Neurofilament elongation into regenerating facial nerve axons

Immunocytochemistry was used to show that neurofilaments advance into regenerating facial nerve axons at 2.5 mm/day, which is less than the rate of axonal elongation (4.3 mm/day), measured from the transport of radiolabeled protein into the axons. Thus, the distal region of the newly-regenerated axons is deficient in neurofilaments, and this was confirmed by electron microscopy. These neurofilament-free regenerating axons could also be detected by immunocytochemistry using antibody to protein B50 (GAP43), a component of growth-cones. Immunoblots of nerve segments, incubated with monoclonal antibodies against the three neurofilament proteins, showed that all three proteins were present in the neurofilaments elongating into the regenerating axons, and confirmed the more distal extensions of B50 immunoreactivity. These results show that neurofilament immunocytochemistry underestimates the extent of axonal regeneration, and it is suggested that this technique should be employed with caution in regeneration studies. When the facial nerve received a conditioning lesion 7 days prior to a test lesion, axonal regeneration rate increased to 6.0 mm/day, and there was a proportional increase in neurofilament elongation rate to 4.4 mm/day. This occurred in spite of the reduction in cell body neurofilament protein synthesis induced by the lesions. It is concluded that the rate of neurofilament extension into regenerating axons is not governed by cell body synthesis but by local interactions with other cytoskeletal materials which support the increased regeneration rate of conditioned axons.