Caspase activation and neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation

Abstract Background : Connexin 43 (Cx43) are the most widely distributed gap junction proteins in the nervous system. Cx43 enables cell-to-cell communication and plays an important role in ion transport, substrate exchange and delivery of information , which have been implicated in cerebral ischemia injury. Our previous work revealed the relationships between Cx43 and glia-mediated neuroinflammation through the release of ATP in oxygen-glucose deprivation (OGD), which means degradation of Cx43 may improve neuroinflammatory damage during OGD injury . However, the roles of Cx43 degradation and neuroinflammation caused by OGD remain unclear. Methods: We used primary cultured astrocytes treated with OGD as an in vitro model of cerebral ischemia injury and we used middle cerebral artery occlusion (MCAO) model as an in vivo model of cerebral ischemia. HeLa cells were used in overexpression experiments. Cx43 protein levels were determined by western blotting. The interaction between Cx43 and related autophagy receptors was determined by co-immunoprecipitation and immunofluorescence. The gene knockdown (KD) of ATG5, OPTN, NDP52, PINK1 and Cx43 was applied by siRNA transfection. Related cytokines were detected by cytometric bead assay. Results: We found that Cx43 protein levels increased after ischemia in gene KD of ATG5, OPTN, NDP52 and PINK1 primary astrocytes. The interaction of Cx43 with OPTN, NDP52 and PINK1 was increased after cerebral ischemia injury in vitro and vivo. While the interaction was weakened after point mutation of Cx43 at Ser368, Tyr265 and Tyr247. Meanwhile, IL-10 upregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes , while TNF downregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes. Conclusions: Our results suggest that degradation of Cx43 is caused by selective autophagy during ischemia injury and the autophagy degradation of Cx43 plays important roles in neuroinflammation mediated by OGD injury. Treatment targeting Cx43 degradation pathway can improve neuroinflammation responses induced by OGD injury , which provide novel therapeutic strategies and crosstalk between autophagy and neuroinflammation.

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EphrinB2-EphB2 signaling for dendrite protection after neuronal ischemia in vivo and oxygen-glucose deprivation in vitro

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x20973119 ◽

2020 ◽

pp. 0271678X2097311

Author(s):

Zhanyang Yu ◽

Wenlu Li ◽

Jing Lan ◽

Kazuhide Hayakawa ◽

Xunming Ji ◽

...

Keyword(s):

Cerebral Ischemia ◽

Mouse Model ◽

Therapeutic Potential ◽

Focal Cerebral Ischemia ◽

Ischemic Injury ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Neuron Soma

In order to rescue neuronal function, neuroprotection should be required not only for the neuron soma but also the dendrites. Here, we propose the hypothesis that ephrin-B2-EphB2 signaling may be involved in dendritic degeneration after ischemic injury. A mouse model of focal cerebral ischemia with middle cerebral artery occlusion (MCAO) method was used for EphB2 signaling test in vivo. Primary cortical neuron culture and oxygen-glucose deprivation were used to assess EphB2 signaling in vitro. siRNA and soluble ephrin-B2 ectodomain were used to block ephrin-B2-Ephb2 signaling. In the mouse model of focal cerebral ischemia and in neurons subjected to oxygen-glucose deprivation, clustering of ephrin-B2 with its receptor EphB2 was detected. Phosphorylation of EphB2 suggested activation of this signaling pathway. RNA silencing of EphB2 prevented neuronal death and preserved dendritic length. To assess therapeutic potential, we compared the soluble EphB2 ectodomain with the NMDA antagonist MK801 in neurons after oxygen-glucose deprivation. Both agents equally reduced lactate dehydrogenase release as a general marker of neurotoxicity. However, only soluble EphB2 ectodomain protected the dendrites. These findings provide a proof of concept that ephrin-B2-EphB2 signaling may represent a novel therapeutic target to protect both the neuron soma as well as dendrites against ischemic injury.

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A novel therapeutic approach using peripheral blood mononuclear cells preconditioned by oxygen-glucose deprivation

Scientific Reports ◽

10.1038/s41598-019-53418-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Masahiro Hatakeyama ◽

Masato Kanazawa ◽

Itaru Ninomiya ◽

Kaoru Omae ◽

Yasuko Kimura ◽

...

Keyword(s):

Cerebral Ischemia ◽

Therapeutic Strategy ◽

Mononuclear Cells ◽

Focal Cerebral Ischemia ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Peripheral Blood Mononuclear ◽

Novel Therapeutic

AbstractCell therapies that invoke pleiotropic mechanisms may facilitate functional recovery in patients with stroke. Based on previous experiments using microglia preconditioned by oxygen-glucose deprivation, we hypothesized that the administration of peripheral blood mononuclear cells (PBMCs) preconditioned by oxygen-glucose deprivation (OGD-PBMCs) to be a therapeutic strategy for ischemic stroke. Here, OGD-PBMCs were identified to secrete remodelling factors, including the vascular endothelial growth factor and transforming growth factor-β in vitro, while intra-arterial administration of OGD-PBMCs at 7 days after focal cerebral ischemia prompted expression of such factors in the brain parenchyma at 28 days following focal cerebral ischemia in vivo. Furthermore, administration of OGD-PBMCs induced an increasing number of stage-specific embryonic antigen-3-positive cells both in vitro and in vivo. Finally, it was found to prompt angiogenesis and axonal outgrowth, and functional recovery after cerebral ischemia. In conclusion, the administration of OGD-PBMCs might be a novel therapeutic strategy against ischemic stroke.

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LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽

10.1007/s13577-021-00527-x ◽

2021 ◽

Author(s):

Jiaying Zhu ◽

Zhu Zhu ◽

Yipin Ren ◽

Yukang Dong ◽

Yaqi Li ◽

...

Keyword(s):

Cerebral Ischemia ◽

Nuclear Translocation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Mice Model ◽

Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

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Effect of Ischemia In vivo and Oxygen-Glucose Deprivation In vitro on NOS Pools in the Spinal Cord: Comparative Study

Cellular and Molecular Neurobiology ◽

10.1007/s10571-006-9032-1 ◽

2006 ◽

Vol 26 (7-8) ◽

pp. 1279-1292 ◽

Cited By ~ 4

Author(s):

Mária Kolesárová ◽

Jaroslav Pavel ◽

Nadežda Lukáčová ◽

Dalibor Kolesár ◽

Jozef Maršala

Keyword(s):

Spinal Cord ◽

Comparative Study ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation

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Generation and Characterization of Smac/DIABLO-Deficient Mice

Molecular and Cellular Biology ◽

10.1128/mcb.22.10.3509-3517.2002 ◽

2002 ◽

Vol 22 (10) ◽

pp. 3509-3517 ◽

Cited By ~ 120

Author(s):

Hitoshi Okada ◽

Woong-Kyung Suh ◽

Jianping Jin ◽

Minna Woo ◽

Chunying Du ◽

...

Keyword(s):

Physiological Function ◽

Es Cells ◽

Embryonic Stem ◽

Caspase Activation ◽

Proapoptotic Protein ◽

Apoptosis Proteins ◽

Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

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The Rapid Decrease in Astrocyte-Associated Dystroglycan Expression by Focal Cerebral Ischemia is Protease-Dependent

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600585 ◽

2007 ◽

Vol 28 (4) ◽

pp. 812-823 ◽

Cited By ~ 54

Author(s):

Richard Milner ◽

Stephanie Hung ◽

Xiaoyun Wang ◽

Maria Spatz ◽

Gregory J del Zoppo

Keyword(s):

Cerebral Ischemia ◽

Basal Lamina ◽

Focal Cerebral Ischemia ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Receptor Expression ◽

Cerebral Microvessels ◽

The Impact

During focal cerebral ischemia, the detachment of astrocytes from the microvascular basal lamina is not completely explained by known integrin receptor expression changes. Here, the impact of experimental ischemia (oxygen—glucose deprivation (OGD)) on dystroglycan expression by murine endothelial cells and astrocytes grown on vascular matrix laminin, perlecan, or collagen and the impact of middle cerebral artery occlusion on αβ-dystroglycan within cerebral microvessels of the nonhuman primate were examined. Dystroglycan was expressed on all cerebral microvessels in cortical gray and white matter, and the striatum. Astrocyte adhesion to basal lamina proteins was managed in part by α-dystroglycan, while ischemia significantly reduced expression of dystroglycan both in vivo and in vitro. Furthermore, dystroglycan and integrin α6β4 expressions on astrocyte end-feet decreased in parallel both in vivo and in vitro. The rapid loss of astrocyte dystroglycan during OGD appears protease-dependent, involving an matrix metalloproteinase-like activity. This may explain the rapid detachment of astrocytes from the microvascular basal lamina during ischemic injury, which could contribute to significant changes in microvascular integrity.

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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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Neuroprotective Effects of Propofol in Models of Cerebral Ischemia

Anesthesiology ◽

10.1097/00000542-200601000-00014 ◽

2006 ◽

Vol 104 (1) ◽

pp. 80-89 ◽

Cited By ~ 66

Author(s):

Chiara Adembri ◽

Luna Venturi ◽

Alessia Tani ◽

Alberto Chiarugi ◽

Elena Gramigni ◽

...

Keyword(s):

Cerebral Ischemia ◽

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Mitochondrial Swelling ◽

Oxygen Glucose Deprivation ◽

Neuroprotective Effects ◽

Cerebral Artery Occlusion

Background Propofol (2,6-diisopropylphenol) has been shown to attenuate neuronal injury in a number of experimental conditions, but studies in models of cerebral ischemia have yielded conflicting results. Moreover, the mechanisms involved in its neuroprotective effects are yet unclear. Methods The authors evaluated the neuroprotective effects of propofol in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. To investigate its possible mechanism of action, the authors then examined whether propofol could reduce Ca2+-induced rat brain mitochondrial swelling, an index of mitochondrial membrane permeability, as well as the mitochondrial swelling evoked by oxygen-glucose deprivation in CA1 pyramidal cells by transmission electron microscopy. Finally, they evaluated whether propofol could attenuate the infarct size and improve the neurobehavioral outcome in rats subjected to permanent middle cerebral artery occlusion in vivo. Results When present in the incubation medium during oxygen-glucose deprivation and the subsequent 24 h recovery period, propofol (10-100 microM) attenuated CA1 injury in hippocampal slices in vitro. Ca2+-induced brain mitochondrial swelling was prevented by 30-100 microM propofol, and so were the ultrastructural mitochondrial changes in CA1 pyramidal cells exposed to oxygen-glucose deprivation. Twenty-four hours after permanent middle cerebral artery occlusion, propofol (100 mg/kg, intraperitoneal) reduced the infarct size by approximately 30% when administered immediately after and up to 30 min after the occlusion. Finally, propofol administered within 30 min after middle cerebral artery occlusion was unable to affect the global neurobehavioral score but significantly preserved spontaneous activity in ischemic rats. Conclusions These results show that propofol, at clinically relevant concentrations, is neuroprotective in models of cerebral ischemia in vitro and in vivo and that it may act by preventing the increase in neuronal mitochondrial swelling.

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