Ischemia-induced Blood-brain Barrier Research Articles

The combination model of serum occludin and clinical risk factors improved the efficacy for predicting hemorrhagic transformation in stroke patients with recanalization

Backgroundand Purpose: Hemorrhagic transformation (HT) is one of the severe complications in acute ischemic stroke, especially for the patients who undergo recanalization treatment. It is crucial to screen patients who have high risk of HT before recanalization. However, current prediction models based on clinical factors are not ideal for clinical practice. Serum occludin, a biomarker for cerebral ischemia-induced blood-brain barrier disruption, has potential for predicting HT. This study was to investigate whether the combination of serum occludin and clinical risk factors improved the efficacy of predicting HT. MethodsThis was a single-center prospective observational study. Baseline clinical data and blood samples of recanalization patients were collected upon admission to our hospital. The level of serum occludin was measured using enzyme-linked immunosorbent assay. The diagnosis of HT was confirmed by CT scans within 36 h post recanalization. ResultsA total of 324 patients with recanalization were enrolled and 68 patients presented HT occurrence. HT patients had the higher level of baseline occludin than patients without HT (p < 0.001). Multivariate regression analysis showed that serum occludin level, Alberta Stroke Program Early CT Scores and endovascular therapy were independent risk factors (p < 0.05) for HT after adjusting potential confounders. The combination of serum occludin and clinical risk factors significantly improved the accuracy of predicting HT [area under the curve (AUC, 0.821 vs 0.701, p < 0.001), and net reclassification improvement (31.1 %), integrated discrimination improvement (21.5 %), p < 0.001] compared to a model employing only clinical risk factors. The modified AUC (0.806) of combined model based on 10-fold-cross-validation was still higher than clinical risk model (0.701). ConclusionThe combination of serum occludin and clinical risk factors significantly improved the prediction efficacy for HT, providing a novel potential prediction model to screen for patients with high risk of HT before recanalization in acute ischemic stroke.

Mesenchymal Stem Cells Overexpressing FGF21 Preserve Blood-Brain Barrier Integrity in Experimental Ischemic Stroke.

Blood-brain barrier (BBB) disruption is a prominent pathophysiological mechanism in stroke. Transplantation of mesenchymal stem cells (MSCs) preserves BBB integrity following ischemic stroke. Fibroblast growth factor 21 (FGF21) has been shown to be a potent neuroprotective agent that reduces neuroinflammation and protects against BBB leakage. In this study, we assessed the effects of transplantation of MSCs overexpressing FGF21 (MSCs-FGF21) on ischemia-induced neurological deficits and BBB breakdown. MSCs-FGF21 was injected into the rat brain via the intracerebroventricular route 24 h after middle cerebral artery occlusion (MCAO) surgery. The behavioral performance was assessed using modified neurological severity scores and Y-maze tests. BBB disruption was measured using Evans blue staining, IgG extravasation, and brain water content. The levels of tight junction proteins, aquaporin 4, and neuroinflammatory markers were analyzed by western blotting and immunohistochemistry. The activity of matrix metalloproteinase-9 (MMP-9) was determined using gelatin zymography. At day-5 after MCAO surgery, intraventricular injection of MSCs-FGF21 was found to significantly mitigate the neurological deficits and BBB disruption. The MCAO-induced loss of tight junction proteins, including ZO-1, occludin, and claudin-5, and upregulation of the edema inducer, aquaporin 4, were also remarkably inhibited. In addition, brain infarct volume, pro-inflammatory protein expression, and MMP-9 activation were effectively suppressed. These MCAO-induced changes were only marginally improved by treatment with MSCs-mCherry, which did not overexpress FGF21. Overexpression of FGF21 dramatically improved the therapeutic efficacy of MSCs in treating ischemic stroke. Given its multiple benefits and long therapeutic window, MSC-FGF21 therapy may be a promising treatment strategy for ischemic stroke.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood–brain barrier in acute ischemic stroke

Blood–brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Nicotinamide Mononucleotide Adenylyltransferase 1 Regulates Cerebral Ischemia-Induced Blood-Brain Barrier Disruption Through NAD+/SIRT1 Signaling Pathway.

The molecular mechanisms of blood-brain barrier (BBB) disruption in the early stage after ischemic stroke are poorly understood. In the present study, we investigated the potential role of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) in ischemia-induced BBB damage using an animal middle cerebral artery occlusion (MCAO) model of ischemic stroke. Recombinant human NMNAT1 (rh-NMNAT1) was administered intranasally and Sirtuin 1 (SIRT1) siRNA was administered by intracerebroventricular injection. Our results indicate that rh-NMNAT1 reduced infarct volume, improved functional outcome, and decreased BBB permeability in mice after ischemic stroke. Furthermore, rh-NMNAT1 prevented the loss of tight junction proteins (occludin and claudin-5) and reduced cell apoptosis in ischemic microvessels. NMNAT1-mediated BBB permeability was correlated with the elevation of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and SIRT1 level in brain microvascular endothelial cells. In addition, rh-NMNAT1 treatment significantly decreased the levels of acetylated nuclear factor-κB, acetylated p53, and matrix metalloproteinase-9 in ischemic microvessels. Moreover, the protective effects of rh-NMNAT1 could be reversed by SIRT1 siRNA. In conclusion, these findings indicate that rh-NMNAT1 protects BBB integrity after cerebral ischemia via the NAD+/SIRT1 signaling pathway in brain microvascular endothelial cells. NMNAT1 may be a novel potential therapeutic target for reducing BBB disruption after ischemic stroke.

Aqueous extract of Terminalia arjuna bark attenuates blood brain barrier disruption in rat model of transient focal cerebral ischemia

BackgroundTerminalia arjuna (TA) is a widely used medicinal plant in Indian traditional medicinal system. In the Ayurvedic system of medicine it is used as a cardiotonic and also well documented in experimental studies for its efficacy in various cardiac ailments. Few recent studies have reported its neuroprotective effects in rat model of cerebral ischemia. However, the effect of TA on cerebral ischemia induced Blood brain barrier (BBB) damage is still unclear. PurposeThe present study is aimed to evaluate the ameliorative effect of aqueous extract of Terminalia arjuna (aeTA) against transient focal cerebral ischemia induced BBB damage. Study designAdult male Sprague-dawley rats (270–300g) were randomly divided into three groups: Sham, Lesion (Middle cerebral artery occlusion for 2 hours) and aeTA pre-treatment at a dose of 500 mg /kg body weight/day orally for 15 days prior to MCAo. Materials and MethodsAfter 24 h of ischemic-reperfusion injury, the rats were assessed for behavioral deficits by Modified neurological severity scoring (mNSS), infarct measurement by Triphenyl tetrazolium chloride (TTC) staining, BBB integrity by Evans blue (EB) staining, protein and mRNA expressions of TJ proteins (Claudin5 & Occludin), MMPs (MMP2 & MMP9) and TIMPs (TIMP1 & TIMP2) through western blot analysis and real time PCR. ResultsThe pre-treatment with aeTA for 15 days exhibited significant improvement in neurological outcome and reduced infarct volume compared with the MCAo rats. The aeTA treatment also reduced the BBB damage which was confirmed through decreased EB extravasations, significant reduction in protein levels of MMPs (MMP2 &MMP9) and maintaining the protein levels of tight junction proteins. We also found that aeTA treatment downregulated the mRNA levels of MMPs and upregulated mRNA expression levels of TIMPs and tight junction proteins (Claudin5 & Occludin). ConclusionThese results demonstrate the neuroprotective property of aeTA treatment by reducing the MMPs mediated BBB damage after focal cerebral ischemia.

DDAH-1, via regulation of ADMA levels, protects against ischemia-induced blood-brain barrier leakage

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) is an important regulator of nitric oxide (NO) metabolism that has been implicated in the pathogenesis of cardiovascular diseases. Nevertheless, its role in cerebral ischemia still needs to be elucidated. Herein, we examined the expression of DDAH-1 in the brain of rat by double-label immunofluorescence staining. DDAH-1 knock-out (DDAH-1−/−) and wild-type rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, neurological scores, TTC staining and TUNEL assay were used to evaluate neurological damages. 3 and 7-days infarct outcomes were also shown. Blood-brain-barrier (BBB) permeability was examined via Evans blue extravasation and tight junction (TJ) proteins expression and mRNA levels by western blot and RT-qPCR. The levels of plasma asymmetric dimethylarginine (ADMA), NO and ADMA in brain tissue were also assessed. In addition, supplementation of L-arginine to DDAH-1−/− rats was used to explore its role in regulating NO. DDAH-1 was abundantly distributed in cerebral cortex and basal nuclei, and mainly expressed in neurons and endothelial cells. DDAH-1−/− rats showed aggravated neurological damage and BBB disruption, including decrease of TJ proteins expression but indistinguishable mRNA levels after MCAO/R. DDAH-1 depletion and neurological damages were accompanied with increased ADMA levels and decreased NO concentrations. The supplementation with L-arginine partly restored the neurological damages and BBB disruption. To sum up, DDAH-1 revealed to have a protective role in ischemia stroke (IS) and IS-induced leakage of BBB via decreasing ADMA level and possibly via preventing TJ proteins degradation.Dimethylarginine dimethylamino hydrolase-1 (DDAH-1), as the critical enzyme responsible for asymmetric dimethylarginine (ADMA) degradation, serves as a protective factor for ischemic stroke. DDAH-1 protects ischemia-induced disruption of blood-brain-barrier via regulating ADMA level and preventing tight junction proteins degradation. The supplementation of L-arginine helps restore the function of DDAH-1.

Therapeutic Effects of Decursin and Angelica gigas Nakai Root Extract in Gerbil Brain after Transient Ischemia via Protecting BBB Leakage and Astrocyte Endfeet Damage.

Angelica gigas Nakai root contains decursin which exerts beneficial properties such as anti-amnesic and anti-inflammatory activities. Until now, however, the neuroprotective effects of decursin against transient ischemic injury in the forebrain have been insufficiently investigated. Here, we revealed that post-treatment with decursin and the root extract saved pyramidal neurons in the hippocampus following transient ischemia for 5 min in gerbil forebrain. Through high-performance liquid chromatography, we defined that decursin was contained in the extract as 7.3 ± 0.2%. Based on this, we post-treated with 350 mg/kg of extract, which is the corresponding dosage of 25 mg/kg of decursin that exerted neuroprotection in gerbil hippocampus against the ischemia. In addition, behavioral tests were conducted to evaluate ischemia-induced dysfunctions via tests of spatial memory (by the 8-arm radial maze test) and learning memory (by the passive avoidance test), and post-treatment with the extract and decursin attenuated ischemia-induced memory impairments. Furthermore, we carried out histochemistry, immunohistochemistry, and double immunohistofluorescence. Pyramidal neurons located in the subfield cornu ammonis 1 (CA1) among the hippocampal subfields were dead at 5 days after the ischemia; however, treatment with the extract and decursin saved the pyramidal neurons after ischemia. Immunoglobulin G (IgG, an indicator of extravasation), which is not found in the parenchyma in normal brain tissue, was apparently shown in CA1 parenchyma from 2 days after the ischemia, but IgG leakage was dramatically attenuated in the CA1 parenchyma treated with the extract and decursin. Furthermore, astrocyte endfeet, which are a component of the blood–brain barrier (BBB), were severely damaged at 5 days after the ischemia; however, post-treatment with the extract and decursin dramatically attenuated the damage of the endfeet. In brief, therapeutic treatment of the extract of Angelica gigas Nakai root and decursin after 5 min transient forebrain ischemia protected hippocampal neurons from the ischemia, showing that ischemia-induced BBB leakage and damage of astrocyte endfeet was significantly attenuated by the extract and decursin. Based on these findings, we suggest that Angelica gigas Nakai root containing decursin can be employed as a pharmaceutical composition to develop a therapeutic strategy for brain ischemic injury.

A novel PGAM5 inhibitor LFHP-1c protects blood–brain barrier integrity in ischemic stroke

Blood–brain barrier (BBB) damage after ischemia significantly influences stroke outcome. Compound LFHP-1c was previously discovered with neuroprotective role in stroke model, but its mechanism of action on protection of BBB disruption after stroke remains unknown. Here, we show that LFHP-1c, as a direct PGAM5 inhibitor, prevented BBB disruption after transient middle cerebral artery occlusion (tMCAO) in rats. Mechanistically, LFHP-1c binding with endothelial PGAM5 not only inhibited the PGAM5 phosphatase activity, but also reduced the interaction of PGAM5 with NRF2, which facilitated nuclear translocation of NRF2 to prevent BBB disruption from ischemia. Furthermore, LFHP-1c administration by targeting PGAM5 shows a trend toward reduced infarct volume, brain edema and neurological deficits in nonhuman primate Macaca fascicularis model with tMCAO. Thus, our study identifies compound LFHP-1c as a firstly direct PGAM5 inhibitor showing amelioration of ischemia-induced BBB disruption in vitro and in vivo, and provides a potentially therapeutics for brain ischemic stroke.

Occludin degradation makes brain microvascular endothelial cells more vulnerable to reperfusion injury in vitro.

Intracerebral hemorrhage is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke, which occurs as a consequence of endothelial cell death at the blood-brain barrier (BBB) during thrombolytic reperfusion. We have previously shown that cerebral ischemia-induced rapid occludin degradation and BBB disruption. Here we demonstrated an important role of occludin degradation in facilitating the evolution of ischemic endothelial cells toward death. Cultured brain microvascular endothelial cells (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or over-expression status before exposing to reoxygenation (R) or TNF-α treatment. Cell death was assessed by measuring lactate dehydrogenase release, TUNEL staining, and flow cytometry analysis. Inhibition of OGD-induced occludin degradation with SB-3CT or over-expression of occludin with occludin AAV both significantly attenuated OGD/R-induced apoptosis and pyroptosis in bEnd.3 cells. Consistently, knockdown of occludin with siRNA potentiated TNF-α-induced apoptosis, supporting an important role of occludin integrity in endothelial cell survival. Similar results were observed for pyroptosis, in which occludin knockdown with siRNA led to a significant augmentation of cytokines secretion, inflammasome activation, and pyroptosis occurrence in TNF-α-treated bEnd.3 cells. Lastly, up-regulation of c-Yes, PI3K/AKT, and ERK concurrently occurred with occludin degradation after OGD/R or TNF-α treatment, and the level of these proteins were further increased when inhibition of occludin degradation or over-expression of occludin. These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion-associated stress stimuli.

MicroRNA-30a regulates acute cerebral ischemia-induced blood–brain barrier damage through ZnT4/zinc pathway

The mechanism of early blood–brain barrier (BBB) disruption after stroke has been intensively studied but still not fully understood. Here, we report that microRNA-30a (miR-30a) could mediate BBB damage using both cellular and animal models of ischemic stroke. In the experiments in vitro, inhibition of miR-30a decreased BBB permeability, prevented the degradation of tight junction proteins, and reduced intracellular free zinc in endothelial cells. We found that the zinc transporter ZnT4 was a direct target of negative regulation by miR-30a, and ZnT4/zinc signaling pathway contributed significantly to miR-30a-mediated BBB damage. Consistent with these in vitro findings, treatment with miR-30a inhibitor reduced zinc accumulation, increased the expression of ZnT4, and prevented the loss of tight junction proteins in microvessels of ischemic animals. Furthermore, inhibition of miR-30a, even at 90 min post onset of middle cerebral artery occlusion, prevented BBB damage, reduced infarct volume, and ameliorated neurological deficits. Together, our findings provide novel insights into the mechanisms of cerebral ischemia-induced BBB disruption and indicate miR-30a as a regulator of BBB function that can be an effective therapeutic target for ischemic stroke.

Ginsenoside Rd attenuates blood-brain barrier damage by suppressing proteasome-mediated signaling after transient forebrain ischemia.

Ischemic stroke damages the blood-brain barrier (BBB), which leads to brain edema and increases the risk of intracranial hemorrhage. Proteasome inhibition has been found to protect the BBB against cerebral ischemia by suppressing neuroinflammation-mediated matrix metalloproteases-9 (MMP-9) activation. We recently showed that ginsenoside Rd (Rd), a major active ingredient of Panax ginseng, could suppress proteasome-mediated inflammation and be efficient for treating ischemic stroke but downstream mechanisms were still unidentified. For this purpose, Sprague-Dawley rats were subjected to focal cerebral ischemic injury. The activity of proteasome and its downstream effectors nuclear factor-kappa B (NF-κB) and MMP-9 were evaluated. Rd reduced the activity of 20S proteasome in a cell-free assay and inhibited proteasome activity in brain lysates after ischemic stroke. Rd administration suppressed ischemic injury-induced NF-κB activity and IκB degradation mediated by the proteasome. Moreover, Rd reduced the activity and level of MMP-9, a downstream effector of NF-κB, and protected against BBB damage as evidenced by reduced Evan's Blue leakage and brain edema after cerebral ischemic injury. Jointly, these data demonstrate that ginsenoside Rd attenuates the pathogenesis of cerebral ischemia-induced BBB damage, probably by inhibiting proteasome activity and sequentially suppressing NF-κB/MMP-9 pathway.

Effects of nicorandil on neurobehavioral function, BBB integrity, edema and stereological parameters of the brain in the sub-acute phase of stroke in a rat model

Blood-brain barrier (BBB) disruption, inflammation, and cell death are the pathogenic mechanisms of cerebral ischemia/reperfusion (I/R) injury. Nicorandil protects ischemic injury via some of these mechanisms. The aim of this study was to investigate the therapeutic effects of this drug on the brain ischemia after transient middle cerebral artery occlusion (MCAO) and clarify the NF-jB and Nrf2-dependent mechanisms modulated by this drug. Sixty-six rats were randomized into sham, MCAO and MCAO + nicorandil groups with oral gavage for 3 days. Cerebral I/R injury were induced by a transient MCAO for 1 h and neurobehavioral scores were performed for 3 days. In addition to measurement of BBB disruption and brain water content, the total and infarct volume, density, and total number of neurons, non-neurons and dead neurons in the right cortex were estimated by unbiased stereological methods. RT-PCR was performed to analyze the expression levels of NFjB and Nrf2. Although nicorandil treatment in the sub-acute brain ischemia did not have a prominent effect on neurobehavioral function and number of neurons, non-neurons and dead neurons probably through up-regulation of NF-jB, it, however, improved ischemia-induced BBB disruption and brain edema and showed a significant reduction in the infarction volume probably through up-regulation of Nrf2.

TMEM16A Inhibition Preserves Blood-Brain Barrier Integrity After Ischemic Stroke.

The inflammatory response plays a pivotal role in Blood–Brain Barrier (BBB) destruction following ischemic brain injury. Enhanced leukocyte adhesion to vascular endothelial cells is an essential event in the inflammatory process. TMEM16A, a newly discovered protein regulating calcium-activated chloride channels, is widely expressed in eukaryotes. Recent studies have suggested that upregulated expression of TMEM16A is associated with the occurrence and development of many diseases. However, the role of TMEM16A in regulating BBB integrity after ischemic stroke has not been fully investigated. In this study, we found that TMEM16A is mainly expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. Caccinh-A01, an TMEM16A inhibitor that reduced its upregulation, attenuated brain infarct size and neurological deficits after ischemic stroke. ICAM-1 and MPO expression and BBB permeability were decreased after TMEM16A inhibitor administration. In addition, TMEM16A silencing rescued oxygen-glucose deprivation/reoxygenation (OGD/R)-induced transendothelial permeability in vitro accompanied by decreased ICAM-1 expression and leukocyte adhesion. Furthermore, our mechanistic study showed that TMEM16A knockdown alleviated NF-κB activation and nuclear translocation, indicating that TMEM16A knockdown downregulated OGD/R-induced ICAM-1 expression in an NF-κB-dependent manner. Finally, NF-κB inhibitor treatment also alleviated OGD/ R-induced BBB permeability, confirming that activated NF-κB and increased ICAM-1 are essential factors involved in ischemia-induced BBB damage. Thus, our research provides a promising treatment strategy against BBB destruction after ischemic stroke, and TMEM16A may become a potential target for the treatment of ischemic stroke.

Zinc accumulation in mitochondria promotes ischemia-induced BBB disruption through Drp1-dependent mitochondria fission

High concentration of zinc has been reported to act as a critical mediator of neuronal death in the ischemic brain. Our previous studies showed that labile zinc accumulates in cerebromicrovessels and contributes to blood–brain barrier (BBB) permeability increase after cerebral ischemia. However, the role of mitochondrial zinc in ischemia-induced BBB permeability alteration is still unclear. In this study, we showed that ischemia/reperfusion induced free zinc accumulation in endothelial cells (ECs), resulting in increased generation of reactive oxygen species (ROS) in both cultured ECs and in microvessels isolated from the brain of ischemic rats. Furthermore, we found that zinc was highly accumulated in mitochondria, leading to mitochondrial ROS generation under the ischemic condition. Moreover, zinc overload in mitochondria resulted in the collapse of the network of mitochondria, which was mediated through Dynamin-related protein-1 (Drp-1) dependent mitochondrial fission pathway. Finally, the zinc overload in mitochondria activated matrix metalloproteinase-2 and led to ischemia-induced BBB permeability increase. This study demonstrated that zinc-ROS pathway in mitochondria contributes to the ischemia-induced BBB disruption via Drp-1 dependent mitochondrial fission pathway.

Inhibition of HIF-1α Reduced Blood Brain Barrier Damage by Regulating MMP-2 and VEGF During Acute Cerebral Ischemia.

Increase of blood brain barrier (BBB) permeability after acute ischemia stroke is a predictor to intracerebral hemorrhage transformation (HT) for tissue plasminogen activator (tPA) thrombolysis and post-endovascular treatment. Previous studies showed that 2-h ischemia induced damage of BBB integrity and matrix metalloproteinase-2 (MMP-2) made major contribution to this disruption. A recent study showed that blocking β2-adrenergic receptor (β2-AR) alleviated ischemia-induced BBB injury by reducing hypoxia-inducible factor-1 alpha (HIF-1α) level. In this study, we sought to investigate the interaction of HIF-1α with MMP-2 and vascular endothelial growth factor (VEGF) in BBB injury after acute ischemia stroke. Rat suture middle cerebral artery occlusion (MCAO) model was used to mimic ischemia condition. Our results showed that ischemia produced BBB damage and MMP-2/9 upregulation was colocalized with Rhodamine-dextran leakage. Pretreatment with YC-1, a HIF-1α inhibitor, alleviated 2-h ischemia-induced BBB injury significantly accompanied by decrease of MMP-2 upregulation. In addition, YC-1 also prevented VEGF-induced BBB damage. Of note, VEGF was shown to be colocalized with neurons but not astrocytes. Taken together, BBB damage was reduced by inhibition of interaction of HIF-1α with MMP-2 and VEGF during acute cerebral ischemia. These findings provide mechanisms underlying BBB damage after acute ischemia stroke and may help reduce thrombolysis- and post-endovascular treatment-related cerebral hemorrhage.

MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

Blood-brain barrier (BBB) disruption plays a critical role in brain injury induced by cerebral ischemia, and preserving BBB integrity during ischemia could alleviate cerebral injury. We examined the role of miR-130a in ischemic BBB disruption by using models of rat middle cerebral artery occlusion and cell oxygen-glucose deprivation. We found that ischemia significantly increased microRNA-130a (miR-130a) level and that miR-130a was predominantly from brain microvascular endothelial cells. Antagomir-130a, an antagonist of miR-130a, could attenuate brain edema, lower BBB permeability, reduce infarct volume, and improve neurologic function. MiR-130a overexpression induced by miR-130a mimic increased monolayer permeability, and intercellular inhibition of miR-130a by a miR-130a inhibitor suppressed oxygen-glucose deprivation-induced increase in monolayer permeability. Moreover, dual luciferase reporter system showed that Homeobox A5 was the direct target of miR-130a. MiR-130a, by inhibiting Homeobox A5 expression, could down-regulate occludin, thereby increasing BBB permeability. Our results suggested that miR-130a might be implicated in ischemia-induced BBB dysfunction and serve as a target for the treatment of ischemic stroke.-Wang, Y., Wang, M.-D., Xia, Y.-P., Gao, Y., Zhu, Y.-Y., Chen, S.-C., Mao, L., He, Q.-W., Yue, Z.-Y., Hu, B. MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

β2-Adrenergic Receptor-Mediated HIF-1α Upregulation Mediates Blood Brain Barrier Damage in Acute Cerebral Ischemia.

Disruption of the blood brain barrier (BBB) within the thrombolytic time window is an antecedent event to intracerebral hemorrhage in ischemic stroke. Our recent studies showed that 2-h cerebral ischemia induced BBB damage in non-infarcted area and secreted matrix metalloproteinase-2 (MMP-2) accounted for this disruption. However, the factors that affect MMP-2 secretion and regulate BBB damage remains unknown. Since hypoxia-inducible factor-1 alpha (HIF-1α) was discovered as a mater regulator in hypoxia, we sought to investigate the roles of HIF-1α in BBB damage as well as the factors regulating HIF-1α expression in the ischemic brain. in vivo rat middle cerebral artery occlusion (MCAO) and in vitro oxygen glucose deprivation (OGD) models were used to mimic ischemia. Pretreatment with HIF-1α inhibitor YC-1 significantly inhibited 2-h MCAO-induced BBB damage, which was accompanied by suppressed occludin degradation and vascular endothelial growth factor (VEGF) mRNA upregulation. Interestingly, β2-adrenergic receptor (β2-AR) antagonist ICI 118551 attenuated ischemia-induced BBB damage by regulating HIF-1α expression. Double immunostaining showed that HIF-1α was upregulated in ischemic neurons but not in astrocytes andendothelial cells. Of note, HIF-1α inhibition with inhibitor YC-1 or siRNA significantly prevented OGD-induced VEGF upregulation as well as the secretion of VEGF and MMP-2 in neurons. More importantly, blocking β2-AR with ICI 118551 suppressedHIF-1α upregulation in ischemic neurons and attenuated occludin degradation induced by the conditioned media of OGD-treatedneurons. Taken together, blockade of β2-AR-mediated HIF-1α upregulation mediates BBB damage during acute cerebral ischemia. These findings provide new mechanistic understanding of early BBB damage in ischemic stroke and may help reduce thrombolysis-related hemorrhagic complications.

Role of glycogen synthase kinase 3 in ischemia-induced blood-brain barrier disruption in aged female rats.

Estrogen receptors have protective effects against ischemic brain injury. However, the molecular mechanisms underlying this phenomenon have yet to be well studied. Given that inhibition of glycogen synthase kinase (GSK3) can reduce cerebralischemia/reperfusion injury, we hypothesized that estrogen receptors-mediated protective effects against ischemia-induced blood-brain barrier (BBB) disruption involve inhibition of GSK3. Thus, we evaluated GSK3 expression in the brain of ovariectomized female rats, and examined the effects of intracerebroventricular pre-treatments of SB216763, GSK3 inhibitor, on BBB permeability following middle cerebral artery occlusion (MCAO). We also examined the role of specific estrogen receptor subtype in regulation of GSK3 expression and BBB permeability after MCAO. We found that ovariectomized female rats exhibited increased mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ), and increased protein levels of GSK3β but not GSK3α in brain cortical areas. Furthermore, intracerebroventricular pre-treatments of SB216763 dose-dependently attenuated brain infarction volume, brain water contents, neurological deficits, and BBB disruption, and increased tight junction protein ZO-1 and occludin expression at 24h following MCAO. Finally, activation of ERβ but not ERα dose-dependently decreased GSK3β expression at 24h following MCAO. This was associated with increased tight junction protein expression and improved neurological scores. Thus, our study suggested that activation of ERβ may protect against brain ischemia-induced BBB disruption by inhibiting GSK3β-mediated signaling.

Pretreatment with 17β-Estradiol Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Disruption in Aged Rats: Involvement of Antioxidant Signaling

Background/Aims: Blood-brain barrier (BBB) disruption is often induced in brain injury and particularly associated with cerebral ischemia in stroke. Estradiol (17β-estradiol 3-benzoate, E2), an endogenous steroid hormone, has been reported to play a protective role against cerebrovascular pathologies. Here we aimed to determine the potential effects of E2 on the integrity of BBB and brain damage following middle cerebral artery occlusion (MCAO). Methods: Aged (24- month-old) ovariectomized female rats were administered E2 for 2 months through daily intraperitoneal injections prior to induction of MCAO. Results: Compared to vehicle controls, E2 had a dose-dependent effect in reducing the severity of brain injuries while maintaining the integrity of the BBB in aged rats. The neuroprotective effects of E2 were associated with the rescued tight junction loss, decreased oxidative stress, and attenuated ERK activation in the ischemic brains. Conclusion: Our data suggest a beneficial role of E2 in aged stroke patients, associated with a protective effect of E2 against BBB disruption in aging-related brain ischemia.

Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway.

Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen–glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1–10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.