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 was conducted on 15 male Sprague Dawley rats that were 12 weeks old.
<ol>
	<li>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.</li>
	<li>Check the animals&#39; 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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Surgery
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Identify the tibial nerve and its bifurcation. 
	NOTE: The bifurcation is usually visible with a lighter line longitudinally over the nerve.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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%.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Post-surgical treatment
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Start with baseline measures at least 1 week before surgery. Make sure three independent baseline measurements are performed on separate days.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Von Frey testing of the neuroma site
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Start with baseline measures at least 1 week before surgery. Make sure three independent baseline measurements are performed on separate days.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
7. Specimen recovery for histology and preparation
NOTE: Histological examination is performed 12 weeks after initial surgery.
<ol>
	<li>Induce anesthesia and prepare the animals as described in steps 2.2, 2.3, and 2.4.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors report to have no conflict of interest. Although this research work was partly financed by Axogen, the company had no influence on the execution of the study and on the results.

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><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 &amp;amp; Johnson</td><td>1854G</td><td>IMPORTANT: the color should be blue or black</td></tr><tr><td>Ethilon 8.0</td><td>Johnson &amp;amp; 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&amp;nbsp;</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&amp;nbsp;</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 ...

surgery-behavioral-testing-tibial-neuroma-transposition-model

Research

JoVE Journal

Neuroscience

1.8K Views.  University Medical Center Utrecht, Utrecht University. 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.

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

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

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

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. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Medicine

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Bioengineering

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

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

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

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.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. Recently, this surgery was creatively applied in the clinical treatment of brain injury and achieved substantial rehabilitation of the paralyzed arm. This functional recovery after the surgery suggests that peripheral sensorimotor intervention induces profound neuroplasticity to compensate for the loss of function after brain damage; however, the underlying neural mechanism is poorly understood. Therefore, an emergent clinical animal model is required. Here, we simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice. Neuroanatomical, electrophysiological, and behavioral experiments helped identify that the transferred nerves of these mice successfully reinnervated the impaired forelimb and contributed to accelerating motor recovery after brain injury. Therefore, the mouse model revealed the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

We simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice, contributing to the study of the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...