Protection from neuronal damage induced by combined oxygen and glucose deprivation in organotypic hippocampal cultures by glutamate receptor antagonists

doi:10.1016/0006-8993(95)00519-V

Brain Research

Volume 687, Issues 1–2, 31 July 1995, Pages 167-174

https://doi.org/10.1016/0006-8993(95)00519-V Get rights and content

Abstract

Organotypic hippocampal cultures were exposed to defined periods (30 and 60 min) of combined oxygen and glucose deprivation, mimicking transient ischemic conditions. The involvement of different glutamate receptors in individual hippocampal subfields (CA1, CA3 and dentate gyrus) was studied using antagonists of NMDA (dizocilpine) and AMPA/kainate receptors (CNQX and GYKI 52466). Staining with the fluorescent dye propidium iodide (PI) allowed detection of damaged cells. For quantitative determination of neuronalv damage, fluorescence intensity was measured after a 22 h recovery period and was related to maximal fluorescence intensity measured after fixation and PI restaining of the cultures at the end of the experiment. Dizocilpine (10 μM), CNQX (100 μM) and GYKI 52466 (100 μM) provided complete protection in CA1, CA3 and dentate gyrus following the moderate ischemic insult, when the antagonists were present permanently. This indicates that none of the ionotropic glutamate receptor subtypes dominated toxicity in the most sensitive subpopulation of neurons. When applied only during the recovery period protection with dizocilpine (10 μM) or CNQX (100 μM) was drastically reduced by about 60% in the most sensitive area (CA1), but only slightly by 15% in CA3. Therefore the onset of irreversible damage seems to occur earlier in CA1 than in CA3. Blockade of AMPA/kainate receptors by GYKI 52466 (100 μM) offered no neuroprotection if the compounds was applied only during the recovery period. Combined blockade of NMDA and AMPA/kainate receptors during the recovery period only, did not significantly enhance the protection compared to blockade of either receptor alone. By increasing the period of oxygen and glucose deprivation to 60 min, protection by permanent blockade of NMDA or AMPA/kainate receptors was lost completely in CA1 and was significant in CA3 and dentate gyrus only after NMDA receptor blockade by dizocilpine (∼ 50% protection). When a combination of dizocilpine and CNQX or GYKI 52466 was applied permanently, significantly protection could be shown in all hippocampal areas, being about 65% for dizocilpine plus CNQX and almost complete for dizocilpine plus GYKI 52466. These findings indicate that in vitro combined blockade of NMDA and AMPA/kainate receptors gains importance in protecting neurons from degeneration following prolonged oxygen and glucose deprivation periods. When the receptors are blocked only during recovery, no significant protection was obtained in any hippocampal area, indicating that irreversible neuronal damage, mediated by ionotropic glutamate receptors, is manifested already during the 60 min deprivation period.

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Citation Excerpt :
This failure concept also applies to the well-known neuroprotective effect of extracellular acidity against OGD in cultured neurons (Fig. 1E) (Giffard et al., 1990; Tombaugh and Sapolsky, 1990), ascribed primarily to proton inhibition of NMDA receptors (Tang et al., 1990), since neuroprotection dissipates with increased OGD duration (Kaku et al., 1993). This concept applies to other NMDARA's (Obeidat et al., 2000), and extends to in vivo studies (Buchan et al., 1991; Nellgård et al., 1991; Strasser and Fischer, 1995). Fifth, antagonists of other receptors also display these trait of decreasing effectiveness with longer durations of OGD, thereby requiring higher concentrations, which nonetheless plateau and eventually are rendered ineffective for a sufficiently severe insult (Strasser and Fischer, 1995; Pringle et al., 1997).
The goal of this study was to identify cocktails of drugs able to protect cultured rodent cortical neurons against increasing durations of oxygen-glucose deprivation (OGD). As expected, a cocktail composed of an NMDA and AMPA receptor antagonists and a voltage gated Ca²⁺ channel blocker (MK-801, CNQX and nifedipine, respectively) provided complete neuroprotection against mild OGD. Increasingly longer durations of OGD necessitated increasing the doses of MK-801 and CNQX, until these cocktails ultimately failed to provide neuroprotection against supra-lethal OGD, even at maximal drug concentrations. Surprisingly, supplementation of any of these cocktails with blockers of TRPM7 channels for increasing OGD durations was not neuroprotective, unless these blockers possessed the ability to inhibit NMDA receptors. Supplementation of the maximally effective cocktail with other NMDA receptor antagonists augmented neuroprotection, suggesting insufficient NMDAR blockade by MK-801. Substitution of MK-801 in cocktails with high concentrations of a glycine site NMDA receptor antagonist caused the greatest improvements in neuroprotection, with the more potent SM-31900 superior to L689,560. Substitution of CQNX in cocktails with AMPA receptor antagonists at high concentrations also improved neuroprotection, particularly with the combination of SYM2206 and NBQX. The most neuroprotective cocktail was thus composed of SM-31900, SYM2206, NBQX, nifedipine and the antioxidant trolox. Thus, the cumulative properties of antagonist potency and concentration in a cocktail dictate neuroprotective efficacy. The central target of supra-lethal OGD is excitotoxicity, which must be blocked to the greatest extent possible to minimize ion influx.
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We previously showed that JM-20, a novel 1,5-benzodiazepine fused to a dihydropyridine moiety, possessed an anxiolytic profile similar to diazepam and strong neuroprotective activity in different cell models relevant to cerebral ischemia. Here, we investigated whether JM-20 protects against ischemic neuronal damage in vitro and in vivo. The effects of JM-20 were evaluated on hippocampal slices subjected to oxygen and glucose deprivation (OGD). For in vivo studies, Wistar rats were subjected 90 min of middle cerebral artery occlusion (MCAo) and oral administration of JM-20 at 2, 4 and 8 mg/kg 1 h following reperfusion. Twenty-four hours after cerebral blood flow restoration, neurological deficits were scored, and the infarct volume, histopathological changes in cortex, number of hippocampal and striatal neurons, and glutamate/aspartate concentrations in the cerebrospinal fluid were measured. Susceptibility to brain mitochondrial swelling, membrane potential dissipation, H₂O₂ generation, cytochrome c release, Ca²⁺ accumulation, and morphological changes in the organelles were assessed 24 h post-ischemia. In vitro, JM-20 (1 and 10 μM) administered during reperfusion significantly reduced cell death in hippocampal slices subjected to OGD. In vivo, JM-20 treatment (4 and 8 mg/kg) significantly decreased neurological deficit scores, edema formation, total infarct volumes and histological alterations in different brain regions. JM-20 treatment also protected brain mitochondria from ischemic damage, most likely by preventing Ca²⁺ accumulation in organelles. Moreover, an 8-mg/kg JM-20 dose reduced glutamate and aspartate concentrations in cerebrospinal fluid and the deleterious effects of MCAo even when delivered 8 h after blood flow restoration. These results suggest that in rats, JM-20 is a robust neuroprotective agent against ischemia/reperfusion injury with a wide therapeutic window. Our findings support the further examination of potential clinical JM-20 use to treat acute ischemic stroke.
Neuroprotection of Persea major extract against oxygen and glucose deprivation in hippocampal slices involves increased glutamate uptake and modulation of A1 and A<inf>2A</inf> adenosine receptors
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Ischemic stroke is characterised by a lack of oxygen and glucose in the brain, leading to excessive glutamate release and neuronal cell death. Adenosine is produced in response to ATP depletion and acts as an endogenous neuromodulator that reduces excitotoxicity. Persea major (Meins.) L.E. Kopp (Lauraceae) is a medical plant that is indigenous to South Brazil, and the rural population has used it medicinally due to its anti-inflammatory properties. The aim of this study was to evaluate the neuroprotective effect of Persea major methanolic extract against oxygen and glucose deprivation and re-oxygenation as well as to determine its underlying mechanism of action in hippocampal brain slices. Persea major methanolic extract (0.5 mg/ml) has a neuroprotective effect on hippocampal slices when added before or during 15 min of oxygen and glucose deprivation or 2 h of re-oxygenation. Hippocampal slices subjected to oxygen and glucose deprivation and re-oxygenation showed significantly reduced glutamate uptake, and the addition of Persea major methanolic extract in the re-oxygenation period counteracted the reduction of glutamate uptake. The presence of A1 or A_2A, but not A_2B or A3 receptor antagonists, abolished the neuroprotective effect of Persea major methanolic extract. In conclusion, the neuroprotective effect of Persea major methanolic extract involves augmentation of glutamate uptake and modulation of A1 and A_2B adenosine receptors.
Nicotine and estrogen synergistically exacerbate cerebral ischemic injury
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The greater incidence of myocardial infarction, cardiac arrest, and ischemic stroke among women who smoke and use oral contraception (OC) compared to women who do not smoke and who do or do not use OC may be due in part to how nicotine influences endocrine function in women. For example, we recently demonstrated that chronic exposure to nicotine, the addictive agent in tobacco smoke responsible for the elevated risk of cardiac arrest, abolishes the endogenous or exogenous 17β-estradiol-conferred protection of the hippocampus against global cerebral ischemia (a potential outcome of cardiac arrest) in naive or ovariectomized female rats. In the current study we examined the hypotheses that (1) a synergistic deleterious effect of nicotine plus oral contraceptives exacerbates post-ischemic hippocampal damage in female rats, and (2) nicotine directly inhibits estrogen-mediated intracellular signaling in the hippocampus. To test first hypothesis and to simulate smoking behavior-induced nicotine levels in the human body, we implanted osmotic pumps containing nicotine in the female rats for 16 days. Furthermore, we mimicked the use of oral contraceptives in females by administering oral contraceptives orally to the rat. Rats exposed to either nicotine alone or in combination with oral contraceptives were subjected to an episode of cerebral ischemia and the resultant brain damage was quantified. These results showed for the first time that nicotine with oral contraceptives did indeed exacerbate post-ischemic CA1 damage as compared to nicotine alone in naive female rats. In ex vivo hippocampal slice cultures, we found that nicotine alone or with 17β-estradiol directly hinders estrogen receptors-mediated phosphorylation of cyclic-AMP element binding protein, a process required for neuronal survival and also exacerbates ischemic damage. Thus, nicotine can affect the outcome of cerebral ischemia by influencing brain endocrine function directly rather than through indirect systemic effects.
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Research reportProtection from neuronal damage induced by combined oxygen and glucose deprivation in organotypic hippocampal cultures by glutamate receptor antagonists

Abstract

Neurosci. Lett.

Dev. Brain Res.

Neuroscience

Trends Neurosci.

Brain Res.

Brain Res.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Brain Res.

J. Neurosci. Methods

Brain Res.

Brain Res.

Neuron

Brain Res.

Neurosci. Lett.

Eur. J. Pharmacol.

J. Neurosci. Methods

J. Neurosci. Methods

Neuroscience

Brain Res.

Exp. Neurol.

The excitotoxin hypothesis in relation to cerebral ischemia

Cerebrovasc. Brain Metab. Rev.

Elevation of the extracellular concentrations of glutamate and asparate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis

J. Neurochem.

The N-methyl-d-aspartate antagonist MK-801, fails to protect against neuronal damage caused by transient severe forebrain ischemia in adult rats

J. Neurosci.

Research report
Protection from neuronal damage induced by combined oxygen and glucose deprivation in organotypic hippocampal cultures by glutamate receptor antagonists