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.
