脑源性神经营养因子基因转染修复大鼠坐骨神经损伤的组织学观察及功能评价

Effect of adenovirus-mediated BDNF transfer on sciatic nerve injury in rats and functional evaluation

目的观察重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后的脊髓前角运动神经元存活、神经生长和功能恢复情况。方法将90只成年Wistar大鼠分成3组,AxCA-BDNF转染组(硅胶管内置AxCA-BDNF原液)、BDNF组(硅胶管内置BDNF溶液)和对照组(硅胶管内置空白病毒稀释液),每组30只,另取5只Wistar大鼠作为正常组。应用组织学图像分析技术对脊髓前角运动神经元和新生神经纤维进行计数,对新生神经髓鞘厚度进行测量分析;应用足印分析和电生理检查技术,从功能角度判定BDNF基因转移的生物效应。结果在3、7、14d及1个月4个时相点,光镜、电镜病理组织学检查和图像分析证实,AxCA-BDNF转染组和BDNF组脊髓前角运动神经元存活的数量、新生神经纤维数目及其髓鞘厚度均明显优于对照组(P<0.01);HRP逆行示踪也证实神经联接的再形成优于对照组(P<0.01);在坐骨神经功能指数测定、神经传导速度和复合肌肉动作电位振幅等神经功能恢复指标上,AxCA-BDNF转染组和BDNF组均优于对照组(P<0.01)。结论应用腺病毒介导的BDNF基因转移,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元,大鼠的神经功能得到了明显改善。

Objective To observe the peripheral nerve regeneration of injured sciatic nerve after adenovirus-mediated gene transfer of brain-derived neurotrophic factor （BDNF） in adult rats. Methods Totally 90 Wistar rats were inflicted to sciatic nerve injury by cutting off a nervous fragment of 10 mm. and then divided into 3 group. A silicone tube containing AxCA-BDNF solution, BDNF solution, or AxCA buffer was grafted as a support to bridge adult rat sciatic nerve gap for the 3 groups correspondingly, Another 5 rats with no any injury served as normal control. In 3, 7, 14 d and 1 month after injury, the rats were sacrificed and their lumbar vertebra （L3 to L6） were resected for frozen section. The number of new nerve fibers and motoneurons in anterior horn of the spinal cord was observed with transmission electron microscopy （TEM）, histomorphometry and image analysis, and the myelin sheath thickness of the new nerve fibers was measured. The sciatic nerve function recovery was evaluated with sciatic function index （SFI） and the electrophysiological parameters, including the nerve conduction velocity （NCV） and compound muscle action potential （CMVP） amplitude. Results There were more survival neurons, new nerve fibers and thicker myelin sheath in AxCA-BDNF group and BDNF group than in the control group （P0.01）. Retrograde axonal transport of HRP tracer indicated that the injured sciatic nerve reconnected with neurons in spinal cord were better in AxCA-BDNF group and BDNF group than in the control group（P0.01）. So were the SFI, NCV and CMVP amplitude （P0.01） in the 2 BDNF groups. Conclusion Adenovirus-mediated gene transfer of BDNF in vivo not only promotes nerve regeneration but also protects the neurons in the spinal cord for sciatic nerve injury in adult rats.