BBB Model Research Articles

Effects of BDNF-loaded poly (lactide-co-glycolide) nanoparticle on hBMEC cell viability and its effectiveness in penetrating the BBB model

Event Abstract Back to Event Effects of BDNF-loaded poly (lactide-co-glycolide) nanoparticle on hBMEC cell viability and its effectiveness in penetrating the BBB model Siti N. Kamarudin1*, Gabriele R A Froemming2, Minaketan Tripathy3, Igor Iezhitsa4, Renad N Alyautdin5 and Nafeeza Mohd Ismail1 1 University Teknologi Mara (UiTM), Pharmacology/ Faculty of Medicine, Malaysia 2 University Teknologi Mara (UiTM), IPPerForM / Faculty of Medicine, Malaysia 3 University Teknologi Mara (UiTM), Fundamental of Pharmaceutics / Faculty of Pharmacy, Malaysia 4 University Teknologi Mara (UiTM), Centre for Neuroscience Research, Faculty of Medicine, Malaysia 5 Ministry of Health Russian Federation , Scientific Centre for Expertise of Medical Application Products, Russia Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier due to their extensive bioavailability, better encapsulation, controlled release properties and low cytotoxicity. Brain Derived Neurotrophic Factor (BDNF) is beneficial to brain tissue especially after ischemic injury promoting the survival of mature neuron cells. The objectives of this research were: 1) to prepare BDNF-loaded PLGA NPs coated with Apolipoprotein E (ApoE) to enhance BBB crossing via LDL-receptor mediated endocytosis; 2) to evaluate in vitro intracellular uptake and possible cytotoxicity of NPs using hBMEC cell line. Cell viability was determined using MTS-assay and cellular uptake of the NPs by the hBMEC cells was investigated using coumarin 6 as a fluorescent probe. For cells toxicity experiments, 5×104 cells per well were cultured in 96-well plates. After 4 hrs incubation with PLGA particles, 20 µl CellTiter 96s Aqueous One Solution was added directly to the cells and the formation of formazan was measured. Percentage of viability has been calculated relative to untreated control. For penetration study, 1×105 cells per well were cultured in 2 well chamber slides. After 4 hrs incubation, the nuclei were stained with DAPI; the cells were fixed and then imaged by CLSM to visualize the internalization of NPs. Totally 50 cells were analyzed using Leica Qwin software. The fluorescence intensity of the coumarin was measured according to its wavelength. Cell viability increased by 10.4% and 10.1% compared to control after exposure of hBMEC to PLGA-BDNF and PLGA-BDNF-ApoE respectively (p<0.001). Cellular uptake of PLGA-BDNF-ApoE was found to be significantly higher than PLGA-BDNF. In conclusion, PLGA-BDNF nanoparticles does not cause toxicity to hBMEC and was able to penetrate the cells. The penetration was enhanced by addition of ApoE on the surface of PLGA nanoparticles. Acknowledgements We would like to acknowledge the financial support from grant 600-RMI/ERGS 5/3 (36/2013) by MOE, Malaysia. Keywords: Blood-Brain Barrier, Vitamin E, Toxicity, in vitro, nanoparticle Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kamarudin SN, Froemming G, Tripathy M, Iezhitsa I, Alyautdin R and Mohd Ismail N (2016). Effects of BDNF-loaded poly (lactide-co-glycolide) nanoparticle on hBMEC cell viability and its effectiveness in penetrating the BBB model. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00153 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Siti N Kamarudin, University Teknologi Mara (UiTM), Pharmacology/ Faculty of Medicine, Sg Buloh, Selangor, Malaysia, syafika84@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Siti N Kamarudin Gabriele R A Froemming Minaketan Tripathy Igor Iezhitsa Renad N Alyautdin Nafeeza Mohd Ismail Google Siti N Kamarudin Gabriele R A Froemming Minaketan Tripathy Igor Iezhitsa Renad N Alyautdin Nafeeza Mohd Ismail Google Scholar Siti N Kamarudin Gabriele R A Froemming Minaketan Tripathy Igor Iezhitsa Renad N Alyautdin Nafeeza Mohd Ismail PubMed Siti N Kamarudin Gabriele R A Froemming Minaketan Tripathy Igor Iezhitsa Renad N Alyautdin Nafeeza Mohd Ismail Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

BMET-27CEREBRAL METASTASIS OF BREAST CANCER IS FACILITATED BY THE T CELL RESPONSE INVOLVING GBP1

INTRODUCTION: Cerebral metastasis usually defines the terminal stage of disease for women suffering from disseminated breast cancer. Understanding the molecular basis of the tumor cells crossing the blood-brain barrier is crucial to prevent metastasis to the brain. THE AIM: To identify pathways involved in the development of cerebral metastasis in breast cancer patients. MATERIAL AND METHOD: The expressional profiles of FFPE primary breast cancer samples of patients who suffered from estrogen receptor negative breast cancer with cerebral metastases were compared to the profiles of women with metastases to other sites. Illumina WG-DASL RNA platform was used. The most prominent emerging pathway was validated by immunohistochemistry. Functional studies using human breast cancer cell lines in an in vitro BBB model were carried out, and by using mass spectrometry proteins responsible for crossing of tumor cells through the BBB were identified. The identified proteins were validated in the original samples used for discovery. RESULTS: The T cell response pathway was the most significant for the formation of cerebral metastasis of breast cancers. Immunohistochemistry confirmed the presence of higher numbers of T cells in the tumor samples of women who developed cerebral metastases. Following co-culturing with T cells, the ability of tumor cells to cross the BBB significantly increased. Proteomics comparison identified 18 candidate proteins expressed in the tumor cells which are associated with the ability of crossing the BBB. Searching for these proteins in the original gene expression profiles of the primary breast cancer samples highlighted the involvement of GBP1 protein in brain metastasis, and the expression of the GBP1 protein was confirmed to be exclusively expressed in samples of patients who developed brain metastases. CONCLUSION: T cell response mediates ER- breast cancer metastasis to the brain. This discovery may open new therapeutic opportunities based on the immune therapy field.

Mechanism of brain targeting by dexibuprofen prodrugs modified with ethanolamine-related structures.

The first molecular insights into how prodrugs modified with ethanolamine-related structures target the brain were generated using an in vitro BBB model and in situ perfusion technique. Prodrugs were delivered safely and efficiently to the brain through tight interaction with the anionic membrane of brain capillary endothelial cells, observed as a shift in zeta potential, followed by uptake into the cells. Prodrugs III and IV carrying primary and secondary amine modifications appeared to enter the brain via energy-independent passive diffusion. In contrast, besides the passive diffusion, prodrugs I and II carrying tertiary amine modifications also appeared to enter via an active process that was energy and pH dependent but was independent of sodium or membrane potential. This active process involved, at least in part, the pyrilamine-sensitive H(+)/OC antiporter, for which the N,N-diethyl-based compound II showed a much lower affinity than the N,N-dimethyl-based compound I, likely due to steric hindrance. These new insights into brain-targeting mechanisms may help guide efforts to design new prodrugs.

F021003 衝撃を受ける細胞・血管の振る舞い : 衝撃を受ける脳神経細胞および血液脳関門(BBB)の機能評価

The presentation is aimed for the prospective contribution of biological experiment to TBI research. The axonal injury of nerve cells and collapse of blood brain barrier system are spotlighted. With a background of rapid development of computational mechanics, injury threshold for impulsive load and morphological change after injury of cells and blood vessels are becoming more important. The injury threshold of nerve cells with direction controlled axon which was stretched uniaxially was evaluated. And BBB model exposed to impulsive pressure fluctuation was evaluated by measuring the TEER.

Nanoparticles and the blood-brain barrier: advancing from in-vitro models towards therapeutic significance.

The blood-brain barrier is a unique cell-based restrictive barrier that prevents the entry of many substances, including most therapeutics, into the central nervous system. A wide range of nanoparticulate delivery systems have been investigated with the aim of targeting therapeutics (drugs, nucleic acids, proteins) to the brain following administration by various routes. This review provides a comprehensive description of the design and formulation of these nanoparticles including the rationale behind individual approaches. In addition, the ability of currently available in-vitro BBB models to accurately predict the in-vivo performance of targeted nanoparticles is critically assessed.

Monocyte mediated brain targeting delivery of macromolecular drug for the therapy of depression

Leukocytes can cross intact blood-brain barrier under healthy conditions and in many neurological diseases, including psychiatric diseases. In present study, a cyclic RGD (cRGD) peptide with high affinity for integrin receptors of leukocytes was used to modify liposomes. The cRGD-modified liposomes (cRGDL) showed high affinity for monocytes in vitro and in vivo and co-migrated across in vitro BBB model with THP-1. The trefoil factor 3 (TFF3), a macromolecular drug, was rapidly and persistently delivered to brain for at least 12 h when loaded into cRGDL while 2.8-fold increase in drug concentration in basolateral amygdala regions related to depression was observed. A systemic administration of cRGDL-TFF3 mimicked antidepressant-like effect of direct intra-basolateral amygdala administration of TFF3 solution in rats subjected to chronic mild stress. The effective dual-brain targeting delivery resulting from the combination and co-migration of cRGDL with leukocyte cross BBB may be a promising strategy for targeted brain delivery. From the Clinical EditorIn an effort to treat depression, brain targeted delivery via monocyte-cRGD liposome complexes capable of crossing the intact BBB was performed in this study in a murine model. Similar approaches may be helpful in the treatment of other neuropsychiatric conditions.

Functional Characteristics and Molecular Mechanism of a New scFv Antibody Against Aβ42 Oligomers and Immature Protofibrils.

Amyloid β peptide (Aβ42) is a major determinant of Alzheimer's disease (AD). In this study, we studied a novel single-chain variable fragment (scFv), AS, generated from an antibody library of AD patients, which recognized and bound specifically to medium-size amyloid β peptide (Aβ42) oligomers and immature protofibrils (25-55 kDa) and, more importantly, reduced their level by blocking their formation or inducing their disassembly. Consequently, scFv AS ameliorated or prevented their cytotoxicity and protected SH-SY5Y cells and primary cultured neurons in vitro from their damage in a concentration-dependent manner. Comparison of its cytotoxicity-inhibiting and cytotoxicity-neutralizing activities indicated that scFv AS displayed its protective effect on target cells mainly due to its cytotoxicity-inhibitory activity though it could also neutralize the cytotoxicity. We also found that scFv AS could efficiently cross the in vitro BBB model with a delivery efficiency of over 70% after a 60-min post-administration. The scFv AS was a monovalent antibody with an affinity constant (KD) of 5.5 × 10(-6) M and a binding threshold of 6.25 × 10(-4) μM for Aβ42 oligomers. The molecular docking simulations of Aβ42 to scFv AS revealed that scFv AS tends to approached Aβ42 oligomers and immature protofibrils mainly by their hydrophobic interaction and then drew Aβ42 molecule into the gap between VL and VH domains of scFv AS by hydrophilic interaction between scFv AS and the N-terminal region (residues 1-15) of Aβ42 and the hydrophobic interactions between scFv AS and the middle region (residues 20-33) of Aβ42. The combination of scFv AS with Aβ42 was realized likely through an induced-fit process.

Examining the role of the temporo-parietal network in memory, imagery, and viewpoint transformations.

The traditional view of the medial temporal lobe (MTL) focuses on its role in episodic memory. However, some of the underlying functions of the MTL can be ascertained from its wider role in supporting spatial cognition in concert with parietal and prefrontal regions. The MTL is strongly implicated in the formation of enduring allocentric representations (e.g., O'Keefe, 1976; King et al., 2002; Ekstrom et al., 2003). According to our BBB model (Byrne et al., 2007), these representations must interact with head-centered and body-centered representations in posterior parietal cortex via a transformation circuit involving retrosplenial areas. Egocentric sensory representations in parietal areas can then cue the recall of allocentric spatial representations in long-term memory and, conversely, the products of retrieval in MTL can generate mental imagery within a parietal “window.” Such imagery is necessarily egocentric and forms part of visuospatial working memory, in which it can be manipulated for the purpose of planning/imagining the future. Recent fMRI evidence (Lambrey et al., 2012; Zhang et al., 2012) supports the BBB model. To further test the model, we had participants learn the locations of objects in a virtual scene and tested their spatial memory under conditions that impose varying demands on the transformation circuit. We analyzed how brain activity correlated with accuracy in judging the direction of an object (1) from visuospatial working memory (we assume transient working memory due to the order of tasks and the absence of change in viewpoint, but long-term memory retrieval is also possible), (2) after a rotation of viewpoint, or (3) after a rotation and translation of viewpoint (judgment of relative direction). We found performance-related activity in both tasks requiring viewpoint rotation (ROT and JRD, i.e., conditions 2 and 3) in the core medial temporal to medial parietal circuit identified by the BBB model. These results are consistent with the predictions of the BBB model, and shed further light on the neural mechanisms underlying spatial memory, mental imagery and viewpoint transformations.

Temporal expression of transporters and receptors in a rat primary co-culture blood–brain barrier model

1. The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines.2. In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin–Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model.3. Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system.4. Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells.5. We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model.

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

In Vitro Models of the Blood–Brain Barrier for the Study of Drug Delivery to the Brain

The most important obstacle to the drug delivery into the brain is the presence of the blood-brain barrier, which limits the traffic of substances between the blood and the nervous tissue. Therefore, adequate in vitro models need to be developed in order to characterize the penetration properties of drug candidates into the central nervous system. This review article summarizes the presently used and the most promising in vitro BBB models based on the culture of brain endothelial cells. Robust models can be obtained using primary porcine brain endothelial cells and rodent coculture models, which have low paracellular permeability and express functional efflux transporters, showing good correlation of drug penetration data with in vivo results. Models mimicking the in vivo anatomophysiological complexity of the BBB are also available, including triple coculture (culture of brain endothelial cells in the presence of pericytes and astrocytes), dynamic, and microfluidic models; however, these are not suitable for rapid, high throughput studies. Potent human cell lines would be needed for easily available and reproducible models which avoid interspecies differences.

『脳毛細血管のin vitroでの再現』 血液脳関門（BBB）構成細胞の単離とin vitro BBBモデルの再構築

『脳毛細血管のin vitroでの再現』 血液脳関門（BBB）構成細胞の単離とin vitro BBBモデルの再構築

Mono and Dually Decorated Nanoliposomes for Brain Targeting, In Vitro and In Vivo Studies

Mono- and dual-decorated (DUAL) liposomes (LIP) were prepared, by immobilization of MAb against transferrin (TfR[OX26 or RI7217]) and/or a peptide analogue of ApoΕ3 (APOe) -to target low-density lipoprotein receptor(LPR)-, characterized physicochemically and investigated for BBB-targeting, in-vitro and in-vivo. Human microvascular endothelial cells (hCMEC/D3) were used as BBB model, and brain targeting was studied by in-vivo imaging of DiR-labelled formulations (at two doses and surface ligand densities), followed by ex-vivo organ imaging. LIP diameter was between 100nm and 150nm, their stability was good and they were non-cytotoxic. LIP uptake and transport across the hCMEC/D3 cell monolayer was significantly affected by decoration with APOe or MAb, the DUAL exerting an additive effect. Intact vesicle-transcytosis was confirmed by equal transport of hydrophilic and lipophilic labels. In-vivo and ex-vivo results confirmed MAb and DUAL-LIP increased brain targeting compared to non-targeted PEG-LIPs, but not for APOe (also targeting ability of DUAL-LIP was not higher than MAb-LIP). The contradiction between in-vitro and in-vivo results was overruled when in-vitro studies (uptake and monolayer transport) were carried out in presence of serum proteins, revealing their important role in targeted-nanoformulation performance. A peptide analogue of ApoΕ3 was found to target BBB and increase the targeting potential of TfR-MAb decorated LIP, in-vitro, but not in-vivo, indicating that different types of ligands (small peptides and antibodies) are affected differently by in-vivo applying conditions. In-vitro tests, carried out in presence of serum proteins, may be a helpful predictive "targetability" tool.

A Simple Method for Assessing Free Brain/Free Plasma Ratios Using an In Vitro Model of the Blood Brain Barrier

Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.

Detrimental Effect of Electromagnetic Pulse Exposure on Permeability of In Vitro Blood-brain-barrier Model

ObjectiveTo study the effect of electromagnetic pulse (EMP) exposure on permeability of in vitro blood-brain-barrier (BBB) model. MethodsAn in vitro BBB model, established by co-culturing brain microvascular endothelial cells (BMVEC) and astroglial cells (AC) isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance (TEER) and the horseradish peroxidase (HRP) transmission at different time points. Levels of BBB tight junction-related proteins were measured at 0, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. ResultsThe TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. ConclusionEMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability.

Influence of borneol and muscone on geniposide transport through MDCK and MDCK-MDR1 cells as blood–brain barrier in vitro model

The objective of this study was (1) to characterize geniposide transport through MDCK and MDCK-MDR1 cell lines to confirm its transport mechanism and (2) to evaluate the effect of borneol and muscone as enhancers of geniposide transport in the BBB models so as to explore the enhancement mechanism. Transport studies of geniposide were performed in both directions, from apical to basolateral and from basolateral to apical sides. Drug concentrations were analyzed by HPLC. Geniposide showed relatively poor absorption in MDCK and MDCK-MDR1 cells, apparent permeability coefficients ranging from 0.323×10(-6) to 0.422×10(-6) cm/s. The in vitro experiments showed that geniposide transport in both directions was not concentration dependent and saturable, indicating purely passive diffusion. The efflux ratio of geniposide was less than 2 in the two cell models, which suggested that geniposide was not P-gp substrates. Geniposide transport in both directions significantly increased when co-administrated with increasing concentrations of borneol and muscone. Actin staining results indicated that borneol and muscone increased geniposide transport in the BBB models may attribute to disassembly effect on tight junction integrity.

NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for neuromyelitis optica (NMO). However, the molecular mechanism of NMO still remains unclear.The purpose of this study was to identify the possible humoral mechanisms responsible for the occurrence of astrocytic damage. Human primary astrocytes (AST) were immortalized by retroviral vectors harboring temperature-sensitive SV40 T antigen gene and AQP4 cDNA (M23), designated as hAST-AQP4. The effects of NMO sera on the content and localization of AQP4, including cytotoxicity and astrocytic morphology, were evaluated. In addition, this study examined whether the amount and localization of AQP4 protein in astrocytes were influenced by direct contact with the immortalized human brain microvascular endothelial cell line, TY09. NMO sera alone induced cytotoxicity and addition of complement had a more harmful effect on hAST-AQP4. NMO sera also decreased AQP4 mRNA and protein. NMO sera alone up-regulated TNFα and IL-6 in astrocytes and co-incubation with anti-TNFα and anti-IL-6 neutralizing antibodies blocked both the cytotoxicity and reduction of AQP4 in astrocytes. In the experiment using the in vitro BBB models, AQP4 protein mainly localized at the astrocytic membrane after co-culture with TY09, in contact with TY09. The future elucidation of factors that up-regulate AQP4 in astrocytes presumably released by blood brain barrier forming endothelial cells and that block the production of inflammatory cytokines may therefore lead to the development of a novel therapeutic strategy.

Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells

It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx—highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry.

Application of in vitro BBB model to measure permeability of nanoparticles

In both pharmaceutical and toxicological fields, one of major issues has been the possibility of nanoparticle uptake to central nerve system. For the safe use of nanoparticles, it is integral to evaluate the permeability of nanoparticles through BBB. In our collaborative research group reported that a few nanoparticles accumulated in brain in animal experiment, as an in vitro model, we applied commercially available cell-based BBB model for establishing evaluation method, which is quick, quantitative and equivalent to in vivo assay. We assayed 30–1500 nm silica and surface charge dependent Qdots. Our results showed the size-dependency and the surface modification dependency. We compared our assay to several animal experiments. There are both equivalence and discrepancy with animal experiments. Our BBB model can be useful tools for evaluating size-dependent permeability, but not for surface modification-dependent permeability. Our BBB assay is non-serum assay and we have not adequately reflected the serum-related interaction between nanoparticles and cell surfaces. To clear up the discrepancy of our BBB model, serum-based assay and low-concentration detection will be needed.

C213 in vitro BBBモデルを用いた衝撃圧力負荷後の経内皮電気抵抗値測定(C2-3 生体計測2)

C213 in vitro BBBモデルを用いた衝撃圧力負荷後の経内皮電気抵抗値測定(C2-3 生体計測2)