Staal J A, Dickson T C, Chung R S, Vickers J C
NeuroRepair Group, Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia.
Dev Neurobiol. 2007 Dec;67(14):1831-42. doi: 10.1002/dneu.20552.
Following central nervous system trauma, diffuse axonal injury and secondary axotomy result from a cascade of cellular alterations including cytoskeletal and mitochondrial disruption. We have examined the link between intracellular changes following mild/moderate axonal stretch injury and secondary axotomy in rat cortical neurons cultured to relative maturity (21 days in vitro). Axon bundles were transiently stretched to a strain level between 103% and 106% using controlled pressurized fluid. Double-immunohistochemical analysis of neurofilaments, neuronal spectrin, alpha-internexin, cytochrome-c, and ubiquitin was conducted at 24-, 48-, 72-, and 96-h postinjury. Stretch injury resulted in delayed cytoskeletal damage, maximal at 48-h postinjury. Accumulation of cytochrome-c and ubiquitin was also evident at 48 h following injury and colocalized to axonal regions of cytoskeletal disruption. Pretreatment of cultures with cyclosporin-A, an inhibitor of calcineurin and the mitochondrial membrane transitional pore, reduced the degree of cytoskeletal damage in stretch-injured axonal bundles. At 48-h postinjury, 20% of untreated cultures demonstrated secondary axotomy, whereas cyclosporin A-treated axon bundles remained intact. By 72-h postinjury, 50% of control preparations and 7% of cyclosporin A-treated axonal bundles had progressed to secondary axotomy, respectively. Statistical analyses demonstrated a significant (p < 0.05) reduction in secondary axotomy between treated and untreated cultures. In summary, these results suggest that cyclosporin-A reduces progressive cytoskeletal damage and secondary axotomy following transient axonal stretch injury in vitro.
中枢神经系统创伤后，弥漫性轴索损伤和继发性轴突切断是由一系列细胞改变引起的，包括细胞骨架和线粒体破坏。我们研究了在体外培养至相对成熟（体外培养21天）的大鼠皮质神经元中，轻度/中度轴突拉伸损伤后细胞内变化与继发性轴突切断之间的联系。使用可控加压流体将轴突束短暂拉伸至103%至106%的应变水平。在损伤后24、48、72和96小时进行神经丝、神经元血影蛋白、α-中间丝蛋白、细胞色素c和泛素的双重免疫组织化学分析。拉伸损伤导致细胞骨架损伤延迟，在损伤后48小时达到最大值。损伤后48小时，细胞色素c和泛素的积累也很明显，并与细胞骨架破坏的轴突区域共定位。用环孢素A（一种钙调神经磷酸酶和线粒体膜过渡孔的抑制剂）预处理培养物，可降低拉伸损伤轴突束中细胞骨架损伤的程度。在损伤后48小时，20%的未处理培养物出现继发性轴突切断，而环孢素A处理的轴突束保持完整。到损伤后72小时，分别有50%的对照制剂和7%的环孢素A处理的轴突束进展为继发性轴突切断。统计分析表明，处理组和未处理组培养物之间继发性轴突切断的发生率有显著降低（p<0.05）。总之，这些结果表明，环孢素A可减少体外短暂轴突拉伸损伤后的进行性细胞骨架损伤和继发性轴突切断。
