The overall goal of this video, is to introduce a novel tissue displacement-based contusive spinal cord injury model, that can produce a consistent contusive spinal cord injury in adult mice. This contusion model can help answer key questions in the experimental spinal cord injury field. The main advantage of this technique, is that it produces very consistent spinal cord injury.
The implications of this technique extended towards therapy of spinal cord injury because this model provides a reliable and a reproducible tools to study the mechanisms of injury and effects of experimental therapies. Generally, individuals new to this method will struggle because they're not familiar with the novel impactor. Visual demonstration of this method is critical as several steps are difficult to learn because of manual and software operations.
Begin by bringing the autoclave surgical instruments and metal spine stabilizer to the clean surgical table. Warm a heating pad to 37 degrees celsius. Then, place the heating pad on the operating table and cover with sterile surgical drapes.
10 week old female, C57BL6J mice are used for this study. After administering an intraperitoneal injection of ketamine and xylazine, check for an adequate level of anesthesia by the absence of any response to paw pinch induced nociception stimulation. Administer buprenorphine, an analgesic agent and carprofen, a non-steroidal anti-inflammatory drug subcutaneously.
Use electric clippers to shave the hair over the thoracolumbar spine. Scrub the skin with betadine solution and 70%alcohol wipes. Apply ophthalmic ointment to the corneas, to protect the eyes from drying.
After making a 1.5 centimeter mid line skin incision on the back of the animal over the ninth to 11th thoracic vertebral laminae, push the subcutaneous adipose tissue rostrally. Using fine scissors, fine forceps and an agricola retractor, dissect the paraspinal muscles away from spinous processes and laminae toward the lateral fascets on each side. Position the mouse on the U-shaped container of the stabilizer, clamp the stainless steel arms beneath the exposed facets of the t10 vertebra bilaterally and tighten the arms with the thumb screws attached to them.
Use a micro rongeur to remove the t10 spinous process and lamina that exposes the dura model overlying the spinal cord. Place the U-shaped container with the mouse on the stage of the impactor and fix the stage in place by tightening the thumb screws of the mount. To set up the impactor, first turn the knob of the pressure regulator on the nitrogen tank to set the compressed nitrogen to 20 pounds per square inch or a 138 kilopascals for the study.
Sterilize the 1.2 milliliter impact tip with 70%alcohol. Then, open the software package on the computer. Push button one of the control box to activate the impactor tip into a full extended position.
On the computer screen, under set zero level, click start reading, to measure the distance to the fully extended plunger tip. The distance will be shown as the range parameter in this zone. Then click on set zero and a number in milliliters will show up in the zero parameter box.
Next, push button one on the control box, to withdraw the impactor tip. Then, unlock the fastening screw, pull the screw to move the tip away from the laser beam path and turn the screw 90 degrees clockwise to lock. Now move the mount stage, by adjusting the frontal and lateral micro drivers to aim the laser beam onto the center of the exposed dorsal spinal cord.
After the injury spot is targeted, measure tissue distance by clicking the start reading button under set injury level. Use the vertical micro driver to slowly adjust the distance between the sensor and spinal cord, until the desired displacement parameter is reached. This is under set injury level, in the injury parameter box.
When the desired injury displacement is reached, record the tissue distance shown in the range parameter box, then, define the desired displacement or injury by subtracting the tissue distance or range from the tip distance at zero. Then click set injury under set injury level. Next, unlock the screw by turning it 90 degrees anti-clockwise.
Push the impact tip back into the laser beam trajectory and lock the screw by turning it 90 degrees clockwise, click the run button under run experiment to execute the impact. The parameter boxes will show the injury time in seconds, force in microvolts, velocity in meters per second and injury displacement in millimeters. After all injury data are recorded and saved, remove the U-shaped trough with the mouse from the stage.
Then, visually confirm the spinal cord injury under a surgical microscope. After suturing the incision in layers, inject the animal with one millimeter of 0.9%saline subcutaneously for hydration and place it onto a temperature controlled pad until consciousness is regained. Then house the mouse in a clean cage with easily accessible water and food.
These data show inconsistent injury parameters, demonstrate that the impactor can produce reliable and reproducible injury. This representative cross section stained with cresyl violet-eosin, shows the t10 region of the spinal cord after sham injury with the 0 millimeter displacement. This image shows the epicenter of a mild contusive spinal cord injury at t10, using the device with 0.2 millimeter displacement.
This moderate injury was achieved using 0.5 millimeter displacement. Finally, this severe injury is a result of a 0.8 millimeter displacement. These images demonstrate that this novel impactor can produce graded contusion injury.
Once mastered, this technique can be done in 30 minutes if performed properly. While attempting this procedure, it's important to remember to set zero for the impactor tip in spinal cord targets. After its development, this technique paved the way for researchers in the field of spinal cord injury, to explore mechanisms and therapies in mice, rats and non-human primates.
After watching this video, you should have a good understanding of how to use this impactor to produce contusive spinal cord injury.
