Novel Surgical Rodent Model for Studying Neuroma Pain Treatment Options using Targeted Muscle Reinnervation Through the Saphenous Nerve

We developed a reproducible sensory nerve red model to study neuroma pain and test surgical treatments. Microsurgery, nerve stimulation and standardized testing support accurate translational assessments. To begin, place the anesthetized animal on its back on a heating pad, with its head turned opposite to the surgeon's position.

Apply a sterile ophthalmic ointment to both eyes to prevent dryness caused by gas anesthesia. Now, shave the surgical site from the thigh to the ankle using an electric razor. After shaving, use the adhesive side of a piece of tape to remove any remaining loose hairs to ensure optimal skin preparation.

Then disinfect the shaved area using three alternating rounds of 0.5%Chlorhexidine digluconate applied with a sterile gauze pad. Allow each application to air dry completely before applying the next round. Then confirm the depth of anesthesia by gently pinching the web space of the animal's foot with forceps.

Rotate the operative leg laterally to expose the surgical site. Under 6x microscope magnification, mark the incision line with a sterile skin marker. Using a one milliliter syringe with a 25 gauge needle, inject 0.05 milliliters of 1%lidocaine subcutaneously along the marked line.

Then create an eight to 10 millimeter longitudinal dermal incision over the medial hind limb using a number 15 scalpel blade. Identify the superficial neurovascular bundle, including the saphenous artery, vein, and nerve. Use a fine needle to isolate the saphenous nerve and prevent accidental damage.

To perform needle dissection precisely, orient the bevel of a fine needle towards the operator and gently slide the tip along the tissue plane. Maintain the bevel angle to create a sharp underside edge, allowing accurate separation of delicate connective tissue without applying excessive traction or pressure. Use electrocautery if required to control minor bleeding from adjacent vessels.

Change the microscope magnification to 16 times. When approaching the adductor muscles medially, carefully separate the tissue until a perpendicularly crossing vein, the vena caudalis femoris becomes visible. Identify the accompanying Ramus Nervi Ophthalmici centrally within the dissection area.

Continue dissecting the adductors longitudinally towards the medial motor branch of the tibial nerve, which innervates the gastrocnemius muscle. Perform meticulous dissection medially or laterally to preserve the integrity of the vein before establishing the coaptation field. Then apply a one milliamp your stimulation using the nerve stimulator to the motor branch of the tibial nerve, and confirm contraction of the gastrocnemius muscle to verify correct neural response.

During preparation of the recipient nerve, dissect precisely along its natural trajectory. After increasing the Isoflurane anesthesia to 5%transect the nerve using microsurgical scissors less than three millimeters from the muscle surface. Then reduce the Isoflurane anesthesia back to two to 3%Position the coaptation site close to the motor branch's entry point into the muscle to promote optimal axonal regeneration.

Tunnel the saphenous nerve through the adductor muscles to reach the medial motor branch of the tibial nerve. Then using 110 nylon sutures, coapt the two nerves with two fine stitches, ensuring the sutures do not obstruct the interface for effective neural connection. For untreated controls, after tunneling, do not perform coaptation.

Instead, transpose the distal stump of the saphenous nerve and secure it to the gastrocnemius muscle fascia. Now adjust the microscope magnification back to 6x before starting skin closure. Approximate the epidermal edges with 80 sutures using an intradermal technique, maintaining minimal tension on the wound.

Finally, clean the skin with 0.9%sodium chloride using a sterile cotton swab to remove debris and ensure optimal healing. Postoperative mechanical sensitivity testing revealed group differences in the tibial dermatome, where animals with untreated saphenous nerve transection exhibited the highest sensitivity across the six week follow up period. The targeted muscle reinnervation group showed a consistent trend toward reduced tibial sensitivity, with divergence between groups most evident at week two.

Measurements of sural dermatome sensitivity showed no significant differences between groups throughout the observation period. Histological examination showed that untreated saphenous neuromas exhibited disorganized axonal sprouting, visible as irregular swirling and fragmentation of axons within and around the lesion site. In contrast, targeted muscle reinnervation samples displayed organized nerve fascicles extending into adjacent muscle tissue, indicating more controlled axonal regeneration and reduced neuroma formation.

No sensory model existed in rodents. Our protocol breaches the gap between preclinical and clinical nerve pain research. Using a purely sensory nerve to a motor branch improves clinical relevance and also allows comparison between different nerve pain strategies.