中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (12): 2173-2182.doi: 10.4103/1673-5374.262601

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

基于定制解剖学的神经导管修复兔坐骨神经缺损：体外和体内实验评价

  

  • 出版日期:2019-12-15 发布日期:2019-12-15
  • 基金资助:

    国家自然科学基金(81360270，81560357，31670986）的支持; 中国卫生部手部重建重点实验室; 中国周边神经与显微外科上海市重点实验室（17DZ2270500）；中国广东省科技计划项目（2014B020227001，2017A050501017）

Application of custom anatomy-based nerve conduits on rabbit sciatic nerve defects: in vitro and in vivo evaluations

Yamuhanmode·Alike 1 , Maimaiaili·Yushan 1 , Ajimu·Keremu 2 , Alimujiang·Abulaiti 1 , Zhen-Hui Liu 1 , Wei Fu 1 , Li-Wei Yan 3, 4, 5 , Aihemaitijiang·Yusufu 1 , Qing-Tang Zhu 3, 4, 5   

  1. 1 Department of Microrepair and Reconstruction, the First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang Uygur Autonomous Region, China
    2 Orthopedic Center, the First People’s Hospital of Kashgar, Kashi, Xinjiang Uygur Autonomous Region, China
    3 Department of Microsurgery and Orthopedic Trauma, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
    4 Center for Peripheral Nerve Tissue Engineering and Technology Research, Guangzhou, Guangdong Province, China
    5 Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, Guangdong Province, China
  • Online:2019-12-15 Published:2019-12-15
  • Contact: Aihemaitijiang·Yusufu, MD, PhD, AYusufu@foxmail.com; Qing-Tang Zhu, MD, PhD, zhuqingt@mail.sysu.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81360270, 81560357 (both to AY), and 31670986 (to QTZ); the Key Laboratory of Hand Reconstruction, Ministry of Health, China; the Shanghai Key Laboratory of Peripheral Nerve and Microsurgery of China, No. 17DZ2270500 (to AY); the Science and Technology Project of Guangdong Province of China, No. 2014B020227001, 2017A050501017 (both to QTZ).

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摘要：

神经移植物中不同来源的神经纤维彼此间交叉缠绕是再生神经纤维错配的主要原因，这种错配的神经纤维是影响神经损伤后肢体功能恢复效果的关键因素之一。课题组前期的研究结果表明，通过改善移植物仿生性能可提高再生神经纤维配对准确性。为此，本次实验设计采用碘液及冷冻干燥法在高分辨Micro-CT下观察了兔坐骨神经中段(25 mm)3D拓扑结构，并根据上述结果设计制备了一系列1-，2-，3-及4-通道仿生神经导管(CANC)。在CANC的匹配指数、管腔表面积及机械性能评估后，显露并完全切取新西兰大白兔中段坐骨神经(缺损长度10 mm)，采用自体神经移植及CANC移植修复上述神经缺损模型。在术后12，24周，采用组织学切片、神经电生理检测、腓肠肌湿质量、行为学观测检测方法评估神经恢复情况。为了解神经纤维再生的准确性，采用荧光逆行实验方法检测荧光标记脊髓前角运动神经元数目变化。结果发现：(1)坐骨神经在高分辨Micro-CT显影的最佳条件是经过40%卢戈氏液染色3 d后冻干2 d；(2) 体外实验显示, 3-神经导管组匹配指数最高，随后依次为2-，1-及4- CANC组；1-CANC组管腔表面积最低； 3-、4-CANC组机械性能(横向压缩力，抗弯强度)均优于1-CANC组；(3)体内实验显示，3-CANC组在神经功能恢复方面(再生神经纤维形态、最大复合动作电位波幅、腓肠肌湿质量百分比、自噬严重等级、踝关节指数)均优于其他3个神经导管组，并达到了与自体坐骨神经移植相近的治疗效果；(4)神经再生准确性检测结果提示，2-，3-及4- CANC组快蓝-核黄(FB-NY-)标记神经元百分比均低于1-CANC组，说明新生神经束交错较少；(5)上述数据说明，采用碘液及冷冻干燥法Micro-CT观察兔坐骨神经缺损是一套切实可行的研究方法，以此为依据设计的CANC导管可用于修复兔坐骨神经缺损。相比1-CANC，3-CANC的匹配指数及管腔表面积更高，其通过减少新生神经束交错可提高神经再生的准确性。所有实验用动物饲养及实验操作规范经新疆医科大学第一附属医院伦理委员会2017-04-04审批(伦理审批号：IACUC20170315-02)。

orcid: 0000-0002-7200-5113 (Yamuhanmode·Alike)
          0000-0003-4632-1216 (Maimaiaili·Yushan)
          0000-0002-7735-9762 (Alimujiang·Abulaiti)
          0000-0003-3798-2303 (Aihemaitijiang·Yusufu)
          0000-0001-6256-1602 (Qing-Tang Zhu)

关键词: 通道仿生神经导管, 错配, 碘液及冷冻干燥法, 高分辨, 显微断层扫描, 仿生, 定制化, 兔坐骨神经, 体外实验, 体内实验, 神经再生

Abstract:

The intermingling of regenerated nerve fibers inside nerve grafts is the main reason for mismatched nerve fibers. This is one of the key factors affecting limb function recovery after nerve injury. Previous research has shown that the accuracy of axon regeneration can be improved by a bionic structural implant. To this aim, iodine and freeze-drying high-resolution micro-computed tomography was performed to visualize the 3D topography of the New Zealand rabbit sciatic nerve (25 mm). A series of 1-, 2-, 3-, and 4-custom anatomy-based nerve conduits (CANCs) were fabricated based on the anatomical structure of the nerve fascicle. The match index, luminal surface, and mechanical properties of CANCs were evaluated before implanting in a 10-mm gap of the sciatic nerve. Recovery was evaluated by histomorphometric analyses, electrophysiological study, gastrocnemius muscle weight recovery ratio, and behavioral assessments at 12 and 24 weeks postoperatively. The accuracy of nerve regeneration was determined by changes in fluorescence-labeled profile number during simultaneous retrograde tracing. Our results showed that the optimal preprocessing condition for high-resolution micro-computed tomography visualization was treatment of the sciatic nerve with 40% Lugol’s solution for 3 days followed by lyophilization for 2 days. In vitro experiments demonstrated that the match index was highest in the 3-CANC group, followed by the 2-, 1-, and 4-CANC groups. The luminal surface was lowest in the 1-CANC group. Mechanical properties (transverse compressive and bending properties) were higher in the 3-and 4-CANC groups than in the 1-CANC group. In vivo experiments demonstrated that the recovery (morphology of regenerated fibers, compound muscle action potential, gastrocnemius muscle weight recovery ratio, pain-related autotomy behaviors, and range of motion) in the 3-CANC group was superior to the other CANC groups, and achieved the same therapeutic effect as the autograft. The simultaneous retrograde tracing results showed that the percentages of double-labeled profiles of the 2-, 3-, and 4-CANC groups were comparatively lower than that of the 1-CANC group, which indicates that regenerated nerve fascicles were less intermingled in the 2-, 3-, and 4-CANC groups. These findings demonstrate that the visualization of the rabbit sciatic nerve can be achieved by iodine and freeze-drying high-resolution micro-computed tomography, and that this method can be used to design CANCs with different channels that are based on the anatomical structure of the nerve. Compared with the 1-CANC, 3-CANC had a higher match index and luminal surface, and improved the accuracy of nerve regeneration by limiting the intermingling of the regenerated fascicles. All procedures were approved by the Animal Care and Use Committee, Xinjiang Medical University, China on April 4, 2017 (ethics approval No. IACUC20170315-02).

Key words: nerve regeneration, nerve conduits, mismatch, iodine and freeze-drying, high-resolution, micro-computed tomography, bio-mimic, custom, rabbit sciatic nerve, in vitro, in vivo, neural regeneration