Blood-brain Barrier Disruption In Mice Research Articles

Pharmacological targeting of coagulation factor XI attenuates experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is the most common causes of non-traumatic disability in young adults worldwide. MS pathophysiologies include the formation of inflammatory lesions, axonal damage and demyelination, and blood brain barrier (BBB) disruption. Coagulation proteins, including factor (F)XII, can serve as important mediators of the adaptive immune response during neuroinflammation. Indeed, plasma FXII levels are increased during relapse in relapsing-remitting MS patients, and previous studies showed that reducing FXII levels was protective in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Our objective was to determine if pharmacological targeting of FXI, a major substrate of activated FXII (FXIIa), improves neurological function and attenuates CNS damage in the setting of EAE. EAE was induced in male mice using murine myelin oligodendrocyte glycoprotein peptides combined with heat-inactivated Mycobacterium tuberculosis and pertussis toxin. Upon onset of symptoms, mice were treated every other day intravenously with anti-FXI antibody, 14E11, or saline. Disease scores were recorded daily until euthanasia for ex vivo analyses of inflammation. Compared to the vehicle control, 14E11 treatment reduced the clinical severity of EAE and total mononuclear cells, including CD11b+CD45high macrophage/microglia and CD4+ T cell numbers in brain. Following pharmacological targeting of FXI, BBB disruption was reduced, as measured by decreased axonal damage and fibrin(ogen) accumulation in the spinal cord. These data demonstrate that pharmacological inhibition of FXI reduces disease severity, immune cell migration, axonal damage, and BBB disruption in mice with EAE. Thus, therapeutic agents targeting FXI and FXII may provide a useful approach for treating autoimmune and neurologic disorders.

Blocking C3d+/GFAP+ A1 Astrocyte Conversion with Semaglutide Attenuates Blood-Brain Barrier Disruption in Mice after Ischemic Stroke.

Astrocytes play an essential role in the modulation of blood-brain barrier function. Neurological diseases induce the transformation of astrocytes into a neurotoxic A1 phenotype, exacerbating brain injury. However, the effect of A1 astrocytes on the BBB dysfunction after stroke is unknown. Adult male ICR mice (n=97) were subjected to 90-minute transient middle cerebral artery occlusion (tMCAO). Immunohistochemical staining of A1 (C3d) and A2 (S100A10) was performed to characterize phenotypic changes in astrocytes over time after tMCAO. The glucagon-like peptide-1 receptor agonist semaglutide was intraperitoneally injected into mice to inhibit A1 astrocytes. Infarct volume, atrophy volume, neurobehavioral outcomes, and BBB permeability were evaluated. RNA-seq was adopted to explore the potential targets and signaling pathways of A1 astrocyte-induced BBB dysfunction. Astrocytic C3d expression was increased, while expression of S100A10 was decreased in the first two weeks after tMCAO, reflecting a shift in the astrocytic phenotype. Semaglutide treatment reduced the expression of CD16/32 in microglia and C3d in astrocytes after ischemic stroke (p<0.05). Ischemia-induced brain infarct volume, atrophy volume and neuroinflammation were reduced in the semaglutide-treated mice, and neurobehavioral outcomes were improved compared to control mice (p<0.05). We further demonstrated that semaglutide treatment reduced the gap formation of tight junction proteins ZO-1, claudin-5 and occludin, as well as IgG leakage three days following tMCAO (p<0.05). In vitro experiments revealed that A1 astrocyte-conditioned medium disrupted BBB integrity. RNA-seq showed that A1 astrocytes were enriched in inflammatory factors and chemokines and significantly modulated the TNF and chemokine signaling pathways, which are closely related to barrier damage. We concluded that astrocytes undergo a phenotypic shift over time after ischemic stroke. C3d+/GFAP+ astrocytes aggravate BBB disruption, suggesting that inhibiting C3d+/GFAP+ astrocyte formation represents a novel strategy for the treatment of ischemic stroke.

New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD.

Background and ObjectivesTo evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model.MethodsWe constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti–IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases.ResultsIn vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG–induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability.DiscussionThese results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.

Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.

Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1β and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.

Non-Invasive Low Pulsed Electrical Fields for Inducing BBB Disruption in Mice-Feasibility Demonstration.

The blood–brain barrier (BBB) is a major hurdle for the treatment of central nervous system disorders, limiting passage of both small and large therapeutic agents from the blood stream into the brain. Thus, means for inducing BBB disruption (BBBd) are urgently needed. Here, we studied the application of low pulsed electrical fields (PEFs) for inducing BBBd in mice. Mice were treated by low PEFs using electrodes pressed against both sides of the skull (100–400 square 50 µs pulses at 4 Hz with different voltages). BBBd as a function of treatment parameters was evaluated using MRI-based treatment response assessment maps (TRAMs) and Evans blue extravasation. A 3D numerical model of the mouse brain and electrodes was constructed using finite element software, simulating the electric fields distribution in the brain and ensuring no significant temperature elevation. BBBd was demonstrated immediately after treatment and significant linear regressions were found between treatment parameters and the extent of BBBd. The maximal induced electric field in the mice brains, calculated by the numerical model, ranged between 62.4 and 187.2 V/cm for the minimal and maximal applied voltages. These results demonstrate the feasibility of inducing significant BBBd using non-invasive low PEFs, well below the threshold for electroporation.

Transcriptomic response of brain tissue to focused ultrasound-mediated blood-brain barrier disruption depends strongly on anesthesia.

Focused ultrasound (FUS) mediated blood–brain barrier disruption (BBBD) targets the delivery of systemically‐administered therapeutics to the central nervous system. Preclinical investigations of BBBD have been performed on different anesthetic backgrounds; however, the influence of the choice of anesthetic on the molecular response to BBBD is unknown, despite its potential to critically affect interpretation of experimental therapeutic outcomes. Here, using bulk RNA sequencing, we comprehensively examined the transcriptomic response of both normal brain tissue and brain tissue exposed to FUS‐induced BBBD in mice anesthetized with either isoflurane with medical air (Iso) or ketamine/dexmedetomidine (KD). In normal murine brain tissue, Iso alone elicited minimal differential gene expression (DGE) and repressed pathways associated with neuronal signaling. KD alone, however, led to massive DGE and enrichment of pathways associated with protein synthesis. In brain tissue exposed to BBBD (1 MHz, 0.5 Hz pulse repetition frequency, 0.4 MPa peak‐negative pressure), we systematically evaluated the relative effects of anesthesia, microbubbles, and FUS on the transcriptome. Of particular interest, we observed that gene sets associated with sterile inflammatory responses and cell–cell junctional activity were induced by BBBD, regardless of the choice of anesthesia. Meanwhile, gene sets associated with metabolism, platelet activity, tissue repair, and signaling pathways, were differentially affected by BBBD, with a strong dependence on the anesthetic. We conclude that the underlying transcriptomic response to FUS‐mediated BBBD may be powerfully influenced by anesthesia. These findings raise considerations for the translation of FUS‐BBBD delivery approaches that impact, in particular, metabolism, tissue repair, and intracellular signaling.

Relationship Between Hematoma Expansion Induced by Hypertension and Hyperglycemia and Blood-brain Barrier Disruption in Mice and Its Possible Mechanism: Role of Aquaporin-4 and Connexin43.

We aimed to select an optimized hematoma expansion (HE) model and investigate the possible mechanism of blood-brain barrier (BBB) damage in mice. The results showed that HE occurred in the group with hypertension combined with hyperglycemia (HH-HE) from 3 to 72h after intracerebral hemorrhage; this was accompanied by neurological deficits and hardly influenced the survival rate. The receiver operating characteristic curve suggested the criterion for this model was hematoma volume expansion ≥ 45.0%. Meanwhile, HH-HE aggravated BBB disruption. A protector of the BBB reduced HH-HE, while a BBB disruptor induced a further HH-HE. Aquaporin-4 (AQP4) knock-out led to larger hematoma volume and more severe BBB disruption. Furthermore, hematoma volume and BBB disruption were reduced by multiple connexin43 (Cx43) inhibitors in the wild-type group but not in the AQP4 knock-out group. In conclusion, the optimized HE model is induced by hypertension and hyperglycemia with the criterion of hematoma volume expanding ≥ 45.0%. HH-HE leads to BBB disruption, which is dependent on AQP4 and Cx43.

TIMP1 preserves the blood–brain barrier through interacting with CD63/integrin β1 complex and regulating downstream FAK/RhoA signaling

Blood–brain barrier (BBB) breakdown and the associated microvascular hyperpermeability are hallmark features of several neurological disorders, including traumatic brain injury (TBI). However, there is no viable therapeutic strategy to rescue BBB function. Tissue inhibitor of metalloproteinase-1 (TIMP1) has been considered to be beneficial for vascular integrity, but the molecular mechanisms underlying the functions of TIMP1 remain elusive. Here, we report that TIMP1 executes a protective role on neuroprotective function via ameliorating BBB disruption in mice with experimental TBI. In human brain microvessel endothelial cells (HBMECs) exposed to hypoxia and inflammation injury, the recombinant TIMP1 (rTIMP1) treatment maintained integrity of junctional proteins and trans-endothelial tightness. Mechanistically, TIMP1 interacts with CD63/integrin β1 complex and activates downstream FAK signaling, leading to attenuation of RhoA activation and F-actin depolymerization for endothelial cells structure stabilization. Notably, these effects depend on CD63/integrin β1 complex, instead of the MMP-inhibitory function. Together, our results identified a novel MMP-independent function of TIMP1 in regulating endothelial barrier integrity. Therapeutic interventions targeting TIMP1 and its downstream signaling may be beneficial to protect BBB function following brain injury and neurological disorders.

Validation of Cadherin HAV6 Peptide in the Transient Modulation of the Blood-Brain Barrier for the Treatment of Brain Tumors.

The blood-brain barrier (BBB) poses a major obstacle by preventing potential therapeutic agents from reaching their intended brain targets at sufficient concentrations. While transient disruption of the BBB has been used to enhance chemotherapeutic efficacy in treating brain tumors, limitations in terms of magnitude and duration of BBB disruption exist. In the present study, the preliminary safety and efficacy profile of HAV6, a peptide that binds to the external domains of cadherin, to transiently open the BBB and improve the delivery of a therapeutic agent, was evaluated in a murine brain tumor model. Transient opening of the BBB in response to HAV6 peptide administration was quantitatively characterized using both a gadolinium magnetic resonance imaging (MRI) contrast agent and adenanthin (Ade), the intended therapeutic agent. The effects of HAV6 peptide on BBB integrity and the efficacy of concurrent administration of HAV6 peptide and the small molecule inhibitor, Ade, in the growth and progression of an orthotopic medulloblastoma mouse model using human D425 tumor cells was examined. Systemic administration of HAV6 peptide caused transient, reversible disruption of BBB in mice. Increases in BBB permeability produced by HAV6 were rapid in onset and observed in all regions of the brain examined. Concurrent administration of HAV6 peptide with Ade, a BBB impermeable inhibitor of Peroxiredoxin-1, caused reduced tumor growth and increased survival in mice bearing medulloblastoma. The rapid onset and transient nature of the BBB modulation produced with the HAV6 peptide along with its uniform disruption and biocompatibility is well-suited for CNS drug delivery applications, especially in the treatment of brain tumors.

Transcranial Photoacoustic Detection of Blood-Brain Barrier Disruption Following Focused Ultrasound-Mediated Nanoparticle Delivery.

Blood-brain barrier disruption (BBBD) is of interest for treating neurodegenerative diseases and tumors by enhancing drug delivery. Focused ultrasound (FUS) is a powerful method to alleviate BBB challenges; however, the detection of BBB opening by non-invasive methods remains limited. The purpose of this work is to demonstrate that 3D transcranial color Doppler (3DCD) and photoacoustic imaging (PAI) combined with custom-made nanoparticle (NP)-mediated FUS delivery can detect BBBD in mice. We use MRI and stereotactic ultrasound-mediated BBBD to create and confirm four openings in the left hemisphere and inject intravenously indocyanine green (ICG) and three sizes (40nm, 100nm, and 240nm in diameter) of fluorophore-labeled NPs. We use PAI and fluorescent imaging (FI) to assess the spatial distribution of ICG/NPs in tissues. A reversible 41 ± 12% (n = 8) decrease in diameter of the left posterior cerebral artery (PCA) relative to the right after FUS treatment is found using CD images. The spectral unmixing of photoacoustic images of the in vivo (2h post FUS), perfused, and ex vivo brain reveals a consistent distribution pattern of ICG and NPs at *FUS locations. Ex vivo spectrally unmixed photoacoustic images show that the opening width is, on average, 1.18 ± 0.12mm and spread laterally 0.49 ± 0.05mm which correlated well with the BBB opening locations on MR images. In vivo PAI confirms a deposit of NPs in tissues for hours and potentially days, is less sensitive to NPs of lower absorbance at a depth greater than 3mm and too noisy with NPs above an absorbance of 85.4. FI correlates well with ex vivo PAI to a depth of 3mm in tissues for small NPs and 4.74mm for large NPs. 3DCD can monitor BBBD over time by detecting reversible anatomical changes in the PCA. In vivo 3DPAI at 15MHz combined with circulating ICG and/or NPs with suitable properties can assess BBB opening 2h post FUS.

Early Blood-Brain Barrier Disruption in Ischemic Stroke Initiates Multifocally Around Capillaries/Venules.

Detection and localization of the early phase of blood-brain barrier disruption (BBBD) in vivo during cerebral ischemia/reperfusion injury remain a major challenge but may be a relevant outcome parameter in stroke. We studied early BBBD in mice after transient middle cerebral artery occlusion by multimodal, high-field (9.4T) in vivo magnetic resonance imaging, including the contrast agent gadofluorineM as an albumin-binding tracer. GadofluorineM contrast-enhanced magnetic resonance imaging was performed to determine BBBD at 2, 6, and 24 hours after reperfusion. BBBD was confirmed and localized along the microvascular tree by using fluorescent gadofluorineM and immunofluorescence stainings (cluster of differentiation 31, ephrin type-B receptor 4, alpha smooth muscle actin, ionized calcium binding adaptor molecule 1). GadofluorineM contrast-enhanced magnetic resonance imaging revealed a multifocal spatial distribution of early BBBD and its close association with the microvasculature at a resolution of 40 μm. GadofluorineM leakage was closely associated with ephrin type-B receptor 4-positive but not alpha smooth muscle actin-positive vessels. The multifocal pattern of early BBBD (already at 2 hours after reperfusion) thus occurred in the distal capillary and venular microvascular bed. These multifocal zones showed distinct imaging signs indicative of early vasogenic edema. The total volume of multifocal early BBBD accurately predicted infarct size at 24 hours after reperfusion. Early BBBD in focal cerebral ischemia initiates multifocally in the distal capillary and venular bed of the cerebral microvasculature. It is closely associated with perimicrovascular vasogenic edema and microglial activation and predicts the extent of final infarction.

Inhibition of 2-AG hydrolysis differentially regulates blood brain barrier permeability after injury

BackgroundAcute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential.MethodsInduction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2.ResultsHere, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult.ConclusionsOur results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.

Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice

BackgroundIschemic stroke induced matrixmetallo-proteinase-9 (MMP-9) upregulation, which increased blood-brain barrier permeability. Studies demonstrated that mesenchymal stem cell therapy protected blood-brain barrier disruption from several cerebrovascular diseases. However, the underlying mechanism was largely unknown. We therefore hypothesized that mesenchymal stem cells reduced blood-brain barrier destruction by inhibiting matrixmetallo-proteinase-9 and it was related to intercellular adhesion molecule-1 (ICAM-1).MethodsAdult ICR male mice (n = 118) underwent 90-min middle cerebral artery occlusion and received 2 × 105 mesenchymal stem cell transplantation. Neurobehavioral outcome, infarct volume, and blood-brain barrier permeability were measured after ischemia. The relationship between myeloperoxidase (MPO) activity and ICAM-1 release was further determined.ResultsWe found that intracranial injection of mesenchymal stem cells reduced infarct volume and improved behavioral function in experimental stroke models (p < 0.05). IgG leakage, tight junction protein loss, and inflammatory cytokines IL-1β, IL-6, and TNF-α reduced in mesenchymal stem cell-treated mice compared to the control group following ischemia (p < 0.05). After transplantation, MMP-9 was decreased in protein and activity levels as compared with controls (p < 0.05). Furthermore, myeloperoxidase-positive cells and myeloperoxidase activity were decreased in mesenchymal stem cell-treated mice (p < 0.05).ConclusionThe results showed that mesenchymal stem cell therapy attenuated blood-brain barrier disruption in mice after ischemia. Mesenchymal stem cells attenuated the upward trend of MMP-9 and potentially via downregulating ICAM-1 in endothelial cells. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) pathway may influence MMP-9 expression of neutrophils and resident cells, and ICAM-1 acted as a key factor in the paracrine actions of mesenchymal stem cell.

Therapeutic time window for conivaptan treatment against stroke-evoked brain edema and blood-brain barrier disruption in mice.

BackgroundIschemic stroke is often complicated by brain edema, disruption of blood-brain barrier (BBB), and uncontrolled release of arginine-vasopressin (AVP). Conivaptan, a V1a and V2 receptor antagonist, reduces brain edema and minimizes damage to the blood-brain barrier after stroke. Most stroke patients do not receive treatment immediately after the onset of brain ischemia. Delays in therapy initiation may worsen stroke outcomes. Therefore, we designed a translational study to explore the therapeutic time window for conivaptan administration.MethodsMice were treated with conivaptan beginning 3, 5, or 20 hours after 60-minute focal middle cerebral artery occlusion. Treatments were administered by continuous IV infusion for a total of 48 hours. Brain edema and blood-brain barrier (BBB) disruption were evaluated at endpoint.ResultsConivaptan therapy initiated at 3 hours following ischemia reduced edema in the ipsilateral hemisphere, which corresponded with improvements in neurological deficits. Stroke-triggered BBB disruption was also reduced in mice when conivaptan treatments were initiated at 3 hours of reperfusion. However, 5 and 20-hour delays of conivaptan administration failed to reduce edema or protect BBB.ConclusionTiming of conivaptan administration is important for successful reduction of brain edema and BBB disruption. Our experimental data open new possibilities to repurpose conivaptan, and make an important “bench-to-bedside translation” of the results into clinical practice.

Sesamin alleviates blood-brain barrier disruption in mice with experimental traumatic brain injury.

Sesamin, a major lignan of sesame oil, was reported to have neuroprotective effects in several brain injury models. However, its protective action in maintaining blood-brain barrier (BBB) integrity has not been studied. In this study we investigated the effects of sesamin on the BBB in a mouse model of traumatic brain injury (TBI) and explored the underlying mechanisms. Adult male C57BL/6 mice were subjected to a controlled cortical impact (CCI) injury and then received sesamin (30 mg·kg-1·d-1, ip). The mice were euthanized on the 1st and 3rd days after CCI injury and samples were collected for analysis. Sesamin treatment significantly attenuated CCI-induced brain edema on the 1st and 3rd days after the injury, evidenced by the decreases in water content, tissue hemoglobin levels, Evans blue extravasation and AQP4 expression levels in the ipsilateral cortical tissue compared with the vehicle-treated group. Furthermore, sesamin treatment significantly alleviated CCI-induced loss of the tight junction proteins ZO-1 and occludin in the brain tissues. The neuroprotective mechanisms of sesamin were further explored in cultured mouse brain microvascular bEnd.3 cells subjected to biaxial stretch injury (SI). Pretreatment with sesamin (50 μmol/L) significantly alleviated SI-induced loss of ZO-1 in bEnd.3 cells. Furthermore, we revealed that pretreatment with sesamin significantly attenuated SI-induced oxidative stress and early-stage apoptosis in bEnd.3 cells by decreasing the activation of ERK, p-38 and caspase-3. In conclusion, sesamin alleviates BBB disruption at least partly through its anti-oxidative and anti-apoptotic effects on endothelial cells in CCI injury. These findings suggest that sesamin may be a promising potential therapeutic intervention for preventing disruption of the BBB after TBI.

Curcumin attenuates blood-brain barrier disruption after subarachnoid hemorrhage in mice

BackgroundEarly brain injury, one of the most important mechanisms underlying subarachnoid hemorrhage (SAH), comprises edema formation and blood-brain barrier (BBB) disruption. Curcumin, an active extract from the rhizomes of Curcuma longa, alleviates neuroinflammation by as yet unknown neuroprotective mechanisms. In this study, we examined whether curcumin treatment ameliorates SAH-induced brain edema and BBB permeability changes, as well as the mechanisms underlying this phenomenon. MethodsWe induced SAH in mice via endovascular perforation, administered curcumin 15 min after surgery and evaluated neurologic scores, brain water content, Evans blue extravasation, Western blot assay results, and immunohistochemical analysis results 24 h after surgery. ResultsCurcumin significantly improved neurologic scores and reduced brain water content in treated mice compared with SAH mice. Furthermore, curcumin decreased Evans blue extravasation, matrix metallopeptidase-9 expression, and the number of Iba-1-positive microglia in treated mice compared with SAH mice. At last, curcumin treatment increased the expression of the tight junction proteins zonula occludens-1 and occludin in treated mice compared with vehicle-treated and sample SAH mice. ConclusionsWe demonstrated that curcumin inhibits microglial activation and matrix metallopeptidase-9 expression, thereby reducing brain edema and attenuating post-SAH BBB disruption in mice.

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice.

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.

BackgroundDisruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.MethodsWe used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using 14C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to 14C-sucrose and radioactive albumin.ResultsIn comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with 14C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and 14C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.ConclusionsThe BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

Arginine-Vasopressin Receptor Blocker Conivaptan Reduces Brain Edema and Blood-Brain Barrier Disruption after Experimental Stroke in Mice.

BackgroundStroke is a major cause of morbidity and mortality. Stroke is complicated by brain edema and blood-brain barrier (BBB) disruption, and is often accompanied by increased release of arginine-vasopressin (AVP). AVP acts through V1a and V2 receptors to trigger hyponatremia, vasospasm, and platelet aggregation which can exacerbate brain edema. The AVP receptor blockers conivaptan (V1a and V2) and tolvaptan (V2) are used to correct hyponatremia, but their effect on post-ischemic brain edema and BBB disruption remains to be elucidated. Therefore, we conducted this study to investigate if these drugs can prevent brain edema and BBB disruption in mice after stroke.MethodsExperimental mice underwent the filament model of middle cerebral artery occlusion (MCAO) with reperfusion. Mice were treated with conivaptan, tolvaptan, or vehicle. Treatments were initiated immediately at reperfusion and administered IV (conivaptan) or orally (tolvaptan) for 48 hours. Physiological variables, neurological deficit scores (NDS), plasma and urine sodium and osmolality were recorded. Brain water content (BWC) and Evans Blue (EB) extravasation index were evaluated at the end point.ResultsBoth conivaptan and tolvaptan produced aquaresis as indicated by changes in plasma and urine sodium levels. However plasma and urine osmolality was changed only by conivaptan. Unlike tolvaptan, conivaptan improved NDS and reduced BWC in the ipsilateral hemisphere: from 81.66 ± 0.43% (vehicle) to 78.28 ± 0.48% (conivaptan, 0.2 mg, p < 0.05 vs vehicle). Conivaptan also attenuated the EB extravasation from 1.22 ± 0.08 (vehicle) to 1.01 ± 0.02 (conivaptan, 0.2 mg, p < 0.05).ConclusionContinuous IV infusion with conivaptan for 48 hours after experimental stroke reduces brain edema, and BBB disruption. Conivaptan but not tolvaptan may potentially be used in patients to prevent brain edema after stroke.

Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion.

BackgroundMetformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.MethodsNinety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.ResultsInfarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.ConclusionsMetformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0177-4) contains supplementary material, which is available to authorized users.