Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

This method is a tool to investigate mechanisms involved in release of CGRP from the trigeminal vascular system. The main advantage of this method is the ability to divide the trigeminal vascular system into three different sites and assess release of the CGRP within each distinct site. Demonstrating the procedure will be myself and Inger Jansen-Olesen, a senior scientist from our group.

To begin, prepare the rat tissue by removing the skin and the muscle around the head and neck using scissors. Next, use a bone trimmer and a pair of scissors to separate the lower jaws from the head. Now expose the spinal cord and brainstem by inserting a bone trimmer caudally into the vertebrae’s dorsal part and remove it.

Then cut the caudal part of the cranium by the borders of the occipital and interparietal bones to remove these bone structures, exposing the cerebellum. To isolate the TNC running caudally approximately 13 to 16 millimeters from the bregma on each side, cut the dorsolateral part of the brainstem with spring scissors. That is followed by immersing the left and right side TNC into SIF.

Next, cut the head midsaggitally to divide the cranium into two using a saw. Now carefully remove the brain without touching the dura mater attached to the cranium using a spatula. To isolate the TG, cut it including its branches around the visual borders with the mandibular branch entering the foramen ovale and the ophthalmic and maxillary branches entering the skull.

Then immerse the cranium halves and the TGs in SIF. To prepare the mouse tissue, remove the skin and the muscle around the head and neck using scissors. Now expose the spinal cord and brainstem by inserting a pair of scissors caudally into the vertebrae’s dorsal part and remove it.

Then cut the caudal part of the cranium by the borders of the occipital and interparietal bones to remove these bone structures exposing the cerebellum. Cut the parietal bone midsaggitally and remove the bone to expose the cerebrum. Carefully remove the cerebellum to expose the brainstem using a spatula.

Isolate the TNC containing part of the brainstem with spring scissors, followed by immersing the brainstem into SIF. Now carefully remove the brain using a spatula and cut the trigeminal nerve where it enters the brainstem. To isolate the TG, cut it including its branches around the visual borders with the mandibular branch where it enters the foramen ovale and ophthalmic and maxillary branches entering the skull.

Next, immerse TGs in SIF. For easy exchange of SIF, add a toll lid to the plastic containers and start washing the rat and mouse tissue in SIF for 30 minutes by replacing SIF every five minutes. Following 30 minutes of washing at room temperature, transfer rat TNC halves and rat TGs to separate microcentrifuge tube caps with 350 microliters of SIF.

Transfer the mouse brainstem with TNC to a microcentrifuge tube cap with 250 microliters of SIF. Finally, transfer the two mouse TGs to a microcentrifuge tube cap with 250 microliters of SIF. Place the rat skull halves on a six-well culture plate and fill the skull with 400 microliters of SIF.

Place rat skulls in microcentrifuge tube caps with rat and mouse tissue in a humidified incubator at 37 degrees Celsius. Replace SIF using a pipette every five minutes for 20 minutes without touching the tissue. Prepare microcentrifuge tubes for sample collection by appropriate labeling.

Then add 50 microliters of 10 strength EIA buffer to each microcentrifuge tube. Next, prepare the test compound solution and vehicle solution by diluting in SIF for all concentrations. Following the last wash, add 250 microliters of SIF to mouse TG and TNC, 350 microliters SIF to rat TG and TNC, and 400 microliters SIF to each rat skull.

After 10 minutes of incubation, collect 200 microliters of the sample in a pre-labeled microcentrifuge tube with 50 microliters of 10 strength EIA buffer to enable measurement of the basal CGRP release. Discard the remaining liquid and immediately store the samples at minus 20 degrees Celsius. Add the test compound in the corresponding vehicle in increasing concentrations, starting with the lowest concentration, and incubate for 10 minutes.

After 10 minutes of incubation, collect 200 microliters of the sample in a pre-labeled microcentrifuge tube with 50 microliters of 10 strength EIA buffer. Discard the remaining liquid and add the second lowest concentration to the tissue. Immediately store the samples at minus 20 degrees Celsius and repeat this procedure with the remaining concentration.

To perform a positive control for the experiment, add the positive control to the tissue at the end of the protocol. After an incubation period of 10 minutes, collect a 200 microliters sample in a microcentrifuge tube with 50 microliters of 10 strength EIA buffer. The concentrations of CGRP released in the collected samples are measured by using an EIA kit while following the manufacturer’s instructions provided with the EIA kit.

In the rat, capsaicin exposure induced a significant CGRP release from dura mater and TG compared to the vehicle. In the dura mater, the maximum release of CGRP was found at one micromolar of capsaicin. And in TG, the maximum CGRP release was found at 10 micromolar of capsaicin.

When analyzed with a one-way ANOVA, glibenclamide show no effect on basal CGRP release from dura mater and TG.Glibenclamide significantly reduced the capsaicin-induced CGRP release in dura mater by 40%and TG by 39%compared to capsaicin with the vehicle when analyzed with a one-way ANOVA. The transient receptor potential Ankyrin-1 agonist super cinnamaldehyde was found to release CGRP in a concentration-dependent manner from the TG with 1, 10, and 100 micromolar of super cinnamaldehyde resulting in 9%52%and 69%increased release of CGRP compared to vehicle respectively when analyzed with two-way ANOVA. The increased release of CGRP was absent in TG from the transient receptor potential Ankyrin-1 knockout mice where exposure to 1, 10, and 100 micromolar of super cinnamaldehyde resulting in 11%minus 13%and 9%change in the release of CGRP compared to vehicle respectively when analyzed with two-way ANOVA.

It is important to be careful not to touch the tissue during sample collection and to be precise when timing the incubation time for all samples. If adequate ELISA kits are available, it is possible to apply this procedure to measure the release of other peptides present in the trigeminal vascular system.