The overall goal of this study is to examine the effect of spinal cord injury on the dynamics of pressure ulcer development and healing in an animal model. The main advantage of this technique is that it does not impair animal movements, and is very reproducible. This model provides a platform to study the dynamics of wound healing and test various therapeutic strategies for SCI patient.
Before beginning the procedure ensure proper anesthesia by the lack of response to a tail pinch. Shave the hair over the dorsum with an electric clipper. Then apply the depilatory cream, and leave for three minutes to remove the remaining hair.
After three minutes remove the depilatory cream with the wet scrub and wash the dorsum similarly. And then return the animals to their cages. The next day anesthetize the animals and apply the ophthalmic ointment to the corneas to protect the eyes from drying during the surgical procedure.
Then, scrub the animal skin three times each with betadine scrub and 70%ethanol. Before incision, use a syringe with a 25 gauge needle to inject 100 microliters of 0.125%bupivacaine around the incision site as analgesia. Count back from the floating ribs that correspond to T13 to identify T8 to T12.
After creating a one to 1.5 centimeter incision, along the midline, on the back, at the level of T8 to T12 vertebrae clear the subcutaneous adipose tissue to access the paraspinal muscles. And then dissect them slowly to expose the spinous processes and lamina on both sides. It is very critical to do this procedure very carefully to avoid excessive bleeding and any injury to the spinal cord.
Perform a laminectomy of the T9 to T10 vertebrae to expose the spinal cord by gently peeling off the spinal lamina using micro dissecting forceps. Use forceps to secure and lift the spinal column at T8 to exaggerate the spinal curvature. Then, use fine scissors to section the spinal cord between the T9 and T10 vertebrae all the way to the floor of the vertebral canal to ensure complete transection.
After observing the complete transection under a surgical microscope apply a piece of subcutaneous fat over the laminectomy site to provide additional protection to the spinal cord prior to surgical site closure. Finally, close the wound and suture the paravertebral muscles and superficial fascia. Then close the skin using suture clips.
Immediately after the SCI surgery scrub the back of the animal with betadine and 70%alcohol. For a pressure ulcer below the spinal cord injury site use a 25 gauge needle to inject 10 microliters of 0.125%bupivacaine solution in an ellipse in the dorsal skin, near the sacrum, with points 0.5 to one centimeter apart. Gently lift a skin fold on the back of the mouse and sandwich it between two magnetic disks.
Immediately after magnet application return the animals to single cages placed onto a heating pad until full consciousness is regained. After 12 hours of magnet application lightly anesthetize the animal with isoflurane and remove the magnets. Take a photograph of the wound sites to record the initial appearance of the pressure ulcer at the day zero time point.
Cover the wound with transparent dressing film to avoid drying or contamination. Single-house the animals following the procedure. Immediately after surgery inject the animal with one milliliter of 0.9%saline for hydration and buprenorphine SR for analgesia.
Perform manual bladder evacuation twice a day. Remove the surgical clips seven days after spinal cord injury surgery. Collect wounded skin samples from the euthanized mouse at the desired time point after spinal cord injury and skin pressure ulcer induction.
Fix in 10%formalin for 24 hours. After 24 hours transfer to 70%ethanol and store at four degrees Celsius until sectioning. This image depicts the disappearance of the epidermis in a control animal by day three of post-magnet removal.
Arrows indicate the epidermal wound edges located where the epithelial lining thins out. In the control animal the epidermis reappears by day seven. For wounds created below the level of the SCI, in SCI mice, significantly slower migration is seen.
Here, the figure depicts slower wound healing and closure in the SCI mice as compared to the control mice. Immunostaining for Ki67, a marker of proliferating cells indicated by the red arrows, revealed less proliferation in the SCI group. Staining for CD31, a marker for blood vessels indicated by the red arrows, revealed lower blood vessel density in the SCI group.
Finally, immunostaining for alpha smooth muscle actin revealed lower levels of this protein in the skin wounds from SCI mice. While attempting this procedure it is important to remember to place the magnets properly to develop two circular wounds separated by intact skin. This will reduce the variability in wound development and healing.
Skin pressure ulcers in spinal cord injured patients impart significant costs to the U.S.healthcare system. This animal model provides a platform for testing various therapeutic strategies to aid pressure ulcer healing in spinal cord injured patients.
