Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats

This method helps finding better treatments for neuroma pain. The advantage of this technique is that you can measure both neuroma pain and generalized pain independently in the same rep model. The TNT model is particularly fitted to the testing of neuroma pain, as the neuroma itself can be mechanically stimulated with the Von Frey monofilaments.

Begin by placing the anesthetized rat on its back with its head either to the left or right. Place the leg to be operated on near the surgeon. Exorotate the lower hind limb so the medial malleolus faces upward.

Before placing the rat under a stereo surgical microscope with 6x magnification, disinfect the shaved area with at least three alternating rounds of iodine-based scrub and alcohol. Locate the knee and gently make a longitudinal incision of two to three centimeters over the medial side of the hind paw from midcalf 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 flies 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 planter 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 flushed 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 4-0, then 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. To stimulate the sterile nerve for hypersensitivity, apply the monofilament on the lateral side close to the hair border without touching the foot pads.

To stimulate the tibial nerve for hyposensitivity, apply the monofilament in the middle of the planter surface of the hind paw. For neuroma site testing, hold the rats with their nose pointed toward the elbow fold. If the rat is held in the right hand, his left hind paw should hang freely between the right thumb and index finger.

After verifying that the rat is calm and comfortable while holding, at baseline, place the 15-gram monofilament gently on the pretibial surface of the exposed hind paw. After the surgery, place the 15-gram monofilament on the visible suture and apply sufficient force to the monofilament to bend the hair and hold for one second. Von Frey of the neuroma site showed that at baseline, rats responded to 10%to 15%of the applications of a 15-gram monofilament.

One week after the tibial neuroma transposition surgery, the response rate increased from 45%to 50%On the contralateral side, the responses after surgery were similar to the baseline. Around 20%of the rats did not develop a painful neuroma and the response rate did not increase compared to the baseline. At the end of the transected and transposed tibial nerve stump 12 weeks after surgery, all rats developed a neuroma.

Transection of the tibial nerve reduced mechanical sensitivity at the middle of the planter side of the hind paw innervated by the tibial nerve. Hyposensitivity present at one week after surgery was significantly different from the contralateral side and baseline from three weeks after surgery and remained until 12 weeks after surgery. At the lateral part of the planter side of the hind paw innervated by the intact sural nerve, the rats developed significantly different mechanical hypersensitivity from the contralateral side and baseline from one week after surgery.

It persisted for 12 weeks after surgery. At the contralateral paw, mechanical sensitivity was not affected compared to baseline in the areas innervated either by the sural or tibial nerve. Remember to create an off-length of the tibial nerve for range of motion for lateral transposition.

Also remember to mark this neuroma site with a dark suture.