DIRECT NEUROTOXICITY OF LOCAL ANESTHETICS

本建议的目的是确定脊髓(鞘内)利多卡因直接神经毒性的机制。其他局部麻醉药有大约1/10,000的持续腰骶神经毒性风险。产生手术麻醉的剂量的利多卡因持续脊麻的风险增加约1/200，单次注射脊麻的风险增加约1/1300。在多项大型研究中，接受脊髓利多卡因治疗的患者中有16%-40%会出现短暂性神经症状(TNS)，表现为臀部和腿部疼痛。利多卡因在动物模型中也有神经毒性，该动物模型用5%的利多卡因进行下肢麻醉，在细胞培养和体外神经研究中也是如此。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、位相显微镜和流式细胞术，并使用适当的、特定的荧光探针。分离的细胞裂解物的免疫印迹和荧光分析也将被使用。从大鼠背根神经节来源的ND7细胞系将被用作神经元损伤的模型系统。有两个假设将作为特定的目标进行检验：(1)利多卡因干扰维持正常细胞质钙(Ca~(2+))的多种机制，导致毒性水平增加。初步数据显示，利多卡因显著提高了细胞内的钙离子浓度(5-7倍于5%的利多卡因；60分钟内细胞死亡超过15%)。我们将测试利多卡因对细胞内钙升高的可能机制的影响，包括细胞外缓冲液的内流、内质网的释放和线粒体的释放。利多卡因诱导的细胞钙升高与轴突损伤、质膜起泡以及因坏死和凋亡导致的神经元死亡之间的因果关系将被确定。(2)利多卡因激活多种线粒体损伤机制。初步数据显示，利多卡因有明显降低全细胞线粒体膜电位的作用。利多卡因降低三角洲的潜在机制将被测试：原突触线粒体解偶联，抑制线粒体呼吸，诱导线粒体通透性转变。利多卡因的作用也将在基于线粒体的细胞死亡机制上进行测试：线粒体细胞色素c的释放和半胱氨酸氨基转移酶的激活。线粒体损伤的每种机制对钙的依赖性将通过在正常水平钳制和不钳制钙的实验来确定。

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.