| We studied the regeneration of peripheral neurites by culturing adult mouse dorsal root ganglia (DRG) explants on top of unfixed, longitudinal cryosections of adult mouse sciatic nerve. DRG neurites are able to grow on cryosections of both uninjured and post-injury (distal segment) sciatic nerves. However, on uninjured nerves, DRG neurites begin to degenerate shortly after contact with the substrate while neurites growing on post-injury nerve substrate remain healthy for up to 9 days in culture, implying that the sciatic nerve environment undergoes a transformation during Wallerian degeneration such that it becomes supportive of stable neurite outgrowth. Using a semi-quantitative scoring system, we found that such capability to support stable outgrowth peaks at 8 days after injury, but gradually decreases at 10 days until it disappears by 20 days after injury, even when regenerating neurites are prevented from entering the distal nerve segment. Macrophages, which begin to accumulate in the distal segment by 4 days after injury, is a likely candidate cell type responsible for the transformation of the post-injury nerve environment since uninjured nerve cryosections can be rendered supportive of stable outgrowth by pre-incubation with macrophage-conditioned medium. The activity of the macrophage-conditioned medium has protein-like biochemical properties and is secreted in increased amount on activation of macrophages. Two hypotheses may account for the molecular mechanism of post-injury nerve transformation: (1) A presumptive "degeneration signal" molecule normally present in the adult nerve is removed by macrophages after injury; (2) A presumptive "permissive signal" molecule is laid down, directly or indirectly, by infiltrating macrophages in the post-injury nerve. Several lines of evidence appear to favor the "degeneration signal" model but not the "permissive signal" model. First, as macrophages are known to secrete several matrix-degrading proteases during tissue remodeling, normal nerve cryosections could be transformed by brief treatment with trypsin, a non-specific protease. Second, a battery of growth factors secreted by macrophages failed to mimic the effect of macrophage-conditioned medium. Third, even in the presence of combinations of neurotrophic factors, regenerating neurites degenerated on normal nerve cryosections, implying that the support of stable outgrowth is not induced via neurotrophic factors. |
