Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

The overall goal of this procedure is to perform an infraorbital nerve injury in mice to study the development of trigeminal neuropathic pain and to test new treatment strategies. Isolated phase growing behavior after infraorbital nerve ligation is one of the few animal models to study spontaneous neuropathic pain, but its use has long been hampered by the complexity of performing an infraorbital nerve ligation. Compared to sensory reflex based pain testing such as von Frey or withdrawal responses from heat or cold stimulation.

Measuring spontaneous or ongoing pain has not received sufficient attention, considering that this is an important clinical symptom affecting quality of life. The distal approach to the infraorbital nerve has the advantage of being a surgically less demanding procedure than the intraorbital approach. It is minimally invasive and can be performed without using a stereotaxic frame.

To begin, gently shave the buccal hair between the whisker pad and the eye of an anesthetized mouse. Fix the head of the mouse in a stereotaxic frame or otherwise fixate the head. Apply ointment on both eyes to avoid drying.

Scrub the shaved head area with alcohol and then with Betadine. Under a dissecting microscope, make a four millimeter skin incision perpendicular to the midline. Approximately halfway between the edge of the whisker pad and the eye just rostral to the infraorbital foramen and centered around the line between the center of the eye and the center of the whisker pad.

To expose the infraorbital nerve, bluntly separate the superficial connective tissue minimizing musculature damage and avoiding the motor nerve fibers. Using a rotation motion, slip the head of a hooked ligation aid under the infraorbital nerve. Place the chromic gut ligature through the hole on the tip of the ligation aid, and retract the ligation aid so that the ligature remains under the infraorbital nerve with both ends of the ligature equidistant from the nerve.

Tie a slip knot from both ends of the ligature and slide it against the infraorbital nerve. Slide the knot further to constrict the infraorbital nerve. Then place a normal knot on top of the slip knot.

Cut the ends of the ligature leaving approximately 1.5 millimeters of free ends to prevent the knot from becoming undone. Close the skin incision using 6-0 synthetic absorbable sutures. Allow the mouse to recover on a heated pad or under an infrared heating lamp.

To begin, carry a single mouse from the housing to the test room in a covered plastic cage without bedding material. Place the cage in front of a video camera, then place a mirror to view the mouse's face when its back is toward the camera. Record the mouse's behavior for 10 minutes.

After recording, clean the observation cage before recording the next animal. Have an observer blind to the experimental conditions to analyze the recorded behavior of the mouse. Note each face grooming episode during the 10 minute recording.

Make a distinction between isolated face grooming and face grooming behaviors during body grooming. Determine the number of face grooming episodes with a four second cutoff criterion. The time between grooming actions of less than four seconds is defined as a pause within a single episode.

A time greater than four seconds is defined as a full interruption of grooming actions between two episodes. Place the mouse tail in a soft silicone clamp and attach the clamp magnetically to a metal plate on the table. Place a three walled plastic holder over the mouse so that only the head protrudes from the container.

Place a weight on top of the holder to keep it in place. Use a grated series of four von Frey hairs to apply force to the mouse. Habituate the mouse to the restrainer and reaching movements for 10 minutes.

When the mouse is relaxed, slowly apply the lightest von Frey hair within the infraorbital nerve territory near the center of the vibrancy until it bends. Score the mouse response to the stimulation into one of the following categories. Calculate the mean score for ipsilateral responses to the four von Frey hairs, then calculate mean scores for the contralateral sides.

Distal infraorbital nerve chronic constriction injury mice exhibited a significant postoperative increase in isolated face grooming time and episodes peaking in the first week, and then gradually decreasing, remaining elevated for six weeks. Initially, distal infraorbital nerve chronic constriction injury mice showed almost complete unresponsiveness to mechanical stimulation, which during subsequent weeks transformed into hyperresponsiveness lasting for six weeks. A slight increase in responsiveness to contralateral mechanical stimulation was also observed.