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.

<table><tbody><tr><td>Ketamine (7.2 mg/mL)/Xylazine (0.475 mg/mL)/Acepromazine</td><td>Patterson Veterinary</td><td>07-890-8598/07-869-7632/07-808-1947</td><td>Anesthetic agent</td></tr><tr><td>Buprenorphine(0.03 mg/mL)</td><td>Patterson Veterinary</td><td>07-891-9756</td><td>Pain relief agent</td></tr><tr><td>Carprofen</td><td>Patterson Veterinary</td><td>07-844-7425</td><td>antibiotic agent</td></tr><tr><td>Purdue Products Betadine Surgerical Scrub</td><td>Fisher Scientific</td><td>19-027132</td><td>for sterilizing skin</td></tr><tr><td>Dukal Gauze Sponges</td><td>Fisher Scientific</td><td>22-415-490</td><td>for sterilizing skin</td></tr><tr><td>Decon Ethanol 200 Proof</td><td>Fisher Scientific</td><td>04-355-450</td><td>for sterilizing skin</td></tr><tr><td>1 mL NORM-JECT</td><td>HENKE SASS WOLF</td><td>D-78532</td><td>for anethesia/pain relief/antibiotic agent injection</td></tr><tr><td>10 mL Syringe</td><td>TERUMO</td><td>REF SS-10L</td><td>for saline injection</td></tr><tr><td>Artificial Tears Eye Ointment</td><td>Webster Veterinary</td><td>07-870-5261</td><td>provent eyes from dry</td></tr><tr><td>Antiobiotic Ointment</td><td>Webster Veterinary</td><td>07-877-0876</td><td>provent surgery cut from infection</td></tr><tr><td>Cotton Tipped Applicators</td><td>Fisher Scientific</td><td>1006015</td><td>stop bleeding</td></tr><tr><td>Instrument Sterilizer</td><td>Fine Science Tools</td><td>18000-50</td><td>for sterilizing surgery tool</td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11223-20</td><td>grasp tissue</td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10003-12</td><td>skin cut</td></tr><tr><td>Scalpel Blade #15</td><td>Fisher Scientific</td><td>10015-00</td><td>skin cut</td></tr><tr><td>Hemostat</td><td>Fine Science Tools</td><td>13004-14</td><td>stop bleeding</td></tr><tr><td>Rongeur</td><td>Fine Science Tools</td><td>16021-14</td><td>laminectomy</td></tr><tr><td>Agricola Retractor</td><td>Fine Science Tools</td><td>17005-04</td><td>keep the surgery view open</td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14040-10</td><td>for muscle seperated from spine</td></tr><tr><td>Sterile sutures</td><td>Fine Science Tools</td><td>12051-10</td><td>skin closure</td></tr><tr><td>Mouse Vertebral stabilizer</td><td>Louisville Impactor System</td><td>N/A</td><td>Stabilize and expose the vertebra</td></tr><tr><td>LISA</td><td>Louisville Impactor System</td><td>N/A</td><td>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>

Watch this Scientific Journal Video about A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice at JoVE.com

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

11.1K Views.  Indiana University School of Medicine.  Wij introduceren een weefselverplaatsingsgebaseerd contusief ruggenmergbeseringsmodel dat een consequente contusieve ruggengraatbesering in volwassen muizen kan veroorzaken. 

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

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 ...

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 ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Neuroscience

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.

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

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