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>
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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. 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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 ...

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