Effects of NMDA and ferrous sulfate on oxidation and cell death in primary neuronal cultures

Abstract

Excessive oxidative radical production has been implicated in a variety of neurodegerative processes including NMDA ( N-methyl- D-aspartate) mediated excitotoxicity. To determine the relationship of oxidation to NMDA-receptor mediated neuronal death, we exposed rat primary cortical neuronal cultures to ferrous sulfate and the fluorescent dyes dichlorofluorescin diacetate (H 2DCF) and propidium iodide (PI) to monitor reactive oxygen species (ROS) and cell death, respectively in the same cultures. Ferrous sulfate (FeSO 4) caused a dose-dependent increase in cellular oxidation with an ED 50 of approximately 136 μM. Levels of oxidation increased over time reaching maximum levels between 15 and 25 min. Ferrous sulfate (ED 50 ≈ 241 μM) treatment for 25 min caused a delayed and progressive neuronal death that was comparable to NMDA (100 μM, 25 min) delayed neuronal death. NMDA (100 μM, 25 min) alone did not result in measurable increases of DCF fluorescence. However, when combined with 40 μM FeSO 4, NMDA dose-dependently increased H 2DCF fluorescence. Despite the increase in DCF oxidation, combinations of FeSO 4 with NMDA did not synergize or accelerate NMDA-receptor mediated or glutamate-mediated excitotoxicity. Although excessive amounts FeSO 4 induced oxidation can cause delayed neuronal death, these findings suggest that oxidative stress is not the key factor in triggering the NMDA mediated excitotoxic cascade.