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Abstract
Introduction
Protocol
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Neuroscience

乙醇诱导的颈椎交交型神经节阻滞在促进犬下肺泡神经再生中的应用

Published: November 30, 2018
doi:
10.3791/58039
, , , ,
1Department of Dental Anesthesia,Nippon Dental University Hospital at Tokyo, 2Department of Dental Anesthesiology,Nippon Dental University School of Life Dentistry at Tokyo, 3Department of Bioartificial Organs, Institute for Frontier Medical Science,Kyoto University

Summary

我们评价了颈椎交感神经节阻滞对人工神经导管修复神经的影响。雄性猎狗分别在左下牙槽神经的10毫米缝隙上植入人工神经;左颈椎交感神经节通过侧开手术注射99.5% 乙醇, 阻断了颈椎交感神经节。

Abstract

聚甘酸胶原蛋白 (pga-c) 管是一种生物吸收神经管, 充满了多室结构的胶原蛋白, 由薄胶原蛋白膜组成。使用这些试管治疗受损的下牙槽神经 (ian) 时, 取得了良好的临床效果。使用 pga-c 管成功神经再生的一个关键因素是周围组织的血液供应。颈椎交感神经节阻滞 (csgb) 在头部和颈部造成交感神经阻滞, 从而增加该地区的血液流动。为确保充分发挥作用, 必须连续几周使用局部麻醉剂每天1至 2次;这在创建用于研究这一技术的动物模型时带来了挑战。为了解决这一限制, 我们在口腔面部区域长期增加血液流动的犬类模型中开发了乙醇诱导的 csgb。我们研究了通过 pga-c 管植入物进行的 ian 再生是否可以通过该模型得到提高。14个比格尔斯分别在左 ian 的10毫米缝隙上植入了一个 pga-c 管。ian 位于下颌骨管内, 周围是骨头, 因此我们选择了压电手术, 包括超声波, 进行骨处理, 以尽量减少神经和血管损伤的风险。这种方法获得了良好的手术效果。手术一周后, 其中7只狗通过注射乙醇离开了 csgb。乙醇诱导的 csgb 改善了神经再生, 提示血流的增加有效地促进了 ian 缺陷中的神经再生。这种犬类模型可以为进一步研究 csgb 的长期效果做出贡献。

Introduction

在许多情况下, 下牙槽神经 (ian) 的创伤是医源性的, 经常是由拔除第三磨牙或放置牙种植体 1,2,3引起的。ian 的损伤会导致热和触觉的缺陷, 以及感觉异常、麻醉、麻醉和异位。神经损伤不仅通过保守治疗治疗, 还通过其他方法治疗, 包括缝合和自体移植。然而, 这些方法有缺点, 其中往往包括缺乏症状改善和神经缺陷的捐献者部位 4,5,6。

人工神经-多糖酸胶原蛋白 (pga-c) 管最初是在日本发展起来的。它是一个生物吸收管, 其内腔充满海绵状胶原蛋白7。在动物实验中, 该管用于增强有腹膜神经缺损的猎狗的神经再生, 并被证明比自体神经移植促进了更高水平的恢复 8.pga-c 管在周围神经损伤患者中的临床应用始于2002年。此外, 在治疗三叉神经病变 (ian 和舌神经)9,10,11方面也取得了良好的临床效果。使用 pga-c 管成功神经再生的一个关键因素是周围组织的血液供应 8。颈椎交感神经节阻滞 (csgb) 在头部和颈部区域造成交感神经阻滞, 并增加血液流向各自神经支配区域12;因此, 它已被用于治疗复杂的区域疼痛综合征和循环系统功能不全 13,14,15。然而, 只有少数实验研究 csgb 在增加血液流动方面的有效性为16,17。为了确保足够的 csgb 效率, 封锁必须与局部麻醉剂一起应用, 每天一次或两次, 为期数周, 从而在生成动物模型以研究这一技术时构成挑战。为了解决这一限制, 在之前的一项研究中, 我们开发了一个犬类模型, 长期增加了口腔面部区域的血液流动。该模型是通过注入99.5% 乙醇来执行 csgb 生成的。我们用激光多普勒血流测量和红外热像仪每周评估口腔黏膜血流和鼻皮肤温度, 为期12周。我们发现, 在这个模型中, 口腔面部区域的血液流动增加了7-10周。

在本研究中, 我们评估了乙醇诱导的 csgb 对神经再生的影响。
pga-c 管被植入左边的 ian 有10毫米缝隙的猎狗体内。一周后, csgb 通过注射乙醇进行。手术三个月后, 我们进行了各种电生理、组织学和形态学研究, 以评估 csgb 对神经再生的影响。我们提供了一个详细的协议, 用于利用 pga-c 管和乙醇诱导的 csgb 重建。

Protocol

这项研究是根据《动物护理和使用指导原则》进行的, 并得到京都大学动物研究委员会的批准 (日本京都; 授权号: r-16-16)。为尽量减少动物痛苦, 我们尽了一切努力, 本报告的所有章节都遵守《动物研究:活体实验报告》的准则。 1. pga-c 管的制造 要通过可吸收的聚乙酸 (pga) 管制造人工神经导管, 请使用装有48个主轴和 5个 pga 纤维的管状编织机, 该编织机由26个细丝?…

Representative Results

我们观察到, 在左 csgb 后 1周, 被阻塞一侧的面部皮肤温度增加 (图 8)。 重建后 3个月, 重建区的 pga-c 管被吸收, 下肺泡神经再生在重建和重建 + csgb 群 (图 9a, b)18中观察到。 snap 在重建 + csgb 和仅重建组的重?…

Discussion

我们提出了一种有效的方法, 以共同作用, 生物可吸收的神经管结合乙醇诱导的 csgb, 进行 ian 再生。在这项研究中, 我们使用了狗, 因为其他动物模型, 如老鼠、老鼠和兔子, 预期寿命短, 身体体积小, 因此不能用来进行精确的外科手术。由于 ian 位于下颌骨周围的下颌骨管内, 因此在进行神经重建时, 需要一种手术技术来避免神经和血管损伤。这个程序的一个重要的技术提示是小心地取出下颌骨骨板, ?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了京都大学前沿医学研究所生物人工器官系的支持。我们要感谢前沿医学研究所的兽医工作人员。

Materials

NMP Collagen PS Nippon Meatpackers 301-84621 Atelocollagen extracted from young porcine skin by enzyme treatment
Surgical clippers Roboz Surgical Instrument Company RC-5903
Disposable scalpel (No.15) Kai medical  219ABBZX00073000
VarioSurg3 Nakanishi VS3-LED-HPSC, E1133 Piezoelectric surgery for bone processing
4-0 nylon sutures Ethicon 8881H
8-0 nylon sutures Ethicon 2775G
Isepamicin sulfate Nichi-Iko 620005641
Disposable scalpel (No.10) Kai medical  219ABBZX00073000
30-gauge needle  Nipro 1134
1-0 absorbable stitches Ethicon J347H
3-0 Nylon stitches Ethicon 8872H
Neo Thermo NEC Avio TVS-700 Infrared thermography 
Neuropack Σ  NIHON KOHDEN MEB-5504 Orthodromic recorder for electrophysiological recording
Toluidine Blue  Sigma-Aldrich  T3260-5G
Light microscope Keyence BZ-9000
Mouse anti-human neurofilament protein monoclonal antibody DAKO N1591
Polyclonal rabbit anti-S100 antibody DAKO Z0311
Transmission electron microscopy Hitachi High Technologies Hitachi H-7000
Dynamic cell count Keyence BZ-H1C Software for morphological evaluation
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Tags

EthanolCervical Sympathetic Ganglion BlockCanine Inferior Alveolar NerveNerve RegenerationArtificial Nerve ConduitPiezoelectric SurgeryOrofacial NeuropathyInferior Alveolar Nerve ReconstructionNerve And Blood Vessel DamageNerve TransectionNerve TubeNerve SuturingComputed Tomography ImagingLocal AnesthesiaAnalgesiaSurgical Procedure
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Shionoya, Y., Sunada, K., Tsujimoto, G., Shigeno, K., Nakamura, T. Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve. J. Vis. Exp. (141), e58039, doi:10.3791/58039 (2018).
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