RNAi-mediated ephrin-B2 silencing attenuates astroglial-fibrotic scar formation and improves spinal cord axon growth.

Aims: Astroglial-fibrotic scar formation following central nervous system injury can help repair blood-brain barrier and seal the lesion, whereas it also represents a strong barrier for axonal regeneration. Intensive preclinical efforts have been made to eliminate/reduce the inhibitory part and, in the meantime, preserve the beneficial role of astroglial-fibrotic scar. Methods: In this study, we established an in vitro system, in which coculture of astrocytes and meningeal fibroblasts was treated with exogenous transforming growth factor-beta 1 (TGF-beta 1) to form astroglial-fibrotic scar-like cell clusters, and thereby evaluated the efficacy of RNAi targeting ephrin-B2 in preventing scar formation from the very beginning. We further tested the effect of RNAi-based mitigation of astroglial-fibrotic scar on spinal axon outgrowth on a custom-made microfluidic platform. Results: We found that siRNA targeting ephrin-B2 significantly reduced both the number and the diameter of cell clusters induced by TGF-beta 1 and diminished the expression of aggrecan and versican in the coculture, and allowed for significantly longer extension of outgrowing spinal cord axons into astroglial-fibrotic scar as assessed on the microfluidic platform. Conclusions: These results suggest that astroglial-fibrotic scar formation and particularly the expression of aggrecan and versican could be mitigated by ephrin-B2 specific siRNA, thus improving the microenvironment for spinal axon regeneration.