This work was supported by the NIH grants K08NS065157 (to T.A.F.) In addition, the Penn Center for Musculoskeletal Disorders, Award Number P30AR050950 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases supported this work (T.A.F. and Steven S. Scherer). Finally, Shriners Pediatric Research Center Seed funding (T.A.F) supported this work. We would like to acknowledge Dr. Young-Jin Son for initially demonstrating the whole mount procedure and Amy A. Kim for her assistance in producing the sketches of Figure 1.

1. Animal Subjects
1.1. Treatment
<ol>
	<li>All animal procedures should be performed with the approval of the local Institution's Animal Care and Ethics Committee and in accordance with the Use and Committee and National Institutes of Health guidelines, with measures taken to minimize pain and discomfort.</li>
	<li>Our mice were housed under temperature-controlled conditions on a 12-hour reverse light and dark cycle, while fed mouse chow and water ad libitum.</li>
	<li>For studies of adult regeneration, mice should be at least 6 weeks of age when the sciatic nerve crush is performed. This age is beyond the time at which pruning of polyneural neuromuscular junctions take place.</li>
	<li>In these experiments, we used 6-8 week old C57BL/6 mice acquired from Charles River. When comparing regeneration after sciatic nerve crush (i.e.: rate of axon growth) mouse strains should be the same, as differences in axonal regeneration have been noted between different inbred strains15,16. If genetically manipulated mice are to be used, littermate controls are most appropriate.</li>
</ol>
1.2. Surgical Preparation
<ol>
	<li>Animals are deeply anesthetized for surgery using a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. Each animal also receives a subcutaneous injection of meloxicam (10 mg/kg) to minimize post-operative pain.</li>
	<li>Both hindquarters are carefully shaved using a surgical clippers (Roboz, RC-5903) and depilation is completed with Nair hair removal cream (found at local pharmacy).</li>
	<li>Skin is cleansed using sterile cotton tipped applicators and betadine surgical scrub (Fisher Scientific, 19066452).</li>
	<li>Ophthalmic ointment (Fisher Scientific, 19082795) is applied to the eyes using sterile cotton tipped applicators.</li>
	<li>The mouse is placed on a clean stainless steel plate, under which has been placed a pre-heated homeothermic blanket system (Harvard Apparatus, 507222F). Animal temperature is maintained at 37°C.</li>
	<li>All limbs are taped down, with care taken to position the hind limbs symmetrically so that the knee joint makes a right angle with the body (Figure 1, panel A).</li>
	<li>The surgical field is covered with a sterile drape. All instruments are sterilized by autoclave or hot bead sterilization (Fine Science Tools, 18000-45) and the surgeon wears a mask, gown, and sterile gloves.</li>
</ol>
2. Reproducible Sciatic Nerve Crush
<ol>
	<li>After preparation, a semi-circular incision across midline (Figure 1) is made in the skin. The skin is gently dissected from the underlying musculature, and folded over to remain out of the way during the procedure. It is kept moist using applications of 0.1 mL sterile saline (Hospira, 0409-4888-20) during the procedure.</li>
	<li>Opening the fascial plane between the gluteus maximus and the anterior head of the biceps femoris reveals the sciatic nerve (Figure 1, panel A). For a surgical control, the contralateral sciatic nerve should be exposed and mobilized, but left intact. The gluteal musculature is then re-opposed and sutured using a 6-0 braided silk, non-absorbable sutures (Roboz, SUT-1073-11).</li>
	<li>The experimental sciatic nerve is then exposed in the same fashion, with retractors in place to ease visualization (Figure 1, panel B). The retractors are sterilized prior to use. 
	Note: although retractor systems are commercially available, they are often quite expensive. We were able to make a satisfactory retractor system using inexpensive hardware supplies and insect pins (see Materials section).</li>
	<li>The sciatic nerve is then gently freed from the surrounding connective tissue using iridectomy scissors.</li>
	<li>Using a fine 5/45 (Fine Science Tools, 11251-35) forceps, the nerve is placed on the bottom jaw of a super-fine hemostatic forceps (Fine Science Tools, 13020-12). The three fascicles are sequentially aligned, not on top of each other (Figure 1, inset B). The hemostatic forceps have been engraved with a mark at 1.5 mm from their tip. The outermost portion of the sciatic is placed in line with this mark before crush. This ensures a crush of uniform width, and that the nerve does not extend beyond the jaws of the hemostatic forceps when flattened due to the crushing force. If the nerve extends beyond the forceps tip the nerve will only be partially crushed.</li>
	<li>The crush is made perpendicular to the nerve at 45 mm from the third toe, as measured by a thread that approximates the path of the sciatic nerve. The nerve is crushed once for 15 seconds at 3 clicks of the hemostatic forceps. Care is taken not to stretch the nerve. When the hemostats are re-opened, the entire nerve should be translucent at the crush site.</li>
	<li>A second pair of hemostatic forceps (identical to the first) that has been pre-dipped in powdered carbon (Fisher Scientific, C272-500) is used to mark the crush site. The nerve is crushed at the same crush site for 15 seconds at 3 clicks. No carbon marking should extend beyond the boundary of the initial crush. This is particularly important if precise marking of the crush site is required. Prior to use, carbon powder is sterilized by exposure to UV light for two hours, and thereafter is handled using sterile technique.
	<ol>
		<li>To pre-dip the forceps in carbon but preclude widespread carbon at the surgical site, the forceps are opened in powdered carbon, then gently closed (but not clicked) shut, and the carbon on the outside of the hemostats is wiped off using sterile gauze. The forceps are checked under at least 3x magnification to verify that the crushing surfaces are evenly coated in powdered carbon. If necessary, they are re-dipped and wiped.</li>
	</ol>
	</li>
	<li>The gluteal musculature is re-opposed and sutured in the same way as the contralateral side.</li>
	<li>Finally, the skin incision is closed using 9 mm reflex clips (World Precision Instruments, 500346; Applier: 500345). If 9 mm reflex clips are found to restrict movement, smaller reflex clips or 6-0 sutures (Roboz, SUT-1073-11) can be used instead.</li>
</ol>
3. Post-Operative Care
<ol>
	<li>Following the procedure, animals are placed on a heating pad at 37°C until they show signs of movement.</li>
	<li>They are then moved back to their home cage, where water and food are readily accessible on the floor in the form of Hydrogel and wetted food.</li>
</ol>
4. Semi-Thin Preparation
<ol>
	<li>Following an overdose of pentobarbital (300 mg/kg) the leg musculature is removed to expose the sciatic nerve*. With the nerve remaining in situ, the hindquarter is immersed in 2% paraformaldehyde and 2% gluteraldehyde in 0.1 M phosphate buffer on ice for 30 minutes. 
	*If completing whole mount muscle preparation, muscles are harvested before exposing the sciatic nerve. </li>
	<li>The nerve is carefully removed, with care taken to only handle the proximal end. Then the nerve is post-fixed in the same fixative for three additional hours.</li>
	<li>After fixation the nerve is rinsed three times in 0.1 M phosphate buffer.</li>
	<li>The nerve is immersed in 2% osmium tetroxide in 0.1 M phosphate buffer for one hour.</li>
	<li>The nerve is then dehydrated by sequential immersion in increasingly concentrated ethanol (50%, 70%, 80%, 95%, 100%, 100%, 100%). Each immersion is 15 minutes.</li>
	<li>The dehydrated nerve is incubated twice in propylene oxide for three minutes each.</li>
	<li>Then the nerve is immersed in a 1:1 mixture of propylene oxide and Embed 812 for at least 6 hours (usually overnight).</li>
	<li>Then the nerve is immersed in a 2:1 mixture of propylene oxide and Embed 812 overnight.</li>
	<li>Finally, the nerve is immersed in pure Embed 812 for six hours, then embedded in the appropriate mold and baked at 60°C for 48 hours.</li>
	<li>1.0 μm sections are cut from the distal nerve stump at a set distance from the crush site using an Ultracut UCT ultramicrotome (Leica) with a glass knife and stained with toluidine blue. Thin sections may also be produced for examination of ultrastructure by electron microscopy.</li>
</ol>
5. Whole Mount Muscle Preparation
<ol>
	<li>First mice are sacrificed with an overdose of pentobarbital (300 mg/kg) and the hind limbs are removed at the knees.</li>
	<li>Four muscles in the hind limb are removed for analysis: the tibialis anterior (TA), extensor digitorum longus (EDL), soleus, and peroneus longus.</li>
	<li>All muscles are removed via careful dissection and pinned through their connective tissue to a black sylgard (Fisher Scientific, NC9492579) coated dish. They are rinsed in PBS, and then fixed in 4% paraformaldehyde for 30 minutes.</li>
	<li>The TA is then rinsed in PBS, embedded in O.C.T (Fisher Scientific, 14-373-65), and quickly frozen in a bath of acetone and dry ice.
	<ol>
		<li>It is stored at -80 °C for thin sectioning in case whole mount preparation fails. In general, the TA is too thick for adequate whole mounts.</li>
	</ol>
	</li>
	<li>The EDL, soleus, and peroneus longus are rinsed for 3 x 10 minutes in PBS, placed in 0.1 M glycine* (Fisher Scientific, AC12007-0010; diluted in PBS) for 30 minutes, and rinsed 3 x 10 minutes in PBS again. They are then quenched in ice cold 100% methanol for exactly 5 minutes at -20°C, rinsed 3 x 10 minutes in PBS, and bathed in fluorescently conjugated alpha-bungarotoxin (diluted 1:200 in PBS) for 30 minutes. The muscles are rinsed for 3 x 10 minutes in PBS, followed by a 1-hour block in 2 % BSA (KPL, 50-61-00) and 0.2 % Triton X-100 (Dot Scientific Incorporated, 9002-93-1) in PBS*, and incubated overnight while rocking at 4 °C in a cocktail of primary antibodies diluted in the same 2% BSA/0.2% Triton block. 
	*Glycine and blocking solutions are made on the same day as muscle harvesting and stirred for 30-60 minutes at room temperature prior to use. 
	<ol>
		<li>To mark axons and neuromuscular synapses, we use a combination of a mouse monoclonal neurofilament marker (Covance, SMI-312R; diluted 1:1000), a mouse monoclonal synaptic vesicle marker ( SV2 DSHB, diluted 1:1000), and rhodamine conjugated alpha-bungarotoxin (Sigma- Aldrich, diluted 1:200). To mark reactive Schwann cells we use rabbit anti-GAP-43 (Novus Biologicals, NB300-143, diluted 1/500). The primary antibodies are visualized with an IgG1 subtype specific fluorescein conjugated goat anti-mouse secondary antibody and DyLight 649 conjugated donkey anti-rabbit (Jackson ImmunoResearch, diluted 1:200).</li>
	</ol>
	</li>
	<li>The following day, the muscles are rinsed in PBS for 3 x 10 minutes, and incubated in secondary antibodies diluted 1:200 (in 2% BSA/0.2% Triton blocking solution). They are then rinsed 2 x 10 minutes in PBS, followed by a 5-minute immersion in DAPI (Invitrogen, D3571; diluted to 300 nM in deionized water), and another 10-minute rinse in PBS.</li>
	<li>Each muscle is then dissected on a black sylgard (Fisher Scientific, NC9492579) coated petri dish. Initially the tendons are removed at their insertion points into the muscle, and then the muscles are thinned by peeling away interior muscle fibers (Figure 3, panels A-C). Care is taken to preserve exterior surface of the muscle, including the endplate bands (Figure 3, panels D-F). The resulting muscles are mounted on plus slides (Fisher Scientific, 12-550-15) with vectashield (Vector Laboratories, H-1000) and 22 x 40 mm glass coverslips (Fisher Scientific, 12-548-5C) sealed by clear nail polish on two sides. When properly mounted on the slide the endplate band contacts the coverslip.</li>
</ol>
6. Measuring Regeneration
<ol>
	<li>Regeneration may be estimated in all three muscles (peroneus, EDL, and soleus). We often examine regeneration fourteen days after nerve crush, a time at which a substantial percentage of NMJs are re-innervated. Both earlier and later time points are also suitable, depending upon the scientific question being asked. In each muscle, re-innervation of the entire endplate band is examined. Only en face surface NMJs are scored. In this fashion, at least 200 NMJs per muscle may be quickly examined.</li>
	<li>To score muscle re-innervation we determine a "re-innervation ratio". The denominator is the number of denervated neuromuscular junctions as labeled by α-bungarotoxin binding and Schwann cell GAP-43 immunoreactivity. The numerator is the number of re-innervated NMJs as labeled by neurofilament/SV2 immunoreactivity. If needed, NMJs may be classified as either partially (incomplete coverage of α-bungarotoxin by SV2) or fully (complete coverage of α-bungarotoxin by SV2) re-innervated. For examples of re-innervated and denervated NMJs see Figure 3, panels G and H.</li>
</ol>
7. Representative Results
<img alt="Anatomical diagram of rodent leg dissection, highlighting gluteus maximus and biceps femoris." src="/files/ftp_upload/3606/3606fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Schematic of hind limb anatomy important to nerve crush. A. A semi-circular skin incision has been made revealing the underlying musculature. B. The gluteal musculature has been separated, and the sciatic nerve revealed (step 2.4 above). An arrow denotes the approximate crush site. Retractor placement is shown as a general guide, and is adjusted during each surgery to ease visualization of the sciatic and approach of the hemostatic forceps for crush. B (inset): Placement of the sciatic on the lower jaw of the hemostatic forceps just before crush (step 2.5 above). Separate fascicles are labeled to demonstrate that they are adjacent horizontally, but not vertically, during the crush. Although three fascicles are labeled in this diagram, one may also see a fourth fascicle, the articular branch of the peroneal. For a more detailed anatomical drawing of the branching patterns of the sciatic nerve fascicles distal to the crush point, please refer to Greene's Anatomy of the Rat, Figure 18817.
<img alt="Histology and nerve regeneration; electron micrographs highlighting nerve fiber structure and repair process." src="/files/ftp_upload/3606/3606fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. A carbon-marked crush site in situ and toluidine blue-stained, semi-thin sections of crushed sciatic nerve. A. An example of a crush site in situ (left hind limb). Black carbon denotes the crush site. The asterisk marks a branch of the tibial nerve that innervates thigh musculature and serves as a useful landmark during crush surgery. The tibial division of the sciatic nerve is outlined in blue, the peroneal in green, and the sural in yellow. B. Semi-thin sections demonstrating a complete crush performed with hemostatic forceps. C. Semi-thin sections demonstrating an incomplete crush performed with angled forceps. In both images degenerating myelin profiles are marked with arrowheads. In panel C arrows mark examples of preserved myelin profiles and a cluster of spared axons. Scale bar is 10 μm.
<img alt="Muscle biomechanics diagrams and cellular fluorescence microscopy for structural analysis." src="/files/ftp_upload/3606/3606fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Whole mount muscle schematic and representative NMJs. A-C: Rendered images of EDL (A), peroneus longus (B), and soleus (C) muscles after removal from hind limb. Muscles shown are from right hind limb. Knee and ankle anatomical orientation are also included for reference. Tendons are colored white and outlined in a solid black line when positioned on top of the muscle. They are outlined in a black dashed line when they extend beneath the muscle. Cuts are shown in red dashed lines. Muscle is peeled away from cut sites, as indicated by blue arrows, and subsequently thinned. After thinning, the muscle is separated from the pins by cutting around what remains of the pinned tendons. D-F: Rendered images from whole mount muscles after removal of connective tissue and subsequent thinning. The endplate bands are outlined on each muscle. G-H: Soleus whole mount muscle fourteen days after sciatic nerve crush demonstrating re-innervated (G) and denervated (H) neuromuscular junctions. In these panels, NMJs, axons, and Schwann cells are visualized as described above in section 5.5.1. Acetylcholine receptors are red, axons green, and Schwann cell processes blue. In panel G, the arrows indicate areas of the NMJ that have been re-innervated by an axon. In panel H arrowheads indicate GAP-43 positive Schwann cell processes. Note the absence of axons. Scale bar is 10 μm.

<ol><li>Pan, Y. A., Misgeld, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+neurotoxic+and+neuroprotective+agents+on+peripheral+nerve+regeneration+assayed+by+time-lapse+imaging+in+vivo%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.</a> J. Neurosci. 23, 11479-11488 (2003).</li>
<li>Magill, C., Tong, A., Kawamura, D., Hayashi, A., Hunter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reinnervation+of+the+tibialis+anterior+following+sciatic+nerve+crush+injury%3A+A+confocal+microscopic+study+in+transgenic+mice%5BTitle%5D)+AND+%22Exp.+Neurol%22%5BJournal%5D)">Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice.</a> Exp. Neurol. 207, 64-74 (2007).</li>
<li>Amado, S., Simñes, M. J., Armada-da-Silva, P. A. S., Luís, A. L., Shirosaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+hybrid+chitosan+membranes+and+N1E-115+cells+for+promoting+nerve+regeneration+in+an+axonotmesis+rat+model%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.</a> Biomaterials. 29, 4409-4419 (2008).</li>
<li>Luís, A. L., Rodrigues, J. M., Geuna, S., Amado, S., Simðes, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+cell+transplantation+effects+on+sciatic+nerve+regeneration+after+a+standardized+crush+injury+in+the+rat%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat.</a> Microsurgery. 28, 458-470 (2008).</li>
<li>Luís, A. L., Amado, S., Geuna, S., Rodrigues, J. M., Simðes, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+functional+and+morphological+assessment+of+a+standardized+rat+sciatic+nerve+crush+injury+with+a+non-serrated+clamp%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.</a> J. Neurosci. Methods. 163, 92-104 (2007).</li>
<li>Baptista, A. F., de Souza Gomes, J. R., Oliveira, J. T., Santos, S. M. G., Vannier-Santos, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+approach+to+assess+function+after+sciatic+nerve+lesion+in+the+mouse+-+adaptation+of+the+sciatic+static+index%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">A new approach to assess function after sciatic nerve lesion in the mouse - adaptation of the sciatic static index.</a> J. Neurosci. Methods. 161, 259-264 (2007).</li>
<li>Ronchi, G., Raimondo, S., Varejão, A. S. P., Tos, P., Perroteau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardized+crush+injury+of+the+mouse+median+nerve%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Standardized crush injury of the mouse median nerve.</a> J. Neurosci. Methods. 188, 71-75 (2010).</li>
<li>Berg, A., Zelano, J., Cullheim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netrin+G-2+ligand+mRNA+is+downregulated+in+spinal+motoneurons+after+sciatic+nerve+lesion%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">Netrin G-2 ligand mRNA is downregulated in spinal motoneurons after sciatic nerve lesion.</a> Neuroreport. 21, 782-785 (2010).</li>
<li>Girolami, E. I., Bouhy, D., Haber, M., Johnson, H., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+expression+and+potential+role+of+SOCS1+and+SOCS3+in+Wallerian+degeneration+in+injured+peripheral+nerve%5BTitle%5D)+AND+%22Exp.+Neurol%22%5BJournal%5D)">Differential expression and potential role of SOCS1 and SOCS3 in Wallerian degeneration in injured peripheral nerve.</a> Exp. Neurol. 223, 173-182 (2010).</li>
<li>Hossain-Ibrahim, M. K., Rezajooi, K., Stallcup, W. B., Lieberman, A. R., Anderson, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+axonal+regeneration+in+the+central+and+peripheral+nervous+systems+of+the+NG2-deficient+mouse%5BTitle%5D)+AND+%22BMC+Neurosci%22%5BJournal%5D)">Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.</a> BMC Neurosci. 8, 80-80 (2007).</li>
<li>Thornton, M. R., Mantovani, C., Birchall, M. A., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+N-CAM+and+N-cadherin+expression+in+axotomized+and+crushed+rat+sciatic+nerve%5BTitle%5D)+AND+%22J.+Anat%22%5BJournal%5D)">Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.</a> J. Anat. 206, 69-78 (2005).</li>
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</ol>

We have nothing to disclose.

We have presented a method to obtain a reliably complete sciatic nerve crush with precise marking of the crush site. As previously mentioned, sciatic nerve crush is a common model of peripheral nerve injury in mice and rats. Although each method of crush has its advantages and disadvantages, we found this method produced a complete crush that was easily marked with a minimum of special equipment (e.g. special clamps, etc).
Crush Methods
The most common instrument use...

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.

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

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

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 ...

reproducible-mouse-sciatic-nerve-crush-subsequent-assessment

Research

JoVE Journal

Neuroscience

33.7K Views.  Temple University. In dit rapport beschrijven we een methode om de muis heupzenuw te verpletteren. Deze methode maakt gebruik van direct beschikbaar hemostatische tang en gemakkelijk en reproduceerbaar maakt complete heupzenuw crush. Daarnaast beschrijven we een methode om de spieren hele mounts geschikt voor analyse van de zenuw regeneratie na heupzenuw verliefd te bereiden.

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

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

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

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

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

Medicine

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

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.

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 ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

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

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

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

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

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 ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

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

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

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

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

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

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

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

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

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 ...

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

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