Abstracts

Rationale: Sustained status epilepticus occurring early-in-life can prevent typical hippocampal injury ensued by a subsequent seizure at a later age. Attenuation of cell death is prominent in the hippocampal CA1, the region most sensitive to seizure-induced injury in the developing brain. To get a better understanding of the mechanism of this type of neuroprotection, we developed a “two hit” model in dissociated cultures of the rat hippocampus to allow a test of the hypothesis that exposure of immature neurons during a resistant period (5-7 DIV) to toxic doses of glutamate or N-methyl-D-aspartic acid (NMDA) may prevent neurons from dying following a second exposure at a later age, when they become highly sensitive to glutamate (14 DIV). Methods: Dissociated hippocampal cultures were prepared from E17 embryos and exposed to varied does of glutamate or NMDA for 48 hrs at 5 DIV and again at 14 DIV for 5 or 15 minutes, respectively. NeuN immunohistochemistry was used to measure neurotoxicity 48 hrs after the second exposure and cell counts were compared with cultures with a single exposure at 14 DIV or changed medium. Results: After the first exposure to NMDA (100 µM), no difference in morphology was noted but there was a decrease in total number of surviving neurons (71.03±4.4%). After the second exposure, NMDA pretreatment increased the proportion of surviving cells compared to one exposure at 14 DIV (from 19.4±3.3 to 53±6.2%). Similarly, high (500 µM) doses of glutamate killed half of the NeuN+ neurons (52±8.6%) after the first treatment, whereas moderate (200-250 µM) doses killed one third (33.1± 5.7%). Two exposures to 500 µm glutamate killed most cultured neurons by rapid apoptosis (akin 500 µm glutamate at 14 DIV). However, lower doses of glutamate (200-250 µM) followed by higher doses (500 µM) spared a significant population (35.3±1.2%). Partial protection occurred despite near control levels of NMDA and AMPA receptor protein expression. Interestingly, calbindin positive interneurons were nearly absent after the first exposure to 500 µM glutamate, partially depleted after 200-250 µM doses, but unchanged after NMDA application regardless of number of treatments. Conclusions: The data suggest that early exposure of excitatory amino acids, even at high doses, can induce a long-lasting pre-conditioning tolerance. Furthermore, the density of interneurons and specific calcium binding proteins appear critical for neuronal survival.