All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Anesthesia and preparation
- Place the animal in the anesthesia chamber and induce general anesthesia via 1.8% isoflurane and 0.9 L/min oxygen.
- Confirm an adequate plane of anesthesia via an assessment of spontaneous breathing and an evaluation of consciousness by assessing the animal's grimace response to a toe pinch.
- Apply eye lubricant bilaterally to guard against corneal irritation or dryness.
- Shave the operative site(s) with a razor or automatic clipper.
- Establish a method for rat identification at this time, either via an ear tag or tail label/marking.
- Administer a subcutaneous injection of 0.05 mg/kg buprenorphine along the animal's back for prophylaxis against postoperative pain.
2. Surgical approach and injury patterns
- Transfer the animal to the operating table and continue the gas flow via a nosecone. Ensure that a warming pad is positioned underneath the animal and the sterile field to maintain its body temperature.
- Place sterilized gauze (rolled up and fastened with tape) on the rat's neck to use as a neck roll; this will enhance the surgical field's exposure. Note that the animal's appropriate positioning is paramount for efficient nerve identification and dissection.
- Prepare the animal's facial skin for the procedure. To ensure disinfection, scrub the surgical site three times with chlorhexidine or an iodine-based solution alternating with 70% ethanol.
- Plan and mark the surgical incision if desired. Manipulate the ipsilateral ear in an anterior-posterior direction to determine the natural folding of the postauricular skin.
- Fashion a 4-5 mm incision in the postauricular crease using sharp iris scissors or a number 15 blade. This can be expanded later in the procedure as necessary.
- Bluntly dissect through the immediate subcutaneous fascia and place a micro-Weitlaner retractor to enhance exposure. Note that there may be small caliber blood vessels in this area; these are best avoided by retracting superiorly or inferiorly via the Weitlaner retractor.
- 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 its insertion point to reveal the tendon of the anterior digastric belly. Note that the tendon appears 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. Note that the subsequently exposed region is the three-dimensional space where the main trunk of the facial nerve lies.
NOTE: This region is bounded superiorly and medially by the skull base, laterally by the anterior digastric muscle, posteromedially by the ear canal, and inferiorly by the structures of the neck, including the superficial temporal artery.
- After adequate exposure, identify the main trunk of the facial nerve as it travels inferiorly from underneath the tendon of the digastric muscle, where it exits the stylomastoid foramen from the skull base. Note that the nerve appears as a pearly white cord encased in the animal's parotid-masseteric fascia. Practice caution when further exposing the nerve for the following reasons.
- Avoid aggressive dissection, or perpendicular spreads, to guard against stretch-mediated neuropraxia injury.
- Avoid aggressive posteriorly and medially directed dissection to guard against violating the thin tissues overlying the ear canal, as this could introduce middle ear flora into the surgical field.
- Avoid damaging the superficial temporal artery through broad medially and inferiorly directed dissection. Note that an injury will be identified by brisk, pulsatile bleeding.
- If the artery is injured, apply prompt pressure with a cotton-tipped applicator or sterile gauze via forceps. Hemostatic agents or liquid fibrin sealant can be placed in near proximity. Keep in mind that the animal may require a subcutaneous injection of 0.9% sterile saline for fluid stabilization.
- Trace the main trunk distally by dissecting along the nerve in an inferior direction, distally from the exit of the stylomastoid foramen.
- Extend the original incision to expose the nerve and its branches fully. Avoid disrupting the parotid gland, as this could result in postoperative sialocele.
- Induce the desired injury patterns as follows.
- For a crush injury, use smooth-surfaced jeweler's forceps to firmly grasp the nerve and compress it9. Apply constant and reproducible pressure to the nerve for 30 seconds to ensure an appropriate crush injury.
- For a simple transection, grasp the fascia overlying the nerve, or the immediate epineurium, with fine-toothed forceps, and use sharp microscissors to cleanly transect the nerve at the desired point with a single cut. Take care to avoid excess traction on the nerve with the forceps.
- For a nerve gap model, create the desired nerve gap using a similar method to the simple transection injury. Use the sterilized shaft of a cotton-tipped applicator cut to the desired nerve gap length-intraoperatively to ensure similarity of injury pattern between animals.
3. Wound closure
- Irrigate the wound with sterile saline and dry it with sterile gauze.
- Approximate the skin edges in a simple, subcuticular fashion with absorbable sutures, or use skin glue or wound clips, which are also acceptable for wound closure. Place a buried stitch by taking a deep-to-superficial bite of one skin edge and then a subsequent superficial-to-deep bite of the opposite skin edge.
