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.

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
<ol>
	<li>Prior to the surgery, perform baseline measurements according to the Von Frey testing procedure, described below in section 5 and section 6.</li>
</ol>
2. Anesthesia and preparation
NOTE: This study wa...

<ol>
	<li>Stokvis, A., vander Avoort, D. J., van Neck, J. W., Hovius, S. E., Coert, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+neuroma+pain:+a+prospective+follow-up+study.">Surgical management of neuroma pain: a prospective follow-up study.</a> Pain. 151 (3), 862-869 (2010).</li><li>Domeshek, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+of+neuromas+improves+patient-reported+pain,+depression,+and+quality+of+life.">Surgical treatment of neuromas improves patient-reported pain, depression, and quality of life.</a> Plastic and Reconstructive Surgery. 139 (2), 407-418 (2017).</li><li>Lame, I. E., Peters, M. L., Vlaeyen, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+criteria+for+symptomatic+neuroma.">Diagnostic criteria for symptomatic neuroma.</a> Annals of Plastic Surgery. 82 (4), 420-427 (2019).</li><li>Liedgens, H., Obradovic, M., De Courcy, J., Holbrook, T., Jakubanis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+burden+of+illness+study+for+neuropathic+pain+in+Europe.">A burden of illness study for neuropathic pain in Europe.</a> Clinicoeconomics and Outcomes Research. 8, 113-126 (2016).</li><li>Langley, P. C., Van Litsenburg, C., Cappelleri, J. C., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+associated+with+neuropathic+pain+in+Western+Europe.">The burden associated with neuropathic pain in Western Europe.</a> Journal of Medical Economics. 16 (1), 85-95 (2013).</li><li>Dworkin, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+the+clinical+importance+of+group+differences+in+chronic+pain+clinical+trials:+IMMPACT+recommendations.">Interpreting the clinical importance of group differences in chronic pain clinical trials: IMMPACT recommendations.</a> Pain. 146 (3), 238-244 (2009).</li><li>Mackinnon, S. E., Dellon, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+treatment+of+recurrent+dorsoradial+wrist+neuromas.">Results of treatment of recurrent dorsoradial wrist neuromas.</a> Annals of Plastic Surgery. 19 (1), 54-61 (1987).</li><li>Harden, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neuropathic+pain.+Mechanisms,+diagnosis,+and+treatment.">Chronic neuropathic pain. Mechanisms, diagnosis, and treatment.</a> Neurologist. 11 (2), 111-122 (2005).</li><li>Poppler, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+interventions+for+the+treatment+of+painful+neuroma:+a+comparative+meta-analysis.">Surgical interventions for the treatment of painful neuroma: a comparative meta-analysis.</a> Pain. 159 (2), 214-223 (2018).</li><li>Eberlin, K. R., Ducic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+algorithm+for+neuroma+management:+a+changing+treatment+paradigm.">Surgical algorithm for neuroma management: a changing treatment paradigm.</a> Plastic and Reconstructive Surgery Global Open. 6 (10), 1952 (2018).</li><li>Dorsi, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tibial+neuroma+transposition+(TNT)+model+of+neuroma+pain+and+hyperalgesia.">The tibial neuroma transposition (TNT) model of neuroma pain and hyperalgesia.</a> Pain. 134 (3), 320-334 (2008).</li><li>Tork, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+porcine+small+intestine+submucosa+nerve+cap+for+prevention+of+neuromas+and+associated+pain.">Application of a porcine small intestine submucosa nerve cap for prevention of neuromas and associated pain.</a> Tissue Engineering Part A. 26 (9-10), 503-511 (2020).</li><li>Miyazaki, R., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+morphine,+pregabalin,+gabapentin,+and+duloxetine+on+mechanical+allodynia+is+different+from+that+on+neuroma+pain+in+the+rat+neuropathic+pain+model.">The efficacy of morphine, pregabalin, gabapentin, and duloxetine on mechanical allodynia is different from that on neuroma pain in the rat neuropathic pain model.</a> Anesthesia and Analgesia. 115 (1), 182-188 (2012).</li><li>Tian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+training+reduces+neuroma+pain+by+regulating+neurotrophins.">Swimming training reduces neuroma pain by regulating neurotrophins.</a> Medicine and Science in Sports Exercise. 50 (1), 54-61 (2018).</li><li>Decosterd, I., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spared+nerve+injury:+an+animal+model+of+persistent+peripheral+neuropathic+pain.">Spared nerve injury: an animal model of persistent peripheral neuropathic pain.</a> Pain. 87 (2), 149-158 (2000).</li><li>Poublon, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomical+relationship+of+the+superficial+radial+nerve+and+the+lateral+antebrachial+cutaneous+nerve:+A+possible+factor+in+persistent+neuropathic+pain.">The anatomical relationship of the superficial radial nerve and the lateral antebrachial cutaneous nerve: A possible factor in persistent neuropathic pain.</a> Journal of Plastic, Reconstructive and Aesthetic Surgery. 68 (2), 237-242 (2015).</li><li>Dixon, W. 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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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aesthesio</td>
			<td>Linton Instrumentation</td>
			<td>514007 until 514015</td>
			<td>0.6 g until 15 g monofilaments</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Local Veterinary Pharmacy</td>
			<td>n/a</td>
			<td>The local veterinary pharmacy makes caprofen dilution</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Nobamed</td>
			<td>974255</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Interchema BV</td>
			<td>400406</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 4.0</td>
			<td>Johnson &#38; Johnson</td>
			<td>1854G</td>
			<td>IMPORTANT: the color should be blue or black</td>
		</tr>
		<tr>
			<td>Ethilon 8.0</td>
			<td>Johnson &#38; Johnson</td>
			<td>BV130-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflo, isoflurane Zoetis</td>
			<td>Dechra Veterinary Products</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh bottom cages</td>
			<td>StoeltingCo</td>
			<td>57816 and 57824</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder&#160;</td>
			<td>Fine Science Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Fine Science Tools</td>
			<td>15019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tweezers</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Trademed</td>
			<td>H7 1000-FRE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12004-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment&#160;</td>
			<td>Local Veterinary Pharmacy</td>
			<td>n/a</td>
			<td>The local veterinary pharmacy makes the ophthalmic ointment</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo surgical microscope</td>
			<td>Leica</td>
			<td>A60 F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile sheet with hole</td>
			<td>Evercare OneMed</td>
			<td>1555-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade nr.15</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11617-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgery and behavioral testing in the tibial neuroma transposition model in rats

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.

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% (&#177; 13%) of the 25 applications of a 15 g monofilament. The response rate increased to 45%-50% (&#177; 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 ...

Watch this Scientific Journal Video about Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats at JoVE.com

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

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.

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia ...

This method helps finding better treatments for neuroma pain. The advantage of this technique is that you can measure both neuroma pain and generalized pain independently in the same rep model. The TNT model is particularly fitted to the testing of neuroma pain, as the neuroma itself can be mechanically stimulated with the Von Frey monofilaments.Begin by placing the anesthetized rat on its back with its head either to the left or right. Place the leg to be operated on near the surgeon. Exorotate the lower hind limb so the medial malleolus faces upward.Before placing the rat under a stereo surgical microscope with 6x magnification, disinfect the shaved area with at least three alternating rounds of iodine-based scrub and alcohol. Locate the knee and gently make a longitudinal incision of two to three centimeters over the medial side of the hind paw from midcalf to ankle using a scalpel. If needed, open the skin and subcutis further with microscissors until the muscle layers are visible.Identify the superficial neurovascular bundle as two to three white, purple, or red lines that can move freely over the muscle layers. Keep the neurovascular bundle intact. Dissect the posterior from this bundle.Next, dissect to open the fascia in between the gastrocnemius muscles. Between the fascia of the muscles, the deeper neurovascular bundle with the tibial nerve can be found, which is about three times the size of the superficial neurovascular bundle. Use the tibial bone as an additional landmark, as the tibial nerve flies just posterior to the tibial bone.To dissect the tibial nerve free from the surrounding vascular bundles, bluntly move the tibial nerve and cut the exposed tissue that shows some stretch while moving the tibial nerve. Once the whole tibial nerve is exposed, place the muscle layers back to avoid dehydration of the nerve until the dissection of the pretibial skin from the subcutaneous muscle. To dissect the pretibial skin from the subcutaneous muscle layer and make a subcutaneous tunnel, hold the skin up and push the blunt tip into the tissue parallel to the skin using a blunt microsurgery tool.Ensure that the end of the tunnel is located pretibially or laterally for easy accessibility of the area for testing of the neuroma. After cutting the tibial nerve at the distal end near the ankle, change the microscope magnification to 10x or 16x and identify the epineurium of the tibial nerve proximal to the cut made near the ankle. Or, in the case of a more proximal bifurcation of the tibial nerve, identify the epineurium of both the medial and lateral planter branches proximal to the cut made near the ankle.Carefully place an 8-0 nylon suture through the epineurium of the proximal nerve end by holding the epineurium with tweezers and putting the needle between the nerve and epineurium with a bite of approximately 0.5 millimeters. Take a bite with the needle subcutaneously at the end of the subcutaneous tunnel and pull the suture to transpose the nerve laterally into the subcutaneous tunnel. Fix the suture by making a knot.Place a thicker suture of 4-0 with a dark color flushed to the fixated nerve end without penetrating the skin. Ensure that the suture is visible outside the skin. Check if 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, then on the 8-0 suture. Change the magnification of the microscope back to 6x and close the skin with intraepidermal sutures using the 8-0 suture. Gently clean the skin with 0.9%sodium chloride using a cotton swab.If the room is cold, place a heating pad under a part of a clean cage and place the rat in the cage under a paper towel in a comfortable position. Ensure easy access to food and water. To stimulate the sterile nerve for hypersensitivity, apply the monofilament on the lateral side close to the hair border without touching the foot pads.To stimulate the tibial nerve for hyposensitivity, apply the monofilament in the middle of the planter surface of the hind paw. For neuroma site testing, hold the rats with their nose pointed toward the elbow fold. If the rat is held in the right hand, his left hind paw should hang freely between the right thumb and index finger.After verifying that the rat is calm and comfortable while holding, at baseline, place the 15-gram monofilament gently on the pretibial surface of the exposed hind paw. After the surgery, place the 15-gram monofilament on the visible suture and apply sufficient force to the monofilament to bend the hair and hold for one second. Von Frey of the neuroma site showed that at baseline, rats responded to 10%to 15%of the applications of a 15-gram monofilament.One week after the tibial neuroma transposition surgery, the response rate increased from 45%to 50%On the contralateral side, the responses after surgery were similar to the baseline. Around 20%of the rats did not develop a painful neuroma and the response rate did not increase compared to the baseline. At the end of the transected and transposed tibial nerve stump 12 weeks after surgery, all rats developed a neuroma.Transection of the tibial nerve reduced mechanical sensitivity at the middle of the planter side of the hind paw innervated by the tibial nerve. Hyposensitivity present at one week after surgery was significantly different from the contralateral side and baseline from three weeks after surgery and remained until 12 weeks after surgery. At the lateral part of the planter side of the hind paw innervated by the intact sural nerve, the rats developed significantly different mechanical hypersensitivity from the contralateral side and baseline from one week after surgery.It persisted for 12 weeks after surgery. At the contralateral paw, mechanical sensitivity was not affected compared to baseline in the areas innervated either by the sural or tibial nerve. Remember to create an off-length of the tibial nerve for range of motion for lateral transposition.Also remember to mark this neuroma site with a dark suture.

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Neuroscience

1.7K ビュー.  University Medical Center Utrecht, Utrecht University. この方法は、神経腫の痛みに対するより良い治療法を見つけるのに役立ちます。この手法の利点は、同じ担当者モデルで神経腫の痛みと全般性の痛みの両方を個別に測定できることです。TNTモデルは、神経腫自体がVon Freyモノフィラメントで機械的に刺激できるため、神経腫の痛みの検査に特に適しています。麻酔をかけたラットを背中に置き、頭を左または右に?...