The goals of this project are to use tissue culture methods, quantitative light and electron microscopy, in situ hybridization, immunocytochemistry, and biochemical techniques to study cellular and molecular mechanisms of myelin formation, breakdown and regeneration. In nerve lesions involving injury to myelinated fibers, return of function depends on successful early interactions of regenerating axons with Schwann cells. Last year, we showed that supernatants prepared from proximal nerve segments 24-48 hours after transection significantly increased mitosis of cultured Schwann cells and also significantly increased their production of laminin, an extracellular matrix component that promotes outgrowth and elongation of regenerating axons after axotomy. When proximal nerve segments were bisected, supernatants prepared from both halves 24 hours after axotomy increased proliferation and laminin production of cultured Schwann cells. This year, we used ELISA assays, Northern blots, receptor binding assays and PCR to investigate the source and composition of the factor(s) in the proximal stump supernatants which are responsible for their early effects on Schwann cells located where nerve regeneration begins. Studies of the supernatants showed that the factor(s) were heat labile and probably were polypeptides of relatively low molecular weight. Assays, using specific antibodies, showed that supernatants prepared from both proximal and distal nerve segments had higher levels of calcitonin gene-related peptide (CGRP) and growth-associated protein 43 (GAP-43) than controls, but there was little difference in levels seen in proximal and distal segments, indicating that neither peptide was responsible for the effect we observed. Tests of other neuropeptides as well as other Schwann cell mitogens are in progress.
