PROTECTION FROM NEURONAL DAMAGE-INDUCED BY COMBINED OXYGEN AND GLUCOSE DEPRIVATION IN ORGANOTYPIC HIPPOCAMPAL CULTURES BY GLUTAMATE-RECEPTOR ANTAGONISTS

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 neuronal 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 mu M), CNQX (100 mu M) and GYKI 52466 (100 mu M) 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 mu M) or CNQX (100 mu M) 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 mu M) offered no neuroprotection if the compound 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 (similar to 50% protection). When a combination of dizocilpine and CNQX or GYKI 52466 was applied permanently, significant 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.