The objective of this proposal is to determine the mechanism of direct neurotoxicity of spinal (intrathecal) lidocaine. Other local anesthetics have a risk of persistent lumbosacral neurotoxicity of approximately 1 in 10,000. Lidocaine at doses producing surgical anesthesia has an increased risk of approximately 1 in 200 via continuous spinal anesthesia, and approximately 1 in 1300 via single injection spinal anesthesia. Transient neurologic symptoms (TNS) of buttock and leg pain occur in 16-40 percent of patients receiving spinal lidocaine in multiple large studies. Lidocaine is also neurotoxic in an animal model that produces lower extremity anesthesia with 5 percent lidocaine, and in cell culture and in vitro nerve studies. To address the mechanism of this neurotoxicity, most studies will be conducted at the level of the single cell, using digitized video fluorescence microscopy, phase microscopy, and flow cytometry, with appropriate, specific, fluorescent probes. Immunoblotting and fluorogenic assays of fractionated cell lysates will also be utilized. The ND7 cell line, derived from rat dorsal root ganglion, will be used as a model system for neuronal injury. Two hypotheses will be tested as specific aims: (1) Lidocaine interferes with multiple mechanisms for maintaining normal cytoplasmic calcium (Ca2+cyt), causing an increase to toxic levels. Preliminary data show a marked effect of lidocaine to elevate Ca2+cyt (5-7 fold with 5 percent lidocaine; greater than 15 percent cell death within 60 min). The effect of lidocaine on possible mechanisms of Ca2+cyt elevation will be tested, including influx from extracellular buffer, release from endoplasmic reticulum, and release from mitochondria. The causal relationship of lidocaine-induced Ca2+cyt elevation to neurite injury, plasma membrane blebbing, and neuronal death by necrosis and apoptosis will be determined. (2) Lidocaine activates multiple mechanisms of mitochondrial injury. Preliminary data show a marked effect of lidocaine to decrease mitochondrial membrane potential deltapsi in whole cells. Potential mechanisms of decreased deltapsi by lidocaine will be tested: protonophoric mitochondrial uncoupling, inhibition of mitochondrial respiration, and induction of the mitochondrial permeability transition. The effect of lidocaine will also be tested on mitochondrially based mechanisms of cell death: release of mitochondrial cytochrome c, and caspase activation. The Ca2+cyt dependence of each mechanism of mitochondrial injury will be determined by experiments with and without Ca2+cyt clamped at a normal level.
