Name: surgery and behavioral testing in the tibial neuroma transposition model in rats
Uploaded: 2023-01-06T13:40:00
Description: Watch this Scientific Journal Video about Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats at JoVE.com

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. W., Kleef, M., Patijn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+of+life+in+chronic+pain+is+more+associated+with+beliefs+about+pain,+than+with+pain+intensity.">Quality of life in chronic pain is more associated with beliefs about pain, than with pain intensity.</a> European Journal of Pain. 9 (1), 15-24 (2005).</li><li>Koch, H., Haas, F., Hubmer, M., Rappl, T., Scharnagl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+painful+neuroma+by+resection+and+nerve+stump+transplantation+into+a+vein.">Treatment of painful neuroma by resection and nerve stump transplantation into a vein.</a> Annals of Plastic Surgery. 51 (1), 45-50 (2003).</li><li>Fisher, G. T., Boswick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroma+formation+following+digital+amputations.">Neuroma formation following digital amputations.</a> Journal of Trauma. 23 (2), 136-142 (1983).</li><li>Bowen, J. B., Ruter, D., Wee, C., West, J., Valerio, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+muscle+reinnervation+technique+in+below-knee+amputation.">Targeted muscle reinnervation technique in below-knee amputation.</a> Plastic and Reconstructive Surgery. 143 (1), 309-312 (2019).</li><li>Jensen, T. S., Krebs, B., Nielsen, J., Rasmussen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phantom+limb,+phantom+pain+and+stump+pain+in+amputees+during+the+first+6+months+following+limb+amputation.">Phantom limb, phantom pain and stump pain in amputees during the first 6 months following limb amputation.</a> Pain. 17 (3), 243-256 (1983).</li><li>Woo, S. L., 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=Regenerative+peripheral+nerve+interfaces+for+the+treatment+of+postamputation+neuroma+pain:+a+pilot+study.">Regenerative peripheral nerve interfaces for the treatment of postamputation neuroma pain: a pilot study.</a> Plastic and Reconstructive Surgery Global Open. 4 (12), 1038 (2016).</li><li>Arnold, D. 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=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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annual Review of Pharmacology and Toxicology. 20 (1), 441-462 (1980).</li><li>Austin, P. J., Wu, A., Moalem-Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+constriction+of+the+sciatic+nerve+and+pain+hypersensitivity+testing+in+rats.">Chronic constriction of the sciatic nerve and pain hypersensitivity testing in rats.</a> Journal of Visualized Experiments. (61), e3393 (2012).</li></ol>

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 ...

Research

JoVE Journal

Neuroscience

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...