Immunochemical expression of proliferative cell nuclear antigen in aging cultured astrocytes

Cell differentiation degree and mitotic activity were sequentially assessed by immunoperoxidase labeling of glial fibrillary acidic protein (GFAP) and proliferative cell nuclear antigen (PCNA), respectively, in rat brain cultured astrocytes maintained up to 60 days in vitro (DIV) of first subculture, or weekly passaged until their 12th subculture. Cell count was performed through a 0.01 mm2 section reticule and morphometric analysis with a stereological grid. The number of double immunoreactive cells peaked by 2 DIV to achieve its lowest value at 60 DIV. At 24 hs of cell seeding of successive passages, such values peaked by the 6th subculture to gradually decrease thereafter. Increasing cell hypertrophy was found during the long-term first subculture but not after passaging. At the end of the observation period, doubly immunolabeled astrocytes were still recorded, thus evidencing retention of proliferative potential despite aging.