tPA protects mouse and human cultured OLs from apoptotic cell death induced by TFD independently of its classical receptors. (a) Cultured mouse OLs were deprived of trophic factors for 24 h. Photomicrographs show representative fields after A2B5 immunostaining (green) and Hoechst 33258 nuclear costaining (blue). Filled arrowhead, A2B5 cell lacking cell processes; empty arrowhead, A2B5 cell lacking cell processes and chromatin condensation (representative image, n = 3). Bar, 100 µm. OL death (percentage) was estimated by measuring the LDH release in the media. (b and c) Mouse OL death was estimated after 24 h of TFD (black bar) and cotreatment with 50 or 100 µM caspase 3 inhibitor (Ac-DEVD-CHO; b, gray bars; mean + SEM; n = 12.) or 0.2–20 µg/ml tPA (c, light gray bars; mean + SEM; n = 12). (d and e) Human OL death (percentage) was estimated after 72 h of TFD (black bar) and cotreatment with increasing doses of tPA (0.2–20 µg/ml; d, light gray bars; mean + SEM; n = 12) in the presence (e, dark gray bar) or absence (e, light gray bar) of 1 µM of the inhibitor of proteolytic activity of tPA, tPAStop (mean + SEM, n = 12). (f) Mouse OL death (percentage) was estimated after 24 h of TFD (black bar) and cotreatment with 10 µg/ml tPA in the presence (dark gray bars) or absence (light gray bar) of the following drugs, as indicated: MK801, RAP, and εACA (mean + SEM; n = 8, three independent plates). *, significantly (P < 0.01) different from TFD.