Apoptosis Is Not Enhanced in Primary Mixed Neuronal/Glial Cultures Protected by Isoflurane Against N-Methyl-d-Aspartate Excitotoxicity

Abstract

Volatile anesthetics reduce acute excitotoxic cell death in primary neuronal/glial cultures. We hypothesized that cells protected by isoflurane against N-methyl-d-aspartate (NMDA)-induced necrosis would instead become apoptotic. Primary mixed neuronal/glial cultures prepared from fetal rat brain were exposed to dissolved isoflurane (0 mM, 0.4 mM [1.8 minimum alveolar anesthetic concentration], or 1.6 mM [7 minimum alveolar anesthetic concentration]) and NMDA (0 or 100 microM) at 37 degrees C for 30 min. Dizocilpine (10 microM) plus 100 microM NMDA served as a positive control. Necrosis and apoptosis were assessed at 24 and/or 48 h after exposure by using Hoechst/propidium iodide staining, terminal-deoxynucleotidyl transferase end-nick labeling, DNA fragmentation enzyme-linked immunoabsorbence, and caspase-3 activity assays. NMDA increased the number of necrotic cells. Isoflurane (1.6 mM) and dizocilpine partially reduced cellular necrosis but did not increase the number of morphologically apoptotic or apoptotic-like cells resulting from exposure to 100 microM NMDA at 24 h. At 48 h, no evidence was found to indicate that cells protected by isoflurane had become apoptotic or apoptotic-like. However, cells protected by dizocilpine against necrosis showed evidence of caspase-3-mediated apoptosis. These in vitro data do not support the hypothesis that isoflurane protection against acute excitotoxic necrosis results in apoptosis.