Effect of cytosine arabinoside on rat cerebral explants: fibroblasts and reactive microglial cells as the primary cellular barrier to neurite growth

Method
In an attempt to determine whether if increased neurite outgrowth is related to a decreased incidence of fibroblast cells, rat cerebral explants were culture with and without cytosine arabinoside (AraC). After 7 to 10 days of treatment in vitro, the explants were viewed using transmission electron microscoty. For the AraC-treated explants, there was a significant decrease in the incidence of fibroblast as well as a significant increase of epithelial cells. This suggests that AraC can be used to increase neurite growth through decreasing the amount of fibroblast cells.

Summary
Cerebral explants that were treated with AraC from 4 to 7 or 7 to 10 days in vitro (DIV) exhibited an increase in neurite outgrowth and a decrease in non-neural cells. However, for the explants treated from 7 to 10 DIV, the effect of AraC on the non-neural cell population is not clear. By withholding AraC until day 7, the cells would be more likely to divide and therefore incorporate the addition of AraC. Fibroblastic-reactive cells are especially susceptive because they have the shortest cell cycle of the non-neural cells. Therefore, they investigated if the increased neurite outgrowth for cerebral explants treated with AraC from days 7 to 10 was caused by the limit of division of the fibroblast cells.



Fig. 1. Electron micrographs of the outgrowth zone of cortical explants at 18 DIV cultured in (a) serum (control) medium and (b) the presence of AraC from 4 to 7 DIV. In both conditions viable neurites (n) are surrounded by non-neuronal cells (as, astrocyte; e, epithelial cell; f, fibroblast).


Explants from 20-day-old fetal rates were cultured in serum medium (control) or in serum medium with 10^-5 M AraC from 7 to 10 DIV. The same criteria were used to identify non-neural profiles of astrocytic and epithelial cells. At 18 DIV, the cultures were sectioned and viewed with an electron microscope. The explants had some noticeable elongated fibroblast cells that dilated rougth endoplasmic reticulum on one side of the nucleus and a long cisternae on the other side. Because of their narrow cisternae, the fibroblast cells could be described as fibroblastic-reactive rather than fibroblastic-resting microglial cells. Fibroblastic-reactive cells are often found after injury.

The non-neural cells in the control and AraC-treated explants were quantified through utilizing grid overlays and taking mean values from eleven analyzed 'micrographs'. The results suggest that the increased neurite outgrowth for AraC-treated explants may be due to a limit of division by fibroblastic-reactive cells. For example, by Day 18, there are no more fibroblastic-reactive cells but the epithelial cells have significantly increased. Increased neurite growth is associated with decreased astrocytes and fibroblast cells, but this study indicates that an enhanced neurite repsonse occurs even when astrocytes are present at levels similar to a control level (Table 1).



Table 1: This table shows the difference between control and AraC-treated explants.


The neurite outgrowth for the AraC-treated explants increased by 156% and 116% in the 4 to 7 DIV and 7 to 10 DIV, respectively. This suggests that the increased neurite outgrowth is caused by the reduced presence of fibroblast cells rather than the astrocytes. To conclude, this study shows that a decrease in fibroblastic-reactive cells, rather than astrocytes, is a leading cause of neurite growth.

It may be said that this study does not provide direct evidence for the relationship between increased neurite outgrowth and limited non-neural cell division. AraC may possibly enhance neurite growth through limiting neuronal cell division, and the decrease in non-neural cells may just be a coincidence. However, since this study used 20-day-old fetal rats, it is unlikely that the neurons proliferated much. Furthermore, the control and AraC-treated explants show an insignificant difference of neural cells.


References:
Oorschot, D.E. and D.G. Jones, “Effect of cytosine arabinoside on rat cerebral explants: fibroblasts and reactive microglial cells as the primary cellular barrier to neurite growth”, University of Otago, Dundein: April 1989. < http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T0G-485H7HN-145-1&_cdi=4862&_user=4420&_pii=0304394089901018&_orig=search&_coverDate=07%2F31%2F1989&_sk=998979997&view=c&wchp=dGLzVtz-zSkWA&md5=1219dde321ea4cb09d1d8f8f7b421f89&ie=/sdarticle.pdf>
Oorschot, D.E. and D.G. Jones, “Non-neuronal cell proliferation in tissue culture: Implications for axonal regeneration in the central nervous system” Brain Research, Volume 368, Issue 1, 12 March 1986 < http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6SYR-484FDK6-15N-1&_cdi=4841&_user=4420&_pii=0006899386910413&_orig=search&_coverDate=03%2F12%2F1986&_sk=996319998&view=c&wchp=dGLbVlW-zSkzk&md5=4115cf2cc2efe6f5eed8a144486e4a6a&ie=/sdarticle.pdf>