Rat Hindlimb Below-Knee Amputation Model and Immediate Targeted Muscle Reinnervation

A research examines pain related to nerve injury and amputation and how rerouting nerves can affect that pain. Amputation limits standard pain assessments. This protocol uses modified and alternative tests to measure post-amputation pain behaviors.

To begin, obtain a Sprague Dawley rat aged eight to 10 weeks and weighing 250 to 300 grams. Turn on the warming pad and prepare a sterile surgical field. Arrange all the sterile tools and materials required for the surgery.

After anesthetizing the rat with isoflurane at a 4%induction and a one to 2%maintenance dose, shave the area beginning at the lower back and continuing along the full length of the hind limb. Clean the surgical site with Povidone iodine antiseptic solution, followed by wiping it with an alcohol prep pad. Repeat this procedure two more times.

Use a tuberculin syringe to inject one milligram per kilogram meloxicam subcutaneously. Use a ruler to mark the knee joint line. And make two additional marks, two centimeters and three centimeters distal to the knee.

Use a scalpel to make a circumferential skin incision three centimeters distal to the knee. Make a blunt and sharp dissection to circumferentially separate the skin from the underlying muscles until it retracts freely. Repeat the undermining process through the deeper muscle layers to dissect down to the bone while preserving tissue planes.

Use a marker to mark the tibia and fibula one centimeter proximal to the circumferential incision. Use small bone scissors to transect the tibia and fibula at the marked location. Use cautery to control bleeding and achieve hemostasis.

Examine the bone end and blunt or trim sharp edges as needed. Use a 4-O absorbable braided suture to approximate the anterior edges of the muscle compartments over the tibia, and secure it with four to six evenly spaced interrupted stitches. Line up and close the anterior and posterior muscle compartments together over the bone to form a muscle pad.

Close the skin laterally with interrupted 4-O absorbable braided sutures, while avoiding placement on the weight-bearing stump. Apply skin adhesive along the incision line. Make a 1.5 to two centimeter longitudinal gluteal splitting incision, approximately five millimeters posterior to the femur.

Use blunt dissection to separate the gluteal and hamstring muscles, and expose the sciatic nerve proximal to its trifurcation. Identify the proximal motor branches, including the large branch beneath the caudal femoralis or CFM and its divisions. Elevate the CFM with forceps and divide it.

Use fine microsurgical Dumont forceps to neuralize the semimembranosis and biceps femoris divisions by blunt dissection. Grasp the epinurium and open it longitudinally using fine curved spring scissors. Then separate the nerve branches.

Preserve the caudofemoralis branch and divide the remaining branches near the sciatic trunk. Curve the motor branches inferiorly to facilitate coaptation. Use curved spring scissors to open the epinurium and divide it to neuralize the sciatic trifurcation.

Use fine forceps to gently separate the nerve branches. Use forceps to grip each of the three branches, common peroneal, tibial, and sural, five to 10 millimeters distal to the trifurcation. While applying tension, transect each nerve distally and excise five to 10 millimeters of the segment.

Use 10-0 nylon sutures to perform the coaptations by first connecting the common perineal nerve to the semimembranosis. Use at least three knots per suture. Ensure tension-free placement, avoiding over-tightening and creasing the nerve, and lay out the coaptations without sharp angulations.

Then connect the tibial nerve to the large biceps femoris branch, and connect the sural nerve to the small biceps femoris branch. For amputation without target muscle re-innervation, after exposing the sciatic branches, use a 5-O silk suture and forceps to ligate the common peroneal, tibial, and sural nerves, ensuring the ligature creases the nerve and is secured with four square knots. Gently pull each nerve proximally using the knot and transect one to two millimeters distal to the ligature.

Remove 10 millimeters of the distal nerve segment and then proceed with the amputation as demonstrated previously. After closing the incision, apply skin adhesive along the incision line. Monitor the rat daily for seven days for wound integrity, signs of infection, pain, or distress, and intervene if needed per IACUC guidelines.

Mechanical withdrawal thresholds measured by Von Frey testing were significantly reduced from baseline and remain decreased across post-amputation time points. Pin testing demonstrated a trend toward increased noxious responses over time compared to baseline. Cold hypersensitivity of the stump was robust and remained sustained compared to baseline across all time points.

This protocol allows different nerve management strategies to be assessed in the context of amputation. The biggest challenge while performing this protocol is atraumatically manipulating nerves less than one millimeter in width. Future studies can expand this work to improve models and develop more reliable methods for studying phantom limb pain.