ИСТИНА

Purpose. Currently it stays unknown how acidosis influences the neuronal damage which is caused by the zinc entry resulted from hyperactivation of AMPA/kainate receptors in consequence of brain ischemia/hypoxia. Our study was performed using cultured cerebellar granule neurons (CGNs) subjected simultaneous exposure to zinc/kainate toxicity and acidosis initiated by either acidification of incubation medium or inhibition of Na+/H+ exchanger by EIPA. Methods. Primary CGN cultures were prepared from cerebella of 7-8-day-old Wistar rats, kept in a CO2-incubator (5% CO2/95% air, 98% relative humidity, 36.50C) and maintained there before experiments for 7 days in vitro. After experiments cultures were fixed in ethanol–formaldehyde–acetic acid mixture (7:2:1) and stained with trypan blue. The surviving neuron percentage was estimated by calculation of intact CGNs in five fields of vision under magnification x40. The living cells were loaded with 0.005 mM FluoZin-3 AM followed by triple washing in balanced salt solution and fluorescent analysis of intraneuronal zinc ion concentration ([Zn2+]i) which was quantified using a Cytofluor plate reader (CytoFluor II, PerSeptive Biosystems, USA) or microscope image system (Olympus, Yapan). Results. CGNs were insensitive to ZnCl2 exposure (0.005 mM, 3 h) and slightly sensitive to kainate (0.1 mM, 3 h). Simultaneous treatment of CGNs with kainate+ZnCl2 caused pronounced neuronal death, which was attenuated by external acidosis (pH 6.5) or Na+/H+ exchange blocker EIPA (0.03 mM). [Zn2+]i was raised under ZnCl2 or kainate+ZnCl2 exposure. This raising was significantly decreased by external acidosis but increased by EIPA. Neuronal imaging demonstrated that EIPA causes the release of Zn2+ from intracellular stores. These data imply that external acidosis retard ZnCl2/kainate toxic effect by decreasing Zn2+ entry in neurons while EIPA prevents intraneuronal stores from overload with zinc. Conclusion. The data obtained suggest that in CGN cultures exposed to ZnCl2/kainate toxicity, neuroprotective effect of extracellular acidosis is mediated by decreasing Zn2+ entry in neurons while EIPA prevents intraneuronal stores from a Zn2+ overload. This study was supported by RFBR grants 12-04-00025-a, 14-04-00030-a.