Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. Ischemic stroke was induced in C57Bl/6 mice by 60 minutes transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 hours of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine, IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.

In ischemic stroke (IS) impairment of the blood–brain barrier (BBB) has an important role in the secondary deterioration of neurological function. BBB disruption is associated with ischemia-induced inflammation, brain edema formation, and hemorrhagic infarct transformation, but the underlying mechanisms are incompletely understood. Dysfunction of endothelial cells (EC) may play a central role in this process. Although neuronal NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome upregulation is an established trigger of inflammation in IS, the contribution of its expression in EC is unclear. We here used brain EC, exposed them to oxygen and glucose deprivation (OGD) in vitro, and analyzed their survival depending on inflammasome inhibition with the NLRP3-specific drug MCC950. During OGD, EC death could significantly be reduced when targeting NLRP3, concomitant with diminished endothelial NLRP3 expression. Furthermore, MCC950 led to reduced levels of Caspase 1 (p20) and activated Gasdermin D as markers for pyroptosis. Moreover, inflammasome inhibition reduced the secretion of pro-inflammatory chemokines, cytokines, and matrix metalloproteinase-9 (MMP9) in EC. In a translational approach, IS was induced in C57Bl/6 mice by 60 mins transient middle cerebral artery occlusion and 23 hours of reperfusion. Stroke volume, functional outcome, the BBB integrity, and—in good agreement with the in vitro results—MMP9 secretion as well as EC survival improved significantly in MCC950-treated mice. In conclusion, our results establish the NLRP3 inflammasome as a critical pathogenic effector of stroke-induced BBB disruption by activating inflammatory signaling cascades and pyroptosis in brain EC.

Lymphocytes have been shown to play an important role in the pathophysiology of acute ischemic stroke, but the properties of B cells remain controversial. The aim of this study was to unravel the role of B cells during acute cerebral ischemia using pharmacologic B cell depletion, B cell transgenic mice, and adoptive B cell transfer experiments. Transient middle cerebral artery occlusion (60 min) was induced in wild-type mice treated with an anti-CD20 antibody 24 h before stroke onset, JHD −/− mice and Rag1 −/− mice 24 h after adoptive B cell transfer. Stroke outcome was assessed at days 1 and 3. Infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections, and neurological scores were evaluated. The local inflammatory response was determined by real-time PCR and immunohistochemistry. Apoptosis was analyzed by TUNEL staining, and astrocyte activation was revealed using immunohistochemistry and Western blot. Pharmacologic depletion of B cells did not influence infarct volumes and functional outcome at day 1 after stroke. Additionally, lack of circulating B cells in JHD −/− mice also failed to influence stroke outcome at days 1 and 3. Furthermore, reconstitution of Rag1 −/− mice with B cells had no influence on infarct volumes. Targeting B cells in experimental stroke did not influence lesion volume and functional outcome during the acute phase. Our findings argue against a major pathophysiologic role of B cells during acute ischemic stroke.

Mechanical thrombectomy is a novel treatment option for patients with acute ischemic stroke (AIS). Only a few studies have previously suggested strategies to categorize retrieved clots according to their histologic composition. However, these reports did not analyze potential biomarkers that are of importance in stroke-related inflammation. We therefore histopathologically investigated 37 intracerebral thrombi mechanically retrieved from patients with AIS, and focused on the composition of immune cells and platelets. We also conducted correlation analyses of distinctive morphologic patterns (erythrocytic, serpentine, layered, red, white, mixed appearance) with clinical parameters. Most T cells and monocytes were detected in erythrocytic and red clots, in which the distribution of these cells was random. In contrast, von Willebrand factor (vWF)-positive areas co-localized with regions of fibrin and collagen. While clots with huge amounts of vWF seem to be associated with a high National Institute of Health Stroke Scale score at admission, histologic findings could not predict the clinical outcome at discharge. In summary, we provide the first histologic description of mechanically retrieved intracerebral thrombi regarding biomarkers relevant for inflammation in ischemic stroke.

Ischemic stroke causes a strong inflammatory response that includes T cells, monocytes/macrophages, and neutrophils. Interaction of these immune cells with platelets and endothelial cells facilitates microvascular dysfunction and leads to secondary infarct growth. We recently showed that blocking of platelet glycoprotein (GP) receptor Ib improves stroke outcome without increasing the risk of intracerebral hemorrhage. Until now, it has been unclear whether GPIb only mediates thrombus formation or also contributes to the pathophysiology of local inflammation. Focal cerebral ischemia was induced in C57BL/6 mice by a 60-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab). Rat immunoglobulin G (IgG) Fab was used as control treatment. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 1 after tMCAO. Blocking of GPIb reduced stroke size and improved functional outcome on day 1 after tMCAO without increasing the risk of intracerebral hemorrhage. As expected, disruption of GPIb-mediated pathways in platelets significantly reduced thrombus burden in the cerebral microvasculature. In addition, inhibition of GPIb limited the local inflammatory response in the ischemic brain as indicated by lower numbers of infiltrating T cells and macrophages and lower expression levels of inflammatory cytokines compared with rat IgG Fab-treated controls. In acute ischemic stroke, thrombus formation and inflammation are closely intertwined (“thrombo-inflammation”). Blocking of platelet GPIb can ameliorate thrombo-inflammation.

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018–05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P = .0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P = .013) concentrations were higher compared with self-controls. Local platelet counts were lower (− 10%: 347,582 vs 383,284/µl, P = .0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P = .034), and between CXCL7 and MPO (r = 0.24, P = .048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r = − 0.2523, P = .0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P = .0014) and platelet counts (r = 0.288, P = .0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization.