The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The overall goal of this peripheral nerve injury method is to reliably examine regenerating neuromuscular junctions in mice. This method will help us identify the molecular, cellular, and functional changes associated with regenerating neuromuscular junctions. The main advantage of this procedure is that the rate of neuromuscular junction regeneration can be reliably compared between experimental groups largely in the absence of myogenic changes.

Demonstrating the procedure will be William Dalkin, a medical student in my laboratory. Before beginning the procedure, use 80%ethanol to clean the surgical site, board, and instruments, followed by disinfection with Povidone-iodine. Next, 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 joints 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 one to two 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 five 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 mice expressing fluorescence proteins in the peripheral axons are used, the fluorescence will disappear from the site of injury. If the nerve has been sufficiently damaged, remove the retractors and realign the muscles in the 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. Compressing the nerve for five seconds using a fine forceps results in the disappearance of YFP from the injury site. The epineurium remains contiguous, serving as a conduit for the precise regeneration of axons to their original targets.

In 70 day old female mice, the nerve injury is sufficient to cause degeneration of all axonal segments distal from the neuronal soma, at four days post-crush. By seven days post-crush, the nerve endings are actively reoccupying the vacated post-synaptic sites. On day 12, the nerve endings continue to differentiate into pre-synaptic sites and the neuromuscular junctions are fully reinervated.

There is little variability among mice denervated for the same length of time, demonstrating that fibular nerve crushing can be used as an assay to compare the reinervation of muscles between animals of the same age and sex. As expected, a number of changes occur in the alpha motor axons as they reinervate muscle fibers. The axonal growth cones expand and branch out as they contact post-synaptic sites, growing over specific regions of the post-synapse, and culminating in the near complete juxtaposition of the axonal endings, with the post-synaptic sites.

As demonstrated by quantitative PCR, neuromuscular junction associated genes increase following denervation and decrease as the neuromuscular junctions are reinervated. Once mastered, this technique can be completed in 15 minutes if it is performed properly. While attempting this procedure, it's important to remember to take your time, visualize all of the structures, and double-check that a full crush has been obtained.

Following this procedure, other methods like QPCR can be performed to answer additional questions about what genes are being up or down-regulated. After its development, this technique paved the way for researchers in the field of neurobiology to explore synaptic regenerations in mice. After watching this video, you should have a good understanding of how to perform a fibular nerve crush to examine the rates of neuromuscular junction reinervation in mice.