可视化与钙西通宁基因相关的肽免疫活性内活性与免疫发光和神经追踪
Summary
在这里，我们提出了一个协议，可视化钙素基因相关肽（CGRP）免疫活性神经纤维和血管的空间相关性，分别使用CGRP和phalloidin的免疫荧光和荧光组织化学。此外，这些神经纤维的起源是逆行跟踪荧光神经示踪剂。
Abstract
本研究的目的是利用免疫荧光、三维（3D）重建和逆行追踪技术，研究钙化物基因相关肽（CGRP）免疫感官神经纤维的分布和起源。在这里，神经纤维和血管分别使用CGRP和荧光法洛丁的免疫荧光和组织化学技术进行染色。3D重建证明了杜拉尔CGRP-免疫神经纤维和血管的空间相关性。同时，通过神经追踪技术从颅内杜拉母体中脑膜动脉（MMA）周围区域到三叉神经结节（TG）和颈椎（C）多头根神经痛（DRG）的神经追踪技术检测出CGRP免疫活性神经纤维的起源。此外，还使用双免疫荧光与CGRP一起检查了TG和DPG中FG标记神经元的化学特性。利用透明的全安装样本和3D重建，结果表明，CGRP免疫活性神经纤维和巴洛丁标记动脉一起运行或单独形成一个神经血管网络在3D视图， 而FG标记的神经元被发现在TG的眼科、最大侧和支线分支，以及C2-3 DRG ipsilateral到示踪器应用的一侧，其中一些FG标记的神经元呈现CGRP免疫活性表达。通过这些方法，我们演示了颅内杜拉母体血管周围CGRP免疫活性神经纤维的分布特征，以及TG和DPG这些神经纤维的来源。从方法论的角度看，它为理解颅骨在生理或病理条件下复杂的神经血管结构提供了宝贵的参考。
Introduction
颅骨母体是保护大脑的最外层，含有丰富的血管和不同种类的神经纤维1，2。许多研究表明，敏感颅杜拉母体可能是导致头痛发生的关键因素，涉及异常血管扩张和内向3，4，5。因此，颅内神经血管结构知识对于理解头痛的发病机理，特别是偏头痛的发病机理非常重要。
虽然杜拉内在研究以前与传统的免疫化学，神经纤维和血管的空间相关性在颅杜拉母体较少研究6，7，8，9。为了更详细地揭示杜拉神经血管结构，钙素基因相关肽（CGRP）和巴洛丁被选为分别用免疫荧光和荧光组织化学10染色全山颅骨神经纤维和血管的标记物。这可能是获得神经血管结构的三维（3D）视图的最佳选择。此外，氟金（FG）被应用于颅内杜拉母体中脑膜动脉（MMA）周围区域，以确定CGRP免疫活性神经纤维的来源， 并追溯到三角结节 （TG） 和颈椎 （C） 多面根神经质 （DRGs）， 而 FG 标记的神经元使用免疫荧光与 CGRP 一起进一步检查。
这项研究的目的是为研究颅内杜拉母体的神经血管结构提供一个有效的工具，用于CGRP免疫活性内分和其起源。通过利用透明的全山杜拉母校，结合免疫荧光、逆行追踪、共焦技术和3D重建，我们期望呈现颅杜拉母校神经血管结构的新3D视图。这些方法方法可以进一步用于探索不同头痛的发病机理。
Protocol
本研究经中国医学科学院针灸与莫西布西翁研究所伦理委员会批准（参考号D2018-09-29-1）。所有程序均按照《国家实验室动物护理和使用卫生指南》（1996年.C，华盛顿特区国家学院出版社）执行。这项研究使用了12只成年斯普拉格-道利雄性大鼠（体重220±20克）。动物[许可证号SCXK（JING） 2017-0005]由国家食品和药物管制研究所提供。 1. 大鼠颅杜拉母体的内向 香水</…
Representative Results
颅体母体的神经血管结构在免疫荧光和荧光组织染色与CGRP和phalloidin后，CGRP免疫活性神经纤维和法洛丁标记的杜拉尔动脉和结缔组织在3D模式（图2C，D，E，F）的全山颅骨母体中清晰地表现出来。结果表明，厚和薄的CGRP免疫活性神经纤维与神经动脉平行运行，在血管壁周围，或血管之间（图2D，E，F?…
Discussion
在这项研究中，我们成功地演示了CGRP免疫活性神经纤维在颅尿母体中的分布和起源，使用CGRP抗体和FG神经示踪剂的免疫荧光、3D重建和神经追踪方法，为更好地了解神经血管网络提供了组织学和化学证据。
众所周知，CGRP在偏头痛4、17的发病机因中起着至关重要的作用。结果表明，增加CGRP可导致血管扩张和神经原性炎症，导致沿三?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由中国国家重点研发项目（项目代码2019YFC1709103;2018YFC1707804号）和中国国家自然科学基金（项目代码81774211;第81774432号项目代码，第81801561号）支持。
Materials
| Alexa Fluor 488 donkey anti-mouse IgG (H+L) | Invitrogen by Thermo Fisher Scientific | A21202 | Protect from light; RRID: AB_141607 |
| Brain stereotaxis instrument | Narishige | SR-50 | |
| CellSens Dimension | Olympus | Version 1.1 | Software of fluorescent microscope |
| Confocal imaging system | Olympus | FV1200 | |
| Fluorogold (FG) | Fluorochrome | 52-9400 | Protect from light |
| Fluorescent imaging system | Olympus | BX53 | |
| Freezing microtome | Thermo | Microm International GmbH | |
| Olympus FV10-ASW 4.2a | Olympus | Version 4.2 | Confocal image processing software system |
| Micro Drill | Saeyang Microtech | Marathon-N7 | |
| Mouse anti-CGRP | Abcam | ab81887 | RRID: AB_1658411 |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | |
| Phalloidin 568 | Molecular Probes | A12380 | Protect from light |
| Photoshop and Illustration | Adobe | CS6 | Photo editing software |
| Rabbit anti- Fluorogold | Abcam | ab153 | RRID: AB_90738 |
| Sprague Dawley | National Institutes for Food and Drug Control | SCXK (JING) 2014-0013 | |
| Superfrost plus microscope slides | Thermo | #4951PLUS-001 | 25x75x1mm |
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Cite This Article
Wang, J., Xu, D., Cui, J., She, C., Wang, H., Wu, S., Zou, L., Zhang, J., Bai, W. Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing. J. Vis. Exp. (167), e61742, doi:10.3791/61742 (2021).