Blood-brain barrier-on-a-chip for brain disease modeling and drug testing

The blood-brain barrier (BBB) is an interface between cerebral blood and the brain parenchyma. As a gate keeper, BBB regulates passage of nutrients and exogeneous compounds. Owing to this highly selective barrier, many drugs targeting brain diseases are not likely to pass through the BBB. Thus, a large amount of time and cost have been paid for the development of BBB targeted therapeutics. However, many drugs validated in in vitro models and animal models have failed in clinical trials primarily due to the lack of an appropriate BBB model. Human BBB has a unique cellular architecture. Different physiologies between human and animal BBB hinder the prediction of drug responses. Therefore, a more physiologically relevant alternative BBB model needs to be developed. In this review, we summarize major features of human BBB and current BBB models and describe organ-on-chip models for BBB modeling and their applications in neurological complications.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Bioengineered microfluidic blood-brain barrier models in oncology research

Metastasis is the major reason for most brain tumors with up to a 50% chance of occurrence in patients with other types of malignancies. Brain metastasis occurs if cancer cells succeed to cross the ‘blood-brain barrier’ (BBB). Moreover, changes in the structure and function of BBB can lead to the onset and progression of diseases including neurological disorders and brain-metastases. Generating BBB models with structural and functional features of intact BBB is highly important to better understand the molecular mechanism of such ailments and finding novel therapeutic agents targeting them. Hence, researchers are developing novel in vitro BBB platforms that can recapitulate the structural and functional characteristics of BBB. Brain endothelial cells-based in vitro BBB models have thus been developed to investigate the mechanism of brain metastasis through BBB and facilitate the testing of brain targeted anticancer drugs. Bioengineered constructs integrated with microfluidic platforms are vital tools for recapitulating the features of BBB in vitro closely as possible. In this review, we outline the fundamentals of BBB biology, recent developments in the microfluidic BBB platforms, and provide a concise discussion of diverse types of bioengineered BBB models with an emphasis on the application of them in brain metastasis and cancer research in general. We also provide insights into the challenges and prospects of the current bioengineered microfluidic platforms in cancer research.     

P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

FLZ, a novel anti-Parkinson''s disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson''s conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.     

The adaptation of the blood-brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy

Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) are increased in the maternal circulation during pregnancy. These factors may increase blood-brain barrier (BBB) permeability, yet brain edema does not normally occur during pregnancy. We therefore hypothesized that in pregnancy, the BBB adapts to high levels of these permeability factors. We investigated the influence of pregnancy-related circulating factors on VEGF-induced BBB permeability by perfusing cerebral veins with plasma from nonpregnant (NP) or late-pregnant (LP) rats (n=6/group) and measuring permeability in response to VEGF. The effect of VEGF, PLGF, and VEGF-receptor (VEGFR) activation on BBB permeability was also determined. Results showed that VEGF significantly increased permeability (×10(7) μm(3)/min) from 9.7 ± 3.5 to 21.0 ± 1.5 (P<0.05) in NP veins exposed to NP plasma, that was prevented when LP veins were exposed to LP plasma; (9.7±3.8; P>0.05). Both LP plasma and soluble FMS-like tyrosine-kinase 1 (sFlt1) in NP plasma abolished VEGF-induced BBB permeability in NP veins (9.5±2.9 and 12±2.6; P>0.05). PLGF significantly increased BBB permeability in NP plasma (18±1.4; P<0.05), and required only VEGFR1 activation, whereas VEGF-induced BBB permeability required both VEGFR1 and VEGFR2. Our findings suggest that VEGF and PLGF enhance BBB permeability through different VEGFR pathways and that circulating sFlt1 prevents VEGF- and PLGF-induced BBB permeability during pregnancy.     

Interleukin-1β Induces Blood–Brain Barrier Disruption by Downregulating Sonic Hedgehog in Astrocytes

The blood–brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.     

Potential Role of Cerebral Glutathione in the Maintenance of Blood-Brain Barrier Integrity in Rat

Using the model of glutathione (GSH) depletion, possible role of GSH in the maintenance of blood-brain barrier (BBB) integrity was evaluated in rats. Administration (ip) of GSH depletors, diethyl maleate (DEM, 1–4 mmol/kg), phorone (2–3 mmol/kg) and 2-cyclohexene-1-one (CHX, 1 mmol/kg), to male adults was found to deplete brain and liver GSH and increase the BBB permeability to micromolecular tracers (sodium fluorescein and [¹⁴C]sucrose) in a dose-dependent manner at 2h. However, BBB permeability to macromolecular tracers such as horseradish peroxidase and Evan's blue remained unaltered. It was also shown that observed BBB permeability dysfunction was associated with brain GSH depletion. A lower magnitude of BBB increase in rat neonates, as compared to adults, indicated a possible bigger role of GSH in the BBB function of mature brain. The treatment with N-acetylcysteine, methionine and GSH provided a partial to full protection against DEM-induced brain (microvessel) GSH depletion and BBB dysfunction; however, the treatment with -tocopherol, ascorbic acid and turmeric were not effective. Our studies showed that cerebral GSH plays an important role in maintaining the functional BBB integrity.     

Neurological diseases at the blood-brain barrier: Stemming new scientific paradigms using patient-derived induced pluripotent cells

The blood-brain barrier (BBB) is a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by a specific basement membrane interacting with astrocytes, neurons, and pericytes. The BBB plays an essential function in the maintenance of brain homeostasis, by providing a physical and chemical barrier against pathogens and xenobiotics. Although the disruption of the BBB occurs with several neurological disorders, the scarcity of patient material source and lack of reliability of current in vitro models hindered our ability to model the BBB during such neurological conditions. The development of novel in vitro models based on patient-derived stem cells opened new venues in modeling the human BBB in vitro, by being more accurate than existing in vitro models, but also bringing such models closer to the in vivo setting. In addition, patient-derived models of the BBB opens the avenue to address the contribution of genetic factors commonly associated with certain neurological diseases on the BBB pathophysiology. This review provides a comprehensive understanding of the BBB, the current development of stem cell-based models in the field, the current challenges and limitations of such models.     

A GABA-EEG test of the blood-brain barrier near epileptic foci

The permeability of the blood-brain barrier (BBB) to gamma-aminobutyric acid (GABA) in the region of an epileptic focus may be assessed by infusing GABA and measuring a change in epileptic spike activity on the EEG. GABA does not cross the normal BBB but will suppress epileptic spike activity when it does cross where the BBB is damaged. 9 alumina-cobalt experimental epileptic foci were all initially suppressible, but 7 then became unsuppressible . When the foci were irradiated to lower the BBB, all 7 became temporarily suppressible. The experiments demonstrate that (1) epileptic foci can be equally active both with the BBB 'open' and 'closed'; (2) the intravenous GABA-EEG test can detect whether the BBB near the epileptic focus is open to GABA, and (3) anatomic tests of BBB integrity (in these experiments intravenous trypan blue) cannot determine if whether BBB near the focus is 'open' to GABA. Since the intravenous GABA-EEG test reveals the permeability of the BBB in the immediate environment of the epileptic focus, it may be very useful in the selection of a susceptible therapeutic group for inhibitory amino acid therapy.     

Mouse models of neurological disorders: A view from the blood–brain barrier

The number of disease models that involve an aspect of blood–brain barrier (BBB) dysregulation have increased tremendously. The main factors contributing to this expansion have been an increased number of diseases in which the BBB is known to be involved, an increase in the known functions of the BBB, and an increase in the number of models and tools with which those diverse functions can be studied. In many cases, the BBB may be a target of disease; current thinking would include hypertensive encephalopathy and perhaps stroke in this category. Another category are those diseases in which special attributes of the BBB may predispose to disease; for example, the ability of a pathogen to cross the BBB often depends on the pathogen's ability to invoke transcytotic pathways in the brain endothelial or choroid plexus cell. Of special interest are those diseases in which the BBB may be the primary seat of disease or play a major role in the onset or progression of the disease. An increasing number of diseases are so categorized in which BBB dysfunction or dysregulation plays a major role; this review highlights such roles for the BBB including those proposed for Alzheimer's disease and obesity.     

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.     

ApoE deficiency leads to a progressive age-dependent blood-brain barrier leakage

Previously, we reported a defect in the blood-brain barrier (BBB) of apolipoprotein E-deficient (apoE–/–) mice (24). Here, we investigate BBB permeability in wild-type (WT) and apoE–/– mice as a function of age. Both WT and apoE–/– mice showed significantly increased cortical BBB leakage with age. However, in apoE–/– mice, the leakage increased at a 3.7x higher rate compared with WT mice. Surprisingly, the cerebellum showed significantly more leakage than other brain regions across age, while there was no difference between the two hemispheres. To determine the contribution of tissue- vs. blood-borne apoE to vascular permeability, we generated chimeric mice by bone marrow transplantation and measured their BBB leakage. These experiments suggest that both blood- and tissue-derived apoE are equally important for BBB function. In sum, we find an age-dependent defect in the BBB that is exacerbated in apoE–/– mice. Since vascular defects are found in patients with age-related neurodegenerative diseases, such as Alzheimer's, age-related BBB leakage could underlie these defects and may thus be an important contributor to the cumulative neuronal damage of these diseases. apolipoprotein E; aging; age-related neurodegeneration; inflammation     

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.     

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.     

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification

The blood–brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma‐targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high‐throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d‐ amino acids, this approach screens only protease‐resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state‐of‐the‐art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix‐and‐split technique to generate a library based on the following: Ac‐d‐ Arg‐XXXXX‐NH₂, where X were: d‐ Ala (a), d‐ Arg (r), d‐ Ile (i), d‐ Glu (e), d‐ Ser (s), d‐ Trp (w) or d‐ Pro (p). The assays used comprised the in vitro cell‐based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two‐step mass spectrometry approach combining LTQ‐Orbitrap and Q‐trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease‐resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Noninvasive, transient and selective blood-brain barrier opening in non-human primates in vivo

The blood-brain barrier (BBB) is a specialized vascular system that impedes entry of all large and the vast majority of small molecules including the most potent central nervous system (CNS) disease therapeutic agents from entering from the lumen into the brain parenchyma. Microbubble-enhanced, focused ultrasound (ME-FUS) has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. For the first time, the feasibility of transcranial ME-FUS BBB opening in non-human primates is demonstrated with subsequent BBB recovery. Sonications were combined with two different types of microbubbles (customized 4–5 µm and Definity®). 3T MRI was used to confirm the BBB disruption and to assess brain damage.     

Improved Method for the Preparation of a Human Cell-based,Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.     

Frequent blood-brain barrier disruption in the human cerebral cortex

1. The blood–brain barrier (BBB) protects the brain from circulating xenobiotic agents. The pathophysiology, time span, spatial pattern, and pathophysiological consequences of BBB disruptions are not known.2. Here, we report the quantification of BBB disruption by measuring enhancement levels in computerized tomography brain images.3. Pathological diffuse enhancement associated with elevated albumin levels in the cerebrospinal fluid (CSF) was observed in the cerebral cortex of 28 out of 43 patients, but not in controls. Four patients displayed weeks-long focal BBB impairment. In 19 other patients, BBB disruption was significantly associated with elevated blood pressure, body temperature, serum cortisol, and stress-associated CSF readthrough acetylcholinesterase. Multielectrode electroencephalography revealed enhanced slow-wave activities in areas of focal BBB disruption. Thus, quantification of BBB disruption using minimally invasive procedures, demonstrated correlations with molecular, clinical, and physiological stress-associated indices.4. These sequelae accompany a wide range of neurological disorders, suggesting that persistent, detrimental BBB disruption is considerably more frequent than previously assumed.     

载药纳米粒作为脑靶向给药系统的研究

血脑屏障使大部分的活性药物很难由血液进入脑内发挥作用。载药纳米粒具有脑靶向性,可显著提高药物在脑内浓度,成为药物突破血脑屏障的有效途径。本文综述了近年来载药纳米粒透过血脑屏障的研究进展,并对纳米粒载中药入脑提出展望。