Attenuated Blood-brain Barrier Disruption Research Articles

Edaravone dexborneol protects against cerebral ischemia/reperfusion-induced blood-brain barrier damage by inhibiting ferroptosis via activation of nrf-2/HO-1/GPX4 signaling

PurposeFerroptosis has recently been recognized as a mechanism of cerebral ischemia-reperfusion (I/R) injury, attributed to blood-brain barrier (BBB) disruption. Edaravone dexboneol (Eda.B) is a novel neuroprotective agent widely employed in ischemic stroke, which is composed of edaravone (Eda) and dexborneol. This study aimed to investigate the protective effects of Eda.B on the BBB in cerebral I/R and explore its potential mechanisms. MethodsTransient middle cerebral artery occlusion (tMCAO) Sprague-Dawley-rats model was used. Rats were randomly assigned to sham-operated group (sham, n = 20), model group (tMCAO, n = 20), Eda.B group (Eda.B, n = 20), Eda group (Eda, n = 20) and dexborneol group (dexborneol, n = 20), and Eda.B + Zinc protoporphyria group (Eda.B + ZnPP, n = 5). Infarct area, cellular apoptosis and neurofunctional recovery were accessed through TTC staining, TUNEL staining, and modified Garcia scoring system, respectively. BBB integrity was evaluated via Evans blue staining. Nuclear factor E2 related factor 2 (Nrf-2)/heme oxygenase 1 (HO-1)/glutathione peroxidase 4 (GPX4) signaling were qualified by Western blot. Transmission electron microscopy (TEM) revealed alterations in ipsilateral brain tissue among groups. Glutathione (GSH) and malondialdehyde (MDA) levels, and Fe2+ tissue content determination were detected. ResultsEda.B effectively improved neurological deficits, diminished infarct area and cellular apoptosis, as well as ameliorated BBB integrity in tMCAO rats. Further, Eda.B significantly inhibited ferroptosis, as evidenced by ameliorated pathological features of mitochondria, down-regulated of MDA and Fe2+ levels and up-regulated GSH content. Mechanistically, Eda.B attenuated BBB disruption via Nrf-2-mediated ferroptosis, promoting nuclear translocation of Nrf-2, increasing HO-1, GPX4 expression, alleviating the loss of zonula occludens 1 (ZO-1) and occludin as well as decreasing 4-hydroxynonenal (4-HNE) level. ConclusionsThis study revealed for the first time that Eda.B safeguarded the BBB from cerebral I/R injury by inhibiting ferroptosis through the activation of the Nrf-2/HO-1/GPX4 axis, providing a novel insight into the neuroprotective effect of Eda.B in cerebral I/R.

Development of a therapeutic monoclonal antibody against circulating adipocyte fatty acid binding protein to treat ischaemic stroke.

Adipocyte fatty acid-binding protein (A-FABP) exacerbates cerebral ischaemia injury by disrupting the blood-brain barrier (BBB) through inducing expression of MMP-9. Circulating A-FABP levels positively correlate with infarct size in stroke patients. We hypothesized that targeting circulating A-FABP by a neutralizing antibody would alleviate ischaemic stroke outcome. Monoclonal antibodies (mAbs) against A-FABP were generated using mouse hybridoma techniques. Binding affinities of a generated mAb named 6H2 towards various FABPs were determined using Biacore. Molecular docking studies were performed to characterize the 6H2-A-FABP complex structure and epitope. The therapeutic potential and safety of 6H2 were evaluated in mice with transient middle cerebral artery occlusion (MCAO) and healthy mice, respectively. Replenishment of recombinant A-FABP exaggerated the stroke outcome in A-FABP-deficient mice. 6H2 exhibited nanomolar to picomolar affinities to human and mouse A-FABP, respectively, with minimal cross-reactivities with heart and epidermal FABPs. 6H2 effectively neutralized JNK/c-Jun activation elicited by A-FABP and reduced MMP-9 production in macrophages. Molecular docking suggested that 6H2 interacts with the "lid" of the fatty acid binding pocket of A-FABP, thus likely hindering the binding of its substrates. In mice with transient MCAO, 6H2 significantly attenuated BBB disruption, cerebral oedema, infarction, neurological deficits, and decreased mortality associated with reduced cytokine and MMP-9 production. Chronic 6H2 treatment showed no obvious adverse effects in healthy mice. These results establish circulating A-FABP as a viable therapeutic target for ischaemic stroke, and provide a highly promising antibody drug candidate with high affinity and specificity.

YangXueQingNaoWan attenuated blood brain barrier disruption after thrombolysis with tissue plasminogen activator in ischemia stroke

Ethnopharmacological relevantYangXueQingNaoWan (YXQNW), a compound Chinese medicine, has been widely used for dizziness, irritability, insomnia, and dreaminess caused by blood deficiency and liver hyperactivity in China. However, whether YXQNW can inhibit cerebral microvascular exudation and cerebral hemorrhage (CH) caused by blood brain barrier (BBB) damage after tissue plasminogen activator (tPA) still unknown. Aim of the researchTo observe the effect of YXQNW on cerebral microvascular exudation and CH after tPA and investigate its mechanism in protecting BBB. Materials and methodsMale C57BL/6 N mice suffered from ischemia stroke by mechanical detachment of carotid artery thrombi with the stimulation of ferric chloride. Then mice were treated with tPA (10 mg/kg) and/or YXQNW (0.72 g/kg) at 4.5 h. Cerebral blood flow (CBF), infarct size, survival rate, neurological scores, gait analysis, Evans blue extravasation, cerebral water content, fluorescein isothiocyanate-labeled albumin leakage, hemorrhage, junction and basement membrane proteins expression, leukocyte adhesion and matrix metalloproteinases (MMPs) expression were evaluated 24 h after tPA. Proteomics was used to identify target proteins. ResultsYXQNW inhibited cerebral infarction, neurobehavioral deficits, decreased survival, Evans blue leakage, albumin leakage, cerebral water content and CH after tPA thrombolysis; improved CBF, low-expression and degradation of junction proteins, basement membrane proteins, Arhgap21 and its downstream α-catenin and β-catenin proteins expression; and suppressed the increase of adherent leukocytes and the release of MMP-9 derived from macrophage. ConclusionYXQNW relieved BBB damage and attenuated cerebral microvascular exudation and CH after tPA thrombolysis. The effect of YXQNW on cerebral microvascular exudation was associated with the inhibition of the low-expression of junction proteins, especially AJs mediated by Rho GTPase-activating protein 21 (Arhgap21), while the effect on CH was associated with the inhibition of leukocyte adhesion, the release of MMP-9 derived from macrophage, and low-expression and degradation of collagen IV and laminin in the vascular basement membrane.

Inhibiting PI3K/Akt-Signaling Pathway Improves Neurobehavior Changes in Anti-NMDAR Encephalitis Mice by Ameliorating Blood–Brain Barrier Disruption and Neuronal Damage

The disruption of the blood–brain barrier (BBB) is hypothesized to be involved in the progression of anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, but its mechanism is still unclear. Recently, the phosphatidylinositol 3-kinase (PI3K)/threonine kinase (Akt) pathway is involved in the regulation of the BBB in various diseases. This study is aimed to investigate the mechanism of BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. Female C57BL/6J mice were actively immunized to establish an anti-NMDAR encephalitis mouse model and evaluate the neurobehavior changes of mice. To study its potential mechanism, LY294002 (PI3K inhibitor, 8 mg/kg) and Recilisib (PI3K agonist, 10 mg/kg) were treated by intraperitoneal injection, respectively. Anti-NMDAR encephalitis mice showed neurological deficits, increased BBB permeability, open endothelial tight junctions (TJs), and decreased expression of TJ-related proteins zonula occludens (ZO)-1 and Claudin-5. However, administration of PI3K inhibitor significantly reduced the expression of p-PI3K and p-Akt, improved neurobehavior function, decreased BBB permeability, and upregulated the expressions of ZO-1 and Claudin-5. Furthermore, PI3K inhibition reversed the decline of NMDAR NR1 in the membranes of hippocampal neurons, which reduced the loss of neuron-specific nucleoprotein (NeuN) and microtubule-associated protein 2 (MAP2). In contrast, administration of the PI3K agonist Recilisib showed a tendency to exacerbate BBB breakdown and neurological deficits. Our results showed that the activation of PI3K/Akt, along with the changes in TJ-related proteins ZO-1 and Claudin-5, may be closely related to BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. PI3K inhibition attenuates BBB disruption and neuronal damage in mice, thereby improving neurobehavior.

Endogenous FGF21 attenuates blood-brain barrier disruption in penumbra after delayed recanalization in MCAO rats through FGFR1/PI3K/Akt pathway.

Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO). Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting. The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO. Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.

Inactivation of microglia dampens blood-brain barrier permeability and loss of dopaminergic neurons in paraquat-lesioned mice.

Prior studies indicated the involvement of neuroinflammation in the dopaminergic neurodegeneration in mice of paraquat (PQ)-induced Parkinson's disease (PD), but the underlying mechanisms remain to be elucidated. The present study explored whether microglia-mediated inflammation disrupted blood-brain barrier (BBB) and its related mechanism. C57BL/6 mice were injected intraperitoneally with PQ, twice a week for six weeks, following with or without minocycline (intraperitoneal injection, once every two days). The microglial activation, BBB permeability, expression of tight junctions (TJs) proteins and matrix metalloproteinase (MMP), as well as the loss of dopaminergic neurons and neurological deficits assessment, were evaluated. Minocycline efficiently restrained nigral microglial activation induced by PQ in mice. PQ-induced increase of EB content in the brain and excessive expression of zonula occludin-1 (ZO-1), claudin-5 and occludin were significantly dampened by minocycline treatment. Inhibition of microglial activation by minocycline greatly ameliorated the loss of dopaminergic neurons and neurological dysfunctions in PQ-exposed mice. Also, microglial inactivation downregulated the expression of MMP-2/9 in PQ-lesioned mice. These findings suggested the potential protection of suppressing microglia-mediated neuroinflammation against dopaminergic neurodegeneration through attenuating BBB disruption in a mouse of PQ-induced PD, and MMP-2/9 might involve in the contribution, which needs to be verified in future study.

MiR-671-5p Upregulation Attenuates Blood–Brain Barrier Disruption in the Ischemia Stroke Model Via the NF-кB/MMP-9 Signaling Pathway

Blood-brain barrier (BBB) disruption can induce further hemorrhagic transformation in ischemic stroke (IS). miR-671-5p, a micro-RNA, is abundant in the cortex of mammalian brains. Herein, we investigated the roles and potential mechanisms for the effects of miR-671-5p on BBB permeability in IS. Results showed that miR-671-5p levels were significantly downregulated in the cerebral cortex of middle cerebral artery occlusion/reperfusion (MCAO/R) C57/BL6 mice in vivo. miR-671-5p agomir administration via right intracerebroventricular injection significantly reduced infarct volume, improved neurological deficits, the axon of neurons and nerve fiber, attenuated cell injury and apoptosis, as well as reduced BBB permeability in MCAO/R mice. Treatment with miR-671-5p agomir alleviated tight junction proteins degradation, including claudin, occludin, and ZO-1 in MCAO/R mice, and these effects were reversed following NF-κB overexpression. Bend.3 brain endothelial cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) treatment in vivo, and then miR-671-5p agomir was transfected into the cells. This resulted in reduction of cytotoxicity, improved cell viability, trans-endothelial electrical resistance, reduced fluorescein sodium permeability, and inhibited tight junction degradation in Bend.3 OGD/R cells. However, these effects were reversed following NF-κB overexpression. These results demonstrated that upregulation of miR-671-5p in IS models in vivo and in vitro alleviated BBB permeability by targeting NF-κB/MMP-9. In summary, miR-671-5p is a potential therapeutic target for protecting BBB permeability in IS to minimize cerebral hemorrhage transformation.

Blood-Brain Barrier Rescue by Roflumilast After Transient Global Cerebral Ischemia in Rats.

Phosphodiesterase 4 inhibitors (PDE4-I), which selectively increase cyclic adenosine monophosphate (cAMP) levels, have shown neuroprotective effects after several neurological injuries inducing blood-brain barrier (BBB) damage including local/focal cerebral ischemia. The present investigated whether roflumilast confers BBB neuroprotection in the hippocampus after transient global cerebral ischemia (TGCI) in rats. TGCI resulted in whole BBB disruption as measured by the increase of Evans blue (EB) and IgG extravasation, neurodegeneration, and downregulation of claudin-5 and endothelial nitric oxide synthase (eNOS) levels in the CA1 hippocampal subfield of ischemic rats. Roflumilast attenuated BBB disruption and restored the levels of eNOS in the CA1 hippocampal area. Moreover, roflumilast increased the levels of B2 cell lymphoma (BcL-2) and neuron-glial antigen-2 (NG2) in the CA1 subfield after global ischemia in rats. The protective effects of roflumilast against TGCI-induced BBB breakdown might involve preservation of BBB integrity, vascularization and angiogenesis, and myelin repair.

Myeloid-derived MIF drives RIPK1-mediated cerebromicrovascular endothelial cell death to exacerbate ischemic brain injury

Macrophage migration inhibitory factor (MIF) is a multifaced protein that plays important roles in multiple inflammatory conditions. However, the role of MIF in endothelial cell (EC) death under inflammatory condition remains largely unknown. Here we show that MIF actively promotes receptor-interacting protein kinase 1 (RIPK1)-mediated cell death under oxygen-glucose deprivation condition. MIF expression is induced by surgical trauma in peripheral myeloid cells both in perioperative humans and mice. We demonstrate that MIF-loaded myeloid cells induced by peripheral surgery adhere to the brain ECs after distal middle cerebral artery occlusion (dMCAO) and exacerbate the blood-brain barrier (BBB) disruption. Genetic depletion of myeloid-derived MIF in perioperative ischemic stroke (PIS) mice with MCAO following a surgical insult leads to significant reduction in ECs apoptosis and necroptosis and the associated BBB disruption. The adoptive transfer of peripheral blood mononuclear cells (PBMC) from surgical MIFΔLyz2 mice to wild-type (WT) MCAO mice also shows reduced ECs apoptosis and necroptosis compared to the transfer of PBMC from surgical MIFf l/f l mice to MCAO recipients. The genetic inhibition of RIPK1 also attenuates BBB disruption and ECs death compared to that of WT mice in PIS. The administration of MIF inhibitor (ISO-1) and RIPK1 inhibitor (Nec-1s) can both reduce the brain EC death and neurological deficits following PIS. We conclude that myeloid-derived MIF promotes ECs apoptosis and necroptosis through RIPK1kinase-dependent pathway. The above findings may provide insights into the mechanism as how peripheral inflammation promotes the pathology in central nervous system.

M2 Microglial Extracellular Vesicles Attenuated Blood-brain Barrier Disruption via MiR-23a-5p in Cerebral Ischemic Mice.

Protecting the integrity of the blood-brain barrier (BBB) is crucial for maintaining brain homeostasis after ischemic stroke. Previous studies showed that M2 microglial extracellular vesicles (EVs) played a neuroprotective role in cerebral ischemia. However, the role of M2 microglial EVs in maintaining BBB integrity is unclear. Therefore, we explored the mechanisms of M2 microglial EVs in regulating BBB integrity. To identify microglial EVs, we used nanoparticle tracking analysis, transmission electron microscopy, and western blot analysis. Adult male ICR mice were subjected to 90-min middle cerebral artery occlusion (MCAO), followed by the injection of PKH26-labeled M2 microglial EVs via the tail vein. After MCAO, we assessed brain infarct and edema volume, as well as modified neurological severity score. BBB integrity was measured by assessing IgG leakage. The effects of M2 microglial EVs on astrocytes and endothelial cells were also examined. To investigate the molecular mechanisms, we performed RNA sequencing, miR-23a-5p knockdown, and luciferase reporter assays. Our results showed that PKH26-labeled microglial EVs were mainly taken up by neurons and glial cells. M2 microglial EVs treatment decreased brain infarct and edema volume, modified neurological severity score, and IgG leakage, while increasing the ZO-1, occludin, and claudin-5 expression after MCAO. Knockdown of miR-23a-5p reversed these effects. RNA sequencing revealed that the TNF, MMP3 and NFκB signaling pathway involved in regulating BBB integrity. Luciferase reporter assay showed that miR-23a-5p could bind to the 3' UTR of TNF. M2 microglial EVs-derived miR-23a-5p decreased TNF, MMP3 and NFκB p65 expression in astrocytes after oxygen-glucose deprivation, thereby increasing ZO-1 and Claudin-5 expression in bEnd.3 cells. In conclusion, our findings demonstrated that M2 microglial EVs attenuated BBB disruption after cerebral ischemia by delivering miR-23a-5p, which targeted TNF and regulated MMP3 and NFκB p65 expression.

Neuronal Serpina3n is an endogenous protector against blood brain barrier damage following cerebral ischemic stroke

Ischemic stroke results in blood-brain barrier (BBB) disruption, during which the reciprocal interaction between ischemic neurons and components of the BBB appears to play a critical role. However, the underlying mechanisms for BBB protection remain largely unknown. In this study, we found that Serpina3n, a serine protease inhibitor, was significantly upregulated in the ischemic brain, predominantly in ischemic neurons from 6 hours to 3 days after stroke. Using neuron-specific adeno-associated virus (AAV), intranasal delivery of recombinant protein, and immune-deficient Rag1−/− mice, we demonstrated that Serpina3n attenuated BBB disruption and immune cell infiltration following stroke by inhibiting the activity of granzyme B (GZMB) and neutrophil elastase (NE) secreted by T cells and neutrophils. Furthermore, we found that intranasal delivery of rSerpina3n significantly attenuated the neurologic deficits after stroke. In conclusion, Serpina3n is a novel ischemic neuron-derived proteinase inhibitor that counterbalances BBB disruption induced by peripheral T cell and neutrophil infiltration after ischemic stroke. These findings reveal a novel endogenous protective mechanism against BBB damage with Serpina3n being a potential therapeutic target in ischemic stroke.

Therapeutic Hypothermia Combined with Hydrogen Sulfide Treatment Attenuated Early Blood–Brain Barrier Disruption and Brain Edema Induced by Cardiac Arrest and Resuscitation in Rat Model

Brain injury remains a major problem in patients suffering cardiac arrest (CA). Disruption of the blood–brain barrier (BBB) is an important factor leading to brain injury. Therapeutic hypothermia is widely accepted to limit neurological impairment. However, the efficacy is incomplete. Hydrogen sulfide (H2S), a signaling gas molecule, has protective effects after cerebral ischemia reperfusion injury. This study showed that combination of hypothermia and H2S after resuscitation was more beneficial for attenuated BBB disruption and brain edema than that of hypothermia or H2S treatment alone. CA was induced by ventricular fibrillation for 4 min. Hypothermia was performed by applying alcohol and ice bags to the body surface under anesthesia. We used sodium hydrosulphide (NaHS) as the H2S donor. We found that global brain ischemia induced by CA and cardiopulmonary resuscitation (CPR) resulted in brain edema and BBB disruption; Hypothermia or H2S treatment diminished brain edema, decreased the permeability and preserved the structure of BBB during the early period of CA and resuscitation, and more importantly, improved the neurologic function, increased the 7-day survival rate after resuscitation; the combination of hypothermia and H2S treatment was more beneficial than that of hypothermia or H2S treatment alone. The beneficial effects were associated with the inhibition of matrix metalloproteinase-9 expression, attenuated the degradation of the tight junction protein occludin, and subsequently protected the structure of BBB. These findings suggest that combined use of therapeutic hypothermia and hydrogen sulfide treatment during resuscitation of CA patients could be a potential strategy to improve clinical outcomes and survival rate.

Inhibition of interaction between ROCK1 and Rubicon restores autophagy in endothelial cells and attenuates brain injury after prolonged ischemia.

Acute ischemic stroke (AIS) induces cerebral endothelial cell death resulting in the breakdown of the blood-brain barrier (BBB). Endothelial cell autophagy acts as a protective mechanism against cell death. Autophagy is activated in the very early stages of ischemic stroke and declines after prolonged ischemia. Previous studies have shown that Rubicon can inhibit autophagy. The current study aimed to investigate whether continuous long-term ischemia can inhibit autophagy in endothelial cells after ischemic stroke by regulating the function of Rubicon and its underlying mechanism. Wild-type male C57BL/6J mice were subjected to transient middle cerebral artery occlusion (tMCAO). ROCK1, ROCK2, and NOX2 inhibitors were injected into male mice 1h before the onset of tMCAO. Disease severity and BBB permeability were evaluated. bEnd.3 cells were cultured in vitro and subjected to oxygen-glucose deprivation (OGD). bEnd.3 cells were pretreated with or without ROCK1, ROCK2, or NOX2 inhibitors overnight and then subjected to OGD. Cell viability and permeability were also evaluated. The expression of Rubicon, ROCK1, and autophagy-related proteins were analyzed. Increased BBB permeability was correlated with Rubicon expression in tMCAO mice and Rubicon was upregulated in endothelial cells subjected to OGD. Autophagy was inhibited in endothelial cells after long-term OGD treatment and knockdown of Rubicon expression restored autophagy and viability in endothelial cells subjected to 6-h OGD. ROCK1 inhibition decreased the interaction between Beclin1 and Rubicon and restored cell viability and autophagy suppressed by 6-h OGD treatment in endothelial cells. Additionally, ROCK1 inhibition suppressed Rubicon, attenuated BBB disruption, and brain injury induced by prolonged ischemia in 6-h tMCAO mice. Prolonged ischemia induced the death of brain endothelial cells and the breakdown of the BBB, thus aggravating brain injury by increasing the interaction of ROCK1 and Rubicon with Beclin1 while inhibiting canonical autophagy. Inhibition of ROCK1 signaling in endothelial cells could be a promising therapeutic strategy to prolong the therapeutic time window in AIS.

Fibroblasts repair blood-brain barrier damage and hemorrhagic brain injury via TIMP2.

The function of fibroblasts in intracerebral hemorrhage (ICH) remains elusive. By targeting Col1α1, a fibroblast-specific marker, we generate mice with ablated Col1α1+ fibroblasts. These mutants show exacerbated blood-brain barrier (BBB) damage, enlarged injury volume, and worse neurological function, highlighting a beneficial role of Col1α1+ fibroblasts in ICH. Echoing these findings, fibroblasts significantly decrease endothelial permeability in an invitro ICH model. Next, we demonstrate that fibroblasts promote BBB integrity in ICH mainly via up-regulating tight junction proteins without affecting transcytosis-associated proteins, indicating a paracellular rather than transcellular mechanism. A subsequent mechanistic study reveals that the BBB-protective effect of fibroblasts is partially mediated by TIMP metallopeptidase inhibitor 2 (TIMP2). Furthermore, we find that exogenous TIMP2 attenuates BBB disruption in these mutants after ICH. These results suggest that Col1α1+ fibroblasts repair BBB damage in ICH via the paracellular pathway in a TIMP2-dependent manner, and that Col1α1+ fibroblasts and TIMP2 may be targeted in ICH treatment.

Mesenchymal stem cell-derived extracellular vesicles attenuate tPA-induced blood–brain barrier disruption in murine ischemic stroke models

Intracerebral hemorrhage following blood–brain barrier (BBB) disruption resulting from thrombolysis of ischemic stroke with tissue plasminogen activator (tPA) remains a critical clinical problem. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are promising nanotherapeutic agents that have the potential to repair the BBB after ischemic stroke; however, whether they can attenuate BBB disruption and hemorrhagic transformation after tPA thrombolysis is largely unknown. Here, we observed that MSC-EVs efficiently passed through the BBB and selectively accumulated in injured brain regions in ischemic stroke model mice in real time using aggregation-induced emission luminogens (AIEgens), which exhibit better tracking ability than the commercially available tracer DiR. Moreover, tPA administration promoted the homing of MSC-EVs to the ischemic brain and increased the uptake of MSC-EVs by astrocytes. Furthermore, the accumulated MSC-EVs attenuated the tPA-induced disruption of BBB integrity and alleviated hemorrhage by inhibiting astrocyte activation and inflammation. Mechanistically, miR-125b-5p delivered by MSC-EVs played an indispensable role in maintaining BBB integrity by targeting Toll-like receptor 4 (TLR4) and inhibiting nuclear transcription factor-kappaB (NF-κB) signaling in astrocytes. This study provides a noninvasive method for real-time tracking of MSC-EVs in the ischemic brain after tPA treatment and highlights the potential of MSC-EVs as thrombolytic adjuvants for ischemic stroke. Statement of significanceAlthough tPA thrombolysis is the most effective pharmaceutical strategy for acute ischemic stroke, its clinical application and therapeutic efficacy are challenged by tPA-induced BBB disruption and hemorrhagic transformation. Our study demonstrated that MSC-EVs can act as an attractive thrombolytic adjuvant to repair the BBB and improve thrombolysis in a mouse ischemic stroke model. Notably, by labeling MSC-EVs with AIEgens, we achieved accurate real-time imaging of MSC-EVs in the ischemic brain and therapeutic visualization. MSC-EVs inhibit astrocyte activation and associated inflammation through miR-125b-5p/TLR4/NF-κB pathway. Consequently, we revealed that MSC-EVs combined with tPA thrombolysis may be a promising approach for the treatment of ischemic stroke in clinical setting.

Abstract 073: Age-Dependent Hypertension-Related Cognitive Impairment And Neuroinflammation In Sprague Dawley Rats Can Be Reversed By An AT 1 R Antagonism

Aim: We hypothesize paraventricular nucleus (PVN)-specific blood brain barrier (BBB) disruption and neuroinflammation increase hypertension and sympathoexcitation with age, promoting cognitive impairment, that can be attenuated by an Angiotensin II Type 1 Receptor (AT 1 R)-dependent mechanism in the aging Sprague Dawley (SD) rat. Methods: In male and female SD rats aged 3, 8 and 16 months old (MO) (N=6/gp) and 16 MO male rats treated with losartan (21 days; sc 3 mg/kg/day), blood pressure (BP; femoral artery cannulation), sympathetic tone to the vasculature (iv hexamethonium) and plasma NE (ELISA) was measured. Memory function was assessed by the novel object recognition task. BBB disruption was assessed via FITC extravasation and IHC/IF was performed for microglia (CD11b/c), astrocytes (GFAP), IL-6 and TNF-α in the PVN. Results: Aged male, but not female, SD rats develop HTN, sympathoexcitation, and cognitive impairment. PVN neuroinflammation, proinflammatory cytokine production and BBB disruption increased in male, but not female, rats with age. Losartan significantly lowered BP, reduced sympathoexcitation, attenuated BBB disruption and neuroinflammation, and reversed cognitive impairment in aged male rats. Conclusions: Our data suggest there is a potential protective role of female sex steroids in preventing the development of age-dependent HTN which is associated with PVN neuroinflammation. Concurrent with recent clinical findings, we found lowering blood pressure improved cognitive function and, thus, an AT 1 R antagonism represents a new therapeutic modality to improve cognitive performance in hypertensive individuals.

Selective sphingosine-1-phosphate receptor 1 modulator attenuates blood–brain barrier disruption following traumatic brain injury by inhibiting vesicular transcytosis

BackgroundTraumatic brain injury (TBI) provokes secondary pathological damage, such as damage to the blood–brain barrier (BBB), ischaemia and inflammation. Major facilitator superfamily domain-containing 2a (Mfsd2a) has been demonstrated to be critical in limiting the increase in BBB vesicle transcytosis following brain injury. Recent studies suggest that a novel and selective modulator of the sphingosine-1-phosphate receptor 1 (S1P1), CYM-5442, maintains the integrity of the BBB by restricting vesicle transcytosis during acute ischaemic stroke. In the current study, we investigated whether CYM-5442, evaluated in a short-term study, could protect the brains of mice with acute-stage TBI by reversing the increase in vesicle transport due to reduced Mfsd2a expression after TBI.MethodsWe used the well-characterized model of TBI caused by controlled cortical impact. CYM-5442 (0.3, 1, 3 mg/kg) was intraperitoneally injected 30 min after surgery for 7 consecutive days. To investigate the effect of CYM-5442 on vesicle transcytosis, we downregulated and upregulated Mfsd2a expression using a specific AAV prior to evaluation of the TBI model. MRI scanning, cerebral blood flow, circulating blood counts, ELISA, TEM, WB, and immunostaining evaluations were performed after brain injury.ResultsCYM-5442 significantly attenuated neurological deficits and reduced brain oedema in TBI mice. CYM-5442 transiently suppressed lymphocyte trafficking but did not induce persistent lymphocytopenia. After TBI, the levels of Mfsd2a were decreased significantly, while the levels of CAV-1 and albumin were increased. In addition, Mfsd2a deficiency caused inadequate sphingosine-1-phosphate (S1P) transport in the brain parenchyma, and the regulation of BBB permeability by Mfsd2a after TBI was shown to be related to changes in vesicle transcytosis. Downregulation of Mfsd2a in mice markedly increased the BBB permeability, neurological deficit scores, and brain water contents after TBI. Intervention with CYM-5442 after TBI protected the BBB by significantly reducing the vesicle transcytosis of cerebrovascular endothelial cells.ConclusionIn addition to transiently suppressing lymphocytes, CYM-5442 alleviated the neurological deficits, cerebral edema and protective BBB permeability in TBI mice by reducing the vesicle transcytosis of cerebrovascular endothelial cells.

Andrographolide Attenuates Blood-Brain Barrier Disruption, Neuronal Apoptosis, and Oxidative Stress Through Activation of Nrf2/HO-1 Signaling Pathway in Subarachnoid Hemorrhage.

Andrographolide (Andro), a diterpene of the labdane family extracted from the Asian plant Andrographis paniculata, is neuroprotective against stroke and Alzheimer's disease. However, whether Andro protected the brain against subarachnoid hemorrhage (SAH) was still unknown. Thus, we explored whether Andro attenuated blood-brain barrier (BBB) disruption and neuronal apoptosis and inhibited oxidative stress to protect the brain against SAH both in vitro and in vivo and detected underlying mechanisms of Andro's neuroprotective effects in the present study. Oxyhemoglobin (OxyHb)-treated neuronal PC12 cells were used as an in vitro model. An in vivo model was established using Sprague-Dawley rats. Moreover, we used an inhibitor of heme oxygenase-1 (HO-1) (ZnPPIX) in vitro and in vivo experiments to evaluate whether the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade acted as one protective molecular mechanism of Andro against SAH. Our results revealed that, in vitro, Andro increased cell viability, inhibited apoptosis, and activated Nrf2/HO-1 cascade of neuronal PC12 cells treated with OxyHb. In vivo, Andro attenuated the neurological dysfunction, neuronal apoptosis, BBB disruption, brain edema, and oxidative stress and activated the Nrf2/HO-1 pathway. ZnPPIX reversed the effects of Andro in vitro and in vivo. Our research suggested that Andro alleviated BBB disruption, neuronal apoptosis, and oxidative stress in SAH, possibly via the Nrf2/HO-1 signaling pathway.

Activation of the sigma-1 receptor attenuates blood–brain barrier disruption by inhibiting amyloid deposition in Alzheimer’s disease mice

The sigma-1 receptor is an important target for drug development in several neuropsychiatric diseases, including Alzheimer’s disease (AD). Accumulating evidence has shown that the integrity and functional activity of the blood–brain barrier (BBB) in AD are impaired, which is closely related to the movement of amyloid beta (Aβ) across the BBB and the formation of Aβ plaques. In this study, we investigated the effects of sigma-1 receptor activation on BBB disruption and Aβ levels in AD mice. We found that PRE-084, a sigma-1 receptor agonist, attenuated learning and memory deficits in Aβ–injected mice, significantly increased levels of low-density lipoprotein receptor-related protein 1 (LRP-1), and lowered the Aβ level synergistically in the brain. Moreover, the upregulation of vascular endothelial growth factor (VEGF) levels through the sigma-1 receptor may be involved in the reduction of the BBB permeability by PRE-084. The identification of this previously unexplored role of the sigma-1 receptor in alleviating BBB disruption via upregulating the levels of VEGF and LRP-1 in AD suggests that reversing BBB dysfunction through sigma-1 receptor activation may be a promising treatment for AD.

Transcranial focused ultrasound stimulation reduces vasogenic edema after middle cerebral artery occlusion in mice.

Blood-brain barrier (BBB) disruption underlies the vasogenic edema and neuronal cell death induced by acute ischemic stroke. Reducing this disruption has therapeutic potential. Transcranial focused ultrasound stimulation has shown neuromodulatory and neuroprotective effects in various brain diseases including ischemic stroke. Ultrasound stimulation can reduce inflammation and promote angiogenesis and neural circuit remodeling. However, its effect on the BBB in the acute phase of ischemic stroke is unknown. In this study of mice subjected to middle cerebral artery occlusion for 90 minutes, low-intensity low-frequency (0.5 MHz) transcranial focused ultrasound stimulation was applied 2, 4, and 8 hours after occlusion. Ultrasound stimulation reduced edema volume, improved neurobehavioral outcomes, improved BBB integrity (enhanced tight junction protein ZO-1 expression and reduced IgG leakage), and reduced secretion of the inflammatory factors tumor necrosis factor-α and activation of matrix metalloproteinase-9 in the ischemic brain. Our results show that low-intensity ultrasound stimulation attenuated BBB disruption and edema formation, which suggests it may have therapeutic use in ischemic brain disease as a protector of BBB integrity.