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

Summary

This protocol demonstrates how to perform a below-knee amputation in rats and to immediately perform targeted muscle reinnervation (iTMR) to investigate strategies to treat and prevent amputation-related pain and to better understand pain mechanisms associated with amputation.

Abstract

Amputation-related pain reduces patient quality of life, contributes to opioid use, and can reduce functional rehabilitation and prosthesis use. Preclinical models that reliably produce post-amputation pain while preserving mobility are essential for studying the biological mechanisms underlying these outcomes. Here, we present a detailed, reproducible, rodent surgical protocol using Sprague-Dawley rats to model hindlimb below-knee amputation. The procedure involves isolating and transecting the sciatic nerve branches, including the tibial, common peroneal, and sural nerves, producing sustained post-amputation nerve pathology and pain-related behaviors. Rats recover well from this procedure and can ambulate immediately post-op with no difficulty eating or drinking. Animals can subsequently be evaluated with reflexive and spontaneous pain measures. The pain behaviors resulting from this procedure are robust over time, and standard reflexive pain behaviors can be obtained from the stump. Importantly, preserved mobility enables longitudinal behavioral testing without confounding motor impairment. While this protocol is centered on amputation, it may be readily adapted to incorporate targeted muscle reinnervation (TMR) as a post-amputation intervention by coapting transected nerves to nearby motor branches of the nerves to the biceps femoris and semimembranosus, enabling investigation of reinnervation interventions and their effects on post-amputation pain. We have successfully used this model previously to evaluate sex-specific differences in pain behaviors following amputation and to study the effects of TMR. This protocol provides a robust and flexible platform for investigating amputation-related pain, neuroma formation, and functional outcomes, with applicability to translational studies aimed at improving post-amputation pain.

Introduction

Chronic pain is one of the most debilitating complications following major limb amputation, affecting up to 70% of patients^1,2,3. Conventional pain management strategies such as pharmacological interventions, physical therapy, nerve blocks, and psychological therapies often provide limited or transient relief⁴. Thus, there remains a significant clinical need for effective long-term pain management strategies.

Targeted muscle reinnervation (TMR), initially developed to enhance control of myoelectric prostheses, has emerged as a promising approach to alleviate pain following limb amputation⁵. The surgical technique involves redirecting transected mixed motor-sensory nerves to nearby motor branches. Clinically, TMR has shown substantial reductions in phantom limb pain, residual limb pain, and neuroma-related symptoms^6,7,8. Regenerative peripheral nerve interfaces (RPNI) are another surgical intervention used for nerve injury and amputation-related pain. Amputated nerve ends are sewn to and then wrapped in a free (denervated, avascular) muscle graft⁹. The amputated axons neurotize the denervated neuromuscular junctions to create muscle contraction in the graft that can be used to create a signal for myoprosthesis control. In contrast to TMR, RPNI has been extensively studied in a rodent model. Similar to TMR, RPNI has been shown to prevent neuroma formation and reduce pain associated with nerve transection injury^10,11. Clinically, RPNI has been shown to reduce both phantom and residual limb pain in amputees¹².

Despite clinical adoption, the biological mechanisms underlying the analgesic effects of TMR and RPNI remain poorly understood. Preclinical models are crucial for elucidating these mechanisms. Prior work has utilized nerve transection models with modification to superficialize the nerve to directly test for neuroma pain^10,11. These models can quantify neuromatous pain with direct stimulation and neuropathic pain by assessing spinal reflex behaviors in the intact nerve distribution remaining in the foot. The spared nerve injury, for example, maintains the sural nerve, and the tibial and common peroneal nerves are transected. Pain behavior testing is performed on the lateral foot¹³. The hindlimb amputation model represents the most severe possible injury to the limb. As such, there is widespread neuronal loss and the significant inflammatory and healing demands associated with an injury of that nature. When there is a nerve injury, the healthy neurons in the DRG help maintain the injured neurons. With amputation, the population of available healthy neurons is greatly reduced, and for each injured nerve, there is greater neuronal loss than if only a single nerve had been injured^14,15,16. Prior reports of rodent hindlimb amputation survival surgery are rare^17,18. However, there are advantages versus an upper limb amputation, such as the abundance of historical data examining pain and regeneration related to the sciatic nerve, as well as multiple ambulatory behavioral tests^19,20.

To address this gap, we recently developed a rat hindlimb amputation model incorporating immediate TMR (Figure 1). The technique for RPNI creation has been previously published and can be adapted to the hindlimb amputation model²¹. This model closely mirrors clinical procedures, allowing us to evaluate various pain behaviors, neuroma formation, and neuron preservation (Figure 2)²². Our work has demonstrated significant analgesic benefits to TMR over standard amputation, neuroma prevention, and demonstrated sexually dimorphic responses in cold sensitivity^22,23.

The protocol presented here provides a detailed, standardized visual demonstration of the surgical technique, ensuring reproducibility and consistency in experimental approaches. By clearly illustrating key steps, including nerve isolation, precise transection, and tension-free nerve coaptation, this protocol offers researchers a robust framework for investigating the mechanisms underlying TMR-mediated analgesia and neuroma prevention.

Protocol

All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC) and comply with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

1. Preoperative preparation

NOTE: Male and female Sprague-Dawley rats (Charles River Laboratories), aged 8–10 weeks and weighing 250–300 g, were used.

1. Turn on the warming pad and prepare a sterile surgical field. Place a sterile pad perpendicular to the body of the rat for optimal exposure.
2. Arrange all surgical tools and materials: sterile saline solution (0.9% sodium chloride), povidone-iodine antiseptic solution, meloxicam, cautery, 10-0 micro suture, 4-0 absorbable braided sutures, ruler, and skin adhesive.
   ​NOTE: All surgical instruments should be sterilized by autoclaving or hot bead sterilization.
3. Induce anesthesia by placing the rat in an induction chamber with 4% isoflurane, oxygen at 10 L/min, and air at 60%. After 4 min or once unresponsive, switch to a nose cone and adjust isoflurane to 1.5%–2.0% depending on rat size.
4. Shave the surgical site from the lower back through the entire hindlimb. Perform a triple prep with alternating povidone-iodine antiseptic solution and alcohol swabs, ending with alcohol.
5. Administer 1 mg/kg meloxicam subcutaneously using a tuberculin syringe (typically 0.2 mL for a 200 g rat).
   NOTE: Throughout the procedure, the animal is monitored for toe and tail pinch responsiveness and respiratory rate. As needed, the flow rate of isoflurane is adjusted or the procedure is stopped to intervene.

2. Below-knee amputation (Figure 1)

1. Use a ruler to mark the knee joint line and make two additional marks 2 cm and 3 cm distal to the knee.
2. Make a circumferential skin incision 3 cm distal to the knee using a scalpel.
3. Use blunt and sharp dissection to separate the skin circumferentially from the underlying musculature until it retracts freely.
4. Repeat this undermining process with the underlying muscles, carefully dissecting down to the bone while preserving tissue planes. Mark the tibia/fibula 1 cm proximal to the circumferential incision using a sterile cautery or marker.
5. Use small bone scissors to transect the tibia and fibula at the marked location. Use cautery to achieve hemostasis. Examine the bone end for sharp edges and blunt or trim as needed.
6. Using a 4-0 absorbable braided suture, close the anterior and posterior muscle compartments together over the bone to create a muscle pad. Begin by suturing the front edge over the tibia, the primary pressure point. Typically, 4–6 interrupted sutures are used.
7. Close the skin laterally with interrupted 4-0 absorbable braided sutures, avoiding placement directly on the weight-bearing stump. Apply skin adhesive along the incision line.

3. TMR intervention surgery (Figure 1)

1. Make a 1.5–2 cm longitudinal gluteal splitting incision approximately 5 mm posterior to the femur.
2. Use blunt dissection to separate the gluteal and hamstring muscles, exposing the sciatic nerve just proximal to its trifurcation into the tibial, common peroneal (CP), and sural nerves.
3. Identify proximal motor branches: one large branch travels beneath the caudofemoralis (CFM), giving rise to the branch to CFM, semimembranosus (SM), and biceps femoris (BF).
4. Elevate the CFM with a cotton swab and divide it. Neurolyze the SM and BF branches carefully with blunt dissection under a surgical microscope using microsurgical fine Dumont forceps.
   1. Use forceps to grab the epineurium and, with fine curved spring scissors, open the epineurium and divide it longitudinally, and separate the different branches from each other.
   2. Preserve the CFM branch and divide the others near the sciatic trunk. Curve motor branches inferiorly to facilitate coaptation.
5. Neurolyze the sciatic trifurcation by opening the epineurium and dividing it with fine curved spring scissors. Gently tease the nerve branches apart with fine forceps. Grip each of the three branches (CP, tibial, sural) 5–10 mm distal to the trifurcation using forceps.
6. Place tension and transect each nerve as distally as possible. Gripping the cut distal end, make a clean cut to the proximal end by excising at least 5–10 mm.
7. Perform the coaptations with 10-0 nylon suture: the CP to SM using one suture, tibial to the large BF branch using one to two sutures, and sural to the small BF branch using one suture. Use at least 3 knots per suture, ensure tension-free placement, avoid tightening enough to crease the nerve, and lay out coaptations without sharp angulation.

4. Amputation-only group

1. Ligate CP, tibial, and sural nerves using a 5-0 silk suture. Handle the silk with Dumont suture tying forceps. Ensure the ligature creases the nerve and is secured with 4 square knots.
2. Gently pull each nerve proximally using the knot and transect 1–2 mm distal to the ligature. Remove 10 mm of the distal nerve segment.
3. Proceed with amputation as detailed in section 2.

5. Postoperative care

1. Return the rat to a warmed cage until ambulatory. Provide a second meloxicam dose (0.2 mL) subcutaneously 24 h later.
2. Monitor daily for 7 days for wound integrity, signs of infection, pain or distress, and intervene as necessary per IACUC guidelines.

Results

This hindlimb amputation model reliably induced neuropathic pain-related behaviors while preserving ambulation and exploratory capacity in rats. As shown in Figure 2, transection of the peripheral nerve during amputation without a distal target results in disorganized axonal growth and neuroma formation at the ligation site, visualized by B-tubulin and calcitonin gene-related peptide (CGRP) immunostaining. In contrast, amputated nerves treated with TMR demonstrated organized axonal growth in...

Discussion

This protocol details essential surgical steps to ensure reproducibility of the hindlimb amputation model and demonstrates preservation of ambulation and behavioral engagement in post-amputation testing. Key surgical principles, including careful anatomic identification, minimal nerve manipulation, and tension-free handling of transected nerves, are emphasized to ensure consistency and reproducibility across surgeons. These technical considerations are critical for reliably inducing post-amputation nerve pathology and mi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-0 nylon microsuture (Arosurgical)	AROSurgical	T04A10N07-13	Fine nerve coaptation suture. 10-0 micro suture.
4-0 polyglycolic acid suture	Ethicon	AQ1268	Closure suture for muscle and skin. Absorbable 4-0 suture.
5-0 silk surgical suture	Generic	Generic	Nerve ligation suture
Adult Sprague-Dawley rats	Charles River Laboratories	Generic	Experimental animal
Alcohol prep pads	Dukal Corporation	Generic	Skin antiseptic
Betadine 10%	Avrio Health LP	Generic	Antiseptic
Bone scissors	FST	16030	Bone cutting instrument
Cautery device	FST	18010-00	Hemostasis device
Cotton tip applicator (ProAdvantage)	ProAdvantage	Ref 76100	Tissue manipulation
Curved microdissector scissors	FST	15019-10	Fine scissors for neurolysis
Forceps (fine Dumont)	FST	11254-20	Nerve handling instrument
Forceps (suture tying Dumont)	FST	11063-07	Suture handling instrument
Gauze USP type VII (Cardinal Health)	Cardinal Health	Ref 2146	Maintains sterile field
Heating pad warmer (TC1000 temperature controller)	Generic	TC1000	Temperature control device
Induction chamber (Braintree Scientific)	Braintree Scientific Inc.	Generic	For anesthesia induction
Isoflurane (Isospire)	Phoenix	Generic	Anesthetic agent
Meloxicam (Ostilox)	VetONE	5 mg/mL	Analgesic agent
Ruler	Generic	Generic	Measurement tool for anatomical landmarks
Sterile saline	Baxter	0.9 Sodium Lot Y425131	Irrigation solution
Surgical blade 15	FST	10015-00	Skin incision tool
Tuberculin syringe	Generic	Generic	Medication administration
Vaporizer (Surgivet model 100)	Surgivet	Model 100 S/N W2211123	For anesthesia maintenance
Vetbond tissue adhesive	World Precision Instruments	Generic	Skin adhesive