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 ...

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

Research

JoVE Journal

Neuroscience

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...