中国修复重建外科杂志

中国修复重建外科杂志

胶原支架结合脑源性神经营养因子移植促进全横断脊髓损伤大鼠轴突再生及运动功能恢复的研究

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目的评价胶原支架结合脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)移植修复大鼠全横断脊髓损伤的效果。方法将 32 只成年雌性 SD 大鼠随机分成 4 组(n=8):A 组为假手术组,只暴露 T9、T10 段脊髓;B、C、D 组切除长 4 mm 的 T9、T10 段脊髓后,C、D 组分别于损伤处植入相应长度的线性有序胶原支架(linear ordered collagen scaffolds,LOCS)和结合了胶原结合结构域(collagen binding domain,CBD)-BDNF 的 LOCS。术前及术后 3 个月内每周对大鼠进行 BBB 运动功能评分。术后 3 个月,实施神经电生理检测各组大鼠运动诱发电位(motor evoked potential,MEP);然后于 L2 段脊髓组织注射荧光金(fluorogold,FG)实施逆行示踪,1 周后取大鼠大脑及胸、腰段脊髓组织,脱水后观察脊髓组织形态;取包含损伤区的胸、腰段脊髓组织作切片。其中,脊髓冠状切片于激光共聚焦显微镜下进行观察,计算 FG 阳性细胞积分吸光度(IA)值;胸段脊髓组织水平切片采用免疫荧光染色,观察全横断脊髓损伤造模情况、脊髓损伤区轴突再生情况、D 组再生轴突的突触形成情况。结果术后各时间点 B、C、D 组 BBB 评分均显著低于 A 组(P<0.05);术后 2~12 周 D 组 BBB 评分均明显高于 B、C 组(P<0.05)。电生理检测示,B 组未观测到 MEP;C、D 组 MEP 潜伏期显著长于 A 组,C 组显著长于 D 组,差异均有统计学意义(P<0.05)。脊髓组织形态观察示,B 组脊髓损伤区域向两端延伸,损伤部位组织破坏严重;C、D 组脊髓形态恢复较好,D 组更接近正常组织形态。逆行示踪结果显示,各组大鼠损伤区以下的腰段脊髓灰质中均充满了 FG 阳性细胞;在损伤区以上的胸段脊髓中,A 组 FG 阳性区域IA 值显著大于 B、C、D 组(P<0.05),C、D 组大于 B 组(P<0.05),C、D 组间差异无统计学意义(P>0.05)。免疫荧光染色示,自同一脊髓背侧至腹侧选出的组织切片显示了明显异于正常组织的全横断脊髓损伤区域。A 组 NF 阳性轴突数明显多于 B、C、D 组,C、D 组多于 B 组,D 组多于 C 组,差异均有统计学意义(P<0.05)。结论LOCS 结合 CBD-BDNF 移植可以促进大鼠全横断脊髓损伤后轴突再生以及后肢运动功能恢复。

ObjectiveTo evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor (BDNF) on the repair of transected spinal cord injury in rats.MethodsThirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A (sham operation group), T9, T10 segments of the spinal cord was only exposed; group B, 4-mm T9, T10 segments of the spinal cord were resected; group C, 4-mm T9, T10 segments of the spinal cord were resected and linear ordered collagen scaffolds (LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T9, T10 segments of the spinal cord were resected and LOCS with collagen binding domain (CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan (BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential (MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold (FG) at the L2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance (IA) value of FG-positive cells. Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.ResultsDuring 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A (P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C (P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A (P<0.05), and in group C than in group D (P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends, and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, theIA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05).ConclusionThe combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.

关键词: 胶原支架; 胶原结合结构域; 脑源性神经营养因子; 脊髓损伤; 运动功能; 大鼠

Key words: Collagen scaffold; collagen binding domain; brain-derived neurotrophic factor; spinal cord injury; motor function; rat

引用本文: 陈曦, 范永恒, 肖志峰, 李星, 杨斌, 赵燕南, 侯祥林, 韩素芳, 戴建武. 胶原支架结合脑源性神经营养因子移植促进全横断脊髓损伤大鼠轴突再生及运动功能恢复的研究. 中国修复重建外科杂志, 2018, 32(6): 650-659. doi: 10.7507/1002-1892.201803094 复制

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