A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery

This protocol can establish mouse models that undergo crossing C7 nerve transfer surgery, which can help to study the internal neuro mechanisms underlying this surgery. So main advantage of this protocol is that it can improve the success rate of direct nerve and smooths and it simulates clinical surgery very well. This protocol provides a good mass model for the research.

Of course, neuro transfer and the research results could be applied to accelerate the recovery of patients after surgeries. After making a transverse incision on the superior edge of the clavicle and exposing the supraclavicular fossa and sternum, incise the sternum from head to tail along the middle line without damaging the pleura, heart, and blood vessels. With the help of two small customized retractors made of acupuncture needles, pull the sternum gently before retracting the sternohyoid muscle over the trachea and esophagus.

At the lateral edge of the left internal jugular vein, pull the fascia and adipose tissue outward to expose the left brachial plexus. Then look for the superior trunk composed of the C five and C six nerves having three branches. Identify the middle trunk composed of the C7 nerve and inferior trunk along the upper trunk up to the tail of the mouse.

Dissect the anterior and posterior division of the middle trunk distally to the division to cord level under the clavicle. Use Vannas Spring Scissors to resect the C7 nerve at its merger points with the lateral cord and posterior cord, and then trim the C7 nerve so that the length of each division is similar. Next, sever the anterior scalene muscle without damaging the phrenic nerve at the level of the C six segment and expose the C7 nerve root.

Then pull out the C7 nerve and remove the left C six lamina ventralis. Harvest the right C7 nerve as described earlier. Before transferring the left C7 nerve, remove the muscular longus coli beside the vertebra bodies partially on both sides.

Then separate and expand the space between the trachea, esophagus, and vertebral body. Once done, send a loop made of 7-0 nylon suture from the right side of the vertebral body to the left side through the prespinal root, hitch the left C7 nerve with a nylon suture loop and guide the nerve to the right side via the prespinal root. After retracting the trachea and esophagus, co apt 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 four to five stitches to coaptate the nerve strongly. When suturing is done, irrigate the wound with sterile normal saline, followed by drying with sterile gauze, then suture the sternum and close the skin using 5-0 monofilament sutures. In the representative analysis, a successfully regenerated C7 nerve is displayed.

Starting at four weeks post CC7 surgery, the myelin sheath thickness of the transferred C7 nerve gradually increased and was almost comparable to that in the control group at eight weeks post CC7 surgery. The electromyography analysis after the contralateral C7 nerve transfer indicated the nerve regeneration by the action potential recorded at the pectoralis major and electromyography of the extensor digitorum. At three weeks after the surgery, compound muscle action potential or CMAPs was emerged in the triceps brachii and increased at four and eight weeks.

The mean amplitude of the pectoralis major was significantly increased from four weeks to eight weeks. Similarly, the mean amplitude of the triceps, brachii, and extensor digitorum showed significant improvement at eight weeks. The cholera toxin subunit B or CTB retrograde labeling showed that the transferred C7 nerve contains motor fibers from the ventral horn and sensory fibers from the dorsal root ganglia of the spinal cord.

Additionally, the behavioral changes in the animals after CC7 surgery were monitored. In the cylinder tests, the TBI plus CC7 group showed a significantly higher usage rate of the impaired forelimb than the TBI group at both four and eight weeks post CC7 surgery. In the grid walking tests, the TBI plus CC7 group showed a lower error rate than the TBI group at four weeks post CC7 surgery.

At eight weeks, the error rate of the TBI plus CC7 group was significantly lower than that in the TBI group, indicating that the CC7 surgery improved the motor function of the effected limb. Separating the C7 nerve as light as possible to obtain sufficient nerve lengths is a key to realize the direct anastomosis of bilateral C7 nerve. This technique contributes to illustration of the nerve mechanisms and the layering rehabilitation upon coercing nerve transfer after central and the peripheral nervous system injuries.