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ANALGESIA

Cytotoxicity of Local Anesthetics in Human Neuronal Cells

Rosalia Perez-Castro, MS^*, Sohin Patel, MD^*, Zayra V. Garavito-Aguilar, BS^*, Andrew Rosenberg, MD, Esperanza Recio-Pinto, PhD^*, Jin Zhang, MD^*, Thomas J. J. Blanck, MD, PhD^*, and Fang Xu, PhD^*

From the *Department of Anesthesiology, New York University School of Medicine; and Department of Anesthesiology, NYU Hospital for Joint Diseases, New York, New York.

Address correspondence and reprint requests to Fang Xu, PhD, Department of Anesthesiology, NYU Medical Center, 560 First Ave., Tisch Building, Rm. HE-438, New York, NY 10016. Address e-mail to fang.xu{at}nyumc.org.

Abstract

BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC₅₀ for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity.

METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD® assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA^TM). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca²⁺-responses were also evaluated.

RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD₅₀, the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca²⁺-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca²⁺-evoked cytosolic Ca²⁺-level.

CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons.

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