Non-neuronal cell proliferation in tissue culture: implications for axonal regeneration in the central nervous system

A tissue culture model has been developed to examine the hypothesis that proliferating non-neuronal cells may constitute a physical and/or chemical barrier to regenerating neurons in the central nervous system. Explants from the sensorimotor cortex of 20-day-old fetal rats were cultured in serum medium (control) or serum medium containing 10(-5) M cytosine arabinoside (AraC), a mitotic inhibitor, for varying periods: 2-10, 4-12, 4-10, 4-8 and 4-7 days in vitro (DIV). The center and outgrowth zone of the explants were examined by phase-contrast microscopy at varying intervals between 3 and 18 DIV. The extent of central degeneration was greatest in explants treated with AraC from 2 DIV, and was least in the 4-7 day treated group in which only minimal degeneration was evident at 13 and 18 DIV. In the outgrowth zone at 18 DIV non-neuronal cell proliferation was controlled in the 4-10 day treated explants, although this was accompanied by extensive degeneration of neurites. Further examination of neurite viability, using a neurite viability ratio, revealed that degeneration was first evident at 6 DIV in the 2-10 day treated explants, but not until 9 or 13 DIV in any of the explants exposed to AraC from 4 days onwards. There was minimal degeneration in the 4-7 day treated explants. Electron microscopic examination revealed the presence of atypical inclusions in non-neuronal cells of 4-8 day treated explants, suggesting that the cytotoxic effect of AraC may be due to a disturbance in lipid and/or ganglioside metabolism. Quantitative electron microscopic analysis of the outgrowth zone at 18 DIV revealed a significant increase in the summated area of neuronal tissue (from 7 to 18 microns2/100 microns2) and a decline in the summated area of non-neuronal cells (from 83 to 61 microns2/100 microns2) for explants treated with AraC from 4 to 7 DIV compared to control. Diminishing the potential of non-neuronal cells to act as a barrier by controlling their proliferation may, therefore, be of importance in enhancing the regenerative response of central neurons.