Hains Bryan C, Black Joel A, Waxman Stephen G
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Comp Neurol. 2003 Jun 9;462(3):328-41. doi: 10.1002/cne.10733.
Spinal cord injury (SCI) results in loss of voluntary motor control followed by incomplete recovery, which is partly mediated by the descending corticospinal tract (CST). This system is an important target for therapeutic repair strategies after SCI; however, the question of whether apoptotic cell death occurs in these axotomized neurons remains unanswered. In this study, adult (150-175 g) male Sprague-Dawley rats underwent T9 transection of the dorsal funiculus, which axotomizes the dorsal CST, and introduction of the retrograde tracer Fluoro-Gold into the lesion site. Primary motor cortex (M1) was then examined for evidence of apoptosis weekly for 4 weeks after injury. Axotomized pyramidal cells, identified by retrograde transport of Fluoro-Gold, were found in M1 (57.5 +/- 9.6/median section, 6127 +/- 292 total), and a significant proportion were terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-rhodamine nick end labeling (TUNEL) -positive at 1 week after injury (39.3 +/- 5.6%), compared with animals undergoing sham surgery (1.2 +/- 1.4%). At 2-4 weeks, fewer cells were Fluoro-Gold-positive (24.6 +/- 65.06 to 25.3 +/- 6.4/median section, 2338 +/- 233 to 2393 +/- 124 total), of which very few were TUNEL-positive. In TUNEL-positive cells, Hoechst 33342 staining revealed nuclear morphology consistent with apoptosis, chromatin condensation, and formation of apoptotic bodies. Fluoro-Gold-positive cells showed increased caspase-3 and Bax immunoreactivity. Hematoxylin and eosin staining revealed similar nuclear changes and dystrophic cells. Internucleosomal DNA fragmentation was detected by gel electrophoresis at the 1-week time point. Lesioned animals not receiving Fluoro-Gold exhibited the same markers of apoptosis. These results document, for the first time, features of apoptotic cell death in a proportion of axotomized cortical motor neurons after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI.
脊髓损伤(SCI)会导致自主运动控制丧失,随后是不完全恢复,这部分是由下行皮质脊髓束(CST)介导的。该系统是SCI后治疗性修复策略的重要靶点;然而,这些轴突切断的神经元是否发生凋亡性细胞死亡的问题仍未得到解答。在本研究中,成年(150 - 175克)雄性Sprague-Dawley大鼠接受了T9背侧索横断术,该手术切断了背侧CST,并将逆行示踪剂荧光金引入损伤部位。然后在损伤后4周内每周检查初级运动皮层(M1)是否有凋亡迹象。通过荧光金的逆行运输鉴定出的轴突切断的锥体细胞在M1中被发现(57.5±9.6/中间切片,共6127±292个),与接受假手术的动物相比,损伤后1周有相当比例的细胞是末端脱氧核苷酸转移酶(TdT)介导的脱氧尿苷三磷酸(dUTP)-罗丹明缺口末端标记(TUNEL)阳性(39.3±5.6%),而假手术动物为(1.2±1.4%)。在2 - 4周时,荧光金阳性细胞减少(24.6±65.06至25.3±6.4/中间切片,共2338±233至2393±124个),其中很少有TUNEL阳性细胞。在TUNEL阳性细胞中,Hoechst 33342染色显示核形态与凋亡一致,染色质浓缩,凋亡小体形成。荧光金阳性细胞显示半胱天冬酶-3和Bax免疫反应性增加。苏木精和伊红染色显示类似的核变化和营养不良细胞。在1周时间点通过凝胶电泳检测到核小体间DNA片段化。未接受荧光金的损伤动物表现出相同的凋亡标志物。这些结果首次证明了SCI后一部分轴突切断的皮质运动神经元存在凋亡性细胞死亡的特征,表明防止凋亡可能是SCI再生方法的先决条件。