Neurologische Klinik und Poliklinik
Refine
Has Fulltext
- yes (33)
Is part of the Bibliography
- yes (33)
Year of publication
Document Type
- Journal article (33)
Language
- English (33)
Keywords
- ischemic stroke (11)
- neuroinflammation (5)
- stroke (5)
- middle cerebral artery occlusion (4)
- deep brain stimulation (3)
- immunohistochemistry (3)
- inflammasome (3)
- mesencephalic locomotor region (3)
- neutrophils (3)
- photothrombotic stroke (3)
Institute
- Neurologische Klinik und Poliklinik (33)
- Institut für diagnostische und interventionelle Neuroradiologie (ehem. Abteilung für Neuroradiologie) (12)
- Rudolf-Virchow-Zentrum (8)
- Institut für Experimentelle Biomedizin (7)
- Medizinische Klinik und Poliklinik I (4)
- Deutsches Zentrum für Herzinsuffizienz (DZHI) (3)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (3)
- Institut für Klinische Epidemiologie und Biometrie (2)
- Kinderklinik und Poliklinik (2)
- Medizinische Klinik und Poliklinik II (2)
Sonstige beteiligte Institutionen
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.
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.
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.
High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6–6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke.
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.
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.
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.
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.
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
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.
Deep brain stimulation of the mesencephalic locomotor region (MLR) improves the motor symptoms in Parkinson’s disease and experimental stroke by intervening in the motor cerebral network. Whether high-frequency stimulation (HFS) of the MLR is involved in non-motor processes, such as neuroprotection and inflammation in the area surrounding the photothrombotic lesion, has not been elucidated. This study evaluates whether MLR-HFS exerts an anti-apoptotic and anti-inflammatory effect on the border zone of cerebral photothrombotic stroke. Rats underwent photothrombotic stroke of the right sensorimotor cortex and the implantation of a microelectrode into the ipsilesional MLR. After intervention, either HFS or sham stimulation of the MLR was applied for 24 h. The infarct volumes were calculated from consecutive brain sections. Neuronal apoptosis was analyzed by TUNEL staining. Flow cytometry and immunohistochemistry determined the perilesional inflammatory response. Neuronal apoptosis was significantly reduced in the ischemic penumbra after MLR-HFS, whereas the infarct volumes did not differ between the groups. MLR-HFS significantly reduced the release of cytokines and chemokines within the ischemic penumbra. MLR-HFS is neuroprotective and it reduces pro-inflammatory mediators in the area that surrounds the photothrombotic stroke without changing the number of immune cells, which indicates that MLR-HFS enables the function of inflammatory cells to be altered on a molecular level.
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.
Blood–brain barrier (BBB) disruption is a critical event after ischemic stroke, which results in edema formation and hemorrhagic transformation of infarcted tissue. BBB dysfunction following stroke is partly mediated by proinflammatory agents. We recently have shown that high frequency stimulation of the mesencephalic locomotor region (MLR-HFS) exerts an antiapoptotic and anti-inflammatory effect in the border zone of cerebral photothrombotic stroke in rats. Whether MLR-HFS also has an impact on BBB dysfunction in the early stage of stroke is unknown. In this study, rats were subjected to photothrombotic stroke of the sensorimotor cortex and implantation of a stimulating microelectrode into the ipsilesional MLR. Thereafter, either HFS or sham stimulation of the MLR was applied for 24 h. After scarifying the rats, BBB disruption was assessed by determining albumin extravasation and tight junction integrity (claudin 3, claudin 5, and occludin) using Western blot analyses and immunohistochemistry. In addition, by applying zymography, expression of pro-metalloproteinase-9 (pro-MMP-9) was analyzed. No differences were found regarding infarct size and BBB dysfunction between stimulated and unstimulated animals 24 h after induction of stroke. Our results indicate that MLR-HFS neither improves nor worsens the damaged BBB after stroke. Attenuating cytokines/chemokines in the perilesional area, as mediated by MLR-HFS, tend to play a less significant role in preventing the BBB integrity.
Background and Purpose
Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.
Methods
For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.
Results
Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.
Conclusions
With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
In acute ischemic stroke, thrombus formation and inflammation are closely intertwined (“thrombo-inflammation”). Blocking of platelet GPIb can ameliorate thrombo-inflammation.
Background
Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.
Objective
To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.
Results
In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.
Conclusion
Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.
In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral Photothrombosis of Mice by \(^{19}\)F MRI
(2011)
Background
\(^{19}\)F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared \(^{19}\)F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation.
Methods/Principal Findings
Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT induction led to a strong \(^{19}\)F signal throughout the entire lesion, two hours delayed application resulted in a rim-like \(^{19}\)F signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the different spatial distribution of the \(^{19}\)F markers (infarct core/rim) could be visualized in the same animal. When PFC were applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage.
Conclusion
Our study shows that vessel occlusion can be followed in vivo by \(^{19}\)F and SPIO-enhanced high-field MRI while in vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions in a single MRI measurement.
Unlike other organs the nervous system is secluded from the rest of the organism by the blood brain barrier (BBB) or blood nerve barrier (BNB) preventing passive influx of fluids from the circulation. Similarly, leukocyte entry to the nervous system is tightly controlled. Breakdown of these barriers and cellular inflammation are hallmarks of inflammatory as well as ischemic neurological diseases and thus represent potential therapeutic targets. The spatiotemporal relationship between BBB/BNB disruption and leukocyte infiltration has been a matter of debate. We here review contrast-enhanced magnetic resonance imaging (MRI) as a non-invasive tool to depict barrier dysfunction and its relation to macrophage infiltration in the central and peripheral nervous system under pathological conditions. Novel experimental contrast agents like Gadofluorine M (Gf) allow more sensitive assessment of BBB dysfunction than conventional Gadolinium (Gd)-DTPA enhanced MRI. In addition, Gf facilitates visualization of functional and transient alterations of the BBB remote from lesions. Cellular contrast agents such as superparamagnetic iron oxide particles (SPIO) and perfluorocarbons enable assessment of leukocyte (mainly macrophage) infiltration by MR technology. Combined use of these MR contrast agents disclosed that leukocytes can enter the nervous system independent from a disturbance of the BBB, and vice versa, a dysfunctional BBB/BNB by itself is not sufficient to attract inflammatory cells from the circulation. We will illustrate these basic imaging findings in animal models of multiple sclerosis, cerebral ischemia, and traumatic nerve injury and review corresponding findings in patients.
Background and Purpose
In animal models, von Willebrand factor (VWF) is involved in thrombus formation and propagation of ischemic stroke. However, the pathophysiological relevance of this molecule in humans, and its potential use as a biomarker for the risk and severity of ischemic stroke remains unclear. This study had two aims: to identify predictors of altered VWF levels and to examine whether VWF levels differ between acute cerebrovascular events and chronic cerebrovascular disease (CCD).
Methods
A case–control study was undertaken between 2010 and 2013 at our University clinic. In total, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HV) were included. Blood was taken at days 0, 1, and 3 in patients with AIS or TIA, and once in CCD patients and HV. VWF serum levels were measured and correlated with demographic and clinical parameters by multivariate linear regression and ANOVA.
Results
Patients with CCD (158±46%) had significantly higher VWF levels than HV (113±36%, P<0.001), but lower levels than AIS/TIA patients (200±95%, P<0.001). Age, sex, and stroke severity influenced VWF levels (P<0.05).
Conclusions
VWF levels differed across disease subtypes and patient characteristics. Our study confirms increased VWF levels as a risk factor for cerebrovascular disease and, moreover, suggests that it may represent a potential biomarker for stroke severity, warranting further investigation.
Background: Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. Methodology/Principal Findings: Focal cerebral ischemia was induced in Vasp2/2 mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan’s Blue tracer was applied to visualize the extent of blood-brainbarrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p,0.05) and edema volumes (1.7 mm360.5 mm3 versus 0.8 mm360.4 mm3; p,0.0001) were significantly enhanced in Vasp2/2 mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm3617.3 mm3 versus 39.3 mm3610.7 mm3, respectively; p,0.01) and a non significant trend (p.0.05) towards worse neurological outcomes. Conclusion: Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.
Background: Thrombus formation is a key step in the pathophysiology of acute ischemic stroke and results from the activation of the coagulation cascade. Thrombin plays a central role in this coagulation system and contributes to thrombus stability via activation of thrombin-activatable fibrinolysis inhibitor (TAFIa). TAFIa counteracts endogenous fibrinolysis at different stages and elevated TAFI levels are a risk factor for thrombotic events including ischemic stroke. Although substantial in vitro data on the influence of TAFI on the coagulation-fibrinolysis-system exist, investigations on the consequences of TAFI inhibition in animal models of cerebral ischemia are still lacking. In the present study we analyzed stroke development and post stroke functional outcome in TAFI-/- mice. Methodology/Principal Findings: TAFI-/- mice and wild-type controls were subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 hours, functional outcome scores were assessed and infarct volumes weremeasured from 2,3,5-Triphenyltetrazoliumchloride (TTC)-stained brain slices. 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. Infarct volumes and functional outcomes did not significantly differ between TAFI-/- mice and controls (p.0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. TAFI deficiency also had no influence on intracerebral fibrin(ogen) formation after tMCAO. Conclusion: Our study shows that TAFI does not play a major role for thrombus formation and neuronal degeneration after ischemic brain challenge.
Background: An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function. Methodology/Principal Findings: As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time. Conclusion: Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.
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.