Immunocytochemical and morphological effects of short-term stimulation of cultured astrocytes

Both image analysis at light microscopy level and ultrastructural characterization by transmission electron microscopy were employed to evaluate the differentiation stage in young cultured mouse astrocytes after 1-day exposure to dBcAMP, a chemical compound known to induce cell activation. The aim was to validate an experimental model of stimulated astrocytes preserving the properties of recently seeded cells, thus avoiding the overlapping effects of in vitro aging. Differentiated astrocytes, as evidenced by GFAP labeling by streptavidin-peroxidase, doubled their number in treated cultures (45%) versus controls (23%). In addition, a significant increase in process-bearing astrocytes (elongated and remified forms) to the detriment of immature polygonal astrocytes, was recorded. No noticeable changes were found in cell perimeter, but cell area displayed a significant reduction in labeled surface of astrocytes undergoing morphological differentiation. Concomitantly, electron microscopy showed that radially organized bundles of numerous intermediate filaments compatible with GFAP replaced the few scattered structures observed in control cultures. However, methodological caution is advisable as regards the relevance of this in vitro counterpart of in situ reactive astrocytes, since cell plasticity is recognized to depend on culture conditions. At any rate, present quantitative results demonstrate that GFAP-positive cell percentage and cell area measurement are adequate parameters of early immunocytochemical and morphological differentiation, respectively, and thus contribute to a better histometric characterization of an easily available substrate to discriminate the wide variety of factors involved in CNS response to injury.