ROCK inhibitors block t-PA/Plgn-induced changes in myosin phosphorylation and morphology in mouse astrocytes and reduce t-PA/Plgn–mediated permeability increase in a human in vitro BBB model. (A) Immunofluorescence images of primary mouse astrocytes treated for 24 hours with vehicle or t-PA (50nM) + Plgn (20nM) in the presence or absence of the Rho kinase inhibitor HA1077 (20μM). GFAP is represented in gray and nuclei in red. Addition of HA1077 fully blocked t-PA/Plgn–induced morphologic changes. Scale bars represent 100 μm. Bottom: representative Western blot demonstrating blockade of t-PA (50nM) + Plgn (100nM)–mediated increase in pMLC in primary mouse astrocytes by HA1077 assessed 2 hours after stimulation of primary mouse astrocytes. (B) Representative phase contrast images showing the blocking effect of Y27632 (20μM) on t-PA (50nM) + Plgn (20nM)–induced morphologic changes in primary mouse astrocytes 24 hours after stimulation. Scale bars represent 100 μm. Bottom: representative Western blot analysis showing a complete inhibition of t-PA (50nM) + Plgn (100nM)–mediated increase in pMLC by Y27632 and by the Rho inhibitor C3 exoenzyme assessed 2 hours after stimulation of primary mouse astrocytes. (C) t-PA (250nM) was added to the luminal chamber of the in vitro human BBB model alone or in combination with HA1077 (20μM, added to both luminal and abluminal chambers). Permeability was assessed after 24 hours. As shown, HA1077 significantly attenuated the ability of t-PA to increase permeability (n = 5). (D) t-PA (25nM) and Plgn (50nM) were added to the luminal chamber of the in vitro human BBB model alone or in combination with HA1077 (20μM, added to both luminal and abluminal chambers) and RAP (0.5μM). Permeability was assessed after 24 hours. As shown, HA1077 significantly reduced the ability of t-PA/Plgn to increase permeability, whereas combination of HA1077 and RAP fully blocked the t-PA/Plgn effect (n = 5).