Functional and biochemical analysis of CNS-relevant neurotrophic activity in the lesioned sciatic nerve of adult rats.

Prolonged posttraumatic survival and axonal elongation of adult retinal ganglion cells (RGC) depends drastically on the external supply of neurotrophic factors or on grafting of factor-producing peripheral nerve pieces to the site of lesion. As reported previously, an exudate collected in vivo from the regenerating sciatic nerve (ScN) of the rat, enhances axonal regeneration of the axotomized adult retinal ganglion cells (RGC) in vitro and retards their degeneration after optic nerve transection in vivo. In the present study, we analyzed the pool of secretable proteins during the posttraumatic phase of sciatic nerve injury and tested their neurotrophic potential on axotomized RGC. The proteins were accumulated within a silicon tube implanted for 1 week in situ. The sciatic nerve was either ligated to degenerate or crushed to regenerate or left intact. The conditioned media, collected according to the three protocols, were compared with regard to their neurotrophic activities both in vivo and in vitro. Proteins were labelled with [35S]-methionine and analyzed biochemically and autoradiographically. All samples had neuroprotective effects when injected into the vitreous body at the time of optic nerve transection and had neuritogenic effects in organ cultures. The number of regenerating RGC axons in vitro could be increased by a factor of 2.8 when comparing regeneration-associated exudate with that from non-crushed nerve and by a factor of 1.9 when evaluating the exudate from degenerating but not regenerating SCN. The neurotrophic activities could not be attributed to a certain molecule, because about 80 newly synthesized proteins have been observed to appear in the conditioned medium of the regenerating ScN and half of them were not contained in the exudates derived under control conditions. Immunoblots detected nerve growth factor (NGF), basic fibroblast growth factor (FGF-2), and transforming growth factor beta (TGF-b) in the conditioned media. In contrast, ciliary neurotrophic factor (CNTF) was neither produced de novo nor present in detectable amounts in silver-stained SDS gels Together these results indicate 1. that lesion of a peripheral nerve induces the production of factors which exert direct or indirect neurotrophic effects on CNS neurons, 2. that at least some of these active components do not require axonal regeneration in the peripheral nerve, and 3. that neurotrophins, FGF and TGFb may in part be responsible for the effects.