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Confocal microscopic visualization of MK-801-induced cytoplasmic vacuoles in vitro
1Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Abstract:
We examined neuroprotective and neurotoxic effects of the NMDA antagonist, MK-801, in primary cell cultures derived from embryonic mouse neocortex. Brief deprivation of oxygen and glucose, or direct application of N-methyl-D-aspartate (NMDA), resulted in acute neuronal swelling followed by neuronal death during the next day. This excitotoxic neuronal injury could be blocked by inclusion of a wide variety of NMDA antagonists in the cell culture medium. MK-801 attenuated neuronal death in the low micromolar range; 1 to 10 microM concentrations were sufficient to maximally reduce injury from NMDA toxicity, oxygen deprivation, or combined deprivation of oxygen and glucose. MK-801 alone caused no apparent toxicity at these concentrations in exposures of 24 to 48 hours. However, 24-hour exposure to 100 microM MK-801 resulted in appearance of cytoplasmic vacuoles, which could be visualized with immunofluorescence against the microtubule associated protein, MAP2, together with laser scanning confocal microscopy. Thus, at concentrations sufficient to block NMDA receptors, MK-801 is neuroprotective rather than neurotoxic for cortical neurons in vitro. This model system may provide a method to examine cellular mechanisms underlying the neurotoxicity of MK-801 at very high concentrations.
Insights
The NMDA antagonist MK-801 protects against excitotoxic neuronal injury at low concentrations. However, high concentrations of MK-801 may cause neurotoxicity, indicating a concentration-dependent effect.
Area of Science:
- Neuroscience
- Cell Biology
Background:
- Excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, contributes to neuronal injury.
- NMDA antagonists are investigated for neuroprotective potential.
Purpose of the Study:
- To evaluate the neuroprotective and neurotoxic effects of the NMDA antagonist MK-801.
- To assess MK-801's efficacy in preventing excitotoxic neuronal damage in vitro.
Main Methods:
- Primary mouse neocortical cell cultures were used.
- Neuronal injury was induced by oxygen-glucose deprivation or NMDA application.
- The effects of MK-801 at various concentrations were analyzed using microscopy and immunofluorescence.
Main Results:
- MK-801 demonstrated neuroprotection against NMDA toxicity and oxygen-glucose deprivation at low micromolar concentrations (1-10 microM).
- MK-801 showed no apparent toxicity at protective concentrations (1-10 microM) after 24-48 hours of exposure.
- High concentration (100 microM) MK-801 exposure for 24 hours induced cytoplasmic vacuoles in neurons.
Conclusions:
- MK-801 exhibits neuroprotective properties at concentrations effective for NMDA receptor blockade.
- High-dose MK-801 may induce neurotoxicity, suggesting a dose-dependent biphasic effect.
- This in vitro model is suitable for investigating the mechanisms of MK-801 neurotoxicity at elevated concentrations.