The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.
1. Preparing Animals for Surgery
<ol>
	<li>Anesthetize mice with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) via subcutaneous inguinal injection with a sterile 1 ml insulin syringe. Carrier solution contains a mixture of 0.9% saline, 17.4 mg/ml ketamine, and 2.6 mg/ml xylazine. Place animals back in cages while waiting for medication to take effect. 
	NOTE: If the loading dose does not provide sufficient anesthesia for the duration of the procedure, an additional 25% of the loading dose may be injected.</li>
	<li>Monitor animals post injection to check for steady respiratory rates and appropriate depression of arousal levels. Check arousal level with a hind foot pinch, which should elicit no response when sufficiently anesthetized. 
	NOTE: This usually takes 3-5 min for a young adult mouse averaging 25 to 30 g. If the animal is still responsive after 10 min post injection, an additional 25% of the anesthetic loading dose may be injected.</li>
	<li>Apply petrolatum and light mineral oil ophthalmic ointment to the animal&#39;s eyes to prevent dryness. Remove animals from cage and place on a clean, flat surface. Shave the desired hind limb from foot to pelvis using an electric hair trimmer, exposing only the lateral aspect of the limb.</li>
	<li>Apply a chemical hair remover to the shaved site for 1 min. Manually remove the hair using laboratory wipes. Clean the depilated area with laboratory wipe soaked in ethanol.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Sterilize surgical instruments via autoclave or other appropriate method. Clean the surgical site and surgical board with 80% ethanol/H20. Disinfect the surgical site with proviodine.&#160;Place mouse on surgical board and align with limb restraints. Keep the target hind limb in an anatomically natural position with the knee joint slightly extended without internal or external rotation.</li>
	<li>Place animal and board under the surgical microscope. Orient to the proper incision site via palpation of superficial landmarks, specifically the bony knee joint and the ridge between the tibialis anterior and gastrocnemius muscles.</li>
	<li>Make an approximately 3 cm incision through the skin using a scalpel or spring scissors while using general forceps for gripping. Make the incision perpendicular to the underlying course of the common fibular nerve.</li>
	<li>Continue the incision through the superficial fascia, exposing the biceps femoris and vastus lateralis muscles. Separate these muscles by cutting through the connecting deep fascia. A 1-2 cm cut should be sufficient.</li>
	<li>Retract the biceps femoris muscle caudally using mechanical retractors, revealing the common fibular nerve.</li>
	<li>Trace the nerve proximally until its intersection with the tendon of the lateral head of the gastrocnemius muscle is found. Note: Exposure may require additional manipulation of the retracted skin and muscle. This intersection is used as the stable landmark for the nerve injury.</li>
	<li>Grasp the nerve with a fine forceps, aligning the tips in a parallel fashion to the lateral border of the gastrocnemius tendon. Crush the common fibular nerve by applying steady, hard pressure for 5 sec.</li>
	<li>Corroborate full crush of the nerve by visual inspection through the surgical scope. It will appear translucent at the site of injury. If using mice expressing fluorescence proteins in peripheral axons, the fluorescence will disappear from the site of injury.</li>
	<li>Remove retractors and realign muscles in their anatomic positions. Close the incision site with 6-0 silk sutures. 1-3 simple interrupted sutures is sufficient. Place recuperating mouse on a heating pad in a clean cage.</li>
	<li>Monitor all animals for 2 hr post-operation to check for breathing and any adverse reactions to the anesthesia. Administer an initial dose of buprenorphine 0.05-0.10 mg/kg via subcutaneous inguinal injection immediately following recovery from surgery. Give 3 additional doses every 12 hr over the next 48 hr. After full recovery, return mice to the animal care facility.</li>
</ol>
3. Isolation and Staining of Extensor Digitorum Longus (EDL) Muscles
<ol>
	<li>Sacrifice animals using isoflurane. Dispense 0.5 ml liquid isoflurane into a 50 ml tube packed with absorbent labwipes. Place the uncapped tube with the animal in a sealed 2,500 cm3 chamber. At least 4 min of exposure is sufficient. Test for loss of bilateral palpebral, toe-pinch, and tail-pinch reflexes to ensure that each animal is unconscious before proceeding with perfusion.</li>
	<li>Transcardially perfuse16 animals first with 10 ml 0.1M PBS, then 25 ml of 4% paraformaldehyde in 0.1M PBS (pH 7.4). Heparin (30 units/20 g animal weight) may be added with the PBS (10 units/ml) to prevent blood clotting in small capillary beds, improving perfusion results.</li>
	<li>Remove the skin covering hind limbs by using scissors to cut transversally through the skin around the circumference of the abdomen. Peel down the skin past the hind limbs and feet using forceps.</li>
	<li>Remove superficial fascia of hind limbs by grasping and peeling with forceps. If using mice expressing fluorescence proteins in peripheral axons, post-fix whole mice overnight in 4% PFA in 50 ml tubes. Rinse three times with PBS. 
	NOTE: Fixed mice can be stored in PBS at 4 &#176;C. If not, skip this step and proceed to step 3.6 without post-fixing animals.</li>
	<li>Dissect out EDL muscles18 from mouse hind limbs, being sure to keep proximal and distal tendons as intact as possible.</li>
	<li>Incubate EDL muscles in blocking buffer (1x PBS containing 0.5% Triton X-100, 3% BSA and 5% goat serum) for at least 1 hr.</li>
	<li>To visualize motor axons and their nerve terminals, place muscles in tubes containing neurofilament (1:1,000) and synaptotagmin-2 (1:250) antibodies diluted in blocking buffer for 3 days. Wash muscles three times with 1x PBS and 10 min each time. Note: Skip this step if using mice expressing fluorescence proteins (XFP) in peripheral axons.</li>
	<li>Stain the contralateral uninjured EDL as a positive control for complete NMJ innervation. Negative controls should include an EDL obtained at 4 days post-injury, a time point where the NMJ is completely denervated, as well as an EDL stained with secondary antibody only.</li>
	<li>Incubate muscles with appropriate fluorescently tagged secondary antibodies to detect neurofilament and synaptotagmin-2 for 1 day. Wash muscles three times with 1x PBS and 10 min each time. Note: This step can be carried out together with step 3.10. Skip this step if using mice expressing fluorescence proteins in peripheral axons.</li>
	<li>To visualize the postsynaptic region of the NMJ, incubate muscles with 5 &#181;g/ml Alexa-555 conjugated alpha-bungarotoxin diluted in blocking buffer for at least 2 hr. Wash muscles three times with 1x PBS and 10 min each time.</li>
	<li>To mount whole muscles on positively charged glass slides, place the muscle directly on the slide, add a few drops of glycerol based mounting medium on the slide and cover with a coverslip. Press the coverslip against the slide to flatten the muscle. Soak off mounting media from the perimeter of the slide and coverslip using laboratory wipes. Apply nail polish to seal the edges between the coverslip and slide.</li>
</ol>
4. Imaging and Data Analysis
<ol>
	<li>To analyze the structure of NMJs, image the EDL muscle using a confocal laser scanning microscope equipped to excite 488, 555 and 633 nm light and capture the emitted light with 20X and 40X objectives.</li>
	<li>To visualize whole NMJs, create maximum intensity projection images of optical sections spaced 1 to 2 &#956;m apart extending from the lowest to highest visible regions of the NMJ. Create maximum intensity projections using commercially available imaging software.</li>
	<li>To determine rates of reinnervation, categorize NMJs as: 1) completely denervated = postsynaptic site is completely devoid of contact with axon, less than 5% colocalization between the axon and AChRs. 2) Partially innervated = the axon partially overlaps the postsynapse, 5-95% colocalization between the axon and AChRs. 3) Full innervation = nearly perfect apposition between the pre- and post-synapse, greater than 95% colocalization between the axon and AChRs. Exclude NMJs that lie perpendicular to the imaging plane or are not fully visualized in the image. Note: In all these experiments, at least 3 animals and 50 NMJs per animal were examined. Results were deemed significant using a student t-test with a P value of less than 0.05.</li>
	<li>To blind the operator, separate individuals can perform the surgery and image analysis. Without knowledge of the treatment groups, the analyzer can be objective with NMJ scoring. Alternatively, images can be randomized and presented to the operator for analysis without knowledge of the source animal.</li>
</ol>
5. Quantitative PCR
<ol>
	<li>Sacrifice animals using isoflurane and cervical dislocation. Remove skin and superficial fascia covering leg muscles according to step 3.3. Dissect tibialis anterior and EDL muscles according to step 3.4.</li>
	<li>Flash freeze the entire tibialis anterior and EDL muscles in a 1.5 ml tube over liquid nitrogen. Remove tissue from tube and place in a pre-chilled mortar partially submerged in liquid nitrogen. Grind frozen muscle into a fine powder using a mortar and pestle.</li>
	<li>Dissolve frozen muscle powder into a commercially available RNA extraction reagent and perform RNA extraction and genomic DNA removal with a commercially available kit according to manufacturer&#39;s instructions.</li>
	<li>Perform reverse transcription with a commercially available reverse transcriptase mix according to manufacturer&#39;s instructions.</li>
	<li>Perform qPCR using a commercially available kit using appropriate housekeeping genes (see table of materials). Use a commercially available quantitative PCR thermal cycler to carry out PCR (see table of materials).</li>
	<li>Set annealing temperature to 58 &#176;C. Adjust additional cycling parameters to specifications of the manufacturer of taq polymerase/SYBR green mix. Include a final melt curve step in thermal cycler program consisting of 0.5 &#176;C incremental increases from 65 &#176;C to 95 &#176;C to test for primer specificity and primer dimer formation.</li>
	<li>Determine relative mRNA expression levels by the 2-&#916;&#916;CT method21 using 18S RNA as the control gene.</li>
</ol>

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J., Alton, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+age+impairs+the+response+of+the+neuromuscular+junction+to+nerve+transection+and+repair:+An+experimental+study+in+rats.">How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats.</a> J Orthop Res. 27 (3), 385-393 (2009).</li><li>Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+neuromuscular+innervation.">Age-related changes in neuromuscular innervation.</a> Muscle Nerve Suppl. 5, S83-S87 (1997).</li><li>Valdez, G., Tapia, J. C., Lichtman, J. W., Fox, M. A., Sanes, J. 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The authors have nothing to disclose.

The method presented in this manuscript provides unique opportunities to identify mechanisms involved in repairing neuromuscular junctions (NMJ). This method involves crushing the common fibular nerve as it passes over the gastrocnemius tendon near the knee. We show that after only 5 sec of nerve compression with a forceps, complete degeneration is noted by 4 days after injury. In young adult mice, alpha-motor axons begin to reinnervate previous synaptic sites in the extensor digitorum longus muscle (EDL) at 7 days post-...

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an &#945;-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate1. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of &#945;-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles and peripheral nerves2-5. These insults often result in degeneration of presynaptic nerve endings, leaving muscles denervated and significantly altering motor skills. For this reason, the identification of molecules that function to maintain and repair the NMJ has become a priority. Because peripheral nerves regenerate and reinnervate targets, peripheral nerve injury models have been used to identify molecular changes associated with regenerating NMJs. 
Peripheral nerve injury models often involve either completely cutting or crushing specific nerve branches6. Following a cut, the endoneurial tube has to be reformed, delaying axonal regeneration and reinnervation of target cells and tissues. The severity of this type of injury also causes axons to meander away from their original path, resulting in their failure to reach original targets. This is in contrast to nerves injured via crush where the endoneurium remains contiguous, providing a path for efficient and proper regrowth of regenerating axons. It also allows axons to find and reinnervate their original muscle fiber partners. Irrespective of injury model, there are a number of cellular and molecular changes that must occur for axons to regenerate and reinnervate targets. After an injury, the nerve segment proximal to the target is broken down and removed via a process termed Wallerian Degeneration7. This process involves reprogramming and de-differentiation of Schwann cells into non-myelinating cells that secrete regenerative factors, clear myelin, and recruit macrophages to the site of injury8. Macrophages in turn complete the clearance of myelin and axonal debris, which would otherwise impede growth of the regenerating axon9. In parallel, motor and sensory neurons activate mechanisms needed to promote regeneration of their severed axons. Once the regenerating axon reaches the target, it must transform from a growth cone to a nerve ending capable of properly transmitting (for motor axons) or receiving (for sensory axons) information10. In this regard, alpha-motor axons undergo a series of well-orchestrated changes that culminate in their growth cone differentiating into a fully functional presynaptic nerve ending that nearly perfectly opposes the post-synaptic site on the target muscle fiber11. 
The sciatic, tibial and accessory nerves have been the primary choices for studying axonal and NMJ regeneration12-14. However, there are a number of drawbacks when using these models to examine cellular and molecular changes associated with regenerating NMJs between animals and under different conditions. Firstly, the sciatic nerve supplies the majority of the muscles of the hind limb, with injury significantly limiting both movement and sensation. It is therefore not possible to use this method to study the impact of exercise alone or in combination with other factors. Additionally, the sciatic nerve is a rather thick structure and thus requires a large amount of compressive force to fully injure all axons. This in turn may result in complete transection of the more superficial axons while leaving the endoneurial tube of deeper lying axons intact, introducing significant variability in the rate and fidelity of regeneration among these axons. Complete transection of this nerve is even less desirable given that many axons will fail to reconnect with the same muscle fibers. Complicating matters, the sciatic nerve possesses intrinsic anatomic variability, both in the number and site of origin of its terminal nerve branches. It is therefore very difficult to lesion the same site. While the tibial nerve is smaller and more amenable to crush injuries, there is also no readily available landmark to serve as a lesion site for this nerve branch. 
The accessory nerve branch (part of cranial nerve XI) that supplies the sternocleidomastoid muscle has also been used to study regeneration of NMJs15. This nerve is particularly attractive because NMJs in the sternocleidomastoid muscle can be more readily imaged in live animals compared to NMJs in other muscles. But similar to the sciatic and tibial nerves, there is no specific landmark that can be used to injure this nerve in the same location, limiting it as a model for comparing regeneration of NMJs among individual animals of an experimental cohort. An inconsistent lesion site introduces variability in the rates of NMJ reinnervation. Due to these shortcomings, the procedure presented here utilizes the injury of a different peripheral nerve branch to examine regenerating NMJs. 
The common fibular nerve, also called the common peroneal nerve, contains many features that make it a reliable nerve to examine regeneration of NMJs between animals and across different treatments. The common fibular nerve has a predictable anatomic course as it runs over the tendon of the lateral head of the gastrocnemius muscle in the knee, the intersection serving as a stable landmark for lesions. The nerve is accessed through a small and minimally invasive incision near but anatomically segregated from the muscles of interest. The findings presented here demonstrate that regenerating motor axons begin to reform NMJs in the extensor digitorum longus (EDL) muscle 8 days after crushing the fibular nerve in 70 days old young adult female mice. Importantly, the pattern and rate of reinnervation is consistent among animals of the same age and sex and therefore provide a reliable injury model that will significantly hasten our understanding of the cellular and molecular changes required to maintain and repair NMJs.

<table><tbody><tr><td>Ketamine</td><td>VetOne</td><td>501072</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Inc.&amp;nbsp;</td><td>003437&amp;nbsp;</td><td></td></tr><tr><td>Buprenorphine</td><td>&amp;nbsp;Zoopharm</td><td>1Z-73000-150910&amp;nbsp;</td><td></td></tr><tr><td>Nair</td><td>Nair</td><td></td><td></td></tr><tr><td>Kim-wipes</td><td>Kimtech</td><td>34155</td><td></td></tr><tr><td>Electric Razor</td><td>Braintree Scientific</td><td>CLP-64800</td><td></td></tr><tr><td>80% EtOH/H&lt;sub&gt;2&lt;/sub&gt;0</td><td></td><td></td><td></td></tr><tr><td>10% Proviodine</td><td></td><td></td><td></td></tr><tr><td>1 ml Insulin Syringe</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Vannas</td><td>91500-09</td><td></td></tr><tr><td>No. 15 scalpel</td><td>Braintree Scientific</td><td>SSS 15</td><td></td></tr><tr><td>#5 Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>6-0 silk suture on reverse cutting needle</td><td>&amp;nbsp;Suture Express</td><td>752B&amp;nbsp;</td><td></td></tr><tr><td>Rodent Heating Pad</td><td>Braintree Scientific</td><td>AP-R-18.5</td><td></td></tr><tr><td>Alexa 555 conjugated alpha-BTX</td><td>Molecular Probes</td><td>B35451</td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>Olympus Stereo Zoom Microscope</td><td>Olympus</td><td>562037192</td><td></td></tr><tr><td>Zeiss 700 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Variable-flow peristaltic perfusion pump</td><td>Fisher Scientific</td><td>13-876-3</td><td></td></tr><tr><td>Aurum Total RNA Mini Kit</td><td>Bio-Rad</td><td>7326820</td><td></td></tr><tr><td>Bio-Rad iScript RT Supermix</td><td>Bio-Rad</td><td>1708840</td><td></td></tr><tr><td>SsoFast Evagreen Supermix</td><td>Bio-Rad</td><td>1725200</td><td></td></tr><tr><td>Bio-Rad CFX96</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>Puralube Vet ointment</td><td>Puralube</td><td>1621</td><td></td></tr><tr><td>Synaptotagmin-2 antibody</td><td>Antibodies-Online</td><td>ABIN401605</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Antibodies-Online</td><td>ABIN2475842</td><td></td></tr></tbody></table>

the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

The common fibular nerve, also called the common peroneal nerve, arises from the sciatic nerve above the popliteal fossa, where it swings around the head of the fibula to the anterior aspect of the leg (Figure 1A). There it branches into the superficial and deep fibular nerves, together supplying the dorsiflexors of the foot and toes (anterior tibialis, extensor digitorum longus and brevis, and extensor halluces longus muscles), and the everters of the foot (peroneus musc...

Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

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Research

JoVE Journal

Neuroscience

15.6K Views.  Virginia Tech. We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle. 

Video: The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

Bioengineering

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Medicine

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 ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

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 ...

Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

The present protocol describes traumatic peripheral nerve injuries (TPNIs), including precisely calibrated crush, strictly aligned and misaligned laceration, as well as grafted and non-grafted gaps of the sciatic nerve in mice. Custom-designed sensors are developed to gauge nerve trauma, induced with commonly available tools to ensure reproducible post-TPNI outcomes.

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve ...

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.

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 ...