1. Set up: Frame and Clamp Design for Holding the Animal
<ol>
	<li>The frame and clamp to hold the animal was custom-designed to accommodate the Infinite Horizon (IH) Spinal Cord Injury Impactor.</li>
	<li>The base of the frame is an aluminum platform cut into the following dimensions (30.2 cm x 20.3 cm x 1.3 cm) in order to fit into the table guide bracket that comes standard with the IH device (Figure 1A).</li>
	<li>Four Flexaframe support foot plates (Fisher Scientific, Toronto, ON) are attached to the platform and eight Flexaframe support rods (Fisher Scientific, Toronto, ON, 30.5 cm) are assembled using eight Flexaframe support connectors (Figure 1B).</li>
	<li>Two additional Flexaframe support connectors, attached to the middle two rods, house the custom made holds for the clamp (Figure 1B).</li>
	<li>The angle by which the animal's spinal cord is rotated with respect to vertical is established by keeping one of the horizontal rod in place and varying the height of the other horizontal rod (Figure 2).</li>
	<li>The clamp is 35.6 mm long, 25.4 mm high and with a 7.6 mm jaw designed to rigidly grab hold underneath the transverse process from C4 to C6 (Figure 1C). Additional details on the clamp design have been described previously by Choo et al., 2009.</li>
</ol>
2. Surgery
<ol>
	<li>Male Sprague Dawley rats (Charles River Laboratories) weighing 300-350 g were anesthetized by isoflurane (4% for induction and 2% for maintenance) in oxygen (1 L/min).</li>
	<li>Once the animals are under the plane of anesthesia, the animal is placed in a stereotaxic frame (Kopf, Tujunga, CA).</li>
	<li>To minimize bleeding during the surgical procedure 0.4 mL of lidocaine (20&thinsp;mg/mL; Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Canada) with epinephrine is injected intramuscularly around the surgical site in the dorsal neck region. </li>
	<li>A 4-5 cm dorsal midline incision is made using a sterile scalpel (#15), starting from the base of the skull and extending caudally.</li>
	<li>Sterile Adson forceps are used to bluntly dissect through the dorsal musculature to reach the spine, and a sterile Alm retractor (Fine Science Tools, North Vancouver, BC) is inserted to keep the muscles spread apart.</li>
	<li>Using the #15 scalpel, the muscles overlaying the laminae of C4-C7 are scraped off, starting in the midline and sweeping them out laterally.</li>
	<li>An incision with a sterile scalpel (#15) is made to the muscle attached to the transverse processes on both side of the spine in order to fit the clamp underneath the transverse processes of C4 to C6.</li>
	<li>With a sterile fine tipped Friedman-Pearson Rongeur (Fine Science Tools, North Vancouver, BC), the left C5 lamina is carefully removed to visualize the dura and the spinal cord.</li>
	<li>A rod with a diameter of 1.5 mm is slid under the arms to prop the animal up, thereby raising the spine slightly and facilitating clamp insertion.</li>
	<li>Mount the jaw of the sterile clamp onto lateral transverse processes of C4 to C6 and tighten the screws.</li>
	<li>Remove the Alm retractor.</li>
</ol>
3. Spinal Cord Injury
<ol>
	<li>After the clamp is mounted on the animal, the animal is moved to the IH impactor.</li>
	<li>The clamp is inserted into the metal holders on the two middle rods of the frame that have been secured at an angle of 22.5&deg; off of horizontal (Figure 2).</li>
	<li>The scissor jack that provides stable height adjustments, (VWR, Mississauga, ON) is raised up until the animal lays flat.</li>
	<li>Ensure that the clamp is horizontal by placing a small cylinder level on top of the clamp and by tightening the screws. It is important that all the screws are tight and the set up is rigid without any movement.</li>
	<li>The remainder of the procedure is conducted under a microscope (Leica MZ8).</li>
	<li>Lower and aim the impactor tip (15 mm in diameter, with rounded-off edges ) using the vertical adjustment knob and the two horizontal adjustment knobs on the IH impactor until the center of the impactor tip is hovering above the apex of C6 spinous process.</li>
	<li>Once the impactor tip is centered, turn the y-axis adjustment knob one and two fifth turn (1.4 mm) to move the tip laterally to the left side and the x axis horizontal adjustment knob to move the impactor tip to the center of C5.</li>
	<li>Lower the tip until it is just above the dura to verify that the impactor tip is aiming the lateral half of the grey matter.</li>
	<li>Turn the vertical adjustment knob two turns to raise the tip 4 mm above the dura.</li>
	<li>Ensure that the impact area is dry by using a cotton swab or stick.</li>
	<li>Set the desired force to 150 kdyn on the program and click "Start Experiment" to trigger the impactor.</li>
	<li>After the injury, the wound is closed in layers with 5-0 vicryl sutures. Buprenorphine (0.03&thinsp;mg/kg SC, Temgesic; Schering-Plough Corporation, Kenilworth, NJ) and Saline (10 mL) is administered subcutaneously before and twice daily for two days after surgery. Animals are closely monitored twice a day for 2 weeks and one a week for 6 weeks post-injury. </li>
</ol>
4. Representative Results
Twenty Nine male Sprague Dawley rats (Charles River Laboratories) weighing 300-350 g were injured at a force setting of 150 kdyn. The impactor tip was aimed 1.4 mm lateral to midline, at an angle of 22.5&deg; off of vertical. The mean actual force was 155.55 &plusmn; 0.73 kdyn. The mean displacement was 1512.72 &plusmn; 27.86 &mu;m and the velocity was 120.24 &plusmn; 0.52 mm/s (Figure 3).
Behavior Outcome Measures
Functional recovery was assessed using the horizontal ladder test, cylinder rearing test, grooming test, and modified Montoya staircase test1. Animals were trained before injury and assessed at 2, 4 and 6 weeks post-injury. There were significant forelimb impairments sustained throughout the experimental period.
Horizontal Ladder test. Before the injury, animals made only 4.75 &plusmn; 0.73% errors on the ipsilateral forelimb while traversing across the irregularly spaced horizontal ladder. Following the injury, the animals demonstrated marked increases in the percentage of forelimb errors. The ipsilateral forelimb percent errors were 26.97 &plusmn; 2.92%, 26.23 &plusmn; 2.84% and 22.06 &plusmn; 2.05% at 2, 4 and 6 weeks post-injury, respectively (Figure 4A). Importantly, the forelimb impairment on this test was sustained for the 6 weeks.
Cylinder Rearing test. The percentage of ipsilateral forelimb (left + both) usage during exploration decreased significantly after SCI. Prior to the injury, the animals used the ipsilateral forelimb 75.12 &plusmn; 2.25%. After the injury the animals used the ipsilateral forelimb 8.59 &plusmn; 1.80% at 2 weeks, 14.25 &plusmn; 2.65% at 4 weeks and 11.76 &plusmn; 2.66% at 6 weeks (Figure 4B).
Modified Montoya Staircase test. The number of pellets retrieved with the ipsilateral forelimb decreased dramatically after the injury. Prior to the injury, the animals collected 84.85 &plusmn; 2.88% of the food rewards. However, at 2, 4 and 6 weeks post-injury, the animals retrieved only 30.91 &plusmn; 4.03%, 28.94 &plusmn; 4.38% and 25.86 &plusmn; 3.09% of the pellets (Figure 4C).
Grooming test. There were dramatic decreases in the grooming scores after injury. After SCI, the ipsilateral grooming scores were 2 weeks: 2.00 &plusmn; 0.17, 4 weeks: 1.83 &plusmn; 0.17 and 6 weeks: 1.79 &plusmn; 0.11 (Figure 4D).
Histological Outcomes
White matter and Grey matter sparing. An example of a spinal cord injury at a force of 150 kdyn, angle of 22.5&deg; and lateral aim of 1.4 mm is presented in Figure 5. The injury resulted in substantial damage to the grey and white matter on the ipsilateral side. Both corticospinal and rubrospinal tracts were injured and 23 out of 29 animals had parenchymal damage contained on the ipsilateral side. The longitudinal extent of the damage of the white and grey matter was 2400 rostrally and 2400 caudally (Figure 6). When adding the sections to provide a gross estimation of the "cumulative spread" of white and grey matter sparing (2000 &mu;m rostral and caudal to the epicenter), the ipsilateral side had only 51.8% of the white matter spared and 39.7% of grey matter remaining compared to the contralateral side (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/3313/3313fig1.jpg" /> 
Figure 1. Infinite Horizon spinal cord injury impactor. A. Infinite Horizon impactor and overall set up. B. Frame set up. C. Close-up image of the clamp for holding cervical transverse processes. D. Specification (unit: inch) of the clamp. Suggested tolerance &lt;0.002 in (Choo et al. 2009). <a href="https://www.jove.com/files/ftp_upload/3313/3313fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3313/3313fig2.jpg" /> 
Figure 2. A. Illustration and corresponding frame set up to achieve spinal cord rotation of 0&deg; (neutral) or B. 22.5&deg; with the lateral aim of 1.4 mm.
<img alt="Figure 3" src="/files/ftp_upload/3313/3313fig3.jpg" /> 
Figure 3. Representative force and displacement graphs for Infinite Horizon impactor. The arrow indicates the time that the impactor tip has reached 20 kdyn and the point when the recording of displacement is initiated. The actual force is read from the peak of the force vs. time curve and the corresponding displacement is calculated. The graphs below depict a typical 150 kdyn contusion, A. Displacement vs. time graph, B. Force vs. Time graph. These graphs show that the actual force achieved was 152 kdyn, and the impactor displacement into the cord was measured to be 1287 &mu;m. <a href="https://www.jove.com/files/ftp_upload/3313/3313fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/3313/3313fig4.jpg" /> 
Figure 4. Behavioral assessments for force of 150 kdyn, impactor angulation of 22.5&deg; and aim of 1.4 mm left of midline. A. Horizontal ladder test. B. Cylinder rearing test. C. Modified Montoya staircase test. D. Grooming test. The ipsilateral forelimb resulted in significant and sustained impairments compared to the contralateral forelimb.
<img alt="Figure 5" src="/files/ftp_upload/3313/3313fig5.jpg" /> 
Figure 5. Representative images of the injured spinal cord from rostral 1600 &mu;m to caudal 1600 &mu;m.
<img alt="Figure 6" src="/files/ftp_upload/3313/3313fig6.jpg" /> 
Figure 6. Histological assessments. A. Percent white and grey matter spared. Matter around 2000 &mu;m of the epicenter of damage was largely spared. B. Cumulative white and grey matter spared within 2000 &mu;m of the epicenter of damage. The ipsilateral side had significantly less spared white and grey matter compared to the contralateral side. <a href="https://www.jove.com/files/ftp_upload/3313/3313fig6large.jpg" target="_blank">Click here to view larger figure</a>.

<ol>
	<li>Lee, J. H., Tigchelaar, S., Liu, J., Stammers, A. M., Streijger, F., 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=Lack+of+neuroprotective+effects+of+simvastatin+and+minocycline+in+a+model+of+cervical+spinal+cord+injury.">Lack of neuroprotective effects of simvastatin and minocycline in a model of cervical spinal cord injury.</a> Exp. Neurol. 225, 219-230 (2010).</li><li>Kwon, B. K., Hillyer, J., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+research+in+spinal+cord+injury:+a+survey+of+opinion+from+the+SCI+community.">Translational research in spinal cord injury: a survey of opinion from the SCI community.</a> J. Neurotrauma. 27, 21-33 (2010).</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, 36-54 (2006).</li><li>Kwon, B. K., Borisoff, J. F., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targets+for+therapeutic+intervention+after+spinal+cord+injury.">Molecular targets for therapeutic intervention after spinal cord injury.</a> Mol. Interv. 2, 244-258 (2002).</li><li>Allen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+of+experimental+lesions+of+spinal+cord+equivalent+to+crush+injury+of+fracture+dislocation.">Surgery of experimental lesions of spinal cord equivalent to crush injury of fracture dislocation.</a> J. Am. Med. Assoc. 57, 878-880 (1911).</li><li>Basso, D. M., 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=Graded+histological+and+locomotor+outcomes+after+spinal+cord+contusion+using+the+NYU+weight-drop+device+versus+transection.">Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.</a> Exp Neurol. 139, 244-256 (1996).</li><li>Gruner, J. 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+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, 123-128 (1992).</li><li>Choo, A. M., Liu, J., Liu, Z., Dvorak, M., Tetzlaff, W., Oxland, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+spinal+cord+contusion,+dislocation,+and+distraction:+characterization+of+vertebral+clamps,+injury+severities,+and+node+of+Ranvier+deformations.">Modeling spinal cord contusion, dislocation, and distraction: characterization of vertebral clamps, injury severities, and node of Ranvier deformations.</a> J. Neurosci. Methods. 181, 6-17 (2009).</li><li>Whishaw, I. Q., Piecharka, D. M., Drever, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+and+partial+lesions+of+the+pyramidal+tract+in+the+rat+affect+qualitative+measures+of+skilled+movements:+impairment+in+fixations+as+a+model+for+clumsy+behavior.">Complete and partial lesions of the pyramidal tract in the rat affect qualitative measures of skilled movements: impairment in fixations as a model for clumsy behavior.</a> Neural. Plast. 10, 77-92 (2003).</li><li>Jones, T. A., Schallert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overgrowth+and+pruning+of+dendrites+in+adult+rats+recovering+from+neocortical+damage.">Overgrowth and pruning of dendrites in adult rats recovering from neocortical damage.</a> Brain Res. 581, 156-160 (1992).</li><li>Liu, Y., Kim, D., Himes, B. T., Chow, S. Y., Schallert, T., Murray, M., Tessler, A., Fischer, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplants+of+fibroblasts+genetically+modified+to+express+BDNF+promote+regeneration+of+adult+rat+rubrospinal+axons+and+recovery+of+forelimb+function.">Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.</a> J. Neurosci. 19, 4370-4387 (1999).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Soblosky, J. S., Song, J. H., Dinh, D. 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, 1-13 (2001).</li><li>McKenna, J. E., Prusky, G. T., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+motoneuron+topography+reflects+the+proximodistal+organization+of+muscles+and+movements+of+the+rat+forelimb:+a+retrograde+carbocyanine+dye+analysis.">Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis.</a> J. Comp. Neurol. 419, 286-296 (2000).</li><li>Sandrow, H. R., Shumsky, J. S., Amin, A., Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspiration+of+a+cervical+spinal+contusion+injury+in+preparation+for+delayed+peripheral+nerve+grafting+does+not+impair+forelimb+behavior+or+axon+regeneration.">Aspiration of a cervical spinal contusion injury in preparation for delayed peripheral nerve grafting does not impair forelimb behavior or axon regeneration.</a> Exp. Neurol. 210, 489-500 (2008).</li><li>Popovich, P. G., Lemeshow, S., Gensel, J. C., Tovar, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+evaluation+of+the+effects+of+glibenclamide+on+reducing+progressive+hemorrhagic+necrosis+after+cervical+spinal+cord+injury.">Independent evaluation of the effects of glibenclamide on reducing progressive hemorrhagic necrosis after cervical spinal cord injury.</a> Exp. Neurol. 233, 615-622 (2012).</li><li>Dunham, K. A., Siriphorn, A., Chompoopong, S., Floyd, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+graded+cervical+hemicontusion+spinal+cord+injury+model+in+adult+male+rats.">Characterization of a graded cervical hemicontusion spinal cord injury model in adult male rats.</a> J. Neurotrauma. 27, 2091-2106 (2010).</li><li>Lee, J. H., Roy, J., Sohn, H. M., Cheong, M., Liu, J., Stammers, A. T., 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=Magnesium+in+a+polyethylene+glycol+formulation+provides+neuroprotection+after+unilateral+cervical+spinal+cord+injury.">Magnesium in a polyethylene glycol formulation provides neuroprotection after unilateral cervical spinal cord injury.</a> Spine (Phila Pa 1976). 35, 2041-2048 (2010).</li><li>Alstermark, B., Isa, T., Lundberg, A., Pettersson, L. G., Tantisira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+low+pyramidal+lesions+on+forelimb+movements+in+the+cat.">The effect of low pyramidal lesions on forelimb movements in the cat.</a> Neurosci. Res. 7, 71-75 (1989).</li></ol>

No conflicts of interest declared.

In this paper, we describe a cervical unilateral contusion model using the Infinite Horizon (IH) impactor at a force of 150 kdyn, an angle of 22.5&deg; off of vertical, and a lateral aim of 1.4 mm from the midline. With these settings, we were able to produce sustained behavioral deficits in the ipsilateral forelimb with parenchymal disruption contained largely to the ipsilateral side, where it appeared that considerable damage occurred to the regions where the rubrospinal, reticulospinal, vestibulospinal and corticospin...

<table border="1">
<tbody>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Infinite Horizon Impactor</td>
 <td>Precision Systems and Instrumentation</td>
 <td>IH-0400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aluminum metal sheet</td>
 <td>Metalsupermarlets.com</td>
 <td>APT6061/500 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flexaframe support foot plates</td>
 <td>Fishers Scientific</td>
 <td>1466625Q </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flexaframe support rods</td>
 <td>Fishers Scientific</td>
 <td>1466610GQ </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flexaframe Support Connectors</td>
 <td>Fishers Scientific</td>
 <td>1466620Q </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Clamp1</td>
 <td>Custom made</td>
 <td>&nbsp;</td>
 <td>Choo et al., 2009</td>
 </tr>
 <tr>
 <td>Metal holders</td>
 <td>Custom made</td>
 <td>&nbsp;</td>
 <td>See above</td>
 </tr>
 <tr>
 <td>Impactor tip</td>
 <td>Custom made</td>
 <td>&nbsp;</td>
 <td>Diameter: 1.15 mm</td>
 </tr>
 <tr>
 <td>Stereotaxic frame</td>
 <td>David Kopf Instruments</td>
 <td>Model 900</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cylinder Level</td>
 <td>YIJIA TOOLS</td>
 <td>YJ-SL0620 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>Model #: MZ8</td>
 </tr>
 <tr>
 <td>Laboratory scissor jack</td>
 <td>VWR</td>
 <td>12620-902</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor at JoVE.com

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

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 ...

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19.3K Views.  University of British Columbia. 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.

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

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 ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

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.

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 ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

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 ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

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 ...