BDA皮质脊髓束神经顺行示踪在大鼠脊髓损伤模型中的应用

目的本研究采用生物素标记葡聚糖(Biotin Dextran Amine,BDA)顺行示踪技术来观察大鼠皮质脊髓束(CST)在中枢神经系统中的走行及脊髓损伤后的表现特征。方法20只雌性成年Sprague-Dawley大鼠,分为脊髓损伤组(n=10)和损伤对照组(n=10)。在相当于T7椎板水平用做好标记的显微剪刀剪断脊髓的后2/3。对照组动物术中仅咬除棘突、椎板,不切断脊髓。术后第15 d,所有动物通过立体定向开颅,将10％BDA溶液注入右侧的感觉运动区皮质内。BDA注射2周后,取出大脑和脊髓组织,采用自由漂浮法行BDA染色显影。实验动物于脊髓损伤术前、术后3d、1周、2周、4周采用Basso、Beatlie、Bresnahan(BBB)评分法测量运动功能,所得数据采用两组均数比较t检验进行统计学处理。结果1.脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);2.BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在中脑、桥脑及延髓的腹侧面行走,但在锥体交叉后皮质脊髓束主要(约99％)在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;在对照组皮质脊髓束纤维染色可一直延伸至L1水平。结论BDA顺行神经

BDA anterograde corticospinal cord neuronal tracing in the rat model of spinal cord injury

Objective To explore the courser of rat corticospinal tract (CST) in the center neural system and manifestations after spinal cord injury by the Biotin Dextran Amine(BDA) anterograde neuronal tracing technique. Methods 20 adult female Sprague-Dawley rats were divided randomly into the spinal cord injury group (n=10) and the control group (n=10). The laminectomy was performed on all animals at spinal levels of T7. In the spinal cord injury group, the dorsal 2/3 of the spinal cord was cut with a pair of microscissors to sever the dorsal parts of the corticospinal tracts(CST).. In the control group, only spinous process and lamina of vertebra were cut instead of a laminectomy. 15 days after surgery, animals in both groups were placed in the stereotaxic frame. The craniotomy was performed on the right side of the skull. 10% BDA was injected into the right sensorimotor cortex. 2 weeks later, animals were sacrificed. The brain and spinal cord were used for BDA staining by the free-floating method. Behavior test was carried out before spinal cord surgery and 3 days, 1 week, 2 weeks and 4 weeks after surgery using the BBB scores. The results were analyzed by students t-test. Results 1. All animals in the injury group showed a paralysis on their hindlimbs. The BBB scores were much lower in the injury group than that in the control group. There was a significant statistically difference between them (P<0.01). 2. BDA anterograde neuronal tracing technique showed pyramidal cells and their axons were positive-stained. The BDA staining positive corticospinal tract fibers were located ipsilateraly in the ventral part of mesencephalon, pons and medulla oblongata level. After the pyramidal decussation, strong labeled CST axons were located contralaterally in the posterio funiculus of the white matter down the whole spinal cord. In the spinal cord injury animals the BDA stained CST axon stopped at the injury level, while the BDA stained CST axon can be seen until L1 level in spinal cord of the control animals; Conclusion BDA anterograde neuronal tracing technique can be used as a reliable method for morphological studies in CNS regeneration after spinal cord injury.