This study was supported by the project of National Key R&#38;D Program of China (Project Code no. 2019YFC1709103; no. 2018YFC1707804) and National Natural Science Foundation of China (Project Code no. 81774211; no. 81774432; no. 81801561).

This study was approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (reference number D2018-09-29-1). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Twelve adult Sprague-Dawley male rats (weight 220 &#177; 20 g) were used in this study. Animals [license number SCXK (JING) 2017-0005] were provided by the National Instit...

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In this study, we have successfully demonstrated the distribution and the origin of CGRP-immunoreactive nerve fibers in the cranial dura mater using immunofluorescence, 3D reconstruction and neural tracing approaches with CGRP antibody and FG neural tracer, providing the histological and chemical evidences to better understand the dural neurovascular network.
As it was known, CGRP plays a critical role in the pathogenesis of migraine4,17</su...

The cranial dura mater is the outermost layer of meninges to protect the brain and contains plentiful blood vessels and different kinds of nerve fibers1,2. Many studies have shown that sensitized cranial dura mater may be the key factor leading to the occurrence of headaches, involving the abnormal vasodilation and innervation3,4,5. Thus, the knowledge of neurovascular structure in the cranial dura mater is important for understanding the pathogenesis of headaches, especially for migraine.
Although the dura innervation has been previously studied with the conventional immunohistochemistry, the spatial correlation of nerve fibers and blood vessels in the cranial dura mater were less studied6,7,8,9. In order to reveal the dural neurovascular structure in more detail, calcitonin gene-related peptide (CGRP) and phalloidin were selected as the markers for respectively staining the dural nerve fibers and blood vessels in the whole-mount cranial dura mater with immunofluorescence and fluorescent histochemistry10. It may be an optimal choice to obtain a three-dimensional (3D) view of neurovascular structure. Additionally, fluorogold (FG) was applied on the area around middle meningeal artery (MMA) in the cranial dura mater to determine the origin of CGRP-immunoreactive nerve fibers, and traced to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs), while the FG-labeled neurons were further examined together with CGRP using immunofluorescence.
The aim of this study was to provide an effective tool for investigating the neurovascular structure in the cranial dura mater for the CGRP-immunoreactive innervation and its origin. By taking the advantage of the transparent whole-mount dura mater and combining the immunofluorescence, retrograde tracing, confocal techniques, and 3D reconstruction, we expected to present a novel 3D view of the neurovascular structure in the cranial dura mater. These methodological approaches may be further served for exploring the pathogenesis of different headaches.

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	<tbody>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse IgG (H+L)</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>A21202</td>
			<td>Protect from light; RRID: AB_141607</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>CellSens Dimension</td>
			<td>Olympus</td>
			<td>Version 1.1</td>
			<td>Software of fluorescent microscope</td>
		</tr>
		<tr>
			<td>Confocal imaging system</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogold (FG)</td>
			<td>Fluorochrome</td>
			<td>52-9400</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Fluorescent imaging system</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International&#160;GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus FV10-ASW 4.2a</td>
			<td>Olympus</td>
			<td>Version 4.2</td>
			<td>Confocal image processing software system</td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-CGRP</td>
			<td>Abcam</td>
			<td>ab81887</td>
			<td>RRID: AB_1658411</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin 568</td>
			<td>Molecular&#160;Probes</td>
			<td>A12380</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Photoshop and&#160; Illustration</td>
			<td>Adobe</td>
			<td>CS6</td>
			<td>Photo editing software</td>
		</tr>
		<tr>
			<td>Rabbit anti- Fluorogold</td>
			<td>Abcam</td>
			<td>ab153</td>
			<td>RRID: AB_90738</td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>National Institutes for Food and Drug Control</td>
			<td>SCXK (JING) 2014-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
	</tbody>
</table>

visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Neurovascular structure of the cranial dura mater 
After immunofluorescent and fluorescent histochemical staining with CGRP and phalloidin, CGRP-immunoreactive nerve fibers and phalloidin-labeled dural arterioles and connective tissues were clearly demonstrated throughout the whole-mount cranial dura mater in a 3D pattern (Figure 2C,D,E,F). It was shown that both thick and thin CGRP-immunoreactive nerve fibers run in par...

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Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...

This protocol may provide a variable methodological reference for understanding the complicated neurovascular structure of the cranial dura mater on the physiological or pathological condition. Taking advantage of transparent whole mount dura mater, the spacial correlation of dural CGRP immunoreactive nerve fibers and blood vessels can be demonstrated in a 3D view. After euthanizing a rat, transcardially perfused it with 100 milliliters of 0.9%normal saline followed by 250 to 300 milliliters of 4%paraformaldehyde in 0.1 molar phosphate buffer.When finished, remove the head skin and open the skull to expose the dura mater and dorsal side of the brain. Dissect the cranial dura mater along the brainstem to the olfactory bulb in a whole mount pattern. Rinse the cranial dura mater in 0.1 molar phosphate buffer for about one minute and incubate it in a blocking solution.Transfer the dura mater into mouse anti-CGRP antibody overnight. On the following day, wash the dura mater three times in 0.1 molar phosphate buffer. Incubate the dura mater in a mixed solution of donkey anti-mouse Alexa Fluor 488 secondary antibody and phalloidin 568 in 0.1.Molar phosphate buffer containing 1%normal donkey serum and 0.5%Triton X-100 for 1.5 hours at room temperature. Then, wash the dura mater three times in 0.1 molar phosphate buffer, trim the edges, and mount it on microscope slides. Put on cover slips with 50%glycerin before observation.Image the whole mount dura mater with a fluorescent microscope, then take images of the CGRP immunoreactive nerve fibers and phalloidin-labeled blood vessels in the dura mater using a confocal microscope. Shave the rat's head with an electric razor, then put blunt ear bars to the rat and place it on the stereotaxic device. Position the mouth holder and apply ophthalmic ointment on the eyes.Clean the surgical side of the head skin using 75%ethanol and make an incision along the midline of the scalp. Bluntly remove the periosteum and muscle tissues away from the skull using sterile cotton tipped applicators. Drill a small hole using a burr drill with a round tip bit on the left parietal and temporal bones above the middle meningeal artery.Build a bank around the hole with dental silicate cement and add two microliters of fluorogold into the hole around the middle meningeal artery with a 10 microliter microsyringe. Cover the hole with a small piece of hemostatic sponge. Put a piece of paraffin film on the hole and seal the edges with bone wax.Suture the wound with sterile thread. Keep the rats in a warm area until they have fully recovered. Then return the rats back to their cages.After seven survival days, perfuse these rats with 0.9%normal saline followed by 4%paraformaldehyde in 0.1 molar phosphate buffer. Dissect out the TG and cervical one to four DRGs, then cut them at the thickness of 30 micrometers on a cryostat microtome system in the sagittal direction. And mount the sections on silane coated glass slides.Circle the sections with a histochemical pen and incubate them for 30 minutes in blocking solution. Transfer the samples into the solution of rabbit anti-fluorogold and mouse anti-CGRP antibody and incubate at four degrees Celsius overnight. On the next day, wash the sections three times in 0.1 molar phosphate buffer.Incubate the washed sections in a mixed solution of donkey anti-rabbit Alexa Fluor 594 and donkey anti-mouse Alexa Fluor 488 secondary antibody. Then wash them three times in 0.1 molar phosphate buffer and apply cover slips with 50%glycerin for observation. Take images of the fluorogold labeled neurons in TG and DRGs under UV illumination using a fluorescent microscope.Then, capture images of the fluorogold and CGRP labeled neurons in TG and DRGs. After immunofluorescent and fluorescent histochemical staining with CGRP and phalloidin, CGRP immunoreactive nerve fibers and phalloidin labeled dural arterioles and connective tissues were clearly visualized throughout the whole mount cranial dura mater in a 3D pattern. Both thick and thin CGRP immunoreactive nerve fibers run in parallel to the dural arterials around the vascular wall or between the blood vessels.Seven days after fluorogold application on the region of the middle meningeal artery and the rat cranial dura mater, the fluorogold labeled neurons were detected in TG and cervical DRGs on the ipsilateral side of the tracer application, which was directly observed under the UV illumination of the fluorescent microscopy. Fluorogold labeled neurons were found in all three branches of TG, with higher concentrations on the ophthalmic and maxillary divisions and with lower concentrations on the mandibular division. Some of the fluorogold labeled neurons were also observed in the cervical two to three DRGs.These double immunofluorescences representative photographs show sensory neurons in TG and DRG. The labeled neurons with FG, CGRP, and both FG and CGRP were visualized by double immunofluorescence. When attempting this protocol, remember to dissect out the cranial dura mater completely and paste it on a slide in a whole mount pattern.

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3.8K visualizações.  China Academy of Chinese Medical Sciences. Este protocolo pode fornecer uma referência metodológica variável para compreender a complicada estrutura neurovascular da dura mater craniana na condição fisiológica ou patológica. Aproveitando-se de toda a montagem transparente dura mater, a correlação espacial de fibras nervosas imunoreativas dural CGRP e vasos sanguíneos pode ser demonstrada em uma visão 3D. Depois de eutanásia de um rato, transcardialmente perfundiu-o com 100 mililitros de soro fisiológico 0,9% normal seguid...

Vídeo: Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing