoxia can prevent such damage, thus preventing behavioral impairments in postnatal as well as in senescent rats.

Topics: Aging; Animals; Animals, Newborn; Avoidance Learning; Behavior, Animal; Dizocilpine Maleate; Hypoxia; Male; Rats; Rats, Inbred Strains

The phencyclidine analogue [3H](1-[2-thienyl]cyclohexyl)piperidine (3H-TCP) binds to the ion channel associated with the N-methyl-D-aspartate receptor channel complex. In vitro autoradiography indicates that the distribution of 3H-TCP binding in brain closely parallels that of [3H]glutamate binding to the N-methyl-D-aspartate receptor. In nine 7-day-old rats, an acute focal hypoxic-ischemic insult produced by unilateral carotid artery ligation and subsequent exposure to 8% oxygen acutely reduced 3H-TCP binding ipsilateral to the ligation by 30% in the CA1, by 27% in the CA3, by 26% in the dentate gyrus, and by 17% in the striatum compared with values from the contralateral hemisphere. In 10 littermates that received 1 mg/kg of the neuroprotective noncompetitive N-methyl-D-aspartate antagonist MK-801 immediately before hypoxic exposure, the regional distribution of 3H-TCP binding in hypoxic-ischemic brain was relatively preserved and there were no interhemispheric asymmetries in 3H-TCP binding densities. In addition, in three unoperated rats decapitated 24 hours after MK-801 treatment, 3H-TCP binding was reduced by 15-35%; similar bilateral suppression of 3H-TCP binding was detected in MK-801-treated ligates. Our data indicate that 3H-TCP autoradiography can be used to assay the efficacy of neuroprotective agents in this experimental model of perinatal stroke.

Topics: Animals; Animals, Newborn; Aspartic Acid; Autoradiography; Binding Sites; Brain; Brain Ischemia; Dibenzocycloheptenes; Dizocilpine Maleate; Glutamates; Glutamic Acid; Hypoxia; N-Methylaspartate; Phencyclidine; Rats; Rats, Inbred Strains; Tritium

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Topics: Animals; Binding Sites; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Fetus; Glycine; Hypoxia; Kinetics; Kynurenic Acid; L-Lactate Dehydrogenase; Nerve Degeneration; Neurons; Phosphopyruvate Hydratase; Pyrrolidinones; Rats; Receptors, N-Methyl-D-Aspartate

The effect of the N-methyl-D-aspartate antagonists, aminophosphonovaleric acid and MK-801, on irreversible transmission loss subsequent to anoxia was examined using the hippocampal slice preparation. A population spike was recorded from either the dentate granule cells or the CA1 pyramidal cells and the amplitude of this spike was compared before and 60 min following anoxia. After 10 min of anoxia the dentate granule cells recovered to 16 +/- 7% (mean +/- SE) of their preanoxic level when untreated and to 54 +/- 15% when treated with aminophosphonovaleric acid (APV). In slices treated with MK-801 the population spikes recorded from dentate granule cells recovered to 85 +/- 4% of their preanoxic level after 10 min of anoxia. Untreated CA1 pyramidal cells recovered to 8 +/- 3% of their preanoxic amplitude after 5 min of anoxia; they recovered to 59 +/- 6% when treated with MK-801 and 31 +/- 13% when treated with APV. The recovery of slices treated with the drugs was significantly different from that of untreated slices. ATP levels were measured in both the dentate and the CA1 region of slices. ATP in both regions fell less during anoxia when the slices were pretreated with either APV or MK-801. These differences between drug-treated and untreated tissue were significant with APV and MK-801. These differences between drug-treated and untreated tissue were significant with APV and MK-801 in dentate tissue after 10 min of anoxia and with MK-801 in CA1 tissue after 5 min of anoxia. This reduced fall in ATP during anoxia was accompanied by better physiological recovery after anoxia. We conclude that these NMDA antagonists provide protection against anoxic damage to dentate granule and CA1 pyramidal cells in this in vitro hippocampal preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; Adenosine Triphosphate; Animals; Anticonvulsants; Aspartic Acid; Dibenzocycloheptenes; Dizocilpine Maleate; Evoked Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Male; N-Methylaspartate; Neurotoxins; Pyramidal Tracts; Rats; Rats, Inbred Strains; Valine

We evaluated the neuroprotective effect of MK-801, a noncompetitive, selective N-methyl-D-aspartate receptor antagonist, in a neonatal hypoxic-ischemic animal model. Seven-day-old rats underwent bilateral ligation of the carotid arteries followed by exposure to an 8% oxygen atmosphere for 1 hr. We sacrificed the animals 72 hrs later and assessed the hypoxic-ischemic brain damage histologically. MK-801 (10 mg/kg), administered IP 0.5 hr before the hypoxia, completely prevented hypoxic-ischemic infarction in cerebral cortex, while treatment immediately and 1 hr after the end of the hypoxia resulted in 76% and 52% reduction in the infarcted area, respectively. MK-801, given 0.5 hr before and immediately after the insult, reduced striatal damage and, given 0.5 hr before, attenuated neuronal necrosis in hippocampal regions. These results show that in neonates MK-801 is neuroprotective even when administered up to 1 hr after the end of a hypoxic-ischemic insult.

Topics: Animals; Animals, Newborn; Brain; Brain Damage, Chronic; Brain Ischemia; Dibenzocycloheptenes; Dizocilpine Maleate; Hypoxia; Rats; Rats, Inbred Strains