Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model

Abstract

Background: To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+cyt), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity. Methods: Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+cyt and time of necrotic cell death (plasma membrane lysis) at the single neuron level. Results: Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca2+cyt. Lidocaine 0.5-5% and bupivacaine 0.125-0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+cyt that was transient and returned to near baseline within 10 min. Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+cyt and death in some neurons during the 60 min exposure period. Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+cyt, consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+cyt. The later sustained increase in Ca2+cyt seen with 2.5 and 5% lidocaine was prevented in Ca2+-free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane. Conclusions: In this model, lidocaine greater than 2.5% elevates Ca2+cyt to toxic levels. Bupivacaine and lower concentrations of lidocaine transiently alter Ca2+cyt homeostasis for several minutes, but without an immediate neurotoxic effect within 60 min.