Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The overall goal of this procedure is to ex sodomize the facial nerve to study peripheral nerve damage. The facial nerve originates in the pons of the brainstem and exits the skull via the stylo. Mastoid foramen the nerve then branches into its major components.

Once exposed, the nerve is ex autotomized at its exit from the stylo mastoid foramen. Two major landmarks help locate the facial nerve. The first is the ear canal, which is composed of pearly white cartilaginous plates connected by a thin membrane.

The other is the anterior belly of the digastric muscle, which lies on top of the nerve and its exit from the stylo mastoid foramen. After the facial nerve is injured, the scientist can study multiple factors such as mo neuron death or gene expression. The facial nerve injury model is one of the most well characterized peripheral nerve injury models.

Advantages of this technique are its simplicity, high reproducibility, and lack of effect on vital functions from the subsequent facial paralysis. Additionally, due to the highly symmetric nature of the nervous system, the uninjured side can serve as a paired internal control for your experiments. To maintain the use of aseptic techniques throughout this procedure, be sure to have all the tools autoclaved and have a glass bead sterilizer prepared to sterilize tools between operations.

Prepare the surgical bench by placing an absorbent pad over a heating pad, setting up the microscope and taping the nose cone so it aligns with the edge of the visual field. After anesthetizing the mouse, ensure that the mouse does not have any physical response to manipulations, including a toe pinch. Then transfer the gas flow from the anesthesia chamber to the nose cone and apply off thalamic ointment.

Position the mouse on its left side over surgical pads with a heated pad below. Continually monitor the animal's breathing and adjust the ISO fluorine flow as needed to maintain the anesthesia. Next to expose the surgical site, tape the right ear to the nose cone.

The posterior auricular vein should be seen traveling across the ear. Next, wet the fur with 70%ethanol and shave the sight. After removing the fur, clean the skin with three alternate scrubs of iodine and 70%ethanol with a sight thoroughly cleaned.

Proceed with the Surgery. Make sure to position the mouth square on its side with the ear vein traveling horizontally. This will make finding the facial nerve much easier.

To determine the location of the first incision, trace the posterior auricular vein from the ear coddly to the area posterior of the ear protuberance. Then using spring scissors, make a four millimeter incision, two to three millimeters posterior to the protuberance for teaching purposes. This incision will be enlarged to expose the underlying anatomy.

Now make a blunt dissection of the subcutaneous fat and fascia. Blood vessels and muscle can be easily damaged by scissors. If bleeding occurs.

Apply pressure with a sterile cotton swab for at least 30 seconds. If significant fluid loss has occurred, inject up to half a milliliter of saline intraperitoneal with a 25 or 27 gauge needle. Now locate the facial nerve using the key landmarks.

The spinal accessory nerve is superficial to the facial nerve and can be seen traveling from the coddle portion of the skull. To innervate the trapezius muscle, identify the cartilaginous ear canal. It is pearly white and can be seen rostral to the facial nerve.

The facial nerve wraps around the coddle portion of the ear canal. Then identify the muscle belly of the anterior digastric muscle that lies on top of and coddle to the facial nerve. Dissect under this structure to expose the facial nerve.

It is a solid white structure, adhered to a layer of underlying fascia. Identify the main branches of the facial nerve. Trace them dorsally to their origin from the stylo mastoid foramen.

Use fine forceps to hold open the site while using the tip of the spring scissors to dissect along the facial nerves path. The bony syl mastoid for Raymond can be felt by the forceps. Now, identify the facial nerve trunk to crush the nerve.

Compress it with the fine forceps for 30 seconds with constant pressure. This will sever all the axons. Then repeat the compression at a perpendicular angle for another 30 seconds.

Keep the pressure consistent between experiments. Now that the relevant anatomical landmarks have been identified, I'll demonstrate the more refined surgical technique. Using a smaller incision, Trace the posterior auricular vein from the ear coddly to the area posterior of the ear protuberance, and make a four millimeter incision, two to three millimeters posterior to the protuberance.

Now bluntly, dissect through the subcutaneous fat and fascia utilizing the three landmarks, the spinal accessory nerve, ear canal, and anterior belly of the digastric muscle. Uncover the facial nerve. Trace the facial nerve back towards the stylo mastoid foramen to reach the facial nerve trunk to transect the nerve, stabilize it gently with fine forceps and cut with scissors.

Avoid applying too much traction on the nerve, or it may uls from the brainstem. Then move the stumps apart or remove a segment of the nerve ensuring no reconnection can occur. Lastly, carefully inspect the nerve and confirm that it is entirely severed.

After the ex sodomy, return all the fat and muscles to their approximate positions. Then close the wound with a 7.5 millimeter wound, clip sutures or cyanoacrylate glue. If necessary, apply analgesics at this time, turn off the anesthesia and return the mouse to an empty cage with without bedding.

While the animal recovers, examine it for signs of facial paralysis, the whiskers are paralyzed. If they're angled back towards the cheek, the nose will appear deviated and the eye will not blink in response to an air puff. Now, if the animal is female, house her with another female.

But if male house him alone until the eye blink function returns apply off thalamic ointment daily to prevent corneal complications. Meanwhile, monitor the mouse for signs of infection or other complications until the surgical site is healed. There is significant variation in motor neuron loss after ex sodomy based on the age and genetic background of the mice in wild type animals that receive a facial nerve transection at eight weeks of age.

About 86%of the motor neurons survive at 28 days. Postex otomy. There was significantly more cell loss observed in the SOD one mouse model of a myotrophic lateral sclerosis, as well as in the immunodeficient recombination activating gene two knockout mouse laser capture.

Microdissection was used to isolate material from the facial motor nucleus. The entire facial motor nucleus could be captured. Alternatively, the sub nuclei could be collected separately.

The technique has great precision. Motor neurons can be captured individually and the remaining neuro pill can also be collected for analysis afterwards. Quantitative PCR was used to examine gene expression in the sub nucle samples of the ventral medial sub nucleus, shown as the dashed line and ventral lateral sub nucleus shown as the solid line expression of four genes associated with the nerve regeneration response was measured.

Some differences were found between the sub nuclei of these. Gap 43 expression showed the greatest difference between the sub nuclei. Once this technique is mastered, the surgery can be completed in just a couple of minutes.

It is important to remember to position the animal correctly, make the incision in the correct location, and use the anatomical landmarks to locate the facial nerve. It is easy to get lost in the head and neck region, so if you feel lost, just locate the cartilaginous ear canal and trace it posteriorly to find the facial nerve.