Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model.
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 Ca. 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.
Johnson
Michael E
ME
Anesthesiology Department, Division of Gastroenterology and Hepatology, Mayo Clinic & Foundation, 200 Southwest First Street, Rochester, MN 55905, USA. johnson.michael@mayo.edu
Saenz
J Armando
JA
DaSilva
Assir Daniel
AD
Uhl
Cindy B
CB
Gores
Gregory J
GJ
D000779:Anesthetics, Local / Q000633:toxicity*; D000818:Animals; D002045:Bupivacaine / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D008012:Lidocaine / Q000633:toxicity*; D009336:Necrosis; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051381:Rats
D000779:Anesthetics, Local; D008012:Lidocaine; D002118:Calcium; D002045:Bupivacaine