Deficiency of Factor VII activating protease alters the outcome of ischemic stroke in mice.

Factor VII activating protease (FSAP) is a circulating protease with a putative role in hemostasis, remodeling and inflammation. A polymorphism giving rise to low proteolytic activity has been associated with an increased risk of stroke and carotid stenosis. To date, no in vivo studies or mechanistic information is available to explain these results. Based on the polymorphism data we hypothesize that a lack of endogenous FSAP will increase the severity of stroke. Stroke was induced by applying thrombin in the middle cerebral artery in wild-type (WT) and FSAP(-/-) mice. Increased stroke volume and worsened neurological deficit were observed in FSAP(-/-) mice. Raised levels of FSAP protein were detected in the infarcted area of WT mice together with enhanced leukocyte infiltration and apoptosis in FSAP(-/-) mice. There was a concomitant increase in the activation of the NFκB pathway and decrease in expression of the PI3K/AKT pathway proteins. At a cellular level, FSAP increased cell survival and decreased apoptosis in primary cortical neurons and astrocytes exposed to tPA/NMDA excitotoxicity or oxygen glucose deprivation (OGD)/reoxygenation, respectively. This was mediated via the PI3K/AKT pathway with involvement of the protease activated receptor-1. To corroborate the human epidemiological data, which link FSAP with stroke, we now show that the lack of FSAP in mice worsens the outcome of stroke. In the absence of FSAP there was a stronger inflammatory response and lower cell survival due to insufficient activation of the PI3K/AKT pathway.

3D Transcranial ultrasound localization microscopy reveals major arteries in the sheep brain.

Cerebral circulation ensures the proper functioning of the entire human body, and its interruption, i.e. stroke, leads to irreversible damage. However, tools for observing cerebral circulation are still lacking. Although MRI and CT scans serve as conventional methods, their accessibility remains a challenge, prompting exploration into alternative, portable, and non-ionizing imaging solutions like ultrasound with reduced costs. While Ultrasound Localization Microscopy (ULM) displays potential in high-resolution vessel imaging, its 2D constraints limit its emergency utility. This study delves into the feasibility of 3D ULM with multiplexed probe for transcranial vessel imaging in sheep brains, emulating human skull characteristics. Three sheep underwent 3D ULM imaging, compared with angiographic MRI, while skull characterization was conducted in vivo using ultrashort bone MRI sequences and ex vivo via micro CT. The study showcased 3D ULM's ability to highlight vessels, down to the Circle of Willis, yet within a confined 3D field-of-view. Future enhancements in signal, aberration correction, and human trials hold promise for a portable, volumetric, transcranial ultrasound angiography system.

Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism.

BACKGROUND: The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. METHODS AND RESULTS: The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. CONCLUSIONS: A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis.

Lack of secondary microthrombosis after thrombin-induced stroke in mice and non-human primates.

BACKGROUND: Secondary microthrombosis is a major pathophysiologic mechanism leading to brain damage following transient mechanical vascular occlusion (TMVO), the most widely used experimental stroke model. Whether secondary microthrombosis also occurs in non-TMVO stroke models represents an important issue for clinical translation of antimicrothrombosis therapeutic strategies. OBJECTIVES: To assess the occurrence and the pathogenic role of secondary microthrombosis in two thrombin-induced stroke models in mice and non-human primates (Macaca mulatta). METHODS: Stroke was induced in mice and non-human primates by intra-arterial administration of recombinant thrombin. This method induces the formation of a fibrin-rich thrombus, which is spontaneously dissolved in the following hours by the endogenous fibrinolytic system. Perfusion-weighted imaging and fluorescent-lectin microangiography were performed after recanalization to detect secondary microthrombosis. Moreover, to investigate its pathogenic role, thrombin-induced stroke was induced in bradykinin receptor B1 (B1R) knockout mice, which are protected from the thromboinflammation responsible for secondary microthrombosis in TMVO models. RESULTS: Reperfusion was stable and complete in all mice and non-human primates tested, revealing no secondary decrease in cerebral blood flow. No evidence of secondary microthrombosis was found in the two models. Accordingly, deficiency in B1R did not protect the mice from brain damage after thrombin-induced stroke. CONCLUSIONS: Our data demonstrate that secondary microthrombosis does not occur after thrombin-induced stroke. In view of this, the pathophysiologic roles of hematologic players promoting or protecting against secondary microthrombosis (such as factor XII, von Willebrand factor, and T cells) deserve to be re-evaluated in non-TMVO stroke models.