Fig. 3.

Effect of apyrase and nucleotide P2 receptor antagonists on the growth and survival of glial cells in the scratched area of retinal monolayer cultures. Retinal cultures at E8C7 were scratched and treated with 2.5 U/mL apyrase. a, b Representative micrographs of scratched cultures cultivated in the absence or presence of apyrase for 3 days. c Effect of apyrase on the growth of glial cells over the scratched area as determined by quantifying the progressive decrease in the area free of cells in the scratched cultures. d Inhibition of the growth of glial cells induced by apyrase as determined by measuring the progressive increase of the border area with glial cells in scratched cultures. e Density of glia nuclei at the growing border of the cultures 3 days after the scratch. Density of nuclei was determined by the number of DAPI⁺ nuclei divided by the area of the border. f Representative micrographs showing apoptotic cells (arrows) in scratched cultures that were treated with 2.5 U/mL of apyrase for 6 h or 3 days, fixed, and processed by TUNEL assays as described in the “Materials and methods” section. Anti-BrdU (green) and propidium iodide (red) were used to label cells. g Effect of P2 receptor antagonists on the growth of glial cells over the scratched area. Retinal cultures at E8C7 were scratched and treated with 100 μM suramin or PPADS, 40 μM Reactive Blue 2 (RB-2), or 30 μM MRS 2179 for the next 3 days. The area free of cells was determined in 10 fields per culture as described in the “Materials and methods” section. Data represent the mean ± SEM of six separate experiments performed in duplicate (c) or three to eight separate experiments performed in duplicate (d, e, g). ***p < 0.001 compared to control cultures just after the scratch at time 0 (c, g) or control cultures 1 day after the scratch (d). ^# p < 0.5, ^## p < 0.01, and ^### p < 0.001 compared to control cultures at the corresponding time after the scratch of the cultures. Scale bar = 30 μm in a and b and 10 μm in f