scholarly journals NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in vitro and protects blood–brain barrier integrity in murine stroke

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Maximilian Bellut ◽  
Lena Papp ◽  
Michael Bieber ◽  
Peter Kraft ◽  
Guido Stoll ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Nlrp3 Inflammasome ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Brain Barrier ◽  
Edema Formation ◽  
Pyrin Domain ◽  
Volume Functional ◽  
Blood Brain

AbstractIn 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.

scholarly journals Cerebromicrovascular endothelial cells are resistant to l-glutamate

2008 ◽  
Vol 295 (4) ◽  
pp. R1099-R1108 ◽  
Author(s):  
Ferenc Domoki ◽  
Béla Kis ◽  
Tamás Gáspár ◽  
Ferenc Bari ◽  
David W. Busija
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Primary Cultures ◽  
Glucose Deprivation ◽  
Brain Barrier ◽  
Cranial Window ◽  
Blood Brain ◽  
Microvascular Cells

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1–10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca2+ ionophore calcimycin (5 μM), but not l-glut (1 mM), increased intracellular Ca2+ levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.

scholarly journals Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model

1996 ◽  
Vol 1 (4) ◽  
pp. E3 ◽  
Author(s):  
Kevin R. Lee ◽  
Nobuyuki Kawai ◽  
Seoung Kim ◽  
Oren Sagher ◽  
Julian T. Hoff
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Intracerebral Hemorrhage ◽  
Blood Brain Barrier ◽  
Brain Slices ◽  
Brain Barrier ◽  
C6 Glioma Cells ◽  
Edema Formation ◽  
Blood Brain

Recently, the authors showed that thrombin contributes to the formation of brain edema following intracerebral hemorrhage. The current study examines whether the action of thrombin is due to an effect on cerebral blood flow (CBF), vasoreactivity, blood-brain barrier (BBB) function, or cell viability. In vivo solutions of thrombin were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; CBF and BBB permeability were measured. The actions of thrombin on vasoreactivity were examined in vitro by superfusing thrombin on cortical brain slices while monitoring microvessel diameter with videomicroscopy. In separate experiments C6 glioma cells were exposed to various concentrations of thrombin and lactate dehydrogenase release, a marker of cell death, was measured. The results indicate that thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. The authors conclude that cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage.

High glucose-induced effects on Na+-K+-Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCβII and SPAK/OSR1

2021 ◽  
Author(s):  
Nicholas R. Klug ◽  
Olga V. Chechneva ◽  
Benjamin Y. Hung ◽  
Martha E. O'Donnell
Keyword(s):  
Blood Brain Barrier ◽  
High Glucose ◽  
Artery Occlusion ◽  
Brain Barrier ◽  
Microvascular Endothelial Cell ◽  
Edema Formation ◽  
Kinase C ◽  
Blood Brain ◽  
Transport Of Ions ◽  
Cerebral Artery Occlusion

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCβII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCβII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCβII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCβII abundance or phosphorylation (activity) after 24 or 48 hr HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCβII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

scholarly journals Mice deficient in endothelial α5 integrin are profoundly resistant to experimental ischemic stroke

2016 ◽  
Vol 37 (1) ◽  
pp. 85-96 ◽  
Author(s):  
Jill Roberts ◽  
Leon de Hoog ◽  
Gregory J Bix
Keyword(s):  
Endothelial Cell ◽  
Blood Brain Barrier ◽  
Infarct Volume ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Brain Barrier ◽  
Barrier Integrity ◽  
Blood Brain ◽  
Blood Brain Barrier Integrity

Stroke is a disease in dire need of better therapies. We have previously shown that a fragment of the extracellular matrix proteoglycan, perlecan, has beneficial effects following cerebral ischemia via the α5β1 integrin receptor. We now report that endothelial cell selective α5 integrin deficient mice (α5 KO) are profoundly resistant to ischemic infarct after transient middle cerebral artery occlusion. Specifically, α5 KOs had little to no infarct 2–3 days post-stroke, whereas controls had an increase in mean infarct volume over the same time period as expected. Functional outcome is also improved in the α5 KOs compared with controls. Importantly, no differences in cerebrovascular anatomy or collateral blood flow were noted that could account for this difference in ischemic injury. Rather, we demonstrate that α5 KOs have increased blood-brain barrier integrity (increased expression of claudin-5, and absent brain parenchymal IgG extravasation) after stroke compared with controls, which could explain their resistance to ischemic injury. Additionally, inhibition of α5 integrin in vitro leads to decreased permeability of brain endothelial cells following oxygen-glucose deprivation. Together, these findings indicate endothelial cell α5 integrin plays an important role in stroke outcome and blood-brain barrier integrity, suggesting that α5 integrin could be a novel therapeutic target for stroke.

scholarly journals Stroke-Induced Brain Parenchymal Injury Drives Blood–Brain Barrier Early Leakage Kinetics: A Combined in Vivo/in Vitro Study

2013 ◽  
Vol 34 (1) ◽  
pp. 95-107 ◽  
Author(s):  
Mélanie Kuntz ◽  
Caroline Mysiorek ◽  
Olivier Pétrault ◽  
Maud Pétrault ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Glial Cells ◽  
Glucose Deprivation ◽  
Brain Barrier ◽  
Imaging Method ◽  
Early Reperfusion ◽  
Blood Brain ◽  
Brain Parenchymal

The disappointing clinical outcomes of neuroprotectants challenge the relevance of preclinical stroke models and data in defining early cerebrovascular events as potential therapeutic targets. The kinetics of blood–brain barrier (BBB) leakage after reperfusion and the link with parenchymal lesion remain debated. By using in vivo and in vitro approaches, we conducted a kinetic analysis of BBB dysfunction during early reperfusion. After 60 minutes of middle cerebral artery occlusion followed by reperfusion times up to 24 hours in mice, a non-invasive magnetic resonance imaging method, through an original sequence of diffusion-weighted imaging, determined brain water mobility in microvascular compartments ( D∗) apart from parenchymal compartments (apparent diffusion coefficient). An increase in D∗ found at 4 hours post reperfusion concurred with the onset of both Evans blue/Dextran extravasations and in vitro BBB opening under oxygen-glucose deprivation and reoxygenation ( R). The BBB leakage coincided with an emerging cell death in brain tissue as well as in activated glial cells in vitro. The co-culture of BBB endothelial and glial cells evidenced a recovery of endothelium tightness when glial cells were absent or non-injured during R. Preserving the ischemic brain parenchymal cells within 4 hours of reperfusion may improve therapeutic strategies for cerebrovascular protection against stroke.

Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model

1997 ◽  
Vol 86 (2) ◽  
pp. 272-278 ◽  
Author(s):  
Kevin R. Lee ◽  
Nobuyuki Kawai ◽  
Seoung Kim ◽  
Oren Sagher ◽  
Julian T. Hoff
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Intracerebral Hemorrhage ◽  
Blood Brain Barrier ◽  
Brain Slices ◽  
Brain Barrier ◽  
C6 Glioma Cells ◽  
Edema Formation ◽  
Blood Brain

✓ Recently, the authors showed that thrombin contributes to the formation of brain edema following intracerebral hemorrhage. The current study examines whether the action of thrombin is due to an effect on cerebral blood flow (CBF), vasoreactivity, blood-brain barrier (BBB) function, or cell viability. In vivo solutions of thrombin were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; CBF and BBB permeability were measured. The actions of thrombin on vasoreactivity were examined in vitro by superfusing thrombin on cortical brain slices while monitoring microvessel diameter with videomicroscopy. In separate experiments C6 glioma cells were exposed to various concentrations of thrombin, and lactate dehydrogenase release, a marker of cell death, was measured. The results indicate that thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. The authors conclude that cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage.

scholarly journals Maf1 Ameliorates Sepsis-Associated Encephalopathy by Suppressing the NF-kB/NLRP3 Inflammasome Signaling Pathway

2020 ◽  
Vol 11 ◽  
Author(s):  
Shenglong Chen ◽  
Chaogang Tang ◽  
Hongguang Ding ◽  
Zhonghua Wang ◽  
Xinqiang Liu ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Inflammatory Cytokines ◽  
Nlrp3 Inflammasome ◽  
Brain Barrier ◽  
Blue Dye ◽  
Inflammasome Activation ◽  
Blood Brain ◽  
Pro Inflammatory Cytokines

BackgroundThe NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome has been identified as an important mediator of blood–brain-barrier disruption in sepsis-associated encephalopathy (SAE). However, no information is available concerning the critical upstream regulators of SAE.MethodsLipopolysaccharide (LPS) was used to establish an in vitro model of blood–brain barrier (BBB) disruption and an in vivo model of SAE. Disruption of BBB integrity was assessed by measuring the expression levels of tight-junction proteins. NLRP3 inflammasome activation, pro-inflammatory cytokines levels, and neuroapoptosis were measured using biochemical assays. Finally, the FITC-dextran Transwell assay and Evan’s blue dye assay were used to assess the effect of Maf1 on LPS-induced endothelial permeability in vitro and in vivo.ResultsWe found that Maf1 significantly suppressed the brain inflammatory response and neuroapoptosis induced by LPS in vivo and in vitro. Notably, Maf1 downregulated activation of the NF-κB/p65-induced NLRP3 inflammasome and the expression of pro-inflammatory cytokines. In addition, we found that Maf1 and p65 directly bound to the NLRP3 gene promoter region and competitively regulated the function of NLRP3 in inflammations. Moreover, overexpression of NLRP3 reversed the effects of p65 on BBB integrity, apoptosis, and inflammation in response to LPS. Our study revealed novel role for Maf1 in regulating NF-κB-mediated inflammasome formation, which plays a prominent role in SAE.ConclusionsRegulation of Maf1 might be a therapeutic strategy for SAE and other neurodegenerative diseases associated with inflammation.

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