Identification and characterization of glial and neuronal cells in vitro and in bone marrow

Frentzou, Georgia Alkistis (2002) Identification and characterization of glial and neuronal cells in vitro and in bone marrow. Masters thesis, University of Central Lancashire.

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This study attempts to identify and characterize glial cells and neuronal cells in avian brain and mammalian bone marrow, respectively. The project has four major objectives: (a) to culture and maintain avian glial and neuronal cells up to 20 days, (b) to examine cell cultures using selective cytological markers for gliaj and nerve cells, (c) to investigate the effects of the steroid hormone oestrogen on the growth of glial cells and (d) to investigate the presence of neuronal cell bodies in mammalian bone marrow using alternative approaches.
Glial cells were grown for 20 days, supplemented with oestrogen after the 10th day. Control cultures were grown for 20 days without oestrogen supplement. Consequently, the cultures were subjected in GFAP, Gal C, NSE and PR antibodies. The results have shown that oestrogen had no significant effect on the development of the cells. Staining of glial cells with Haematoxylin and Eosin and taking measurements for cell diameter, nucleus diameter and number of projections, the results demonstrated significant differences in the nucleus diameter and the number of projections in the cells treated with estrogen compared to non-treated cells. The neuronal cells were cultured for 10-15 days. Comparing to the glial cell cultures, it was found that neuronal cells were more difficult to grow and more vulnerable to contamination even when a modified medium was used to grow the cells. However, the results show that they can express immunoreactivity for NSE but not for either GFAP or Gal C. The expression of NSE was crucial since it positively showed that the cells grown were neuronal cells in nature. The final aim of this study was to identify and localize the innervation pattern within the bone marrow and the possible existence of neuronal cells bodies. Using either male or female young mice as animal model, perfused femurs were decalcified, sectioned with a cryostat and subsequently, stained using immunocytochemistry for different markers,
including Gal C, GFAP, NSE and antityrosine hydroxylase. Furthermore, sections were subjected to silver stain, which is capable in recognising neuronal cells bodies. In addition, Dii neuronal tracer was applied to post-fixed tissue for the same reason. The results showed that the tissue had NSE immunoreactivity revealing an extensive network of neuronal fibers running among the bone marrow. However, there was no expression of Gal C, GFAP or antityrosine hydroxylase. The results also revealed the presence of neuronal fibers within the bone marrow, running alongside with blood vessels. Unfortunately, no neuronal cell bodies were found in this present investigation. In conclusion, the results of this study have shown that (1) oestrogen can have marked effects on the morphology of glial cells and (2) neuronal fibers are present in bone marrow and they seem to be located near blood vessels.

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