Craniocerebral injury promotes sciatic nerve regeneration

doi:10.3969/j.issn.2095-4344.2016.27.016

Abstract

Abstract:

BACKGROUND: Studies have shown that craniocerebral injury can promote the repair of sciatic nerve injury in rats, but its precise mechanism remains unclear.
OBJECTIVE: To further explore the action mechanism of craniocerebral injury on the repair of sciatic nerve injury using morphology and histology.
METHODS: Sixty specific-pathogen-free healthy male Sprague-Dawley rats were randomly divided into two groups. Rats with craniocerebral injury and sciatic nerve injury were considered as the experimental group. Rats with simple sciatic nerve injury were considered as the control group. Classical Feeney method was used in models of craniocerebral injury and SunderlandV sciatic nerve injury. At 8 and 12 weeks after modeling, sciatic nerve index was detected. Masson staining and NF200 immunofluorescence staining were used to observe the nerve regeneration at the anstomotic site. Transmission electron microscope was used to observe the number of regenerative axons.
RESULTS AND CONCLUSION: At 8 and 12 weeks after modeling, compared with the control group, gait and sciatic nerve index recovered better in the experimental group. In the experimental group, Masson staining showed fewer nerve membrane collagen fibers, and the axon arranged neatly. NF200 immunohistochemistry showed that in the experimental group, the density of regenerated nerves was high, and nerves were regularly distributed. Transmission electron microscopy showed that in the experimental group, regenerative axons were regularly arranged, collagen scar was less, and myelin layer arranged regularly. Results suggested that the craniocerebral injury in rats may promote the repair of peripheral nerve injury by reducing scar collagen in nerve endings.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Brain Injuries, Peripheral Nerves, Nerve Regeneration, Cicatrix, Tissue Engineering

CLC Number:

R318

He Xin-ze, Wang Wei, Ma Jian-jun, Hu Tie-min, Yu Chang-yu, Gao Yun-feng, Cheng Xing-long, Wang Pei . Craniocerebral injury promotes sciatic nerve regeneration[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(27): 4061-4067.

Figures/Tables 2

2.1 实验动物数量分析实验选用大鼠60只，分为2组，实验过程无脱失，全部进入结果分析。 2.2 模型创新性/材料、方法的改进根据Feeney法[12] 20 g击锤沿外周导管(导管隔5 mm开窗)从30 cm高处自由坠落冲击撞杆，造成中度脑挫裂伤；在10倍手外科显微镜下完全切断坐骨神经，造成SunderlandV型坐骨神经损伤。 2.3 模型的稳定性脑损伤位置在冠状线后1.5 mm、中线偏右5 mm处，用20 g击锤沿外周导管从30 cm高处自由坠落的强度冲击撞杆，位置准确，打击强度相同；在10倍手外科显微镜下完全切断坐骨神经，应用9-0无损伤线采用外膜缝合法缝合坐骨神经，造成Sunderland V型坐骨神经损伤，损伤程度确切，神经吻合均有同一术者完成，模型稳定。 2.4 颅脑损伤对坐骨神经损伤大鼠大体状态的影响造模后所有动物均存活，未发现切口感染情况。造模后实验组大鼠活动减少、食欲差，1周后逐渐恢复。两组大鼠左下肢均主动屈曲活动受限，左足屈趾、足畸形，行走时足跟着地，未见自食现象；造模后4周内，两组大鼠左足出现红肿、顽固性溃疡。造模后6周，实验组大鼠左足部溃疡面收缩，开始好转；对照组大鼠左足部溃疡未见明显好转；8周时，实验组大鼠左足部溃疡瘢痕愈合，对照组大鼠左足部溃疡开始缩小。12周时，2组大鼠左足部溃疡均基本痊愈。取材前观察吻合口周围，缝线无脱落，未见明显神经瘤形成。 2.5 大鼠坐骨神经功能指数暗箱测试结果显示，造模后第4周，2组大鼠神经功能指数接近(P > 0.05)；从第4周实验组大鼠神经功能指数开始降低，随时间的延长降低的幅度减缓，第8，12周时，实验组大鼠神经功能指数均低于对照组(P < 0.01，表1)。 2.6 Masson三色法染色神经组织切片Masson胶原纤维特殊染色后，在光镜下胶原纤维染成绿色，神经纤维呈红色，细胞核呈蓝黑色。在正常神经组织中，胶原纤维均匀分布在神经外膜、束膜、内膜，并呈波浪形随神经纤维排列。周围神经损伤后，胶原纤维在吻合口处明显增生。造模后8周，镜下可见对照组在吻合口处胶原纤维增生明显多于实验组。神经纤维稀少，且排列紊乱，神经纤维走行失去波浪状结构(图1)。造模后12周实验组可见神将纤维增生明显，胶原纤维较少，对照组胶原纤维增生明显多于实验组，神将纤维增生较实验组少，且被大量胶原纤维阻隔；造模后12周实验组神经纤维与胶原纤维均匀再生，胶原纤维随神经纤维呈波浪状走形，对照组神经纤维与胶原纤维增生欠均匀，走形欠规整。 2.7 轴突再生相关神经丝蛋白200(NF200)表达免疫组织化学NF200阳性标准为神经节细胞、神经纤维细胞胞质内见金黄色颗粒。坐骨神经冰冻切片免疫荧光观察 (图2)。结果显示，实验组与对照组造模后8，12周各组标本中均见NF200阳性颗粒。实验组术后8周NF200阳性颗粒数目明显多于对照组、再生神经纤维排列规则；造模后12周，NF200阳性颗粒数目明显较对照组减少，阳性颗粒密度均匀，神经再生纤维排列整齐、规律。实验组在再生神经的密度、排列规整程度等方面均要优于对照组，表明实验组损伤的神经得到了更快、更好的修复和再生，神经纤维再生的质量较高。 2.8 透射电镜观察周围神经损伤后，神经功能的恢复涉及神经元胞体、轴突及末梢效应器，轴突的再生能直接反映损伤处神经的修复程度，因此本研究直接计数再生轴突数目，评价神经纤维再生的情况(表2)。颅脑损伤后轴突再生数目明显多于对照组。再生神经超微结构正常神经以有髓纤维为主，髓鞘及轴突多近似圆形；髓鞘厚度均匀，板层清晰；轴突充满整个髓鞘，微丝、微管丰富，线粒体无水肿；许旺细胞形态饱满，核清晰，细胞器丰富。造模后8，12周两组均有不同数量的有髓神经纤维再生，造模后12周有髓神经纤维数量较8周明显增多，结构更清晰，层次更分明，神经纤维排列整齐，髓鞘板层厚。造模后8周实验组神经纤维排列整齐，有髓神经纤维界限较清楚，细胞器形态正常，新生髓鞘板层均匀、整齐，组织间胶原纤维较少；对照组神经轴索杂乱排列，新生髓鞘板层薄，有髓鞘变形及脱髓鞘表现，胶原纤维含量较多。造模后12周实验组较对照组新生髓板板层排列均匀，组织间胶原纤维含量少，造模后8，12周实验组再生的有髓纤维数量、结构层次清晰度、髓鞘排列情况以及细胞器恢复情况均好于对照组，组织间胶原纤维含量少于对照组(图3)。"

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