Increased activity of Rho kinase contributes to hemoglobin-induced early disruption of the blood-brain barrier in vivo after the occurrence of intracerebral hemorrhage.

This study is to examine whether the activation of Rho kinase (ROCK) accounts for hemoglobin (Hb)-induced disruption of blood-brain barrier (BBB) after the occurrence of intracerebral hemorrhage. A model of intracerebral injection of Hb was established in rats. Changes in the levels of mRNA of RhoA, ROCK2 and matrix metalloproteinase-9 (MMP-9) were measured using quantitative real-time polymerase chain reaction. Protein expression of RhoA, ROCK2, claudin-5 and MMP-9, as well as ROCK activity, were determined using Western blotting. Immunohistochemical assay was performed to visualize the expression of RhoA, ROCK2, claudin-5 and MMP-9 in endothelial cells. Hb injection produced a significant increase in BBB permeability and water content in the brain. Significant reduction of claudin-5 expression was detected by Western blotting and immunofluorescence in Hb group. The levels of RhoA and ROCK2 were significantly up-regulated from 6 h to 12 h after Hb injection and were concomitant with the increase in ROCK activity. Immunofluorescence double staining showed enhanced p-myosin light chain immunoreactivity but diminished claudin-5 staining in endothelial cells. Significant up-regulation of MMP-9 expression was detected after Hb injection, and statistical analyses further confirmed a positive correlation of MMP-9 expression with ROCK activity. The results showed that ROCK was activated in endothelial cells by Hb. This may account for the early disruption of the BBB via up-regulation of p-myosin light chain expression and aggravation of injuries to TJ proteins. The activation of ROCK may also increase MMP-9 expression, thereby leading to further BBB disruption.