Cyclic Mechanical Stretch Stress Enhances BDNF and NT-3 immunoreactivity in Cultured Spinal Cord Neuronal, Glial cells

研究设计.本研究旨在探讨周期性牵张应力对体外培养大鼠脊髓细胞神经营养因子合成的影响。应用免疫组织化学方法评价机械应力对体外培养的神经元和神经胶质细胞的神经营养反应。近年来，在实验性脊髓损伤动物模型上研究了内源性神经营养因子（BDNF、NT-3）的功能、来源和诱导动力学。据我们所知，目前还没有研究，检查生物学和病理学反应的机械应力来源于脊髓的培养细胞。培养的脊髓细胞分离自14天的Sprague-Dawley大鼠胚胎。为了向细胞提供机械应力，使用FX-3000 TM-Flexercell应变单元（Flexercell International Corp.，麦基斯波特，宾夕法尼亚州，美国）。在开始施加刺激后，观察细胞形态和免疫反应性随时间的变化（0、1、2、6、12和48小时）。应激6 h以上，神经元存活数减少，细胞膜增厚，胞间质扩张。微管相关蛋白（MAP）-2阳性细胞数随应激时间的延长而减少，胶质细胞酸性蛋白GFAP阳性细胞数相对稳定。在星形胶质细胞中，抗GFAP/BDNF和NT-3抗体阳性细胞的数量随着应激时间的推移而相对保持不变，但随着时间的推移，观察显示剩余的抗GFAP阳性细胞的免疫反应性较高。我们的研究结果表明，反应性星形胶质细胞增加神经营养因子的表达，机械应力，可能性，反应性星形胶质细胞对机械应力在培养的细胞参与延长生存和机械修复。

Study Design. A rat model designed to investigate the effects of applied cyclic stretch stress on neurotrophic factor synthesis in the rat spinal cord cells in vitro.Objective. To evaluate the neurotrophic response in vitro neuron and glia cell under mechanical stress using immunohistological analysis.Summary of Background Data. Recent studies conducted in animals with experimental spinal cord damage examined the function, source and dynamics of induction of endogenious neurotrophic factors including brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3. To our knowledge, there are no studies that examined biological and pathological responses to mechanical stress in cultured cells derived from spinal cord.Methods. The cultured spinal cord cells were isolated from 14 day Sprague-Dawley rat embryos. For providing mechanical stress to cells, FX-3000TM-Flexercell Strain Unit (Flexercell International Corp., McKeesport, PA, USA) was used. After starting application of stimulation, the cell morphology and immunoreactivity was observed over time (0, 1, 2, 6, 12 and 48 hours).Results. Decrease in the number of surviving neuron, thickening of cell membrane and extention of intercellular substance were observed after providing stress to cells for 6 hours or more. The number of microtubule-associated protein (MAP)-2 positive cells decreases with time, while the number of glial fibrillary acidic protein GFAP positive cells was comparatively kept against stress. For astrocytes, the number of anti-GFAP/BDNF and NT-3 antibody positive cells was comparatively kept with the lapse of stress time, but observation over time revealed a high immuno-reactivity of the remaining anti-GFAP positive cells.Conclusion. Our results indicate that reactive astrocytes increase the expression of neurotrophic factors to the mechanical stress, a possibility that reactive astrocytes against mechanical stress in cultured cells are involved in the prolongation of survival and mechanical repair.