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对细胞的毒性。局麻药对神经元有直接的神经毒性，大剂量的局麻药可引起生长锥塌陷。然而，长期暴露于小浓度局部麻醉剂的影响从未被研究过。我们研究了小浓度丁卡因是否会减缓鸡胚背根神经节的神经突生长。通过神经突生长速率试验、定量形态学试验、生长锥塌陷试验和Western blot试验，在显微镜下观察丁卡因的作用。当丁卡因浓度大于5 μ m时，神经突生长明显延迟24和48 h。10和20 μ m丁卡因处理后24和48 h，生长球果丝状足收缩，丝状足数量显著减少。在施用5、10和20 μ m丁卡因后48 h，肌动蛋白在细胞体中的数量增加，与对神经突和锥体的影响相反。综上所述，持续暴露于小浓度丁卡因可延缓神经突生长，减少丝状足数量，降低肌动蛋白含量。第二项研究是为了确定异丙酚是否对周围、视网膜和自主神经元有神经毒性作用，以及哪些神经元特别容易受到异丙酚的损伤。从第8天的鸡胚中分离出背根神经节、视网膜神经节细胞层和交感神经节链，培养20小时。之后，加入不同浓度的异丙酚[5-300微米（0.9-53微米× mL(-1)）]，研究其对这三种神经组织的影响。用生长锥塌陷法定量检测形态学变化。采用高效液相色谱法测定异丙酚浓度。异丙酚诱导生长锥塌陷和神经突破坏。三种类型的神经元在应用异丙酚后2小时表现出显著不同的剂量-反应关系（P < 0.001），但在应用后24小时则无显著差异。在暴露于100微米异丙酚长达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，尽管暴露于24小时后未观察到可逆性。虽然异丙酚的临床安全性已被充分证明，但高浓度异丙酚对体外生长的神经元具有潜在的神经毒性。由此得出神经生长锥和神经突对外界环境敏感的结论。一些外用营养因子和/或释放营养因子的细胞，可能有效促进神经再生。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