EFFECTS OF DC FIELDS ON VERTEBRATE NEURONS

直流场对脊椎动物神经元的影响

基本信息

批准号：
6107391
负责人：
MICHAEL E MCGINNIS
金额：
$ 5.64万
依托单位：
SPELMAN COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-09-01 至 2000-01-31
项目状态：
已结题

项目摘要

DC electric fields are thought to stimulate axonal regeneration and growing axons, and so have been suggested as a method for inducing axonal regeneration following nerve injuries. If true, the application of such fields could be important in treating nerve injuries. Although several studies have reported the use of DC fields as a treatment for spinal and peripheral nerve injuries in animals, there are a variety of concerns with each of these studies that have left he general scientific community skeptical of the findings. In contrast, the general phenomena of axonal orientation, growth, and migration towards an electric field in cultaure are well established. While it has been possible to unequivocally demonstrate galvanic responses in vitro, comparable effects have been difficult to produce reliably in vivo. DC fields have low information content and can only provide cells with a directional or stimulatory cue. Either of these cues would be mediated by biasing one side of the cell in terms of ion channels, membrane receptors, membrane potential, etc. In the uniform environment of a culture dish, an applied field may be the only asymmetric influence, and thus may have a significant effect on cellular behavior. In in vivo condiitons, however, there a variety of competing signals for cell behavior such as gradients of diffusible factors, cell to cell interactions, and gradients of substrate adhesiveness. In this situation, the slight directional bias supplied by an electric field may be inconsequential. The long term objectives of the proposed study are to determine the mechanism of the effect of applied fields on cells in vitro and to determine if such a mechanism is functional in the in vivo situation. Specific aims are to; 1. compare the galvanic response of chick neurons grown on simple and complex substrates. 2. determine the effect of developmental stage on the response. 3. examine the effects of cell type and substrate source on the response. 4. to develop the means for systematically and reliable performing such experiments. To test this hypothesis, the galvanic response of neurons will be examined in complex culture conditions such as slice culture. These conditions retain the control, accessibility, and manipulability of the in vitro condition but provide a complex environment ot simulate the natural in vivo situation more closely. The basic method is to acquire time lapse videos of individual cells responding to applied DC fields. The cells will either be fluorescently labeled cells seeded on top of a complex substrate, or cells within a tissue slice that have been dye filled by microinjections. Using fluorescence videomicroscopy, images of individual cells will be made at high magnification. Stepper motors ont he microscope stage will move between many preselected cells and acquire images at regulate time intervals. These images will be recombined into a video sequence detailing the behavior of each cell that will then be statistically analyzed.

直流电场被认为可以刺激轴突再生和生长轴突，因此被建议作为诱导轴突的方法神经损伤后的再生。如果属实，则应用此类磁场对于治疗神经损伤可能很重要。虽然有几个研究报告了使用直流电场来治疗脊柱和动物的周围神经损伤，存在多种问题这些研究中的每一项都离开了整个科学界对研究结果表示怀疑。相比之下，轴突的一般现象文化中向电场的定向、生长和迁移已经建立良好。虽然已经可以明确地证明体外电反应，类似的效果已经难以在体内可靠地产生。 DC 场信息量低内容，只能为细胞提供方向性或刺激性提示。这些线索中的任何一个都可以通过偏向细胞的一侧来介导离子通道、膜受体、膜电位等方面。培养皿的统一环境，应用领域可能是唯一的不对称影响，因此可能对细胞产生重大影响行为。然而，在体内条件下，存在多种竞争性的细胞行为的信号，例如扩散因子的梯度、细胞到细胞相互作用和基质粘附性梯度。在这个在这种情况下，电场提供的轻微方向性偏置可能是无关紧要的。拟议研究的长期目标是确定应用场对体外细胞的影响机制确定这种机制在体内情况下是否起作用。具体目标是： 1. 比较在简单和复杂的基材。 2. 确定发育阶段对反应的影响。 3. 检查细胞类型和底物来源对反应的影响。 4. 开发系统、可靠地执行此类操作的方法实验。为了检验这一假设，将检查神经元的电反应在复杂的培养条件下，例如切片培养。这些条件保留体外实验的控制性、可及性和可操作性条件但提供一个复杂的环境或模拟自然的体内情况更加密切。基本方法是获取延时视频单个细胞对施加的直流场做出反应。细胞将要么是接种在复杂基质顶部的荧光标记细胞，或组织切片内的细胞已通过显微注射填充染料。使用荧光视频显微镜，将制作单个细胞的图像在高倍率下。显微镜载物台上的步进电机将移动在许多预选的细胞之间并在规定的时间获取图像间隔。这些图像将被重新组合成一个视频序列，详细说明然后对每个细胞的行为进行统计分析。