Astrocytes derived from long-term primary cultures recapitulate features of astrogliosis as they re-enter the cell division cycle

Abstract

We investigated whether the initiation of cell cycling by astrocytes prolonged quiescence in long-term primary cultures is associated with immunocytochemical changes that characterize reactive astrogliosis. Primary cultures of newborn rat brain were maintained for >2 months in a stable quiescent state. Partially synchronous transition through a single cell cycle was achieved by trypsinization and replating, and then after 2–3 days, by 48 h of serum depletion and serum shift-up to 10% (time 0). At time 0, the percentages of cells decorated by monoclonal antibodies specific for bromodeoxyuridine (BrDU) after a 2.5 h pulse, and for glial fibrillary acidic protein (GFAP) and vimentin (VIM) were respectively8 ± 2, 15 ± 4and10 ± 3. By 24 h (S phase), 64 ± 7% of nuclei were (BrDU+), and percentages of (GFAP+) and (VIM+) cells were19 ± 4and87 ± 12, respectively. Dual label immunofluorescence showed that >75% of (GFAP+) cells were indeed (VIM+/GFAP+) at 24 h, and that the percentages of (VIM+), (BrDU+) and (VIM+/BrDU+) cells were equivalent for the duration of the first cell cycle (36–48 h). By 72–96 h, (VIM+) cells decreased to <10%, and (BrDU+) cells numbered32 ± 8%, while (GFAP+) cells increased to around 90%. Ran-2 immunofluorescence at 96 h identified virtually all of the cells as type 1 astrocytes. Thus, astrocytes after months of quiescence give rise to cells that recapitulate a VIM/GFAP transition in a manner resembling astrogliosis, and do so relation to progression through a single cell cycle. In addition, the correlation between BrDU and VIM immunofluorescence in cultured astroglia suggests that some elements of gliosis may be cell cycle phase-specific.