Study design: Experimental study of spinal cord injury using an organotypic slice culture.

Objective: To clarify the mechanism of corticospinal axon regeneration following transplantation of neural progenitor cells (NPCs) in the injured spinal cord.

Summary of background data: Several mechanisms underlying central nervous system regeneration after transplantation of NPCs have been proposed; however, the precise mechanism has not been clarified. Previously, we demonstrated that transplanted NPCs secreted humoral factors that in turn promoted corticospinal axon growth using the unique organotypic coculture system involving brain cortex and spinal cord from neonatal rats.

Methods: Cultured NPCs were immunostained with antibodies against neurotrophic factors including brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, nerve growth factor (NGF), and ciliary neurotrophic factor (CNTF) both before and after differentiation. To evaluate corticospinal axon growth quantitatively, we used the organotypic coculture system. The dissected brain cortex and spinal cord obtained from neonatal rats were aligned next to each other and cultured on a membrane. NPCs were transplanted onto the cocultures. Furthermore, neutralizing antibodies against BDNF, NT-3, NGF, or CNTF were added to the cocultures. Axon growth from the brain cortex into the spinal cord was assessed quantitatively using anterograde axon tracing with DiI.

Results: The cultured NPCs were positively immunostained by antibodies against BDNF, NT3, NGF, and CTNF both before and after differentiation. Transplantation of NPCs promoted axon growth from the brain cortex into the spinal cord. The axon growth promoted by NPCs was significantly suppressed by the addition of neutralizing antibodies against BDNF, NT-3, and NGF but not CNTF.

Conclusion: The neurotrophic factors, BDNF, NT-3, and NGF, secreted by transplanted NPCs, were involved in the promotion of corticospinal axon growth after transplantation of NPCs.