This protocol is the first to introduce a standardized contusion type injury in axolotl spinal cord regeneration research. The main advantage of this technique is that it mimics the trauma seen in the everyday clinic, enhancing the likelihood that a future finding will be able to be translated. The contusion injury device can be utilized in any other small animal model of spinal cord injury.
The surgical technique is difficult, especially the laminectomy. Therefore, it is advisable to train the procedure on a large number of animals ex vivo before progressing to in vivo surgery. To begin, set up a surgical table by placing a standard Petri dish under a stereo microscope, on top of a surgical textile cloth.
Place the anesthetized axolotl in the prone position on the Petri dish, and wrap it in paper towels so that the tail is exposed. Identify the hind limbs and use a pair of micro scissors to make the first incision just caudal to them. Make the vertical incision from the keel until the bony prominence of the spinous processes are felt.
Extend the cut laterally, so the incision traverses the entire width of the tail, and grasps the spinous processes with forceps to ensure proper depth. Then extend the vertical incision one millimeter below the spinous process on both sides. Place the animal on one side, and starting from the ventral point of the vertical incision, make a horizontal incision of approximately 15 millimeters for animals that are 10 to 20 grams in weight.
Use the scissors to dissect immediately through the horizontal incision until the vertebral column is felt in the midline. Then turn the animal on to the other side and repeat the process. After dissecting in the deep medial plane from both sides, dissect through the midline to connect the two horizontal incisions.
Then, move the free piece of tail and keel to one side to expose the spinous processes. Place the animal in the prone position with the head facing the surgeon's nondominant side, and use a pair of forceps to grasp the spinous processes, just caudal to the hind limbs. Apply a gentle lift up and towards the animal's head.
Place the blades of a pair of micro scissors horizontal around the process and gently cut it, exposing the spinal cord. Grasp the spinous process caudal to the one just removed, and repeat the process. It is imperative that you keep a constant lift on the spinous process to ensure that it does not inflict damage to the spinal cord when removed.
If the exposed area is not wide enough, use two pairs of forceps to grasp the laminae on both sides of the spinal cord and twist them laterally with a gentle movement. When removing the spinous process, it might be necessary to do this in multiple steps. Therefore, exposing the spinal cord may take several minutes.
To introduce a contusion type injury, use a Petri dish to transfer the animal to the trauma unit and shine a flashlight on the spinal cord. Then, place the contusion trauma unit cylinder above the exposed spinal cord using the micro adjusters, and lower the cylinder until it is level with the laminae. Attach the falling rod to the electro magnet, and place the desired falling height adjustment cylinder on the trauma unit.
Next, place the falling rod on the cylinder, turn off the electromagnet to drop the rod on the exposed spinal cord, and use the height adjustment screw to lift the rod back up. If introducing a sharp injury, cut the spinal cord in a perfect vertical cut, then repeat the cuts two millimeters to the caudal side of the body. Feel the blades of the scissors scraping along the ventral part of the spinal canal to ensure that the cuts are complete, and lift the piece of spinal cord from the canal.
To close the surgical wound, place 10 point O nylon sutures from the most caudal part of the horizontal incision closing the wounds in one layer and working towards the vertical part of the incision. When an angle is reached, turn the Petri dish and suture the other horizontal incision, then set sutures on the vertical incisions. Prior to terminating anesthesia, use a high frequency ultrasound to acquire images of the injury.
Align the tip of the transducer with the animal'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. Identify the injury site and acquire images according to the manuscript directions. When finished, return the axolotl to anesthetic free solution by submerging the Petri dish in five centimeter deep freshwater and letting the animal slide off.
The spinal cord injuries created using this protocol were validated using hematoxylin and eosin staining on injured and sham axolotls. To confirm regeneration, histological sections were prepared nine weeks post injury. The images showed a reestablished spinal cord connection in the injured animals.
Injury and regeneration we're also followed by examining neurological function. The animal's reaction to stimulation of the tail was scored with higher scores indicating higher tactile and nociceptive sensory functions. Among the injured animals, there was a loss of neurological function three weeks post injury, and a gradual restoration within nine weeks.
The spinal cord injury was also visualized using ultrasonic graphic imaging, which allowed for visualization of the dorsal artery as a marker of vessel integrity. Hi-field MRI scans were performed immediately after injury and at three, six, and nine weeks. These scans were able to visualize edema and restoration of spinal cord integrity.
The scans immediately post injury, were prone to noise. Patient and meticulous surgery will pave the way for a successful result after laminectomy without premature damage to the spinal cord. Following this procedure, several in vivo methods can be applied, such as imaging and electrophysiological techniques.
