Name: induction of complete transection-type spinal cord injury in mice
Uploaded: 2020-05-06T14:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Induction of Complete Transection-Type Spinal Cord Injury in Mice at JoVE.com

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 1 L/min oxygen. Confirm appropriate anaesthetization by establishing a lack of pain reflex in tail and hind paws.</li>
	</ol>
	</li>
	<li>&#160;Administer buprenorphine (0.03 mg/kg body weight) for analgesia and enrofloxacin (10 mg/kg body weight) for antibiotic cover, subcutaneously according to body weight. Meloxicam (2 mg/kg body weight) can be given for long term analgesia if required.&#160;</li>
	<li>Keep the animal&#8217;s body temperature steady at 37 &#176;C with a heating pad
	<ol>
		<li>Shave the back fur to expose the surgical area over the dorsal spine. Remove the shaved fur from surgical area before sterilizing the incision site. Sterilize the shaved area with sterile cotton swabs soaked in povidone iodine antiseptic liquid and surgical spirit.</li>
	</ol>
	</li>
	<li>Tape the paws of the mouse to the surgical area to stabilize the animal (Figure 1A). Place an oval window drape over mouse (Figure 1B).</li>
</ol>
2. Laminectomy
<ol>
	<li>Make a vertical midline incision at the T10 vertebral level using a surgical scalpel.
	<ol>
		<li>Locate the spinous process of T10 vertebra to determine the site of laminectomy. The body of the vertebra lies slightly cranial to the tip of the spinous process8 (see Figure 2). The tip rests approximately at the midpoint of the T11 vertebra8.</li>
		<li>Make the incision ~2.5 cm long, so that the spine of T10 vertebra is approximately at the middle of the length of the incision.</li>
	</ol>
	</li>
	<li>Reflect the skin and retract with retractors.
	<ol>
		<li>Use the straight forceps to lift the skin from the underlying fascia. This will create space for the retractors to be placed; these will keep the surgical field open.</li>
	</ol>
	</li>
	<li>Perform blunt dissection of subcutaneous tissue and fascia to expose the spinous processes.
	<ol>
		<li>Use the blunt edge of the scalpel to make a small midline incision in the subcutaneous tissue and underlying fascia to expose the spines of T9&#8211;T11 vertebrae. Use the fine tip forceps (non-sharp) to perform blunt dissection and reflect the fascia.</li>
	</ol>
	</li>
	<li>Split and separate the para-spinous muscles to expose the laminae.
	<ol>
		<li>Use the blunt tip of the scalpel to split the dorsal trunk muscles and para-spinous muscles along the spines of the T9&#8211;T11 vertebrae. Use the blunt fine tip forceps to perform blunt dissection of the muscles in layers and expose the laminae of the vertebrae. This should minimize any bleeding. 
		NOTE: If there is any bleeding, use warmed saline (37 &#176;C) irrigation and cotton swabs to control it and clear blood from the surgical field.</li>
		<li>Use the same forceps to make small pockets around the transverse processes of T10 vertebra. Use the curved forceps to stabilize the T10 vertebral body by hooking its prongs under the transverse processes, in the pockets created (Figure 1C).</li>
		<li>Thoroughly rinse the T10 laminae with warm saline and gently wipe clean with cotton swabs to clearly visualize the bony surface. Make sure that no muscle/ligament attachments remain along the surface bilaterally.</li>
	</ol>
	</li>
	<li>Use a drill with a fine tip (0.55 mm diameter, 7 mm length) to break the laminae bilaterally.
	<ol>
		<li>Use the tip of the drill to trace a vertical path from T9&#8211;T10 intervertebral space to T10&#8211;T11 intervertebral space along both the T10 laminae, without turning the drill on. This is to ensure that the drill bit does not catch any tissue (Figure 1D).</li>
		<li>Now turning the drill on, slowly and carefully dig a vertical trench on the right lamina of the T10 vertebra. This part of the laminectomy should create a precise surgical defect throughout the thickness of the bone in a straight vertical line. Maintain the grip with the curved forceps to keep the vertebra stable.</li>
	</ol>
	</li>
	<li>Make sure that the tip of the drill does not penetrate through the bone and injure the spinal cord. Repeat the same process on the left side of the lamina, keeping the vertebra stable with the curved forceps. Irrigate with warm saline to wash away any remaining bone fragments.</li>
	<li>Lift and remove the posterior part of the neural arch (Figure 1F).
	<ol>
		<li>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. Irrigate and swab again if there is any bleeding, to clearly visualize the spinal cord exposed under the laminectomy window (Figure 1E).</li>
	</ol>
	</li>
</ol>
3. Transection
<ol>
	<li>Induce the spinal cord injury by transection of the exposed cord with a single slice of the blade.
	<ol>
		<li>Use the narrow, round cutting edged blade to slice the cord at the center of the laminectomy window. Ensure to sweep the lateral recesses of the spinal column to induce a complete transection injury (Figure 1G).</li>
	</ol>
	</li>
	<li>Confirm completeness of the transection injury using the blunt fine tip forceps and remove any remaining connections at the transection site.</li>
	<li>Control the bleeding if any, before closing the surgical layers.</li>
	<li>Use the warm saline to irrigate and clear any bleeding that occurs from the transected cord stumps. Use a cotton swab to apply gentle pressure to achieve hemostasis if needed. Take care not to compress the spinal cord.</li>
</ol>
4. Closure and immediate post-operative care
<ol>
	<li>Bring the muscles together and suture in layer.
	<ol>
		<li>Once hemostasis is achieved at the transection site, release the curved forceps grip on the T10 vertebrae. Bring the edges of dissected muscles together along the midline to achieve good apposition.</li>
		<li>Suture the muscles in a layer using 5-0 polyglactin 910 absorbable sutures. Make sure that the natural curvature of the spine does not cause any tension at the suture line or open up the sutures, exposing laminectomy site.</li>
	</ol>
	</li>
	<li>Close subcutaneous tissue and skin.
	<ol>
		<li>Use 5-0 non-absorbable silk sutures to close the skin incision. Make sure that there is no bleeding, clots or debris remaining under the skin before closure. A final irrigation with warm saline may be necessary at this step.</li>
	</ol>
	</li>
	<li>Stop the anesthesia. Observe the animal for 10&#8211;30 min until recovery. The animal should remain on the heating pad for this duration. Provide water gel and hydrated food in the cage.</li>
	<li>For post-operative care, include buprenorphine (twice daily), enrofloxacin (once daily) for the first two days prophylactically. Additionally, manually empty the bladder at least twice daily, adhering to the animal ethics committee&#8217;s guidelines. 
	NOTE: The animals in this experiment were assessed twice daily for their general health and wellbeing, which included checking for persisting pain (to give additional doses of buprenorphine) or infection (additional enrofloxacin), their nutrition and hydration status (give injectable fluids if dehydrated) and any signs of autotomy (provide wound care if minor autotomy). It is strongly recommended that these aspects of post-operative care, including the euthanasia decisions must be determined with the guidance of the institutional animal ethics committee.</li>
</ol>
5. Evaluation of the injury model
<ol>
	<li>Establishing the loss of motor function.
	<ol>
		<li>Assess motor behavior on the injured mice 2, 7, 14, 21 and 28 days after the injury in an open field using the Basso Mouse Scale (BMS) to determine the loss of function9 (Figure 3C&#8211;E).</li>
	</ol>
	</li>
	<li>Histological confirmation of injury
	<ol>
		<li>Harvest the injured spinal cords with the vertebral columns after euthanasia (done with carbon dioxide in this experiment, as approved by the animal ethics committee).</li>
		<li>Fix the tissue by overnight submersion in 4% paraformaldehyde and decalcify the bones by treatment with 20% ethylene diamine tetraacetic acid (EDTA) over 3 weeks, replacing fresh EDTA every 48 h.</li>
		<li>Prepare the decalcified spines for cryo-sectioning and cut them into 30 &#181;m thick sections.</li>
		<li>Mount the sections on gelatin-coated glass slides for immuno-staining with anti-GFAP antibodies and Hoechst 33342.</li>
		<li>Image the slides on a fluorescent microscope (Figure 3A) and perform measurements of the injury size using image analysis software (e.g., Nikon analysis software - NIS Elements [Figure 3B]).</li>
	</ol>
	</li>
</ol>

<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 completeness of the injury, and enhanced ability to control bleeding and achieve hemostasis. The advantage of this method is that the protocol can be modified for use at any other vertebral levels other than T10, as well as to perform other injury types such as hemi-sections, partial dorsal transections, dorsal root avulsions, crush and contusions.
A major component of this protocol is that it employs the use of a fine-tip drill. While the use of the drill may require a high skill level and more extensive training, it achieves a clean and complete laminectomy. Another crucial factor is the use of a narrow-bladed knife for transection, instead of micro-scissors. This results in less unwanted collateral damage as compared to using scissors. Conversely, however, if too much lateral pressure is exerted, the blade can cause some injury to the vertebral body. The described protocol may require the surgeon to perform some troubleshooting. If the laminectomy is not performed properly, there may be bone spurs remaining, which can restrict access to the laminectomy window. Inserting one of the prongs of the fine tip forceps can enable the surgeon to grasp and break off any remaining bone spurs. However, care must be taken to not injure the exposed spinal cord in the process. If the laminectomy results in a jagged bone edge, the drilling can be performed again to straighten the laminectomy margin. This may be impractical if the laminectomy window is already wide enough, in which case, the transection should be performed without tampering with the laminectomy site.
It is strongly recommended that the users practice the laminectomy procedures at least 8&#8211;10 times at the relevant spinal level in a cadaveric dissection prior to attempting in a live survival surgery. Although, the drill holding and maneuvering techniques are simple, they may require the users to get acquainted with the equipment. Here, we also provide some useful advice to help with spine and hand stabilization during the drilling procedure. If the user is right handed, the spine should be stabilized by using the curved forceps with the left hand so that the forceps approach the spine from the cranial aspect. This keeps the caudal aspect of the spine clear to approach with the drill held in the right (or the predominant) hand. The drill should be held with a pincer grip between thumb, index and middle fingers. The hand should be well supported along the medial edge of the wrist and the outstretched fifth finger. Keeping the arm completely adducted so that the elbow touches the body may help achieve better control over the drill grip during practice. The drilling action should only involve motion at the fingers holding the drill and not at the wrist, not unlike using a pen for writing.

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><tbody><tr><td>Baytril injectable 50 mg/mL, 50 mL</td><td>Provet</td><td>BAYT I</td><td>Post-operative care drug</td></tr><tr><td>Betadine 500 mL</td><td>Provet</td><td>BETA AS</td><td>Consumable</td></tr><tr><td>Castroviejo needle holder, locking</td><td>ProSciTech</td><td>T149C</td><td>Reusable</td></tr><tr><td>Ceramic zirconia blade, round with sharp sides, single edge, angled</td><td>ProSciTech</td><td>TXD101A-X</td><td>Reusable</td></tr><tr><td>Cotton swabs (5pcs)</td><td>Multigate</td><td>21-893</td><td>Consumable</td></tr><tr><td>Dremel Micro</td><td>DREMEL</td><td>8050-N/18</td><td>Cordless rotary tool</td></tr><tr><td>Dressing forceps fine</td><td>Multigate</td><td>06-306</td><td>Single use disposable</td></tr><tr><td>Drill bits</td><td>Kemmer Pr&amp;auml;zision</td><td>SM 32 M 0550 070</td><td>Reusable</td></tr><tr><td>Dumont #7b forceps</td><td>Fine Science Tools</td><td>11270-20</td><td>Reusable</td></tr><tr><td>Dumont tweezers, style 5</td><td>ProSciTech</td><td>T05-822</td><td>Reusable</td></tr><tr><td>Fur trimmer</td><td>WAHL</td><td>WA9884-312</td><td>Zero Overlap Hair Trimmer</td></tr><tr><td>Iris scissors, Ti, sharp tips, straight, 90mm</td><td>ProSciTech</td><td>TY-3032</td><td>Reusable</td></tr><tr><td>Isoflurane isothesia NXT 250</td><td>Provet</td><td>ISOF 00 HS</td><td>Anaesthetic agent</td></tr><tr><td>Colibri Retractor - 4cm</td><td>Fine Science Tools</td><td>17000-04</td><td>Reusable</td></tr><tr><td>Scalpel handle</td><td>ProSciTech</td><td>T133</td><td>Reusable</td></tr><tr><td>Signature latex surgical gloves size 7.5</td><td>Medline</td><td>MSG5475</td><td>Consumable</td></tr><tr><td>Sodium Chloride 0.9%</td><td>STS</td><td>PHA19042005</td><td>Consumable</td></tr><tr><td>Sterile Dressing Pack</td><td>Multigate</td><td>08-709</td><td>Single use disposable</td></tr><tr><td>Sterile Fluid Impervious Drape 60x60 cm</td><td>Multigate</td><td>29-220</td><td>Single use disposable</td></tr><tr><td>Surgical spirit 100 mL</td><td>Provet</td><td># SURG SP</td><td>Consumable</td></tr><tr><td>Suture Material - SILK BLK 45CM 5/0 FS-2</td><td>Johnson &amp;amp; Johnson Medical</td><td>682G</td><td>Silk Suture</td></tr><tr><td>Suture Material - Vicryl 70CM 5-0 S/A FS-2</td><td>Johnson &amp;amp; Johnson Medical</td><td>VCP421H</td><td>Vicryl Suture</td></tr><tr><td>Temgesic 0.3 mg in 1 mL, x 5 ampoules (class S8 drug)</td><td>Provet</td><td>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 laminectomy window as shown by the rectangle. The resulting laminectomy window is clear and allows direct visualization and access to the spinal cord (Figure 1E, highlighted zone). The schematic concept of this process is explained in Figure 1F. The narrow transection blade can easily fit through the discrete laminectomy window (Figure 1G) and in a smooth swiping motion, a complete transection type injury can be created (Figure 1H&#8211;I). Thus, this method causes minimal muscle dissection, minimal collateral bone damage, and results in a stable complete transection type injury with minimal blood loss. Despite the induction of severe SCI in the animals, the surgical procedure and post-operative care resulted in high survival rates of the animals. All animals reported for this manuscript survived the spinal cord surgery; and subsequent surgeries by our lab have resulted in a survival rate of &#62;99%.
To assess whether this method for inducing transection-type SCI in mice was reproducible and consistent, we analyzed the injured spinal cord using histology/immunohistochemistry and behavioral testing (n = 8 animals) (Figure 2). Immunolabeling against the astrocyte marker glial fibrillary acidic protein (GFAP) demarcated the boundary of the intact spinal cord, with the injury site being the region between the cord stumps when viewed with longitudinal sections (Figure 3A). A consistent-sized defect was induced at the transection site, with an average minimal distance of 550.4 &#177; 17.3 &#181;m (Figure 3B). Behavioral data deploying the Basso Mouse Scale (BMS)9 of an open field test showed that the injured mice exhibit no hind limb movement after the injury (Figure 3C). This is represented by a score of 0 on the BMS for up to 28 days post-injury. Thus, the protocol produces complete and reliable transection-type injury resulting in complete loss of function below the injury level and does not lead to spontaneous reversal of the paralysis (Figure 3D,E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61131/61131fig1.jpg" /> 
Figure 1: Key steps in the transection injury protocol. (A) Animal set up and stabilization prior to surgery. Schematic and photograph from the surgery are both shown. (B) Schematic and photograph showing incision, retractor placement and blunt dissection to expose the deep muscle layers. (C) Stabilization of spine with forceps and exposing laminae of T10 vertebra. The rectangle in the photograph highlights laminae and spinous process of the T10 vertebra. (D) Fine tip drill to perform laminectomy. The rectangle in this photograph traces the laminectomy window. (E) Complete laminectomy window highlighted by the rectangle, within which the spinal cord is clearly visible. (F) Series of schematic drawings showing the drilling concept to perform a complete laminectomy, in a cross-sectional orientation. (G) This photograph depicts the use of a fine blade transection knife to perform the complete transection type injury, and in (H) the complete injury is visible inside the rectangle, as a dark red transverse line across the spinal cord. (I) Schematic showing the overall view of the laminectomy and injury site. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61131/61131fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61131/61131fig2.jpg" /> 
Figure 2: A schematic explaining the vertebral landmark identification for T10. The spinous process of T10 vertebra is one of the most prominent landmarks that can be palpated at the natural curvature of the thoracic spine. At this point the spinous processes change the morphology so that the tips of the spinous processes cranial from T10 point caudally, and the tips of the spinous processes caudal from T10 point cranially. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61131/61131fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61131/61131fig3.jpg" /> 
Figure 3: Representative results from transection type injury induced in C57BL/6 mice. (A) A longitudinal section of the spinal cord reveals the complete transection-type injury.Tissue was imaged on an inverted microscope. Anti-GFAP immunolabelling labels astrocytes (red) while nuclei are labeled with Hoechst 33342 (blue). The injury gap using a linear&#160;measurement of the shortest point was 550 &#181;m. Scale bar = 200 &#181;m. IS = injury site, IVD = intervertebral disc, SC = spinal cord, VB = vertebral body. (B) Injury size was measured in 8 animals. The mean injury size was 550.4 &#177; 17.3 &#181;m with maximum being 577.8 &#181;m and minimum 525.4 &#181;m. (C) Motor behavior scored on Basso Mouse Scale (BMS), which was 9 in all mice prior to the injury and remained at 0 for 4 weeks after the injury, indicating a complete loss of motor function below the injury site (n = 8 mice). (D) Gait of a healthy mouse before the injury. (E) Gait of the same mouse after the injury. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61131/61131fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</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, ...

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Universidad San Sebastian

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Neuroscience

8.1K Views.  Griffith University. 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.

Video: Induction of Complete Transection-Type Spinal Cord Injury in Mice

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Medicine

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

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

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

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

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

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 Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.

Induction of Complete Transection-Type Spinal Cord Injury in Mice

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Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Controlled Cervical Laceration Injury in Mice

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

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

A Neonatal Mouse Spinal Cord Compression Injury Model

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform