This work was supported by the National Institutes of Health [NS36350, NS52290, and NS50243 to X&#8211;M.X.]; Mari Hulman George Endowment Fund; the State of Indiana; and a Ruth L. Kirschstein National Research Service Award (NRSA) 1F31NS071863 to C.L.W.

1. Exposure of the Cervical Spinal Laminae
<ol>
	<li>Clean the surgical surface with 70% ethanol, pre-warmed with a heating pad. Cover the surface with a sterile surgical drape before placing sterile gauze, cotton swabs, and autoclaved surgical tools in the surgery area. &#160;Use a microbead sterilizer for inter-surgery sterilization of surgical tools.&#160;</li>
	<li>Anesthetize the rat with ketamine (87.7 mg/kg)/xylazine (12.3 mg/kg) intraperitoneally (IP). &#160;The proper plane of anaesthesia is reached when the animal ceases to respond to a toe pinch stimulus.&#160;Subcutaneously inject 0.01-0.05 mg/kg Buprenorphine and 5 mg/kg Carprofen prior to surgical procedure.&#160; Buprenorphine should then be administered every 8-12 hr&#160;and Carprofen once daily, for the first 4-7 days following surgery.</li>
	<li>&#160;Apply protective ointment to the eyes of the animal to prevent corneal drying during surgery.</li>
	<li>Shave the surgical area on the dorsal surface of the rat from the mid-thoracic region to the back of the head with clippers. &#160;Remove shaved fur using a vacuum equipped with a HEPA filter.</li>
	<li>Apply betadine solution to the shaved area as a surgical scrub then clean the area with 70% isopropyl alcohol wipes.</li>
	<li>Use a scalpel blade to perform a 3-4 cm midline incision in the skin from the base of the head caudally to mid-thorax.</li>
	<li>Identify the midline of the fascia and subcutaneous muscles anterior to the hibernating gland at the lower neck; cut through the trapezius and other muscles along the midline to reduce hemorrhage. &#160;&#160;</li>
	<li>&#160;Find the midline of two regions of adipose tissue underlying the muscles; cut the paraspinous muscles caudally strictly along the midline, and separate muscle layers using a small tissue retractor until the level of the thoracic T2 spinous process is reached.</li>
	<li>Identify and cut away the muscle connected to the T2 spinous process to utilize this structure as an anatomic landmark. &#160;</li>
	<li>&#160;Remove the cartilaginous tip of the T2 spinous process to improve visibility of the cervical vertebrae.</li>
	<li>&#160;Separate the paraspinal muscles laterally from the spinous processes and laminae of C4-T1; however, spare the muscle covering on the C3 lamina to prevent bleeding. &#160;</li>
	<li>&#160;Cut the muscles over the laminae from C4-T1 laterally towards the facets on both sides of the spinal column.</li>
	<li>&#160;After the spinal laminae are exposed, place the animal on its ventral surface in the U-shaped channel of the stabilizer.</li>
	<li>&#160;Identify the C5 vertebra by counting the spinous processes rostrally from the T2 landmark to T1, C7, C6, and finally C5.</li>
</ol>
2. Stabilizing the Vertebrae and Performing the Impact Injury
<ol>
	<li>Position the two stainless steel arms of the stabilizer to suspend the animal by placing the serrated edges of the arms underneath the lateral facets of the C5-6 vertebrae (Figure 1C).&#160; After securing the arms with vertebrae in place (Figure 2B), adjust the stabilizing apparatus to ensure the vertebral column is level and centered. &#160;Finally, lock the arms by tightening the thumbscrews of the stabilizer.</li>
	<li>Cut the ligaments between the spinal processes and laminae at C4-5 and C5-6 to identify the margin of the C5 lamina.</li>
	<li>Using a micro-rongeur, clip away the half of the lamina on the right at C5 as intended for SCI (Figure 5C-E). &#160;After laminectomy, transport the animal with the stabilizer under the injury device.&#160; Secure the animal together with stabilizer on a mount (Figure 3A-C) to precisely align the plunger on the spinal cord target using a lateral micro-manipulator (Figure 3).</li>
	<li>Under high magnification, locate the C5 and C6 dorsal root entry zones (DREZs) on the exposed dorsal spinal cord surface without durotomy. &#160;Aim the plunger at the middle of the two identified DREZs and halfway between the midline and the lateral edge of the spinal cord (Figure 5B).</li>
	<li>Using an NYU/MASCIS impactor device with a 2.5 mm diameter tip (Figure 3A &#38; B), produce a&#160;C5 hemi-contusion &#160;(Figure 5B &#38; E) by a 10 g rod x 12.5 mm height drop (Figure 2A).</li>
	<li>Verify the injury visually by bruising on the spinal cord (Figure. 5E, arrow) and check the injury parameters provided by the NYU software12,17 (Figure 6).</li>
	<li>Suture muscles and soft tissue with sterile 4-0 vicryl suture, then close the skin incision with surgical staples (EZ Clips).</li>
	<li>Apply antibacterial ointment to the surgical site.</li>
	<li>Administer 5.0 ml of sterile 0.9% saline subcutaneously to the animal for hydration.</li>
	<li>Place the animal in a heat-controlled environment (recirculating hot water padcage on a heating pad)&#160;) with moist food provided on the bedding (changed daily), and a water bottle with a long spout for easy access placed on the floor of the cage. &#160;Provide care to ensure adequate recovery before returning the animal to the home cage.</li>
	<li>As this is a unilateral cervical contusive injury, the animal will likely lose function of the ipsilateral forelimb, transiently, which begins to recover during the first few weeks after injury.&#160; However, contralateral function should remain intact, thus the animal should be able to eat and drink without impairment, and have only minor impairment in locomotion and grooming.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electromechanical+spinal+injury+technique+with+dynamic+sensitivity.">An electromechanical spinal injury technique with dynamic sensitivity.</a> J. Neurotrauma. 9 (3), 187-195 (1992).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, J. C., Anderson, D. K., Faden, A. I., Gruner, J. A., Holford, T. R., Hsu, C. Y., Noble, L. J., Nockels, R., Perot, P. L., Salzman, S. K., Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MASCIS+evaluation+of+open+field+locomotor+scores:+effects+of+experience+and+teamwork+on+reliability.+Multicenter+Animal+Spinal+Cord+Injury+Study.">MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study.</a> J. Neurotrauma. 13 (7), 343-359 (1996).</li><li>Jakeman, L. B., Guan, Z., Wei, P., Ponnappan, R., Dzwonczyk, R., Popovich, P. G., Stokes, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+spinal+cord+injury+produced+by+controlled+contusion+in+mouse.">Traumatic spinal cord injury produced by controlled contusion in mouse.</a> J. Neurotrauma. 17 (4), 299-319 (2000).</li><li>Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+contusion+models.">Spinal cord contusion models.</a> Prog. Brain Res. 137, 231-255 (2002).</li><li>Ghasemlou, N., Kerr, B. J., 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=Tissue+displacement+and+impact+force+are+important+contributors+to+outcome+after+spinal+cord+contusion+injury.+Exp.+Neurol.">Tissue displacement and impact force are important contributors to outcome after spinal cord contusion injury. Exp. Neurol.</a> 196 (1), 9-17 (2005).</li><li>DeVivo, M. J., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+new+injuries,+prevalent+cases,+and+aging+with+spinal+cord+injury.">Trends in new injuries, prevalent cases, and aging with spinal cord injury.</a> Arch. Phys. Med. Rehabil. 92 (3), 332-338 (2011).</li><li>Onifer, S. M., Rodr&#237;guez, J. F., Santiago, D. I., Benitez, J. C., Kim, D. T., Brunschwig, J. P., Pacheco, J. T., Perrone, J. V., Llorente, O., Hesse, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+unilateral+cervical+spinal+cord+injury+in+the+rat:+evaluation+of+forelimb+recovery+and+histological+effects.">Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects.</a> Behav. Brain Res. 119 (1), 1-13 (2001).</li><li>Pearse, D. D., Lo, T. P., Cho, K. S., Lynch, M. P., Garg, M. S., Marcillo, A. E., Sanchez, A. R., Cruz, Y., Dietrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+and+behavioral+characterization+of+a+novel+cervical+spinal+cord+displacement+contusion+injury+in+the+rat.">Histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat.</a> J. Neurotrauma. 22 (6), 680-702 (2005).</li><li>Gensel, J. C., Tovar, C. A., Hamers, F. P., Deibert, R. J., Beattie, M. S., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+histological+characterization+of+unilateral+cervical+spinal+cord+contusion+injury+in+rats.">Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats.</a> J. Neurotrauma. 23 (1), 36-54 (2006).</li><li>Anderson, K. D., Sharp, K. G., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+cervical+contusion+spinal+cord+injury+in+rats.">Bilateral cervical contusion spinal cord injury in rats.</a> Exp. Neurol. 220 (1), 9-22 (2009).</li><li>Gruner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monitored+contusion+model+of+spinal+cord+injury+in+the+rat.">A monitored contusion model of spinal cord injury in the rat.</a> J. Neurotrauma. 9 (2), 123-126 (1992).</li><li>Scheff, S. W., Rabchevsky, A. G., Fugaccia, I., Main, J. A., Lumpp, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+modeling+of+spinal+cord+injury:+characterization+of+a+force-defined+injury+device.">Experimental modeling of spinal cord injury: characterization of a force-defined injury device.</a> J. Neurotrauma. 20 (2), 179-193 (2003).</li><li>Zhang, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+contusion+based+on+precise+vertebral+stabilization+and+tissue+displacement+measured+by+combined+assessment+to+discriminate+small+functional+differences.">Spinal cord contusion based on precise vertebral stabilization and tissue displacement measured by combined assessment to discriminate small functional differences.</a> J Neurotrauma. 25 (10), 1227-1240 (2008).</li><li>Walker, C. L., Walker, M. J., Liu, N. K., Risberg, E. C., Gao, X., Chen, J., Xu, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+bisperoxovanadium+activates+Akt/mTOR,+reduces+autophagy,+and+enhances+recovery+following+cervical+spinal+cord+injury.">Systemic bisperoxovanadium activates Akt/mTOR, reduces autophagy, and enhances recovery following cervical spinal cord injury.</a> PLoS One. 7 (1), e30012 (2012).</li><li>Irvine, K. A., Ferguson, A. R., Mitchell, K. D., Beattie, S. B., Beattie, M. S., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+assessing+proximal+and+distal+forelimb+function+in+the+rat:+the+Irvine,+Beatties+and+Bresnahan+(IBB)+forelimb+scale.">A novel method for assessing proximal and distal forelimb function in the rat: the Irvine, Beatties and Bresnahan (IBB) forelimb scale.</a> J. Vis. Exp. (46), 2246 (2010).</li><li>Martinez, M., Brezun, J. M., Bonnier, L., Xerri, C. <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+rating+scale+for+open-field+evaluation+of+behavioral+recovery+after+cervical+spinal+cord+injury+in+rats.">A new rating scale for open-field evaluation of behavioral recovery after cervical spinal cord injury in rats.</a> J Neurotrauma. 26 (7), 1043-1053 (2009).</li><li>Cao, Q., Zhang, Y. P., Iannotti, C., DeVries, W. H., Xu, X. M., Shields, C. B., Whittemore, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+electrophysiological+changes+after+graded+traumatic+spinal+cord+injury+in+adult+rat.">Functional and electrophysiological changes after graded traumatic spinal cord injury in adult rat.</a> Exp. Neurol. 191 Suppl 1, S3-S16 (2005).</li><li>Lee, J. H., Streijger, F., Tigchelaar, S., Maloon, M., Liu, J., Tetzlaff, W., Kwon, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Contusive+Model+of+Unilateral+Cervical+Spinal+Cord+Injury+Using+the+Infinite+Horizon+Impactor.">A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor.</a> J. Vis. Exp. (65), e3313 (2012).</li><li>Zhang, Y. P., Walker, M. J., Shields, L. B. E., Wang, X., Walker, C. L., Xu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Cervical+Laceration+Injury+in+Mice.">Controlled Cervical Laceration Injury in Mice.</a> J. Vis. Exp. (75), e50030 (2013).</li></ol>

We have nothing to disclose.

Here we have demonstrated a cervical spine stabilization method for producing unilateral contusive SCI at C5. &#160;This stabilization method increases the precision of the trauma anatomically and produces consistent functional deficits13,18.&#160; In other models that rely on dorsal clamping of the spinous processes, the risk of spinous process damage or detachment of the clamps from the vertebra is quite high. &#160;These models may also allow considerable spine shifting and yielding from the contusion force and the flexible nature of the spine and vertebral columns (Figure 1A and B).&#160; The tissue yielding alters the plunger-tissue contact time and results in unpredictable injury force (Figure 1A-B &#38; 6B). &#160;Our described vertebral stabilization also provides other benefits to the surgical preparation: 1) this method fully stabilizes the vertebrae centered at C5 under the surgical microscope which increases the accuracy of laminectomies (Figure 1C); 2) the animal mounted within the U-shaped stabilizer can be taken directly from the surgical location to the customized mounting attachment, which avoids the procedure of remounting the animal on SCI devices and saves time; and 3) stabilizing the vertebrae at the injury level and directly dorsal and caudal to the intended site of injury can greatly diminish the body movement caused by respiration, another measure to &#160;reduce variability.
The primary advantage of utilizing this stabilization method is the reduced amount of yielding, or ventral movement of the spinal cord and column upon impact.&#160; Based on simple physics of a contusion injury, the force and energy of the impact will transfer from the rod to the spinal cord, ideally with the cord absorbing this energy at the site of impact.&#160; However, if the spine yields beneath the cord, as is possible in the dorsal spinous clamping method (Figure 1A &#38; B), the actual force applied to the cord is decreased and variable, depending upon the degree of yield.&#160;
Although this video illustrates the entire procedure of a cervical contusive SCI model, the essence of this article is the introduction of the spinal stabilization method we use in various applications in our lab, specifically for SCI studies. &#160;A modified version of this stabilization device and method has been used on mice SCI23. &#160;This simple method of spine stabilization is highly useful for SCI research, and we have previously used this method and equipment to perform thoracic contusion as well as laceration SCI models. &#160;Another lab has recently described a variation of this form of stabilization for cervical injury in this journal22.&#160; In summary, we introduce this novel vertebral stabilization method to several surgical procedures for generating reproducible experimental SCI ranging from laminectomy to injury production.&#160; The benefits of this stabilization device are not limited to cervical spinal cord contusion, as this stabilization method has been adapted for a wide variety of experiments such as intra-spinal injections, cellular transplantation, CSF collection from the cisterna magna, hemisection and transection injuries, thoracic contusion injuries, in vivo imaging employing two-photon microscopy, and spinal electrophysiological recording. &#160;Increasing the quality of the spinal surgical and injury procedures and reducing the experimental variability will help provide insight into true mechanisms of injury and recovery, and screen the effects of different therapies on the devastating disorder of SCI.

Consistent and replicable mechanical force on target spinal tissue is critical for minimizing functional and histological variability and for establishing successful contusive spinal cord injury (SCI) models1-7.&#160; The amount of force applied to a target region of the spinal cord depends on the methods utilized for spine stabilization.&#160; Positional shifting of the target spine during contact between the impact plunger and the spinal cord alters the resultant injury force.&#160; The cervical contusive SCI model is a more clinically relevant model than other forms of SCI, as approximately 50% of human SCI cases occur at this level8, and several SCI studies have been performed using animal cervical injury models9-14.&#160; Although contusive SCI models often utilize some form of stabilization by clamping the spinal processes anterior and posterior to the injury site, this preparation is difficult for producing cervical SCI. &#160;As shown in this demonstration, the stabilization method we developed is advantageous in its ability to increase both the quality and reproducibility of contusion injury.&#160; Particularly, this method of vertebral stabilization was established in an attempt to amend the shortcomings and challenges of other models: 1) variability in vertebral yielding under the impact force may occur by clamping adjacent dorsal spinous processes rostral and caudal to the laminectomy. The degree of vertebral shifting is dependent on the number of vertebral joints between the impact and the vertebrae being stabilized (Figure 1). Therefore, the more joints involved the less stable the spine becomes; 2) the dorsal spinous processes are fragile and cause clamp failure as a result of spinous process fracture or the clamp slipping off of the process; and 3) the spinous processes on these vertebrae are extremely short between the C3 to T1 vertebrae compared to those of the thoracic vertebrae, which makes it difficult using traditional clamps to grasp the spinous processes for stabilizing the cervical spine.&#160;&#160;&#160;&#160;
Here we describe a novel method of stabilizing the spine for producing C5 contusive SCI in adult female Sprague-Dawley rats. &#160;This method can be used for stabilization of other levels of the vertebral column and spinal cord, &#160;and conjugates well with other contusive SCI devices, including the New York University/Multicenter Animal Spinal Cord Injury Study (NYU/MASCIS) impactor15 (Figure 2), Precision Systems and Instrumentation, LLC Infinite Horizon (IH) device16, Ohio State University/Electromagnetic Spinal Cord Injury Device1, and the Louisville Injury System Apparatus (LISA)17, allowing for widespread use in SCI research.

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		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Surgical Stabilizer</td>
			<td>Custom Manufactured</td>
			<td>N/A</td>
			<td>Contact Y. Ping Zhang for details. (yipingzhang50@gmail.com)</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Vertebral Stabilization Bars (clawed endfeet)</td>
			<td>Custom Manufactured</td>
			<td>N/A</td>
			<td>Contact Y. Ping Zhang for details. (yipingzhang50@gmail.com)</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;">NYU/MASCIS Impactor Device</td>
			<td>Custom Manufactured</td>
			<td style="width:341px;">W. M. Keck Center for Collaborative Neuroscience 
			Rutgers, The State University of New Jersey 
			e-mail: impactor@biology.rutgers.edu</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel vertebral stabilization method for producing  contusive spinal cord injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Upon following this protocol, consistent and reproducible cervical hemi-contusive SCI is produced (Figure 5 &#38; 6). The use of a vertebral stabilizer to stabilize the lateral processes of the same vertebra at the level intended for SCI allows for such satisfactory results. Using this method, not only the target C5 vertebra, but also adjacent C4 and C6 are rigidly fixed.
The NYU/MASCIS software provides a read-out with a graph set on an x and y-axis, and supports the use of our vertebral stabilization method, and equipment (Figure 6). This method of stabilization reduces injury variability that can result from the downward shifting of the target tissue and spinal column (Figure 1). Following injury, a clear unilateral bluish hematoma centered between the C5 and C6 DREZs is visible (Figure 5E). These injury parameters are consistent from animal to animal according to the readout provided by the NYU/MASCIS software (Figure 6).
As the cervical hemi-contusion produces clear forelimb deficits, this model is ideal for assessing forelimb functional abilities such as reaching, grooming13, and object manipulation18-19. As hindlimb motor deficits are less prominent, the Basso, Beattie and Bresnahan (BBB) locomotor scoring scale4 is not appropriate for use in this model. The functional outcome following injury is most noticeable in the ipsilateral forepaw extensor deficits, in which the rat exhibits a &#8220;clubbed&#8221; fist with all digits flexed18. All animals exposed to the same injury severity and level of the spinal cord should exhibit similar deficits to the ipsilateral forelimb illustrated in this protocol, upon correct injury. Animals improperly injured may present with very different manifestation and duration of the deficits13,18.
Histologically, this model produces extensive gray and white matter damage at the injury epicenter and rostral and caudal to the site of injury, leading to considerable lesion and cavity formation contained almost exclusively within the injured side of the spinal cord. A large, primarily astrocyte-based glial scar forms at the lesion borders with massive neuronal death18.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig1highres.jpg" width="500" /> 
Figure 1: Illustration of spine flexibility during contusive SCI with different clamping methods. Figures A and B show flexibility or &#8220;yield&#8221; of the spine when spinous processes are clamped dorsally, allowing for improper impacts and inconsistent data. The illustration shown in A displays much more flexibility upon impact (red dashed line and large curved arrows) compared to that shown in B (smaller curved arrows), as the clamps are farther from the site of laminectomy and injury. Figure C shows lateral stabilization with our described device with the stabilizing arm securely tightened under the transverse process of the vertebra where the site of impact will be performed. There is no flexibility of the spine during this procedure, as the vertebra of interest is completely stabilized.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig2highres.jpg" width="500" /> 
Figure 2: NYU/MASCIS impactor and custom stabilization container. Figure A displays the parts and features of the NYU/MASCIS spinal cord injury device, with multiple rod height settings for injury severity (inset). Figures B and C illustrate the U-shaped container that holds the rat, and the serrated stabilization arms that securely stabilize the vertebral column during surgery and injury (designed and produced by Y.P. Zhang).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig3highres.jpg" width="500" /> 
Figure 3: Custom mounting system and lateral microadjuster on the NYU/MASCIS impactor. Figure A details the different components of the custom mounting system for the U-shaped rat stabilizer for spinal cord injury. Note the lateral microadjuster in figure A, crucial for precise alignment of the rat spinal cord for injury. Figures B and C provide further depiction of the stabilizer without (B) and with the U-shaped rat container (C) with respect to other important components of the injury device (mounting system designed and produced by Y.P. Zhang).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig4highres.jpg" width="500" /> 
Figure 4: Measurements of the individual components of the surgical stabilization device and attachments. Each component of the custom stabilization system is highlighted to show the dimensions and scale (A, C, and D). Thoracic stabilization arms (B) are shown to display the potential application of this device for use in different spinal surgical models.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig5highres.jpg" width="500" /> 
Figure 5: Surgical landmarks and preparation for cervical hemi-contusion spinal cord injury. Figures A and B portray the correct landmarks for proper impact alignment on the exposed rat spinal cord. The appropriate impact point is directly between the C5 and C6 dorsal nerve roots (rostral-caudal) and the midline and lateral edges of the spinal cord (B). Figures C-E show, in higher magnification, the process of exposing the desired half of the cervical spinal cord for injury, through careful unilateral laminectomy. Also, figures D and E demonstrate the cord immediately before and after spinal cord contusion injury. Note the visible hemorrhage (E) caused by the impact (black arrow).
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/50149/50149fig6highres.jpg" width="500" /> 
Figure 6:. Examples of acceptable versus unacceptable data readouts following impact with the NYU/MASCIS impactor. The top graph (A) and top data set (C) illustrate a readout of a very good impact, with data measurements of &#8220;% error&#8221; for impact rod velocity, initial height, and starting time, as indicated with the red arrow and underline. All values fall well within the window of acceptable error. Conversely, the bottom panel demonstrates data produced by an improper impact caused by improper stabilization of the spinal column (B) and error during &#8220;zeroing&#8221; of the impactor rod and tip onto the spinal cord surface, prior to setting the height of the impactor rod (C). Note the considerable error indicated for the initial height and start time of the impactor drop, as indicated by the red arrow and underline. The software also provides a warning that error has been detected for these parameters (bottom of panel C).

Watch this Scientific Journal Video about A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury at JoVE.com

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

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Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...