TY - JOUR A1 - Bellut, Maximilian A1 - Papp, Lena A1 - Bieber, Michael A1 - Kraft, Peter A1 - Stoll, Guido A1 - Schuhmann, Michael K. T1 - NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in-vitro and protects blood-brain barrier integrity in murine stroke JF - Cell Death & Disease N2 - 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. KW - inflammasome KW - preclinical research KW - stroke Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265693 VL - 13 ER - TY - JOUR A1 - Bittner, Stefan A1 - Bobak, Nicole A1 - Hofmann, Majella-Sophie A1 - Schuhmann, Michael K. A1 - Ruck, Tobias A1 - Göbel, Kerstin A1 - Brück, Wolfgang A1 - Wiendl, Heinz A1 - Meuth, Sven G. T1 - Murine K\(_{2P}\)5.1 Deficiency Has No Impact on Autoimmune Neuroinflammation due to Compensatory K\(_{2P}\)3.1-and K\(_{V}\)1.3-Dependent Mechanisms JF - International Journal of Molecular Sciences N2 - Lymphocytes express potassium channels that regulate physiological cell functions, such as activation, proliferation and migration. Expression levels of K\(_{2P}\)5.1(TASK2; KCNK5) channels belonging to the family of two-pore domain potassium channels have previously been correlated to the activity of autoreactive T lymphocytes in patients with multiple sclerosis and rheumatoid arthritis. In humans, K\(_{2P}\)5.1 channels are upregulated upon T cell stimulation and influence T cell effector functions. However, a further clinical translation of targeting K\(_{2P}\)5.1 is currently hampered by a lack of highly selective inhibitors, making it necessary to evaluate the impact of KCNK5 in established preclinical animal disease models. We here demonstrate that K\(_{2P}\)5.1 knockout (K\(_{2P}\)5.1\(^{-/-}\) mice display no significant alterations concerning T cell cytokine production, proliferation rates, surface marker molecules or signaling pathways. In an experimental model of autoimmune neuroinflammation, K\(_{2P}\)5.1\(^{-/-}\) mice show a comparable disease course to wild-type animals and no major changes in the peripheral immune system or CNS compartment. A compensatory upregulation of the potassium channels K\(_{2P}\)3.1 and K\(_{V}\)1.3 seems to counterbalance the deletion of K\(_{2P}\)5.1. As an alternative model mimicking autoimmune neuroinflammation, experimental autoimmune encephalomyelitis in the common marmoset has been proposed, especially for testing the efficacy of new potential drugs. Initial experiments show that K\(_{2P}\)5.1 is functionally expressed on marmoset T lymphocytes, opening up the possibility for assessing future K\(_{2P}\)5.1-targeting drugs. KW - domain potassium channels KW - volume regulation KW - multiple-sclerosis KW - potassium channels KW - multiple sclerosis KW - ion channels KW - K+ channel KW - T lymphocytes KW - up-regulation KW - TASK2 KW - K2P channels KW - B cells KW - ph KW - K\(_{2P}\)5.1 KW - KCNK5 KW - autoimmune neuroinflammation KW - EAE Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151454 VL - 16 SP - 16880 EP - 16896 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Papp, Lena A1 - Stoll, Guido A1 - Blum, Robert A1 - Volkmann, Jens A1 - Fluri, Felix T1 - Mesencephalic electrical stimulation reduces neuroinflammation after photothrombotic stroke in rats by targeting the cholinergic anti-inflammatory pathway JF - International Journal of Molecular Sciences N2 - Inflammation is crucial in the pathophysiology of stroke and thus a promising therapeutic target. High-frequency stimulation (HFS) of the mesencephalic locomotor region (MLR) reduces perilesional inflammation after photothrombotic stroke (PTS). However, the underlying mechanism is not completely understood. Since distinct neural and immune cells respond to electrical stimulation by releasing acetylcholine, we hypothesize that HFS might trigger the cholinergic anti-inflammatory pathway via activation of the α7 nicotinic acetylcholine receptor (α7nAchR). To test this hypothesis, rats underwent PTS and implantation of a microelectrode into the MLR. Three hours after intervention, either HFS or sham-stimulation of the MLR was applied for 24 h. IFN-γ, TNF-α, and IL-1α were quantified by cytometric bead array. Choline acetyltransferase (ChAT)\(^+\) CD4\(^+\)-cells and α7nAchR\(^+\)-cells were quantified visually using immunohistochemistry. Phosphorylation of NFĸB, ERK1/2, Akt, and Stat3 was determined by Western blot analyses. IFN-γ, TNF-α, and IL-1α were decreased in the perilesional area of stimulated rats compared to controls. The number of ChAT\(^+\) CD4\(^+\)-cells increased after MLR-HFS, whereas the amount of α7nAchR\(^+\)-cells was similar in both groups. Phospho-ERK1/2 was reduced significantly in stimulated rats. The present study suggests that MLR-HFS may trigger anti-inflammatory processes within the perilesional area by modulating the cholinergic system, probably via activation of the α7nAchR. KW - photothrombotic stroke KW - deep brain stimulation KW - mesencephalic locomotor region KW - neuroinflammation KW - choline acetyltransferase KW - alpha-7 nicotinic acetylcholine receptor Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259099 SN - 1422-0067 VL - 22 IS - 3 ER - TY - JOUR A1 - Kollikowski, Alexander M. A1 - Schuhmann, Michael K. A1 - Nieswandt, Bernhard A1 - Müllges, Wolfgang A1 - Stoll, Guido A1 - Pham, Mirko T1 - Local Leukocyte Invasion during Hyperacute Human Ischemic Stroke JF - Annals of Neurology N2 - 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 KW - neurology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212168 VL - 87 IS - 3 ER - TY - JOUR A1 - Göb, Vanessa A1 - Voll, Maximilian G. A1 - Zimmermann, Lena A1 - Hemmen, Katharina A1 - Stoll, Guido A1 - Nieswandt, Bernhard A1 - Schuhmann, Michael K. A1 - Heinze, Katrin G. A1 - Stegner, David T1 - Infarct growth precedes cerebral thrombosis following experimental stroke in mice JF - Scientific Reports N2 - 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. KW - cerebrovascular disorders KW - thrombosis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265791 VL - 11 IS - 1 ER - TY - JOUR A1 - Essig, Fabian A1 - Kollikowski, Alexander M. A1 - Pham, Mirko A1 - Solymosi, László A1 - Stoll, Guido A1 - Haeusler, Karl Georg A1 - Kraft, Peter A1 - Schuhmann, Michael K. T1 - Immunohistological analysis of neutrophils and neutrophil extracellular traps in human thrombemboli causing acute ischemic stroke JF - International Journal of Molecular Sciences N2 - 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. KW - acute ischemic stroke KW - thrombemboli KW - neutrophils KW - NETs KW - immunohistochemistry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236192 SN - 1422-0067 VL - 21 IS - 19 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Gunreben, Ignaz A1 - Kleinschnitz, Christoph A1 - Kraft, Peter T1 - Immunohistochemical Analysis of Cerebral Thrombi Retrieved by Mechanical Thrombectomy from Patients with Acute Ischemic Stroke JF - International Journal of Molecular Sciences N2 - 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. KW - thrombus formation KW - immune cells KW - lymphocytes KW - mechanical thrombectomy KW - ischemic stroke KW - inflammation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166206 VL - 17 IS - 3 ER - TY - JOUR A1 - Strinitz, Marc A1 - Pham, Mirko A1 - März, Alexander G. A1 - Feick, Jörn A1 - Weidner, Franziska A1 - Vogt, Marius L. A1 - Essig, Fabian A1 - Neugebauer, Hermann A1 - Stoll, Guido A1 - Schuhmann, Michael K. A1 - Kollikowski, Alexander M. T1 - Immune cells invade the collateral circulation during human stroke: prospective replication and extension JF - International Journal of Molecular Sciences N2 - 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. KW - ischemic stroke KW - cerebral ischemia KW - mechanical thrombectomy KW - large vessel occlusion KW - leukocytes KW - neutrophils KW - collateral circulation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284281 SN - 1422-0067 VL - 22 IS - 17 ER - TY - JOUR A1 - Kraft, Peter A1 - Schuhmann, Michael K. A1 - Garz, Cornelia A1 - Jandke, Solveig A1 - Urlaub, Daniela A1 - Mencl, Stine A1 - Zernecke, Alma A1 - Heinze, Hans-Jochen A1 - Carare, Roxana O. A1 - Kleinschnitz, Christoph A1 - Schreiber, Stefanie T1 - Hypercholesterolemia induced cerebral small vessel disease JF - PLoS ONE N2 - Background While hypercholesterolemia plays a causative role for the development of ischemic stroke in large vessels, its significance for cerebral small vessel disease (CSVD) remains unclear. We thus aimed to understand the detailed relationship between hypercholesterolemia and CSVD using the well described Ldlr\(^{−/-}\) mouse model. Methods We used Ldlr\(^{−/-}\) mice (n = 16) and wild-type (WT) mice (n = 15) at the age of 6 and 12 months. Ldlr\(^{−/-}\) mice develop high plasma cholesterol levels following a high fat diet. We analyzed cerebral capillaries and arterioles for intravascular erythrocyte accumulations, thrombotic vessel occlusions, blood-brain barrier (BBB) dysfunction and microbleeds. Results We found a significant increase in the number of erythrocyte stases in 6 months old Ldlr\(^{−/-}\) mice compared to all other groups (P < 0.05). Ldlr\(^{−/-}\) animals aged 12 months showed the highest number of thrombotic occlusions while in WT animals hardly any occlusions could be observed (P < 0.001). Compared to WT mice, Ldlr\(^{−/-}\) mice did not display significant gray matter BBB breakdown. Microhemorrhages were observed in one Ldlr\(^{−/-}\) mouse that was 6 months old. Results did not differ when considering subcortical and cortical regions. Conclusions In Ldlr\(^{−/-}\) mice, hypercholesterolemia is related to a thrombotic CSVD phenotype, which is different from hypertension-related CSVD that associates with a hemorrhagic CSVD phenotype. Our data demonstrate a relationship between hypercholesterolemia and the development of CSVD. Ldlr\(^{−/-}\) mice appear to be an adequate animal model for research into CSVD. KW - hypercholesterolemia KW - cerebral small vessel disease KW - mouse model KW - histology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170493 VL - 12 IS - 8 ER - TY - JOUR A1 - Haarmann, Axel A1 - Schuhmann, Michael K. A1 - Silwedel, Christine A1 - Monoranu, Camelia-Maria A1 - Stoll, Guido A1 - Buttmann, Mathias T1 - Human brain endothelial CXCR2 is inflammation-inducible and mediates CXCL5- and CXCL8-triggered paraendothelial barrier breakdown JF - International Journal of Molecular Science N2 - Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood–brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood–brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood–brain barrier stabilization. KW - blood–brain barrier KW - multiple sclerosis KW - human cerebral endothelial cells KW - CXCR2 KW - CXCL5 KW - CXCL8 KW - interleukin-8 KW - SB332235 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201297 SN - 1422-0067 VL - 20 IS - 3 ER -