Refining in vitro neurotoxicity testing--the development of blood-brain barrier models

The purpose of this paper is to review the current state of development of advanced in vitro blood-brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.     

Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

Impact of chronic smoking on traumatic brain microvascular injury: An in vitro study

Traumatic brain injury (TBI) is a major reason of cerebrovascular and neurological damage. Premorbid conditions such as tobacco smoking (TS) can worsen post-TBI injuries by promoting vascular endothelial impairments. Indeed, TS-induced oxidative stress (OS) and inflammation can hamper the blood-brain barrier (BBB) endothelium. This study evaluated the subsequence of chronic TS exposure on BBB endothelial cells in an established in vitro model of traumatic cell injury. Experiments were conducted on confluent TS-exposed mouse brain microvascular endothelial cells (mBMEC-P5) following scratch injury. The expression of BBB integrity–associated tight junction (TJ) proteins was assessed by immunofluorescence imaging (IF), Western blotting (WB) and quantitative RT-PCR. We evaluated reactive oxygen species (ROS) generation, the nuclear factor 2–related (Nrf2) with its downstream effectors and several inflammatory markers. Thrombomodulin expression was used to assess the endothelial haemostatic response to injury and TS exposure. Our results show that TS significantly decreased Nrf2, thrombomodulin and TJ expression in the BBB endothelium injury models while increased OS and inflammation compared to parallel TS-free cultures. These data suggest that chronic TS exposure exacerbates traumatic endothelial injury and abrogates the protective antioxidative cell responses. The downstream effect was a more significant decline of BBB endothelial viability, which could aggravate subsequent neurological impairments.     

Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

(1) The blood–brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.     

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.     

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.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.     

The blood-brain barrier permeability of 18β-glycyrrhetinic Acid, a major metabolite of glycyrrhizin in glycyrrhiza root, a constituent of the traditional Japanese medicine yokukansan

18β-Glycyrrhetinic acid (GA) is a major metabolite of glycyrrhizin (GL), which is one of the components of glycyrrhiza root, a constituent herb of the traditional Japanese medicine yokukansan. It is well known that most GL is metabolized to GA in the intestine by bacteria. A previous in vitro study using cultured rat cortical astrocytes suggested that GA activates glutamate transport, which is a putative mechanism of the psychotropic effect of yokukansan. To activate the glutamate transport in the brain, GA must be absorbed into the blood after oral administration of yokukansan and then cross the blood-brain barrier (BBB) to reach the brain. However, there is no data on the BBB permeability of GA derived from yokukansan. In the present study, the BBB permeability of GA was investigated in both in vivo and in vitro studies. In the in vivo study, GA was detected in the plasma, brain, and cerebrospinal fluid of rats orally administered yokukansan. In the in vitro study using a BBB model composed of co-culture of endothelial cells, pericytes, and astrocytes, the permeability rate and apparent permeability coefficient of GA were found to be 13.3?±?0.5?% and 16.5?±?0.7 × 10(-6) cm/s. These in vivo and in vitro results suggest that GL in orally administered yokukansan is absorbed into the blood as GA, and then reaches the brain through the BBB. This evidence further supports the possibility that GA is an active component in the psychotropic effect of yokukansan.     

Characterization and use of human brain microvascular endothelial cells to examine β-amyloid exchange in the blood-brain barrier

Alzheimer’s disease (AD) is characterized by excessive cerebrovascular deposition of the β-amyloid peptide (Aβ). The investigation of Aβ transport across the blood-brain barrier (BBB) has been hindered by inherent limitations in the cellular systems currently used to model the BBB, such as insufficient barrier properties and poor reproducibility. In addition, many of the existing models are not of human or brain origin and are often arduous to establish and maintain. Thus, we characterized an in vitro model of the BBB employing human brain microvascular endothelial cells (HBMEC) and evaluated its utility to investigate Aβ exchange at the blood-brain interface. Our HBMEC model offers an ease of culture compared with primary isolated or coculture BBB models and is more representative of the human brain endothelium than many of the cell lines currently used to study the BBB. In our studies, the HBMEC model exhibited barrier properties comparable to existing BBB models as evidenced by the restricted permeability of a known paracellular marker. In addition, using a simple and rapid fluormetric assay, we showed that antagonism of key Aβ transport proteins significantly altered the bi-directional transcytosis of fluorescein-Aβ (1–42) across the HBMEC model. Moreover, the magnitude of these effects was consistent with reports in the literature using the same ligands in existing in vitro models of the BBB. These studies establish the HBMEC as a representative in vitro model of the BBB and offer a rapid fluorometric method of assessing Aβ exchange between the periphery and the brain.     

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.     

Alterations in Transendothelial Electrical Resistance by Vasoactive Agonists and Cyclic AMP in a Blood-Brain Barrier Model System

We have previously reported that the co-culture of endothelial and glioma cell lines provides an in vitro model for investigating properties of the blood-brain barrier (BBB). To characterise the model system further we have investigated the effects of vasoactive substances implicated in increases in BBB permeability. Additionally, we have also examined whether activation of cyclic AMP signalling pathways, which elevate cerebral endothelial cell barrier function, similarly modulate our model system. ATP, histamine, bradykinin, and serotonin significantly decreased model BBB transendothelial electrical resistance and manipulations which elevate cyclic AMP enhanced culture resistance. These data indicate that our model BBB system responds in a manner characteristic of cerebral microvascular endothelial cells and the BBB in vivo. These data further emphasize the usefulness of our model system.     

Shiga toxin-2 enhances heat-shock-induced apoptotic cell death in cultured and primary glial cells

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.     

The Noncompetitive AMPAR Antagonist Perampanel Abrogates Brain Endothelial Cell Permeability in Response to Ischemia: Involvement of Claudin-5

The blood–brain barrier (BBB) is formed by brain endothelial cells, and decreased BBB integrity contributes to vasogenic cerebral edema and increased mortality after stroke. In the present study, we investigated the protective effect of perampanel, an orally active noncompetitive AMPA receptor antagonist, on BBB permeability in an in vitro ischemia model in murine brain endothelial cells (mBECs). The results showed that perampanel significantly attenuated oxygen glucose deprivation (OGD)-induced loss of cell viability, release of lactate dehydrogenase, and apoptotic cell death in a dose-dependent manner. Perampanel treatment did not alter the expression and surface distribution of various glutamate receptors. Furthermore, the results of calcium imaging showed that perampanel had no effect on OGD-induced increase in intracellular Ca²⁺ concentrations. Treatment with perampanel markedly reduced the paracellular permeability of mBECs after OGD in different time points, as measured by transepithelial electrical resistance assay. In addition, the expression of claudin-5 at protein level, but not at mRNA level, was increased by perampanel treatment after OGD. Knockdown of claudin-5 partially prevented perampanel-induced protection in cell viability and BBB integrity in OGD-injured mBECs. These data show that the noncompetitive AMPA receptor antagonist perampanel affords protection against ischemic stroke through caludin-5 mediated regulation of BBB permeability.     

Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier

The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.     

LSR/angulin-1 is a tricellular tight junction protein involved in blood–brain barrier formation

The blood–brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage.     

A new blood–brain barrier model using primary rat brain endothelial cells,pericytes and astrocytes

Blood–brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400 Ω cm² on average, while the endothelial permeability coefficients (P_e) for fluorescein was in the range of 3 × 10^?6 cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R² = 0.89) was found between in vitro P_e values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.