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Oxidative stress has been implicated in neuronal death caused by cerebral ischemia or some neurologic disorders. Chemical hypoxia (term defining the simulation by using respiratory inhibitors) chosen as in vitro ischemic model, was induced in primary cultures of rat cerebellar granule neurons by inhibitors of mitochondrial electron transport such as rotenone or paraquat (complex I), 3-nitropropionic acid (3-NPA, complex II), antimycin A (complex III), or sodium azide (complex IV). All compounds caused neuronal death determined by trypan blue staining and MTT-test. On the other hand, neurotoxicity of rotenone and paraquat but not of 3-NPA, antimycin or azide was significantly abolished by menadione (vitamin K3, 2-methyl-1,4-naphthoquinone). This neuroprotective effect of menadione was associated with a decrease of rotenone-induced free radical production.

Pretreatment with 10 microM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 microM clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca(2+) overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca(2+) overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.

Treatment of cultured rat cerebellar granular cells with calmodulin antagonist bifonazole (10 mM) during oxygen-glucose deprivation or exposure to glutamate (75 mM) prevented neuronal death. However, addition of bifonazole after glutamate treatment promoted neuronal death. Calmodulin antagonists trifluoperazine and thioridazine had no protective effects, while thioridazine even potentiated the toxic effect of glutamate.

Rhodamine 123 staining, light and electron microscopy were used to evaluate the ultrastructural and functional state of cultured cerebellar granule cells after short treatment with the solution where NaCl was substituted by sucrose (sucrose balance salt medium, SBSM). Cell exposure to SBSM for 20 min resulted in the fact that mitochondria in the neurons lost their ability to sequester rhodamine 123. This effect could be prevented by: (i) non-competitive N-methyl-D-aspartate (NMDA) receptor channel blocker, 10(-5) M MK-801; (ii) a competitive specific antagonist of NMDA glutamate receptors, 0.25 x 10(-3) M D,L-2-amino-7-phosphonoheptanoate (APH); (iii) 10(-3) M cobalt chloride; (iv) removal of Ca2+ from the medium. Low Na+ in the Ca2+-containing medium caused considerable mitochondrial swelling in granule cells. However, the same treatment in the absence of calcium ions in the medium abolished the deleterious effect of SBSM on the neuronal mitochondrial structure and functions. It is suggested that (i) the exposure of cultured cerebellar granule cells to SBSM leads to a release of endogenous glutamate from cells; (ii) Ca2+ ions potentially de-energizing neuronal mitochondria enter the neuron preferentially through the NMDA channels rather than through the Na+/Ca2+ exchanger; (iii) mitochondrial swelling in granule cells is highly Ca2+-dependent; (iv) cellular overload with sodium ions can activate mitochondrial Na+/Ca2+ exchanger and thus prevent permeability transition pore opening in mitochondria.

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (100 microM) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na+, K+-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10-100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.

Using primary cultures of cerebellar granule cells from 4-6-day old Wistar rats we showed the protective effect of vitamin E against kainate-induced neurotoxicity. The preincubation of 7-8-day old cultures with 5 x 10(-4) M alpha-tocopherol solution significantly (on 10-20%) reduces the number of damaged granule cells. As vitamin E takes part in stabilization of membrane lipids the data presented allows us to suggest that one of the possible mechanisms of neuronal injury includes lipid oxidation of the neuronal membranes which leads to additional influx of Ca2+ and results in neuronal death.