Multiple neurotrophic factors including NGF-like activity in nerve regeneration chamber fluids.

Silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve of adult rats. Neurotrophic activities in cell-free fluids collected from the chambers were determined using bioassays for survival of embryonic chick ciliary and sympathetic neurons in culture. Separation by molecular exclusion HPLC of the components of fluids collected 1, 2 or 3 days after implantation revealed the presence of a multitude of neurotrophic factors differing in their molecular weights, specificity towards the two types of neurons, and time course. Antiserum to nerve growth factor partially blocked sympathetic activity of fluids collected at 1 day. Affinity purified antibody was also effective and completely eliminated bioactivity of HPLC fractions corresponding to the molecular weight of nerve growth factor. The presence in the fluids of 13-18 and 20-32 kD components active towards ciliary neurons is consistent with the release of fibroblast growth factor and ciliary neurotrophic factor respectively. The stimulation of sympathetic neurons by the 13-18 kD material, and also by 4-6 and 7-11 kD components cannot be entirely accounted for by known factors. This study demonstrates that a number of neurotrophic factors, which differ in their specificity towards sympathetic and parasympathetic neurons, are made available to the region of axonal regrowth over the first few days of regeneration. Contrary to earlier reports, nerve growth factor-like activity was shown to be present in nerve regeneration chambers.

Blot and culture analysis of neuronotrophic factors in nerve regeneration chamber fluids.

The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19-30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.