This study demonstrates a surgical technique for spinal cord injury in rats, focusing on the use of a piezoelectric polymer conduit to facilitate axon regeneration. The method involves the transection of the spinal cord and the application of Schwann cells to promote nerve fiber growth.
Take an anesthetized rat with exposed vertebrae, with the spinous processes and laminae excised from thoracic levels T7 to T9.
Cut the dorsal and ventral roots above and below T8.
Apply saline to help stop bleeding and remove the liquid using a sponge.
Transect the spinal cord between T8 and T9 to sever nerve fibers.
Place compressed foam in the gap and apply saline.
Once the bleeding stops, remove the foam and clear residual fluid.
Slide a pre-prepared piezoelectric polymer conduit, with windows open, over the rostral spinal cord stump.
Insert the caudal stump into the conduit’s opposite end.
Inject Schwann cells, suspended in an injectable matrix, through the conduit windows.
Close the windows securely.
Schwann cells populate the conduit and secrete neurotrophic factors that support axon growth.
The conduit provides structural guidance, while its piezoelectric properties generate bioelectric cues for neurite outgrowth.
Gradually, axons regenerate across the bridge, restoring continuity between the stumps.
When all of the laminae have been removed, confirm that the gaps between the transverse processes are visible, especially at T8, and examine the bones along both sides between T7 and T9 to confirm there are no outwardly protruding bone fragments, and the dorsal roots should become visible. Using angled spring scissors, cut the dorsal and ventral roots above and below T8. And add saline to the spinal cord to help stop the bleeding.
Next, place the angled spring scissors above the spinal cord in the gaps between the transverse processes at T8, and make one cut to completely sever the nerve tissue. Place a small piece of compressed foam into the resulting 2 to 2.5 millimeter gap, and immediately add saline to the area.
While waiting for the severed cord stumps to reach hemostasis, cut absorption triangles into thin, long pieces and remove a conduit from PBS storage. Place some triangles into the conduit to remove excess PBS and confirm that the pre-cut windows are open.
Next, remove the medium from a GFP Schwann cell pellet and resuspend the Schwann cells in 10 microliters of cold DMEM/F-12. Add 10 microliters of cold injectable gel to the cell suspension and mix well with repeated pipetting. Then, place the cell suspension on ice. When the cord stumps are ready, remove the compressed foam and then use long pieces of the absorption triangles to remove the saline and blood from the laminectomy area.
Then, use a micro spatula to gently lift the rostral stump and slip the conduit over the stump with the windows facing up. Taking care that the entire stump is inserted and that there is no excess bleeding into the conduit. Gently lift the caudal stump and slip the other end of the conduit over it. Taking care that the entire stump is inserted and that the windows are on the dorsal surface.
Then, use a micropipette equipped with a Western blot loading tip to inject 20 microliters of the GFP Schwann cell injectable matrix mixture into the conduit through one of the pre-cut windows. And close the windows.
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