scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

scholarly journals 3D hydrogel models of the neurovascular unit to investigate blood-brain barrier dysfunction

2021 ◽  
Author(s):  
Geoffrey Potjewyd ◽  
Katherine Kellett ◽  
Nigel M Hooper
Keyword(s):  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Brain Parenchyma ◽  
Physical Models ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Blood Brain

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood-brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer’s disease. The ability to differentiate induced pluripotent stem cells (iPSCs) to the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs, or to design cell-based therapies.

Effects of Chlorpyrifos Exposure on Acetylcholine Metabolism across a Model Blood-Brain Barrier

2019 ◽  
Vol MA2019-02 (55) ◽  
pp. 2426-2426
Author(s):  
Ethan S. McClain ◽  
Dusty R. Miller ◽  
Jacquelyn A Brown ◽  
John P Wikswo ◽  
David E. Cliffel
Keyword(s):  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Acetylcholinesterase Activity ◽  
Cell Types ◽  
Electrochemical Analysis ◽  
Brain Barrier ◽  
Agricultural Industry ◽  
Tight Junction Formation ◽  
Blood Brain

Organophosphate (OP) compounds, used throughout the agricultural industry as insecticides, are known to directly and irreparably alter brain function in humans. Exposure to OPs decreases acetylcholinesterase activity and leads to a buildup of acetylcholine, with chronic exposure to sub-lethal levels inducing neuropathy. This buildup of acetylcholine can be monitored through electrochemical methods to study the effects of OP toxicity. The microclinical analyzer (µCA), an in vitro microfluidic device allowing for electrochemical analysis using a screen-printed electrode, can be modified with enzymes to detect acetylcholine. Using the µCA in combination with the neurovascular unit (NVU), an organotypic model of the blood-brain barrier (BBB), can provide a better understanding of the BBB forms, functions, and responds to insults. The NVU supports all the cell types necessary for proper BBB formation (endothelial cells, astrocytes, pericytes, and neurons) and provides the flow-created shear forces for mature tight junction formation. The µCA and NVU were used study the effects of chlorpyrifos on acetylcholine concentrations present across the BBB. Understanding the effects of OP like chlorpyrifos on neurotoxicity can contributes to the assessment and treatment of chronic and acute exposure and inform policy decisions around the uses of OP pesticides in the agricultural industry.

Abstract TP86: The Ischemic Blood-brain Barrier In A Dish: Use Of Patient-derived Stem Cells To Model The Response Of The Neurovascular Unit To Oxygen-glucose Deprivation Stress

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Shyanne Page ◽  
Ronak Patel ◽  
Abraham Alahmad
Keyword(s):  
Cell Death ◽  
Ischemic Stroke ◽  
Blood Brain Barrier ◽  
Barrier Function ◽  
Neurovascular Unit ◽  
Barrier Properties ◽  
Cell Types ◽  
Brain Barrier ◽  
Blood Brain

The blood-brain barrier (BBB) constitutes a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes and neurons. During ischemic stroke injury, the BBB constitutes the first responding element resulting in the opening of the BBB and eventually neural cell death by excitotoxicity. A better understanding of the cellular mechanisms underlying the opening of the BBB during ischemic stroke is essential to identify targets to restore such barrier function after injury. Current in vitro models of the human BBB, based on primary or immortalized BMECs monocultures, display poor barrier properties but also lack one or two cellular components of the neurovascular unit.In this study, we designed an integrative in vitro model of the BBB by generating BMECs, astrocytes and neurons using patient-derived BMECs from two iPSC lines (IMR90-c4 and CTR66M). We were able to obtain all three cell types from these two cell lines. iPSC-derived BMECs showed barrier properties similar or better barrier function than hCMEC/D3 monolayer (an immortalized adult somatic BMEC). Furthermore, iPSC—derived astrocytes were capable to induce barrier properties in BMECs upon co-cultures. whereas iPSC-derived neurons were capable to form extensive and branched neurites. Upon OGD stress, iPSC-derived BMECs showed a disruption of their barrier function as early as 6 hours of OGD stress and showed a complete disruption by 24 hours. Such disruption was reversed by reoxygenation. Interestingly such barrier disruption occurs through a VEGF-independent mechanism. In the other hand, iPSC-derived neurons showed a significant decrease in cell metabolic activity preceding neurites pruning. Finally, astrocytes showed the most robust phenotype, as we noted no cell death by 24 hours OGD.In this study, we demonstrated the ability to differentiate three cell types from the same patient in two iPSC lines. We also demonstrated the ability of these cells to respond to OGD/reoxygenation stress in agreement with the current literature. We are currently investigating the molecular mechanisms by which OGD/reoxygenation drive the cellular response in these cell types.

scholarly journals Nitric Oxide Isoenzymes Regulate Lipopolysaccharide-Enhanced Insulin Transport across the Blood-Brain Barrier

Endocrinology ◽  
2008 ◽  
Vol 149 (4) ◽  
pp. 1514-1523 ◽  
Author(s):  
William A. Banks ◽  
Shinya Dohgu ◽  
Jessica L. Lynch ◽  
Melissa A. Fleegal-DeMotta ◽  
Michelle A. Erickson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Brain Barrier ◽  
Mrna Levels ◽  
Insulin Transport ◽  
Blood Brain ◽  
Inducible Nos

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

scholarly journals Design of Experiment Based Optimization of an in Vitro Direct Contact Triculture Blood Brain Barrier Model for Permeability Screening

2021 ◽  
Author(s):  
Kelsey E Lubin ◽  
Gregory T. Knipp
Keyword(s):  
Endothelial Cell ◽  
Blood Brain Barrier ◽  
Direct Contact ◽  
Barrier Properties ◽  
Cell Types ◽  
Brain Barrier ◽  
Seeding Density ◽  
Blood Brain

Abstract Background: The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie the brain microvessel endothelial cells (BMEC). In vivo relevant direct contact between astrocytes, pericytes, and BMECS, to our knowledge, has not been established in conventional Transwell® based in vitro screening models of the BBB. We hypothesize that a design of experiments (DOE) optimized direct contact layered triculture model will offer more in vivo relevance for screening in comparison to indirect models. Methods: Plating conditions including the seeding density of all three cell types, matrix protein, and culture time were assessed in DOEP. DOEP was followed by DOEM1 and DOEM2 to assess the influence of medium additives on barrier properties. The permeability of 4 kD dextran, a paracellular marker, was the measured response to arrive at the optimal plating conditions. The optimized model was further assessed for p-glycoprotein function using a substrate and inhibitor along with a set of BBB paracellular and transcellular markers at varying permeation rates.Results: DOEP revealed that length of culture post endothelial cell plating correlated highest with paracellular tightness. In addition, seeding density of the endothelial cell layer influenced paracellular tightness at earlier times of culture, and its impact decreased as culture is extended. Medium additives had varying effects on barrier properties as seen from DOEM1 and DOEM2. At optimal conditions, the model revealed P-gp function along with the ability to differentiate between BBB positive and negative permeants. Conclusions: We have demonstrated that the implementation of DOE based optimization for biologically based systems is an expedited method to establish multi-component in vitro cell models. The direct contact BBB triculture model reveals that the physiologically relevant layering of the three cell types is a practical method of culture to establish a screening model compared to indirect plating methods that incorporate physical barriers between cell types. Additionally, the ability of the model to differentiate between BBB positive and negative permeants suggests that this model may be an enhanced screening tool for potential neuroactive compounds.

scholarly journals Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B0AT2 and y+LAT1 functional cooperation

2021 ◽  
pp. 0271678X2110395
Author(s):  
Mehdi Taslimifar ◽  
Martin Faltys ◽  
Vartan Kurtcuoglu ◽  
François Verrey ◽  
Victoria Makrides
Keyword(s):  
Amino Acid ◽  
Blood Brain Barrier ◽  
Cell Model ◽  
Cell Types ◽  
Brain Barrier ◽  
In Vivo Studies ◽  
Amino Acid Transporters ◽  
Blood Brain

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple “receptors” may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B0AT2 (B0AT2), and SLC7A7/y+LAT1 (y+LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y+LAT1), facilitative diffusion (LAT4), and sodium symporter (B0AT2) transporters.

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

2005 ◽  
Vol 289 (5) ◽  
pp. H2012-H2019 ◽  
Author(s):  
Melissa A. Fleegal ◽  
Sharon Hom ◽  
Lindsay K. Borg ◽  
Thomas P. Davis
Keyword(s):  
Blood Brain Barrier ◽  
Paracellular Permeability ◽  
Cell Permeability ◽  
Brain Barrier ◽  
Cerebral Microvessels ◽  
Permeability Changes ◽  
Blood Brain ◽  
Brain Microvessel

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

Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models

2003 ◽  
Vol 31 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Hanna Tähti ◽  
Heidi Nevala ◽  
Tarja Toimela
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Three Dimensional ◽  
Brain Barrier ◽  
Culture Methods ◽  
Blood Brain ◽  
Capillary Endothelial Cells ◽  
In Vitro Bbb Model

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.

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