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。局麻药在大剂量作用于神经元时，具有直接的神经毒性，并可引起生长锥塌陷。然而，长期接触小浓度局麻药的影响从未被研究过。我们研究了小浓度丁卡因对鸡胚背根神经节中神经突起生长的抑制作用。在显微镜下观察丁卡因的作用，并通过轴突生长速率分析、定量形态分析、生长锥体塌陷实验和Western印迹分析来观察丁卡因对神经元的影响。丁卡因浓度大于5微米时，给药后24、48h轴突生长明显延迟。10和20微米丁卡因处理24和48h后，生长球果的丝状伪足回缩，数量显著减少。胞体内肌动蛋白的数量增加，而对神经突起和GR…的影响相反在应用5、10和20微米丁卡因后48小时，肌动蛋白减少。总之，持续暴露于小浓度丁卡因可延缓神经突起的生长，减少丝状足的数量，减少肌动蛋白的含量。第二项研究是为了确定异丙酚是否对外周、视网膜和自主神经有神经毒性作用，以及哪些神经元特别容易受到异丙酚的损伤。从8日龄鸡胚胎分离背根神经节、视网膜神经节细胞层和交感神经节链，培养20h。然后，加入不同浓度的异丙酚[5~300微米(0.9~53微克×毫升(-1)])，观察其对这三种类型神经元组织的影响。生长锥体塌陷实验定量观察细胞形态变化。用高效液相色谱法测定丙泊酚浓度。异丙酚引起生长锥体塌陷和轴突破坏。三种类型的神经元在应用异丙酚(P&lt；0.001)后2小时呈现明显不同的剂量-反应关系，但在应用后24小时无明显差异。在暴露于100微米异丙酚6小时后，所有三种类型的神经元的生长锥体塌陷效应至少是部分可逆的，尽管在暴露24小时后观察不到可逆性。虽然异丙酚的临床安全性已经得到了很好的证明，但高浓度的异丙酚对体外培养的生长神经元具有潜在的神经毒性。一些外用营养因子和/或释放营养因子的细胞可能有效地促进神经再生。较少

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