Tight Junction Degradation Research Articles

Sanguinarine-based isoquinoline alkaloids modulated the gut-brain axis and enhanced growth performance and gut integrity in natural heat stress broiler chickens

Persistent hot and humid tropical climate induces heat stress (HS), negatively impacting gut integrity with inflammation and tight junction degradation in broilers. As integrating operations of the digestive tract and hypothalamus in avian are regulated by feed intake, the effect of HS may be eased by feed supplementation. Herein, the effect of isoquinoline alkaloids (IQ) supplementation from Macleaya cordata on integrating gut-brain axis and gut integrity was assessed. 720 male broilers (Ross 308) were treated with IQ at null (control), 60 (IQ60), and 100 mg/kg (IQ100). They were reared in open production house between April and May in Southern Thailand with 33.0–38.0°C (average 35.0°C), 75–80% relative humidity (RH), and 160–200 heat stress index (HI). From day 1 to 42, IQ supplementation positively affected growth performance where body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly higher in birds fed IQs than in the control group (P<0.05), and feed conversion ratio (FCR) was significantly lower in the IQ group (P<0.05). IQ improved gut integrity by reducing inflammation with significant down-regulation of nitric oxide synthase (iNOS), cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), and nuclear factor κB (NF-κB) in the ileum (P<0.05). It strengthened tight junction by upregulates occludin (OCLD), claudin-1 (CLDN1), tight junction proteins-1 (TJP-1) and mucin-2 (MUC-2) in the jejunum (P<0.05). As a result, serum fluorescein isothiocyanate-dextran (FITC-d) and corticosterone levels were significantly lower (P<0.05). In terms of the gut-brain axis, IQ supplementation reduces ghrelin and cholecystokinin (CCK) mRNA expression in the jejunum and hypothalamus (P<0.05), which downregulated the expression of pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotropin-releasing hormone (CRH), and melanocortin four receptor (MC4R). It was the first evidence that IQ influences on modulating the gut-brain axis to suppress anorexigenic regulation in broilers with HS exposure.

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.

Abstract P3002: Endothelial Trpm2 Deletion Attenuates Ischemic Stroke By Preventing Blood-brain Barrier Disruption

Introduction: Endothelial hyperpermeability is a hallmark of brain damage at the early stage of ischemic stroke. The subsequent blood-brain barrier (BBB) disruption drives the initial pathological changes and aggregates neuronal death. Ca 2+ overload is an important cause of endothelial dysfunction. TRPM2 is a Ca 2+ -permeable ion channel activated by oxidative stress, and oxidative stress is a critical contributor for endothelial hyperpermeability during ischemic stroke. Hypothesis: We hypothesized that TRPM2-mediated Ca 2+ influx plays a key role in causing endothelial hyperpermeability during ischemic stroke. Methods: Specific deletion of Trpm2 in endothelial cells was achieved by crossing Cdh5-cre mice with Trpm2 fl/fl mice. Middle cerebral artery occlusion (MCAO) was performed to simulate ischemic stroke. Tetrazolium chloride and Evans blue assays were used to evaluate infarct size and BBB leakage. Occludin and F4-80 / myeloperoxidase staining were used to detect tight junction degradation and immune cell invasion. Cerebral endothelial cells (CECs) were isolated and cultured. Oxygen-glucose deprivation (OGD) was used to simulate in vitro ischemic conditions. In vitro endothelial leakage assays were used to examine CEC permeability. Rhodamine-123 imaging and Ca 2+ imaging were used to evaluate oxidative stress and Ca 2+ overload in CECs. WB was used to examine CD36 signaling activation and whole-cell current recording was used to detect TRPM2 activation. Results: Specific deletion of Trpm2 in endothelial cells protects mice against MCAO-induced brain injury, which is characterized by reduced infarct size, mitigated plasma extravasation, reduced neutrophil / macrophage invasion and inhibited oxidative stress in the brain. In vitro experiments demonstrated that Trpm2 deletion alleviates ROS production, Ca 2+ overload and endothelial hyperpermeability induced by OGD and CD36 ligand thrombospondin-1 (TSP1). In CECs, activation of CD36 signaling induced by OGD and TSP1 was impaired by TRPM2 knockout. In transfected HEK293T cells, OGD and TSP1 induce the activation of TRPM2 currents in a CD36-dependent manner. Conclusion: Endothelial specific TRPM2 knockout attenuates ischemic stroke by preserving BBB integrity.

Astrocytic p75NTR expression provoked by ischemic stroke exacerbates the blood-brain barrier disruption.

The disruption of the blood-brain barrier (BBB) plays a critical role in the pathology of ischemic stroke. p75 neurotrophin receptor (p75NTR ) contributes to the disruption of the blood-retinal barrier in retinal ischemia. However, whether p75NTR influences the BBB permeability after acute cerebral ischemia remains unknown. The present study investigated the role and underlying mechanism of p75NTR on BBB integrity in an ischemic stroke mouse model, middle cerebral artery occlusion (MCAO). After 24 h of MCAO, astrocytes and endothelial cells in the infarct-affected brain area up-regulated p75NTR . Genetic p75NTR knockdown (p75NTR+/- ) or pharmacological inhibition of p75NTR using LM11A-31, a selective inhibitor of p75NTR , both attenuated brain damage and BBB leakage in MCAO mice. Astrocyte-specific conditional knockdown of p75NTR mediated with an adeno-associated virus significantly ameliorated BBB disruption and brain tissue damage, as well as the neurological functions after stroke. Further molecular biological examinations indicated that astrocytic p75NTR activated NF-κB and HIF-1α signals, which upregulated the expression of MMP-9 and vascular endothelial growth factor (VEGF), subsequently leading to tight junction degradation after ischemia. As a result, increased leukocyte infiltration and microglia activation exacerbated brain injury after stroke. Overall, our results provide novel insight into the role of astrocytic p75NTR in BBB disruption after acute cerebral ischemia. The p75NTR may therefore be a potential therapeutic target for the treatment of ischemic stroke.

Rotenone impairs learning and memory in mice through microglia-mediated blood brain barrier disruption and neuronal apoptosis

Rotenone is a neurotoxic pesticide widely used in agriculture. Dopaminergic neuron has long been considered as the target of rotenone. We recently reported that rotenone exposure also resulted in hippocampal and cortical neurodegeneration and cognitive dysfunction in mice. However, the mechanisms remain unknown. Here, we elucidated whether blood brain barrier (BBB) disruption and subsequent neuronal apoptosis mediated by microglial activation were involved in rotenone-elicited cognitive impairments. Results showed that rotenone dose-dependently elevated evens blue extravasation, fibrinogen accumulation and reduced expressions of tight junction proteins in the hippocampus and cortex of mice. Interestingly, microglial depletion and inactivation by PLX3397 and minocycline, respectively, markedly attenuated rotenone-elicited increase of BBB permeability, indicating a critical role of microglia. Furthermore, microglial depletion and inactivation were shown to abrogate rotenone-induced activation of matrix metalloproteinases 2 and 9 (MMP-2/-9), two important factors to regulate tight junction degradation and BBB permeability, in mice. Moreover, SB-3CT, a widely used MMP-2/-9 inhibitor, increased BBB integrity and simultaneously elevated expressions of tight junction proteins in rotenone-intoxicated mice. Finally, we found that SB-3CT significantly mitigated rotenone-induced neuronal apoptosis and synaptic loss as well as learning and memory impairments in mice. Altogether, this study revealed that rotenone elicited cognitive impairments in mice through microglia-mediated BBB disruption and neuronal apoptosis via MMP-2/-9, providing a novel aspect for the pathogenesis of pesticide-induced neurotoxicity and Parkinson's disease (PD)-related dementia.

Brain endothelial LRP1 maintains blood\u2013brain barrier integrity

The entry of blood-borne molecules into the brain is restricted by the blood–brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor–related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreERT2 specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity.

TIM2 modulates retinal iron levels and is involved in blood-retinal barrier breakdown

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.

Safe Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Repair is Not Mediated by Tight Junction Degradation

Focused ultrasound (FUS) with microbubbles opens the blood-brain barrier (BBB) to allow targeted drug delivery into the brain; however, the mechanisms of BBB opening and the response of the neurovascular unit at either low acoustic pressures, known to open the BBB transiently, or high pressures that cause brain damage, remain unclear. Employing a transgenic mouse strain where tight junctions (TJs) are labelled with eGFP, we examined TJ strand morphology at 1 and 72 hours post-FUS at both low and high pressures. We find that transient BBB opening due to FUS is not caused by TJ strand damage, unless FUS is used at high pressures, and this is localized primarily in arterioles and capillaries. At high pressures, TJ strands are obliterated and remain unrepaired even at 72 hours, allowing for fibrinogen passage, and persistent microglial activation. Our findings suggest that at low safe pressures, upregulation of transcytosis may likely increase transiently BBB permeability to deliver therapeutics to the brain.

Cycloastragenol upregulates SIRT1 expression, attenuates apoptosis and suppresses neuroinflammation after brain ischemia.

Cycloastragenol (CAG) is the active form of astragaloside IV isolated from Astragalus Radix, which displays multiple pharmacological effects. Silent information regulator 1 (SIRT1), a class III histone deacetylase, has been shown to play an important role in neuroprotection against cerebral ischemia. In this study, we investigated whether CAG protected against ischemic brain injury and, if so, whether the beneficial effects were associated with the regulation of SIRT1 in the ischemic brain. Mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. CAG (5, 10, 20 mg/kg) was injected intraperitoneally at the onset of reperfusion, 12 h later and then twice daily for up to three days. CAG dose-dependently reduced brain infarct volume, significantly ameliorated functional deficits, and prevented neuronal cell loss in MCAO mice. Meanwhile, CAG significantly reduced matrix metalloproteinase-9 activity, prevented tight junction degradation and subsequently ameliorated blood-brain barrier disruption. Moreover, CAG significantly upregulated SIRT1 expression in the ischemic brain but did not directly activate its enzymatic activity. Concomitant with SIRT1 upregulation, CAG reduced p53 acetylation and the ratio of Bax to Bcl-2 in the ischemic brain. CAG also inhibited NF-κB p65 nuclear translocation. As a result, CAG suppressed the mRNA expression of pro-inflammatory cytokines, including TNF-α and IL-1β, and inhibited the activation of microglia and astrocytes in the ischemic brain. Our findings suggest that CAG is neuroprotective against ischemic brain injury in mice and that its beneficial effect may involve SIRT1 upregulation and the inhibition of apoptosis and neuroinflammation in the ischemic brain.

Protease-Activated Receptors 2-Antagonist Suppresses Asthma by Inhibiting Reactive Oxygen Species-Thymic Stromal Lymphopoietin Inflammation and Epithelial Tight Junction Degradation.

PurposeProtease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2.MethodsHuman lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies.ResultsIn A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC.ConclusionsROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.

Endothelial colony-forming cell-derived exosomes restore blood-brain barrier continuity in mice subjected to traumatic brain injury

Traumatic brain injury (TBI) tends to cause disruption of the blood-brain barrier (BBB). Previous studies have shown that intravenously or intracerebroventricularly infused human umbilical cord blood-derived endothelial colony-forming cells (ECFCs) can home to injury sites and improve outcomes in mice subjected to experimental TBI. Several reports have demonstrated that these cells did not incorporate directly into newly formed vasculature but instead stimulated the proliferation and migration of tissue-resident endothelial cells (ECs) via paracrine mechanisms. In the present study, exosomes, which range from 30 to 150 nm in diameter, were isolated from ECFC-conditioned medium. The exosomes were labeled with PKH67 ex vivo, and we observed that they were taken up by ECs with high efficiency after 12 h of incubation. Pretreatment with ECFC-derived exosomes promoted the migration of ECs subjected to scratch injury, and incubating ECs exposed to hypoxia with ECFC-derived exosomes decreased PTEN expression, stimulated AKT phosphorylation and increased tight junction (TJ) protein expression in the cells. Furthermore, in vivo delivery of ECFC-derived exosomes into TBI mice also inhibited PTEN expression and increased AKT expression, changes accompanied by reductions in Evans blue (EB) dye extravasation, brain edema and TJ degradation. These data demonstrated that ECFC-derived exosomes have beneficial effects on BBB integrity in mice with TBI.

German Cockroach Extract Induces Matrix Metalloproteinase-1 Expression, Leading to Tight Junction Disruption in Human Airway Epithelial Cells.

PurposeCockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292).Materials and MethodsmRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting.ResultsGCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration.ConclusionGCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.

Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation

Lymphocytes cross vascular boundaries via either disrupted tight junctions (TJs) or caveolae to induce tissue inflammation. In the CNS, Th17 lymphocytes cross the blood-brain barrier (BBB) before Th1 cells; yet this differential crossing is poorly understood. We have used intravital two-photon imaging of the spinal cord in wild-type and caveolae-deficient mice with fluorescently labeled endothelial tight junctions to determine how tight junction remodeling and caveolae regulate CNS entry of lymphocytes during the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. We find that dynamic tight junction remodeling occurs early in EAE but does not depend upon caveolar transport. Moreover, Th1, but not Th17, lymphocytes are significantly reduced in the inflamed CNS of mice lacking caveolae. Therefore, tight junction remodeling facilitates Th17 migration across the BBB, whereas caveolae promote Th1 entry into the CNS. Moreover, therapies that target both tight junction degradation and caveolar transcytosis may limit lymphocyte infiltration during inflammation.

Selective inhibition of brain endothelial Rho-kinase-2 provides optimal protection of an in vitro blood-brain barrier from tissue-type plasminogen activator and plasmin.

Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an in vitro human model. ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.

Overexpression of caveolin-1 attenuates brain edema by inhibiting tight junction degradation.

Cerebral edema from the disruption of the blood-brain barrier (BBB) after cerebral ischemia is a major cause of morbidity and mortality as well as a common event in patients with stroke. Caveolins (Cavs) are thought to regulate BBB functions. Here, we report for the first time that Cav-1 overexpression (OE) decreased brain edema from BBB disruption following ischemic insult. Edema volumes and Cav-1 expression levels were measured following photothrombosis and middle cerebral artery occlusion (MCAO). Endothelial cells that were transduced with a Cav-1 lentiviral expression vector were transplanted into rats. BBB permeability was quantified with Evans blue extravasation. Edema volume was determined from measures of the extravasation area, brain water content, and average fluorescence intensity after Cy5.5 injections. Tight junction (TJ) protein expression was measured with immunoblotting. Cav-1 expression levels and vasogenic brain edema correlated strongly after ischemic insult. Cav-1 expression and BBB disruption peaked 3 d after the MCAO. In addition, intravenous administration of endothelial cells expressing Cav-1 effectively increased the Cav-1 levels 3 d after the MCAO ischemic insult. Importantly, Cav-1 OE ameliorated the vasogenic edema by inhibiting the degradation of TJ protein expression in the acute phase of ischemic stroke. These results suggested that Cav-1 OE protected the integrity of the BBB mainly by preventing the degradation of TJ proteins in rats. These findings need to be confirmed in a clinical setting in human subjects.

Equine Tight Junctions: Tissue-Specific Localization and Expression of Junction Adhesion Molecule-A, Zona Occludens-1, and Occludin

Tight junctions regulate physiological homeostasis by controlling paracellular transportation and forming physical barriers. Equus ferus caballus, the domestic horse, is an economically important animal and studies on equine cell-to-cell junctions are insufficient. In fact, common equine diseases such as colic, skin carcinoma, and West Nile disease are related to tight junction degradation. In this study, expression and distribution of tight junction proteins including junction adhesion molecule-A, zona occludens-1, and occludin were examined to understand the physiological characteristics of equine tight junctions. Protein expression of these three factors was quantified by Western blotting using proteins extracted from liver, kidney, lung, duodenum, heart, testis, ovary, and uterus. Localization of the tight junction proteins was observed in those tissues via immunohistochemistry. Expression of the proteins was primarily detected in the epithelium and endothelium. Expression pattern in equine tissues for the three tight junction proteins tested is different from that reported in mice or humans. Results from this study can help elucidate the organization of tight junctions in equine tissues as well as mechanisms underlying tight junction-related disease in horse.

Partially purified components of Uncaria sinensis attenuate blood brain barrier disruption after ischemic brain injury in mice.

BackgroundUncaria sinensis (US) has long been used in traditional Korean medicine to relieve various nervous-related symptoms and cardiovascular disease. We recently showed the neuroprotective and cerebrovascular protective effects of US on cerebral ischemia; however, its effects on the blood–brain barrier (BBB) are poorly understood. In this study, the effects of partially purified components of US (PPUS) on BBB disruption were investigated in mice subjected to ischemic brain injury.MethodsFocal cerebral ischemia was induced in C57BL/6J mice by photothrombotic cortical ischemia. PPUS was injected intraperitoneally 30 min before ischemic insults. Infarct volume, neurological score, wire-grip test, Evans blue leakage and brain water content were then examined 24 h after ischemic brain injury.ResultsInfarct volume was significantly reduced and neurological deficit and motor deficit were greatly improved in PPUS-pretreated mice relative to those treated with vehicle following photothrombotic cortical ischemia. Brain edema-induced change of Evans blue extravasation and water content in the ipsilateral hemisphere were alleviated by treatment with PPUS. In addition, PPUS significantly reduced ischemic brain injury-induced degradation of tight junction proteins and elevation of matrix metalloproteinase-9 (MMP-9).ConclusionsPPUS prevents cerebral ischemic damage by BBB protection, and these effects were associated with inhibition of tight junction degradation and MMP-9 induction.

Abstract T P250: Role of Autophagy in Blood-Brain Barrier Disruption and Tight Junction Degradation Under Ischemic Condition

During ischemic stroke, the integrity of blood-brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged amplifying ischemic neuronal damage. There have been attempts to identify the exact mechanism ischemic BBB disruption to minimize brain damage under ischemic stroke. Autophagy is catabolic process which involves degradation and recycling of damaged or unnecessary organelles. However, excessive autophagy can induce cell damage and death under pathological conditions such as ischemia. In this study, we evaluated if autophagy is a key mechanism of BBB dysfunction under ischemic stroke. In vitro BBB model of bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), an ischemic mimic condition. After exposure to OGD for 18 hours, cell viability was significantly decreased and cellular permeability was impaired. The conversion of LC3-I to LC3-II and puncta of LC3 in bEnd.3 were increased, demonstrating that autophagy is induced under ischemic condition. Modulation of autophagy by 3-methyladenine, an autophagy inhibitor, reversed the conversion of LC3 as well as decreased cell viability, suggesting that autophagy involves in ischemic BBB damage. The level of occludin, a tight junction protein in BBB, was decreased after OGD, and this was reversed by inhibition of autophagy. Our findings showed that induction of autophagy might contribute to increased permeability through occludin degradation in brain endothelial cells under ischemia, providing a new mechanism of BBB disruption in ischemic stroke.

Valproic Acid Attenuates Blood–Brain Barrier Disruption in a Rat Model of Transient Focal Cerebral Ischemia: The Roles of HDAC and MMP-9 Inhibition

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is known to protect against cerebral ischemia. The effects of VPA on blood-brain barrier (BBB) disruption were investigated in rats subjected to transient middle cerebral artery occlusion (MCAO). Postischemic VPA treatment remarkably attenuated MCAO-induced BBB disruption and brain edema. Meanwhile, VPA significantly reduced MCAO-induced elevation of matrix metalloproteinase-9 (MMP-9), degradation of tight junction proteins, and nuclear translocation of nuclear factor-κB (NF-κB). Sodium butyrate, another HDAC inhibitor, mimicked these effects of VPA. Our findings suggest that BBB protection by VPA involves HDAC inhibition-mediated suppression of NF-κB activation, MMP-9 induction, and tight junction degradation.

Degradation of tight junctions in HT29, a human colon adenocarcinoma cell line

The process of tight junction degradation was followed in a cell line of human colon adenocarcinoma. Tight junctions are degraded by 2 mechanisms: (1) breakdown of junctional elements to intramembrane particles; (2) bleb formation by which tight-junctional elements are internalized into the cytoplasm or excluded into the medium. It is suggested that the first mechanism allows preservation of membrane particles for re-use, whereas the second is a mechanism by which the cells eliminate unneeded junctional elements.