Electromagnetic fields (GSM 1800) do not alter blood-brain barrier permeability to sucrose in models in vitro with high barrier tightness

We previously reported that electromagnetic fields (EMFs) [GSM 1800 standard (Global System for Mobile Communications, 1800 MHz)] increased sucrose permeation across the blood-brain barrier (BBB) in vitro. The cell culture model used in our previous study was comprised of rat astrocytes in coculture with porcine brain microvascular endothelial cells (PBECs). In this study, after optimization of cell culture conditions, distinctly improved barrier tightness was observed, accompanied by a loss of susceptibility to EMF-related effects on BBB permeability. Cell cultures were exposed for 1-5 days at an average specific absorption rate (SAR) of 0.3 W/kg in the identical exposure system as described before. To quantify barrier tightness, sucrose permeation across exposed PBEC was measured and compared to values of sham exposed cells and to a control group. Additionally, observations in the BBB coculture system were complemented by similar experiments using two other in vitro models, composed of PBEC monocultures with or without serum. These three models display distinctly higher barrier tightness than the previously used system. In all three BBB models, sucrose permeation across the cell layers remained unaffected by exposure to a GSM 1800 field for up to 5 days. We thus could not confirm enhanced permeability of the BBB in vitro after EMF exposure as reported before since the in vitro barrier tightness in these experiments is now more like that of the in vivo situation.     

Effects of Universal Mobile Telecommunications System (UMTS) electromagnetic fields on the blood-brain barrier in vitro

The extensive use of mobile phone communication has raised public concerns about adverse health effects of radiofrequency (RF) electromagnetic fields (EMFs) in recent years. A central issue in this discussion is the question whether EMFs enhance the permeability of the blood-brain barrier (BBB). Here we report an investigation on the influence of a generic UMTS (Universal Mobile Telecommunications System) signal on barrier tightness, transport processes and the morphology of porcine brain microvascular endothelial cell cultures (PBEC) serving as an in vitro model of the BBB. An exposure device with integrated online monitoring system was developed for simultaneous exposure and measuring of transendothelial electrical resistance (TEER) to determine the tightness of the BBB. PBEC were exposed continuously for up to 84 h at an average electric-field strength of 3.4-34 V/m (maximum 1.8 W/kg) ensuring athermal conditions. We did not find any evidence of RF-field-induced disturbance of the function of the BBB. After and during exposure, the tightness of the BBB quantified by 14C-sucrose and serum albumin permeation as well as by TEER remained unchanged compared to sham-exposed cultures. Permeation of transporter substrates at the BBB as well as the localization and integrity of the tight-junction proteins occludin and ZO1 were not affected either.     

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood–brain barrier

α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo .     

Permeability properties of a three-cell type in vitro model of blood-brain barrier

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood-brain barrier (BBB) properties if co-cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of 3H-sucrose able to cross the BCEC layer in this three-cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decreases even more. By Western analysis, we also found that, in addition to the canonical 60 kDa occludin, anti-occludin antibodies recognize a smaller protein of 48 kDa which accumulates during rat brain development. Interestingly this latter protein is present at higher amounts in endothelial cells cultured in the presence of both astrocytes and neurons, that is in those conditions in which sucrose permeation studies indicate formation of BBB.     

The Susceptibility of Cerebral Endothelial Cells to Astroglial Induction of Blood-Brain Barrier Enzymes Depends on Their Proliferative State

Primary cultures of brain capillary endothelial cells (BCECs) were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro. Enzymatic activities of gamma-glutamyltranspeptidase (gamma-GT) and alkaline phosphatase (ALP) were taken as indicators for the expression of the BBB phenotype. We were able to show that a coculture system with a direct cell-cell contact between astroglial cells and BCECs is the necessary precondition for an increase of these enzyme activities that are lost in pure BCEC cultures. Coculture with both astrocytes and C6-glioma cells reestablishes the BBB phenotype whereas conditioned media as well as an astrocyte-derived extracellular matrix were ineffective. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs. Cells in an early highly proliferative culture phase were stimulated to express an enzymatic activity level similar to the in vivo situation. Confluent BCEC monolayers were not induced at all. With the ALP we observed a spatial induction within a BCEC colony. Astrocyte-induced ALP activity was first observed at an outer belt of BCEC colonies in direct contact with the astrocyte layer. However, this signal is transferred to the center of the colony with time in culture. We conclude that direct contact of BCECs with astroglial cells is necessary for the induction of the BBB phenotype in cultured BCECs and that this signal may be transferred from induced to noninduced BCECs.     

Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity

In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood–brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effect on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances ¹⁴C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4 h. The observed disruption of the barrier could also be confirmed by ¹⁴C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4 h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.     

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for ³H-inulin and ¹⁴C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins -catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.This work is dedicated to the memory of Werner Risau (died 13.12.1998), who initiated this collaboration     

Endothelium-derived relaxing factor contributes to the regulation of endothelial permeability.

To determine whether endothelium-derived relaxing factor (EDRF) contributes to the regulation of endothelial permeability, the transendothelial flux of 14C-sucrose, a marker for the paracellular pathway across endothelial monolayers (Oliver, J. Cell. Physiol. 145:536-548, 1990), was examined in monolayers of bovine aortic endothelial cells grown on collagen-coated filters. The permeability coefficient of 14C-sucrose was significantly decreased by 10(-3) M 8-Bromoguanosine 3',5'-cyclic monophosphate or by 5 x 10(-6) M glyceryl trinitrate, an activator of soluble guanylate cyclase. Depletion of L-arginine from endothelial monolayers increased 14C-sucrose permeability from 3.21 +/- 0.59 to 3.88 +/- 0.50 x 10(-5) cm.sec-1 (mean +/- SEM; n = 6; P < 0.05). The acute administration of 5 x 10(-4) M L-arginine to monolayers depleted of this amino acid decreased 14C-sucrose permeability from 2.91 +/- 0.27 to 2.52 +/- 0.26 x 10(-5) cm.sec-1 (n = 11; P < 0.05). 14C-sucrose permeability was increased by 10(-7) M bradykinin and this effect was enhanced by the presence of each one of the following compounds: 10(-5) M methylene blue, 4 x 10(-6) M oxyhemoglobin, 5 x 10(-4) M NG-methyl-L-arginine or 5 x 10(-4) M N omega-nitro-L-arginine. These results suggest that EDRF contributes to the sealing of the endothelial monolayer and that EDRF released by bradykinin acts as a feedback inhibitor attenuating the increase in endothelial permeability induced by this peptide. Because endothelial cells have the ability to contract and relax and possess guanylate cyclase responsive to nitric oxide, our results suggest that EDRF decreases 14C-sucrose permeability by relaxing endothelial cells, thereby narrowing the width of endothelial junctions.     

Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions.

To determine whether the endothelial paracellular pathway is regulated, the effect of intracellular messengers on the transendothelial flux of inert radiolabeled molecules of diverse molecular size was examined in bovine aortic endothelial cells grown on collagen-coated filters. The endothelial monolayers showed a modest electrical resistance (21 +/- 10 delta.cm2; m +/- SD) and restricted the passage to 14C-sucrose, 3H-inulin, 14C-dextran (70 kDa), and 125I-polyvinyl pyrrolidone (125I-PVP, 360 kDa) according to their molecular mass. 8-Bromoadenosine 3'-5' cyclic monophosphate (8-Br-cAMP) reduced by more than 30% the permeability coefficients of 14C-sucrose and 3H-inulin but had no effect on the permeability of 125I-PVP. The permeabilities of 14C-sucrose and of 14C-inulin were strikingly increased by activating protein kinase C (PKC) by phorbol 12-myristate-13-acetate or sn-1,2-dioctanoly-glycerol whereas the latter compound had no effect on the permeability of 125I-PVP. In addition, the permeability of 14C-sucrose was unchanged by a phorbol ester that does not activate PKC. Increasing intracellular calcium with ionomycin had no effect on the permeability of 14C-sucrose. None of these maneuvers significantly affected the protein content of the endothelial monolayers. The results indicate that 8-Br-cAMP and PKC activators modulate a pathway across the endothelial monolayer that excludes 125I-PVP (360 kDa) but readily accepts 14C-sucrose and 3H-inulin, suggesting that this pathway is the paracellular pathway. Hence, low molecular weight molecules such as sucrose and inulin can be used to probe the behavior of the paracellular pathway of endothelial monolayers grown in vitro. The results also indicate that the paracellular pathway in endothelium is regulated and suggest that endothelial junctions can be closed by simulating adenylate cyclase and opened by stimulating protein kinase C.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Lack of effects of 1439 MHz electromagnetic near field exposure on the blood-brain barrier in immature and young rats

Possible effects of 1439 MHz electromagnetic near field (EMF) exposure on the blood-brain barrier (BBB) were investigated using immature (4 weeks old) and young (10 weeks old) rats, equivalent in age to the time when the BBB development is completed and the young adult, respectively. Alteration of BBB related genes, such as those encoding p-glycoprotein, aquaporin-4, and claudin-5, was assessed at the protein and mRNA levels in the brain after local exposure of the head to EMF at 0, 2, and 6 W/kg specific energy absorption rates (SARs) for 90 min/day for 1 or 2 weeks. Although expression of the 3 genes was clearly decreased after administration of 1,3-dinitrobenzene (DNB) as a positive control, when compared with the control values, there were no pathologically relevant differences with the EMF at any exposure levels at either age. Vascular permeability, monitored with reference to transfer of FITC-dextran, FD20, was not affected by EMF exposure. Thus, these findings suggest that local exposure of the head to 1439 MHz EMF exerts no adverse effects on the BBB in immature and young rats.     

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.     

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.     

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apolipoprotein E regulates the integrity of tight junctions in an isoform-dependent manner in an in vitro blood-brain barrier model

Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ε4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3- and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC)η in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKCη, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner.     

The Blood–Brain Barrier Permeability of Geissoschizine Methyl Ether in Uncaria Hook,a Galenical Constituent of the Traditional Japanese Medicine <Emphasis Type="Italic">Yokukansan</Emphasis>

Geissoschizine methyl ether (GM) in Uncaria hook, a galenical constituent of yokukansan is thought to be one of active components in the psychotropic effect of yokukansan, a traditional Japanese medicine (kampo medicine). However, there is no data on the blood–brain barrier (BBB) permeability of Uncaria hook-derived alkaloids containing GM. In this study, we investigated the BBB permeability of seven Uncaria hook alkaloids (GM, isocorynoxeine, isorhynchophylline, hirsuteine, hirsutine, rhynchophylline, and corynoxeine) using in vivo and in vitro methods. In the in vivo experiment, seven alkaloids in the plasma and brain of rats orally administered with yokukansan were measured by liquid chromatography–mass spectroscopy/mass spectrometric multiple reaction monitoring assay. In the in vitro experiment, the BBB permeability of seven alkaloids were examined using the BBB model composed of co-culture of endothelial cells, pericytes, and astrocytes. In the in vivo study, six components containing GM but not isocorynoxeine were detected in the plasma, and three (GM, hirsuteine, and corynoxeine) of components were detected in the brain. The in vitro BBB permeability data indicated that seven alkaloids were able to cross brain endothelial cells in culture conditions and that the BBB permeability of GM was higher than those of the other six alkaloids. These results suggest that target ingredient GM in yokukansan administered orally is absorbed into the blood and then reaches the brain through the BBB. This evidence further supports the possibility that GM is an active component in the psychotropic effect of yokukansan.     

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.     

In vitro study of the effects of Angiostrongylus cantonensis larvae extracts on apoptosis and dysfunction in the blood-brain barrier (BBB)

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.     

MiR-539 Targets MMP-9 to Regulate the Permeability of Blood–Brain Barrier in Ischemia/Reperfusion Injury of Brain

Cerebral ischemia/reperfusion (I/R) injury severely threatens human life, while the potential mechanism underlying it is still need further exploration. The rat model of cerebral I/R injury was established using middle cerebral artery occlusion (MCAO). The rat microvascular endothelial cell line bEND.3 was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic condition in vitro. Evans blue was performed to determine the blood–brain barrier (BBB) permeability. Real-time PCR and western blot were performed to determine gene expression in mRNA and protein level, individually. Luciferase reporter assay was conducted to determine the relationship between miR-539 and MMP-9. The infarct volume and BBB permeability of cerebral (I/R) rats were significantly greater than Sham group. The expression of miR-539 was decreased, while MMP-9 was increased in the brain tissues of I/R injury rats and OGD/R pretreated bEND.3. Up-regulated miR-539 in OGD/R pretreated bEND.3 significantly promoted the BBB permeability. MiR-539 targets MMP-9 to regulate its expression. OGD/R treatment significantly promoted the BBB permeability in bEND.3, miR-539 mimic transfection abolished the effects of OGD/R, while co-transfected with pcDNA-MMP-9 abolished the effects of miR-539 mimic. MiR-539 targets MMP-9 and further regulates the BBB permeability in cerebral I/R injury.     

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.