Characterization of a microfluidic in vitro model of the blood-brain barrier (μBBB)

The blood-brain barrier (BBB), a unique selective barrier for the central nervous system (CNS), hinders the passage of most compounds to the CNS, complicating drug development. Innovative in vitro models of the BBB can provide useful insights into its role in CNS disease progression and drug delivery. Static transwell models lack fluidic shear stress, while the conventional dynamic in vitro BBB lacks a thin dual cell layer interface. To address both limitations, we developed a microfluidic blood-brain barrier (μBBB) which closely mimics the in vivo BBB with a dynamic environment and a comparatively thin culture membrane (10 μm). To test validity of the fabricated BBB model, μBBBs were cultured with b.End3 endothelial cells, both with and without co-cultured C8-D1A astrocytes, and their key properties were tested with optical imaging, trans-endothelial electrical resistance (TEER), and permeability assays. The resultant imaging of ZO-1 revealed clearly expressed tight junctions in b.End3 cells, Live/Dead assays indicated high cell viability, and astrocytic morphology of C8-D1A cells were confirmed by ESEM and GFAP immunostains. By day 3 of endothelial culture, TEER levels typically exceeded 250 Ω cm(2) in μBBB co-cultures, and 25 Ω cm(2) for transwell co-cultures. Instantaneous transient drop in TEER in response to histamine exposure was observed in real-time, followed by recovery, implying stability of the fabricated μBBB model. Resultant permeability coefficients were comparable to previous BBB models, and were significantly increased at higher pH (>10). These results demonstrate that the developed μBBB system is a valid model for some studies of BBB function and drug delivery.     

Influence of H<Subscript>2</Subscript>O and SO<Subscript>2</Subscript> on the activity of deposited cobalt oxide catalysts in the processes of reduction of nitrogen(I), (II) oxides with carbon monoxide and C<Subscript>3</Subscript>-C<Subscript>4</Subscript> alkanes

It is shown that palladium–cobalt oxide–cerium catalyst deposited on cordierite catalyzes the reduction of nitrogen(II) oxide with carbon monoxide, and cobalt–iron catalysts in simultaneous reduction of NO + N₂O with C₃-C₄ alkanes retained high activity in the presence of water vapor and sulfur dioxide. The Pd-Co₃O₄/cordierite catalyst exceeds the Pt-Co₃O₄/codierite catalyst in the conversion of NO and CO in the reaction mixture CO + NO + O₂ + H₂O + SO₂. Modification of the Pd-Co₃O₄/cordierite with cerium oxide considerably increases its sulfur resistance.     

A Comparative Study of the PFP and BAB Models in Predicting the Surface and Transport Properties of Liquid Ternary Systems

Measurements of the surface tension, viscosity and density were carried out for three ternary and their nine contributing binary liquid systems, namely: (1) toluene + cyclohexane + carbon tetrachloride and its contributing binaries (toluene + cyclohexane), (cyclohexane + carbon tetrachloride), and (carbon tetrachloride + toluene); (2) benzene + cyclohexane  + toluene and its contributing binaries (benzene + cyclohexane), (cyclohexane  + toluene), and (toluene + benzene); and (3) carbon tetrachloride + cyclohexane + benzene and its contributing binaries, (carbon tetrachloride + cyclohexane), (cyclohexane + benzene) and (benzene + carbon tetrachloride), all at 298.15 K over a wide range of compositions. The surface and transport properties of these liquid ternary systems were also studied theoretically by two liquid models, those of PFP (Prigogine-Flory-Patterson) and BAB (Bertrand-Acree-Bruchfield). The excess surface thermodynamic and excess transport properties predicted with the use of these two models agree well with the experimental results. An attempt has also been made to explain the nature of the molecular interactions and forces involved in these liquid ternary systems.     

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.     

An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane

The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.     

Effect of human astrocytes on the characteristics of human brain-microvascular endothelial cells in the blood-brain barrier

A blood-brain barrier (BBB) model in vitro was established by cultivating human brain-microvascular endothelial cells (HBMECs) with the regulation of human astrocytes (HAs) (HBMEC/HA). Astrocyte-conditioned medium (ACM) was employed to constitute a confluent monolayer of HBMECs without directly conjugated HAs. HBMECs exhibited an orientated multiplication on the supporting membrane; while HAs grew in an overlapping fashion. In addition, HBMECs could propagate over the membrane pore, and the end-feet of HAs extended into the membrane pore to improve the integral feature of the BBB. HBMEC/HA demonstrated a high transendothelial electrical resistance (TEER) about 230 Ω cm2 and low permeability of propidium iodide (PI) about 4 × 10?? cm/s. The order in TEER was HBMEC/HA>HBMECs with 100% ACM>HBMECs with 50% ACM > HBMECs. The reverse order was valid for the permeability of PI and uptake of calcein-AM by HBMECs. The tranwell culture of HBMECs and HAs displays appropriate characteristics of the BBB and can be applied to estimate the delivery efficiency of therapeutic chemicals for the brain-related disease.     

Evaluating the effects of estradiol on endothelial nitric oxide stimulated by erythrocyte-derived ATP using a microfluidic approach

Recently, estrogens have been reported to have protective effects against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Although the molecular mechanism for such a protective effect is currently incomplete, we hypothesized that estradiol may reduce the release of ATP from erythrocytes (ERYs), thereby lowering the production of nitric oxide (NO) by endothelial cells. Here, we report on the use of a microfluidic device to investigate the direct effects of the estrogen estradiol on endothelial cell nitric oxide production. In addition, the incorporation of a thin polycarbonate membrane into the device enabled the passage of ERYs through the device to determine indirect effects of estradiol on NO production that may be meditated by ERYs. When these ERYs were incubated with increasing concentrations of estradiol, the NO production from the endothelial cells was attenuated to a value that was only 59 ± 7% of ERYs in the absence of estradiol. This decrease in NO production coincides with reductions in ERY-derived ATP release in the presence of estradiol. Estradiol is typically reported to have NO-stimulating effects; however, such reports have employed in vitro experimental designs that include only a single cell type. To demonstrate the potential importance of this attenuation of ATP from ERYs, results from a small-scale study show that the ATP release obtained from healthy controls was 138 ± 21 nM (n = 18) while the release from the ERYs obtained from people with MS was 375 ± 51 nM (n = 11). The studies reported here involving multiple cells types (endothelial cells and ERYs) may lead to a reappraisal of the in vivo activities of estradiol.     

Liquid–(Solid + Liquid) Transitions in a Two-Component System of (CH 3 )CCl 3 + CCl 4

Liquid–(solid + liquid) transitions are studied in (CH3)CCl3 + CCl4 by using the Landau phenomelogical model. The Gibbs energy is expanded in terms of the orientational disorder (OD) parameters for the transitions of the liquid–(rhombohedral + liquid) and liquid–(face-centered cubic + liquid) in a two component system of (CH3)CCl3 + CCl4. From the Gibbs energy, the phase line equations are derived for the transitions studied and they are fitted to the observed T–X phase diagram of (CH3)CCl3 + CCl4 for the concentration (X) CCl4. Temperature and concentration dependences of the OD parameters (Ψ and η) and the inverse susceptibility ($$\chi_{\psi }^{ - 1}$$ and $$\chi_{\eta }^{ - 1}$$) for the two transitions of interest, are predicted by using the melting curves of (CH3)CCl3 + CCl4 on the basis of the Landau phenomenological model. Our predictions, which can be compared with the experimental data, indicate that the first order transition of the liquid–(solid + liquid), in particular, for (CH3)CCl3 + CCl4 can be described satisfactorily by the Landau mean field model.     

Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood–brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.     

Ionic processes in solid-electrolyte passivating films on lithium

The electrochemical behaviour of a Li electrode in solutions of LiAlCl₄ in thionyl chloride, LiBF₄ in γ-butyrolactone and LiClO₄ in the mixed solvent propylene carbonate (PC) + dimethoxyethane (DME) in the process of cell storage has been investigated by the methods of electrode impedance spectroscopy and pulse voltammetry. Analogous studies have been carried out in PC + DME solution with the Li electrode coated with a specially formed protecting film of Li₂CO₃. The results have been compared with those obtained earlier for other lithium electrochemical systems. The general regularities of the Li electrode electrochemical kinetics attributed to the process of Li⁺ ion transport through a passivating film coating a lithium surface have been discussed. Received: 22 February 1999 / Accepted: 20 June 1999     

Influence of Amino Acid Composition and Phosphorylation on the Ion Yields of Peptides in MALDI-MS

The influence of arginine (Arg), lysine (Lys), and phenylalanine (Phe) residues and phosphorylation on the molecular ion yields of model peptides have been quantitatively studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive- and negative-ion mode. The results obtained from these experiments have been interpreted from the standpoint of two different components, namely, desorption and ionization, on the basis of the physicochemical properties of constituent amino acids of the model peptides. The presence of basic residues such as Arg and Lys enhanced the ion yields of protonated molecules [M + H]⁺. An N-terminal rather than a C-terminal Arg residue was advantageous for the formation of both [M + H]⁺ and [M – H]^–. The presence of the Phe residue resulted in the increase of the ion yields of both [M + H]⁺ and [M – H]^–. In contrast, the presence of phosphate group(s) contributed to the suppression of the yields of both [M + H]⁺ and [M – H]^– due to the loss of phosphate group. The detection limits for both [M + H]⁺ and [M – H]^– of model peptides have been evaluated.     

Quaternary Liquid–Liquid Equilibria for Fuel Additive Systems Containing Diethyl Carbonate or 1,1-Dimethylethyl Methyl Ether at 298.15 K and Atmospheric Pressure

Liquid–liquid equilibrium tie line data were determined for three quaternary systems water + ethanol + diethyl carbonate+n-heptane, water + ethanol + 1,1-dimethylethyl methyl ether + diethyl carbonate, and water + 1,1-dimethylethyl methyl ether + diethyl carbonate+n-heptane at 298.15 K and atmospheric pressure. The experimental liquid–liquid equilibria results have been correlated using a modified UNIQUAC model and an extended UNIQUAC model, both with multicomponent interaction parameters in addition to the binary ones.     

Impedance Based Nanotoxicity Assessment of Graphene Nanomaterials at the Cellular and Tissue Level

The interest in graphene for biomedical applications has grown substantially in the past few years creating a need for biocompatibility testing. Biomedical engineering applications using graphene such as biosensing devices, microbial detection, disease diagnosis, and drug delivery systems are progressing rapidly, perhaps overlooking any possible hazards as graphene nanomaterials may interact with biological materials differently than other graphitic materials such as carbon nanotubes and fullerenes. As a potential application for graphene is drug delivery, the toxicity of graphene was tested against an in vitro model of the blood brain barrier (BBB) by measuring trans-endothelial-electrical resistance (TEER). A new approach in terms of electrical impedance sensing was also utilized to kinetically analyze the cytotoxicity of graphene nanomaterials towards the BBB model's individual components, rat astrocytes (CRL-2006) and mouse endothelial cells (CRL-2583), in real time by measuring the impedimetric response. Graphene showed little or no toxicity toward both individual cell types as the resistance measurements were similar to those of the control and further, graphene did not interrupt the integrity of the BBB model as a whole showing the biocompatibility of graphene and the broad potential of using these new nanomaterials for biomedical applications.     

Regulation of endocytosis into human brain-microvascular endothelial cells by inhibition of efflux proteins

The expressions of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) in the blood-brain barrier (BBB) were regulated by verapamil and probenecid. The two protein interveners inhibited the efflux-pumping capability for permeating calcein-AM in the monolayer of human brain-microvascular endothelial cells (HBMECs). The immunochemical staining revealed that the order in the integrity of tight junction was human astrocyte (HA)-regulated HBMECs>HBMECs cultured with 100% astrocyte-conditioned medium (ACM)>HBMECs cultured with 50% ACM>HBMECs. The viability of HBMECs was higher than 94% when the concentrations of verapamil and probenecid were lower than 50 μM and 1000 μM, respectively. The culture using ACM negligibly affected the activity of P-gp and MRPs on HBMECs after the suppression with verapamil and/or probenecid. However, the double culture of HA-regulated HBMECs promoted the quantity of P-gp and MRPs and reduced the endocytosis of calcein-AM. The inhibitive and endocytotic analysis can unveil the role of HAs in the protein expressions on HBMECs for establishing a reliable BBB model in vitro.     

Solid–Liquid Phase Equilibria in the Ternary Systems (LiBO 2 + NaBO 2 + H 2 O) and (LiBO 2 + KBO 2 + H 2 O) at 288.15 K and 0.1 MPa

Solid–liquid phase equilibria for the two aqueous systems (LiBO2 + NaBO2 + H2O) and (LiBO2 + KBO2 + H2O) at T = 288.15 K and p = 0.1 MPa were determined using the isothermal dissolution equilibrium method. The experimental results show that the phase diagrams consist of one two-salt co-saturated invariant point, two univariant solubility isotherms, and three crystallization fields. The two systems belong to simple co-saturated type, and neither double salt nor solid solution were found. The densities change regularly as the sodium metaborate (potassium metaborate) concentration increases in solution, and reach their maximum values at the invariant point. Based on the Pitzer and its extended Harvie–Møller–Weare (HMW) model, the solubilities for the ternary systems at 288.15 K were represented, and the calculated results agree well with the experimental values.     

A neutral polysaccharide from <Emphasis Type="Italic">Glycyrrhiza inflata</Emphasis>

A neutral polysaccharide Gi-A1 was isolated from the roots of Glycyrrhiza inflata Bat. It had a molecular mass of over 2000 kDa and showed [α]_D²⁰ + 81.4° (c 1.05, H₂O). Acid hydrolysis and methylation analysis indicated that Gi-A1 was mainly composed of α-D-glucose, α-L-arabinose, and α-D-galactose with a molar ratio of 8.0:1.8:1.0. It can significantly stimulate spleen cell proliferation in vitro (P < 0.01). Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 13–14, January–February, 2009.     

Liquid–Liquid Equilibria of Oxygenate Fuel Additives with Water: Two Quaternary Aqueous Systems of Diisopropyl Ether + 2,2,4-Trimethylpentane with Methyl <Emphasis Type="Italic">tert</Emphasis>-Butyl Ether or Toluene

Experimental tie-line data for two quaternary systems, water + diisopropyl ether + 2,2,4-trimethylpentane + methyl tert-butyl ether or toluene, were investigated at 298.15 K and atmospheric pressure. The experimental liquid–liquid equilibrium data were correlated using a modified UNIQUAC activity coefficient model with ternary and quaternary parameters, in addition to the binary ones. The calculated results were further compared with those obtained with an extended UNIQUAC model from Nagata [Fluid Phase Equilib. 54, 191–206 (1990)].     

Interactions between cationic mixed micelles and poly(vinyl pyrrolidone)

 The conductances of trimethyltetradecylammonium bromide (TTAB) + trimethylhexa decylammonium bromide (HTAB) and TTAB + trimethyldodecylammonium bromide (DTAB) mixtures over the entire mole fraction range were measured in aqueous poly(vinyl pyrrolidone) (PVP) containing 1–10 wt% PVP at 30 °C. Each conductivity (κ) curve for the TTAB + HTAB mixtures showed two breaks corresponding to two aggregation processes over the whole mole fraction range, except in the case of pure TTAB, where a single break corresponding to the conventional critical micelle concentration (cmc) was observed. In the case of TTAB + DTAB mixtures, each κ curve at a particular mole fraction of TTAB showed only one break, which was quite close to a similar one in pure water. In TTAB + HTAB mixtures, the first break is called the critical aggregation concentration. It is close to the conventional cmc and is attributed to the polymer-free micelle formation, whereas the second break is due to the polymer-bound micellar aggregates. However, no polymer-bound micellar aggregation process was observed in the case of TTAB + DTAB mixtures. Therefore, the presence of micelle–PVP interactions in the TTAB + HTAB case have been attributed to the stronger hydrophobicity of HTAB or TTAB + HTAB micelles in comparison to that of single or mixed micelles of TTAB + DTAB mixtures. From the conductivity data, various micelle parameters in the presence of PVP have been computed and discussed in terms of micelle–polymer interactions. The mixing behavior of TTAB +  HTAB corresponding to the first break, and that of TTAB + DTAB mixtures in the presence of PVP, is close to ideal and is also identical to that in pure water. Received: 26 August 1999 Accepted: 6 November 1999     

Ab initio molecular orbital study of the flavin-catalyzed dehydrogenation reaction of glycine - protein transport channel driving hydride-transfer mechanism

The reaction mechanism of flavin-catalyzed dehydrogenation of glycine has been studied by ab initio molecular orbital calculations using the 6-31G* basis set. 10-Methyl isoalloxazine (10-MIA) has been used as the flavin model compound. The results showed that when we assume a proton transport channel in amino acid oxidase, which is switched on by the substrate anion, the O12-protonated 10-MIA [10-MIAH⁺(O12)] is generated. The main structure of 10-MIAH⁺(O12) is one in which the central ring is expressed by an NAD⁺-like structure, which is favorable for driving the hydride-transfer reaction, i.e., the abstraction of the α-hydrogen of glycine by the hydride-transfer mechanism. We have found that this protonation results in a dramatic lowering of the activation energy of the reaction. The proposed mechanism is summarized as follows: the hydride transfer proceeds via two-electron transfer and synchronous intramolecular proton transfer → intermolecular proton transfer. Received: 10 August 1998 / Accepted: 17 September 1998 / Published online: 8 February 1999     

Excess Molar Volumes,Viscosities and Speeds of Sound of the Ternary Mixture 1-Heptanol (1) + Tetrachloroethylene (2) + Methylcyclohexane (3) at 298.15 K

Densities (ρ), viscosities (η) and speeds of sound (u) of the ternary mixture (1-heptanol + tetrachloroethylene + methylcyclohexane) and the corresponding binary mixtures (1-heptanol + tetrachloroethylene), (1-heptanol + methylcyclohexane) and (tetrachloroethylene + methylcyclohexane) at 298.15 K were measured over the whole composition range. The data obtained are used to calculate the excess molar volumes (V ^E), excess isobaric thermal expansivities (α ^E), viscosity deviations (Δη), excess Gibbs energies of activation of viscous flow (ΔG ^*E) and excess isentropic compressibilities (κ _S ^E) of the binary and ternary mixtures. The data from the binary systems were fitted by the Redlich–Kister equation whereas the best correlation method for the ternary system was found using the Nagata equation. Viscosities, speeds of sound and isentropic compressibilities of the binary and ternary mixtures have been correlated by means of several empirical and semi-empirical equations. The best correlation method for viscosities of binary systems is found using the Iulan et al. equation and for the ternary system using the Heric and McAllister equations. The best correlation method for the speeds of sound and isentropic compressibilities of the binary system (1-heptanol + methylcyclohexane) is found using IMR (Van Deal ideal mixing relation) and for the binary system (tetrachloroethylene + methylcyclohexane) it is found using the NR (Nomoto relation) and for the binary system (1-heptanol + tetrachloroethylene) and the ternary system (1-heptanol + trichloroethylene + methylcyclohexane) it is obtained from the FLT (Jacobson free length theory).