This work was supported in part by NIH NS059622, NS073636, DOD CDMRP W81XWH-12-1-0562; Merit Review Award I01 BX002356 from the U.S. Department of Veterans Affairs; Craig H Neilsen Foundation 296749; Indiana Spinal Cord and Brain Injury Research Foundation and Mari Hulman George Endowment Funds (XMX); Norton Healthcare, Louisville, KY (YPZ); the State of Indiana ISDH 13679 (XW); and the NeuroCures Foundation.

All surgical and animal handling procedures were performed as approved under the Guide for the Care and Use of Laboratory Animals (National Research Council) and the Guidelines of the Indiana University School of Medicine Institutional Animal Care and Use Committee.
1. Preparing the Animal and Performing the T10 Spinal Laminectomy
<ol>
	<li>Sterilize the surgical instruments and metal spine stabilizer in an autoclave. Clean the surgical operating table. Warm a heating pad to 37 &#176;C. Place the heating pad on the operating table and cover it with sterile surgical drapes. Use sterile technique throughout the operation.</li>
	<li>Use female young adult C57/6J mice at 10 weeks old for this study. Anesthetize each animal with an intraperitoneal (i.p.) injection of a ketamine (87.7 mg/kg) and xylazine (12.3 mg/kg) mixture. Confirm complete anesthesia by eliciting no response to a paw pinch-induced nociception stimulation.
	<ol>
		<li>Subcutaneously administer buprenorphine (0.01-0.05 mg/kg), an analgesic agent, and carprofen (5 mg/kg), a non-steroid anti-inflammatory drug.</li>
	</ol>
	</li>
	<li>Shave the hair over the thoracolumbar spine using an electric clipper. Scrub the skin with betadine solution and 70% alcohol wipes.</li>
	<li>Apply ophthalmic ointment to the corneas to protect the eyes from drying during surgery.</li>
	<li>With a scalpel, make a 1.5 cm midline skin incision on the back of the animal to expose the 9th to 11th thoracic vertebral laminae. Push the subcutaneous adipose tissue rostrally. Dissect the paraspinal muscles away from spinous processes and laminae, toward the lateral facets on each side.</li>
	<li>Position the mouse on the U-shaped trough of the stabilizer (Figure 2A and 2B). Bilaterally clamp the stainless steel arms beneath the exposed facets of the T10 vertebra (Figure 4A) and tighten using the thumb screws attached to the arms (Figure 2A).</li>
	<li>Remove the T10 spinous process and lamina (laminectomy) using a micro-rongeur that exposes the dura mater overlying the spinal cord (Figure 4B).</li>
</ol>
2. Performing the T10 Contusion Injury Using the LISA Impactor
<ol>
	<li>Turn the knob of the pressure regulator on the nitrogen tank to set the compressed nitrogen to 20 PSI or 138 kPa (Figure 1A) for this study. 
	NOTE: The pressure is adjustable from 10-120 PSI. A higher pressure will result in a higher-velocity impact. The SCI device tip with a diameter of 1.2 mm is designed for mice, and the tip with a diameter of 2.2 mm is designed for rats. When changing from mice to rats, the larger-diameter tip can be formed by adding a ring to the metal tip (id 1.2 mm/od 2.2 mm). We used the 1.2 mm tip in this mice SCI study. Sterilize the SCI tip before use.</li>
	<li>Turn on the computer to start the software. Push Button 1 (Figure 1B) to activate the impactor tip into a fully-extended position (Figure 3A-1). 
	NOTE: The function of Button 1 is to manually activate or deactivate the pneumatic cylinder.</li>
	<li>Place the U-shaped container with the mouse onto the stage (Figure 2B). Fix the stage in place by tightening the thumb screws of the mount (Figure 2B).</li>
	<li>Under the &#34;SET ZERO LEVEL&#34; zone (green), set the zero level, with a laser sensor measuring the distance to the fully-extended plunger tip, by clicking the &#34;START READING&#34; button (Figure 3A). The distance will be shown in the &#34;Range&#34; parameter in this zone (Figure 3A). Click the &#34;SET ZERO&#34; button (Figure 3A), and a number in mm will appear in the &#34;Zero&#34; parameter box (e.g., 8.951 mm, shown in Figure 3A).</li>
	<li>Push Button 1 (Figure 1B) to withdraw the impactor tip (Figure 3B-1, indicated by an upper arrow) and unlock fastening Screw 1 (Figure 2B). Pull the screw to the right position (Figure 3B-1, indicated by a lateral arrow) to move the tip away from the laser beam path and turn the screw 90&#176; clockwise to lock the screw.</li>
	<li>Move the stage by adjusting the frontal and lateral micro-drivers (Figure 1C) to aim the laser beam onto the center of the exposed dorsal spinal cord. After the injury location is targeted, measure the tissue distance by clicking the &#34;START READING&#34; button under the &#34;SET INJURY LEVEL&#34; zone (blue) (Figure 3B and 3B-1).</li>
	<li>Slowly adjust the distance between the sensor and the spinal cord via the vertical micro-driver (Figure 1C) to reach the desired displacement parameter (e.g., 0.500 mm, shown in the &#34;Injury&#34; parameter box) in the &#34;SET INJURY LEVEL&#34; zone (blue) (Figure 3B).
	<ol>
		<li>When the desired injury displacement is reached, record the tissue distance (e.g., 8.451 mm, shown in the &#34;Range&#34; parameter box) (Figure 3B). Define the desired displacement (Injury) = tip distance (Zero) - tissue distance (Range) (Figure 3B). When the desired injury (e.g., 0.500 mm tissue displacement) is reached (Figure 3B), click the &#34;SET INJURY&#34; button under the &#34;SET INJURY LEVEL&#34; zone to set the injury.</li>
	</ol>
	</li>
	<li>Turn Screw 1 90&#176; counterclockwise to unlock the screw, push the impact tip back into the laser beam trajectory (Figure 3C-1, direction indicated by an arrow), and lock Screw 1 by turning it 90&#176; clockwise.</li>
	<li>Click the Run button under the red &#34;RUN EXPERIMENT&#34; zone (Figure 3C) to execute the impact. The parameter boxes under this zone will show the injury time (s), the force (mV), the velocity (m/s), and the injury displacement (mm) (Figure 3C).</li>
	<li>After all injury data are recorded and saved, remove the U-shaped trough with the mouse from the stage. Visually confirm the spinal cord injury under a surgical microscope (Figure 4C).</li>
	<li>Suture the paravertebral muscles, superficial fascia, and skin using continuous suture with 3-0 silk (Henry Schein, 776-SK).</li>
	<li>Inject the animal with 1 mL of 0.9% saline subcutaneously for hydration and place it onto a temperature-controlled pad until full consciousness has been regained. Place the mouse into a cage with accessible food and water.</li>
	<li>For post-operative care, manually express the bladder until spontaneous bladder voiding returns. &#160;For analgesia, inject Buprenorphine (0.05-2.0 mg/kg, SQ) 8-12 h/day for 2 days. If urinary bladder infection occurs, inject Baytril (SQ, 5-10 mg/kg in 0.1 mL, 1 dose daily) for 7-10 days. If regional/systemic infection occurs, inject Gentamycin (SQ, 5-8 mg/kg, diluted in 1 mL sterile saline, every 8-12 h) for 4 days.</li>
	<li>Remove the suture threads at 14 days post-SCI.&#160;</li>
	<li>On the 42th day post injury, mice will be sacrificed by perfusion. After appropriate anesthesia as 1.2, they will be perfused with 30 ml (0.01M) phosphate buffered saline (PBS) and 30 ml 4% paraformaldehyde in 0.01 M PBS. One centimeter of the spinal cord including the lesion epicenter will be collected and processed for sectioning and histological analysis.</li>
</ol>

<ol>
	<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>Gale, K., Kerasidis, H., Wrathall, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+contusion+in+the+rat:+behavioral+analysis+of+functional+neurologic+impairment.">Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment.</a> Exp. Neurol. 88 (1), 123-134 (1985).</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 (2), 126-128 (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>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=Experimental+spinal+cord+injury:+a+dynamic+and+verifiable+injury+device.">Experimental spinal cord injury: a dynamic and verifiable injury device.</a> J. Neurotrauma. 9 (2), 129-134 (1992).</li><li>Young, W., Chen, J., Xu, X. M., Xu, Z. C., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MASCIS+spinal+cord+contusion+model.">MASCIS spinal cord contusion model.</a> Animal Models of Acute Neurological Injuries. , 411-422 (2009).</li><li>Jakeman, L. B., McTigue, D. M., Walters, P., Stokes, B. T., Chen, J., Xu, X. M., Xu, Z. C., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ohio+State+University+ESCID+spinal+cord+contusion+model.">The Ohio State University ESCID spinal cord contusion model.</a> Animal Models of Acute Neurological Injuries. , 433-448 (2009).</li><li>Scheff, S., Roberts, K. N., Chen, J., Xu, X. M., Xu, Z. C., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infinite+Horizon+spinal+cord+contusion+model.">Infinite Horizon spinal cord contusion model.</a> Animal Models of Acute Neurological Injuries. , 423-433 (2009).</li><li>Sances, A., 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=The+biomechanics+of+spinal+injuries.">The biomechanics of spinal injuries.</a> Crit. Rev. Biomed. Eng. 11 (1), 1-76 (1984).</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, M. J., 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=A+novel+vertebral+stabilization+method+for+producing+contusive+spinal+cord+injury.">A novel vertebral stabilization method for producing contusive spinal cord injury.</a> J. Vis. Exp. (95), (2015).</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+lesion+of+spinal+cord+equivalent+to+crush+injury+of+fracture+dislocation+of+spinal+column.+A+preliminary+report.">Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report.</a> J. A. M. A. 57, 878-880 (1911).</li><li>Jakeman, L. B., 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=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>Rivlin, A. S., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+duration+of+acute+spinal+cord+compression+in+a+new+acute+cord+injury+model+in+the+rat.">Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat.</a> Surg. Neurol. 10 (1), 38-43 (1978).</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=Controlled+cervical+laceration+injury+in+mice.">Controlled cervical laceration injury in mice.</a> J. Vis. Exp. (75), (2013).</li><li>Ma, Z., 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=A+controlled+spinal+cord+contusion+for+the+rhesus+macaque+monkey.">A controlled spinal cord contusion for the rhesus macaque monkey.</a> Exp. Neurol. 279, 261-273 (2016).</li></ol>

Christopher B. Shields, M.D. has ownership of the Louisville Injury System Apparatus (LISA) produced by Louisville Impactor System, LLC.

In 1911, Allen described the first weight drop model using a fixed weight to induce injuries on the exposed spinal cords of dogs12. Similar weight drop models have been developed based on the Allen model, including the NYU/MASCIS impactor3,6,13,14. In addition to the weight drop model, other SCI devices have been created. The OSU/ESCID5,7 model uses a tissue displacement mechanism to control injury severity, and the IH model4,8 uses force to create a gradable SCI. In these systems, vertebral stabilization is obtained by clamping the spinous processes rostral and caudal to the injury site. These devices utilize low injury velocities, specifically 0.33 - 0.9 m/s (NYU/MASCIS), 0.148 m/s (OSU/ESCID), and 0.13 m/s (IH). Stabilizing the rostral and caudal spinous processes may cause spine flexibility and spine movement during the impact, which may affect injury accuracy.
The LISA method attempts to overcome the shortcomings of existing models, particularly in regard to spine instability and low injury velocity. This method uses bilateral facet stabilization and avoids movement artifacts associated with the injury. This device utilizes a high-impact velocity that can be set between 0.5-2 m/s11,15. The laser sensor is more advanced than the Ling Vibrator used in the ESCID model and precisely measures the distance from the surface of the spinal cord without requiring any tissue contact. The model was originally developed to produce a rat SCI, and it has now been adapted to produce SCI on mice and on non-human primates16, with modifications.
Spine stabilization reduces variability in all experimental SCI methods, particularly in tissue displacement models. The laser distance sensor determines the magnitude of tissue displacement of the spinal cord during respiratory movements. It is important that the point of the spinal cord on which the laser is focused should be the identical point struck by the impactor. This step is accomplished during the calibration step (Figure 3), when the impactor tip and the laser beam are aligned. A potential weakness of this model is that the magnitude of tissue displacement is measured from the dural surface. Although the thickness of the dura constitutes a negligible difference between animals, significant variability may exist in the subarachnoid space filled with cerebrospinal fluid (CSF). Variability in injury outcomes may occur when producing a very mild contusion injury using a small tissue displacement. Overall, the consistency of the injury is mainly dependent upon the accuracy of tissue displacement and also upon the velocity and tissue contact time of the plunger.
The range of tissue displacement is wide (accuracy: 0-10 &#177; 0.005 mm). Based on previous pilot data and published information in rodents and non-human primates, a displacement of 20% of the anteroposterior diameter of the SC yields a mild SCI, a 30-40% displacement produces a moderate SCI, and a &#62;50% displacement produces severe SCI at a 1 m/s velocity. There will be slight differences depending on the animal species. The dwell time is adjustable from 0 to 5 s using a time relay. In our study, the dwell time was set at 300 ms. This can be easily adjusted to replicate the dwell times of other SCI devices, including the NYU and IH models.
In summary, we have developed a displacement-based model of contusive SCI in adult mice. The model uses a U-shaped stabilizer to stabilize the bilateral spinal facets, avoiding the cord movement artifacts associated with the laser-guided measurement of the cord surface. This model can produce high-velocity cord injuries from 0.5-2 m/s. The laser sensor is more accurate than the conventional method to determine the velocity and the distance to the impact surface. The model can produce injuries of the spinal cord at all levels, from mild to severe. When modified, this device can also produce injuries in rats and large animals such as non-human primates.

The most common spinal cord injury (SCI) occurring in humans is a contusive SCI1. To investigate the mechanisms of injury and the various therapeutic strategies following SCI, a precise, consistent, and reproducible contusive SCI model in rodents is needed.
Many spinal cord contusive injury models with various injury-producing mechanisms have been used in experimental SCI research2,3,4,5,6. Three contusive SCI models &#8211; specifically, the weight drop-based New York University (NYU)/ Multicenter Animal Spinal Cord Injury Studies (MASCIS) impactor3,6, the Ohio State University (OSU) impactor/electromagnetic SCI device (ESCID)5,7, and the Infinite Horizon (IH) impactor4,8 &#8211; are widely accepted in the SCI research field. The NYU/MASCIS impactor or an equivalent produces injury by dropping a fixed weight from different heights onto the target spinal cord to create multiple injury severities3,6. The OSU/ESCID causes injury by inducing tissue displacement5,7. The IH impactor produces injury by applying different forces to the spinal cord4,8. Each impactor uses a different velocity, which is an important parameter that influences the injury outcomes. The NYU/MASCIS apparatus generates velocities ranging from 0.33-0.9 m/s. The IH device has a maximum velocity of 0.13 m/s4. The OSU/ESCID impactor has a fixed velocity of 0.148 m/s5. Notably, the velocities of these models are lower than that observed in clinical velocities, which usually exceed 1.0 m/s9.
Here, we introduce a novel displacement-based contusive SCI device, called the Louisville Injury System Apparatus (LISA), to produce SCI in mice with a high impact velocity10. This system includes a vertebral stabilizer, which firmly stabilizes the vertebra at the site of injury, allowing the production of a constant, reproducible SCI. The laser sensor of the device ensures the precise determination of tissue displacement and the resultant severity of the SCI. The velocity of the plunger at the point of contact with the spinal cord can be adjusted from 0.5 to 2 m/s. These injury parameters closely replicate traumatic SCI seen clinically.

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			<td height="42" style="height:42px;width:216px;">LISA</td>
			<td style="width:219px;">Louisville Impactor System</td>
			<td style="width:248px;">N/A</td>
			<td style="width:247px;">Produce an experimental contusion injury of the spinal cord in mice</td>
		</tr>
	</tbody>
</table>

a tissue displacement-based contusive spinal cord injury model in mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

This device consists of five main components: (1) a body with an impactor tip (Figure 1C), (2) a computer with software (Figure 1B), (3) an electrical control box (Figure 1B), (4) a vertebral stabilizer (Figure 2A), and (5) compressed air for the pneumatic control system (Figure 1A). To induce precise tissue displacement, the system relies on a laser sensor to measure the distance between the fully-extended plunger tip and the targeted spinal cord dorsal surface. The software takes the 4-mm thickness of the tip into account due to the fact that the laser beam only reaches the reflecting surface of the impactor (Figure 2B and Figure 3A-1). There are two positions at which the plunger tip may be placed: (1) in the path of the laser beam (Figure 3A-1) or (2) in a lateral position away from the laser beam (Figure 3B-1). When the plunger is in the laser beam path (Figure 3A-1), it measures the distance from the impactor tip and monitors the velocity of the impactor tip during motion between extension and retraction. When the plunger is in the lateral position away from the laser beam path (Figure 3B-1), the distance between the laser and the spinal cord is measured.
The stabilization of the T10 vertebra using our vertebral stabilizer is an integral component of the procedure (Figure 2A)10,11. Reliable distance measurements using the laser sensor depend on the stability of the target, which can be distorted if motion is present. To determine the accuracy and consistency of this system, 8 mice were subjected to 0.5-mm displacement injuries. These animals showed a displacement variability of &#177; 0.001 mm (&#177; SD), indicating that the system is highly accurate and reproducible. Figure 4 demonstrates the immobilized target vertebrae in the stabilizer (Figure 4A) and the exposed T10 spinal cord prior to (Figure 4B) and after (Figure 4C) contusion under a surgical microscope.
The pressure of the compressed air controls the velocity of the impactor at the moment of injury. Our data demonstrate that the impact velocity is 0.81 &#177; 0.0345 m/s (mean &#177; SD) at a pressure of 138 kPa. The knob (Figure 1B) on the electrical box controls the duration of the tip-cord contact (dwell time) following the injury, and it can be adjusted between 0 and 5,000 ms. The tip-cord dwell time in most experiments is set at 0.32 &#177; 0.0147 s (mean &#177; SD) (Figure 5). Using this device, severity-dependent contusive injuries can be produced with tissue displacements of 0 mm (sham control), 0.2 mm (mild injury), 0.5 mm (moderate injury), and 0.8 mm (severe injury) in adult mice (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54988/54988fig1.jpg" /> 
Figure 1: The Louisville Injury System Apparatus (LISA). (A) The system consists of an impactor, a control system, and a source of compressed air. (B) The control system includes a control box and a laptop computer. The software and control buttons of the control box allow the user to establish injury parameters. (C) The laser sensor is the key component of the device and measures the position of the injury target, the distance from the spinal cord to the sensor, and the injury velocity. The quick down-and-up movement of the impact tip is powered by compressed air. The location of the injury and the severity of the tissue displacement are adjusted by microdrivers, which control movement in three dimensions. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54988/54988fig2.jpg" /> 
Figure 2: The stabilizer and Mouse Holder. (A) The spine stabilizer consists of a U-shaped trough and two metal arms to hold the mouse vertebra. (B) The stabilizer is then mounted on the impactor device. The red line indicates the laser beam path. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54988/54988fig3.jpg" /> 
Figure 3: Method to Produce a Contusive SCI. (A-C) The Graphical User Interface (GUI) software with three injury parameters/zones are shown. (A, A-1) The green zone (SET ZERO LEVEL) calibrates the distance of the plunger tip. The red line indicates the laser beam path. (B, B-1) The blue zone is used to set the injury level (SET INJURY LEVEL). The impactor is lifted up and moved laterally to the right side to allow the laser beam to reach the dorsal surface of the spinal cord to set the zero level. The red line indicates the laser beam path. (C, C-1) Prior to impact, the tip is moved back onto the laser beam path to execute the injury (RUN). The injury parameters are under the red zone (RUN EXPERIMENT). The red line indicates the laser beam path. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54988/54988fig4.jpg" /> 
Figure 4: Injury Exposure and Assessment. (A) The metal arms of the spine stabilizer stabilize the T10 vertebra. (B) T10 laminectomy to expose the spinal cord, with the dorsal vessels clearly seen. (C) The impact-induced contusion (arrow) on the dorsal surface of the spinal cord confirms the injury. Scale bar = 2 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54988/54988fig5.jpg" /> 
Figure 5: Injury Parameters. Consistent injury parameters include tissue displacement (mm), injury velocity (m/s), and tip dwell time (s). N = 8, Mean &#177; SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54988/54988fig6.jpg" /> 
Figure 6: Histologic Assessment. Representative cross sections of spinal cords, stained with Cresyl Violet and Eosin, show displacement-severity-dependent injuries following (A) sham (0 mm), (B) mild (0.2 mm), (C) moderate (0.5 mm), and (D) severe (0.8 mm) contusive SCIs at T10 using the LISA device. Images were taken at the injury epicenter. Scale bar = 500 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54988/54988fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

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Nanyang Technological University

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JoVE Journal

Medicine

11.1K Views.  Indiana University School of Medicine. We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Video: A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

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 ...

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 ...

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 ...