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>

<ol>
	<li>Fu, S. Y., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributing+factors+to+poor+functional+recovery+after+delayed+nerve+repair:+prolonged+denervation.">Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation.</a> J. Neurosci. 15, 3886-3895 (1995).</li><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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+duration+of+muscle+denervation+on+functional+recovery+in+the+rat+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+functional+recovery+of+denervated+skeletal+muscle+after+temporary+sensory+nerve+innervation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+profiling+of+short+and+long+term+denervated+muscle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber+types+in+the+adult+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+heavy+chain+isoforms+in+postnatal+muscle+development+of+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+ligase+nedd4-1+participates+in+denervation-induced+skeletal+muscle+atrophy+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+caspase-3+protects+against+denervation-induced+skeletal+muscle+atrophy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evaluation+of+peripheral+nerve+regeneration+in+the+rat:+walking+track+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+muscle+stimulation+after+immediate+nerve+repair+reduces+muscle+atrophy+without+affecting+reinnervation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3-enhanced+nerve+regeneration+selectively+improves+recovery+of+muscle+fibers+expressing+myosin+heavy+chains+2b.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+airway+smooth+muscle+mass+correlates+with+increased+airway+responsiveness+in+a+murine+model+of+asthma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evaluation+of+complete+sciatic,+peroneal,+and+posterior+tibial+nerve+lesions+in+the+rat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+track+analysis:+utilization+of+individual+footprint+parameters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+rate+of+nerve+regeneration+and+directional+errors+after+sciatic+nerve+injury+in+receptor+protein+tyrosine+phosphatase+sigma+knock-out+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spared+nerve+injury+(SNI)+model+of+induced+mechanical+allodynia+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VGLUT2-dependent+glutamatergic+transmission+in+primary+afferents+is+required+for+intact+nociception+in+both+acute+and+persistent+pain+modalities.">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><li>Thornell, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcopenic+obesity:+satellite+cells+in+the+aging+muscle.">Sarcopenic obesity: satellite cells in the aging muscle.</a> Curr. Opin. Clin. Nutr. Metab. Care. 14, 22-27 (2011).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</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></td>
			<td></td>
			<td></td>
			<td>Equipment</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

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...

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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 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. ...

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24.2K Views.  St Michaels Hospital. 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.  

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

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P&#60;0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split&#160;hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split&#160;hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.

Dissociated atrophy of intrinsic hand muscles, termed the split hand, appears to be a specific feature of amyotrophic lateral sclerosis (ALS).&#160;Consequently, a novel neurodiagnostic test, termed the split hand&#160;index, was developed to quantify the clinical phenomenon of the split hand, which differentiated ALS from mimic disorders.

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

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

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

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.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...