Michael Pedersen, Aarhus University for his expertise and time on developing MRI protocols and setting up the entire project. Peter Agger, Aarhus University for his expertise and time on developing the MRI protocols. Steffen Ringgard, Aarhus University for his expertise and time on developing the MRI protocols. The development of the SCI model in the axolotl was kindly supported by The A.P. M&#248;ller Maersk Foundation, The Riisfort Foundation, The Linex Foundation, and The ELRO Foundation.

All applicable institutional and governmental regulations concerning the ethical use of animals were followed during this study. The study was conducted under the approval id: 2015-15-0201-0061 by the Danish Animal Experiment Inspectorate. Animals were Mexican axolotls (Ambystoma mexicanum, mean body mass &#177; STD: 12.12 g &#177; 1.25 g).
1. Preparation
<ol>
	<li>Prepare axolotl for anesthesia.
	<ol>
		<li>Use high quality non-chemically treated tap water. If unavailable, use 40% Holtfreter&#8217;s solution.</li>
		<li>Dissolve 200 mg of ethyl 4-aminobenzoate (benzocaine) in 3 mL of acetone. Dissolve this solution in 1 L of tap water or 40% Holtfreter&#8217;s solution.</li>
	</ol>
	</li>
	<li>Use a standard Petri dish (100 mm in diameter) placed under a stereo microscope as a surgical table. Place a surgical textile cloth on the Petri dish. 
	NOTE: Using a Petri dish as a surgical area enables moving and rotation of the animal without touching it, ensuring spinal stability during surgery.</li>
	<li>Prepare all sterile microsurgical instruments (i.e., scissors and anatomical forceps).</li>
</ol>
2. Anesthesia
<ol>
	<li>Place the axolotl in a container with benzocaine solution for approximately 45 min to ensure deep and stable anesthesia. 
	NOTE: The given concentration of benzocaine will cause anesthesia in all sizes of axolotls.</li>
	<li>Check for signs of general anesthesia within 30-45 min. These include a complete lack of gill movements, righting reflex, or response to either tactile or painful stimuli (gentle pinching of toe web).</li>
	<li>To maintain anesthesia, wrap the animals in paper towels wetted in the anesthetic solution. Wet these regularly with this solution during the surgical procedure to ensure that the skin and gills are kept moist.</li>
	<li>Recover the animal after the surgery by placing it in a container containing fresh tap water. Observe signs of recovery, such as gill movement and regained righting reflex, within 1 h16.</li>
</ol>
3. Microsurgical Laminectomy
NOTE: The laminectomy is performed under a stereomicroscope.
<ol>
	<li>Place the animal in the prone position on the Petri dish. Wrap it in paper towels so that the tail is exposed. 
	NOTE: The paper towels are excellent for ensuring stability throughout the procedure.</li>
	<li>Identify the hind limbs. Make the first incision just caudal to them.
	<ol>
		<li>With a pair of microscissors, perform a vertical incision from the keel until the bony prominence of the spinous processes are felt. 
		NOTE: Be very careful when grasping the keel and skin with forceps, because these easily inflict damage to the delicate skin.</li>
		<li>Extend the cut laterally, so the incision traverses the entire width of the tail.</li>
		<li>Grasp the spinous process with forceps to ensure the right depth.</li>
		<li>Extend the vertical incisions 1 mm below the spinous process on both sides.</li>
	</ol>
	</li>
	<li>Place the animal on one side to perform ventral and horizontal incisions as stated below.
	<ol>
		<li>With a pair of microscissors, starting from the ventral point of the vertical incision, make a horizontal incision of approximately 15 mm for animals 10-20 g in weight. Make the incision longer for larger animals, and shorter for smaller animals.</li>
		<li>Using the scissors, dissect medially through the horizontal incision until the vertebral column is felt in the midline.</li>
		<li>Repeat steps 3.3, 3.3.1, and 3.3.2 on the other side of the animal.</li>
	</ol>
	</li>
	<li>Having dissected in the deep medial plane from both sides, dissect through the midline, thereby connecting the two horizontal incisions.
	<ol>
		<li>Move the free piece of tail and keel to one side, exposing the spinous processes (Figure 1).</li>
		<li>Fixate the tail piece using wet paper towels.</li>
	</ol>
	</li>
	<li>Place the animal in the prone position again with the head facing the surgeon&#8217;s non-dominant side.
	<ol>
		<li>With a pair of forceps, grasp the spinous processes just caudal to the hind limbs. Apply a gentle lift both up and towards the head of the animal.</li>
		<li>Place the blades of a pair of microscissors horizontal around the process and gently cut it. The lift on the process ensures that it is now removed, exposing the spinal cord.</li>
		<li>Grasp the spinous process just caudal to the one that was just removed and repeat steps 3.5.1 and 3.5.2. 
		NOTE: This should leave an exposed spinal cord corresponding to two vertebral levels. When performing the laminectomy, a white foamy secretion often appears. The spinal cord is easily identified by its distinctive shine, along with a vessel running along the midline.</li>
		<li>Depending on the size of the animal, the exposed area may not be wide enough. Using two pairs of forceps, grasp the laminae on both sides of the spinal cord and twist these laterally with a gentle movement.</li>
	</ol>
	</li>
</ol>
4. Introducing a Contusion Type Injury (Figure 2)
<ol>
	<li>Keep the animal in the prone position.</li>
	<li>Use the Petri dish to transfer the animal to the trauma unit.</li>
	<li>Have an assistant shine a flashlight on the spinal cord.</li>
	<li>Place the contusion trauma unit cylinder above the exposed spinal cord using the microadjusters on the unit. Aim through the cylinder.</li>
	<li>Lower the cylinder until it is level with the laminae.</li>
	<li>Attach the falling rod to the electromagnet. Place the desired falling height adjustment cylinder on the trauma unit.</li>
	<li>Place the falling rod in the cylinder. 
	NOTE: For a blinded study, the surgeon should now leave the room without knowing if the animal will be assigned to an injury or a sham surgery group.</li>
	<li>Turn off the electromagnet. The rod falls to the exposed spinal cord.</li>
	<li>Use the height adjustment screw to lift the rod from the spinal cord.</li>
	<li>Confirm the injury by looking at the spinal cord through the microscope. The injured site will appear darker, and bleeding from the midline vessel will be apparent.</li>
</ol>
5. Introducing a Sharp Injury
NOTE: Perform these steps after 3.5.4.
<ol>
	<li>With a pair of microscissors cut the spinal cord in a perfect vertical cut.</li>
	<li>Repeat the cut 2 mm to the caudal side of the body. 
	NOTE: The length of the removed piece of spinal cord can be adjusted as per the study requirement. However, a 2 mm cut will be regenerable10.</li>
	<li>Ensure that the cuts are complete. Upon completion, feel the blades of the scissors scraping along the ventral part of the spinal canal.</li>
	<li>Lift the 2 mm piece of spinal cord from the spinal canal.</li>
</ol>
6. Closing the Surgical Wound
<ol>
	<li>Return the animal to the surgical table. In a blinded study, reposition the keel so the spinal cord is not visible to the surgeon.</li>
	<li>Keep the animal in the prone position.
	<ol>
		<li>Begin placing 10.0 nylon sutures from the most caudal part of the horizontal incision. Close the wounds in one layer. 
		NOTE: Do not grasp the skin too tight, because it will inflict necrosis.</li>
		<li>Work towards the vertical part of the incision.</li>
		<li>When reaching the angle, turn the Petri dish and suture the other horizontal incision.</li>
		<li>Set sutures on the vertical incisions.</li>
		<li>Do not place sutures in the uppermost part of the keel, because the skin here will not be able to hold.</li>
	</ol>
	</li>
</ol>
7. Returning the Animal to the Anesthetic-free Solution
<ol>
	<li>Lift the Petri dish with the animal and submerge both very gently into fresh water only 5 cm deep and let the animal slide off. 
	NOTE: The shallow water depth ensures that the animal will not attempt to swim to the surface to breathe.</li>
	<li>Do not change the water during the first week.</li>
	<li>When feeding the animals, ensure that the food is placed near the animal&#8217;s head. 
	NOTE: The purpose of these measures is to avoid as much movement as possible during the first week.</li>
</ol>
8. Postoperative Ultrasound
<ol>
	<li>Prior to the termination of anesthesia, use a high frequency ultrasound system to acquire images of the injury that can be used for the construction of three-dimensional images of the SCI site.</li>
	<li>Attach the transducer to a micromanipulator preferably governed by a remote joystick.</li>
	<li>Submerge the anesthetized animal in the prone position into a small container filled with anesthetic solution. 
	NOTE: Fix the animal with miniature sandbags or other equipment to avoid movement during the scanning sequence.</li>
	<li>Align the tip of the transducer with the animal&#8217;s length axis and submerge it into the benzocaine solution until it is only a few millimeters above the keel behind the hind limbs of the animal.</li>
	<li>Identify the SCI site. 
	NOTE: The injury site is easily recognizable due to the missing spinous processes directly above the SCI.</li>
	<li>Optimize the image by adjusting the ultrasound settings. Ensure that the SCI site is in the center of the image. Adjust the field of view (i.e., image depth, depth offset, and image width) to cover the SCI site and adjacent healthy tissue. Adjust the two-dimensional gain to optimize the image contrast.</li>
	<li>By sweeping the ultrasound transducer across the SCI site with an electronically operated micromanipulator, acquire B-mode images covering the SCI site at multiple sagittal cross-sectional slice locations, with consecutive slices with an interslice interval of 50 &#181;m. Acquire cine-images containing 500 frames with a frame rate of ~50 frames/s and a transducer frequency of 40 MHz. 
	NOTE: This setup requires an electronic micromanipulator governed by a remote joystick (step 8.2).</li>
	<li>After finishing the scanning sequence return to step 7.</li>
</ol>

<ol>
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S., Seiger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stockholm+spinal+cord+injury+study:+1.+Medical+problems+in+a+regional+SCI+population.">The Stockholm spinal cord injury study: 1. Medical problems in a regional SCI population.</a> Paraplegia. 33 (6), 308-315 (1995).</li><li>Bjornshave Noe, B., Mikkelsen, E. M., Hansen, R. M., Thygesen, M., Hagen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+traumatic+spinal+cord+injury+in+Denmark,+1990-2012:+a+hospital-based+study.">Incidence of traumatic spinal cord injury in Denmark, 1990-2012: a hospital-based study.</a> Spinal Cord. 53 (6), 436-440 (2015).</li><li>Singh, A., Tetreault, L., Kalsi-Ryan, S., Nouri, A., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+prevalence+and+incidence+of+traumatic+spinal+cord+injury.">Global prevalence and incidence of traumatic spinal cord injury.</a> Clinical Epidemiology. 6, 309-331 (2014).</li><li>Aguayo, A. 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=Degenerative+and+regenerative+responses+of+injured+neurons+in+the+central+nervous+system+of+adult+mammals.">Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 331 (1261), 337-343 (1991).</li><li>Aguayo, A. J., Bjorklund, A., Stenevi, U., Carlstedt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+mesencephalic+neurons+survive+and+extend+long+axons+across+peripheral+nervous+system+grafts+inserted+into+the+adult+rat+striatum.">Fetal mesencephalic neurons survive and extend long axons across peripheral nervous system grafts inserted into the adult rat striatum.</a> Neuroscience Letters. 45 (1), 53-58 (1984).</li><li>Richardson, P. M., Issa, V. M., Aguayo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+long+spinal+axons+in+the+rat.">Regeneration of long spinal axons in the rat.</a> Journal of Neurocytology. 13 (1), 165-182 (1984).</li><li>Butler, E. G., Ward, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+spinal+cord+following+ablation+in+urodele+larvae.">Reconstitution of the spinal cord following ablation in urodele larvae.</a> Journal of Experimental Zoology. 160 (1), 47-65 (1965).</li><li>Diaz Quiroz, J. F., Tsai, E., Coyle, M., Sehm, T., Echeverri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+control+of+miR-125b+levels+is+required+to+create+a+regeneration-permissive+environment+after+spinal+cord+injury:+a+cross-species+comparison+between+salamander+and+rat.">Precise control of miR-125b levels is required to create a regeneration-permissive environment after spinal cord injury: a cross-species comparison between salamander and rat.</a> Disease Model Mechanisms. 7 (6), 601-611 (2014).</li><li>Clarke, J. D., Alexander, R., Holder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+descending+axons+in+the+spinal+cord+of+the+axolotl.">Regeneration of descending axons in the spinal cord of the axolotl.</a> Neuroscience Letters. 89 (1), 1-6 (1988).</li><li>McHedlishvili, L., Mazurov, V., Tanaka, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+central+nervous+system+during+salamander+tail+regeneration+from+the+implanted+neurospheres.">Reconstitution of the central nervous system during salamander tail regeneration from the implanted neurospheres.</a> Methods of Molecular Biology. 916, 197-202 (2012).</li><li>Thygesen, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinically+relevant+blunt+spinal+cord+injury+model+in+the+regeneration+competent+axolotl+(Ambystoma+mexicanum)+tail.">A clinically relevant blunt spinal cord injury model in the regeneration competent axolotl (Ambystoma mexicanum) tail.</a> Experimental Therapeutic Medicine. 17 (3), 2322-2328 (2019).</li><li>Goss, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Regeneration. , (1969).</li><li>Hutchison, C., Pilote, M., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+limb:+a+model+for+bone+development,+regeneration+and+fracture+healing.">The axolotl limb: a model for bone development, regeneration and fracture healing.</a> Bone. 40 (1), 45-56 (2007).</li><li>Thygesen, M. M., Rasmussen, M. M., Madsen, J. G., Pedersen, M., Lauridsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+(2,6-diisopropylphenol)+is+an+applicable+immersion+anesthetic+in+the+axolotl+with+potential+uses+in+hemodynamic+and+neurophysiological+experiments.">Propofol (2,6-diisopropylphenol) is an applicable immersion anesthetic in the axolotl with potential uses in hemodynamic and neurophysiological experiments.</a> Regeneration (Oxford). 4 (3), 124-131 (2017).</li><li>Krogh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Progress+of+Physiology.">The Progress of Physiology.</a> The American Journal of Physiology. 90 (2), 243-251 (1929).</li></ol>

The authors have nothing to disclose.

Because risk of injury to the spinal cord is significant, the critical steps of the protocol are removing the spinous processes and widening of the bony access to the spinal canal if needed. As mentioned in the protocol, removing the most cranial process first is highly recommended. This will mean that the more caudal processes protect the spinal cord from being hit by the scissors. It is recommended to ensure enough surgical access, meaning to not make too small a primary incision. Also, when grasping anything with forc...

The overall goal of this study was to establish a controlled and reproducible microsurgical method for inflicting blunt and sharp SCI to the axolotl (Ambystoma mexicanum), producing a regenerative spinal cord injury model.
SCI is a severe condition that, depending on the level and extent, inflicts neurological disability to the extremities along with impaired bladder and bowel control1,2,3. Most SCI are the result of high energy blunt trauma such as traffic accidents and falls4,5. Sharp injuries are very rare. Therefore, the most common macroscopic injury type is contusions.
The mammalian central nervous system (CNS) is a non-regenerative tissue, hence no restoration of neurological tissue following SCI is seen6,7,8. On the other hand, some animals have an intriguing ability to regenerate tissues, including CNS tissue. One of these animals is the axolotl. It is widely used in studies of regenerative biology and is of interest in spinal cord regeneration, because it is a vertebrate9,10,11,12.
Most SCI studies in the axolotl are performed as either amputation of the entire tail or ablation of a larger part of the spinal cord9,10,11,12. Recently, a new study was published on blunt injuries13 that mimics clinical situations better. Whereas complete appendage amputation in the axolotl results in full regeneration, some non-amputation-based regenerative phenomena are dependent on the critical size defect (CSD)14,15. This means that injuries exceeding a critical threshold are not regenerated. To develop a regenerative model with a higher clinical translational value, this study investigated whether a 2 mm blunt trauma would exceed the CSD limit.
This method is relevant for researchers working on spinal cord regeneration in small animal models, especially in the axolotl. Furthermore, it may be of more general interest, because it exhibits a way of using standard laboratory equipment to develop a blunt trauma mechanism that is suitable for use in small animals in general.

<table>
	<tbody>
		<tr>
			<td>25 g custom falling rod</td>
			<td>custom home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm PVC pipe</td>
			<td>custom home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>67-64-1</td>
			<td>Propanone</td>
		</tr>
		<tr>
			<td>Axolotl (Ambystoma mexicanum)</td>
			<td>Exoterra GmbH</td>
			<td>N/A</td>
			<td>12-22 cm and 10 g - 80 g, All strains (wildtype, melanoid, white, albino, transgenic white with GFP)</td>
		</tr>
		<tr>
			<td>Benzocain</td>
			<td>Sigma-Aldrich</td>
			<td>94-09-7</td>
			<td>ethyl 4-aminobenzoate</td>
		</tr>
		<tr>
			<td>electromaget</td>
			<td>custom home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel 2010</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>Excel 2010 or newer</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>ImageJ 1.5e or newer. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2016.</td>
		</tr>
		<tr>
			<td>kimwipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microsurgical instruments</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Forceps and scissors</td>
		</tr>
		<tr>
			<td>MS550s</td>
			<td>Fujifilm, Visualsonics</td>
			<td>MS550s</td>
			<td>40 MHz center frequency, transducer</td>
		</tr>
		<tr>
			<td>MS700</td>
			<td>Fujifilm, Visualsonics</td>
			<td>MS700</td>
			<td>50 MHz center frequency, transducer</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>any maker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soft cloth</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any piece of soft cloth measuring appromixately 70 x 55 cm^2 e.g. a dish towel</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100</td>
			<td>Fujifilm, Visualsonics</td>
			<td>Vevo 2100</td>
			<td>High frequency ultrasound system</td>
		</tr>
	</tbody>
</table>

contusion spinal cord injury via a microsurgical laminectomy in the regenerative axolotl

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma mexicanum). Most clinical spinal cord injuries occur as high energy blunt traumas, inducing contusion injuries. However, most studies in the axolotl spinal cord have been conducted with sharp traumas. Hence, this study aims to produce a more clinically relevant regenerative model. Due to their impressive ability to regenerate almost any tissue, axolotls are widely used as models in regenerative studies and have been used extensively in spinal cord injury (SCI) studies. In this protocol, the axolotls are anesthetized by submersion in a benzocaine solution. Under the microscope, an angular incision is made bilaterally at a level just caudal to the hind limbs. From this incision, it is possible to dissect and expose the spinous processes. Using forceps and scissors, a two-level laminectomy is performed, exposing the spinal cord. A custom trauma device consisting of a falling rod in a cylinder is constructed, and this device is used to induce a contusion injury to the spinal cord. The incisions are then sutured, and the animal recovers from anesthesia. The surgical approach is successful in exposing the spinal cord. The trauma mechanism can produce contusion injuries to the spinal cord, as confirmed by histology, MRI, and neurological examination. Finally, the spinal cord regenerates from the injury. The critical step of the protocol is removing the spinous processes without inflicting damage to the spinal cord. This step requires training to ensure a safe procedure. Furthermore, wound closure is highly dependent on not inflicting unnecessary damage to the skin during incision. The protocol was performed in a randomized study of 12 animals.

The purpose of the protocol is to produce an SCI that will paralyze the motor and sensory functions caudal to the injury. Because the axolotl is regeneration-competent it restores function within weeks, allowing researchers to study CNS regeneration during a short time span.
Anesthesia was provided for 45 min to all animals, and no episodes of preterm recovery were experienced. All animals recovered within an hour and showed no signs of damage from anesthesia in the following weeks<sup class="...

Watch this Scientific Journal Video about Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl at JoVE.com

Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum).

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma ...

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7.7K Views.  Aarhus University. This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum).

Video: Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

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 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 ...

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

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

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

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

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...