电刺激联合神经营养素3可促进脊髓损伤大鼠内源性神经干细胞的增殖和分化

Electrical stimulation combined with neurotrophin 3 promotes proliferation and differentiation of endogenous neural stem cells after spinal cord injury in rats

背景:由于外源性神经干细胞的获取有限,且容易产生免疫排斥以及伦理问题等严重制约其向临床转化,因此如何激活内源性神经干细胞并促进其生长增殖、分化,成为近期科研工作者究的热点。目的:探讨电刺激联合神经营养素3对大鼠脊髓损伤后内源性神经干细胞增殖及向神经元分化的作用。方法:将96只SD大鼠随机分为假手术组、脊髓损伤组、电刺激组、电刺激+神经营养素3组,每组24只。假手术组仅暴露脊髓,其他3组大鼠应用改良Allen法建立脊髓损伤模型,造模后给予相应措施进行干预。造模后7,14,21,28 d时,以BBB评分评价大鼠后肢运动功能,电生理学检查运动诱发电位潜伏期;造模后28 d取材,进行苏木精-伊红染色观察脊髓病理变化,免疫组化染色观察内源性神经干细胞的增殖和分化情况。实验方案经兰州大学第二医院医学伦理委员会批准。结果与结论:①与假手术组相比,脊髓损伤组大鼠的BBB评分明显降低(P<0.01),脊髓组织可见大量炎症细胞浸润,并存在多个空洞;与脊髓损伤组相比,电刺激组、电刺激+神经营养素3组大鼠后肢功能开始逐渐恢复,电刺激+神经营养素3组BBB评分明显高于电刺激组(P<0.05),上述病理损伤变化明显改善;②脊髓损伤组7,14d及电刺激组大鼠7 d时双后肢运动诱发电位潜伏期均未测出,电刺激组、电刺激+神经营养素3组21,28 d时运动诱发电位潜伏期较模型组缩短(P<0.05),电刺激+神经营养素3组潜伏期缩短更显著(P<0.05);③BrdU和Nestin阳性细胞数、微管相关蛋白2的表达:电刺激+神经营养素3组>电刺激组>脊髓损伤组;胶质纤维酸性蛋白的表达:脊髓损伤组>电刺激组>电刺激+神经营养素3组。结果表明脊髓损伤大鼠经电刺激及神经营养素3干预后,促进内源性神经干细胞增殖和向神经元分化,病理损伤明显减轻,后肢运动功能显著改善。

BACKGROUND:Due to limited access to exogenous neural stem cells,immune rejection and ethical problems,how to activate endogenous neural stem cells and promote their growth,proliferation and differentiation has become an issue of concern.OBJECTIVE:To investigate the effects of electrical stimulation combined with neurotrophin 3 on the proliferation and differentiation of endogenous neural stem cells into neurons after spinal cord injury in rats.METHODS:Ninety-six rats were randomly divided into sham operation(spinal cord exposed only),spinal cord injury,electrical stimulation,and electrical stimulation+neurotrophin groups,24 rats in each group.A rat model of spinal cord injury was established by modified Allen method in the latter three groups.After the model was established,the rats in the four groups were given corresponding treatments.At 7,14,21,and 28 days after modeling,the motor function of hind limbs was evaluated by Basso-Beattie-Bresnahan score.The latency of motor evoked potential was examined by electrophysiology.At 28 days after modeling,samples of the spinal cord were taken for hematoxylin-eosin staining to observe the pathological changes and for immunohistochemical staining to observe the the proliferation and differentiation of endogenous neural stem cells.The study was approved by the Ethics Committee of the Second Hospital of Lanzhou University.RESULTS AND CONCLUSION:(1)Compared with the sham operation group,the Basso-Beattie-Bresnahan score in the spinal cord injury group was significantly decreased(P<0.01),and a large number of inflammatory cells infiltrated into the spinal cord tissues with multiple cavities.Compared with the spinal cord injury group,the hind limb function in the electrical stimulation and electrical stimulation+neurotrophin groups began to recover gradually.Basso-Beattie-Bresnahan score in the electrical stimulation+neurotrophin group was significantly higher than that in the electrical stimulation group(P<0.05).The above pathological changes were significantly improved.(2)No latency of motor evoked potentials in both hind limbs was detected in the spinal cord injury group at 7,14 days and in the electrical stimulation group at 7 days,respectively.At 21 and 28 days,the latency of motor evoked potential was shorter in the electrical stimulation and electrical stimulation+neurotrophin groups than in the spinal cord injury group(P<0.05);and the latency of motor evoked potential in the electrical stimulation+neurotrophin group was shorter than that in the electrical stimulation group(P<0.05).(3)The number of BrdU and Nestin positive cells and the expression of microtubule-associated protein 2 were ranked as follows:electrical stimulation+neurotrophin group>electrical stimulation group>spinal cord injury group.The expression level of glial fibrillary acidic protein was highest in the spinal cord injury group,followed by electrical stimulation group,and lowest in the electrical stimulation+neurotrophin group.These results show that after electrical stimulation plus neurotrophin 3 intervention,endogenous neural stem cells can proliferate and differentiate into neurons.Pathological damage is significantly alleviated and motor function of hind limbs is significantly improved.