Proceedings of The Physiological Society
University of Central Lancashire (2002) J Physiol 543P, S226
Cell cycle analysis of cerebral cortical cells following blockage of the cerebrospinal fluid pathway during development
Clare E. Draper, Jaleel Miyan and Jane Owen-Lynch*
Department of Biomolecular Sciences, University of Manchester Institute of Science & Technology, PO Box 88, Sackville Street, Manchester M60 1QD and *Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LA1 4YH, UK
Cerebrospinal fluid (CSF) is continuously secreted into the brain ventricular system by the choroid plexus. The fluid flows through the ventricular system, passing over all regions of germinal activity. The hydrocephalic Texas (H-Tx) rat is an excellent model for fetal-onset hydrocephalus (Kohn et al. 1981), involving a constriction of the cerebral aqueduct leading to the blockage of CSF flow. Our recent studies have shown that prior to blockage, neurogenesis and migration occur as in normal rats but immediately following obstruction of fluid flow, cell proliferation decreases (Mashayekhi et al. 2002). We have suggested that it is the in vivo environment that is inhibiting their proliferation, since these cells are able to proliferate as normal when placed into culture for 96 h (Draper et al. 2001). The present study aimed at defining where the inhibition may be occurring.
Time-mated pregnant female H-Tx rats were humanely killed and cerebral cortical cells were isolated from the unaffected and affected fetuses (killed by decapitation) at gestation day 20 (2 days post-blockage of CSF flow) and cultured in Neurobasal medium containing B27 supplement (Invitrogen Limited). Samples were fixed in 70 % ethanol at time zero (point of isolation) and following 24, 48 and 96 h in culture and analysed for cell cycle status using a flow cytometer. The proportion of cells in G0/G1, S and G2-M were quantified. There was no significant difference in the number of cells in the G0/G1 and G2-M phases between the unaffected and affected H-Tx cortices. However, there was a significantly greater (Student's two-tailed unpaired t test, P < 0.05) number of affected cells in the S phase at both time zero and following 24 h in culture, 6.9 ± 0.4 and 6.2 ± 0.97 % (mean ± S.E.M., n = 5), respectively, when compared with the unaffected cells, 4.8 ± 0.3 and 2.8 ± 0.5 % (mean ± S.E.M., n = 5), respectively. Following 48 and 96 h in culture, no differences were observed. The data suggest an in vivo inhibition, causing an arrest of cells in the S phase of the cell cycle, which is removed following 48 h in normal culture conditions.
All procedures accord with current UK legislation.
Where applicable, experiments conform with Society ethical requirements