Promotion of nerve regeneration and treatment of chronic pain using slow releasing nerve trophic factors and cellular transplantation.

在第一项研究中，我们从生物学角度评估了局麻药的细胞毒性。局部麻醉药具有直接的神经毒性，当以大浓度应用于神经元时，会诱导生长锥塌陷。然而，长期暴露于小浓度局部麻醉剂的影响从未被研究过。我们研究了在鸡胚背根神经节中，小浓度丁卡因是否会减慢神经突起的生长。丁卡因的影响进行了检查显微镜和神经突起生长速率测定，定量形态学测定，生长锥塌陷测定，和蛋白质印迹分析。当丁卡因浓度大于5 μ M时，应用后24和48小时的神经突生长显著延迟。丝状伪足的生长锥缩回，其数量显着减少24和48小时后，应用10和20 μ M的丁卡因。细胞胞体中肌动蛋白的数量增加，与对神经突和生长的影响相反 关于我们 owth锥，其中肌动蛋白减少48小时后，应用5，10和20 μ M的丁卡因。总之，连续暴露于丁卡因在小浓度延迟神经突起的生长，减少丝状伪足的数量，并降低actin content.The第二项研究进行，以确定是否丙泊酚对外周，视网膜和自主神经元的神经毒性作用，哪些神经元特别容易受到丙泊酚的损伤。从8天鸡胚中分离背根神经节、视网膜神经节细胞层和交感神经节链，培养20小时，然后加入不同浓度的异丙酚[5-300 μ M（0.9-53 μ g × mL（-1））]，研究其对这三种类型神经组织的影响。形态学变化通过生长锥塌陷实验进行定量检测。使用高效液相色谱法测定丙泊酚浓度。异丙酚诱导生长锥塌陷和神经突破坏。在异丙酚作用后2 h，三种类型的神经元的量效关系有显著性差异（P < 0.001），但在24 h时无显著性差异。在暴露于100 μ M丙泊酚长达6小时后，所有三种类型的神经元的生长锥塌陷效应至少部分可逆，尽管在暴露24小时后没有观察到可逆性。虽然丙泊酚的临床安全性已被充分证明，但在高浓度下，丙泊酚对体外生长的神经元具有潜在的神经毒性。从这些观察中，我们得出结论，神经生长锥和神经突是敏感的外部环境。一些外部应用的营养因子和/或释放营养因子的细胞可能有效地促进神经再生。少

In the first study, we assessed local anesthetics toxicity cell biologically. The local anesthetics have direct neurotoxicity and induce growth cone collapse when applied to neurons at large concentrations. However, the effects of prolonged exposure to local anesthetics at a small concentration have never been studied. We examined whether neurite growth was slowed by tetracaine at small concentrations in chick embryo dorsal root ganglions. The effects of tetracaine were examined microscopically and by a neurite growth rate assay, quantitative morphologic assay, growth cone collapse assay, and Western blot assay. Neurite growth 24 and 48 h after application was delayed significantly when tetracaine was applied at a concentration larger than 5 microM. Filopodia of growth cones retracted, and their number was significantly decreased 24 and 48 h after the application of 10 and 20 microM of tetracaine. The quantity of actin in cell bodies increased, contrary to the effect on neurites and gr … More owth cones, where actin decreased 48 h after the application of 5, 10, and 20 microM of tetracaine. In conclusion, continuous exposure to tetracaine at small concentrations delayed neurite growth, reduced the number of filopodia, and decreased actin content.The second study was undertaken to determine whether propofol has neurotoxic effects on peripheral, retinal, and autonomic neurons, and which neurons are particularly liable to injury by propofol. Dorsal root ganglia, retinal ganglion cell layers, and sympathetic ganglion chains were isolated from day eight chick embryos and cultured for 20 hr. Thereafter, propofol was added at various concentrations [5-300 microM (0.9-53 microg x mL (-1) )] to investigate its effects on these three types of neuronal tissue. Morphological changes were examined quantitatively by growth cone collapse assay. Propofol concentrations were measured using high performance liquid chromatography. Propofol induced growth cone collapse and neurite destruction. The three types of neurons tested exhibited significantly different dose-response relationships two hours after the application of propofol (P < 0.001) but not at 24 hr after application. The growth cone-collapsing effect was at least partially reversible in all three types of neurons after exposure to 100 microM propofol up to six hours, though reversibility was not observed after 24-hr exposure. While the clinical safety profile of propofol has been well documented, at high concentrations propofol has potential neurotoxicity on growing neurons in vitro.From these observations, we concluded nerve growth cones and neurites are sensitive outer environment. Some externally applied trophic factors and/or cells that release trophic factors, may be effective to promote nerve regeneration. Less