Effects of freezing a segment of peripheral nerve on subsequent protein release and axonal regeneration in the frog

Previous studies in frogs have shown that axons from the proximal stump of a cut nerve will grow toward the distal stump, possibly in response to diffusible trophic factors produced by cells of the nerve sheath. In the present experiments, the synthesis and release of proteins in vitro, from proximal and distal stumps of frog sciatic nerves, were studied 1, 4, and 14 days after nerve section in vivo. Using two-dimensional gel electrophoresis to separate released proteins, a marked increase in the synthesis of two lipoproteins of 37 and 67 kDa was seen, initially in both proximal and distal stumps, but by 14 days these proteins were produced exclusively by the distal stump. To see if the production of these proteins was correlated with subsequent reinnervation of the distal stump, isolated nerve segments were removed from the frogs and either replaced immediately or frozen (to kill sheath cells) and replaced. After 2 weeks, the pattern of newly synthesized proteins released by both the frozen and nonfrozen nerve segments was similar although freezing severely impaired the reinnervation of the nerve segment. These results suggest that although the 37- and 67-kDa lipoproteins may have a role in nerve regeneration, their presence per se is not sufficient to support the reinnervation of a distal stump of a cut peripheral nerve and that additional factors may therefore be required.