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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
Eight-week-old adult male C57BL/6N mice were used.
1. Preoperative setup
- Ensure an appropriate stock of autoclaved, sterilized surgical instruments, equipment, analgesic medications, and anesthetic medications.
- Ensure adequate working space on an operating table.
- Prepare the operating table using a diaper-covered customized surgical foam board as a bed for the mouse. Fix a warming pad to the foam board with medical tape covered with sterile gauze.
- Create retractors by bending an acupuncture needle using vascular forceps, folding it in half, and then bending the tip of the folded acupuncture needle into a hook. Fix a rubber strip at the end of the acupuncture needle, and use a thumbtack to fix the end of the rubber strip to the foam board.
- Calibrate the stereomicroscope; choose a stereomicroscope with an adequate focus distance. Cover the zoom/focus buttons with sterilized aluminum foil to allow the surgeon to adjust them during the operation. The sterilized aluminum foil was placed on the zoom/focus buttons, allowing the surgeon to use it with sterile gloves.
2. Mouse anesthesia and preparation
- Weigh the mouse and anesthetize it according to its body weight (isoflurane 3%). Ensure that the mouse does not respond when the interdigital spaces of its paw are pinched to confirm the depth of anesthesia. Adequate depth of anesthesia should be maintained throughout the procedure (1% isoflurane).
- Apply ophthalmic ointment bilaterally to the eyes to prevent irritation or drying of the cornea during surgery.
- Prepare the surgical site by shaving the fur on the neck and chest with an automatic clipper. Remove and clean the loose hair.
- Place the mouse in a supine position on the warming pad covered with sterile gauze. Maintain the temperature of the mouse at 37 °C during the whole operation. Fix the mouse with medical tape to cause the forelimbs to abduct horizontally and prevent the hind limbs and tail from moving. A sterilized disposable surgical drape with appropriate opening was placed on the mice.
3. Operative procedure
- Inject tramadol as the pre-emptive analgesia (20 mg/kg, intraperitoneal or i.p.). Mark the transverse incision on the superior edge of the clavicle. Use three cycles of alternating scrubs of iodophor disinfection solution and ethanol to disinfect the surgical site. Confirm the depth of anesthesia with a toe pinch before surgery.
- Working under a microscope, make a 4 mm transverse incision along the mark using a sterile scalpel. Enlarge the incision during the procedure as necessary.
- Bluntly dissect through the subcutaneous fascia and identify the inferior border of the submandibular gland. Pull the submandibular gland upward to expose the supraclavicular fossa and sternum.
NOTE: There might be small-caliber blood vessels in this area. Electrocautery can be used to stop bleeding. - Make a partial median sternotomy incision (~4 mm) by incising the sternum from head to tail along the midline. Protect the pleura, heart, and blood vessels during sternotomy.
- Identify the sternohyoid muscle. Pull the sternum gently with two small customized retractors made of acupuncture needles and identify the sternohyoid muscle, over the trachea and esophagus. Retract this muscle to expose the carotid artery, internal jugular vein, phrenic nerve, vagus nerve, trachea, and esophagus.
NOTE: Gently retract the sternum to avoid open pneumothorax. Unlike in humans, the esophagus of the mouse is not behind the trachea but adjacent to the trachea on the left side. - Identify the left brachial plexus. At the lateral edge of the left internal jugular vein, pull the fascia and adipose tissue outward to expose the brachial plexus. Look for the superior trunk, composed of the Cervical or C5 and C6 nerves, which have three branches. Identify the middle trunk composed of the C7 nerve and the inferior trunk composed of the C8 and Thoracic or T1 nerves, along the upper trunk up to the tail of the mouse.
NOTE: There are longitudinal blood vessels on the surface of the brachial plexus. Use electrocautery to prevent bleeding. When separating the left brachial plexus, protect the chylous canal to avoid a chylous fistula. - Harvest the left C7 nerve. Dissect the anterior division and posterior division of the middle trunk (C7 nerve) distally to the division-to-cord level under the clavicle and block the C7 nerve with 0.1 mL of 2% lidocaine by local infusion into the nerve trunk. Resect the C7 nerve with Vannas spring scissors at its merger points with the lateral cord and posterior cord. Trim the C7 nerve so that the length of each division is similar.
NOTE: The anterior and posterior divisions of the C7 nerve and the anterior and posterior divisions of the upper and lower trunks run for a long distance before confluence, so the C7 nerve should be freed sufficiently before resection. In fact, the C7 nerve is not always divided into two divisions; sometimes, it is divided into three divisions or even into four in rare cases. - Remove the left C6 lamina ventralis. Carefully protect the phrenic nerve and sever the anterior scalene muscle at the level of the C6 segment to expose the C7 nerve root. Cut small branches of the C7 nerve innervating the paraspinal muscle with microforceps. Pull out the C7 nerve gently and excise the C6 lamina ventralis carefully.
NOTE: There is a bony prominence between the medial side of the left carotid artery and the lateral side of the esophagus. This bony prominence is the lamina ventralis of the 6th cervical vertebra. The longitudinal muscle of the lateral edge of the C6 lamina ventralis is the anterior scalene muscle, and the phrenic nerve runs on the surface of the anterior scalene muscle. - Harvest the right C7 nerve. Sever the anterior scalene muscle on the right side, similar to the left side, and transect the right C7 nerve root close to the intervertebral foramen. Dissect the right C7 nerve from its division level.
NOTE: Carefully cut the right C7 nerve to prevent damage to the blood vessels under the nerve. - Transfer the left C7 nerve.
- Remove the muscular longus colli beside the vertebral bodies partially on both sides. Bluntly separate and expand the space between the trachea-esophagus and the vertebral body.
- Send a half-fold 5-0 nylon suture from the right side of the vertebral body to the left side through the prespinal route.
- Hitch the left C7 nerve with an infusion tube and guide the nerve to the right side via the prespinal route.
- Retract the trachea and esophagus gently and coapt the anterior and posterior divisions of the left C7 nerve to the right C7 nerve root without tension using 12-0 Nylon sutures. Suture the epineurium around the nerves with 4-5 stitches to coaptate the nerves strongly.
NOTE: It is crucial to choose a plastic infusion tube of appropriate thickness. Too thin of a tube could damage the nerve, and too thick of a tube could damage the trachea and esophagus. In addition, the space between the trachea-esophagus and the vertebral body is a "V"-shaped space, and cutting part of the muscular longus colli can shorten the transfer pathway.
4. Wound closure
- Irrigate the wound with sterile normal saline and dry it with sterile gauze.
- Suture the sternum and close the skin using 5-0 monofilament sutures.
5. Postoperative care
- Wait for the mouse to wake from anesthesia. Transfer the mouse to a clean cage without bedding material, but warmed with a warming blanket. Observe the mouse until it is ambulatory. Use tramadol (20mg/kg, i.p.) as the postoperative analgesia.
- Place the mice in a recovery cage and monitor it till recovery. Restore the water and diet of the mice after the operation. Monitor the mice postoperatively for signs of impairment or infection every day, including malnourishment, hunched posture, and ruffled fur. At two weeks after surgery, suture removal should occur.
NOTE: Apply erythromycin ointment to the wound surface every day for three consecutive days. - If any complications, such as wound edema, are observed, they should be immediately resolved.