Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O2), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1–100 nM) on astrocyte cell volume using 3- O-methyl-d-[3H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30–180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.

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F-actin microfilament polymerized by jasplakinolide affects the expression of aquaporin-4 in astrocytic swelling after oxygen-glucose deprivation and reoxygenation

Materials Express ◽

10.1166/mex.2020.1658 ◽

2020 ◽

Vol 10 (4) ◽

pp. 563-570

Author(s):

Zhengang Sun ◽

Xinchao Zhang ◽

Lingyun Hu ◽

Xiaoshuai Wang ◽

Zhenjuan Sun ◽

...

Keyword(s):

Cell Volume ◽

Glucose Deprivation ◽

Aquaporin 4 ◽

Strong Increase ◽

Oxygen Glucose Deprivation ◽

Protective Effects ◽

Aqp4 Expression ◽

Actin Microfilament ◽

Astrocytic Swelling ◽

Aqp4 Protein

This study was to determine whether Jasplakinolide (JSK), an actin microfilament polymerization, reduced the astrocytic swelling induced by oxygen-glucose deprivation and reoxygenation (OGD/Reox) and whether this effect could be mediated through the modulation of aquaporin-4 (AQP4) expression. Astrocytes were obtained from rat spinal cord. Cells were subjected to reoxygenation after 5 h OGD. JSK with different concentrations (20, 50, 100, 200 and 400 nM) was added to astrocytes for 12 h before and during OGD/Reox. Then the cell volume and AQP4 expression were analyzed. The results showed that astrocytic swelling occurred with cell volume increasing after OGD/Reox and the severity was significantly attenuated by JSK. A strong increase of AQP4 protein expression was observed after OGD/Reox, reaching a peak at 1.5 h into reoxygenation. The increase in levels of AQP4 protein was significantly reduced by treatment with JSK. In addition, confocal microscopy analysis showed that the F-actin microfilament polymerization by JSK accounted for AQP4 downregulation in the plasma membrane. This study demonstrated that JSK could attenuate astrocytes swelling caused by OGD/Reox, an ischemia and reperfusion-like injury, and part of its protective effects might be related to the reorganization the F-actin microfilament and inhibiting the abundance expression of AQP4 protein.

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Endothelium-targeted deletion of the miR-15a/16-1 cluster ameliorates blood-brain barrier dysfunction in ischemic stroke

Science Signaling ◽

10.1126/scisignal.aay5686 ◽

2020 ◽

Vol 13 (626) ◽

pp. eaay5686 ◽

Cited By ~ 4

Author(s):

Feifei Ma ◽

Ping Sun ◽

Xuejing Zhang ◽

Milton H. Hamblin ◽

Ke-Jie Yin

Keyword(s):

Ischemic Stroke ◽

Blood Brain Barrier ◽

Molecular Mechanisms ◽

Neuronal Loss ◽

Glucose Deprivation ◽

Brain Barrier ◽

Protein Abundance ◽

Barrier Dysfunction ◽

Small Noncoding Rnas ◽

Blood Brain

The blood-brain barrier (BBB) maintains a stable brain microenvironment. Breakdown of BBB integrity during cerebral ischemia initiates a devastating cascade of events that eventually leads to neuronal loss. MicroRNAs are small noncoding RNAs that suppress protein expression, and we previously showed that the miR-15a/16-1 cluster is involved in the pathogenesis of ischemic brain injury. Here, we demonstrated that when subjected to experimentally induced stroke, mice with an endothelial cell (EC)–selective deletion of miR-15a/16-1 had smaller brain infarcts, reduced BBB leakage, and decreased infiltration of peripheral immune cells. These mice also showed reduced infiltration of proinflammatory M1-type microglia/macrophage in the peri-infarct area without changes in the number of resolving M2-type cells. Stroke decreases claudin-5 abundance, and we found that EC-selective miR-15a/16-1 deletion enhanced claudin-5 mRNA and protein abundance in ischemic mouse brains. In cultured mouse brain microvascular ECs (mBMECs), the miR-15a/16-1 cluster directly bound to the 3′ untranslated region (3′UTR) of Claudin-5, and lentivirus-mediated ablation of miR-15a/16-1 diminished oxygen-glucose deprivation (OGD)–induced down-regulation of claudin-5 mRNA and protein abundance and endothelial barrier dysfunction. These findings suggest that genetic deletion of endothelial miR-15a/16-1 suppresses BBB pathologies after ischemic stroke. Elucidating the molecular mechanisms of miR-15a/16-1–mediated BBB dysfunction may enable the discovery of new therapies for ischemic stroke.

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Scoparone protects neuronal cells from oxygen glucose deprivation/reoxygenation injury

RSC Advances ◽

10.1039/c8ra09867k ◽

2019 ◽

Vol 9 (4) ◽

pp. 2302-2308 ◽

Cited By ~ 2

Author(s):

Chunfang Wu ◽

Ting Li ◽

Baihui Zhu ◽

Ruiming Zhu ◽

Youran Zhang ◽

...

Keyword(s):

Ischemic Stroke ◽

Causes Of Death ◽

Neuronal Cells ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

The World ◽

Reoxygenation Injury

Ischemic stroke is one of the leading causes of death and disability in the world.

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Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways

Journal of Neurosurgery ◽

10.3171/2017.5.jns163165 ◽

2019 ◽

Vol 130 (3) ◽

pp. 977-988 ◽

Cited By ~ 10

Author(s):

Yu Shuang Tian ◽

Di Zhong ◽

Qing Qing Liu ◽

Xiu Li Zhao ◽

Hong Xue Sun ◽

...

Keyword(s):

Ischemic Stroke ◽

Inflammatory Mediators ◽

Neurological Deficit ◽

Ischemic Injury ◽

Glucose Deprivation ◽

Luciferase Assay ◽

Oxygen Glucose Deprivation ◽

Tnf Α

OBJECTIVEIschemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke–related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke.METHODSThe JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)–treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)–induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3′ untranslated region (3′UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase–9 [MMP-9], tumor necrosis factor–α [TNF-α], and interleukin-1β [IL-1β]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V–FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits.RESULTSJAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3′UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1β). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit.CONCLUSIONSThese findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.

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Silencing of Long Noncoding RNA Nespas Aggravates Microglial Cell Death and Neuroinflammation in Ischemic Stroke

Stroke ◽

10.1161/strokeaha.118.023376 ◽

2019 ◽

Vol 50 (7) ◽

pp. 1850-1858 ◽

Cited By ~ 14

Author(s):

Yiming Deng ◽

Duanduan Chen ◽

Luyao Wang ◽

Feng Gao ◽

Bo Jin ◽

...

Keyword(s):

Cell Death ◽

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Long Noncoding Rna ◽

Noncoding Rna ◽

Noncoding Rnas ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Oxygen Glucose Deprivation ◽

Cerebral Artery Occlusion

Background and Purpose— Ischemic stroke is one of the leading causes of morbidity and mortality worldwide and a major cause of long-term disability. Recently, long noncoding RNAs have been revealed, which are tightly associated with several human diseases. However, the functions of long noncoding RNAs in ischemic stroke still remain largely unknown. In the current study, for the first time, we investigated the role of long noncoding RNA Nespas in ischemic stroke. Methods— We used in vivo models of middle cerebral artery occlusion and in vitro models of oxygen-glucose deprivation to illustrate the effect of long noncoding RNA Nespas on ischemic stroke. Results— We found expression of Nespas was significantly increased in ischemic cerebral tissues and oxygen-glucose deprivation–treated BV2 cells in a time-dependent manner. Silencing of Nespas aggravated middle cerebral artery occlusion operation–induced IR injury and cell death. In addition, proinflammatory cytokine production and NF-κB (nuclear factor-κB) signaling activation were inhibited by Nespas overexpression. TAK1 (transforming growth factor-β–activated kinase 1) was found to directly interact with Nespas, and TAK1 activation was significantly suppressed by Nespas. At last, we found Nespas-inhibited TRIM8 (tripartite motif 8)-induced K63-linked polyubiquitination of TAK1. Conclusions— We showed that Nespas played anti-inflammatory and antiapoptotic roles in cultured microglial cells after oxygen-glucose deprivation stimulation and in mice after ischemic stroke by inhibiting TRIM8-related K63-linked polyubiquitination of TAK1.

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Expression and Distribution Pattern of Pnn in Ischemic Cerebral Cortex and Cultured Neural Cells Exposed to Oxygen-Glucose Deprivation

Brain Sciences ◽

10.3390/brainsci10100708 ◽

2020 ◽

Vol 10 (10) ◽

pp. 708

Author(s):

Shu-Yuan Hsu ◽

Sujira Mukda ◽

Steve Leu

Keyword(s):

Ischemic Stroke ◽

Mouse Model ◽

Glucose Deprivation ◽

Expression Level ◽

Oxygen Glucose Deprivation ◽

Neural Cells ◽

Nuclear Speckle ◽

Different Types ◽

Cellular Apoptosis ◽

Ischemic Stress

Pinin (Pnn), a multifunctional protein, participates in embryonic development as well as in cellular apoptosis, proliferation, and migration through regulating mRNA alternative splicing and gene transcription. Previous studies have shown that Pnn plays important roles in neural system development and the expression level of Pnn in astrocytes is altered by ischemic stress and associated with cellular apoptosis. In the present study, we further utilized primary cultured rat neurons and astrocytes with oxygen-glucose deprivation (OGD) and a mouse model with middle cerebral artery occlusion (MCAO)-induced ischemic stroke to examine the effect of ischemic stress on Pnn expression and distribution in different types of neural cells. Under normoxia, Pnn is mainly localized in the nuclear speckle of primary cultured neurons. The expression level of Pnn was increased after the OGD treatment and then decreased in the reoxygenation period. Moreover, the cytoplasmic expression of Pnn was observed in neurons with OGD and reoxygenation (OGD/R). Unlike that in neurons, the Pnn expression in astrocytes was decreased after OGD treatment and then gradually increased during the reoxygenation period. Of interest, the nuclear–cytoplasmic translocation of Pnn was not observed in astrocytes with OGD/R. In the MCAO mouse model, the neuronal expression of Pnn in the peri-ischemic region was reduced by three days post induction of ischemic stroke. However, the Pnn expression in astrocytes was not altered. Moreover, the nuclear speckle distribution of Pnn in neurons was also diminished following ischemic stroke. In conclusion, the Pnn expression and distribution after OGD and during reoxygenation showed distinct manners in neurons and astrocytes, implying that Pnn may play different roles in different types of neural cells in the stress response to ischemic injury.

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Relaxin Peptide Hormones Are Protective During the Early Stages of Ischemic Stroke in Male Rats

Endocrinology ◽

10.1210/en.2014-1676 ◽

2014 ◽

Vol 156 (2) ◽

pp. 638-646 ◽

Cited By ~ 9

Author(s):

Lindsay H. Bergeron ◽

Jordan M. Willcox ◽

Faisal J. Alibhai ◽

Barry J. Connell ◽

Tarek M. Saleh ◽

...

Keyword(s):

Nitric Oxide ◽

Ischemic Stroke ◽

Nitric Oxide Synthase ◽

Infarct Size ◽

Endothelial Nitric Oxide Synthase ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Protective Mechanisms ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

The pregnancy hormone relaxin protects tissue from ischemic damage. The ability of relaxin-3, a relaxin paralog, to do so has not been explored. The cerebral expression levels of these peptides and their receptors make them logical targets for study in the ischemic brain. We assessed relaxin peptide-mediated protection, relative relaxin family peptide receptor (RXFP) involvement, and protective mechanisms. Sprague-Dawley rats receiving permanent (pMCAO) or transient middle cerebral artery occlusions (tMCAO) were treated with relaxin peptides, and brains were collected for infarct analysis. Activation of the endothelial nitric oxide synthase pathway was evaluated as a potential protective mechanism. Primary cortical rat astrocytes were exposed to oxygen glucose deprivation and treated with relaxin peptides, and viability was examined. Receptor involvement was explored using RXFP3 antagonist or agonist treatment and real-time PCR. Relaxin and relaxin-3 reduced infarct size after pMCAO. Both peptides activated endothelial nitric oxide synthase. Because relaxin-3 has not previously been associated with this pathway and displays promiscuous RXFP binding, we explored the receptor contribution. Expression of rxfp1 was greater than that of rxfp3 in rat brain, although peptide binding at either receptor resulted in similar overall protection after pMCAO. Only RXFP3 activation reduced infarct size after tMCAO. In astrocytes, rxfp3 gene expression was greater than that of rxfp1. Selective activation of RXFP3 maintained astrocyte viability after oxygen glucose deprivation. Relaxin peptides are protective during the early stages of ischemic stroke. Differential responses among treatments and models suggest that RXFP1 and RXFP3 initiate different protective mechanisms. This preliminary work is a pivotal first step in identifying the clinical implications of relaxin peptides in ischemic stroke.

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Exacerbation of oxygen–glucose deprivation-induced blood–brain barrier disruption: potential pathogenic role of interleukin-9 in ischemic stroke

Clinical Science ◽

10.1042/cs20170984 ◽

2017 ◽

Vol 131 (13) ◽

pp. 1499-1513 ◽

Cited By ~ 5

Author(s):

Sha Tan ◽

Yilong Shan ◽

Yuge Wang ◽

Yinyao Lin ◽

Siyuan Liao ◽

...

Keyword(s):

T Cells ◽

Ischemic Stroke ◽

Blood Brain Barrier ◽

Mononuclear Cells ◽

Biological Effects ◽

Glucose Deprivation ◽

Brain Barrier ◽

Oxygen Glucose Deprivation ◽

Expression Levels ◽

Blood Brain

Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood–brain barrier (BBB) following oxygen–glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.

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Role of long noncoding RNA MEG3/miR-378/GRB2 axis in neuronal autophagy and neurological functional impairment in ischemic stroke

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010946 ◽

2020 ◽

Vol 295 (41) ◽

pp. 14125-14139 ◽

Cited By ~ 2

Author(s):

Hong-Cheng Luo ◽

Ting-Zhuang Yi ◽

Fu-Gao Huang ◽

Ying Wei ◽

Xiao-Peng Luo ◽

...

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Functional Impairment ◽

Molecular Mechanisms ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Neuronal Autophagy

Autophagy has been shown to maintain neural system homeostasis during stroke. However, the molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. This study aims to investigate the regulatory mechanisms of the pathway consisting of MEG3 (maternally expressed gene 3), microRNA-378 (miR-378), and GRB2 (growth factor receptor-bound protein 2) in neuronal autophagy and neurological functional impairment in ischemic stroke. A mouse model of the middle cerebral artery occluded–induced ischemic stroke and an in vitro model of oxygen-glucose deprivation–induced neuronal injury were developed. To understand the role of the MEG3/miR-378/GRB2 axis in the neuronal regulation, the expression of proteins associated with autophagy in neurons was measured by Western blotting analysis, and neuron death was evaluated using a lactate dehydrogenase leakage rate test. First, it was found that the GRB2 gene, up-regulated in middle cerebral artery occluded–operated mice and oxygen-glucose deprivation–exposed neurons, was a target gene of miR-378. Next, miR-378 inhibited neuronal loss and neurological functional impairment in mice, as well as neuronal autophagy and neuronal death by silencing of GRB2. Confirmatory in vitro experiments showed that MEG3 could specifically bind to miR-378 and subsequently up-regulate the expression of GRB2, which in turn suppressed the activation of Akt/mTOR pathway. Taken together, these findings suggested that miR-378 might protect against neuronal autophagy and neurological functional impairment and proposed that a MEG3/miR-378/GRB2 regulatory axis contributed to better understanding of the pathophysiology of ischemic stroke.

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