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....

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% s....

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