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UBC Theses and Dissertations

Neurite inhibiting factors from the developing and mature spinal cord Ethell, Douglas Wayne


The developing spinal cord supports the outgrowth of ascending and descending pathways as well as propriospinal connections between spinal cord segments (Okado and Oppenheim, 1985). During this early embryonic period axonal injury is readily repaired, in part due to the similar environments required by developing and regenerating axons (Shimizu et al., 1990). Soon after completing the development of long ascending and descending tracts the environment of the spinal cord changes such that subsequent damage to axons within longitudinal spinal pathways does not result in regeneration. Several recent studies on the embryonic chick (Shimizu et al, 1990; Hasan et al., 1991, 1993) have established embryonic day (E) 13 as the approximate time when the transition from a "permissive" to "restrictive" environment for spinal cord repair occurs. What developmental changes bring about this physiological loss of regenerative ability remains unclear. Several reports have documented that neurite inhibiting proteins, associated with myelin, are one group of factors preventing axonal repair in the adult CNS (Caroni and Schwab, 1988; Schwab and Caroni, 1988). This dissertation describes an examination of several biochemical changes accompanying the loss of regenerative ability during spinal cord development. Proteins isolated from an individual developing thoracic spinal cord were separated using high resolution 2D gel electrophoresis and compared with proteins expressed at other developmental ages. Initially, ten protein spots were identified as showing consistent changes in expression over a developmental period encompassing the transition. Two of these protein spots were of sufficient abundance to be further isolated for internal amino acid sequencing. One novel protein has been identified (DSP 7) and is currently the subject of cloning experiments. Plasma membranes purified from early embryonic chick and rat spinal cord were shown to provide a supportive substrate for the differentiation and outgrowth of neuroblastoma x glioma hybrid NG108-15 cells, in vitro. However, plasma membranes isolated from spinal cords later in development displayed an increasing neurite inhibiting activity. Components of plasma membranes were further purified and tested as substrates for neurite outgrowth, in vitro. No inhibitory effects were observed when purified lipids alone were used as substrates for neurite outgrowth. Although a specific neurite inhibiting protein was not isolated, a crude lipid isolation protocol co-purified proteins with strong neurite inhibitory effects. Major components of the extracellular matrix, heparan sulfate and chondroitin sulfate proteoglycans, were also examined for changes over the developmental period encompassing the transition from permissive to restrictive periods for spinal cord repair. The ratio of neurite promoting heparan sulfate proteoglycans to neurite inhibiting chondroitin sulfate proteoglycans decreases as spinal cord development proceeds.

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