Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

What events occur within the meninges that contribute to headache in the absence of tissue injury? Unlike prior use of dural stimulation models in rats, it allows the use of the extensive catalog of genetically modified mice. The primary struggle would be locating the intersection of the lambdoidal and sagittal sutures by palpating the skull with the injector.

To begin, create the mouse infusers by modifying a commercially available internal cannula and infuser with a fused silica plastic cap which is adjustable and inserts into a 28-gauge guide cannula. Use a caliper to adjust the fused silica plastic cap on the infuser without bending the infuser. Adjust the infuser to a length of 6 millimeters measured from the tip of the infuser to the edge of the silica plastic cap.

Perform pilot injections for other mouse strains and adjust the length to precisely inject in dura mater. Then, attach the long end of the infuser to plastic tubing of eight inches or more to hold five microliters volume. Insert the tube while ensuring full coverage of the metal part and the top of the plastic stopper located on the infuser to prevent air bubbles from accumulating in the line.

Next, fill the microsyringe and attach the other end of the tubing to a 10-microliter glass microsyringe, ensuring tight sealing over the metal part of the syringe. Then, backfill the syringe with five microliters of PBS or synthetic interstitial fluid or other vehicles of choice to prevent air bubble formation. If needed, flood the line with the vehicle to dissipate preformed bubbles.

Alternatively, pre-fill the syringe, and after connecting, push the fluid through the connected line. Once the vehicle-filled line is working efficiently, load five microliters of the solution into the Hamilton syringe. Once the mouse is completely anesthetized, apply sterile ophthalmic ointment to the eyes and shave the head of the animal, then disinfect the skin with povidone iodine and ethanol.

Steady the head of the animal with one hand and hold the infuser with the drug-loaded syringe in the other. Then, using an infuser, carefully probe, locate, and verify the junction of the sagittal and lambdoidal sutures on the skull of the mouse. Once the suture is located and the infuser is in place, slowly and gently wiggle the in infuser back and forth until the in infuser pierces through the skin and drops down into the junction all the way up to the plastic stopper, ensuring the insertion of the entire 6-millimeter infuser tip into the junction.

Then, slowly push the plunger of the syringe to expel the five microliters of fluid into the dura mater. On the testing day, post-acclimation to the room, remove one mouse along with the cup from the respective chamber. Maintain the cup in the horizontal position for evenly distributed body weight, as the unequal weight distribution may alter or prevent animals'response.

Place the cup with the mouse on an absorbent pad kept on the table below the testing rack. Then, for periorbital von Frey testing, place the 07-gram von Frey filament at the center of the face in between the eyes and apply enough pressure on the filament to cause the von Frey hair to bend into a C-shaped formation. Maintain filament contact for about three to five seconds or until the mouse swipes away the filament using the head or paws.

To follow the Dixon up-down method, apply the next lower-weight filament if the mouse responds to the filament, otherwise, apply higher-weight filament. The procedure was used to examine the effects of dural prolactin on mechanically evoked facial hypersensitivity. The results of the study demonstrated significantly reduced facial withdrawal thresholds in female ICR mice in response to five micrograms of dural prolactin.

A ten-fold lower dose of 5 micrograms of prolactin also showed reduced facial withdrawal thresholds, similar to a high dose of prolactin. The prolactin injections produced spontaneous pain-related behaviors, which were assessed via grimace. Dural 5 micrograms of prolactin caused significant grimacing in female mice and further demonstrated a clear role for dural prolactin in female migraine-like behaviors Behavioral testing using von Frey filaments or facial grimace to determine whether the stimulus, in the absence of injury, causes a headache response.

This technique can now be used to investigate inflammatory based mechanisms in the meninges without concern over caveats that are caused by craniotomies and the subsequent inflammation.