Inducing Fibular Nerve Injury in a Mouse to Study Neuromuscular Junction Regeneration

Place an anesthetized mouse with a shaved hind leg under a surgical microscope.

Incise the skin on the shaved leg, then cut through the connective tissue layers to expose the underlying muscles.

Using retractors, separate the muscles to expose the common fibular nerve.

The nerve contains motor neurons, whose axon terminals connect with muscle fibers to form neuromuscular junctions or NMJs.

Locate the nerve's intersection with the tendon of the gastrocnemius muscle in the knee.

Hold the nerve with forceps and apply pressure to induce injury.

A translucent injury site indicates axonal damage, which leads to NMJ impairment.

Ensure the nerve's outer epineurium layer is intact to guide axonal regeneration.

Remove the retractors and realign the muscles. Suture the incision site and allow the mouse to recover.

Signals from the damaged axons recruit macrophages to clear axonal debris, which supports axonal regeneration and the reestablishment of NMJs.

Place the mouse on the surgical board and confirm a lack of response to toe pinch. Align the limbs within the restraints, with the target hind limb in an anatomically natural position and the knee joint slightly extended without internal or external rotation.

Then transfer the animal under a surgical microscope. And adjust the board until the bony knee joint and the ridge between the tibialis anterior and the gastrocnemius muscles are visible through the objective. Using a scalpel and forceps, make an approximately three-centimeter incision through the skin perpendicular to the underlying course of the common fibular nerve. Continue the incision through the superficial fascia, exposing the biceps femoris and the vastus lateralis muscles, followed by a 1 to 2 centimeter incision through the connecting deep fascia to separate the muscles.

Next, use mechanical retractors to recede the biceps femoris muscle caudally to reveal the common fibular nerve. Trace the nerve proximally until its intersection with the tendon of the lateral head of the gastrocnemius muscle is found. Then use a fine forceps to grasp the nerve, aligning the tips parallel to the lateral border of the gastrocnemius tendon. And apply steady concentrated pressure for 5 seconds to crush the common fibular nerve.

Holding the forceps perpendicular to the fiber, grasp the tissue with the area just behind the tip of the forceps. And apply firm but gentle pressure for a clean, linear crush of the nerve along the border of the tendon.

Visually inspect the nerve through the surgical microscope to confirm a complete crush of the tissue. The nerve will appear translucent at the site of injury. If the nerve has been sufficiently damaged, remove the retractors. And realign the muscles in their anatomic positions. Then use 6-0 silk sutures to close the incision site with one to three simple, interrupted sutures and place the mouse on a heating pad in a clean cage with monitoring until it is fully recovered.