A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.
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机译:提出了一种研究局部轴突拉伸损伤的新模型,该模型使用微流体装置在柔软的聚二甲基硅氧烷薄膜上选择性地培养轴突,该薄膜可以向上偏转以拉伸轴突。在体外生长7天后,对原代皮层神经元施加了非常轻度(0.5%应变)或轻度拉伸损伤(5%应变)。使用损伤后(PI)固定在24小时和72小时的轴突的变性指数(DI,轴突碎片面积与总轴突面积之比)量化远端变性的程度,并免疫标记轴突特异性,微管相关蛋白头在非常轻度损伤后(0.5%),PI后24小时,大多数轴突保持完好且健康,与对照组相比,DI无明显差异,但在PI 72小时,DI显着增加(DI(= 0.11±0.03 )。值得注意的是,在PI 24小时观察到的是在体腔中的树突状珠粒,表明死后退化。当损伤程度增加(拉伸程度为5%,轻度损伤)时,观察到沿受伤轴突的微管碎裂,在24 h PI(DI = 0.17±0.02)和72 h PI(DI = 0.18±)时,DI显着增加。 0.01),相对于未受伤的轴突。在轻度和非常轻度损伤中观察到的反应与在创伤性脑损伤的体内模型中观察到的反应相似,这表明该模型可用于研究神经元损伤,并将为表征神经元的细胞和分子改变提供新的见解。对离散轴突损伤的反应。
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