The Regulation of Glial Glutamate Transporters after in-vitro Ischemic Stroke

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

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In vitro and in silico modeling of endovascular stroke treatments for acute ischemic stroke

Journal of Biomechanics ◽

10.1016/j.jbiomech.2021.110693 ◽

2021 ◽

pp. 110693

Author(s):

Giulia Luraghi ◽

Rachel M. E. Cahalane ◽

Emma van de Ven ◽

Serena Overschie ◽

Frank J. H. Gijsen ◽

...

Keyword(s):

Ischemic Stroke ◽

Acute Ischemic Stroke ◽

In Silico ◽

In Silico Modeling

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Abstract P683: Thrombin Generation in Plasma of Stroke Patients With Atrial Fibrillation

Stroke ◽

10.1161/str.52.suppl_1.p683 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Sarina Falcione ◽

Gina Sykes ◽

Joseph Kamtchum Tatuene ◽

Danielle Munsterman ◽

Twinkle Joy ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ischemic Stroke ◽

Thrombin Generation ◽

Thrombus Formation ◽

Lag Time ◽

Thrombin Time ◽

Stroke Patients ◽

Time To Peak ◽

Generation Capacity

Background and Purpose: Thrombus formation is central to pathophysiology of stroke in patients with atrial fibrillation. Whether factors in plasma contribute to thrombus generation in patients with atrial fibrillation remains unclear. In this study we sought to determine whether plasma contributes to thrombin generation in patients with atrial fibrillation. Methods: There were 78 acute ischemic strokes with atrial fibrillation and 37 non-stroke controls. Plasma thrombin generation was measured by thrombin generation assay, resulting lag time, peak thrombin, time to peak and area under the curve was assessed. Thrombin generation capacity was compared in stroke patients with atrial fibrillation to non-stroke controls. The relationship to anticoagulation was assessed. In vitro, the effect of anticoagulation on plasma thrombin generation was determined. Results: Thrombin generation capacity was increased (shorter lag time and time to peak) in ischemic stroke patients with atrial fibrillation compared to non-stroke atrial-fibrillation controls (p<0.05 and p<0.01, respectively). Anticoagulation decreased plasma induced thrombin generation. Ischemic stroke patients with atrial fibrillation treated with anticoagulation (DOAC or warfarin) had lower plasma induced thrombin generation compared to atrial-fibrillation patients not on anticoagulation (p<0.05). Thrombin generation by plasma could be further reduced by DOAC in an in-vitro assay. Conclusions: Stroke patients with atrial fibrillation have a higher plasma induced thrombin generation compared to atrial fibrillation controls. Factors in plasma such as leukocyte derived tissue factor likely contribute to thrombus formation in patients with atrial fibrillation. As such, components in plasma may represent new targets to reduce thrombus formation and stroke risk in patients with atrial fibrillation.

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The Role of Glutamate Transporters in Neuroprotection after in vitro Stroke and Cocaine Seeking Behavior in vivo

10.17918/00000293 ◽

2020 ◽

Author(s):

Bhumi Patel

Keyword(s):

Glutamate Transporters ◽

Cocaine Seeking ◽

Seeking Behavior

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Senolytic Therapy for Cerebral Ischemia-Reperfusion Injury

International Journal of Molecular Sciences ◽

10.3390/ijms222111967 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11967

Author(s):

Songhyun Lim ◽

Tae Jung Kim ◽

Young-Ju Kim ◽

Cheesue Kim ◽

Sang-Bae Ko ◽

...

Keyword(s):

Ischemic Stroke ◽

Cerebral Ischemia ◽

Inflammatory Cytokines ◽

Cellular Senescence ◽

Therapeutic Potential ◽

Ischemia Reperfusion ◽

Severe Disabilities ◽

Cerebral Ischemia Reperfusion ◽

Cerebral Ir Injury

Ischemic stroke is one of the leading causes of death, and even timely treatment can result in severe disabilities. Reperfusion of the ischemic stroke region and restoration of the blood supply often lead to a series of cellular and biochemical consequences, including generation of reactive oxygen species (ROS), expression of inflammatory cytokines, inflammation, and cerebral cell damage, which is collectively called cerebral ischemia-reperfusion (IR) injury. Since ROS and inflammatory cytokines are involved in cerebral IR injury, injury could involve cellular senescence. Thus, we investigated whether senolytic therapy that eliminates senescent cells could be an effective treatment for cerebral IR injury. To determine whether IR induces neural cell senescence in vitro, astrocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). OGD/R induced astrocyte senescence and senescent cells in OGD/R-injured astrocytes were effectively eliminated in vitro by ABT263, a senolytic agent. IR in rats with intraluminal middle cerebral artery occlusion induced cellular senescence in the ischemic region. The senescent cells in IR-injured rats were effectively eliminated by intravenous injections of ABT263. Importantly, ABT263 treatment significantly reduced the infarct volume and improved neurological function in behavioral tests. This study demonstrated, for the first time, that senolytic therapy has therapeutic potential for cerebral IR injury.

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Janus Kinase Inhibition Ameliorates Ischemic Stroke Injury and Neuroinflammation Through Reducing NLRP3 Inflammasome Activation via JAK2/STAT3 Pathway Inhibition

Frontiers in Immunology ◽

10.3389/fimmu.2021.714943 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hua Zhu ◽

Zhihong Jian ◽

Yi Zhong ◽

Yingze Ye ◽

Yonggang Zhang ◽

...

Keyword(s):

Ischemic Stroke ◽

Brain Tissue ◽

Proinflammatory Cytokines ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Immunofluorescence Staining ◽

Western Blots ◽

Stat3 Pathway ◽

Stat3 Signaling

BackgroundInflammatory responses play a multiphase role in the pathogenesis of cerebral ischemic stroke (IS). Ruxolitinib (Rux), a selective oral JAK 1/2 inhibitor, reduces inflammatory responses via the JAK2/STAT3 pathway. Based on its anti-inflammatory and immunosuppressive effects, we hypothesized that it may have a protective effect against stroke. The aim of this study was to investigate whether inhibition of JAK2 has a neuroprotective effect on ischemic stroke and to explore the potential molecular mechanisms.MethodsRux, MCC950 or vehicle was applied to middle cerebral artery occlusion (MCAO) mice in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. After 3 days of reperfusion, neurological deficit scores, infarct volume and brain water content were assessed. Immunofluorescence staining and western blots were used to measure the expression of NLRP3 inflammasome components. The infiltrating cells were investigated by flow cytometry. Proinflammatory cytokines were assessed by RT-qPCR. The expression of the JAK2/STAT3 pathway was measured by western blots. Local STAT3 deficiency in brain tissue was established with a lentiviral vector carrying STAT3 shRNA, and chromatin immunoprecipitation (ChIP) assays were used to investigate the interplay between NLRP3 and STAT3 signaling.ResultsRux treatment improved neurological scores, decreased the infarct size and ameliorated cerebral edema 3 days after stroke. In addition, immunofluorescence staining and western blots showed that Rux application inhibited the expression of proteins related to the NLRP3 inflammasome and phosphorylated STAT3 (P-STAT3) in neurons and microglia/macrophages. Furthermore, Rux administration inhibited the expression of proinflammatory cytokines, including TNF-α, IFN-γ, HMGB1, IL-1β, IL-2, and IL-6, suggesting that Rux may alleviate IS injury by inhibiting proinflammatory reactions via JAK2/STAT3 signaling pathway regulation. Infiltrating macrophages, B, T, cells were also reduced by Rux. Local STAT3 deficiency in brain tissue decreased histone H3 and H4 acetylation on the NLRP3 promoter and NLRP3 inflammasome component expression, indicating that the NLRP3 inflammasome may be directly regulated by STAT3 signaling. Rux application suppressed lipopolysaccharide (LPS)-induced NLRP3 inflammasome secretion and JAK2/STAT3 pathway activation in the OGD/R model in vitro.ConclusionJAK2 inhibition by Rux in MCAO mice decreased STAT3 phosphorylation, thus inhibiting the expression of downstream proinflammatory cytokines and the acetylation of histones H3 and H4 on the NLRP3 promoter, resulting in the downregulation of NLRP3 inflammasome expression.

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Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

10.21203/rs.3.rs-1050647/v1 ◽

2021 ◽

Author(s):

Maximilian Wilmes ◽

Carolina Pinto Espinoza ◽

Peter Ludewig ◽

Arthur Liesz ◽

Annette Nicke ◽

...

Keyword(s):

Ischemic Stroke ◽

Transgenic Mice ◽

Tissue Damage ◽

Calcium Influx ◽

Atp Release ◽

Artery Occlusion ◽

Membrane Pores ◽

Caspase 1 ◽

Ischemic Tissue

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

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