Abstract Body: Introduction
Despite timely recanalization, some ischemic stroke patients treated with endovascular thrombectomy experience persistent neurological deterioration. This has been partly attributed to no-reflow. Experimental stroke models have indicated a critical role for platelets in this process. However, the mechanism and timing of platelet involvement remain unknown.
Methods
Transient middle cerebral artery occlusion was performed in C57Bl/6J mice. Intra-arterial blood was sampled directly from the brain immediately after reperfusion. In a different cohort, intra-venous blood was sampled immediately after reperfusion. A LEGENDplex assay was used to measure 23 inflammatory cytokines. Endothelial cell activation (von Willebrand factor, VWF) was measured through ELISA. We generated mice with endogenously fluorescent platelets and imaged the penumbra with 2-photon intravital microscopy at different times after stroke onset.
Results
Immediately after reperfusion, intra-arterial blood sampled from the brain exhibited a distinct inflammatory cytokine profile compared to intravenous systemic blood samples. IL-1alpha, IL-1beta, and IL-6 were significantly elevated in the intra-arterial blood samples compared to the intravenous blood samples. These cytokines have been reported to influence the release and activity of VWF, a hemostatic protein secreted by activated endothelial cells. VWF levels increased both locally (3-fold) and systemically (2-fold) at the onset of reperfusion compared to baseline. The thrombogenicity of VWF is limited by ADAMTS13. Interestingly, ADAMTS13 activity was strongly reduced (2-fold) locally in the reperfused brain, while it remained normal systemically. These results suggest a prothrombotic imbalance of VWF localized in the reperfused ischemic stroke brain. VWF traps platelets, and excessive VWF activation leads to thrombosis. Therefore, we imaged the reperfused brain in mice using 2-photon intravital microscopy. Immediately after the onset of reperfusion, we observed VWF capturing platelets, resulting in the formation of platelet-rich thrombi that disrupted blood flow in the penumbra. Inhibition of platelet binding to VWF using a monoclonal antibody prevented this and resulted in greatly improved stroke outcomes.
Conclusions
We found that ischemic stroke causes a local cytokine storm which potentiates VWF-mediated platelet accumulation in the reperfused brain contributing to no-reflow and exacerbated stroke outcomes.