Effects of Isoflurane Inhibition of Mitochondrial Complex I on Calcium Removal in Mouse Neuronal Cultures.

Abstract

One mechanism proposed for anesthetic-induced neurotoxicity (AIN) is elevated neuronal calcium, leading to mitochondrial damage and caspase activation. Increased cytosolic calcium could arise from increased entry or decreased removal. The relative importance of these distinct mechanisms is unknown. Isoflurane inhibits mitochondrial complex I and reduces adenosine triphosphate (ATP) at presynaptic terminals leading to synaptic quiescence. The authors hypothesized that mitochondrial inhibition initiates calcium dysregulation in mouse wild-type and mitochondrial mutant neurons, leading to AIN. Presynaptic calcium levels were monitored using VGlut1-GCaMP5 or an endoplasmic reticulum-specific GCaMP6 during electrical stimulations of neuronal cultures. Cultures were stimulated in the presence of isoflurane and blockers or activators of calcium removal. Mitochondrial damage was monitored using MitoView (Biotium, USA). Cleaved caspase induction assessed AIN. In the absence of isoflurane, neuronal stimulation transiently increased presynaptic calcium levels. Isoflurane increased the half-life for calcium decay in wild-type cultures (time [s], unexposed, 14 [10]; exposed, 160 [77]; P = 0.001). Maintaining ATP levels rescued the isoflurane-induced defective removal of calcium (time [s], 30 mM glucose, 16 [14]; n = 8; P = 0.001). Activation of sarcoplasmic endoplasmic reticulum calcium adenosine triphosphatase (SERCA) alleviated the isoflurane-induced defective removal of calcium (time [s], no SERCA activator, 159 [78]; SERCA activator, 36 [18]; P = 0.002). Similar results were seen for mutant cultures exposed to lower, but equipotent, concentrations of isoflurane. Isoflurane induced a SERCA-dependent decrease in uptake of MitoView and an increase in cleaved caspase in wild-type cultures. Isoflurane causes a failure of SERCA-dependent calcium removal by inhibition of mitochondrial production of ATP. The increase in intracellular calcium leads to early signs of cellular toxicity.