DIRECT NEUROTOXICITY OF LOCAL ANESTHETICS

本提案的目的是确定脊髓（鞘内）利多卡因的直接神经毒性机制。 其他局麻药的持续腰骶神经毒性风险约为1/10，000。 在产生手术麻醉的剂量下，利多卡因通过连续脊髓麻醉的风险增加约1/200，通过单次注射脊髓麻醉的风险增加约1/1300。在多项大型研究中，16%-40%接受脊髓利多卡因治疗的患者出现一过性神经系统症状（TNS），包括坐骨神经痛和腿痛。 在用5%利多卡因产生下肢麻醉的动物模型中，以及在细胞培养和体外神经研究中，利多卡因也具有神经毒性。 为了解决这种神经毒性的机制，大多数研究将在单细胞水平上进行，使用数字化视频荧光显微镜，相位显微镜和流式细胞术，使用适当的，特异性的荧光探针。还将使用分级细胞裂解物的免疫印迹和荧光测定。 来自大鼠背根神经节的ND 7细胞系将用作神经元损伤的模型系统。 将检验两个假设作为特定目的：（1）利多卡因干扰维持正常细胞质钙（Ca 2 +cyt）的多种机制，导致毒性水平升高。 初步数据显示，利多卡因对升高Ca 2 +cyt有显著作用（5%利多卡因为5-7倍; 60分钟内细胞死亡率超过15%）。 将检测利多卡因对Ca 2 +cyt升高的可能机制的影响，包括细胞外缓冲液的流入、内质网的释放和线粒体的释放。 将确定利多卡因诱导的Ca 2 +cyt升高与神经突损伤、质膜起泡以及坏死和凋亡引起的神经元死亡之间的因果关系。(2)利多卡因激活线粒体损伤的多种机制。初步数据显示，利多卡因对降低全细胞线粒体膜电位δ具有显著作用。 将测试利多卡因降低Δ Psi的潜在机制：质子转运线粒体解偶联、线粒体呼吸抑制和诱导线粒体渗透性转变。 利多卡因的作用也将在基于神经的细胞死亡机制上进行测试：线粒体细胞色素c的释放和半胱天冬酶激活。 将通过将Ca 2 +cyt夹在正常水平和不夹在正常水平的实验来确定线粒体损伤的每种机制的Ca 2 + cyt依赖性。

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.