Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

The effects of peripheral nerve injury on the content, synthesis, and axonal transport of the class III beta-tubulin protein in adult rat dorsal root ganglion (DRG) neurons were examined. Recent reports of selective increases in the steady-state levels of the beta III-tubulin mRNA during axonal regeneration (Moskowitz et al., 1993) led to the hypothesis that upregulated levels of expression of the beta III-tubulin isotype that alter the composition of neuronal microtubules is important for effective axonal regrowth. If this is the case, the increases in mRNA levels must be translated into increased beta III-tubulin protein levels and subsequently modify the axonal cytoskeleton via axonal transport mechanisms. The present study assessed whether or not this occurs by examining beta III-tubulin protein content in adult rat lumbar DRG neurons at different times (1-14 d) after a distal sciatic nerve crush (approximately 55 mm from the DRG) by Western blotting and immunocytochemistry with a beta III-tubulin specific monoclonal antibody. These studies showed substantial increases in beta III-tubulin content in DRG neurons, as well as in proximal regions of peripheral sensory axons (0-6 mm from the DRG), from 1-2 weeks after a distal nerve injury. Pulse labeling of DRG neurons with 35S-methionine and 35S-cysteine and immunoprecipitation of labeled beta III-tubulin indicated that the synthesis of beta III-tubulin was increased in the DRG after axotomy. Studies of axonal transport, wherein L5 DRG proteins were labeled with 35S-methionine and 35S-cysteine by microinjection, revealed that slow component b(SCb) of axonal transport conveyed more labeled tubulin moving at apparently faster rates through the intact regions of sciatic nerve axons in response to crush injury of the distal sciatic nerve. Immunoprecipitation experiments using proximal peripheral nerve segments showed that SCb in distally injured DRG neurons was enriched in the beta III-tubulin isotype. These findings demonstrate that the augmented synthesis of beta III-tubulin after axotomy alters the composition of the axonally transported cytoskeleton that moves with SCb. The increased amounts and rate of delivery of beta III-tubulin in axons of regenerating DRG neurons suggest that the altered pattern of tubulin gene expression that is initiated by axotomy impacts on the composition and organization of the axonal cytoskeleton in a manner that can facilitate axonal regrowth.