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>

<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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M., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+regeneration.">Peripheral regeneration.</a> Annu Rev Neurosci. 30, 209-233 (2007).</li><li>Darabid, H., Perez-Gonzalez, A. P., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+synaptogenesis:+coordinating+partners+with+multiple+functions.">Neuromuscular synaptogenesis: coordinating partners with multiple functions.</a> Nat. Rev. Neurosci. 15 (11), 703-718 (2014).</li><li>Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sciatic+nerve+injury+model+in+pre-clinical+research.">The sciatic nerve injury model in pre-clinical research.</a> J. Neurosci. Methods. 243, 39-46 (2015).</li><li>Batt, J. A. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>VetOne&#160;</td>
			<td>501072&#160;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd Inc.&#160;</td>
			<td>003437&#160;</td>
		</tr>
		<tr>
			<td>Buprenorphine </td>
			<td>&#160;Zoopharm</td>
			<td>1Z-73000-150910&#160;</td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Nair</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim-wipes</td>
			<td>Kimtech</td>
			<td>34155</td>
		</tr>
		<tr>
			<td>Electric Razor </td>
			<td>Braintree Scientific</td>
			<td>CLP-64800</td>
		</tr>
		<tr>
			<td>80% EtOH/H20</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Proviodine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Insulin Syringe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Vannas</td>
			<td>91500-09</td>
		</tr>
		<tr>
			<td>No. 15 scalpel</td>
			<td>Braintree Scientific</td>
			<td>SSS 15</td>
		</tr>
		<tr>
			<td>#5 Forceps</td>
			<td>Dumont</td>
			<td>11252-00</td>
		</tr>
		<tr>
			<td>6-0 silk suture on reverse cutting needle</td>
			<td>&#160;Suture Express</td>
			<td>752B&#160;</td>
		</tr>
		<tr>
			<td>Rodent Heating Pad</td>
			<td>Braintree Scientific</td>
			<td>AP-R-18.5</td>
		</tr>
		<tr>
			<td>Alexa 555 conjugated alpha-BTX </td>
			<td>Molecular Probes</td>
			<td>B35451</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
		</tr>
		<tr>
			<td>Olympus Stereo Zoom Microscope</td>
			<td>Olympus</td>
			<td>562037192</td>
		</tr>
		<tr>
			<td>Zeiss 700 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable-flow peristaltic perfusion pump</td>
			<td>Fisher Scientific</td>
			<td>13-876-3</td>
		</tr>
		<tr>
			<td>Aurum Total RNA Mini Kit</td>
			<td>Bio-Rad</td>
			<td>7326820</td>
		</tr>
		<tr>
			<td>Bio-Rad iScript RT Supermix</td>
			<td>Bio-Rad</td>
			<td>1708840</td>
		</tr>
		<tr>
			<td>SsoFast Evagreen Supermix</td>
			<td>Bio-Rad</td>
			<td>1725200</td>
		</tr>
		<tr>
			<td>Bio-Rad CFX96</td>
			<td>Bio-Rad</td>
			<td>1855196</td>
		</tr>
		<tr>
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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

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

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 ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

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, ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...