神经营养因子对成年大鼠脊髓损伤后皮质脊髓束再生的影响

背景胶质细胞源性神经营养因子(GDNF)对脊髓运动神经元的存活有明显的保护作用,但GDNF对成年大鼠脊髓损伤后皮质脊髓束再生是否有促进作用仍不清楚.目的研究胶质细胞源性神经营养因子对脊髓损伤后皮质脊髓束再生的保护作用.设计随机对照实验.地点和材料本实验在第二军医大学神经生物实验室完成.实验动物采用雄性成年SD大鼠64只,体质量250～300g.干预采用Nystrom法制备大鼠胸髓后路压迫损伤模型,压迫质量为50 g,时间为5min,造成大鼠胸段脊髓急性重度损伤.脊髓损伤后24 h,对照组注射6μL阳离子脂质体DC-Chol和2μgpCDNA3的混合物,实验组将6μL DC-Chol和2μg重组质粒pEGFP-GDNF cDNA混合后注入大鼠损伤脊髓.主要观察指标应用RT-PCR技术和荧光显微镜检测GDNF体内转基因后在局部的表达,通过辣根过氧化物酶(HRP)顺行追踪技术和神经微丝(NF)免疫组化活性的变化来评价GDNF基因体内转染对大鼠皮质脊髓束再生的影响.结果GDNF基因体内转染1周后发现在基因注射局部有转录和蛋白水平表达,4周后在脊髓损伤周围仍检测到GDNF蛋白表达.脊髓损伤后4周,实验组脊髓损伤区皮质脊髓束HRP标记明显多于对照组,仅在实验组可见部分神经纤维穿过损伤区至远端5～9mm.损伤区NF阳性轴突数(524.33±80.55/低倍视野)较对照组(309.84±56.65/低倍视野)明显增多(P＜0.01),GFAP阳性细胞数(186.68±16.25)明显少于对照组(239.78±21.44)(P＜0.01).结论阳离子脂质体介导GDNF体内转基因能促进近端皮质脊髓束再生和神经骨架蛋白的修复,提示神经营养因子基因治疗可用来治疗创伤性脊髓损伤.

Effects of neurotrophic factor on cortical spinal tract regeneration after spinal cord injury in adult rats

BACKGROUND: Glial cell line-derived neurotrophic factor(GDNF) has been shown to protect spinal motoneurons, however, it is not clear whether GDNF can promote axonal regeneration after spinal cord injury(SCI) in adult rats.OBJECTIVE: To study the protective effect of GDNF on the regeneration of corticospinal tract in spinal cord injury adult rats.DESIGN: A randomized controlled study was conducted.SETTING and MATERIALS: The experiment was completed in the Laboratory of Neurobiology, Second Military Medical University. Sixty-four male Sprague-Dawley rats with body mass of 250-300 g were used in this study.INTERVENTIONS: The model of acute rats spinal cord compression injury was established according to the method of Nystrom. The compressive load is 50 g for 5 minutes, which caused severe thoracic SCI. 24 hours after injury,2 μg pCDNA3(4 μL) mixed with 6 μL DC-Chol(Control group, n = 32) or 2μg pEGFP-GDNF(4 μL) mixed with 6 μL DC-Chol(GDNF group, n = 32)were injected stereotactically into the injured spinal cord.MAIN OUTCOME MEASURES: The presence of expression of GDNF mRNA and GDNF protein were observed using RT-PCR and fluorescence observation. Using the technique of HRP anterograde tracing and the immunohistochemical reactivity of NF and GFAP in the injured spinal cord, we observed the effect of in vivo GDNF gene transfer on corticospianl tract regeneration.RESULTS: RT-PCR confirmed the increased expression of GDNF mRNA in the injected areas at 7 days after injection. The expression of EGFP-GDNF was observed in the cells around the injection locus by fluorescence microscope at least 4 weeks after injection. Four weeks after GDNF gene transfer,there are more HRP labeling of corticospinal tract axons across the lesion in GDNF group compared with control group. In GDNF group, the maximum distance these labeled axons extended varied in different animals and ranged from 5 mm to about 9 nm from the lesion. The number of NF-positive axons in GDNF group(524. 33 ± 80. 55/low power field) was more than that in the control group(309. 84 ± 56. 65/low power field) ( P ＜ 0. 01) . In GDNF group the number of labeling astrocytes( 186.68 + 16.25) was less than that in the controlgroup(239.78 + 21.44) ( P ＜ 0. 01 ).CONCLUSION: These data demonstrate that in vivo transfer of GDNF cDNA could promote axonal regeneration and facilitate the repair of the cytoskeleton in the injured neurons, suggesting that cationic liposome-mediated delivery of GDNF cDNA may be a practical gene transfer method for traumaric SCI treatment.