The contributing authors for this video are Dr.VOR Krishna, chief resident physician in the Department of Neuroscience, division of Neurosurgery at the Medical University of South Carolina, Dr.Mark Kendi, professor of neuroscience at the Medical University of South Carolina, and career scientist at the South Carolina VA Medical Center, and Hampton Andrews, a second year student in the College of Medicine at the Medical University of South Carolina. This video will demonstrate the preparation of an animal in sterile field for surgery, the surgical procedure itself and the methods of post-surgical care. First, all surgical instruments and supplies must be autoclave prior to setting up the surgical field.
If you're performing more than one procedure, be sure to autoclave additional sets of instruments for each animal. Next, clean the surgical area and all components with alcohol wipes. Once the surgical field is prepared, lay out all your surgical instruments on a sterile drape.
We will demonstrate today's procedure using an adult female spray. Dolly rat. Bring your rats to the lab a few hours before the procedure.
Administer 1.5 milliliters of 0.006 milligrams per milliliter of buprenorphine subcutaneously at least one hour before the procedure, just before anesthesia. Give 0.1 milliliters of subcutaneous arol at four milligrams per kilogram. Then anesthetize app with an intraperitoneal injection of 90 milligrams per kilogram of ketamine and wait for the animal to fall asleep.
Palpate and mark the prominent T 10 spinous process in the thoracic spine. Shave the area surrounding the mark with an electric razor. Then sterilize the area three times with Betadine solution comfortably position the rat on the surgical field and ensure that it is on top of the heating pad.
Confirm anesthesia with the tail pinch. Maintaining counter traction began with an approximate four centimeter incision with a number 10 scalpel blade centered on the T 10 mark. Cut out the loose underlying connective tissue, exposing the muscles and ligaments surrounding the vertebral column.
Use retractors to separate the skin and loose connective tissue. Exposing these muscles and ligaments. Proceed to carefully and patiently dissect fascia and muscle layers away from the spinous processes and lamina of T nine through T 11.
Use retractors to separate the muscle and fascia away from the bone and expose the spinal column while stabilizing the spinous process of T nine sharply. Dissect the interspinous ligament between T nine and T 10 using small scissors. Repeat this process for the interspinous ligament between T 10 and T 11.
Carefully complete the division of ligaments through the ligamentum flavum Using fine tipped raw jores, carefully perform a piecemeal bilateral laminectomy at T 10. Be careful to avoid downward pressure on the thecal sac during bone resection, Completely remove the lamina and spinous process of T 10. Once the spinal cord is exposed and free of bon debris, move the rat into position on the Stereotaxic platform.
Use stereotactic clamps to immobilize the spine by clamping the lateral aspects of the T 11 vertebral body, followed by the lateral aspects of the T nine vertebral body. After securing the spinal column, confirm the settings on the computer controlled impactor. Our model uses a speed of four centimeters a second to a depth of two millimeters.
Extend the tip of the impactor and lower it to a position Just touching the spinal cord, retract the tip. Then lower the impactor two millimeters. Being careful to maintain its position over the cord.
Now fire the piston to create a severe contusion injury. Remove the rat from the stereotactic clamps. Then use gentle pressure with a cotton tip applicator to attain hemostasis.
Using a figure eight technique stitched together muscle and fascial layers with absorbable sutures. Now close the skin using a minimum of two small staples. Once the procedure is completed, place rats in a warm environment for approximately 24 hours post-surgery.
Once the animals are completely awake, administer five milliliters of saline, 1.5 milliliters of buprenorphine and 0.1 milliliters of atri. All subcutaneously continue with buprenorphine twice a day for the next 48 hours and atri once a day for seven days. Bladders should be manually expressed three times a day until bladder function returns.
This will be indicated by less than two milliliters of urine in the early morning. Expressions for three consecutive days. Animals should also be checked during this time for signs of infection.
These include blood purulent, discharge or foul odor in urine, as well as decreased activity, weight loss, and problems with wound healing. If an is found increase dosage or reinitiate antibiotics, you have just seen how to create a survivable severe contusion spinal cord injury in rats. When conducting this procedure, it is important to remember to consistently locate the targeted level of the spinal cord for each rat.
Also clear all bone fragments from the laminectomy site to reduce other possible injury mechanisms. Be sure the rat is positioned comfortably in the stereotactic clamps and constantly monitor breathing throughout the procedure. Check the animal's weight every day post procedure.
This is done to screen for infection, appetite problems, skin breakdown, and post spinal cord injury. Ileus you for watching our video and good luck with your experiments.
