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  - Steinhardt, M. J.
A1  - Wiercinska, E.
A1  - Pham, M.
A1  - Grigoleit, G. U.
A1  - Mazzoni, A.
A1  - Da-Via, M.
A1  - Zhou, X.
A1  - Meckel, K.
A1  - Nickel, K.
A1  - Duell, J.
A1  - Krummenast, F. C.
A1  - Kraus, S.
A1  - Hopkinson, C.
A1  - Weissbrich, B.
A1  - Müllges, W.
A1  - Stoll, G.
A1  - Kortüm, K. M.
A1  - Einsele, H.
A1  - Bonig, H.
A1  - Rasche, L.
T1  - Progressive multifocal leukoencephalopathy in a patient post allo-HCT successfully treated with JC virus specific donor lymphocytes
JF  - Journal of Translational Medicine
N2  - Background 
Progressive multifocal leukoencephalopathy is a demyelinating CNS disorder. Reactivation of John Cunningham virus leads to oligodendrocyte infection with lysis and consequent axonal loss due to demyelination. Patients usually present with confusion and seizures. Late diagnosis and lack of adequate therapy options persistently result in permanent impairment of brain functions. Due to profound T cell depletion, impairment of T-cell function and potent immunosuppressive factors, allogeneic hematopoietic cell transplantation recipients are at high risk for JCV reactivation. To date, PML is almost universally fatal when occurring after allo-HCT. 

Methods 
To optimize therapy specificity, we enriched JCV specific T-cells out of the donor T-cell repertoire from the HLA-identical, anti-JCV-antibody positive family stem cell donor by unstimulated peripheral apheresis [1]. For this, we selected T cells responsive to five JCV peptide libraries via the Cytokine Capture System technology. It enables the enrichment of JCV specific T cells via identification of stimulus-induced interferon gamma secretion. 

Results 
Despite low frequencies of responsive T cells, we succeeded in generating a product containing 20 000 JCV reactive T cells ready for patient infusion. The adoptive cell transfer was performed without complication. Consequently, the clinical course stabilized and the patient slowly went into remission of PML with JCV negative CSF and containment of PML lesion expansion. 

Conclusion 
We report for the first time feasibility of generating T cells with possible anti-JCV activity from a seropositive family donor, a variation of virus specific T-cell therapies suitable for the post allo transplant setting. We also present the unusual case for successful treatment of PML after allo-HCT via virus specific T-cell therapy.
KW  - Myeloma
KW  - JCV
KW  - Prodigy
KW  - CCS
KW  - PML
KW  - Donor lymphocytes
KW  - Adaptive cell transfer
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229307
VL  - 18
ER  - 
TY  - JOUR
A1  - Braeuninger, Stefan
A1  - Kleinschnitz, C.
A1  - Stoll, G.
T1  - Interleukin-18 does not influence infarct volume or functional outcome in the early stage after transient focal brain ischemia in mice
N2  - Interleukin-18 (IL-18) is a proinflammatory cytokine of the interleukin-1 family which is upregulated after cerebral ischemia. The functional role of IL-18 in cerebral ischemia is unknown. In the present study, we compared infarct size in IL-18 knock-out and wild-type mice 24 hours and 48 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). Moreover, the functional outcome was evaluated in a modified Bederson score, foot fault test and grip test. There were no significant differences in infarct size or functional outcome tests between wild-type and IL-18 knock-out mice. These data indicate that the early inflammatory response to cerebral ischemia does not involve IL-18, in contrast to other interleukin-1 family members such as interleukin-1.
KW  - Interleukin-18
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68141
ER  - 
TY  - JOUR
A1  - Kraft, P.
A1  - Schwarz, T.
A1  - Pochet, L.
A1  - Stoll, G.
A1  - Kleinschnitz, Christoph
T1  - COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke
N2  - Background: Anticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)- deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke functional outcome in mice. Methods: C57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Results: Infarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent intracerebral fibrin(ogen) formation. Conclusions: COU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the antithrombotic potential of this novel class of FXII inhibitors can be finally judged.
KW  - Schlaganfall
KW  - Maus
KW  - COU254
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68103
ER  - 
TY  - JOUR
A1  - Pham, Mirko
A1  - Helluy, X.
A1  - Braeuninger, S.
A1  - Jakob, P.
A1  - Stoll, G.
A1  - Kleinschnitz, Christoph
A1  - Bendszus, M.
T1  - Outcome of experimental stroke in C57Bl/6 and Sv/129 mice assessed by multimodal ultra-high field MRI
N2  - Transgenic mice bred on C57Bl/6 or Sv/129 genetic background are frequently used in stroke research. It is well established that variations in cerebrovascular anatomy and hemodynamics can influence stroke outcome in different inbred mouse lines. We compared stroke development in C57Bl/6 and Sv/129 mice in the widely used model of transient middle cerebral artery occlusion (tMCAO) by multimodal ultra-high field magnetic resonance imaging (MRI). C57Bl/6 and Sv/129 mice underwent 60 min of tMCAO and were analyzed by MRI 2 h and 24 h afterwards. Structural and functional images were registered to a standard anatomical template. Probability maps of infarction were rendered by automated segmentation from quantitative T2-relaxometric images. Whole-brain segmentation of infarction was accomplished manually on high-resolution T2-weighted (T2-w) RARE images. Cerebral perfusion (cerebral blood flow, CBF) was measured quantitatively by modified continuous arterial-spin-labeling (CASL) and apparent diffusion coefficients (ADC) by spin-echo diffusion-weighted imaging (DWI). Probabilities of cortical (95.1% ± 3.1 vs. 92.1% ± 2.5; p > 0.05) and subcortical (100% vs. 100%; p > 0.05) infarctions at 24 h were similar in both groups as was the whole-brain volumetric extent of cerebral infarction. In addition, CBF and ADC values did not differ between C57Bl/6 and Sv/129 mice at any time point or region of interest. The C57Bl/6 and Sv/129 genetic background is no major confounding factor of infarct size and cerebral perfusion in the tMCAO model.
KW  - NMR-Tomographie
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68115
ER  - 
TY  - JOUR
A1  - Kleinschnitz, Christoph
A1  - Grund, Henrike
A1  - Wingler, Kirstin
A1  - Armitage, Melanie E.
A1  - Jones, Emma
A1  - Mittal, Manish
A1  - Barit, David
A1  - Schwarz, Tobias
A1  - Geis, Christian
A1  - Kraft, Peter
A1  - Barthel, Konstanze
A1  - Schuhmann, Michael K.
A1  - Herrmann, Alexander M.
A1  - Meuth, Sven G.
A1  - Stoll, Guido
A1  - Meurer, Sabine
A1  - Schrewe, Anja
A1  - Becker, Lore
A1  - Gailus-Durner, Valerie
A1  - Fuchs, Helmut
A1  - Klopstock, Thomas
A1  - de Angelis, Martin Hrabe
A1  - Jandeleit-Dahm, Karin
A1  - Shah, Ajay M.
A1  - Weissmann, Norbert
A1  - Schmidt, Harald H. H. W.
T1  - Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration
N2  - Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox42/2) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox42/2 mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.
KW  - Schlaganfall
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68416
ER  - 
TY  - JOUR
A1  - Kollikowski, Alexander M.
A1  - Pham, Mirko
A1  - März, Alexander G.
A1  - Papp, Lena
A1  - Nieswandt, Bernhard
A1  - Stoll, Guido
A1  - Schuhmann, Michael K.
T1  - Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke
JF  - Translational Stroke Research
N2  - 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.
KW  - chemokines
KW  - CXCL4
KW  - PF4
KW  - CXCL7
KW  - NAP-2
KW  - ischemic stroke
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270194
SN  - 1868-601X
VL  - 13
IS  - 3
ER  - 
TY  - JOUR
A1  - Schuhmann, Michael K.
A1  - Bieber, Michael
A1  - Franke, Maximilian
A1  - Kollikowski, Alexander M.
A1  - Stegner, David
A1  - Heinze, Katrin G.
A1  - Nieswandt, Bernhard
A1  - Pham, Mirko
A1  - Stoll, Guido
T1  - Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice
JF  - Journal of Neuroinflammation
N2  - Background

In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood.

Methods

To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1\(^{−/−}\) mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion.

Results

We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion.

Conclusions

Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.
KW  - ischemic penumbra
KW  - glycoprotein receptor Ib
KW  - T-cells
KW  - ischemic stroke
KW  - thrombo-inflammation
KW  - middle cerebral artery occlusion
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259172
VL  - 18
IS  - 1
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  - Bieber, Michael
A1  - Schuhmann, Michael K.
A1  - Bellut, Maximilian
A1  - Stegner, David
A1  - Heinze, Katrin G.
A1  - Pham, Mirko
A1  - Nieswandt, Bernhard
A1  - Stoll, Guido
T1  - Blockade of platelet glycoprotein Ibα augments neuroprotection in Orai2-deficient mice during middle cerebral artery occlusion
JF  - International Journal of Molecular Sciences
N2  - During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2\(^{-/-}\)) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2\(^{-/-}\) mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2\(^{-/-}\) mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke.
KW  - ischemic penumbra
KW  - Orai2
KW  - glycoprotein receptor Ibα
KW  - ischemic stroke
KW  - thrombo-inflammation
KW  - middle cerebral artery occlusion
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286038
SN  - 1422-0067
VL  - 23
IS  - 16
ER  -