Name: contusion spinal cord injury via a microsurgical laminectomy in the regenerative axolotl
Uploaded: 2019-10-20T14:45:00
Description: Watch this Scientific Journal Video about Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl at JoVE.com

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 unav...

<ol>
	<li>Shavelle, R. M., DeVivo, M. J., Brooks, J. C., Strauss, D. J., Paculdo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+Long-Term+Survival+After+Spinal+Cord+Injury.">Improvements in Long-Term Survival After Spinal Cord Injury.</a> Archives of Physical Medicine and Rehabilitation. 96 (4), 645-651 (2015).</li><li>Hicken, B. L., Putzke, J. D., Richards, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+management+and+quality+of+life+after+spinal+cord+injury.">Bladder management and quality of life after spinal cord injury.</a> American Journal of Physical Medicine &amp; Rehabilitation. 80 (12), 916-922 (2001).</li><li>Levi, R., Hultling, C., Nash, M. 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. 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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 ...

This protocol is the first to introduce a standardized contusion type injury in axolotl spinal cord regeneration research. The main advantage of this technique is that it mimics the trauma seen in the everyday clinic, enhancing the likelihood that a future finding will be able to be translated. The contusion injury device can be utilized in any other small animal model of spinal cord injury.The surgical technique is difficult, especially the laminectomy. Therefore, it is advisable to train the procedure on a large number of animals ex vivo before progressing to in vivo surgery. To begin, set up a surgical table by placing a standard Petri dish under a stereo microscope, on top of a surgical textile cloth.Place the anesthetized axolotl in the prone position on the Petri dish, and wrap it in paper towels so that the tail is exposed. Identify the hind limbs and use a pair of micro scissors to make the first incision just caudal to them. Make the vertical incision from the keel until the bony prominence of the spinous processes are felt.Extend the cut laterally, so the incision traverses the entire width of the tail, and grasps the spinous processes with forceps to ensure proper depth. Then extend the vertical incision one millimeter below the spinous process on both sides. Place the animal on one side, and starting from the ventral point of the vertical incision, make a horizontal incision of approximately 15 millimeters for animals that are 10 to 20 grams in weight.Use the scissors to dissect immediately through the horizontal incision until the vertebral column is felt in the midline. Then turn the animal on to the other side and repeat the process. After dissecting in the deep medial plane from both sides, dissect through the midline to connect the two horizontal incisions.Then, move the free piece of tail and keel to one side to expose the spinous processes. Place the animal in the prone position with the head facing the surgeon's nondominant side, and use a pair of forceps to grasp the spinous processes, just caudal to the hind limbs. Apply a gentle lift up and towards the animal's head.Place the blades of a pair of micro scissors horizontal around the process and gently cut it, exposing the spinal cord. Grasp the spinous process caudal to the one just removed, and repeat the process. It is imperative that you keep a constant lift on the spinous process to ensure that it does not inflict damage to the spinal cord when removed.If the exposed area is not wide enough, use two pairs of forceps to grasp the laminae on both sides of the spinal cord and twist them laterally with a gentle movement. When removing the spinous process, it might be necessary to do this in multiple steps. Therefore, exposing the spinal cord may take several minutes.To introduce a contusion type injury, use a Petri dish to transfer the animal to the trauma unit and shine a flashlight on the spinal cord. Then, place the contusion trauma unit cylinder above the exposed spinal cord using the micro adjusters, and lower the cylinder until it is level with the laminae. Attach the falling rod to the electro magnet, and place the desired falling height adjustment cylinder on the trauma unit.Next, place the falling rod on the cylinder, turn off the electromagnet to drop the rod on the exposed spinal cord, and use the height adjustment screw to lift the rod back up. If introducing a sharp injury, cut the spinal cord in a perfect vertical cut, then repeat the cuts two millimeters to the caudal side of the body. Feel the blades of the scissors scraping along the ventral part of the spinal canal to ensure that the cuts are complete, and lift the piece of spinal cord from the canal.To close the surgical wound, place 10 point O nylon sutures from the most caudal part of the horizontal incision closing the wounds in one layer and working towards the vertical part of the incision. When an angle is reached, turn the Petri dish and suture the other horizontal incision, then set sutures on the vertical incisions. Prior to terminating anesthesia, use a high frequency ultrasound to acquire images of the injury.Align the tip of the transducer with the animal'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. Identify the injury site and acquire images according to the manuscript directions. When finished, return the axolotl to anesthetic free solution by submerging the Petri dish in five centimeter deep freshwater and letting the animal slide off.The spinal cord injuries created using this protocol were validated using hematoxylin and eosin staining on injured and sham axolotls. To confirm regeneration, histological sections were prepared nine weeks post injury. The images showed a reestablished spinal cord connection in the injured animals.Injury and regeneration we're also followed by examining neurological function. The animal's reaction to stimulation of the tail was scored with higher scores indicating higher tactile and nociceptive sensory functions. Among the injured animals, there was a loss of neurological function three weeks post injury, and a gradual restoration within nine weeks.The spinal cord injury was also visualized using ultrasonic graphic imaging, which allowed for visualization of the dorsal artery as a marker of vessel integrity. Hi-field MRI scans were performed immediately after injury and at three, six, and nine weeks. These scans were able to visualize edema and restoration of spinal cord integrity.The scans immediately post injury, were prone to noise. Patient and meticulous surgery will pave the way for a successful result after laminectomy without premature damage to the spinal cord. Following this procedure, several in vivo methods can be applied, such as imaging and electrophysiological techniques.

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