Figure 7.

hμG influence axon regeneration through proinflammatory cytokine-mediated recruitment of the mTOR pathway

hRGCs were cultured in the soma chambers of microfluidic devices till they elaborated axons into the axon chambers and divided in 4 groups. Axons were retrogradely labeled with CTB 488 (pre-axotomy lane) followed by axotomy (axotomy lane) and regeneration was followed for five days post axotomy (post-axotomy lane) (A–D). Only CTB 488⁺ tdT⁺ processes in the axon chambers were regarded regenerative and counted. The proportion of regenerated axons (E) and their length (F) were significantly increased in hμG CM group versus antibody-neutralized hμG CM group demonstrating the influence of cytokines on axon regeneration. That the facilitatory influence of hμG-derived TNF-α and IL-1β involved AKT/mTOR pathway was demonstrated by remarkable abrogation of increase in axon regeneration in the presence of rapamycin (E, F). pS6 staining showed higher levels when exposed to hμG CM, an effect abrogated by neutralization and abolished by rapamycin (G and H). Experiments were carried out in triplicates per group from 3 independent biological replicates. Values expressed as mean ± SEM. Scale bars: 20 (post-axotomy group) and 50 μm (pre-axotomy, Axotomy, pS6).