This protocol can help scientists study outcomes after facial nerve injury and methods to improve regeneration across many different parameters. The main advantage of this technique is that the rat anatomy allows easy access to the facial nerve, and its large scale allows us to study all of the relevant injury patterns. The applications of this technique extend toward the rehabilitation of the patients after facial nerve injury.
As the regenerative potential the rat provides a reproducible and concise experimental model. And this method can provide insight into surgical and medical therapies for facial nerve paresis or paralysis. And it's translatable to other head and neck models or other peripheral nerves.
Operating under a microscope using both hands can be challenging. I recommend practicing using binocular version under the microscope before attempting a facial nerve dissection. After confirming a lack of response to toe pinch, apply ointment to the eyes of the rat and shave the surgical area.
Establish a method for rat identification and place a roll of gauze under the neck. Disinfect the exposed skin with three alternating chlorhexidine and 70%ethanol scrubs and place the rat under a stereo microscope. Manipulate the ipsilateral ear in an anterior-posterior direction to determine the natural folding of the postauricular skin.
Use a number 15 blade to make a two to three millimeter incision in the postauricular crease. The planning and the placement of the incision are the most critical steps for ensuring reliable identification of the facial nerve while minimizing the wound size. Bluntly dissect through the immediate subcutaneous fascia and place a micro-Weitlaner retractor to enhance the tissue exposure.
Identify the anterior digastric muscle as it travels in an inferior-to-superior direction toward its insertion along the skull base. Spread gently through the muscle belly along the muscle insertion point to reveal the tendon of the anterior digastric belly. The tendon will appear as a filmy white process emanating from the muscle with a solid insertion onto the skull base.
After identification of the anterior digastric muscle and its tendon, adjust the Weitlaner retractor to further retract the muscle belly. The exposed region is the three-dimensional space in which the main trunk of the facial nerve lies. Dissect along the nerve in an inferior direction distally from the exit of the stylomastoid foramen.
To induce a crush injury pattern, use smooth-surfaced jeweler's forceps to firmly grasp and compress the nerve applying constant and reproducible pressure for 30 seconds to ensure an appropriate crush injury. For a simple transection, grasp the immediate epineurium overlying the nerve with fine-toothed forceps, and use sharp microscissors to cleanly transect the nerve at the desired point with a single cut. For a nerve gap model, use adjustable calipers to set the desired nerve nerve gap length to ensure similar injury patterns between animals and transect the nerve as just demonstrated.
After the experimental injury has been delivered, Irrigate the wound with sterile saline and dry the tissue with sterile gauze. before closing the skin incision according to institutional guidelines. Here, live imaging of the main trunk of the facial nerve with a crush injury approximately two to three millimeters distal to the branch point of the first PES in an adult transgenic Thy1-GFP rat is shown.
Assessment of the gradual return of fluorescence at one, two, three, and four weeks post injury can be used as a marker for monitoring nerve regeneration in these transgenic animals. Histomorphometric analysis can be used to obtain cross-sectional images of the marginal mandibular division allowing quantification of the axonal diameter, amount of debris, nerve fiber, percentage of nerve, and density measurements. It is important to frequently adjust the placement of the retractor as needed to ensure that the relevant anatomic nerves are visible when identifying the facial nerve.
Following this procedure the relevant nerves and muscles can be harvested for immunohistochemical analysis to assess the degree of regeneration relative to other experimental groups. Following its development, this technique has paved the way for researchers in studying the functional outcomes after facial nerve injury. Specifically, how to effect the phenomenon of synkinesis.
