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.