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This research was supported by the Medical College of Wisconsin Neuroscience Research Center, the Rita Allen Foundation, and the Plastic Surgery Foundation. The authors thank the Comparative Medicine Unit for animal care and support.

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&#8211;10 weeks and weighing 250&#8211;300 g, were used.
<ol>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: All surgical instruments should be sterilized by autoclaving or hot bead sterilization.</li>
	<li>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%&#8211;2.0% depending on rat size.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Below-knee amputation (Figure 1)
<ol>
	<li>Use a ruler to mark the knee joint line and make two additional marks 2 cm and 3 cm distal to the knee.</li>
	<li>Make a circumferential skin incision 3 cm distal to the knee using a scalpel.</li>
	<li>Use blunt and sharp dissection to separate the skin circumferentially from the underlying musculature until it retracts freely.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#8211;6 interrupted sutures are used.</li>
	<li>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.</li>
</ol>
3. TMR intervention surgery (Figure 1)
<ol>
	<li>Make a 1.5&#8211;2 cm longitudinal gluteal splitting incision approximately 5 mm posterior to the femur.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Preserve the CFM branch and divide the others near the sciatic trunk. Curve motor branches inferiorly to facilitate coaptation.</li>
	</ol>
	</li>
	<li>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&#8211;10 mm distal to the trifurcation using forceps.</li>
	<li>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&#8211;10 mm.</li>
	<li>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.</li>
</ol>
4. Amputation-only group
<ol>
	<li>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.</li>
	<li>Gently pull each nerve proximally using the knot and transect 1&#8211;2 mm distal to the ligature. Remove 10 mm of the distal nerve segment.</li>
	<li>Proceed with amputation as detailed in section 2.</li>
</ol>
5. Postoperative care
<ol>
	<li>Return the rat to a warmed cage until ambulatory. Provide a second meloxicam dose (0.2 mL) subcutaneously 24 h later.</li>
	<li>Monitor daily for 7 days for wound integrity, signs of infection, pain or distress, and intervene as necessary per IACUC guidelines.</li>
</ol>

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<li>Resnik, L., Ekerholm, S., Borgia, M., Clark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+national+study+of+veterans+with+major+upper+limb+amputation%3A+survey+methods%2C+participants%2C+and+summary+findings%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A national study of veterans with major upper limb amputation: survey methods, participants, and summary findings.</a> PLoS One. 14 (3), e0213578(2019).</li>
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<li>Kuiken, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+muscle+reinnervation+for+real-time+myoelectric+control+of+multifunction+artificial+arms%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Targeted muscle reinnervation for real-time myoelectric control of multifunction artificial arms.</a> JAMA. 301 (6), 619-628 (2009).</li>
<li>Valerio, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preemptive+treatment+of+phantom+and+residual+limb+pain+with+targeted+muscle+reinnervation+at+the+time+of+major+limb+amputation%5BTitle%5D)+AND+%22J+Am+Coll+Surg%22%5BJournal%5D)">Preemptive treatment of phantom and residual limb pain with targeted muscle reinnervation at the time of major limb amputation.</a> J Am Coll Surg. 228 (3), 217-226 (2019).</li>
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<li>Dumanian, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+muscle+reinnervation+treats+neuroma+and+phantom+pain+in+major+limb+amputees%3A+a+randomized+clinical+trial%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Targeted muscle reinnervation treats neuroma and phantom pain in major limb amputees: a randomized clinical trial.</a> Ann Surg. 270 (2), 238-246 (2019).</li>
<li>Woo, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+peripheral+nerve+interfaces+for+the+treatment+of+postamputation+neuroma+pain%3A+a+pilot+study%5BTitle%5D)+AND+%22Plast+Reconstr+Surg+Glob+Open%22%5BJournal%5D)">Regenerative peripheral nerve interfaces for the treatment of postamputation neuroma pain: a pilot study.</a> Plast Reconstr Surg Glob Open. 4 (12), e1038(2016).</li>
<li>Dehdashtian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexually+dimorphic+pattern+of+pain+mitigation+following+prophylactic+regenerative+peripheral+nerve+interface+%28RPNI%29+in+a+rat+neuroma+model%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Sexually dimorphic pattern of pain mitigation following prophylactic regenerative peripheral nerve interface (RPNI) in a rat neuroma model.</a> Neurosurgery. 93 (5), 1192-1201 (2023).</li>
<li>Senger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+direct+comparison+of+targeted+muscle+reinnervation+and+regenerative+peripheral+nerve+interfaces+to+prevent+neuroma+pain%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">A direct comparison of targeted muscle reinnervation and regenerative peripheral nerve interfaces to prevent neuroma pain.</a> Neurosurgery. 93 (5), 1180-1191 (2023).</li>
<li>Kuniak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prophylactic+regenerative+peripheral+nerve+interfaces+to+prevent+postamputation+pain%5BTitle%5D)+AND+%22Plast+Reconstr+Surg%22%5BJournal%5D)">Prophylactic regenerative peripheral nerve interfaces to prevent postamputation pain.</a> Plast Reconstr Surg. 144 (3), 421e-430e (2019).</li>
<li>Decosterd, I., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spared+nerve+injury%3A+an+animal+model+of+persistent+peripheral+neuropathic+pain%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Spared nerve injury: an animal model of persistent peripheral neuropathic pain.</a> Pain. 87 (2), 149-158 (2000).</li>
<li>Hu, P., McLachlan, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+reactions+of+cutaneous+and+muscle+afferent+neurons+to+peripheral+nerve+transection+in+rats%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Selective reactions of cutaneous and muscle afferent neurons to peripheral nerve transection in rats.</a> J Neurosci. 23 (33), 10559-10567 (2003).</li>
<li>Welin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Survival+and+regeneration+of+cutaneous+and+muscular+afferent+neurons+after+peripheral+nerve+injury+in+adult+rats%5BTitle%5D)+AND+%22Exp+Brain+Res%22%5BJournal%5D)">Survival and regeneration of cutaneous and muscular afferent neurons after peripheral nerve injury in adult rats.</a> Exp Brain Res. 186 (2), 315-323 (2008).</li>
<li>Jivan, S., Novikova, L. N., Wiberg, M., Novikov, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+delayed+nerve+repair+on+neuronal+survival+and+axonal+regeneration+after+seventh+cervical+spinal+nerve+axotomy+in+adult+rats%5BTitle%5D)+AND+%22Exp+Brain+Res%22%5BJournal%5D)">The effects of delayed nerve repair on neuronal survival and axonal regeneration after seventh cervical spinal nerve axotomy in adult rats.</a> Exp Brain Res. 170 (2), 245-254 (2006).</li>
<li>Garg, R., Uygur, S., Cwykiel, J., Siemionow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+targeted+muscle+reinnervation+model+in+hind+limb+amputated+rats%5BTitle%5D)+AND+%22J+Reconstr+Microsurg%22%5BJournal%5D)">Development of targeted muscle reinnervation model in hind limb amputated rats.</a> J Reconstr Microsurg. 34 (7), 509-513 (2018).</li>
<li>Lago, N., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+long-term+regenerative+potential+in+an+experimental+nerve+amputee+model%5BTitle%5D)+AND+%22J+Peripher+Nerv+Syst%22%5BJournal%5D)">Evaluation of the long-term regenerative potential in an experimental nerve amputee model.</a> J Peripher Nerv Syst. 12 (2), 108-120 (2007).</li>
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<li>Sadler, K. E., Mogil, J. S., Stucky, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innovations+and+advances+in+modelling+and+measuring+pain+in+animals%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Innovations and advances in modelling and measuring pain in animals.</a> Nat Rev Neurosci. 23 (2), 70-85 (2022).</li>
<li>Svientek, S. R., Ursu, D. C., Cederna, P. S., Kemp, S. W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+of+the+composite+regenerative+peripheral+nerve+interface+%28C-RPNI%29+in+the+adult+rat%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Fabrication of the composite regenerative peripheral nerve interface (C-RPNI) in the adult rat.</a> J Vis Exp. (156), e60841(2020).</li>
<li>Zepeda, J. L., Mraz, G., Roth, E., Weihrauch, D., Hoben, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+hindlimb+amputation+model+shows+analgesia+and+sexually+dimorphic+cold+hypersensitivity+with+immediate+targeted+muscle+reinnervation%5BTitle%5D)+AND+%22J+Pain%22%5BJournal%5D)">Rat hindlimb amputation model shows analgesia and sexually dimorphic cold hypersensitivity with immediate targeted muscle reinnervation.</a> J Pain. 33, 105460(2025).</li>
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The authors declare no conflicts of interest.

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...

Chronic pain is one of the most debilitating complications following major limb amputation, affecting up to 70% of patients1,2,3. 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 symptoms6,7,8. 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 injury10,11. 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 pain10,11. 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 injured14,15,16. Prior reports of rodent hindlimb amputation survival surgery are rare17,18. 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 tests19,20.
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 sensitivity22,23.
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.

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

rat hindlimb below-knee amputation model and immediate targeted muscle reinnervation

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

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.

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...

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.

Amputation-related pain reduces patient quality of life, contributes to opioid use, and can reduce functional rehabilitation and prosthesis use. ...

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.

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Research

JoVE Journal

Medicine

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Orthotopic Hind-Limb Transplantation in Rats

Source: Brakkee, E. M., et al. Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats. J. Vis. Exp. (2023).This video demonstrates a procedure to generate a rat tibial neuroma transposition (TNT) model. After exposing the tibial nerve in the hind leg of an anesthetized rat, transect the nerve. Next, create a subcutaneous tunnel, thread the severed nerve through the tunnel, and suture it in place. In the absence of a target for reinnervation, disorganized axonal...

Generating a Rat Model of Tibial Neuroma Transposition

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Orthotopic Hind Limb Transplantation in the Mouse

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Partial Heterotopic Hindlimb Transplantation Model in Rats

This work presents a protocol to enhance prosthetic function after selective nerve transfer surgery. Rehabilitation interventions include patient information and selection, support of wound healing, cortical re-activation of sensory-motor areas of the upper limb, training of selective muscle activation, prosthetic handling in daily life, and regular follow-up assessments.

Therapy Interventions for Upper Limb Amputees Undergoing Selective Nerve Transfers

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI).  This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

The protocol outlines the surgical procedure for the treatment of postamputation pain using Targeted Muscle Reinnervation (TMR). TMR will be compared with two other surgical techniques, specifically Regenerative Peripheral Nerve Interface (RPNI) and neuroma excision, followed by immediate burying within muscle under the context of an international, randomized controlled trial.

Targeted Muscle Reinnervation: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain

This protocol presents a reproducible rat model using sensory nerves to mimic human neuroma pain and assess surgical treatments, offering a valuable tool for translational neuroma research.

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

Medical College of Wisconsin.  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.

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

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