Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.
1) Preparation for microscopic surgery
2) Transection of PN to promote degeneration
3) Removal of PN segment
4) Cervical hemisection injury and transplantation
Day 1
Day 21
Day 23
5) Electrophysiological recording of action potential in graft and distal spinal cord
6) Retrograde labeling of neurons that regenerate axons into the PNG and anterograde labeling of axons that regenerate back into the spinal cord
7) Thoracic complete transection injury and transplantation
This offers an alternative approach to the cervical hemisection injury model above. Here a more severe, bilateral injury is produced and segments of peripheral nerve are placed into the lesion cavity to span and rostral and caudal stumps of the injured spinal cord.
Day 1
Day 3
8) Representative Results
When this procedure is optimized the peripheral nerve segment will closely integrate with the host spinal cord. Ascending and descending spinal axons will enter the graft, grow in a relatively straight line parallel to the length of the graft and extend to the distal end of the graft at a rate of approximately 1 mm per day. Upon reaching the distal end, axons will penetrate the adjacent spinal cord if chondroitinase has been used to remove inhibitory proteoglycans from the surrounding scar tissue. Functional connectivity will be determined by electrophysiological and immunocytochemical detection of a response by spinal cord neurons to stimulation of the axons within the graft. Anatomical correlation for functional recovery will be assessed by tracing the number and length of axons that have extended beyond the nerve graft back into the spinal cord.
Significance:
With the peripheral nerve grafting approach described above we have demonstrated that adult motor and sensory neurons will regenerate their injured axonal process for long distances when provided with an appropriate substratum for growth. We have shown that this approach can be carried out as an acute or delayed treatment strategy and that it can be applied successfully to small (mouse, rat) and large (cat) experimental animals. It is important that the functional activity of regenerating axons be tested and the peripheral nerve graft model provides relatively easy access to all of the axons bridging a spinal cord lesion. This is an obvious advantage over other transplantation approaches.
This work was supported by NIH/NINDS Grants NS26380 and NS55976, the Christopher and Dana Reeve Foundation and the Daniel Heumann Fund for Spinal Cord Research. The Drexel University College of Medicine Spinal Cord Research Center provides support for core facilities used to complete this work.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
10-0 silk suture | ArosSurgical | T5A10N10 | ||
6-0 silk suture | McKesson | 2693 | ||
Ampicillin | McKesson | 483549 | ||
Antibody to cFos | Sigma-Aldrich | F7799 | ||
Biotinylated dextran Amine | Invitrogen | D7135 | ||
Buprenorphin (.3mg/ml) | McKesson | 12496075701 | ||
Chondroitinase ABC | Associates of Cape Cod | 100332-1A | ||
Euthasol | Webster Veterinary | 07-805-9296 | ||
Hanks Balanced Salt Solution | Cellgro | 21-021-CV | ||
Isoflurane | Henry Schein | 209-1966 | ||
Michel Wound Clips | Fine Science Tools | 12040-02 | ||
Neurotrace Kit | Invitrogen | N7167 | ||
True Blue | Sigma-Aldrich | T5891 | ||
Xenodine | Webster Veterinary | 92201 | ||
Hot Bead Sterilizer | Fine Science Tools | |||
Forced Exercise Wheel | Lafayette Instruments | |||
TreadScan System | Clever System | |||
Infinite Horizon Impact Device | Precision Systems and Instrumentation | |||
Magnetic Stimulation Device | MagStim Inc. |