Neurosteroid modulation of embryonic neuronal survival in vitro following anoxia

Neurosteroids are synthesized de novo in the brain from cholesterol or peripheral steroid precursors and modulate inhibitory gamma-aminobutyric acid (GABA(A)) and excitatory N-methyl-D-aspartate (NMDA) receptors. Evidence indicates that neurosteroids are neuroprotective and important during neurodevelopment. We tested the hypothesis that neurosteroids increase embryonic neuronal survival following anoxia in rat embryonic day 18 cerebral cortical cultures to examine potential neurosteroid modulation of this insult during early development. Twenty-four hours after plating in serum-free medium, cultures were exposed to DHEA, DHEAS, or allopregnanolone (10(-10), 10(-8), or 10(-6) M), or vehicle, for 24 h (n=9 per treatment condition). Cultures were then subjected to anoxia for 2 h and subsequently reincubated for 24 h prior to neuron immunostaining with microtubule-associated protein 2 (MAP-2) antibody. Supernatant from DHEA and DHEAS-exposed cultures was tested for 17beta-estradiol metabolite formation by radioimmunoassay. DHEA 10(-6) and 10(-8) M significantly increased neuron survival by 85-87% following anoxia. DHEAS 10(-6) M significantly increased neuron survival by 74% following anoxia, but DHEAS 10(-10) M decreased neuron survival after this insult. Allopregnanolone had modest effects on neuron survival that did not attain statistical significance. 17beta-Estradiol concentrations were below the limit of detection in all specimens tested (sensitivity 4.7 nM). Our data indicate that pretreatment with DHEA and DHEAS at physiologically relevant concentrations promotes neuronal survival following anoxia in embryonic rat cerebral cortical cultures, and that these effects are not secondary to 17beta-estradiol metabolite formation. DHEA and DHEAS modulation of anoxia in embryonic neurons may be relevant to disorders of neurodevelopment involving this insult.