Name: Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice (Video) | JoVE Journal
Uploaded: 2013-11-03T19:00:00.000+00:00
Duration: 10 min 50 s
Description: Learn the tibial nerve transection model for studying denervation-induced skeletal muscle atrophy in mice. This validated surgical method enables researchers to monitor progressive muscle mass loss with minimal morbidity.

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5 minutes

This work was supported by grants from the CIHR Neuromuscular Research Partnership (JNM &#x2013; 90959; to J.A.E.B).

Prior to using this model, investigators must have received approval for the surgical protocol from their institution&#39;s animal use governing body. The model is approved by the Research Ethics Board, Hamilton Health Sciences Corporation, McMaster University (AUP # 10-04-24) and is carried out in strict accordance with the recommendations of the Canadian Council on Animal Care.
1. Mouse Preparation
<ol>
	<li>Weigh the mouse. Induce anesthesia with 5% isoflurane or 2% halothane. The circuit used should ensure adequate scavenging of the anesthetic to protect the surgeon. &#160;After 2-3 min the animal&#39;s respiration will slow. Ensure the blink reflex is absent and pinch the &#160;interdigital spaces in the paw to confirm surgical anesthesia (i.e. no response by the mouse). Apply ophthalmic lubricant to the eye, to prevent drying of the cornea during surgery.</li>
	<li>Shave the lateral thigh and buttock from the sciatic notch to the knee and disinfect with proviodine. Shaving will keep the site of the incision hair free to ensure adequate visualization of the surgical field and to minimize interference with nerve dissection and transection. The sciatic notch, which is superior and posterior to the femur, can be identified by palpation.</li>
</ol>
2. Operative Procedure
<ol>
	<li>Reduce inhalational isoflurane to 2% (halothane 1%) and place the mouse on its side (side intended for surgery facing up), under an operating or dissecting microscope. Alternatively surgery can be performed with surgical loupes since 3.5X magnification is satisfactory for an adult (20-25 g) mouse.</li>
	<li>Don sterile gloves. Identify the sciatic notch by palpation. Using a scalpel, incise the skin of the lateral thigh from the sciatic notch to the knee (approximately 1 cm).</li>
	<li>Gently spread the skin. Identify the biceps femoris muscle, which is the flat superficial muscle of the lateral thigh immediately beneath the skin. Using fine scissors, split the biceps femoris muscle along the muscle fibers and hold open with a spring retractor to expose the sciatic nerve and its branches.</li>
	<li>Identify the sciatic nerve immediately deep to the biceps femoris muscle. It can be identified by its characteristic shiny white color and is approximately 0.8 mm in diameter. It runs from the sciatic notch to the knee, branching into the tibial, peroneal and sural nerves at the level of the popliteal fossa.</li>
	<li>Gently separate the tibial from the peroneal and sural nerve branches with ultrafine forceps and spring microdissecting scissors. The tibial nerve is the largest branch and is usually central. It&#39;s important not to crush the nerve while separating the branches. Holding the nerve just on the outer adventicial layer with the ultrafine forceps, and keeping the nerve slack (not taught), will avoid nerve crush and traction injury.</li>
	<li>For complete and lasting denervation, cut the tibial nerve with microdissecting scissors as distally as possible, carefully avoiding the popliteal vessels. Alternatively, for temporary denervation with expected complete reinnervation in 2-4 weeks, the tibial nerve can simply be crushed with ultrafine forceps for 15 sec instead of transected. (Peripheral nerves regrow following injury and will reinnervate target muscle.)</li>
	<li>If complete denervation is required, suture the end of the transected tibial nerve to the anterior surface of the biceps femoris muscle with 10-0 Nylon and re-approximate the biceps femoris with 5-0 Vicryl to prevent aberrant re-innervation of the gastrocnemius and soleus muscles.</li>
	<li>Close the skin with a running 5-0 Vicryl suture.</li>
</ol>
3. Post Operative Care
<ol>
	<li>Turn off the inhalational anesthetic but maintain the flow of oxygen. Administer buprinorphine (or substitute) analgesic subcutaneously.</li>
	<li>Transfer the mouse to a clean cage with no bedding while waking from anesthesia. Keep on a warming blanket within the cage and under direct observation until ambulating.</li>
	<li>Transfer and house in soft bottom cage (not wire) with ample soft bedding.</li>
	<li>Inspect the operative limb daily for condition of the surgical wound and the foot for the development of decubitus heel ulcers or evidence of chewing. Minor problems can be managed with topical antibiotics or antiseptics such as proviodine. Endpoint indicators requiring animal euthanasia are weight loss, evidence of poor self care (ruffled fur), and hunched posture. In addition, animals with major wound disruption or ulcers that do not heal in 1-2 weeks with topical antibiotics or appear to have pain should be sacrificed.</li>
</ol>
4. Denervated Gastrocnemius and Soleus Muscle Harvest
<ol>
	<li>At the post-operative time point desired, weigh the mouse and sacrifice with CO2 overdose.</li>
	<li>Shave the medial aspect of both the operative and contralateral control legs and clean with alcohol. Place the mouse under an operating or dissecting microscope, or alternatively use surgical viewing loupes for magnification.</li>
	<li>On the operative limb, incise the medial calf skin with a scalpel from the ankle to the knee and circumferentially around the ankle. Gently pull the skin with forceps off the muscle and proximally toward the thigh. This exposes all the muscles of the leg. Identify the gastrocnemius muscle, which is the calf muscle that runs from the knee to the ankle on the posterior aspect of the leg and lies immediately underneath the skin. Identify the distal insertion of the biceps femoris muscle, proximal to the gastrocnemius muscle on the medial aspect of the knee. At its distal insertion the biceps femoris appears thin and filmy and overlies the most proximal portion of the gastrocnemius. Using scissors and blunt end dissection, gently separate the distal insertion of the biceps femoris from the gastrocnemius muscle.</li>
	<li>At its distal insertion into the calcaneous, the gastrocnemius muscle tapers into the Achilles tendon. Identify the Achilles tendon, which appears white and sinewy. Hold the Achilles tendon with forceps, taking care not to hold or crush the gastrocnemius muscle, and divide the Achilles tendon from the calcaneus insertion using scissors.</li>
	<li>Still holding the tendon, gently lift the gastrocnemius muscle (pale red) off the deep soleus (deeper red), from the distal insertion towards its origin at the knee (the soleus can be harvested separately).</li>
	<li>Dissect the gastrocnemius off the leg by dividing the origin of the gastrocnemius from the medial and lateral femur chondyles using scissors. Very gentle traction on the muscle facilitates this process. Be careful not to crush the muscle.</li>
	<li>The soleus will now be plainly visible, immediately under the site of the gastrocnemius. Lift the soleus from its insertion on the Achilles tendon to its origin on the posterior calf. If the soleus is inadvertently raised with the gastrocnemius, gently separate it from the harvested specimen. The gastrocnemius (light red) and soleus (dark red) remain easily identifiable in the harvested specimen due to their color differences.</li>
	<li>Weigh the muscles separately on a precision scale.</li>
	<li>Split the muscles vertically, half for snap freezing in liquid nitrogen (for subsequent protein and/or RNA extraction), and half for histology (i.e. morphometric assessment, immunohistology) fixing either in 10% formalin, or frozen fixation in isopentane cooled with liquid nitrogen, as desired.</li>
	<li>Repeat from step 4.3 above on the control, un-operated, side to harvest control gastrocnemius and soleus muscles.</li>
</ol>

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<li>Kobayashi, J., Mackinnon, S. E., Watanabe, O., Ball, D. J., Gu, X. M., Hunter, D. A., Kuzon, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+duration+of+muscle+denervation+on+functional+recovery+in+the+rat+model%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">The effect of duration of muscle denervation on functional recovery in the rat model.</a> Muscle Nerve. 20, 858-866 (1997).</li>
<li>Bain, J. R., Veltri, K. L., Chamberlain, D., Fahnestock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+functional+recovery+of+denervated+skeletal+muscle+after+temporary+sensory+nerve+innervation%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Improved functional recovery of denervated skeletal muscle after temporary sensory nerve innervation.</a> Neuroscience. , 103-503 (2001).</li>
<li>Batt, J., Bain, J., Goncalves, J., Michalski, B., Plant, P., Fahnestock, M., Woodgett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+gene+expression+profiling+of+short+and+long+term+denervated+muscle%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Differential gene expression profiling of short and long term denervated muscle.</a> FASEB J. 20, 115-117 (2006).</li>
<li>Sher, J., Cardasis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+fiber+types+in+the+adult+mouse%5BTitle%5D)+AND+%22Acta+Neurol.+Scand%22%5BJournal%5D)">Skeletal muscle fiber types in the adult mouse.</a> Acta Neurol. Scand. 54, 45-56 (1976).</li>
<li>Agbulut, O., Noirez, P., Beaumont, F., Butler-Browne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+heavy+chain+isoforms+in+postnatal+muscle+development+of+mice%5BTitle%5D)+AND+%22Biol.+Cell%22%5BJournal%5D)">Myosin heavy chain isoforms in postnatal muscle development of mice.</a> Biol. Cell. 95, 399-406 (2003).</li>
<li>Nagpal, P., Plant, P. J., Correa, J., Bain, A., Takeda, M., Kawabe, H., Rotin, D., Bain, J. R., Batt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin+ligase+nedd4-1+participates+in+denervation-induced+skeletal+muscle+atrophy+in+mice%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">The ubiquitin ligase nedd4-1 participates in denervation-induced skeletal muscle atrophy in mice.</a> PLoS ONE. 7, e46427(2012).</li>
<li>Plant, P. J., Bain, J. R., Correa, J. E., Woo, M., Batt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absence+of+caspase-3+protects+against+denervation-induced+skeletal+muscle+atrophy%5BTitle%5D)+AND+%22J.+Appl.+Physiol%22%5BJournal%5D)">Absence of caspase-3 protects against denervation-induced skeletal muscle atrophy.</a> J. Appl. Physiol. 107, 224-234 (2009).</li>
<li>Varejao, A. S., Meek, M. F., Ferreira, A. J., Patricio, J. A., Cabrita, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+evaluation+of+peripheral+nerve+regeneration+in+the+rat%3A+walking+track+analysis%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Functional evaluation of peripheral nerve regeneration in the rat: walking track analysis.</a> J. Neurosci. Methods. 108, 1-9 (2001).</li>
<li>Willand, M. P., Holmes, M., Bain, J., Fahnestock, M., de Bruin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+muscle+stimulation+after+immediate+nerve+repair+reduces+muscle+atrophy+without+affecting+reinnervation%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Electrical muscle stimulation after immediate nerve repair reduces muscle atrophy without affecting reinnervation.</a> Muscle Nerve. 48, 219-225 (2013).</li>
<li>Sterne, G. D., Coulton, G. R., Brown, R. A., Green, C. J., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophin-3-enhanced+nerve+regeneration+selectively+improves+recovery+of+muscle+fibers+expressing+myosin+heavy+chains+2b%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b.</a> J. Cell Biol. 139, 709-715 (1997).</li>
<li>Plant, P. J., North, M. L., Ward, A., Ward, M., Khanna, N., Correa, J., Scott, J. A., Batt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypertrophic+airway+smooth+muscle+mass+correlates+with+increased+airway+responsiveness+in+a+murine+model+of+asthma%5BTitle%5D)+AND+%22Am.+J.+Respir.+Cell+Mol.+Biol%22%5BJournal%5D)">Hypertrophic airway smooth muscle mass correlates with increased airway responsiveness in a murine model of asthma.</a> Am. J. Respir. Cell Mol. Biol. 46, 532-540 (2012).</li>
<li>Bain, J. R., Mackinnon, S. E., Hunter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+evaluation+of+complete+sciatic%2C+peroneal%2C+and+posterior+tibial+nerve+lesions+in+the+rat%5BTitle%5D)+AND+%22Plast.+Reconstr.+Surg%22%5BJournal%5D)">Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat.</a> Plast. Reconstr. Surg. 83, 129-138 (1989).</li>
<li>Hare, G. M., Evans, P. J., Mackinnon, S. E., Best, T. J., Midha, R., Szalai, J. P., Hunter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Walking+track+analysis%3A+utilization+of+individual+footprint+parameters%5BTitle%5D)+AND+%22Ann.+Plast.+Surg%22%5BJournal%5D)">Walking track analysis: utilization of individual footprint parameters.</a> Ann. Plast. Surg. 30, 147-153 (1993).</li>
<li>McLean, J., Batt, J., Doering, L. C., Rotin, D., Bain, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+rate+of+nerve+regeneration+and+directional+errors+after+sciatic+nerve+injury+in+receptor+protein+tyrosine+phosphatase+sigma+knock-out+mice%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Enhanced rate of nerve regeneration and directional errors after sciatic nerve injury in receptor protein tyrosine phosphatase sigma knock-out mice.</a> J. Neurosci. 22, 5481-5491 (2002).</li>
<li>Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+spared+nerve+injury+%28SNI%29+model+of+induced+mechanical+allodynia+in+mice%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">The spared nerve injury (SNI) model of induced mechanical allodynia in mice.</a> J. Vis. Exp. (54), e3092(2011).</li>
<li>Rogoz, K., Lagerstrom, M. C., Dufour, S., Kullander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VGLUT2-dependent+glutamatergic+transmission+in+primary+afferents+is+required+for+intact+nociception+in+both+acute+and+persistent+pain+modalities%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">VGLUT2-dependent glutamatergic transmission in primary afferents is required for intact nociception in both acute and persistent pain modalities.</a> Pain. 153, 1525-1536 (2012).</li>
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No conflicts of interest are declared.

The tibial nerve transection model of denervation-induced skeletal muscle atrophy is a commonly employed and well validated model in rats. We have adapted this model for use in mice, which allows the investigator to take advantage of the existence of genetically engineered mice and study the process of muscle atrophy in vivo in the absence of proteins crucial to the regulation of muscle mass7,8. The gastrocnemius and soleus muscles, both denervated in this model, can be easily and rapidly dissected wi...

Skeletal muscle denervation, due to traumatic peripheral nerve injury, disease or pharmacologic intervention, results in the immediate loss of muscle voluntary contractile function. Muscle concomitantly begins to atrophy and this atrophy is reversible if timely, good-quality reinnervation occurs1,2. In the absence of reinnervation, myofiber atrophy progresses, and irreversible biologic changes in the muscle occur with muscle fibrosis and myofiber death. Here we demonstrate the tibial nerve transection model, a model of denervation-induced skeletal muscle atrophy and fibrosis, in mice. This model enables scientists to study the physiologic, cellular and molecular biologic mechanisms that underlie muscle atrophy in vivo in the gastrocnemius and soleus muscles. While historically used predominantly in rats, more recent application of this model to knockout and transgenic mouse lines specifically, allows investigators to assess the role of their particular protein(s) of interest in the induction, development and maintenance, or alternatively the resolution of, muscle atrophy and fibrosis in vivo.
The tibial nerve is a mixed motor-sensory peripheral nerve in the rodent hindlimb, and is one of the three terminal branches of the sciatic nerve. Transection of the tibial nerve denervates the gastrocnemius, soleus and plantaris muscles (and the three small deep flexor muscles of the foot including tibialis posterior, flexor digitorum longus and flexor hallicus longus), and is a well standardized and validated model in rats3,4. The gastrocnemius and soleus muscles can be easily dissected at serial time points post tibial nerve transection, fixed and processed for assessment of muscle histology and muscle fiber morphometrics, or flash frozen for extraction of muscle RNA and protein for the purpose of studying, for example, the cellular signaling networks regulating muscle atrophy. The gastrocnemius muscle is a mixed fiber type muscle (type I and type II, although predominantly type II) and the soleus muscle is composed of a large proportion of type I fibers, thereby providing both fast and slow twitch muscle for assessment5,6. The tibial nerve transection model is suitable for studying the process of denervation-induced muscle atrophy in both the short term (days)7 and long term (weeks to months)4,8.
In contrast to the sciatic nerve transection model (a second model of denervation-induced muscle atrophy commonly used in rodents), tibial nerve transection induces less morbidity in the animal, making it a more attractive model. Transection of the sciatic nerve denervates all the muscles of the leg (below the knee) and foot, impairing the animal&#39;s ability to ambulate2, whereas transection of the tibial nerve leaves the peroneal and sural nerve branches of the sciatic nerve intact, thus preserving their target muscles and sensory territories. The mouse is unable to plantar flex or invert the foot, but is able to ambulate easily and the weight bears equally on both hind limbs, thereby significantly diminishing the morbidity of the model. Gait analysis studies evaluating walking patterns have been performed in rats following tibial and sciatic nerve injuries and demonstrate that footprint and weight bearing is better preserved with tibial injury9,10. In addition, in the tibial nerve transection model, the peroneal nerve can be mobilized at a later time point and transferred as a source of delayed reinnervation, if the study design requires3. In contrast, delayed reinnervation in the sciatic nerve transection model necessitates the use of a nerve graft to the sciatic nerve deficit, very significantly increasing the technical difficulty of the model and limiting its use to skilled surgeons.
While the tibial nerve transection model requires familiarity of the operator with sterile operative technique in animal surgery, both the tibial nerve and calf muscles it innervates are easily accessible and identifiable for manipulation, so that individuals who are not surgeons, or highly experienced with animal surgery, can readily master this model.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents and Materials&lt;/strong&gt;</td></tr><tr><td>10-0 Nylon suture</td><td>Ethicon</td><td>2850G</td><td></td></tr><tr><td>5-0 Vicryl suture</td><td>Ethicon</td><td>J553G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Spring microdissecting scissors</td><td>Fine Surgical Tools</td><td>15021-15</td><td></td></tr><tr><td>Ultra fine forceps</td><td>Fine Surgical Tools</td><td>11370-40</td><td></td></tr><tr><td>Non locking micro needle holder (driver)</td><td>Fine Surgical Tools</td><td>12076-12</td><td></td></tr><tr><td>Spring retractor</td><td>Fine Surgical Tools</td><td>17000-02</td><td></td></tr></tbody></table>

tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice

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.

Tibial nerve transection denervates the gastrocnemius, soleus and plantaris muscles of the calf. Here we assess the development of atrophy in the gastrocnemius muscle, as a representative muscle. Gastrocnemius muscle was harvested from 2-3 months old C57Black 6 mice (Jackson Laboratories) denervated for 1, 2, or 4 weeks. Muscle weights progressively decrease (Figure 1), as does the cross-sectional area of type II fast twitch muscle fibers (Figure 2), over time. The gastrocnemius is a mix...

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. ...

The overall goal of this video is to demonstrate the tibial nerve transection model of skeletal muscle atrophy in mice. This is accomplished by isolating the sciatic nerve and its three branches, the tibial, syl, and peroneal nerves. Once isolated, the tibial nerve is transected.After denervation, the wound is sealed and the animal's condition is closely monitored. Ultimately, samples are obtained that show a progressive decrease in muscle mass after denervation. The tibial nerve transection model of denervation induced skeletal muscle atrophy is a commonly employed in well validated model in rats, and this has much less morbidity than that associated with sciatic nerve transection.We have adapted this model for use in mice, which we will demonstrate here. This allows the investigator to take advantage of the existence of genetically engineered mice and study the process of muscle atrophy in the absence of proteins that are crucial to the regulation of skeletal muscle mass while inducing minimal morbidity in the animal. First, record the weight of the mouse after confirming adequate anesthesia by toe pinch, identify the sciatic notch by palpation and shave the lateral thigh and buttock from there to the knee.Next, clean the area with repeated applications of alcohol and pro iodine. Place the mouse on its side under an operating or dissecting microscope while wearing sterile gloves. Identify the sciatic notch by palpation.Incise the skin of the lateral thigh from the sciatic notch to the knee. Gently spread the skin open. Identify the biceps femoris muscle, which is the flat superficial muscle of the lateral thigh immediately beneath the skin using fine scissors.Split the biceps femoris muscle along the muscle fibers and insert a spring retractor. To expose the sciatic nerve and its branches, identify the sciatic nerve immediately below the biceps femoris muscle. It can be identified by its characteristic shiny white color and is approximately 0.8 millimeters in diameter.It runs from the sciatic notch to the knee branching into the tibial peroneal and sal nerves at the level of the popliteal fossa. Gently separate the tibial from the peroneal and sal nerve branches with ultra fine forceps and spring micro dissecting scissors. The tibial nerve is the largest branch and is usually central.Gentle handling of the sciatic nerve and its branches are essential to the success of this procedure. Take care that the sal and perineal nerve branches are not damaged or stretched. Should these branches be damaged or stretched, this will denate their targets as well.Holding the nerve just on the outer adventitial layer with the ultra fine forceps and keeping the nerve slack will avoid nerve crush and traction injury for temporary denervation With expected complete reation in two to four weeks, crush the tibial nerve with ultra fine forceps for 15 seconds for complete and lasting denervation. Cut the tibial nerve with the micros dissecting scissors as distally as possible. Carefully avoiding the popliteal vessels if complete denervation is required.Suture the end of the transected tibial nerve to the anterior surface of the biceps femoris muscle with 10 T nylon and re approximate the biceps femoris with five T Vicryl to prevent abberant reation of the gastros and soleus muscles. Once denervation is complete, close the skin with a running five aught Vicryl suture. When ready, turn off the inhaled anesthetic, but maintain the flow of oxygen.After administering an analgesic singly, house the mouse in a warm clean cage with no bedding. Keep the mouse under direct observation until ambulating. When fully recovered, transfer the mouse to a clean, solid bottom cage with ample soft bedding.Inspect the operative limb and the foot daily for the development of decubitus heel ulcers or evidence of chewing minor problems can be managed with topical antibiotics or antiseptics, such as odine. Endpoint indicators requiring euthanasia of the animal are weight loss, evidence of poor self care and hunched posture. In addition, animals with wound disruption or ulcers they do not heal with one to two weeks of wound care should also be sacrificed.In addition, any animals that are in undue pain At the postoperative time point, desired tissues are collected for analysis. After euthanasia, shave the medial aspect of both the operative and contralateral control legs and clean with alcohol working under a dissecting microscope, incise the medial calf skin with a scalpel from the ankle to the knee and circumferentially around the ankle on the operative limb. Next, gently pull the skin off the muscle and proximally toward the thigh.To expose all the muscles of the leg, identify the gastro muscle, which is the calf muscle that runs from the knee to the ankle on the posterior aspect of the leg. Next, locate the distal insertion of the biceps femoris muscle proximal to the gastro muscle on the medial aspect of the knee. At its distal insertion, the biceps femoris appears thin and filmy and overlies the most proximal portion of the gastro using scissors and blunt end dissection.Gently separate the distal insertion of the biceps femoris from the gastros muscle. Add its distal insertion into the calcaneus, the gastros muscle tapers into the achilles tendon. Identify the achilles tendon which appears white and sinewy.Hold the achilles tendon with forceps. Taking care not to hold or crush the gastric muscle and divide the achilles tendon from the calcaneus insertion. Using scissors still holding the tendon gently lift the pale red gastro muscle off the deeper red soleus from the distal insertion towards its origin at the knee, using very gentle traction.To facilitate the process, dissect the gastro off the leg by dividing the origin of the gastro from the medial and lateral femur condyles. Using scissors. Be careful not to crush the muscle.The soleus will now be plainly visible immediately under the sight of the gastros. Lift the sous from its insertion on the achilles tendon to its origin on the posterior calf. Once removed, weigh the muscles separately on a precision scale.Next, split the muscles vertically half for snap freezing and liquid nitrogen and half for histology. Lastly, repeat these procedures on the control unrated side to harvest control gastro and soleus muscles. Gastro muscles were harvested from denervated and contralateral control hind limbs at one, two or four weeks and expressed as a ratio.Denervation induced a progressive loss of muscle mass over time. The cross-sectional area of myo fibers was measured after immuno staining as seen here. Fast twitch type two fibers stain brown and slow twitch type one fibers, stain white type two fast.Twitch fibers demonstrated a progressive decrease in cross-sectional area in the denervated muscle. Over four weeks After watching this video, you should be able to perform the tibial nerve transection in mice. This provides a reproducible, robust and well validated model of denervation induced skeletal muscle atrophy.

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Medicine

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

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

Generating a Rat Model of Tibial Neuroma Transposition

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

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.

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

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

In vivo Measurement of Knee Extensor Muscle Function in Mice

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.

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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.

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

This protocol demonstrates how to perform a below-knee amputation in rats and to immediately perform targeted muscle reinnervation (iTMR) to investigate strategies to treat and prevent amputation-related pain and to better understand pain mechanisms associated with amputation.

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

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

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

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mechanical Analysis of Isolated Skeletal Muscle

Learn the tibial nerve transection model for studying denervation-induced skeletal muscle atrophy in mice. This validated surgical method enables researchers to monitor progressive muscle mass loss with minimal morbidity.

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

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