The overall goal of this procedure is to generate consistent, accurate, and reproducible spinal cord injury using a novel vertebral stabilizer, which can be used with multiple SCI devices. This is accomplished by carefully dissecting the tissue surrounding the cervical vertebrae, using caution to minimize hemorrhage. The second step is to place the animal in the stabilization device and stabilize the vertebrae using the serrated stabilization arms.
Next, perform a laminectomy to expose the targeted region of the spinal cord. The final step is to mount the U-shaped stabilization device to the NYU masses, impactor, and perform SCI Ultimately consistent reproducible contusive spinal cord injury is generated with the novel stabilization device. Today we'll demonstrate the use of our unique surgical stabilization device accompanied by the New York University spinal cord impactor for creating consistent reproducible conducive spinal cord injuries.
We use this stabilization device in our laboratory for generating spinal cord injuries as well as for transplantation and microinjection surgeries and image. Begin this procedure by cleaning the surgical surface with 70%ethanol. Place a heating pad on the surgical area and turn on the heating pad.
Then place the sterile gauze cotton swabs and autoclave surgical tools in the surgery area. After covering the surface with a sterile surgical drape, use a microbead sterilizer for inter surgery sterilization of surgical tools. Also clean the stabilizer with 70%ethanol prior to and between surgeries.
Now anesthetize the rat by intraperitoneal injection with ketamine xylazine, and confirm the anesthesia depth by toe pinch stimulus. Then apply protective ointment to the eyes of the animal to prevent corneal drying. After that, shave the back of the rat from the mid thoracic region to its head.
Then remove the fur using a vacuum equipped with a HEPA filter. Next, apply Betadine to the shaved area as a surgical scrub and clean the area with 70%isopropyl alcohol wipes. Subcutaneously inject 0.01 to 0.05 milligrams per kilogram buprenorphine and five milligrams per kilogram carprofen prior to surgical procedure.
Buprenorphine should then be administered every eight to 12 hours and carprofen once daily for the first four to seven days following surgery Afterward, place the rat onto the Prewarm surgical area. Use a scalpel blade to perform a three to four centimeter midline incision on the skin from the base of the head coddly to midt thorax. Then identify the midline of the fascia and subcutaneous muscles anterior to the hibernating gland at the lower neck.
Cut through the trapezius and other muscles along the midline to reduce hemorrhage. Find the adipose tissue underlying the muscles at the midline. Cut the paraspinous muscles coddly.
Subsequently separate the muscle layers using a small tissue retractor until the level of the thoracic T two spinous process is reached. Then cut away the muscle connected to the T two spinous process and utilize this structure as an anatomic landmark. Next, remove the cartilaginous tip of the T two spinous process to improve visibility of the cervical vertebrae.
Separate the paraspinal muscles laterally from the spinous processes and laminate, however, spare the muscle covering the C3 lamina to prevent bleeding. Next, cut the muscles over the lamini laterally towards the facets on both sides of the spinal column. After the spinal lamini are exposed, place the animal in the U-shaped channel of the stabilizer.
Locate the C five vertebra by counting the spinous processes roly from the T two landmark to T one C seven, C six, and finally C five. In this procedure, position the two stainless steel arms of the stabilizer to suspend the animal by placing the serrated edges of the arms underneath the lateral facets of the C five to six vertebrae. After securing the arms with the vertebrae in place, adjust the stabilizing apparatus to ensure the vertebral column is leveled and centered.
Finally, lock the arms by tightening the thumb screws of the stabilizer there. Then cut the ligaments between the spinal processes and lamini at C four to five and C five to six. In order to locate the margin of the C five lamina using a micro ron, clip away half of the lamina on the right C five for SCI.
After laminectomy, transport the animal with the stabilizer from the surgical table to the injury device. Secure the animal together with the stabilizer on a mount which is affixed to the injury device. Precisely align the plunger with the spinal cord target using a lateral micro manipulator under high magnification.
Locate the C five and C six dorsal root entry zones on the exposed dorsal spinal cord surface without dur otomy. Aim the plunger at the middle of the two identified DRE Zs and halfway between the midline and the lateral edge of the spinal cord. Using an NYU masses impactor device with a 2.5 millimeter diameter tip, produce a C five HEMI contusion by a 10 gram rod at 12.5 millimeter drop height.
Verify the injury visually by the bruise on the spinal cord and check the injury parameters provided by the NYU software. Then suture the muscles and soft tissue and close the skin incision with surgical staples. Apply antibacterial ointment to the surgical site and administer 5.0 milliliters of sterile 0.9%saline subcutaneously to the animal for hydration.
Afterward, place the animal in a heat controlled environment with moist food provided on the bedding and a water bottle with a long spout for easy access placed on the floor of the cage. Provide care to ensure adequate recovery before returning the animal to the home cage. This figure illustrates a readout of a very good impact with data measurements of percent error for impact, rod velocity, initial height, and starting time, all values fall well within the window of acceptable error.
Conversely, this panel demonstrates data produced by an improper impact caused by improper stabilization of the spinal column and the error during zeroing of the impactor rod onto the spinal cord surface. Prior to setting the height of the impactor rod, This stabilization device immobilizes the spinal cord. Yet a great advantage of this device is the mobility it provides for transporting the stabilized animal from a surgical table to a transplantation microinjection station to an impact device or imaging station.
Stabilization of the lateral vertebral processes allows for stabilization of the specific vertebrae of interest, which allows for more reproducible spinal cord injury Consistency and the reproducibility are crucial factors for experimental models of spinal cord injuries in order to minimize variability and optimize clarification of therapy effects, all of which are important for application towards clinical translation and finding a cure for spinal cord injury.
