• acute ischemic stroke
• endovascular treatment
• hemorrhagic transformation
• microRNAs
• oxygen–glucose deprivation/reoxygenation
• parenchymal hematoma

• 81720108014, 81371275 National Natural Science Foundation of China
• 201704020166 Science and Technology Planning Project of Guangzhou City
• 2023B1212060018 Science and Technology Planning Project of Guangdong Province

• Brain pericytes
• Calcium
• Experimental stroke
• Rapamycin
• mTOR

• MR/M022757/1 Medical Research Council
• MR/M022757/1 Medical Research Council
• MR/M022757/1 Medical Research Council
• MR/M022757/1 Medical Research Council
• MR/M022757/1 Medical Research Council
• APP1182153 National Health and Medical Research Council Australia
• APP1137776, APP2003351 National Health and Medical Research Council
• APP1137776, APP2003351 National Health and Medical Research Council
• APP1137776, APP2003351 National Health and Medical Research Council

• Alpha-smooth muscle actin
• Cell culture
• Contractility
• Morphology
• Pericytes

• Humans
• Pericytes/metabolism
• Brain
• Phenotype
• Cell Line

• APP2003351 National Health and Medical Research Council
• APP1137776 National Health and Medical Research Council
• PG2018137 Rebecca L. Cooper Medical Research Foundation
• University of Tasmania

• Biomarkers
• Blood brain barrier
• Ischemic stroke
• β-Actin

• Humans
• Ischemic Stroke
• Actins
• Stroke
• Brain Ischemia
• Biomarkers

• 2022KY1367 Medical Health Science and Technology Project of Zhejiang Provincial Health Commission
• 2018C11044 Planned Projects of Bureau of Science and Technology of Zhoushan

• Hypoperfusion
• Microglia
• Neuroinflammation
• Vascular dementia

• Blood
• Brain barrier
• Hemorrhagic transformation
• Ischemic stroke
• Tissue plasminogen activator

• Animals
• Biomarkers
• Brain Ischemia/complications
• Brain Ischemia/drug therapy
• Humans
• Ischemic Stroke
• Stroke/complications
• Stroke/drug therapy
• Tissue Plasminogen Activator/adverse effects

• 81801174 National Natural Science Foundation of China
• 81571891 National Natural Science Foundation of China

• Blood–brain barrier
• Cerebral edema
• Endothelium
• Neurovascular unit
• Perivascular signaling
• Tight junctions
• Vascular remodeling

Cerebral stroke is a leading cause of death and adult-acquired disability worldwide. To this date, treatment options are limited; hence, the search for new therapeutic approaches continues. Electromagnetic fields (EMFs) affect a wide variety of biological processes and accumulating evidence shows their potential as a treatment for ischemic stroke. Based on their characteristics, they can be divided into stationary, pulsed, and sinusoidal EMF. The aim of this review is to provide an extensive literature overview ranging from in vitro to even clinical studies within the field of ischemic stroke of all EMF types. A thorough comparison between EMF types and their effects is provided, as well as an overview of the signal pathways activated in cell types relevant for ischemic stroke such as neurons, microglia, astrocytes, and endothelial cells. We also discuss which steps have to be taken to improve their therapeutic efficacy in the frame of the clinical translation of this promising therapy.

• cerebral ischemia
• electromagnetic field
• neuroprotection
• neurorehabilitation
• pulsed electromagnetic field
• sinusoidal electromagnetic field
• stroke

Pericytes play essential roles in blood-brain barrier integrity and their dysfunction is implicated in neurological disorders such as stroke although the underlying mechanisms remain unknown. Hypoxia-inducible factor-1 (HIF-1), a master regulator of injury responses, has divergent roles in different cells especially during stress scenarios. On one hand HIF-1 is neuroprotective but on the other it induces vascular permeability. Since pericytes are critical for barrier stability, we asked if pericyte HIF-1 signaling impacts barrier integrity and injury severity in a mouse model of ischemic stroke. We show that pericyte HIF-1 loss of function (LoF) diminishes ischemic damage and barrier permeability at 3 days reperfusion. HIF-1 deficiency preserved barrier integrity by reducing pericyte death thereby maintaining vessel coverage and junctional protein organization, and suppressing vascular remodeling. Importantly, considerable improvements in sensorimotor function were observed in HIF-1 LoF mice indicating that better vascular functionality post stroke improves outcome. Thus, boosting vascular integrity by inhibiting pericytic HIF-1 activation and/or increasing pericyte survival may be a lucrative option to accelerate recovery after severe brain injury.

• Cerebral ischemia
• Pericyte coverage
• Pericyte death
• Vascular permeability

Vascular dysfunction and vasoregression are hallmarks of a variety of inflammatory central nervous system disorders and inflammation-related retinal diseases like diabetic retinopathy. Activation of microglia and the humoral innate immune system are contributing factors. Anti-inflammatory approaches have been proposed as therapies for neurovascular diseases, which include the modulation of microglial activation. The present study aimed at investigating the effects of microglial activation by clodronate-coated liposomes on vasoregression in a model of retinal degeneration. Clodronate treatment over 5 weeks led to an increase in activated CD74⁺ microglia and completely prevented acellular capillaries and pericyte loss. Gene expression analyses indicated that vasoprotection was due to the induction of vasoprotective factors such as Egr1, Stat3, and Ahr while expression of pro-inflammatory genes remained unchanged. We concluded that activated microglia led to a shift toward induction of pleiotropic protective pathways supporting vasoprotection in neurovascular retinal diseases.

• clodronate
• microglia
• pericyte
• retinopathy
• vasoregression

• Blood-brain barrier
• Hemorrhagic transformation
• Neuroinflammation
• Stroke
• Tissue plasminogen activator

The supply of oxygen and nutrients to the brain is vital for its function and requires a complex vascular network that, when disturbed, results in profound neurological dysfunction. As part of the pathology in stroke, endothelial cells die. As endothelial cell death affects the surrounding cellular environment and is a potential target for the treatment and prevention of neurological disorders, we have systematically reviewed important aspects of endothelial cell death with a particular focus on stroke. After screening 2876 publications published between January 1, 2010 and August 7, 2019, we identified 154 records to be included. We found that endothelial cell death occurs rapidly as well as later after the onset of stroke conditions. Among the different cell death mechanisms, apoptosis was the most widely investigated (92 records), followed by autophagy (20 records), while other, more recently defined mechanisms received less attention, such as lysosome-dependent cell death (2 records) and necroptosis (2 records). We also discuss the differential vulnerability of brain cells to injury after stroke and the role of endothelial cell death in the no-reflow phenomenon with a special focus on the microvasculature. Further investigation of the different cell death mechanisms using novel tools and biomarkers will greatly enhance our understanding of endothelial cell death. For this task, at least two markers/criteria are desirable to determine cell death subroutines according to the recommendations of the Nomenclature Committee on Cell Death.

• apoptosis
• autophagy
• ischemia
• lysosome-dependent cell death
• necroptosis
• vasculature

Mobility quantification of single cells and cellular processes in dense cultures is a challenge, because single cell tracking is impossible. We developed a software for cell structure segmentation and implemented 2 algorithms to measure motility speed. Complex algorithms were tested to separate cells and cellular components, an important prerequisite for the acquisition of meaningful motility data. Plasma membrane segmentation was performed to measure membrane contraction dynamics and organelle trafficking. The discriminative performance and sensitivity of the algorithms were tested on different cell types and calibrated on computer-simulated cells to obtain absolute values for cellular velocity. Both motility algorithms had advantages in different experimental setups, depending on the complexity of the cellular movement. The correlation algorithm (COPRAMove) performed best under most tested conditions and appeared less sensitive to variable cell densities, brightness and focus changes than the differentiation algorithm (DiffMove). In summary, our software can be used successfully to analyze and quantify cellular and subcellular movements in dense cell cultures.

• cell motility
• hippocampal neurons
• image analysis
• membrane contraction
• pericytes
• velocity measurement

• Cell culture
• Chemical reaction
• Hypoxia
• Oxygen-glucose deprivation

• Anoxia
• Astrocytes
• Brain endothelial cells
• Erythropoietin
• Neurovascular unit
• Stroke

Microvascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium.

Summary:In vitro systems derived from AD mice can be used to investigate the vulnerability of AD neurons to different neurotoxic challenges, including oxygen glucose deprivation.

• Alzheimer's disease
• Glutamate
• Intraneuronal amyloid
• Neurovascular coupling
• Oxygen glucose deprivation
• Primary neurons

• Animal research
• Brain
• Cell-death cascade
• Clinical trial
• Cost
• Cytoprotection
• Cytoprotective
• Drug discovery
• Hemorrhage
• ICH
• NIHSS
• Neuroprotection
• Neuroprotective
• RIGOR
• SAH
• STAIR
• Stroke
• Translational
• Transparency
• rt-PA

AIM: To investigate the role of activating transcription factor 4 (ATF4) in glucose deprivation (GD) induced colorectal cancer (CRC) drug resistance and the mechanism involved.

METHODS: Chemosensitivity and apoptosis were measured under the GD condition. Inhibition of ATF4 using short hairpin RNA in CRC cells under the GD condition and in ATF4-overexpressing CRC cells was performed to identify the role of ATF4 in the GD induced chemoresistance. Quantitative real-time RT-PCR and Western blot were used to detect the mRNA and protein expression of drug resistance gene 1 (MDR1), respectively.

RESULTS: GD protected CRC cells from drug-induced apoptosis (oxaliplatin and 5-fluorouracil) and induced the expression of ATF4, a key gene of the unfolded protein response. Depletion of ATF4 in CRC cells under the GD condition can induce apoptosis and drug re-sensitization. Similarly, inhibition of ATF4 in the ATF4-overexpressing CRC cells reintroduced therapeutic sensitivity and apoptosis. In addition, increased MDR1 expression was observed in GD-treated CRC cells.

CONCLUSION: These data indicate that GD promotes chemoresistance in CRC cells through up-regulating ATF4 expression.

• Glucose deprivation
• ATF4
• Oxaliplatin
• 5-Fluorouracil
• Chemoresistance

• ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
• Activating Transcription Factor 4/genetics
• Activating Transcription Factor 4/metabolism
• Antineoplastic Combined Chemotherapy Protocols/pharmacology
• Apoptosis/genetics
• Blotting, Western
• Cell Line, Tumor
• Cell Proliferation/drug effects
• Colorectal Neoplasms/metabolism
• Drug Resistance, Neoplasm/genetics
• Endoplasmic Reticulum Chaperone BiP
• Fluorouracil/administration & dosage
• Gene Knockdown Techniques
• Glucose/deficiency
• Glucose/metabolism
• HCT116 Cells
• Heat-Shock Proteins/genetics
• Heat-Shock Proteins/metabolism
• Humans
• Organoplatinum Compounds/administration & dosage
• Oxaliplatin
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• Tumor Microenvironment
• eIF-2 Kinase/genetics
• eIF-2 Kinase/metabolism

• Angiogenesis
• Astrocyte
• Axonal sprouting
• Excitotoxicity
• Neurogenesis
• Oligodendrocyte progenitor

• Brain Ischemia/physiopathology
• Cerebral Infarction/physiopathology
• Humans
• Nerve Regeneration/physiology
• Neural Pathways/physiopathology
• Stroke/drug therapy
• Stroke/physiopathology

• NS081055 NINDS NIH HHS
• R01 NS077521 NINDS NIH HHS
• R01 NS071481 NINDS NIH HHS
• R01 NS081055 NINDS NIH HHS
• NS071481 NINDS NIH HHS
• R01 NS045729 NINDS NIH HHS
• R01 NS085019 NINDS NIH HHS
• NS085019 NINDS NIH HHS
• R01 NS053957 NINDS NIH HHS
• NS077521 NINDS NIH HHS
• National Institute of Neurological Disorders and Stroke (US)
• American Heart Association (US)

• blood-brain barrier
• cerebral ischemia
• cytokines
• macrophages
• microglia
• stroke

• Brain Ischemia/metabolism
• Brain Ischemia/pathology
• Humans
• Macrophage Activation/physiology
• Macrophages/metabolism
• Microglia/metabolism
• Microglia/pathology

Activation of microglia/macrophages following cerebral ischemia may be beneficial or detrimental for the survival of brain cells, an ambiguity in effects that has been explained by findings that ischemia can induce transformation of resting monocytes/macrophages into two different inflammation-related phenotypes, termed M1 and M2. The extent to which this differentiation depends on paracrine signaling from other brain cells is not clear. This study explored if oxygen glucose deprivation (OGD) can trigger expression of phenotype-specific markers in rat microglia/macrophages in primary culture, in absence/low abundance of other brain cells. Time pattern of these changes was assessed and compared to time-pattern that has been revealed in vivo previously. Effects of phenotype-specific cytokines on viability of astrocytes in primary culture during anoxia were also explored.

Primary cultures of rat microglia/macrophages were exposed to 2h OGD and then incubated further under normal conditions; this was considered as a recovery period. Expression of mRNA for specific markers and secretion of phenotype-specific cytokines were explored at different time points by real time PCR and ELISA, respectively. Effects of cytokines that were secreted by microglia in primary culture after OGD on viability of astrocytes were determined.

Expression and secretion of M2 phenotype-specific markers and/or cytokines after OGD increased early after OGD and then decreased in the later stages of the recovery period. Expression and secretion of M1 phenotype-specific markers and cytokines did not show a common time pattern, but there was a tendency for an increase during the recovery period. All M1 phenotype-specific and two out of the three tested M2 phenotype-specific cytokines revealed protective effects on astrocytes during near-anoxia by a marked reduction of apoptosis.

Time-pattern of expression/secretion of phenotype-specific markers suggested that polarization of the brain microglia/macrophages in vitro to M2 and M1 phenotypes were largely independent and likely dependent on signaling from other brain cells, respectively. Time-pattern of polarization to the M2 phenotype partially resembled time-pattern that has been seen in vivo. Effects of M1 phenotype-specific cytokines on primary culture of astrocytes were protective, thus largely opposite to effects that have been observed in vivo.

The online version of this article (doi:10.1186/s12987-015-0002-1) contains supplementary material, which is available to authorized users.

• Astrocytes
• Cerebral ischemia
• Cytokines
• Microglia
• Microglia polarization

Undisturbed functioning of the blood–brain barrier (BBB) crucially depends on paracellular signaling between its associated cells; particularly endothelial cells, pericytes and astrocytes. Hypoxic and ischemic injuries are closely associated with disturbed BBB function and the contribution of perivascular cells to hypoxic/ischemic barrier regulation has gained increased attention. Regardless, detailed information on the basal hypoxic/ischemic responses of the barrier-associated cells is rare and the outcome of such cell-specific responses on BBB modulation is not well understood. This study investigated crucial parameters of hypoxic/ischemic adaptation in order to characterize individual perivascular cell responses to stress conditions.

The brain microvascular endothelial cell line RBE4 (EC cell line) as well as primary rat brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were exposed to 24 and 48 hours of oxygen deprivation at 1% and 0.2% O₂. All primary cells were additionally subjected to combined oxygen and glucose deprivation mimicking ischemia. Central parameters of cellular adaptation and state, such as HIF-1α and HIF-1 target gene induction, actin cytoskeletal architecture, proliferation and cell viability, were compared between the cell types.

We show that endothelial cells exhibit greater responsiveness and sensitivity to oxygen deprivation than ACs and PCs. This higher sensitivity coincided with rapid and significant stabilization of HIF-1α and its downstream targets (VEGF, GLUT-1, MMP-9 and PHD2), early disruption of the actin cytoskeleton and metabolic impairment in conditions where the perivascular cells remain largely unaffected. Additional adaptation (suppression) of proliferation also likely contributes to astrocytic and pericytic tolerance during severe injury conditions. Moreover, unlike the perivascular cells, ECs were incapable of inducing autophagy (monitored via LC3-II and Beclin-1 expression) - a putative protective mechanism. Notably, both ACs and PCs were significantly more susceptible to glucose than oxygen deprivation with ACs proving to be most resistant overall.

In summary this work highlights considerable differences in sensitivity to hypoxic/ischemic injury between microvascular endothelial cells and the perivascular cells. This can have marked impact on barrier stability. Such fundamental knowledge provides an important foundation to better understand the complex cellular interactions at the BBB both physiologically and in injury-related contexts in vivo.

Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.

• C6
• blood-brain barrier
• cerebEND
• in vitro
• ischemia
• oxygen/glucose deprivation
• stroke
• traumatic brain injury