@article{StrinitzPhamMaerzetal.2021, author = {Strinitz, Marc and Pham, Mirko and M{\"a}rz, Alexander G. and Feick, J{\"o}rn and Weidner, Franziska and Vogt, Marius L. and Essig, Fabian and Neugebauer, Hermann and Stoll, Guido and Schuhmann, Michael K. and Kollikowski, Alexander M.}, title = {Immune cells invade the collateral circulation during human stroke: prospective replication and extension}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {17}, issn = {1422-0067}, doi = {10.3390/ijms22179161}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284281}, year = {2021}, abstract = {It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6\%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1\%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R\(^2\) = 0.09696, p = 0.0373) and (4) greater infarct extent (R\(^2\) = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment.}, language = {en} } @article{HaarmannZimmermannBieberetal.2022, author = {Haarmann, Axel and Zimmermann, Lena and Bieber, Michael and Silwedel, Christine and Stoll, Guido and Schuhmann, Michael K.}, title = {Regulation and release of vasoactive endoglin by brain endothelium in response to hypoxia/reoxygenation in stroke}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {13}, issn = {1422-0067}, doi = {10.3390/ijms23137085}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284361}, year = {2022}, abstract = {In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.}, language = {en} } @article{BellutRaimondiHaarmannetal.2022, author = {Bellut, Maximilian and Raimondi, Anthony T. and Haarmann, Axel and Zimmermann, Lena and Stoll, Guido and Schuhmann, Michael K.}, title = {NLRP3 inhibition reduces rt-PA induced endothelial dysfunction under ischemic conditions}, series = {Biomedicines}, volume = {10}, journal = {Biomedicines}, number = {4}, issn = {2227-9059}, doi = {10.3390/biomedicines10040762}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267261}, year = {2022}, abstract = {Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is a mainstay of acute ischemic stroke treatment but is associated with bleeding complications, especially after prolonged large vessel occlusion. Recently, inhibition of the NLRP3 inflammasome led to preserved blood-brain barrier (BBB) integrity in experimental stroke in vivo. To further address the potential of NLRP3 inflammasome inhibition as adjunct stroke treatment we used immortalized brain derived endothelial cells (bEnd5) as an in vitro model of the BBB. We treated bEnd5 with rt-PA in combination with the NLRP3 specific inhibitor MCC950 or vehicle under normoxic as well as ischemic (OGD) conditions. We found that rt-PA exerted a cytotoxic effect on bEnd5 cells under OGD confirming that rt-PA is harmful to the BBB. This detrimental effect could be significantly reduced by MCC950 treatment. Moreover, under ischemic conditions, the Cell Index — a sensible indicator for a patent BBB — and the protein expression of Zonula occludens 1 stabilized after MCC950 treatment. At the same time, the extent of endothelial cell death and NLRP3 expression decreased. In conclusion, NLRP3 inhibition can protect the BBB from rt-PA-induced damage and thereby potentially increase the narrow time window for safe thrombolysis in stroke.}, language = {en} } @article{FrankeBieberStolletal.2021, author = {Franke, Maximilian and Bieber, Michael and Stoll, Guido and Schuhmann, Michael Klaus}, title = {Validity and Efficacy of Methods to Define Blood Brain Barrier Integrity in Experimental Ischemic Strokes: A Comparison of Albumin Western Blot, IgG Western Blot and Albumin Immunofluorescence}, series = {Methods and Protocols}, volume = {4}, journal = {Methods and Protocols}, number = {1}, issn = {2409-9279}, doi = {10.3390/mps4010023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234214}, year = {2021}, abstract = {The clinical and preclinical research of ischemic strokes (IS) is becoming increasingly comprehensive, especially with the emerging evidence of complex thrombotic and inflammatory interactions. Within these, the blood brain barrier (BBB) plays an important role in regulating the cellular interactions at the vascular interface and is therefore the object of many IS-related questions. Consequently, valid, economic and responsible methods to define BBB integrity are necessary. Therefore, we compared the three ex-vivo setups albumin Western blot (WB), IgG WB and albumin intensity measurement (AIM) with regard to validity as well as temporal and economic efficacy. While the informative value of the three methods correlated significantly, the efficacy of the IgG WB dominated.}, language = {en} } @article{BellutPappBieberetal.2022, author = {Bellut, Maximilian and Papp, Lena and Bieber, Michael and Kraft, Peter and Stoll, Guido and Schuhmann, Michael K.}, title = {NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in-vitro and protects blood-brain barrier integrity in murine stroke}, series = {Cell Death \& Disease}, volume = {13}, journal = {Cell Death \& Disease}, doi = {10.1038/s41419-021-04379-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265693}, year = {2022}, abstract = {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.}, language = {en} } @article{KollikowskiPhamMaerzetal.2022, author = {Kollikowski, Alexander M. and Pham, Mirko and M{\"a}rz, Alexander G. and Papp, Lena and Nieswandt, Bernhard and Stoll, Guido and Schuhmann, Michael K.}, title = {Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke}, series = {Translational Stroke Research}, volume = {13}, journal = {Translational Stroke Research}, number = {3}, issn = {1868-601X}, doi = {10.1007/s12975-021-00938-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270194}, pages = {364-369}, year = {2022}, abstract = {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.}, language = {en} } @article{EssigKollikowskiPhametal.2020, author = {Essig, Fabian and Kollikowski, Alexander M. and Pham, Mirko and Solymosi, L{\´a}szl{\´o} and Stoll, Guido and Haeusler, Karl Georg and Kraft, Peter and Schuhmann, Michael K.}, title = {Immunohistological analysis of neutrophils and neutrophil extracellular traps in human thrombemboli causing acute ischemic stroke}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {19}, issn = {1422-0067}, doi = {10.3390/ijms21197387}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236192}, year = {2020}, abstract = {Ischemic stroke caused by thromboembolic occlusion of large cerebral arteries, such as the internal carotid (ICA) and/or the middle cerebral artery (MCA), is treated by mechanical thrombectomy (MT). MT allows salvage of the vessel-occluding thrombemboli, which most frequently originate from the left atrium or the left ventricle of the heart or from sites of plaque rupture within large arteries above the heart. Clot composition may influence the efficacy of (intravenous) thrombolysis and MT, respectively. We analyzed 37 human thrombemboli obtained from acute ischemic stroke patients during MT with special emphasis on histological staining of neutrophils and neutrophil extracellular traps (NETs). We found neutrophils as the main cellular component of cerebral thrombemboli but encountered considerable morphological heterogeneity. Neutrophils accumulated in the border region of fibrin-rich structures indicating possible interaction of neutrophils with distinct structural thrombembolus components. Web-like NETs were found in 35 of 37 thrombemboli in varying amounts. NETs were almost exclusively found within fibrin-rich areas. Importantly, stroke etiology, age and present oral anticoagulation was associated with morphological patterns and the amount of neutrophils. Correlation of histological data and imaging data revealed that relative Hounsfield units of cerebral thrombemboli positively correlated with the amount of red blood cells. In summary, our results demonstrate that neutrophils and NETs are substantial constituents of cerebral thrombemboli and contribute to their structural complexity.}, language = {en} } @article{SchuhmannKraftBieberetal.2019, author = {Schuhmann, Michael K. and Kraft, Peter and Bieber, Michael and Kollikowski, Alexander M. and Schulze, Harald and Nieswandt, Bernhard and Pham, Mirko and Stegner, David and Stoll, Guido}, title = {Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice}, series = {International Journal of Molecular Science}, volume = {20}, journal = {International Journal of Molecular Science}, number = {8}, issn = {1422-0067}, doi = {10.3390/ijms20082019}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201700}, year = {2019}, abstract = {Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{-/-}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.}, language = {en} } @article{KollikowskiSchuhmannNieswandtetal.2020, author = {Kollikowski, Alexander M. and Schuhmann, Michael K. and Nieswandt, Bernhard and M{\"u}llges, Wolfgang and Stoll, Guido and Pham, Mirko}, title = {Local Leukocyte Invasion during Hyperacute Human Ischemic Stroke}, series = {Annals of Neurology}, volume = {87}, journal = {Annals of Neurology}, number = {3}, doi = {10.1002/ana.25665}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212168}, pages = {466-479}, year = {2020}, abstract = {Objective Bridging the gap between experimental stroke and patients by ischemic blood probing during the hyperacute stage of vascular occlusion is crucial to assess the role of inflammation in human stroke and for the development of adjunct treatments beyond recanalization. Methods We prospectively observed 151 consecutive ischemic stroke patients with embolic large vessel occlusion of the anterior circulation who underwent mechanical thrombectomy. In all these patients, we attempted microcatheter aspiration of 3 different arterial blood samples: (1) within the core of the occluded vascular compartment and controlled by (2) carotid and (3) femoral samples obtained under physiological flow conditions. Subsequent laboratory analyses comprised leukocyte counting and differentiation, platelet counting, and the quantification of 13 proinflammatory human chemokines/cytokines. Results Forty patients meeting all clinical, imaging, interventional, and laboratory inclusion criteria could be analyzed, showing that the total number of leukocytes significantly increased under the occlusion condition. This increase was predominantly driven by neutrophils. Significant increases were also apparent for lymphocytes and monocytes, accompanied by locally elevated plasma levels of the T-cell chemoattractant CXCL-11. Finally, we found evidence that short-term clinical outcome (National Institute of Health Stroke Scale at 72 hours) was negatively associated with neutrophil accumulation. Interpretation We provide the first direct human evidence that neutrophils, lymphocytes, and monocytes, accompanied by specific chemokine upregulation, accumulate in the ischemic vasculature during hyperacute stroke and may affect outcome. These findings strongly support experimental evidence that immune cells contribute to acute ischemic brain damage and indicate that ischemic inflammation initiates already during vascular occlusion. Ann Neurol 2020;87:466-479}, language = {en} } @article{GoebVollZimmermannetal.2021, author = {G{\"o}b, Vanessa and Voll, Maximilian G. and Zimmermann, Lena and Hemmen, Katharina and Stoll, Guido and Nieswandt, Bernhard and Schuhmann, Michael K. and Heinze, Katrin G. and Stegner, David}, title = {Infarct growth precedes cerebral thrombosis following experimental stroke in mice}, series = {Scientific Reports}, volume = {11}, journal = {Scientific Reports}, number = {1}, doi = {10.1038/s41598-021-02360-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265791}, year = {2021}, abstract = {Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.}, language = {en} }