The ability to biochemically increase the survival and growth of CNS neurons could have major implications for the treatment of stroke, CNS trauma, and degenerative neurologic diseases. It has been hypothesized that CNS neuronotrophic factors (CNS-NTF) could play a role in CNS regeneration. This project proposes to isolate a CNS-NTF, and develop a method to test its in vivo effects on septal cholinergic neurons after transection of the septal-hippocampal pathway. This system was chosen because it has been previously studied for sprouting and implantation of fetal tissues. Evidence has been obtained for the presence of a protein factor in blood, which supports the survival of a variety of chick and rat CNS neurons in low-density, serum-free cell culture. Standard biochemical approaches to protein purification will be used, including: DEAE chromatography, Sephacryl chromatography, dye affinity gel chromatography, isoelectric focusing, and SDS polyacrylamide gel electrophoresis. Cholinergic septal neurons will be identified in cell cultures of rat septum by anti-CAT immunohistochemistry. Effects of CNS-NTF on neuronal survival, growth as indicated by size, and acetylcholine production will be assessed in vitro. Implanted cannulas attached to an osmotic minipump will be examined as a method of chronically administering CNS-NTF into the CNS of young adult rats. Stability of CNS-NTF and area of distribution within the CNS will be assessed by in vitro assay of brain tissue for CNS-NTF activity after various periods of administration. If this approach is unsuccessful, slow release pellets of ethylene-vinyl acetate copolymer or daily injections through implanted cannulas will be examined. Young adult rats will undergo transection of the fimbria-fornix, and cannulas will be implanted at the levels of the septal nucleus and transection site. Administration of the CNS-NTF will be continued for several weeks. The septal nuclei, region of transection and hippocampus will be histochemically examined for cholinergic neurons by either anti-CAT immunohistochemistry or cholinesterase staining. Portions of the tissue will also be biochemically assayed for choline acetyltransferase activity. Effects of CNS-NTF on survival of septal neurons, sprouting in the region of transection and into the hippocampus, and capacity for acetylcholine synthesis will be assessed.
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