Absence of direct antioxidant effects from volatile anesthetics in primary mixed neuronal-glial cultures

Background: Volatile anesthetics decrease ischemic brain injury. Mechanisms for this protection remain under investigation. The authors hypothesized that volatile anesthetics serve as antioxidants in a neuronal-glial cell culture system, Methods: Primary cortical neuronal-glial cultures were prepared from fetal rat brain. Cultures were exposed to iron, H2O2, or xanthine-xanthine oxidase for 30 min in serum-free media containing dissolved isoflurane (0-3.2 mr), sevoflurane (0-3.6 mr), halothane (0-4.1 mu), n-hexanol, or known antioxidants, Cell damage was assessed by release of lactate dehydrogenase (LDH) and trypan blue exclusion 24 h later. Lipid peroxidation was measured by the production of thiobarbituric acid-reactive substances in a cell-free lipid system. Iron and calcium uptake and mitochondrial depolarization were measured after exposure to iron in the presence or absence of isoflurane. Results: Deferoxamine reduced LDH release caused by H2O2, or xanthine-xanthine oxidase, but the volatile anesthetics had no effect. Iron-induced LDH release was prevented by the volatile anesthetics (maximum effect for halothane = 1.2 mM, isoflurane = 1.2 mM, and sevoflurane = 2.1 mM aqueous phase). When corrected for lipid solubility, the three volatile anesthetics were equipotent against iron-induced LDH release. In the cell-free system, there was no effect of the anesthetics on thiobarbituric acid-reactive substance formation in contrast to Trolox, which provided complete inhibition. Isoflurane (1.2 mM) reduced mean iron uptake by 46% and inhibited mitochondrial depolarization but had no effect on calcium uptake. Conclusions: Volatile anesthetics reduced cell death induced by oxidative stress only in the context of iron challenge. The likely reason for protection against iron toxicity is inhibition of iron uptake and therefore indirect reduction of subsequent intracellular oxidative stress caused by this challenge. These data argue against a primary antioxidant effect of volatile anesthetics.