In vitro combinations of MK-801, an NMDA receptor antagonist, and alpha-phenyl-tert-butyl-nitrone (PBN), a free radical scavenger, have been tested for possible additive neuroprotective effects against anoxia/hypoglycemia (Ax/Hg)-induced neuronal damage. Rat organotypic hippocampal slice cultures were exposed to Ax/Hg for different lengths of time to vary the severity of the insult. Cell death (CD) was assessed using propidium iodide fluorescence and expressed as a percentage of the total neuronal cells present. Pretreatment with PBN alone (500 microM) provided significant protection against moderate ischemic injury and reduced CD from 65% in controls to 2% in the treated group (P < 0.003). A longer ischemic exposure time caused more neuronal damage, which was only slightly reduced by PBN, but significantly reduced by MK-801 (30 microM) (4% CD with MK-801 vs 75% CD in controls; P < 0.0003). With a further increase in the time of ischemic exposure, MK-801 was still protective (33% CD with MK-801 vs 90% CD in controls; P < 0.002), although the combination MK-801 + PBN was more efficient (7% CD with combination, P < 0.01 compared to MK-801 alone). With yet a further increase in the ischemic exposure, PBN or MK-801 alone was not protective; however, a combination of the two still provided significant protection (64% CD with combination vs 100% CD with MK-801 alone; P < 0.01). PBN was protective when administered up to 2 h after Ax/Hg (66% CD in controls vs 36% CD with PBN 500 microM; P < 0.007). The combination MK-801 + PBN was able to increase the therapeutic window up to 3 h (61% CD in controls vs 41% with PBN alone vs 7% with MK-801 + PBN; P < 0.002 compared to PBN alone). In conclusion, the combination of MK-801 and PBN increases both the efficacy and the time window of protection against ischemia.