ASTROCYTES DERIVED FROM LONG-TERM PRIMARY CULTURES RECAPITULATE FEATURES OF ASTROGLIOSIS AS THEY REENTER THE CELL-DIVISION CYCLE

We investigated whether the initiation of cell cycling by astrocytes after 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 respectively 8 +/- 2, 15 +/- 4 and 10 +/- 3. By 24 h (S phase), 64 +/- 7% of nuclei were (BrDU+), and percentages of (GFAP+) and (VIM+) cells were 19 +/- 4 and 87 +/- 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 numbered 32 +/- 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 in 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.