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>

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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 approximately 70 x 55 cm&lt;sup&gt;2&lt;/sup&gt; 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 ...

contusion-spinal-cord-injury-via-microsurgical-laminectomy

Research

JoVE Journal

Medicine

7.7K Views.  Aarhus University. Dit protocol is de eerste om een gestandaardiseerde kneuzing type letsel in axolotl ruggenmerg regeneratie onderzoek te introduceren. Het belangrijkste voordeel van deze techniek is dat het het trauma in de dagelijkse kliniek nabootst, waardoor de kans groter wordt dat een toekomstige bevinding kan worden vertaald. De kneuzing letsel apparaat kan worden gebruikt in een ander klein dier model van dwarslaesie.De chirurgische techniek is moeilijk, vooral de laminectomie. Daarom is het raa...

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

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

Genetics

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 ...

Cryo-injury Induced Heart Regeneration in the Axolotl and Echocardiography and Unbiased Quantitative Histology to Evaluate Regenerative Progression

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration following ventricular resection. The resection model has generated a basic knowledge of the processes involved in cardiac repair. However, the model does not relate well to clinical situations in which tissue damage, apoptosis, necrosis, fibrosis, and hypertrophy are all key detrimental consequences of ischemia-induced myocardial infarctions rather than tissue removal. On the other hand, cryoinjury-induced myocardial infarction resembles ischemia-induced myocardial infarction more closely. Here we provide a detailed description of the cryoinjury procedure in the axolotl (Ambystoma mexicanum), which provides a tool for investigating basic mechanisms in cardiac repair in a tetrapod model. Additionally, we provide quantitative methods for estimating infarction size non-invasively in vivo with echocardiography and for measuring infarction size precisely with unbiased quantitative histology ex vivo.

Here we present a surgery protocol to induce cryoinjury to the ventricular myocardium of the axolotl. Additionally, we present a protocol to non-invasively estimate infarction fraction during the regenerative process with echocardiography and a protocol to precisely measure infarction fraction in the excised heart with unbiased quantitative histology.

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration ...

Developmental Biology

A Neonatal Mouse Spinal Cord Compression Injury Model

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

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

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

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Neuroscience

Induction of Complete Transection-Type Spinal Cord Injury in Mice

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

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

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

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

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. 
This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. 
The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other animals.

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Biology

Spinal Cord Transection In Xenopus laevis Tadpoles

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.

Spinal Cord Transection In Xenopus laevis Tadpoles

Xenopus laevis tadpole spinal cord transection is a relevant injury method to study spinal cord injury and regeneration by making a transverse cut that completely severs the spinal cord at the thoracic level.

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis ...

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

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

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