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

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.