This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.
The overall goal of this surgical intervention procedure is to use a conduit filled with Schwann Cells and injectable matrix to establish a bridge between completely transected spinal cord stumps to promote axon regeneration across the bridge. Our goal is to bridge a spinal cord injury site in the rat to restore function. The injury that we are using is the creation of a complete transection gap.
It is only by using this kind of injury that we can say with assurance that the axons have regenerated when we find them in the injury site. The bridge that we are using is a polymer channel that contains Schwann Cells, Schwann Cells being so important for regeneration of axons. We show in this video how to introduce such a bridge into a complete transection gap.
Investigators new to this method will need practice as the complete transection of the spinal cord and introduction of the conduit can be challenging to beginners. Before beginning the procedure, use a number 10 blade to cut the conduit to five millimeters. This can also be performed under a dissecting microscope if preferred.
Fold the conduit gently along the longitudinal side and use straight edged Vannas scissors to cut four small incisions about 0.4 millimeters long at least one millimeter from the openings and about one millimeter apart. Then, unfold the conduit and cut between the two incisions along the same side to create two side by side windows making sure that the windows can be opened and closed properly along the uncut side. To perform the T7-T9 laminectomy, after confirming a lack of response to toe pinch in an anesthetized 180 to 200 gram female Fischer rat, locate the landmark for the T9-T11 spinous processes.
They will feel like a small triangular bump through the skin. Using a number 10 blade, make a four to five centimeter midline skin incision from T4 to T11 followed by a small incision in the superficial fat layer with curved blunt tipped scissors. After dissecting the fat, use the number 10 blade to locate the T7-T9 spinous processes and use blunt forceps to hold the muscle at about T4.Make an incision in the muscle along each side of the vertebrae from T6 to T10 as close as to the vertebrae as possible to make a clean opening and to cause minimal injury to the animal.
Place the retractor around T7 to T9.Use the number 10 blade to cut the muscles and ligaments between the individual process from T6 to T10 and use a rongeur to remove any muscle or ligaments on the laminae from T7 to T9 as laterally as possible. When the gaps between the transverse processes from T7 to T9 are visible, use the rongeur to remove the T9 spinous process, gently lifting the T8 spinous process with blunt forceps to elevate the T9 lamina at the small opening between the T9 and T10 processes. Then, use the rongeur to remove the lamina as laterally as possible, starting from the opening and moving rostrally at T9.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 two 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 precut 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 precut windows and close the windows.
A cautionary note. It is very important that the cord stumps be handled as quickly and as carefully as possible. A delay may cause swelling of the stumps making it difficult to slip the conduit over the spinal cord creating further injury.
Three weeks after transplantation, confocal fluorescent images of cryostat sagittal spinal tissue sections reveal an even distribution of the Schwann Cells along and within the conduit as well as blood vessels and myelinated axons within the center of the bridge. Axon regeneration is also closely associated with the presence of Schwann Cells, confirming the effectiveness of using a Schwann Cell bridge within a structured conduit to promote axon regeneration along the bridge between the rostral and caudal stumps. Once mastered, this procedure can be completed in 45 minutes when it's performed properly.
Following this procedure, other methods such as EndoRay tracing may be performed to answer additional questions. For example, how many axons regenerate into the bridge and how many of these exit the bridge to reenter the spinal cord? After watching this video, you should have a good understanding of how to transect the spinal cord, insert a conduit, and inject cells within a matrix to create a reliable bridge for axon regeneration and other outcome assessments.
