This article details a surgical procedure to induce and study nerve injury in a rat model. The focus is on the dissection of the tibial nerve and the subsequent regeneration process.
Take an anesthetized rat with one hind leg shaved. Incise from midcalf to ankle, exposing the muscle.
Dissect the connective tissue layer to reveal the tibial nerve.
Create a subcutaneous tunnel beneath the skin. Transect the nerve to induce injury.
Thread the proximal end of the severed nerve through the tunnel and suture it to the tunnel's distal end, positioning the nerve subcutaneously.
Close the incision, clean the area, and allow the rat to recover.
Following transection, the nerve degenerates as axons and myelin sheaths break down.
Denervated Schwann cells release signaling molecules to attract macrophages for debris clearance, following which the severed nerve sends out axonal outgrowths to promote regeneration.
Without proper target structures to reinnervate, disorganized axons form a neuroma, a mass of axons, Schwann cells, collagen fibers, and blood vessels.
Regenerating axons release molecules that excite nearby nociceptors, pain-signaling sensory neurons, leading to neuroma-associated pain signals.
Locate the knee and gently make a longitudinal incision of 2 to three centimeters over the medial side of the hind paw from mid-calf to ankle using a scalpel. If needed, open the skin and subcutis further with microscissors until the muscle layers are visible. Identify the superficial neurovascular bundle as two to three white, purple, or red lines that can move freely over the muscle layers. Keep the neurovascular bundle intact. Dissect the posterior from this bundle.
Next, dissect to open the fascia in between the gastrocnemius muscles. Between the fascia of the muscles, the deeper neurovascular bundle with the tibial nerve can be found, which is about three times the size of the superficial neurovascular bundle. Use the tibial bone as an additional landmark, as the tibial nerve lies just posterior to the tibial bone. To dissect the tibial nerve free from the surrounding vascular bundles, bluntly move the tibial nerve and cut the exposed tissue that shows some stretch while moving the tibial nerve.
Once the whole tibial nerve is exposed, place the muscle layers back to avoid dehydration of the nerve until the dissection of the pretibial skin from the subcutaneous muscle. To dissect the pretibial skin from the subcutaneous muscle layer and make a subcutaneous tunnel, hold the skin up and push the blunt tip into the tissue parallel to the skin, using a blunt microsurgery tool. Ensure that the end of the tunnel is located pretibially or laterally for easy accessibility of the area for testing of the neuroma.
After cutting the tibial nerve at the distal end near the ankle, change the microscope magnification to 10x or 16x and identify the epineurium of the tibial nerve proximal to the cut made near the ankle, or in the case of a more proximal bifurcation of the tibial nerve, identify the epineurium of both the medial and lateral plantar branches proximal to the cut made near the ankle. Carefully place an 8-0 nylon suture through the epineurium of the proximal nerve end by holding the epineurium with tweezers and putting the needle between the nerve and epineurium with a bite of approximately 0.5 millimeters.
Take a bite with the needle subcutaneously at the end of the subcutaneous tunnel, and pull the suture to transpose the nerve laterally into the subcutaneous tunnel. Fix the suture by making a knot. Place a thicker suture of 4-0 with a dark color flush to the fixated nerve end without penetrating the skin. Ensure that the suture is visible outside the skin.
Check if the nerve stays in place after moving the paw and muscles. Cut off the suture ends with a slightly longer suture end on the furrow than on the 8-0 suture. Change the magnification of the microscope back to 6x and close the skin with intraepidermal sutures, using the 8-0 suture. Gently clean the skin with 0.9% sodium chloride using a cotton swab.
If the room is cold, place a heating pad, under a part of a clean cage and place the rat in the cage under a paper towel in a comfortable position. Ensure easy access to food and water.
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