Yap Yiing C, King Anna E, Guijt Rosanne M, Jiang Tongcui, Blizzard Catherine A, Breadmore Michael C, Dickson Tracey C
Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia.
Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia.
PLoS One. 2017 May 4;12(5):e0176997. doi: 10.1371/journal.pone.0176997. eCollection 2017.
Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of βIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased βIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model.
弥漫性轴索损伤是非穿透性创伤性脑损伤（TBI）的标志性病理后果，然而，在细胞水平上，轴索对拉伸损伤的反应尚未完全明确。在此，我们使用最近开发的体外分隔模型，研究了轻度（5%）、极轻度（0.5%）和重复性极轻度（2×0.5%）轴索拉伸损伤对原代皮层神经元的影响。我们发现，与未损伤培养物相比，在损伤后24小时和72小时测量时，极轻度和轻度拉伸损伤导致轴突末端生长锥变小，塌陷结构数量显著增加。免疫细胞化学研究显示，轻度损伤后72小时，轴突生长锥中βIII微管蛋白和F-肌动蛋白的共定位显著高于极轻度损伤后的生长锥，塌陷形态的百分比也更高。有趣的是，在第一次损伤后24小时接受第二次极轻度拉伸损伤的培养物，与损伤后72小时的单次极轻度损伤相比，生长锥塌陷比例进一步增加，βIII微管蛋白和F-肌动蛋白共定位增加。此外，我们的结果表明，使用可穿透脑屏障的埃坡霉素D（EpoD）（100 nM）稳定轴突微管，可显著减少轻度损伤后轴突断裂的数量。总的来说，这些结果表明，对皮层轴突非常局部区域的轻度和极轻度拉伸损伤能够引发以生长锥塌陷和显著异常的细胞骨架重排为特征的退行性反应。此外，重复性极轻度拉伸损伤显著加剧了这种反应。结果表明，拉伸损伤后的轴突退变涉及微管细胞骨架的不稳定，因此用EpoD治疗可减少轴突断裂。这些结果共同有助于更好地理解轻度和重复性TBI的发病机制，并突出了使用体外分隔培养模型，微管靶向药物对神经元远端部分的治疗效果。
