Endogenous plasminogen activator expression after embolic focal cerebral ischemia in mice

Abstract

Urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) play important roles in fibrinolysis, cell migration, tissue destruction, angiogenesis and tissue remodeling. u-PA and t-PA activity in tissue are tightly regulated by plasminogen activator inhibitor-1 (PAI-1). However, little is known of the activity of endogenous plasminogen activators (PAs) and PAI-1 in ischemic brain. To evaluate whether cerebral ischemic injury induces endogenous PAs and PAI-1, we measured PA activity from brain homogenates, and examined the expression of t-PA mRNA, u-PA mRNA and PAI-1 mRNA from brain homogenates in C57BL/6J mice (n=45) weighing 29-35 g in which the middle cerebral artery (MCA) was occluded by a fibrin-rich clot. Brain homogenates were prepared for direct casein zymography from control non-ischemic mice (n=4) and mice at 2 h (n=5), 4 h (n=5), and 24 h (n=4) after MCA occlusion (MCAO). Also, u-PA and t-PA knockout mice at 4 h (n=2, each) after MCAO were used as a negative control for direct casein zymography. Frozen sections for in situ zymography were obtained from control mice (n=2) and mice at 2 h, 4 h, and 24 h (n=2, per time point) after clot occlusion. Brain homogenates were prepared for reverse transcriptase-polymerase chain reaction (RT-PCR) to examine t-PA mRNA, u-PA mRNA and PAI-1 mRNA expression from control non-ischemic mice (n=4) and mice at 2 h (n=5), 4 h (n=5), and 24 h (n=5) after MCAO. By direct casein zymography, u-PA activity increased at 4 h (P<0.05), and 24 h (P<0.05) after stroke in the ischemic hemisphere compared with the non-ischemic mice. Activity of t-PA in ischemic brain was not significantly different from the control group. As measured by in situ zymography, PA activity, most likely u-PA, was present in the ischemic hemisphere. By RT-PCR, expression of PAI-1 mRNA, but not u-PA mRNA and t-PA mRNA, increased 3-, 15- and 25-folds in the ischemic hemisphere at 2 h, 4 h and 24 h after stroke, respectively, compared with control mice. This study demonstrates that PAI-1 mRNA and u-PA activity increase in mouse brain after stroke.