Schwann Cell-Seeded Conduits for Axonal Regeneration After Spinal Cord Injury

Abstract

Source: Lee, Y., et.al. Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap. J. Vis. Exp. (2017)

This video demonstrates the use of a piezoelectric polymer conduit seeded with Schwann cells to promote axon regeneration in a transected rat spinal cord. The conduit provides structural guidance, while its bioelectric properties stimulate neurite outgrowth. Schwann cells within the conduit secrete neurotrophic factors, supporting axonal bridging between the rostral and caudal stumps.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Transection at T8

1. Place the retractor around T7 to T9. Once all the laminae are removed, make sure that the gaps between the transverse processes are visible, especially at T8. Also examine the bones along the length on both sides between T7 to T9 to ensure there are no bone fragments protruding outward.
   NOTE: This gap at T8 is important for performing the complete transection. The vertebrae along the length should be removed for easier conduit insertion.
2. Cut the dorsal and ventral roots above and below T8 using angled spring scissors. Add saline to the spinal cord and wait for the blood to clot.
   NOTE: This step is important for proper conduit insertion.
3. 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 spinal cord. Place a small piece of compressed foam into the resulting 2-2.5 mm gap and add saline to the area immediately.

2. Conduit insertion

1. While waiting for the severed cord stumps to reach hemostasis, take out a conduit from 1x PBS. Cut the absorption triangles into thin long pieces and place them into the conduit to remove excess PBS. Check that the pre-cut windows are open.
2. Remove saline and blood using thin long pieces of absorption triangles. Lift the two stumps of spinal cord using a microspatula to ensure good separation. Gently lift the rostral stump with the microspatula and slip the conduit over the stump with the windows facing oneself. Ensure that the entire stump is inserted and there is no excess bleeding into the conduit.
3. Gently lift the caudal stump and slip the other end of the conduit over the stump. Make sure that the entire stump is inserted and the windows are on the dorsal surface (Figure 1A).

3. Schwann Cell(SC)/ Injectable Matrix Preparation and Injection

1. While waiting for the severed cord to reach hemostasis, remove the medium above the GFP-SC pellet. Resuspend the GFP-SCs in 10 µl of cold DMEM/F12. Add 10 µl of cold injectable gel to the cell suspension and mix well by repeated pipetting. Keep the GFP-SC/DMEM/F12/injectable matrix mixture on ice until conduit insertion is completed.
2. After ensuring that the windows on the dorsal surface are open (Figure 1A), inject 20 µl of the GFP-SC/DMEM/F12/injectable matrix mixture into the conduit through one of the pre-cut windows using a western blot loading tip and a micropipette. If the SC/DMEM/F12/injectable matrix mixture should overfill the conduit, remove the excess with absorption triangles. Close the windows after injection; suturing is not needed (Figure 1B).

Results

Figure 1: Window closing after SC/DMEM/F12/matrix injection. Windows are opened by folding back ea...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's modified Eagle's medium: nutrient mixture F-12	ThermoFisher Scientific	11039-021	"DMEM/F12" in protocol.
Female Fischer rat (160-180g)	Envigo
Vannas scissor, straight	FST	15018-10
Micro Spatula	FST	10089-11	Can be any brand of choice.
Curved scissors with blunt end	FST	14017-18	Can be any brand of choice.
Blunt forceps	FST	11006-12	Can be any brand of choice.
Rongeur	FST	16121-14	Can be any brand of choice.
Angled spring scissors	FST	15006-09	Can be any brand of choice.
Absorption triangles	FST	18105-03	Can be any brand of choice.
Gelfoam	Henry Schein	9083300	"Compressed foam" in protocol.
#10 blades	Sklar	06-3010	Can be any brand of choice.
Matrigel	Corning	354234	"Injectable matrix" in protocol.