Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats

Elisabeth  Maria Brakkee; Erick DeVinney; Niels Eijkelkamp; J. Henk Coert

doi:10.3791/64659

JoVE Journal > Neuroscience

Neurosciences

Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats

Published: January 06, 2023

doi:

10.3791/64659

Elisabeth Maria Brakkee¹, Erick DeVinney², Niels Eijkelkamp³, J. Henk Coert¹

¹Department of Plastic and Reconstructive Surgery,University Medical Center Utrecht, Utrecht University, ²Axogen, ³Center for Translational Immunology,University Medical Center Utrecht, Utrecht University

Summary

This protocol describes the tibial neuroma transposition model, which entails a lesion of the tibial nerve with subsequent transposition of the proximal nerve end toward a subcutaneous pretibial or lateral position. Behavioral testing of neuroma pain and plantar hyperalgesia is quantified using Von Frey monofilaments.

Abstract

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia at the plantar surface of the hind paw innervated by the intact sural nerve. This TNT model is suitable to test therapies for neuroma pain, such as the potential superiority of certain surgical therapies that are already used in the clinic, or to evaluate new drugs and their effect on both pain modalities in the same animal. In this model, a distal lesion (neurotmesis) is made in the tibial nerve, and the proximal nerve end is transposed and fixed subcutaneously and pretibially to enable assessments of the neuroma site with a 15 g Von Frey monofilament. To assess allodynia over the sural nerve, Von Frey monofilaments can be used via the up-down method on the plantar lateral region of the hind paw. After cutting the tibial nerve, mechanical hypersensitivity develops at the neuroma site within 1 week after surgery and persists at least until 12 weeks after surgery. Allodynia at the sural innervated plantar surface develops within 3 weeks after surgery compared to the contralateral limb. At 12 weeks, a neuroma forms on the proximal end of the severed tibial nerve, indicated by dispersion and swirling of axons. For the TNT model surgery, multiple critical (micro)surgical steps need to be followed, and some surgery practice under terminal anesthesia is advised. Compared to other neuropathic pain models, such as the spared nerve injury model, allodynia over the neuroma site can be independently tested from sural nerve hypersensitivity in the TNT model. However, the neuroma site can be tested only in rats, not in mice. The tips and directions provided in this protocol can help research groups working on pain successfully implement the TNT model in their facility.

Introduction

Every wound, varying from simple lacerations to whole limb amputation, is accompanied by varying degrees of peripheral nerve injury. Such nerve injury can result in the formation of a neuroma, a disorganized entanglement of sprouting nerve fibers. Neuromas become painful in 8%-30% of patients, severely impacting their quality of life¹^,²^,³^,⁴^,⁵. After limb amputation, neuroma pain develops in 50% of patients⁶^,⁷^,⁸. Reported symptoms include tenderness, spontaneous pain, allodynia, hyperalgesia, and mechanical or thermal hypersensitivity in the innervated area⁹. When not treated adequately within 1 year, neuroma pain can advance to a chronic pain state, resulting in high societal burden and associated medical costs¹⁰^,¹¹^,¹²^,¹³^,¹⁴. Due to the poor efficacy of current pharmacological interventions, neuroma pain is preferably treated by surgical removal of the painful neuroma, and the nerve treated by various surgical techniques, as described in the literature¹⁵. It is important to note that complete pain relief is rare, pain often worsens over time, and 40% of patients do not benefit from the surgery, indicating that new treatments are needed¹^,¹⁶.

A standardized rat model of neuroma pain aids in understanding the mechanisms that drive neuroma pain, and may help identify new treatments or evaluate existing ones used in the clinic. The tibial neuroma transposition (TNT) model was first described by Dorsi et al. in 2008¹⁷ and has been used by different research groups¹⁸^,¹⁹^,²⁰. The overall goal of this method is to be able to test different treatment techniques for neuroma pain. The advantage of the model over, for example, the spared nerve injury (SNI) model²¹, is that it allows to test allodynia at the neuroma site. This is because the model involves transposing the proximal nerve ending of the tibial nerve to a subcutaneous pretibial position, where it can be probed with von Frey monofilaments. Moreover, allodynia develops at the plantar surface of the hind paw innervated by the intact sural nerve, which can be assessed independently from the neuroma pain in the same animal. This is similar to symptoms of neuroma pain in patients, where persistent neuropathic pain after removal of a painful neuroma is sometimes caused by the neighboring nerves²². Moreover, allodynia over a severed nerve with a neuroma is a different pain modality than allodynia over the intact neighboring nerve. Thus, this model facilitates assessment of the effect of new therapies on both allodynia present at the neuroma site and more widespread neuropathic pain tested in the plantar surface of the hind paw. As the surgery performed to create the TNT model can be challenging, this paper elaborates on the procedure to support researchers implementing the model in their facility.

Protocol

This research was performed in accordance with the IVD (Instantie voor Dierenwelzijn Utrecht) and the guidelines for animal research, project number AVD1150020198824.

1. Von Frey baseline measurements

Prior to the surgery, perform baseline measurements according to the Von Frey testing procedure, described below in section 5 and section 6.

2. Anesthesia and preparation

NOTE: This study was conducted on 15 male Sprague Dawley rats that were 12 weeks old.

Anesthetize the animals by induction with 5% isoflurane and maintain anesthesia with 2%-3% isoflurane.
NOTE: Maintenance with 2% isoflurane usually results in sufficient anesthesia and spontaneous breathing, without the need for tracheal intubation or mechanical ventilation.
Check the animals' reflexes by pinching the foot with tweezers. Be sure that the animal is unresponsive before proceeding. Shave the operative field from knee to ankle with an electric shaver and apply ophthalmic ointment to the eyes to prevent dryness. Inject 0.5 mg/kg of analgesic carprofen subcutaneously in the abdominal region.
Place the anesthetized rat on its back with its head either to the left or right and the leg to be operated on near the surgeon. Exorotate the lower hind limb so that the medial malleolus faces upward. Place the rat under a stereo surgical microscope with 6x magnification.
Disinfect the shaved area with three alternating rounds of iodine-based scrub followed by alcohol. Place a sterile sheet with an operative hole over the leg, so only the operative field is visible. Ensure to maintain these sterile conditions during the surgery.

3. Surgery

Place a small cotton swab below the ankle in order to keep the operative field horizontal. Locate the knee and gently make a longitudinal incision of 1-2 cm using a scalpel over the medial side of the hind paw from mid-calf to ankle. If needed, open the skin and subcutis further with micro scissors until the muscle layers are visible.
Identify the superficial neurovascular bundle as two or three white and a thicker purple/red line, sometimes with minor branches, that can move freely over the muscle layers. Using an electrocautery (see Table of Materials), coagulate any active bleeding or oozing in the operative field. Be careful not to damage the neurovascular bundle.
Bluntly dissect to open the fascia in between the gastrocnemius muscles, just posterior to the superficial neurovascular bundle of 3.2. Between the fascia of the muscles, the tibial nerve can be found. The tibial nerve is about three times the size of the superficial nerve in the neurovascular bundle. Use the tibial bone as an additional landmark (the tibial nerve lies just posterior to the tibial bone).
Identify the tibial nerve and its bifurcation.
NOTE: The bifurcation is usually visible with a lighter line longitudinally over the nerve.
Carefully dissect the tibial nerve free from the surrounding vascular bundles. Perform the dissection by bluntly moving the tibial nerve and cutting the exposed tissue that shows some stretch while moving the tibial nerve.
NOTE: If the tibial nerve is attached to crossing veins after dissection, these veins can be coagulated in order to expose the whole tibial nerve. Be careful to not coagulate the tibial bundle itself.
Expose the tibial nerve proximally until it disappears underneath a crossing muscle layer. At this point, the tibial nerve seems to dive more deeply into the hind paw toward the knee. Expose the tibial nerve distally up to the ankle.
NOTE: When the tibial nerve is exposed more distally, the amount of crossing collagen fibers (i.e., fibers perpendicular to the direction of the nerve fibers) will increase. These collagen fibers need to be cut to enable enough length for transposing the tibial nerve.
1. When the whole tibial nerve is exposed, place the muscle layers back to avoid dehydration of the nerve. If the nerve dehydrates (i.e., it becomes more stiff, dull, and wrinkly), and covering with muscle layers does not suffice, add drops of saline to hydrate it.
Using a blunt microsurgery tool, preferably a micro needle holder, dissect the pretibial skin from the subcutaneous muscle layer in order to make a subcutaneous tunnel. To do this, hold the skin up and push the blunt tip into the tissue, parallel to the skin. Make sure the end of the tunnel is located pretibially or more laterally to ensure easy accessibility of the area for testing of the neuroma.
Increase the isoflurane to 5%. Return to the tibial nerve and expose it (i.e., go back to the place described in step 3.6). Cut the tibial nerve (i.e., both plantar branches) at the most distal level near the ankle. Decrease the isoflurane to the normal level of 2%-3%.
Change the microscope magnification to 10x or 16x. Identify the epineurium of the tibial nerve proximal to the cut made in step 3.8, or in case of a more proximal bifurcation of the tibial nerve, identify the epineurium of both the medial and lateral plantar branches proximal to the cut in step 3.8.
NOTE: The epineurium is whiter and firmer compared to the nerve fibers within, which are more yellow and soft.
Carefully place a 8-0 nylon suture (see Table of Materials) through the epineurium of the proximal nerve end by carefully holding the epineurium with tweezers and putting the needle between the nerve and epineurium with a bite of approximately 0.5 mm. Pull the suture through and take a bite with the needle subcutaneously at the end of the subcutaneous tunnel made in step 3.7. Make a knot, which will transpose the nerve laterally into the subcutaneous tunnel.
NOTE: If both plantar branches share a common epineurium, one suture should suffice. If both plantar branches have their own epineurium, each epineurium should be fixated individually. Avoid placing the suture through the skin; only fix it subcutaneously.
Place a thicker suture with a dark color (preferably a blue or black 4-0 suture) flush to the fixated nerve end, not penetrating the skin. Make sure the suture is visible from the outside of the skin. Check whether the nerve stays in place after moving the paw and muscles. Cut off the suture ends with a slightly longer suture end on the 4-0 than on the 8-0 suture.
Change the magnification of the microscope back to 6x. Close the skin with intraepidermal sutures using the 8-0 suture and gently clean the skin with 0.9% NaCl using a cotton swab.

4. Post-surgical treatment

Place the rat in a clean cage under a paper towel in a comfortable position. If the room is cold, place a heat pad under a part of the cage (only under a part of the cage as the animal should be able to escape the heat when needed). Ensure easy access to food and water.
Do not leave the post-surgical rat unattended until it has regained sufficient consciousness to maintain sternal recumbency. The rat can be returned to the company of other animals when it has fully recovered from anesthesia after surgery. This is usually after 1 h and when the rat exhibits its normal walking pattern and behavior.
At 24 h and 48 h after the surgery, administer a dose of 0.5 mg/kg carprofen subcutaneously (abdominal region) to treat post-surgical pain.

5. Von Frey testing of the plantar side of the hind paws

NOTE: Von Frey testing (step 5 and 6) is performed prior to surgery (for baseline measurement), and from 3 days after the surgery.

Place the rats in mesh wire bottom cages 1 week before baseline measurement, or 2 weeks before the surgery, to ensure acclimatization to the testing environment.
Start with baseline measures at least 1 week before surgery. Make sure three independent baseline measurements are performed on separate days.
Verify that the rats are calm in the mesh wired bottom cages. Apply a series of Von Frey monofilaments with a logarithmic scale perpendicular to the plantar surface of the hind paw.
1. In order to stimulate the sural nerve (hypersensitivity), apply the monofilament on the lateral side close to the hair border. Avoid touching the footpads as these are more sensitive.
2. To stimulate the tibial nerve (hyposensitivity), apply the monofilament in the middle of the plantar surface of the hind paw. If the monofilament is applied in the most medial area, this might also stimulate the saphenous nerve, a branch of the femoral nerve (Figure 1). Avoid touching the footpads.
Start with the 4 g monofilament. Apply sufficient force to the monofilament so that the hair is bending and hold for 3 s, and then check the responses of the animal on the monofilament. A positive response is sudden paw withdrawal, sudden flinching, sudden paw licking, or vocalization. In some cases, the rat moves and tries to find/attack the monofilament.
Choose the next monofilament depending on the response to the stimulus via the up-down method²³. For example, if the rat responds, stimulate next with the 2 g monofilament; if the rat does not respond, stimulate with the 6 g monofilament, and so on. In total, apply 5-10 stimuli depending on the reaction.

6. Von Frey testing of the neuroma site

Handle the animals daily for a minimum of 5-7 days before baseline measures or 2 weeks before surgery. Make sure the animals are held as described in step 6.2, so that they are comfortable with the position.
Hold the rats with their nose pointed toward the elbow fold. If the rat is held in the right hand, their left hind paw should hang freely between the right thumb and index finger (first web space). If the rat is held in the left hand their right hind paw should hang freely between the left thumb and index finger.
Start with baseline measures at least 1 week before surgery. Make sure three independent baseline measurements are performed on separate days.
Verify that the rats are calm and comfortable while being held. At baseline, place the 15 g monofilament gently on the pretibial surface of the exposed hind paw. After surgery, place the 15 g monofilament on the visible suture (e.g., at the location of the neuroma). Apply sufficient force to the monofilament so that the hair is bending and hold for 1 s.
1. Record the reaction to each stimulus. The reaction options include no reaction, slow withdrawal, quick withdrawal, and vocalization. Record the response as 0 points for no reaction and one point for slow withdrawal, quick withdrawal, or vocalization.
Repeat five clusters of five applications of the monofilament, with 2-3 s between each application and 2-3 min or more between the five clusters. In total, each hind paw should have 25 applications of the monofilament with recorded responses.

7. Specimen recovery for histology and preparation

NOTE: Histological examination is performed 12 weeks after initial surgery.

Induce anesthesia and prepare the animals as described in steps 2.2, 2.3, and 2.4.
Gently make an incision of 2-3 cm using a scalpel over the scar that was made by the initial surgery, but be careful not to cut too deep as the nerve is superficially placed.
Determine the position of the neuroma, carefully dissect the neuroma and nerve free from surrounding scar tissue, and place the harvested neuroma in fixative. To evaluate neuroma morphology, the tissue is preferably longitudinally embedded in paraffin or epoxy resin as described by Tork et al.¹⁸.
After harvesting the tissue, euthanize the rats under terminal anesthesia (5% isoflurane) via cardiac puncture or decapitation.
NOTE: It is advised to first harvest the neuroma before killing the rats, because then it is easier to distinguish the neuroma from its surrounding tissue in vivo.

Representative Results

Assessment at the neuroma site showed increased sensitivity to the application of the 15 g von Frey monofilament. At baseline, rats typically responded to 10%-15% (± 13%) of the 25 applications of a 15 g monofilament. The response rate increased to 45%-50% (± 24%) 1 week after TNT surgery. On the contralateral side, the number of responses after surgery was similar to those at baseline (Figure 2A). Around 20% of the rats did not develop a painful neuroma; the response rate did not increase compared to the baseline (Figure 2B). This is comparable to the human situation, where not all patients (50% after amputation) develop pain after the formation of a neuroma. All rats developed a neuroma at the end of the transected and transposed tibial nerve stump 12 weeks after surgery (Figure 3). This neuroma was characterized by swirling axons and microfascicles within collagen depositions.

Transection of the tibial nerve reduced mechanical sensitivity at the middle of the plantar side of the hind paw, innervated by the tibial nerve (Figure 1). The hyposensitivity was present at 1 week after surgery, was significantly different from the contralateral side and baseline from 3 weeks after surgery, and remained until at least 12 weeks after surgery (Figure 4). At the lateral part of the plantar side of the hind paw innervated by the intact sural nerve, the rats developed mechanical hypersensitivity that was significantly different from the contralateral side and baseline from 1 week after surgery (Figure 4). This hypersensitivity persisted to at least 12 weeks after surgery. At the contralateral paw, mechanical sensitivity was not affected compared to baseline in the areas either innervated by the sural or tibial nerve (Figure 4).

Figure 1: Nerve distribution on the plantar side of the hind paw. Red = sural nerve distribution (lateral); purple = tibial nerve distribution (middle); green = saphenous nerve distribution (medial). Please click here to view a larger version of this figure.

Figure 2: Von Frey of the neuroma site. The tibial nerve was transected and the course of mechanical sensitivity over the neuroma site was assessed with a 15 g monofilament applied in five clusters of five applications each, with a total of 25 applications. A response is scored as one point. (A) The neuroma site showed a significantly higher response 1 week after surgery compared to baseline and the contralateral side. N = 15; error bars: standard error of the mean (SEM); mixed model analysis with multiple comparisons and Tukey test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. (B) Individual values of the ipsilateral site show diversity in reaction. Three rats (20%) had a relatively high baseline score, and three rats (20%) did not show any changes in pretibial sensitivity. Please click here to view a larger version of this figure.

Figure 3: Morphology of the neuroma. Histological images of a 12 week old neuroma. (A) Hematoxylin-Eosin staining, (B) Masson's trichrome staining, and (C) Neurofilament staining. Green arrow = tibial nerve just proximal to the neuroma. Orange arrow = neuroma, identified by swirling of axons and diffusion of the fascicles. Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 4: Von Frey of the plantar hind paw (tibial and sural nerve). The tibial nerve was transected and the course of mechanical sensitivity was assessed by Von Frey testing at the plantar surface of the hind paw. The middle part of the ipsilateral operated hind paw innervated by the tibial nerve showed hyposensitivity. The lateral part of the ipsilateral operated hind paw innervated by the sural nerve showed hypersensitivity. The middle and lateral parts of the plantar contralateral hind paw showed no changes in sensitivity compared to the baseline. N = 15; error bars: standard error of the mean (SEM); mixed model analysis with multiple comparisons and Tukey test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Please click here to view a larger version of this figure.

Discussion

Critical steps in the protocol
The TNT model involves cutting the tibial nerve and transposing it laterally and subcutaneously to a pretibial location to enable sensitivity testing of the neuroma, in addition to plantar hyperalgesia over the sural nerve. In the TNT model, it is key that the place of the neuroma is visible to the researchers. Therefore, an albino rat strain is preferred because subcutaneous sutures are easily visible through the skin and the color of the suture should preferably be dark blue or black.

When the surgery is conducted and the tibial nerve is exposed, there is variation in the place (e.g., proximal or distal) of the bifurcation of the tibial nerve. If rats have a proximal bifurcation, it is possible that two nerves can be found (the medial and lateral plantar nerve) proximal to the ankle (Figure 5A), instead of just one tibial nerve (Figure 5B). It is important that both branches are cut and transposed in order to induce plantar hyperalgesia over the sural nerve. One could choose to only transpose one plantar nerve; however, a distinction between the lateral and medial plantar nerve is not easily made on this level and could influence the results. Therefore, it is advised to transpose both nerves. Moreover, some rats might have a more distal bifurcation of the tibial nerve, and transposing only one plantar nerve might be impossible.

Figure 5: Proximal and distal bifurcation of the tibial nerve. Anatomical variation in the level of bifurcation (*) of the tibial nerve. (A) Proximal bifurcation of the tibial nerve; (B) distal bifurcation of the tibial nerve. Abbreviations: MPN = medial plantar nerve, LPN = lateral plantar nerve. Please click here to view a larger version of this figure.

In the original paper by Dorsi et al.¹⁷, a ligature is placed around the proximal tibial nerve end and the nerve is transposed and fixed via this ligature suture. As the ligature around the nerve can induce constriction pain²⁴, an alternative described in this method is to fix the nerve using epineurinal sutures. If the tibial nerve is cut and transposed, it is important that the suture to fix the nerve subcutaneously is placed through the epineurium and not the nerve fascicles themselves, as this could influence pain measures as well. In addition, one should avoid placing the suture through the skin, as rats tend to gnaw any visible sutures, resulting in displacement of the neuroma and thus non-reliable outcomes of pain measures.

When the skin is closed, it is also important to use intra-epidermal sutures to avoid gnawing that will result in an open wound. Moreover, after transposing the tibial nerve, it will be situated in a more superficial layer just under the skin. An open wound in combination with a superficially placed nerve is undesirable.

Von Frey measurements can be performed to test neuroma pain on the neuroma site and to test plantar hyperalgesia over the sural nerve, on the lateral side of the hind paw. The neuroma site is visible after surgery due to the dark color of the suture. For testing the sural nerve hypersensitivity, one location should be chosen where the Von Frey monofilament is applied. This can be proximal or distal to the lateral food pad, but should be approximately the same place during baseline measurements and measurements in the weeks after surgery.

Troubleshooting of the method
If the ratsreact to all stimuli applied to the pretibial area already during baseline measurements, make sure that they are calm and relaxed, and that they are properly acclimatized to the testing area. Repeat baseline measurement until the rats react less to the stimuli. In addition, it is preferable to not wear any perfume while performing measurements. Ideally, rats do not react to the pretibial stimulus when a 15 g monofilament is applied before surgery. However, if the rats are calm, and 50%-100% of the rats still react to the 15 g monofilament, change it to a monofilament that has a 10%-20% response rate during baseline. However, if the monofilament is changed, it is advised to first perform a pilot experiment to test whether the TNT rats react to this lower strength of monofilament. In the initial paper of the TNT model, the neuroma site was measured by applying the monofilament through an opening at the bottom of a Perspex box¹⁷. In pilot experiments, rats were found to react to each stimulus when applied via the bottom of the cage and tended to attack the monofilament. When closely held by the researcher, the rats were in a calm state, resulting in a lower response rate on the monofilament during baseline measurement.

If the proximal tibial nerve end cannot reach far enough in the subcutaneous tunnel, follow the course of the tibial nerve more proximal and remove any collagenous and fatty tissue around the nerve. Cut any minor nerve branches or vessels that keep the nerve fixed to its surrounding. This will give the nerve a wider range of motion to be transposed more laterally. Note that in the original paper by Dorsi et al.¹⁷, the nerve was transposed more laterally. In pilot experiments, it was found to be impossible to reach the lateral position. Therefore, this method describes a pretibial position of the neuroma site.

Limitations of the method
A limitation of the TNT model is that the surgery involves multiple (micro)surgical steps to be followed. Another limitation is that the TNT model is not easily translated to mice. From experience, mice tend to be rather sensitive to stimuli applied at a pretibial area, even with the application of a 0.008 g monofilament.

Significance of the method with respect to existing/alternative methods
In the TNT model, neuroma pain can be tested independently from plantar hyperalgesia over the sural nerve. The latter is also induced in other neuropathic pain models such as the SNI model, but here, neuroma pain cannot be independently tested²¹. In addition, both the tibial and peroneal nerve are cut in the SNI model, resulting in more loss of motor function which results in paralyzed intrinsic muscles of the paw²¹. Because only the tibial nerve is cut at a distal level in the TNT model, the intrinsic muscles of the feet show only a negligible loss of motor function.

Potential applications of the method
Previous research has already shown that the TNT model can be used to test various pain medication, nerve caps, or other surgical tools for neuroma treatment¹⁸^,¹⁹^,²⁰. However, all research groups that are interested in pain could have potential benefit from using the TNT model, as two different pain modalities can be tested in the same animal.

Divulgations

The authors have nothing to disclose.

Acknowledgements

We would like to thank Sabine Versteeg for assisting during microsurgery and Anja van der Sar and Trudy Oosterveld-Romijn from the Common Animal Laboratory (Gemeenschappelijk Dieren Laboratorium) for their help in preparing the microscope and surgical room and taking care of the animals.

This research was funded by Axogen.

Materials

Aesthesio	Linton Instrumentation	514007 until 514015	0.6 g until 15 g monofilaments
Carprofen	Local Veterinary Pharmacy	n/a	The local veterinary pharmacy makes caprofen dilution
Cotton swabs	Nobamed	974255
Electrocautery	Fine Science Tools	18010-00
Ethanol 70%	Interchema BV	400406
Ethilon 4.0	Johnson & Johnson	1854G	IMPORTANT: the color should be blue or black
Ethilon 8.0	Johnson & Johnson	BV130-5
Isoflo, isoflurane Zoetis	Dechra Veterinary Products	B506
Mesh bottom cages	StoeltingCo	57816 and 57824
Micro forceps	Fine Science Tools	11251-35
Micro needle holder	Fine Science Tools	12076-12
Micro scissors	Fine Science Tools	15019-10
Micro tweezers	Fine Science Tools	11254-20
NaCl 0.9%	Trademed	H7 1000-FRE
Needle holder	Fine Science Tools	12004-16
Ophthalmic ointment	Local Veterinary Pharmacy	n/a	The local veterinary pharmacy makes the ophthalmic ointment
Scalpel	Fine Science Tools	10003-12
Scissors	Fine Science Tools	14001-12
Stereo surgical microscope	Leica	A60 F
Sterile sheet with hole	Evercare OneMed	1555-01
Surgical blade nr.15	Fine Science Tools	10015-00
Tweezers	Fine Science Tools	11617-12

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Brakkee, E. M., DeVinney, E., Eijkelkamp, N., Coert, J. H. Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats. J. Vis. Exp. (191), e64659, doi:10.3791/64659 (2023).