Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critical for synaptic plasticity that underlies learning and memory. But, they have also been described as a common source of neuronal damage during stroke and neurodegenerative diseases. Several studies have suggested that cellular location of NMDARs (synaptic or extrasynaptic) is a key parameter controlling their effect on neuronal viability. The aim of the study was to understand the relation between these two pools of receptors and to determine their implication in both beneficial and/or deleterious events related to NMDAR activation. We demonstrated that selective extrasynaptic NMDAR activation, as well as NMDA bath application, does not activate extracellular signal-regulated kinase (ERK) pathways, but induces mitochondrial membrane potential breakdown and triggers cell body and dendrite damages, whereas synaptic NMDAR activation is innocuous and induces a sustained ERK activation. The functional dichotomy between these two NMDAR pools is tightly controlled by glutamate uptake systems. Finally, we demonstrated that the only clinically approved NMDAR antagonist, memantine, preferentially antagonizes extrasynaptic NMDARs. Together, these results suggest that extrasynaptic NMDAR activation contributes to excitotoxicity and that a selective targeting of the extrasynaptic NMDARs represents a promising therapeutic strategy for brain injuries.

Potentiated necrosis of cultured cortical neurons by neurotrophins.

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

3D printed TCP-based scaffold incorporating VEGF-loaded PLGA microspheres for craniofacial tissue engineering.

OBJECTIVE: Vascularization is a critical process during bone regeneration/repair and the lack of tissue vascularization is recognized as a major challenge in applying bone tissue engineering methods for cranial and maxillofacial surgeries. The aim of our study is to fabricate a vascular endothelial growth factor (VEGF)-loaded gelatin/alginate/ß-TCP composite scaffold by 3D printing method using a computer-assisted design (CAD) model. METHODS: The paste, composed of (VEGF-loaded PLGA)-containing gelatin/alginate/ß-TCP in water, was loaded into standard Nordson cartridges and promptly employed for printing the scaffolds. Rheological characterization of various gelatin/alginate/ß-TCP formulations led to an optimized paste as a printable bioink at room temperature. RESULTS: The in vitro release kinetics of the loaded VEGF revealed that the designed scaffolds fulfill the bioavailability of VEGF required for vascularization in the early stages of tissue regeneration. The results were confirmed by two times increment of proliferation of human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds after 10 days. The compressive modulus of the scaffolds, 98±11MPa, was found to be in the range of cancellous bone suggesting their potential application for craniofacial tissue engineering. Osteoblast culture on the scaffolds showed that the construct supports cell viability, adhesion and proliferation. It was found that the ALP activity increased over 50% using VEGF-loaded scaffolds after 2 weeks of culture. SIGNIFICANCE: The 3D printed gelatin/alginate/ß-TCP scaffold with slow releasing of VEGF can be considered as a potential candidate for regeneration of craniofacial defects.

GABAA receptor activation attenuates excitotoxicity but exacerbates oxygen-glucose deprivation-induced neuronal injury in vitro.

We examined the effects of GABA receptor stimulation on the neuronal death induced by exogenously added excitatory amino acids or combined oxygen-glucose deprivation in mouse cortical cell cultures. Death induced by exposure to NMDA, AMPA, or kainate was attenuated by addition of GABA or the GABAA receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. The antiexcitotoxic effect of GABAA receptor agonists was blocked by bicuculline or picrotoxin. In contrast, GABA or muscimol, but not baclofen, markedly increased the neuronal death induced by oxygen-glucose deprivation. Muscimol potentiation of neuronal death was associated with increased glutamate efflux to the bathing medium, and increased cellular 45Ca2+ accumulation; it was blocked by MK-801, but not NBQX, suggesting mediation by NMDA receptors. Bicuculline only weakly attenuated muscimol potentiation of oxygen-glucose deprivation-induced neuronal death, probably because it itself increased this death. Present results raise a note of caution in the proposed use of GABAA receptor stimulation to limit ischemic brain damage in vivo.

Selective activation of group II mGluRs with LY354740 does not prevent neuronal excitotoxicity.

Recent reports have suggested a role for group II metabotropic glutamate receptors (mGluRs) in the attenuation of excitotoxicity. Here we examined the effects of the recently available group II agonist (+)-2-Aminobicyclo[3.1.0]hexane-2-6-dicarboxylic acid (LY354740) on N-methyl-D-aspartate (NMDA)-induced excitotoxic neuronal death, as well as on hypoxic-ischemic neuronal death both in vitro and in vivo. At concentrations shown to be selective for group II mGluRs expressed in cell lines (0.1-100 nM), LY354740 did not attenuate NMDA-mediated neuronal death in vitro or in vivo. Furthermore, LY354740 did not attenuate oxygen-glucose deprivation-induced neuronal death in vitro or ischemic infarction after transient middle cerebral artery occlusion in rats. In addition, the neuroprotective effect of another group II agonist, (S)-4-carboxy-3-phenylglycine (4C3HPG), which has shown injury attenuating effects both in vitro and in vivo, was not blocked by the group II antagonists (2 S)-alpha-ethylglutamic acid (EGLU), (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG), or the group III antagonist (S)-alpha-methyl-3-carboxyphenylalanine (MCPA), suggesting that this neuroprotection may be mediated by other effects such as upon group I mGluRs.

Preincubation with protein synthesis inhibitors protects cortical neurons against oxygen-glucose deprivation-induced death.

Twenty-four hour exposure to cycloheximide produced a concentration-dependent reduction in protein synthesis in mouse cortical cell cultures. Unexpectedly, a 24 h pretreatment with cycloheximide exposure also reduced neuronal vulnerability to subsequent oxygen-glucose deprivation-induced injury, measured both acutely (cell swelling) or after one day (cell lysis). This neuroprotective effect was attenuated if the period of cycloheximide pretreatment was shortened to 8 h, and lost if the pretreatment was shortened to 1 h. A comparable neuroprotective effect was also induced by 24 h pretreatment with another protein synthesis inhibitor, emetine. The neuroprotection induced by pretreatment with cycloheximide or emetine was probably not attributable to reduction of apoptosis: (i) neuronal death under these conditions occurs by N-methyl-D-aspartate receptor-mediated excitotoxic necrosis, not apoptosis; (ii) the same cycloheximide pretreatment did not block staurosporine-induced apoptosis. Also unlikely as an explanation is reduction in postsynaptic vulnerability to excitotoxicity, as death induced by exogenous addition of N-methyl-D-aspartate, kainate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was little affected by cycloheximide pretreatment. Rather, the protective effect of cycloheximide pretreatment was probably explained, at least in part, by marked reduction in the glutamate release induced by oxygen-glucose deprivation.

Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release.

Central neurons exposed to several types of sublethal stress, including ischemia, acquire resistance to injury induced by subsequent ischemic insults, a phenomenon called ischemic preconditioning. We modeled this phenomenon in vitro, utilizing exposure to 45 mM KCl to reduce the vulnerability of cultured murine cortical neurons to subsequent oxygen-glucose deprivation. Twenty-four hours after preconditioning, cultures exhibited enhanced depolarization-induced, tetanus toxin-sensitive GABA release and a modest decrease in glutamate release. Total cellular GABA levels were unaltered. Inhibition of GABA degradation with the GABA transaminase inhibitor (+/-)-gamma-vinyl GABA, or addition of low levels of GABA, muscimol, or chlormethiazole to the bathing medium, mimicked the neuroprotective effect of preconditioning against oxygen-glucose deprivation-induced death. However, neuronal death was enhanced by higher levels of these manipulations, as well as by prior selective destruction of GABAergic neurons by kainate. Finally, selective blockade of GABA(A) receptors during oxygen-glucose deprivation or removal of GABAergic neurons eliminated the neuroprotective effects of prior preconditioning. Taken together, these data predict that presynaptic alterations, specifically enhanced GABA release together with reduced glutamate release, may be important mediators of ischemic preconditioning, but suggest caution in regard to interventions aimed at increasing GABA(A) receptor activation.

Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro.

Mouse cortical cell cultures exposed to transient oxygen-glucose deprivation developed marked acute cell body swelling followed by neurodegeneration, consistent with necrosis-type death. This death was not attenuated by the protein synthesis inhibitor, cycloheximide, but was attenuated by addition of the N-methyl-D-asparate antagonist, MK-801 (dizocilpine maleate), and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. If the deprivation insult was extended to overcome the protective effect of glutamate antagonists, neuronal death resulted that was associated with cell body shrinkage and DNA fragmentation, and was attenuated by cycloheximide. These data suggest that oxygen-glucose deprivation can induce in cortical neurons both excitotoxic necrosis, and apoptosis dependent on new macromolecule synthesis.

Profound systemic hypothermia inhibits the release of neurotransmitter amino acids in spinal cord ischemia.

Profound hypothermia induced with cardiopulmonary bypass has a protective effect on spinal cord function during operations on the thoracoabdominal aorta. The mechanism of this protection remains unknown. It has been proposed that the release of excitatory amino acids in the extracellular space plays a causal role in irreversible neuronal damage. We investigated the changes in extracellular neurotransmitter amino acid concentrations with the use of in vivo microdialysis in a swine model of spinal cord ischemia. All animals underwent left thoracotomy and right atrium-femoral artery cardiopulmonary bypass with additional aortic arch perfusion. Lumbar laminectomies were then done and microdialysis probes were inserted stereotactically in the anterior horn of the second and fourth segments of the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid at a rate of 2 microliters/min and 15-minute samples were assayed by high-performance liquid chromatography. Group 1 animals (n = 6) underwent aortic clamping distal to the left subclavian artery and proximal to the renal arteries for 60 minutes at normothermia (37 degrees C) and group 2 animals (n = 5) were cooled to a rectal temperature of 20 degrees C before application of aortic clamps, maintained at this level during cardiopulmonary bypass until the aorta was unclamped, and then slowly rewarmed to 37 degrees C. Seven amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and five putative inhibitory neurotransmitters (glycine, gamma-aminobutyric acid, serine, adenosine, and taurine). Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. The increase in aspartate concentration was not significant. The extracellular concentrations of glycine and gamma-aminobutyric acid also increased significantly during ischemia and reperfusion. Hypothermia uniformly prevented the release of amino acids in the extracellular space. Glutamate levels remained significantly decreased even after rewarming to normothermia whereas glycine levels returned to baseline values. These results are consistent with a role for excitatory amino acids in the production of ischemic spinal cord injury and suggest that the mechanism of hypothermic protection may be related to decreased release of these amino acids in the ischemic spinal cord.

Neurotrophin-mediated potentiation of neuronal injury.

The neurotrophins are a diverse family of peptides which activate specific tyrosine kinase-linked receptors. Over the past five decades, since the pioneering work of Levi-Montalcini and colleagues, the critical role that neurotrophins play in shaping the developing nervous system has become increasingly established. These molecules, which include the nerve growth factor (NGF)-related peptides, NGF, brain-derived neurotrophic factor (BDNF), NT-4/5 and NT-3, promote differentiation and survival in the developing nervous system, and to a lesser extent in the adult nervous system. As survival-promoting molecules, neurotrophins have been studied as potential neuroprotective agents, and have shown beneficial effects in many model systems. However, a surprising "dark side" to neurotrophin behavior has emerged from some of these studies implying that, under certain pathological conditions, neurotrophins may exacerbate, rather than alleviate, injury. How neurotrophins cause these deleterious consequences is a question which is only beginning to be answered, but initial work supports altered free radical handling or modification of glutamate receptor expression as possible mechanisms underlying these effects. This review will focus on evidence suggesting that neurotrophins may enhance injury under certain circumstances and on the mechanisms behind these injury-promoting aspects.

BDNF or IGF-I potentiates free radical-mediated injury in cortical cell cultures.

Free radical-mediated damage to cultured cortical neurons was induced by a 24 h exposure to Fe2+ (30 microM) or an inhibitor of gamma-glutamylcysteine synthetase, L-buthionine-[S,R]-sulfoximine (BSO, 1 mM). As expected, neuronal death was blocked by inclusion of the free radical scavenger trolox during the Fe2+ or BSO exposure. However, unexpectedly, pretreatment of the cultures with BDNF or IGF-I markedly potentiated neuronal death. This growth factor-potentiated death was still blocked by trolox, but was insensitive to glutamate antagonists. Concurrent addition of cycloheximide with the growth factors prevented injury potentiation. Present findings suggest that growth factors may increase free radical-induced neuronal death by mechanisms dependent upon protein synthesis.

Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?

Neuronal apoptosis induced in cortical cultures by exposure to serum deprivation, staurosporine, nifedipine, or C2-ceramide was assayed by lactate dehydrogenase (LDH) release or inhibition of 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction. The protective effects of neurotrophin-4, Z-Val-Ala-Asp-fluoromethylketone (ZVAD), and cycloheximide against each insult were also assayed. The level of injury for each insult was similar whether determined by LDH release or inhibition of MTT reduction, but effects of anti-apoptotic agents were assay dependent. ZVAD and cycloheximide protected neurons from nifedipine-induced death, when assayed by LDH release, but not MTT reduction. In contrast, only cycloheximide attenuated C2-ceramide-induced LDH release, while ZVAD and cycloheximide actually enhanced the C2-ceramide induced inhibition of MTT reduction. Counting of trypan blue positive cells provided results consistent with values obtained using the LDH assay. These results indicate that both LDH release and MTT reduction accurately determine apoptotic death of neurons. However, the MTT assay does not always correctly quantify neuroprotective effects, this likely reflects differences in the point of the death pathway that the neuroprotective agents act. Therefore, while the MTT assay is of limited value in assessing the efficacy of neuroprotective strategies, it may provide information regarding whether specific anti-apoptotic agents act up or downstream of mitochondrial dysfunction.

Dextrorphan inhibits the release of excitatory amino acids during spinal cord ischemia.

The release of excitatory amino acids, particularly glutamate, into the extracellular space plays a causal role in irreversible neuronal damage after central nervous system ischemia. Dextrorphan, a noncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to provide significant protection against cerebral damage after focal ischemia. We investigated the changes in extracellular neurotransmitter amino acid concentrations using in vivo microdialysis in a swine model of spinal cord ischemia. After lumbar laminectomies were performed, all animals underwent left thoracotomy and right atrial-femoral cardiopulmonary bypass with additional aortic arch perfusion. Microdialysis probes were then inserted stereotactically into the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid and 15-minute samples were assayed using high-performance liquid chromatography. Group 1 animals (n = 9) underwent aortic clamping distal to the left subclavian and proximal to the renal arteries for 60 minutes. Group 2 animals (n = 7) were treated with dextrorphan before application of aortic clamps, and during aortic occlusion and reperfusion. Five amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and three putative inhibitory neurotransmitters (glycine, gamma-amino-butyric acid, and serine). Somatosensory-evoked potentials and motor-evoked potentials were monitored. Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. In animals treated with dextrorphan, glutamate concentrations decreased to one-third of baseline levels before aortic clamping and remained unchanged during ischemia and reperfusion. There was early loss of somatosensory-evoked potentials and motor-evoked potentials during ischemia in group 1 animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Sigma-ligands and non-competitive NMDA antagonists inhibit glutamate release during cerebral ischemia.

Release of glutamate from brain cells is increased during ischemia and is thought to be involved in ischemic damage. In rat hippocampal slices the release of glutamate during 'in vitro ischemia' (anoxia without glucose) is shown to be blocked by two groups of compounds: non-competitive N-methyl-D-aspartate (NMDA) antagonists and sigma ligands. The effects are selective for the ischemic glutamate release, which is independent of extracellular Ca2+. High K+, Ca2+ dependent, induced release of glutamate is not inhibited. NMDA receptor blockade normally does not prevent ischemic transmission damage in the rat hippocampal slice. However, when ischemic glutamate release is attenuated, NMDA receptor antagonists do prevent the damage. This indicates that high levels of glutamate may cause damage via non-NMDA as well as NMDA receptors.

Neurotoxicity of dental amalgam is mediated by zinc.

The use of dental amalgam is controversial largely because it contains mercury. We tested whether amalgam caused toxicity in neuronal cultures and whether that toxicity was caused by mercury. In this study, we used cortical cell cultures to show for the first time that amalgam causes nerve cell toxicity in culture. However, the toxicity was not blocked by the mercury chelator, 2,3-dimercaptopropane-1-sulphonate (DMPS), but was blocked by the metal chelator, calcium disodium ethylenediaminetetraacetate (CaEDTA). DMPS was an effective mercury chelator in this system, since it blocked mercury toxicity. Of the components that comprise amalgam (mercury, zinc, tin, copper, and silver), only zinc neurotoxicity was blocked by CaEDTA. These results indicate that amalgam is toxic to nerve cells in culture by releasing zinc. While zinc is known to be neurotoxic, ingestion of zinc is not a major concern because zinc levels in the body are tightly regulated.

Mechanisms of chlorpyrifos and diazinon induced neurotoxicity in cortical culture.

The main action of organophosphorous insecticides is generally believed to be the inhibition of acetylcholinesterase (AChE). However, these compounds also inhibit many other enzymes, any of which may play a role in their toxicity. We tested the neurotoxic mechanism of two organophosphorous insecticides, chlorpyrifos and diazinon in primary cortical cultures. Exposure to the insecticides caused a concentration-dependent toxicity that could not be directly attributed to the oxon forms of the compounds which caused little toxicity but strongly inhibited AChE. Addition of 1 mM acetylcholine or carbachol actually attenuated the toxicity of chlorpyrifos and diazinon, and the muscarinic receptor antagonist, atropine, and the nicotinic receptor antagonist, mecamylamine, did not attenuate the toxicity of either insecticide. These results strongly suggest that the organophosphorous toxicity observed in this culture system is not mediated by buildup of extracellular acetylcholine resulting from inhibition of AChE. The toxicity of chlorpyrifos was attenuated by antagonists of either the NMDA or AMPA/kainate-type glutamate receptors, but the cell death was potentiated by the caspase inhibitor ZVAD. Diazinon toxicity was not affected by glutamate receptor antagonists, but was attenuated by ZVAD. Chlorpyrifos induced diffuse nuclear staining characteristic of necrosis, while diazinon induced chromatin condensation characteristic of apoptosis. Also, chlorpyrifos exposure increased the levels of extracellular glutamate, while diazinon did not. The results suggest two different mechanisms of neurotoxicity of the insecticides, neither one of which involved acetylcholine. Chlorpyrifos induced a glutamate-mediated excitotoxicity, while diazinon induced apoptotic neuronal death.

Mechanisms of intracellular calcium accumulation in the CA1 region of rat hippocampus during anoxia in vitro.

There is a net movement of calcium into brain cells during anoxia and ischemia. This communication examines the mechanisms of this movement in rat hippocampal slices by analyzing changes in 45Ca2+ distribution. The CA1 pyramidal cells are the most sensitive to anoxic/ischemic damage; therefore, our measurements of Ca2+ and high-energy nucleotides are restricted to this region. The increase in intracellular Ca2+ levels during anoxia is not blocked by the Ca2+ channel blocker cobalt, nor is it blocked by N-methyl-D-aspartate receptor antagonists kynurenic acid, D-2-amino-5-phosphonovaleric acid, or ketamine. Kinetic measurements show that the rate of Ca2+ efflux across the plasmalemma during anoxia is sufficiently decreased to account for the increase in intracellular Ca2+. It thus appears that the net increase in calcium does not result from the opening of voltage-sensitive Ca2+ channels, nor from flux through the N-methyl-D-aspartate channel. Rather, it results from inhibition of the adenosine triphosphate-dependent extrusion mechanism for Ca2+. The relation of this conclusion to mechanisms of anoxic cell damage is discussed.

Calcitonin potentiates oxygen-glucose deprivation-induced neuronal death.

Calcitonin is a hormone that decreases plasma calcium through inhibition of osteolysis. It is used in the treatment of osteoporosis and other bone disorders. Salmon calcitonin is typically utilized in individuals for whom use of estrogen is contraindicated, for example, women at high risk for breast cancer. In addition to actions on bone, calcitonin may have effects on the central nervous system. Receptors for calcitonin are present on central neurons, and salmon calcitonin has been shown to alter neuronal activity. Since salmon calcitonin is used clinically, and it can have actions on neurons, the present studies were designed to determine whether salmon calcitonin could alter death of cortical neurons. The effects of salmon calcitonin on neuronal death induced by exposure of murine cortical cultures to serum deprivation, staurosporine, oxygen-glucose deprivation, kainate, and NMDA were tested. Salmon calcitonin had no effect on apoptotic cell death in cortical cultures. However, acute treatment with salmon calcitonin (1-1000 nM) caused significant potentiation of neuronal death induced by oxygen-glucose deprivation. Similarly, salmon calcitonin potentiated cell death induced by exposure to kainate. In contrast, it did not potentiate cell death induced by exposure to NMDA. In fact, addition of a high concentration (1000 nM) of salmon calcitonin attenuated NMDA toxicity. These results indicate that calcitonin is not a survival factor for cortical neurons; however, it can alter excitotoxic cell death. The most interesting, and disturbing, effect is the ability of low concentrations of salmon calcitonin to potentiate oxygen-glucose deprivation-induced cell death.

Intracellular calcium levels and calcium fluxes in the CA1 region of the rat hippocampal slice during in vitro ischemia: relationship to electrophysiological cell damage.

Five minutes of oxygen and glucose deprivation (termed "in vitro ischemia") causes long-term synaptic transmission failure (LTF) in the CA1 region of the rat hippocampal slice. Dependence of LTF on cell calcium was tested by generating graded reductions in cell Ca. There was a strong correlation between the average level of exchangeable cell Ca in CA1 during ischemia, and the extent of LTF. In standard buffer, exchangeable cell Ca in CA1 increased by 35% after 3 min of ischemia and remained elevated for the entire 5 min of ischemia. Unidirectional Ca influx increased by 35% during the first 2.5 min of ischemia and remained at that level for the next 2.5 min. There were no changes in unidirectional Ca efflux during this period. Thus, the accumulation results from increased influx of Ca. Ca influx during the first 2.5 min of ischemia depended entirely on NMDA channels; it was completely blocked by the noncompetitive NMDA receptor antagonist MK-801. However MK-801 had no effect during the second 2.5 min. This inactivation of NMDA-mediated influx during ischemia appears to result from dephosphorylation. Okadaic acid increased Ca influx during the second 2.5 min of ischemia and this increase was blocked by MK-801. The ischemia-induced Ca influx during the second 2.5 min of ischemia was attenuated 25% by nifedipine (50 microM) and an additional 35% by the Na/Ca exchange inhibitor benzamil (100 microM). The AMPA/kainate antagonist DNQX had no effect on the Ca influx. Antagonists were used to relate Ca influx to LTF. Blockade of enhanced Ca entry during ischemia in standard buffer (2.4 mM Ca) had no effect on LTF, consistent with total cell Ca prior to ischemia being adequate to cause complete LTF. However, MK-801 strongly protected against LTF when the buffer contained 1.2 mM Ca, a more physiological level. MK-801 combined with DNQX prevented transmission damage in standard buffer. Thus, AMPA/kainate receptor activation contributes to ischemic damage, although not by enhancing Ca entry.

Dipyridamole increases oxygen-glucose deprivation-induced injury in cortical cell culture.

BACKGROUND AND PURPOSE: Adenosine transport inhibitors attenuate ischemic central neuronal damage in vivo, but the locus of this protective action is presently unknown. To help address the question of whether adenosine transport inhibitors have a protective effect directly on brain parenchyma, we tested the effect of the adenosine transport inhibitor dipyridamole on neuronal loss induced by oxygen-glucose deprivation in vitro. METHODS: Murine cortical cultures were exposed to combined oxygen and glucose deprivation, N-methyl-D-aspartate, or kainate. The extracellular concentrations of glutamate and adenosine were measured by high-performance liquid chromatography; neuronal cell death was assessed by morphological examination and measurement of lactate dehydrogenase release. RESULTS: Cultures exposed to oxygen-glucose deprivation for 30 to 75 minutes exhibited an insult-dependent increase in extracellular adenosine, followed shortly by an increase in extracellular glutamate and 24 hours later by neuronal death. Addition of the A1 receptor antagonist 8-cyclopentyltheophylline during oxygen-glucose deprivation enhanced both glutamate release and neuronal damage. Addition of 10 mumol/L dipyridamole decreased extracellular adenosine and also enhanced extracellular glutamate and neuronal death. In contrast, dipyridamole increased the levels of extracellular adenosine stimulated by N-methyl-D-aspartate or kainate. CONCLUSIONS: These results are consistent with the idea that endogenous adenosine has a neuroprotective effect directly on cortical cells exposed to oxygen-glucose deprivation. However, inhibition of adenosine transport with dipyridamole was surprisingly not an effective strategy for enhancing this protective effect. The beneficial effects of adenosine transport inhibitors observed in vivo may be mediated indirectly--for example, by effects on the vasculature.