This work was supported by a Griffith University International Student (PhD) stipend to RR, a Perry Cross Foundation Grant to JE and JSJ, a Clem Jones Foundation Grant to JSJ and JE, and a Motor Accident Insurance Commission of Queensland grant to JSJ and JE.

All procedures were carried out with the approval of the Griffith University Animal Ethics Committee (ESK/04/16 AEC and MSC/04/18 AEC) under the guidelines of the National Health and Medical Research Council of Australia.
1. Animal set-up procedure for the surgery
<ol>
	<li>Anesthetize and stabilize the animal.
	<ol>
		<li>Use 8&#8211;10-week old female C57BL/6 mice. Use 5% isoflurane in 1 L/min oxygen for induction of anesthesia. For maintenance of anesthesia, use 1.5&#8211;2% isoflurane in...

<ol>
	<li>Sharif-Alhoseini, 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=Animal+models+of+spinal+cord+injury:+a+systematic+review.">Animal models of spinal cord injury: a systematic review.</a> Spinal Cord. 55 (8), 714-721 (2017).</li><li>Lee, D. H., Lee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+axon+regeneration+after+spinal+cord+injury.">Animal models of axon regeneration after spinal cord injury.</a> Neuroscience Bulletin. 29 (4), 436-444 (2013).</li><li>Sharif-Alhoseini, M., Rahimi-Movaghar, V., Dionyssiotis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topics in Paraplegia. , (2014).</li><li>Talac, R., 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=Animal+models+of+spinal+cord+injury+for+evaluation+of+tissue+engineering+treatment+strategies.">Animal models of spinal cord injury for evaluation of tissue engineering treatment strategies.</a> Biomaterials. 25 (9), 1505-1510 (2004).</li><li>Nakae, A., 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=The+animal+model+of+spinal+cord+injury+as+an+experimental+pain+model.">The animal model of spinal cord injury as an experimental pain model.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 939023 (2011).</li><li>Kwon, B., Oxland, T., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+used+in+spinal+cord+regeneration+research.">Animal models used in spinal cord regeneration research.</a> Spine. 27, 1504-1510 (2002).</li><li>Kundi, S., Bicknell, R., Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+current+mammalian+models.">Spinal cord injury: current mammalian models.</a> American Journal of Neuroscience. (4), 1-12 (2013).</li><li>Harrison, 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=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Basso, D. 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=Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains.">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> Journal of Neurotrauma. 23 (5), 635-659 (2006).</li><li>Seitz, A., Aglow, E., Heber-Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+from+spinal+cord+injury:+a+new+transection+model+in+the+C57Bl/6+mouse.">Recovery from spinal cord injury: a new transection model in the C57Bl/6 mouse.</a> Journal of Neuroscience Research. 67 (3), 337-345 (2002).</li></ol>

The authors have nothing to disclose.

This method induces a complete transection type injury at the T10 vertebral level in mice, which results in complete paraplegia of the animal, below the level of injury. Overall, this method results in minimal bleeding, negligible collateral damage and a stable, reproducible injury. As compared to previously published methods of transection without laminectomy10, this method offers the benefits in terms of direct visualization without manipulating the curvature of the spine, better control over co...

Spinal cord injury (SCI) is a complex medical problem that results in drastic changes in health and lifestyle. There is no cure for SCI, and the pathophysiology of SCI is not thoroughly understood. Animal SCI models, in particular rodent models, offer an invaluable tool for trialing new treatments, and have been used to explore SCI for decades. To date, over 72% of pre-clinical SCI studies have employed rat models, as compared to a mere 16% that have used mice1. Although rats, due to their larger size and tendency to form cavities akin to human SCIs, have traditionally been the preferred model animals for studying novel therapeutic approaches, mice (including many transgenic mouse models) are now being used more frequently to study cellular and molecular mechanisms of the SCI2. The mouse model offers additional benefits in terms of easier handling, faster reproductive rates and lower costs than rats; mice also exhibit a high degree of genomic similarity with humans1,2,3. The major disadvantage of the mouse model has been identified as the significantly smaller size that creates challenges for surgical interventions for creating and treating spinal cord injuries4,5.
There is a gap in the existing literature that highlights the need for a robust and reproducible surgical protocol to induce stable SCI in the mouse model. Therefore, we provide a novel and precise surgical approach in this protocol to overcome these limitations. This protocol provides in-depth guidelines to induce a transection-type injury in mice, as this injury type has been recognized to be the most appropriate to study regenerative and degenerative changes following an injury6, as well as neuroplasticity, neural circuitry and tissue engineering approaches7. We have chosen to induce the injury in the lower thoracic region, since thoracic level SCI is used most commonly in the literature1.

<table border="0" cellpadding="0" cellspacing="0" style="width:1037px;" width="1036">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Baytril injectable 50 mg/mL, 50 mL</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">BAYT I</td>
			<td style="width:184px;">Post-operative care drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Betadine 500 mL</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">BETA AS</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Castroviejo needle holder, locking</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">T149C</td>
			<td>Reusable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:449px;">Ceramic zirconia blade, round with sharp sides, single edge, angled</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">TXD101A-X</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cotton swabs (5pcs)</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">21-893</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dremel Micro</td>
			<td style="width:243px;">DREMEL</td>
			<td style="width:161px;">8050-N/18</td>
			<td>Cordless rotary tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:449px;">Dressing forceps fine</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">06-306</td>
			<td>Single use disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Drill bits</td>
			<td style="width:243px;">Kemmer Pr&#228;zision</td>
			<td style="width:161px;">SM 32 M 0550 070</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dumont #7b forceps</td>
			<td style="width:243px;">Fine Science Tools</td>
			<td style="width:161px;">11270-20</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dumont tweezers, style 5</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">T05-822</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Fur trimmer</td>
			<td style="width:243px;">WAHL</td>
			<td style="width:161px;">WA9884-312</td>
			<td>Zero Overlap Hair Trimmer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Iris scissors, Ti, sharp tips, straight, 90mm</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">TY-3032</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Isoflurane isothesia NXT 250</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">ISOF 00 HS</td>
			<td>Anaesthetic agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Colibri Retractor - 4cm</td>
			<td style="width:243px;">Fine Science Tools</td>
			<td>17000-04</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scalpel handle</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">T133</td>
			<td>Reusable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Signature latex surgical gloves size 7.5</td>
			<td style="width:243px;">Medline</td>
			<td style="width:161px;">MSG5475</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sodium Chloride 0.9%</td>
			<td style="width:243px;">STS</td>
			<td>PHA19042005</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sterile Dressing Pack</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">08-709</td>
			<td>Single use disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sterile Fluid Impervious Drape 60x60 cm</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">29-220</td>
			<td>Single use disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Surgical spirit 100 mL</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;"># SURG SP</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Suture Material - SILK BLK 45CM 5/0 FS-2</td>
			<td style="width:243px;">Johnson &#38; Johnson Medical</td>
			<td style="width:161px;">682G</td>
			<td>Silk Suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Suture Material - Vicryl 70CM 5-0 S/A FS-2</td>
			<td style="width:243px;">Johnson &#38; Johnson Medical</td>
			<td style="width:161px;">VCP421H</td>
			<td>Vicryl Suture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:449px;">Temgesic 0.3 mg in 1 mL, x 5 ampoules (class S8 drug)</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">TEMG I</td>
			<td>Post-operative care drug</td>
		</tr>
	</tbody>
</table>

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.

The resulting method as depicted in Figure 1, involves adequate stabilization of the mouse (Figure 1A) and good visualization of the spine and paraspinous tissue (Figure 1B). Spinous process and laminae can be clearly visualized with minimal muscle dissection and blood loss (Figure 1C, highlighted zone). The fine tip drilling is performed as shown in Figure 1D, to create a ...

Watch this Scientific Journal Video about Induction of Complete Transection-Type Spinal Cord Injury in Mice at JoVE.com

Induction of Complete Transection-Type Spinal Cord Injury in Mice

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, ...

This protocol delivers a precise, surgically controlled injury to induce a full spinal cord transection in mice with a high reproducibility and very high survival rates. The use of a fine drill for the bone injury minimizes the damage to the surrounding tissue, allowing the injury and treatment responses to be modeled with more accuracy. To perform a laminectomy, after confirming a lack of response to pedal reflex in an anesthetized eight to 10 week old female C57 black 6 mouse, shave the back fur to expose the surgical area over the dorsal spine and sterilize the shaved area with sterile cotton swabs soaked in povidone iodine antiseptic liquid and surgical spirit.Using a scalpel, make a vertical midline incision at the T10 vertebral level and use straight forceps to lift the skin from the underlying fascia to facilitate the retractor placement. To expose the spines of the T9 through T11 vertebrae, use the blunt edge of the scalpel to make a small midline incision in the subcutaneous tissue and underlying fascia and use fine tip non-sharp forceps to blunt dissect and reflect the fascia. To expose the laminae, use the blunt tip of the scalpel to split the dorsal trunk and paraspinous muscles along the spines of the T9 through T11 vertebrae and use the blunt fine tip forceps to blunt dissect the muscles in layers to expose the laminae of the vertebrae.Use the same forceps to make small pockets around the transverse processes of the T10 vertebra and hook the prongs of the curved forceps under the transverse processes within the created pockets to stabilize the T10 vertebral body. Thoroughly rinse the T10 laminae with warm saline and use cotton swabs to gently wipe the bony surface clean, taking care that no muscle or ligament attachments remain along the bilateral surface. To break the laminae bilaterally, use a 0.55 millimeter diameter, seven millimeter length drill bit tip to trace a vertical path from the T9/T10 intervertebral space to the T10/T11 intervertebral space along both T10 laminae with the drill off.When the path has been traced, turn on the drill and slowly and carefully make a vertical trench on the right lamina of the T10 vertebra, using the curved forceps to keep the vertebra stable. After repeating the process on the left side of the lamina, irrigate the tissue with warm saline to wash away any remaining bone fragments. Maintaining a good grip on the spine with one hand is essential for this step.It is also important to become comfortable and confident with using the drill. Next, use the angled fine tip forceps to grip the spinous process and remove the whole dorsal segment of the laminae separated by the bilateral drilling. Then, irrigate and swab as necessary to allow a clear visualization of the spinal cord within the laminectomy window.To induce the spinal cord injury within the exposed cord, use a narrow, round cutting edged blade to slice the cord at the center of the laminectomy window, taking care to sweep the lateral recesses of the spinal column to induce a complete transection injury. To confirm completeness of the transection injury, use the blunt fine tip forceps to remove any remaining connections at the transection site. Once hemostasis has been achieved at the transection site, release the curved forcep's grip on the T10 vertebra and bring the edges of the dissected muscles together along the midline to achieve good apposition.Using 5/0 polyglactin 910 absorbable sutures, suture the muscles in layers, making sure that the natural curvature of the spine does not cause any tension at the suture line or open up the sutures. Then, use 5/0 non-absorbable silk sutures to close the subcutaneous tissue and skin, taking care that there is no bleeding, clots, or debris remaining under the skin before closure. To assess whether this method for inducing transection-type spinal cord injury in mice is reproducible and consistent, the injured spinal cord can be analyzed by immunohistochemistry and behavioral testing can be performed on the mice.Immuno-labeling against the astrocyte marker, glial fibrillary acidic protein, facilitates demarcation of the boundary of the intact spinal cord. In this longitudinal section, the injury site located can be visualized between the cord stumps as expected. A consistent size defect can be induced at the transection site with an average minimal distance of 550.4, plus or minus 17.3 micrometers.Behavioral data deploying the Basso Mouse Scale of an open field test shows that the injured mice exhibit no hind limb movement after the injury. Thus, the protocol produces a complete and reliable transection type injury that results in a complete loss of function below the injury level that does not lead to spontaneous reversal of the paralysis. Take care to drill in a straight line with a stable hand, because it can be very tricky to repair the laminectomy window once it has been drilled incorrectly.After spinal cord injury induction, various treatment options can be applied, including drug delivery and cell transplantation to identify potential therapies that improve spinal cord injury recovery.

induction-of-complete-transection-type-spinal-cord-injury-in-mice

Research

JoVE Journal

Neuroscience

7.8K visualizações.  Griffith University. Este protocolo oferece uma lesão precisa e cirurgicamente controlada para induzir uma transeção completa da medula espinhal em camundongos com alta reprodutibilidade e taxas de sobrevivência muito altas. O uso de uma broca fina para a lesão óssea minimiza os danos ao tecido circundante, permitindo que as respostas de lesão e tratamento sejam modeladas com mais precisão. Para realizar uma laminectomia, depois de confirmar a falta de resposta ao reflexo do pedal em um camundongo c57 pre...