Name: reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis
Uploaded: 2012-02-22T12:00:00
Description: Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

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 reg...

<ol>
	<li>Pan, Y. A., Misgeld, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neurotoxic+and+neuroprotective+agents+on+peripheral+nerve+regeneration+assayed+by+time-lapse+imaging+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+the+tibialis+anterior+following+sciatic+nerve+crush+injury:+A+confocal+microscopic+study+in+transgenic+mice.">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., Sim&#241;es, M. J., Armada-da-Silva, P. A. S., Lu&#237;s, A. L., Shirosaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hybrid+chitosan+membranes+and+N1E-115+cells+for+promoting+nerve+regeneration+in+an+axonotmesis+rat+model.">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>Lu&#237;s, A. L., Rodrigues, J. M., Geuna, S., Amado, S., Sim&#240;es, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+cell+transplantation+effects+on+sciatic+nerve+regeneration+after+a+standardized+crush+injury+in+the+rat.">Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat.</a> Microsurgery. 28, 458-470 (2008).</li><li>Lu&#237;s, A. L., Amado, S., Geuna, S., Rodrigues, J. M., Sim&#240;es, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+functional+and+morphological+assessment+of+a+standardized+rat+sciatic+nerve+crush+injury+with+a+non-serrated+clamp.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+assess+function+after+sciatic+nerve+lesion+in+the+mouse+-+adaptation+of+the+sciatic+static+index.">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., Varej&#227;o, A. S. P., Tos, P., Perroteau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+crush+injury+of+the+mouse+median+nerve.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrin+G-2+ligand+mRNA+is+downregulated+in+spinal+motoneurons+after+sciatic+nerve+lesion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+potential+role+of+SOCS1+and+SOCS3+in+Wallerian+degeneration+in+injured+peripheral+nerve.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+axonal+regeneration+in+the+central+and+peripheral+nervous+systems+of+the+NG2-deficient+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+N-CAM+and+N-cadherin+expression+in+axotomized+and+crushed+rat+sciatic+nerve.">Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.</a> J. Anat. 206, 69-78 (2005).</li><li>Beer, G. M., Steurer, J., Meyer, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+nerve+crushes+with+a+non-serrated+clamp.">Standardizing nerve crushes with a non-serrated clamp.</a> J. Reconstr. Microsurg. 17, 531-534 (2001).</li></ol>

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...

<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Mini Clipper with No. 0000 Blade</td>
 <td>Roboz</td>
 <td>RC-5903</td>
 </tr>
 <tr>
 <td>Nair Hair Remover (9 oz.)</td>
 <td>Church and Dwight Co., Inc.</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Betadine Surgical Scrub (1 gallon)</td>
 <td>Fisher Scientific</td>
 <td>19066452</td>
 </tr>
 <tr>
 <td>Ophthalmic Ointment (1 oz.)</td>
 <td>Fisher Scientific</td>
 <td>19082795</td>
 </tr>
 </tbody>
</table>
Surgical Tools:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>FST 250 Hot bead sterilizer</td>
 <td>Fine Science Tools</td>
 <td>18000-45</td>
 </tr>
 <tr>
 <td>Iris Scissors (11 cm long)</td>
 <td>World Precision Instruments</td>
 <td>500216</td>
 </tr>
 <tr>
 <td>Potts-Smith Forceps (Straight; 18cm; 1x2 Teeth)</td>
 <td>Fine Science Tools</td>
 <td>11024-18</td>
 </tr>
 <tr>
 <td>McPherson-Vannas Scissors (5 mm blades) </td>
 <td>World Precision Instruments</td>
 <td>14124-G</td>
 </tr>
 <tr>
 <td>Dumont #5 Forceps &#x2013; Dumoxel Standard Tip</td>
 <td>Fine Science Tools</td>
 <td>11252-30</td>
 </tr>
 <tr>
 <td>Dumont #5/45 Forceps &#x2013; Dumoxel Standard Tip</td>
 <td>Fine Science Tools</td>
 <td>11251-35</td>
 </tr>
 <tr>
 <td>Ultra Fine Hemostats (Straight; Smooth Inside Surfaces) </td>
 <td>Fine Science Tools</td>
 <td>13020-12</td>
 </tr>
 <tr>
 <td>Powdered Activated Carbon (500 g)</td>
 <td>Fisher Scientific</td>
 <td>C272-500</td>
 </tr>
 <tr>
 <td>Size 6-0 Sutures with C-1 Needle (Sterile, Silk, Black, Braided , Non-absorbable; 18&#x201D; Length; Box of 36)</td>
 <td>Roboz</td>
 <td>SUT-1073-11</td>
 </tr>
 <tr>
 <td>Reflex Clip Applier (for 9 mm clips) </td>
 <td>World Precision Instruments</td>
 <td>500345</td>
 </tr>
 <tr>
 <td>9mm Stainless Steel Reflex Clips (100/box) </td>
 <td>World Precision Instruments</td>
 <td>500346</td>
 </tr>
 </tbody>
</table>

Animal Care:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride Injection (Preservative Free; 20 mL)</td>
 <td>Hospira</td>
 <td>0409-4888-20</td>
 </tr>
 <tr>
 <td>Complete Homeothermic Blanket System with Flexible Probe (Medium, 115 VAC, 60 Hz)</td>
 <td>Harvard Apparatus</td>
 <td>507222F</td>
 </tr>
 </tbody>
</table>
Surgical Platform and Retractors:
 (purchased at local hardware store)
<table border="1">
<tbody>
 <tr>
 <td>Name (description/)</td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>Stainless Steel Platform (Milled; ~12&#x201D;x12&#x201D;x1/8&#x201D;)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Button Magnets (Example: Eclipse E825)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Stainless Steel Bolts (3&#x201D; Long; Diameter Determined by Button Magnets)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Stainless Steel Nuts (Sized to Fit Bolts)</td>
 <td>9</td>
 </tr>
 <tr>
 <td>Rubber Bands (Light Tension)</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Insect Pins (Both Ends Bent to Form Hooks)</td>
 <td>3</td>
 </tr>
 </tbody>
</table>
Semi-Thins:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (1 kg)</td>
 <td>Sigma-Aldrich</td>
 <td>P6148-1KG</td>
 </tr>
 <tr>
 <td>Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>S375-500 </td>
 </tr>
 <tr>
 <td>Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>AC38987-0010</td>
 </tr>
 <tr>
 <td>Glutaraldehyde (50%; 10 x 10 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18431</td>
 </tr>
 <tr>
 <td>Osmium Tetroxide (4% Aqueous, 10 x 10 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18465</td>
 </tr>
 <tr>
 <td>Propylene Oxide (450 mL)</td>
 <td>Ted Pella, Inc.</td>
 <td>18601</td>
 </tr>
 <tr>
 <td>Embed 812 (Kit, for hard blocks/high image contrast)</td>
 <td>Electron Microscopy Sciences</td>
 <td>14120</td>
 </tr>
 <tr>
 <td>Toluidine Blue (25 g)</td>
 <td>Ted Pella, Inc.</td>
 <td>19451</td>
 </tr>
 </tbody>
</table>
Whole Mount Muscle Preparation and Immunohistochemistry:
<table border="1">
<tbody>
 <tr>
 <td>Name (description/quantity)</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (1 kg)</td>
 <td>Sigma-Aldrich</td>
 <td>P6148-1KG</td>
 </tr>
 <tr>
 <td>Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>S375-500 </td>
 </tr>
 <tr>
 <td>Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer)</td>
 <td>Fisher Scientific</td>
 <td>AC38987-0010</td>
 </tr>
 <tr>
 <td>10% BSA Diluent/Blocking Solution (200 mL)
 </td>
 <td>Kirkegaard &amp; Perry Laboratories, Inc.</td>
 <td>50-61-00</td>
 </tr>
 <tr>
 <td>Triton X-100 (100 mL)</td>
 <td>Dot Scientific Incorporated</td>
 <td>9002-93-1</td>
 </tr>
 <tr>
 <td>Glycine, 98% (1 kg)</td>
 <td>Fisher Scientific</td>
 <td>AC12007-0010</td>
 </tr>
 <tr>
 <td>Tissue-Tek Cryo-OCT Compound (Case of 12; 4 oz bottles)</td>
 <td>Fisher Scientific</td>
 <td>14-373-65</td>
 </tr>
 <tr>
 <td>Sylgard DOW 170 (2 lb. Kit)</td>
 <td>Fisher Scientific</td>
 <td>NC9492579</td>
 </tr>
 <tr>
 <td>Stainless Steel Insect Pins, Size 1 (100/pkg)</td>
 <td>Fine Science Tools</td>
 <td>26001-40</td>
 </tr>
 <tr>
 <td>Tetramethylrhodamine-A-Bungarotoxin (0.5 mg)</td>
 <td>Sigma-Aldrich</td>
 <td>T0195-.5MG</td>
 </tr>
 <tr>
 <td>Mouse Monoclonal Antibody Against SMI-312 (0.1 mL)</td>
 <td>Covance</td>
 <td>SMI-312R</td>
 </tr>
 <tr>
 <td>Mouse Monoclonal Antibody Against SV2 (0.1 mL)</td>
 <td>Developmental Studies Hybridoma Bank (DSHB)</td>
 <td>SV2</td>
 </tr>
 <tr>
 <td>Rabbit Polyclonal Antibody Against GAP-43</td>
 <td>Novus Biologicals</td>
 <td>NB300-143</td>
 </tr>
 <tr>
 <td>Flourescein Conjugated Goat Anti-Mouse IgG, Fc&#x03B3;&nbsp;Subclass 1 Specific</td>
 <td>Jackson ImmunoResearch</td>
 <td><a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">115-095-205</a></td>
 </tr>
 <tr>
 <td>DyLight 649 Conjugated Donkey Anti-Rabbit IgG</td>
 <td>Jackson ImmunoResearch</td>
 <td><a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a></td>
 </tr>
 <tr>
 <td>4',6-diamidino-2-phenylindole (DAPI, dilactate; 10 mg)</td>
 <td>Invitrogen</td>
 <td>D3571</td>
 </tr>
 <tr>
 <td>Vectashield Mounting Medium (10 mL)</td>
 <td>Vector Laboratories</td>
 <td>H-1000</td>
 </tr>
 <tr>
 <td>Superfrost Plus Microscope Slides (White; Size: 75 x 25 mm; Pack of 144)</td>
 <td>Fisher Scientific</td>
 <td>12-550-15</td>
 </tr>
 <tr>
 <td>Fisherfinest Premium Cover Glasses (Size: 40 x 22 mm; Pack of 1 oz.)</td>
 <td>Fisher Scientific</td>
 <td>12-548-5C</td>
 </tr>
 </tbody>
</table>

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 ...

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