Name: A Contusion Model of Severe Spinal Cord Injury in Rats (Video) | JoVE Journal
Uploaded: 2013-08-17T14:00:00.000+00:00
Duration: 10 min
Description: Learn a contusion model of severe spinal cord injury in rats for neuroscience research. This protocol covers sterile surgical preparation, laminectomy, impactor injury induction, and essential post-operative care procedures.

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5 minutes

The authors are grateful to Dr. N. Banik and Dr. D. Mitchell for their guidance in the development of this model.

1. Preparation Before Spinal Cord Injury
The surgical instruments required for this procedure are scalpel, pickups with and without teeth, hemostats, self retaining retractors, fine tip rongeurs, needle driver, absorbable sutures, and skin clip applicators. Other surgical supplies required are surgical drapes, sterile sheets for surgical field, gauze sponges, cotton-tip applicators, and metallic foil. Autoclave the surgical instruments and supplies prior to the surgery. Use the individual set of instruments and supplies for one animal. Clean the surgical area and apparatus (impactor, light source, stereotactic frame, glass bead sterilizers, and heating pads) with alcohol wipes. 
Open the surgical drapes and use sterile gloves to drape the surgical field carefully avoiding contamination. Open the individual instruments sterilely and carefully place those in the surgical field. Cover the knobs and handles of the apparatus likely needed during the procedure with the sterile metallic foil. Switch-on the glass bead sterilizer to be ready for use during the procedure. 
2. Preparation of the Animals
Bring the rats to the laboratory area few hr before the actual procedure. Administer pain medication at least one hr ahead of the expected procedure (typically buprinorphine 1.5 ml of 0.006 mg/ml subcutaneous). Administer preoperative antibiotics (typically Baytril 4 mg/kg subcutaneous). Anesthetize the rats using 90 mg/kg of Ketamine and wait until there is no toe-pinch response. Palpate the most prominent spinous process in the thoracic spine. This level typically corresponds with T10 spinous process. Mark the location of intended level in relation to T10. In our lab we perform a T10 injury. The following narrative describes the technique of T10 SCI. Shave a 3 cm x 6 cm rectangle longitudinally and centered at T10 level. Clean skin three times with Betadine solution. Apply ophthalmic lubricant on each eye. Transfer the rat in a comfortable position to the surgical field carefully avoiding contamination. Insert a rectal temperature probe to monitor the core temperature and adjust the heating accordingly to keep animal temperature as close to normal (~37.5 &deg;C) as possible. Cover the rat with a surgical drape with a window above the surgical site. 
3. Surgical Procedure
Begin with an approximate 4 cm incision using a #10 blade centered on the T10 mark. Proceed to patiently dissect fascia and muscle layers away from the T9-T11 spinous processes and laminae. Place retractors to retract muscle and fascia away from the bone. While stabilizing the spinous process of T9 sharply divide the interspinous ligament between T9 and T10 using fine scissors. Similarly stabilize the T10 spinous process and divide the interspinous ligament between T10-T11. Use loop magnification to complete the division of ligaments all the way through the ligamentum flavum (Figure 1). The thecal sac is readily apparent once the ligamentum flavum is disconnected. Use fine tip rongeurs to carefully perform piece-meal laminectomy bilaterally at T10. Utmost care is taken to avoid a downward pressure on the thecal sac and inadvertent injury from the rongeur tips. The lamina and spinous process of T10 is completely removed. 
Move the animal into position on stabilization platform. Use stabilizing clamps to immobilize the spine by clamping the lateral aspects of the T11 vertebral body followed by the lateral aspects of the T9 vertebral body. Be careful not to compress the rat into the platform with the stabilizing clamps as this would restrict space for respiratory movements and add unwanted stress the animal. After securing the spinal column settings on the computer-controlled impactor are checked. We typically use impactor tip of 3.0 mm at a speed of 4 cm/s with a depth of 2 mm and a dwell time of 0.3 sec. Extend the impactor tip and lower it until it just touches the cord surface. Retract the tip and lower the device 2 mm towards the spinal cord surface. Release piston at 4 cm/sec to cause severe contusion spinal cord injury. Attain hemostasis using just enough pressure to keep the cotton-tip applicator in place, being careful not to create any unnecessary pressure on the cord. Suture muscle and fascia layers with a figure-8 stitch being careful not to pull the too tight using absorbable sutures. Close the skin with a minimum of 2 small staples; up to 4 or 5 staples may be used if parts of the incision remain open after the first 2 or 3 staples. 
3. Post-procedure Care
Place rats in a warm environment of about 33-35 &deg;C for 24 hr post-surgery. This entails an incubator (while they are unconscious) and a heated cage space once they begin to move. Once rats are fully awake, administer 5ml of saline, 1.5 ml (0.006 mg/ml) of buprinorphine, and 0.1 ml of Baytril all subcutaneously. Continue with buprinorphine subcutaneously twice a day for first 24-48 hr and Baytril once a day for 7 days. Bladders should be manually expressed three times a day until return of bladder function (&lt;2 ml of urine in early morning expression for 3 consecutive days). Animals should also be checked during this time for infection (blood in urine, whitish color, or foul odor), decreased physical activity or problems with wound healing. Presence of infection should be countered with an increased dosage (or re-initiation) of antibiotics in consultation with local veterinarians. Weigh rats daily beginning the day after surgery to assess their recovery.

<ol><li>Blesch, A., Tuszynski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+injury%3A+plasticity%2C+regeneration+and+the+challenge+of+translational+drug+development%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Spinal cord injury: plasticity, regeneration and the challenge of translational drug development.</a> Trends Neurosci. 32, 41-47 (2009).</li>
<li>Dobkin, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curiosity+and+cure%3A+translational+research+strategies+for+neural+repair-mediated+rehabilitation%5BTitle%5D)+AND+%22Dev.+Neurobiol%22%5BJournal%5D)">Curiosity and cure: translational research strategies for neural repair-mediated rehabilitation.</a> Dev. Neurobiol. 67, 1133-1147 (2007).</li>
<li>Fehlings, M. G., Cadotte, D. W., Fehlings, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+systematic+reviews+on+the+treatment+of+acute+spinal+cord+injury%3A+a+foundation+for+best+medical+practice%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">A series of systematic reviews on the treatment of acute spinal cord injury: a foundation for best medical practice.</a> J. Neurotrauma. 28, 1329-1333 (2011).</li>
<li>Iseda, T., Okuda, T., Kane-Goldsmith, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single%2C+high-dose+intraspinal+injection+of+chondroitinase+reduces+glycosaminoglycans+in+injured+spinal+cord+and+promotes+corticospinal+axonal+regrowth+after+hemisection+but+not+contusion%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">Single, high-dose intraspinal injection of chondroitinase reduces glycosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after hemisection but not contusion.</a> J. Neurotrauma. 25, 334-349 (2008).</li>
<li>Khan, T., Havey, R. M., Sayers, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+spinal+cord+contusion+injuries%5BTitle%5D)+AND+%22Laboratory+Animal+Science%22%5BJournal%5D)">Animal models of spinal cord contusion injuries.</a> Laboratory Animal Science. 49, 161-172 (1999).</li>
<li>Kim, B. G., Kang, Y. M., Phi, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantation+of+polymer+scaffolds+seeded+with+neural+stem+cells+in+a+canine+spinal+cord+injury+model%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Implantation of polymer scaffolds seeded with neural stem cells in a canine spinal cord injury model.</a> Cytotherapy. 12, 841-845 (2010).</li>
<li>Kim, J. H., Tu, T. W., Bayly, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+speed+does+not+determine+severity+of+spinal+cord+injury+in+mice+with+fixed+impact+displacement%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Impact speed does not determine severity of spinal cord injury in mice with fixed impact displacement.</a> Journal of Neurotrauma. 26, 1395-1404 (2009).</li>
<li>Kwon, B. K., Hillyer, J., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+research+in+spinal+cord+injury%3A+a+survey+of+opinion+from+the+SCI+community%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">Translational research in spinal cord injury: a survey of opinion from the SCI community.</a> J. Neurotrauma. 27, 21-33 (2010).</li>
<li>Kwon, B. K., Okon, E. B., Tsai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+grading+system+to+evaluate+objectively+the+strength+of+pre-clinical+data+of+acute+neuroprotective+therapies+for+clinical+translation+in+spinal+cord+injury%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">A grading system to evaluate objectively the strength of pre-clinical data of acute neuroprotective therapies for clinical translation in spinal cord injury.</a> J. Neurotrauma. 28, 1525-1543 (2011).</li>
<li>Norenberg, M. D., Smith, J., Marcillo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathology+of+human+spinal+cord+injury%3A+defining+the+problems%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">The pathology of human spinal cord injury: defining the problems.</a> J. Neurotrauma. 21, 429-440 (2004).</li>
<li>Siegenthaler, M. M., Tu, M. K., Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extent+of+myelin+pathology+differs+following+contusion+and+transection+spinal+cord+injury%5BTitle%5D)+AND+%22J.+Neurotrauma%22%5BJournal%5D)">The extent of myelin pathology differs following contusion and transection spinal cord injury.</a> J. Neurotrauma. 24, 1631-1646 (2007).</li>
<li>Talac, R., Friedman, J. A., Moore, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+spinal+cord+injury+for+evaluation+of+tissue+engineering+treatment+strategies%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Animal models of spinal cord injury for evaluation of tissue engineering treatment strategies.</a> Biomaterials. 25, 1505-1510 (2004).</li>
<li>Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+treatment+trials+in+human+spinal+cord+injury%3A+issues%2C+difficulties%2C+and+recommendations%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Review of treatment trials in human spinal cord injury: issues, difficulties, and recommendations.</a> Neurosurgery. 59, 957-982 (2006).</li>
</ol>

Financial disclosure – none Funding disclosure – none.

Several novel treatments have recently shown early promise in the field of SCI research.3 Careful evaluation of these treatments is essential in clinically relevant model of SCI to select strategies with maximum translational potential. A scheme of grading was recently developed to evaluate the strength of preclinical studies.9 This scheme emphasized the importance of utilizing contusion model of severe SCI. Here we describe such a contusion model of severe SCI with consistent lesion volumes and fun...

Choice of appropriate injury model is crucial for preclinical evaluation of novel treatments for spinal cord injury (SCI).1,2,13 In a recent survey of physicians and scientists in the field of neurotrauma contusion model, as opposed to hemisection or complete transection models, was universally accepted to be clinically relevant.8 This opinion is based on the observation that majority of spinal cord injury in humans is contusive in nature.10 The biology of contusion also appears to be different from hemisection or transection models.11 Iseda, et al. compared the effect of intraspinal chondroitinase ABC injection on neuroregeneration separately in hemisection and contusion models.4 Axonal regeneration was observed in the neuronal bridge in hemisection but not the contusion SCI group. The hemisection or complete transection models also create conditions known to exist in only a very small subset of clinical circumstances. For example, several investigators have employed scaffold-based interventions for implantation in the lesion cavity after hemisection or complete transection to promote regeneration.6 This approach becomes clinically irrelevant because creation of a cavity within injured spinal cord is impractical and probably unethical. 
Variability in functional recovery remains a major challenge for the contusion models.5,12 This variability can be minimized by the use of computer-controlled impactor and stabilization of spine before impact for uniform force delivery across the spinal cord volume especially the ventrally located motor pathways. It must be noted that plasticity and collateral contribution from surviving axons is the predominant mechanism of recovery after spinal cord injury.1 Therefore even minor variations in contusion technique may yield significantly different results. To this end we have developed a model of severe spinal cord injury which yields consistent contusion volume and functional recovery comparable with transection models. This model may be utilized for investigating both the neuroprotection and neuroregeneration strategies as a proof of concept for the treatment efficacy.

<table><tbody><tr><td>Computer controlled impactor</td><td>Leica or the Infinite Horizons (formerly OSU) impactor</td><td></td><td></td></tr><tr><td colspan="4">&lt;em&gt;Surgical instruments &lt;/em&gt;</td></tr><tr><td>Scissors</td><td>Fine Science Tools Inc</td><td>14094-11 or 14060-09</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools Inc</td><td>11006-12 and 11027-12 or 11506-12</td><td></td></tr><tr><td>Hemostats</td><td>Fine Science Tools Inc</td><td>13009-12</td><td></td></tr><tr><td>Retractors</td><td>Fine Science Tools Inc</td><td>17011-10</td><td></td></tr><tr><td>Rongeurs</td><td>Fine Science Tools Inc</td><td>16020-14</td><td></td></tr><tr><td>Needle driver</td><td>Fine Science Tools Inc</td><td>12001-13</td><td></td></tr><tr><td>Stereotactic frame</td><td>Leica or RWD Life Science Co. or TSE systems</td><td></td><td></td></tr><tr><td>Buprinorphine</td><td></td><td></td><td></td></tr><tr><td>Baytril</td><td>Bayer</td><td></td><td></td></tr><tr><td>Ketamine</td><td></td><td></td><td></td></tr></tbody></table>

a contusion model of severe spinal cord injury in rats

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.

Lesion Volume
We have obtained large and consistent lesion volumes by following the technique described above. Using a Luxol fast blue staining a mean lesion volume of 2.04 mm3 (95% CI 1.9-2.18) (n = 5 animals) was obtained. Figure 2 shows mean lesion volume with a representative staining using Luxol fast blue through the lesion epicenter.
Functional Scores
The behavioral scores as measured by the Basso, Beattie, Bre...

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 ...

The contributing authors for this video are Dr.VOR Krishna, chief resident physician in the Department of Neuroscience, division of Neurosurgery at the Medical University of South Carolina, Dr.Mark Kendi, professor of neuroscience at the Medical University of South Carolina, and career scientist at the South Carolina VA Medical Center, and Hampton Andrews, a second year student in the College of Medicine at the Medical University of South Carolina. This video will demonstrate the preparation of an animal in sterile field for surgery, the surgical procedure itself and the methods of post-surgical care. First, all surgical instruments and supplies must be autoclave prior to setting up the surgical field.If you're performing more than one procedure, be sure to autoclave additional sets of instruments for each animal. Next, clean the surgical area and all components with alcohol wipes. Once the surgical field is prepared, lay out all your surgical instruments on a sterile drape.We will demonstrate today's procedure using an adult female spray. Dolly rat. Bring your rats to the lab a few hours before the procedure.Administer 1.5 milliliters of 0.006 milligrams per milliliter of buprenorphine subcutaneously at least one hour before the procedure, just before anesthesia. Give 0.1 milliliters of subcutaneous arol at four milligrams per kilogram. Then anesthetize app with an intraperitoneal injection of 90 milligrams per kilogram of ketamine and wait for the animal to fall asleep.Palpate and mark the prominent T 10 spinous process in the thoracic spine. Shave the area surrounding the mark with an electric razor. Then sterilize the area three times with Betadine solution comfortably position the rat on the surgical field and ensure that it is on top of the heating pad.Confirm anesthesia with the tail pinch. Maintaining counter traction began with an approximate four centimeter incision with a number 10 scalpel blade centered on the T 10 mark. Cut out the loose underlying connective tissue, exposing the muscles and ligaments surrounding the vertebral column.Use retractors to separate the skin and loose connective tissue. Exposing these muscles and ligaments. Proceed to carefully and patiently dissect fascia and muscle layers away from the spinous processes and lamina of T nine through T 11.Use retractors to separate the muscle and fascia away from the bone and expose the spinal column while stabilizing the spinous process of T nine sharply. Dissect the interspinous ligament between T nine and T 10 using small scissors. Repeat this process for the interspinous ligament between T 10 and T 11.Carefully complete the division of ligaments through the ligamentum flavum Using fine tipped raw jores, carefully perform a piecemeal bilateral laminectomy at T 10. Be careful to avoid downward pressure on the thecal sac during bone resection, Completely remove the lamina and spinous process of T 10. Once the spinal cord is exposed and free of bon debris, move the rat into position on the Stereotaxic platform.Use stereotactic clamps to immobilize the spine by clamping the lateral aspects of the T 11 vertebral body, followed by the lateral aspects of the T nine vertebral body. After securing the spinal column, confirm the settings on the computer controlled impactor. Our model uses a speed of four centimeters a second to a depth of two millimeters.Extend the tip of the impactor and lower it to a position Just touching the spinal cord, retract the tip. Then lower the impactor two millimeters. Being careful to maintain its position over the cord.Now fire the piston to create a severe contusion injury. Remove the rat from the stereotactic clamps. Then use gentle pressure with a cotton tip applicator to attain hemostasis.Using a figure eight technique stitched together muscle and fascial layers with absorbable sutures. Now close the skin using a minimum of two small staples. Once the procedure is completed, place rats in a warm environment for approximately 24 hours post-surgery.Once the animals are completely awake, administer five milliliters of saline, 1.5 milliliters of buprenorphine and 0.1 milliliters of atri. All subcutaneously continue with buprenorphine twice a day for the next 48 hours and atri once a day for seven days. Bladders should be manually expressed three times a day until bladder function returns.This will be indicated by less than two milliliters of urine in the early morning. Expressions for three consecutive days. Animals should also be checked during this time for signs of infection.These include blood purulent, discharge or foul odor in urine, as well as decreased activity, weight loss, and problems with wound healing. If an is found increase dosage or reinitiate antibiotics, you have just seen how to create a survivable severe contusion spinal cord injury in rats. When conducting this procedure, it is important to remember to consistently locate the targeted level of the spinal cord for each rat.Also clear all bone fragments from the laminectomy site to reduce other possible injury mechanisms. Be sure the rat is positioned comfortably in the stereotactic clamps and constantly monitor breathing throughout the procedure. Check the animal's weight every day post procedure.This is done to screen for infection, appetite problems, skin breakdown, and post spinal cord injury. Ileus you for watching our video and good luck with your experiments.

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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.

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

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.  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.

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

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

Calibrated Forceps Model of Spinal Cord Compression Injury

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

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

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

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A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

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.

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

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.

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

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.

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Learn a contusion model of severe spinal cord injury in rats for neuroscience research. This protocol covers sterile surgical preparation, laminectomy, impactor injury induction, and essential post-operative care procedures.

A Contusion Model of Severe Spinal Cord Injury in Rats

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