EFFECTS OF DC FIELDS ON VERTEBRATE NEURONS

直流场对脊椎动物神经元的影响

• 批准号: 6240324
• 负责人: MICHAEL E MCGINNIS
• 金额: $ 4.82万
• 依托单位: SPELMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240324
• 关键词: PC12 cells; biological signal transduction; cell age; cell growth regulation; cell motility; cell type; chick embryo; dendrites; electric field; electrical measurement; electrostimulus; fluorescence microscopy; ganglion cell; image processing; immunocytochemistry; microinjections; nervous system regeneration; neurons; orientation; periaqueductal gray matter; peripheral nervous system disorders; spinal cord injury; statistics /biometry; tissue /cell culture; video microscopy

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.

直流电场被认为会刺激轴突再生和增长 轴突，因此被建议作为诱导轴突的方法 神经损伤后的再生。 如果是真的，则适用 场对于治疗神经损伤可能很重要。 虽然有几个 研究报道了使用直流场作为脊柱和 动物的周围神经损伤，有多种担忧 这些研究都离开了他一般科学界 对发现的怀疑。 相反，轴突的一般现象 崇拜中的电场的方向，增长和迁移 建立得很好。 虽然有可能明确 在体外证明电能反应，可比较的效果一直是 很难在体内可靠地产生。 DC字段的信息很低 内容，只能为细胞提供方向或刺激提示。 这些提示中的任何一个都会通过偏向细胞的一侧介导 离子通道，膜受体，膜电位等的术语等。 培养皿的统一环境，应用领域可能是唯一的 不对称影响，因此可能对细胞有显着影响 行为。 但是，在体内condiitons中，有各种各样的竞争 细胞行为的信号，例如可扩散因素的梯度，细胞到 细胞相互作用和底物粘附性的梯度。 在这个 情况，电场提供的轻微定向偏差可能是 无关紧要。 拟议研究的长期目标是确定 应用领域对细胞体外和TO细胞影响的作用机制 确定这种机制是否在体内情况下起作用。 具体目标是； 1。比较在简单和 复杂的底物。 2。确定发育阶段对响应的影响。 3。检查细胞类型和底物源对响应的影响。 4。为执行此类执行的系统和可靠的手段 实验。 为了检验这一假设，将检查神经元的电响应 在复杂的培养条件下，例如切片文化。 这些条件 保留体外的控制，可及性和可操作性 条件，但提供一个复杂的环境OT模拟天然体内 情况更加紧密。 基本方法是获取延时视频 对应用DC场的响应的单个单元。 细胞将要么 被荧光标记在复杂底物的顶部播种的细胞，或 在组织切片中已被微分注射填充的细胞。 使用荧光视频显微镜检查，将制作单个细胞的图像 高放大倍数。 步进电动机的显微镜阶段将移动 在许多预选细胞之间并在调节时间获取图像 间隔。 这些图像将重组为视频序列细节 然后将对每个单元的行为进行统计分析。