Involvement of Secondary Induced Thrombus on Hemorrhage Induced by Both Delayed Recanalization and Delayed t-PA Treatment in Murine Ischemic Stroke Models

<b>Background</b>: In the treatment of ischemic stroke, both tissue-type plasminogen activator (t-PA) thrombolytic and endovascular therapy are employed; however, delayed intervention with these therapies increases the risk of hemorrhage. Hemorrhage associated with delayed t-PA treatment involves the activation of plasmin and matrix metalloproteinases (MMPs); however, the detailed mechanisms underlying I/R activation remain unclear. <b>Objectives</b>: This study examined the effects of delayed recanalization on ischemic stroke in a permanent middle cerebral artery (MCA) occlusion (MCA-O) model, and a novel MCA ischemia/reperfusion (I/R) model: 2-h ischemia followed by reperfusion (I/R 2 h), and 4.5-h ischemia followed by reperfusion (I/R 4.5 h). Secondary induced thrombus (SIT) formation, hemorrhage, MMP activity, MMP-9 immunoreactivity, and tomato lectin (TL) staining, as well as the effects of t-PA and heparin treatment were evaluated. <b>Results</b>: SIT formed within 1 h after reperfusion in the I/R 4.5 h model only, while t-PA or heparin treatment reduced SIT formation. Hemorrhage increased with or without t-PA administration in the I/R 4.5 h model, but it was suppressed by heparin pretreatment. MMP activity and MMP-9 immunoreactivity were localized to the SIT. Additionally, a negative staining area for TL was observed in the damaged area, where SIT formed in the I/R 4.5 h model. <b>Conclusions</b>: These results suggest that delayed recanalization induces SIT via glycocalyx degradation, leading to hemorrhage via plasmin/MMP-9 activation by endogenous and exogenous t-PA-mediated fibrinolysis in novel murine models of ischemic stroke. Furthermore, inhibition of SIT formation is beneficial for suppressing hemorrhages associated with delayed recanalization after endovascular or t-PA therapy.