Performing Crush Axotomy on the Facial Nerve in a Mouse Model

Abstract

Source: Olmstead, D. N., et al., Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury. J. Vis. Exp. (2015).

This video demonstrates the facial nerve crush procedure in a mouse, where blunt dissection through key landmarks exposes the facial nerve trunk at the stylomastoid foramen. Forceps are then used to compress the nerve trunk, inducing axonal damage and disrupting neuronal signal transmission.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Surgical Approach

1. Align and focus the stereoscope with the surgical field. Adjust the nose cone and tape it down so that it is positioned along the edge of the visual field.
2. With the mouse lying on its left side, tape the edge of the right ear to the nose cone, exposing the area behind the ear where the incision will be made. Ensure that the posterior auricular vein travels horizontally across the ear. Note that the correct placement of the animal and taping of the ear are crucial in order to quickly find the facial nerve.
3. Wet the fur on and behind the ear with 70% ethanol and shave the surgical site using a razor or scalpel blade. Pre-wetting the fur makes shaving easier in this anatomical location.
4. Clean the skin with an iodine solution, such as Betadine surgical scrub (7.5% povidone-iodine), followed by 70% ethanol. Repeat this cleaning two more times to thoroughly disinfect the area.
5. To determine where to make the incision, trace the posterior auricular vein from the ear caudally to the area posterior to the ear protuberance. Using spring scissors, make a 4 mm incision 2 - 3 mm posterior to the protuberance.
6. Dissect through the subcutaneous fat and fascia using blunt dissection. Avoid direct cutting with the scissors because blood vessels or muscle tissue could be easily damaged.
7. If bleeding occurs, apply pressure to the surgical site with a sterile cotton swab for at least 30 sec. If significant fluid loss occurs, inject the mouse intraperitoneally with up to 0.5 ml of sterile 0.9% saline solution using a 25 or 27 G needle.
8. Use several key landmarks, the spinal accessory nerve, ear canal, and anterior digastric muscle (described below), to locate the facial nerve. Dissect around these landmarks until the branches of the facial nerve are visualized. The nerve will appear as a significant solid white structure when it is revealed, and a layer of fascia adheres it to the underlying structures.
   1. Find the spinal accessory nerve, which travels from the caudal portion of the skull to innervate the trapezius muscle once the subcutaneous fat and fascia have been dissected. The facial nerve is deep to the spinal accessory nerve.
   2. Find the cartilaginous ear canal that looks pearly white and can be seen rostral to the facial nerve.
   3. Find the muscle belly of the anterior digastric muscle that lies on top of and caudal to the facial nerve.
9. When the main branches of the facial nerve are visualized, trace them dorsally to find their origin from the stylomastoid foramen. Using fine-tipped Dumont forceps #5/45 to hold the surgical site open, advance the spring scissor tips following the nerve’s path, then move the forceps dorsally to keep the newly advanced area open.
10. Visualize the trunk of the facial nerve with the zygomatic, buccal, and marginal mandibular branches at this point.
    NOTE: The temporal branch will be found closer to the foramen. The marginal mandibular nerve branches into its upper and lower parts closer to the jaw, thus those nerve branches will not be visible at this level.
    1. If performing a crush injury, use Dumont #5/45 forceps to compress the nerve for 30 sec using constant pressure to sever all axons, then repeat this crush at a second angle perpendicular to the first crush site. Avoid applying variable amounts of pressure during the 30 sec crush, otherwise the injury will be inconsistent between animals.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo Microscope	Leica	M60
Labeling tape	Fisher Scientific	15-952
Vannas-Tübingen Spring Scissors - Straight/Sharp/8.5cm/5mm cutting edge	Fine Science Tools	15003-08	Sterilize before use
Dumont #5/45 Forceps - Standard tips/Angled 45°/Dumoxel/11cm	Fine Science Tools	11251-35	Sterilize before use
0.9% Sodium Chloride Injection, USP	Hospira	0409-4888-10
Betadine, 16 oz, with dispenser	Fisher Scientific	19-027132
70% Ethanol
Glass Bead Sterilizer