METHODS: A mouse model of transection SCI was prepared by forceps clamping, and an adeno-associated virus (AAV) carrying the transcription factors NeuroD1 and Neurogenin-2(Ngn2) was injected in situ into the spinal cord to specifically overexpress these transcription factors in astrocytes close to the injury site. 5-bromo-2´-deoxyuridine (BrdU) was subsequently injected intraperitoneally to continuously track cell regeneration, neuroblasts and immature neurons marker expression, neuronal regeneration, and glial scar regeneration. In addition, immunoprotein blotting was used to measure the levels of transforming growth factor-β (TGF-β) pathway-related protein expression. We also evaluated motor function, sensory function, and the integrity of the blood-spinal cord barrier(BSCB).
RESULTS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord was achieved by specific AAV vectors. This intervention led to a significant increase in cell regeneration and the proportion of cells with neuroblasts and immature neurons cell properties at the injury site(p < 0.0001). Immunofluorescence staining identified astrocytes with neuroblasts and immature neurons cell properties at the site of injury while neuronal marker-specific staining revealed an increased number of mature astrocytes at the injury site. Behavioral assessments showed that the intervention did not improve The BMS (Basso mouse scale) score (p = 0.0726) and gait (p > 0.05), although the treated mice had more sensory sensitivity and greater voluntary motor ability in open field than the non-intervention mice. We observed significant repair of the BSCB at the center of the injury site (p < 0.0001) and a significant improvement in glial scar proliferation. Electrophysiological assessments revealed a significant improvement in spinal nerve conduction (p < 0.0001) while immunostaining revealed that the levels of TGF-β protein at the site of injury in the intervention group were lower than control group (p = 0.0034); in addition, P70 s6 and PP2A related to the TGF-β pathway showed ascending trend (p = 0.0036, p = 0.0152 respectively).
CONCLUSIONS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord after spinal cord injury can reprogram astrocytes into neurons and significantly enhance cell regeneration at the injury site. The reprogramming of astrocytes can lead to tissue repair, thus improving the reduced threshold and increasing voluntary movements. This strategy can also improve the integrity of the blood-spinal cord barrier and enhance nerve conduction function. However, the simple reprogramming of astrocytes cannot lead to significant improvements in the striding function of the lower limbs.
方法:采用钳夹法制备横切SCI小鼠模型,将携带转录因子NeuroD1和Neurogenin-2(Ngn2)的腺相关病毒(AAV)原位注射到脊髓中,以在损伤部位附近的星形胶质细胞中特异性地过表达这些转录因子。随后腹膜内注射5-溴-2'-脱氧尿苷(BrdU)以连续跟踪细胞再生,成神经细胞和未成熟神经元标记表达,神经元再生,和胶质瘢痕再生.此外,免疫蛋白印迹法检测转化生长因子-β(TGF-β)通路相关蛋白表达水平。我们还评估了运动功能,感觉功能,和血脊髓屏障(BSCB)的完整性。
结果:通过特异性AAV载体实现了NeuroD1和Ngn2在脊髓中的原位过表达。这种干预导致细胞再生和在损伤部位具有成神经细胞和未成熟神经元细胞特性的细胞比例显著增加(p<0.0001)。免疫荧光染色在损伤部位鉴定了具有成神经细胞和未成熟神经元细胞特性的星形胶质细胞,而神经元标记特异性染色显示损伤部位成熟星形胶质细胞数量增加。行为评估显示干预并未改善BMS(Basso小鼠量表)评分(p=0.0726)和步态(p>0.05),尽管与未干预的小鼠相比,接受治疗的小鼠在开放视野中具有更高的感觉敏感性和更大的自主运动能力。我们观察到BSCB在损伤部位中心的显著修复(p<0.0001)和神经胶质瘢痕增殖的显著改善。电生理评估显示脊神经传导显著改善(p<0.0001),而免疫染色显示干预组损伤部位TGF-β蛋白水平低于对照组(p=0.0034);与TGF-β通路相关的P70s6和PP2A呈上升趋势(p=0.0036,p=0.0152)。
结论:脊髓损伤后NeuroD1和Ngn2在脊髓中的原位过表达可以将星形胶质细胞重新编程为神经元,并显着增强损伤部位的细胞再生。星形胶质细胞的重编程可以导致组织修复,从而改善降低的门槛,增加自愿运动。该策略还可以改善血脊髓屏障的完整性并增强神经传导功能。然而,星形胶质细胞的简单重编程不能显著改善下肢的跨步功能.