Neurite outgrowth in E18 neurons co-cultured with confluent astrocytes from young adult (3 mo) or old adult (20 mo) cortex. At times indicated,
co-cultures were double immunostained for GFAP and MAP-5. (A) Photomicrographs of immunostaining for MAP-5 (green) and GFAP (red) at 1, 2 and 5 d in
co-cultures; scale bar, 10 m. (B) An example of neurite analysis. Micrographs of culture (double immunostained for GFAP and MAP-5) were digitized into
two fluorescent channels. The area covered by MAP-5 immunopositive neurites was calculated using the following strategy: a semi-automated program using
IPLab imaging software was developed to define the edge of the neurites exclusive of cell bodies. This filtered image was further processed (skeletonized) to
generate a single pixel line. From the one pixel width line, the total neurite length was calculated as shown as MAP-5 immunopositive neurites in (C). (C) During
5 days of co-culture, old astrocytes supported 50% less neurite outgrowth. Neurite outgrowth was measured as the area covered by MAP-5 immunopositive
neurites (Sobel edge detection algorithm); mean ± S.E.M., as % of old at 1d; five experiments;