Blood-brain Barrier Physiology Research Articles

In vitro cerebrovascular modeling in the 21st century: current and prospective technologies.

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-brain Barrier.

Quantitative fluorescence microscopy provides high resolution imaging of passive diffusion and P-gp mediated efflux at the in vivo blood–brain barrier

Quantitative fluorescent microscopy is an emerging technology that has provided significant insight into cellular dye accumulation, organelle function, and tissue physiology. However, historically dyes have only been used to qualitatively or semi-quantitatively (fold change) determine changes in blood–brain barrier (BBB) integrity. Herein, we present a novel method to calculate the blood to brain transfer rates of the dyes rhodamine 123 and Texas red across the in situ BBB. We observed that rhodamine 123 is subject to p-glycoprotein mediated efflux at the rat BBB and can be increased nearly 20-fold with p-glycoprotein inhibition. However, Texas Red appears to not be subject to MRP2 mediated efflux at the rat BBB, agreeing with literature reports suggesting MRP2 may lack functionality at the normal rat BBB. Lastly, we present data demonstrating that once dyes have crossed the BBB, diffusion of the dye molecule is not as instantaneous as has been previously suggested. We propose that future work can now be completed to (1) match BBB transfer coefficients to interstitial diffusion constants and (2) use dyes with specific affinities to cellular organelles or that have specific properties (e.g., subject to efflux transporters) to more fully understand BBB physiology.

Reliable permeability assay system in a microfluidic device mimicking cerebral vasculatures

Since most of the bioavailable drugs are impermeable through the blood-brain barrier (BBB), development of a rapid and reliable permeability assay system has been a challenge in drug discovery targeting central nervous system (CNS). Here, we designed a microfluidic device to monitor the drug permeability into the CNS. Human umbilical vein endothelial cells (HUVECs) were shortly (2 ~ 3 h) incubated with astrocyte-conditioned medium after being trapped on microholes in the microfluidic device and tested for chip-based permeability measurement of drugs. The measured permeability values were highly correlated with those measured by conventional in vitro methods and the brain uptake index representing the quantity of transported substances across the in vivo BBB of rats. Using the microfluidic device, we could easily monitor the effect of hydrogen peroxide on the trans-endothelial permeability, which are consistent with the finding that the same treatment disrupted the formation of tight junctions between endothelial cells. Considering relatively short period of time needed for endothelial cell culture and ability to monitor the BBB physiology continuously, we propose that this novel system can be used as an invaluable first-line tool for CNS-related drug development.

Blood-brain barrier unlocked

The brain is protected by a physiological blood-brain barrier (BBB) against toxins and some metabolites circulating in the blood. At the same time, the BBB limits penetration into the brain of many neuroactive drugs. Efficient ways to increase BBB permeability for delivery of drugs of different chemical nature into the brain are unknown. This work deals with delivery into the brain of 10(-2) M dopamine, a substance that does not penetrate the BBB under normal circumstances. It was studied in two independent experiments: (i) penetration of (3)H-labeled dopamine from its mixture with 10(-5) M H2O2 into hypothalamus and striatum structures of intact rat brain, and (ii) effect of unlabeled dopamine from a mixture with H(2)O(2) on the rat motor activity in a haloperidol catalepsy model. It was shown that (i) at the third minute after nasal application of the dopamine + H(2)O(2) mixture, the dopamine level increases 45-fold in the hypothalamus and almost 30-fold in the striatum and (ii) motility of animals in the catalepsy haloperidol model is recovered 90 sec after intranasal introduction of dopamine. No such effects were observed after replacement of H(2)O(2) by 0.9% NaCl solution. Thus, it was shown on the example of dopamine that its introduction into the nasal cavity simultaneously with H(2)O(2) provides for rapid delivery of the drug into the brain. These results expand our knowledge concerning the biological role of exoROS in modulating BBB permeability and may contribute to the development of a new therapeutic strategy for neurological diseases.

Developmental Aspects of the Intracerebral Microvasculature and Perivascular Spaces: Insights into Brain Response to Late-Life Diseases

The development of the microvasculature of the human cerebral cortex offers insight into the response of the cerebral cortex to later-life brain injury. We describe the 3 basic and distinct components of the developmental anatomy of the cerebral cortical microvascular system. The first compartment is meningeal and, therefore, extracerebral. In addition to the major venous sinuses, arachnoidal arteries, and veins, the pial anastomotic capillary plexus that covers the surface of the developing and adult cerebral cortex represents the source of thepenetrating vessels that become the second component, the intracerebral extrinsic microvascular compartment. During embryogenesis, sprouting vascular elements from pial capillaries pierce the brain's external glial limiting membrane and penetrate the cortex. These vessels, which eventually differentiate into arterioles and venules, are separated from the cortical tissue by the extravascular Virchow-Robin compartment (V-RC) formed between the internal vascular and the external glial basal laminae. The V-RC remains open to the meningeal interstitial spaces and outside the blood-brain barrier (BBB) and acts asa prelymphatic drainage system for removal of substances that cannot be transported into the blood or catabolized intracellularly. The third element is the dense intracerebralintrinsic microvascular compartment. Intracerebral capillary vessels sprout from the perforating vessels, penetrate through the Virchow-Robin glial membrane, and enter the neuropil. Intracerebral capillaries lack smooth muscle and a V-RC and consist only of endothelial cells separated from the intracerebral space by a basal lamina. Their role as the physiological BBB is the exchange of oxygen, glucose, and small molecules. This developmental perspective highlights 3 principles: (a) the V-RC is intimately related to the cortical penetrating arterioles and venules and represents an inefficient protolymphatic system that lacks the anatomic and physiological constituents found in lymphatic beds elsewhere in the body; (b)the anatomic contiguity of the V-RC and the penetrating vascular compartment (arterioles and venules) implies that the pathology in 1 compartment could lead to dysfunction in the others; and (c) the anatomic localization of the immunologic BBB at the level of the penetrating venules might impose constraints on immunologically mediated transport involving the V-RC.

Modelling the Neurovascular Unit and the Blood-Brain Barrier with the Unique Function of Pericytes

The blood-brain barrier (BBB) is a dynamic cellular complex that is responsible for the maintenance of brain homeostasis. To understand the BBB's key cellular and molecular mechanisms, in vitro models combining endothelial cells and astrocytes can be used to reproduce most of the barrier's in vivo features (low paracellular permeability and the expression of specific transporters). However, these models lack pericytes - a poorly characterized cell type which appears to be of crucial importance to understand BBB's function in healthy and diseased states. The present study sought to identify and characterize this cell population - which lacks a specific marker - by comparing its phenotype with that of vascular smooth muscle cells. Even if pericytes and smooth muscle cells shared many markers in vitro, our results showed that they could be distinguished by their different P-glycoprotein expression and γ-glutamyltranspeptidase activity. Two different three-cell-type culture models were described, including pericytes to mimic the neurovascular unit. In the first model, endothelial cells were cultured alone on a filter, away from glial cells and pericytes, allowing endothelial cell phenotype characterization. In the second model, glial cells were at the bottom of the well while pericytes and endothelial cells were cultured together in the filter: close interactions were observed in peg-and-socket contacts. In both models low paracellular permeability and P-glycoprotein functionality were demonstrated. These models are likely to be useful tools for understanding the pericytes' role in BBB physiology and could be of value in investigating the pericytes' influence on BBB in diseased states.

In Vitro Modeling of the Blood-Brain Barrier: Simplicity Versus Complexity

Proper understanding of blood-brain barrier (BBB) regulation is crucial to reduce/prevent its disruption during injury. Since high brain complexity makes interpretation of in vivo data challenging BBB studies are frequently performed using simplified in vitro models. Although such models represent an important and frequently employed alternative for investigation of BBB function and alterations, our ability to translate in vitro findings to in vivo situation remains sub-optimal. Consequently, despite the fact that our knowledge of the cellular and molecular mechanisms underlying BBB physiology and pathophysiology is constantly increasing, our ability to modulate barrier function remains virtually non-existent. Classical in vitro model systems have provided a wealth of knowledge until now, but it is now evident that newer in vitro models that are more representative of the in vivo situation are needed to further our understanding of barrier physiology. This paper will provide an overview of the BBB cellular components and the most frequently used in vitro BBB model systems. I will discuss their advantages and disadvantages, as well as highlight recently developed models that more closely mimic the BBB in vivo.

Neuronal and BBB damage induced by sera from patients with secondary progressive multiple sclerosis

An important component of the pathogenic process of multiple sclerosis (MS) is the blood-brain barrier (BBB) damage. We recently set an in vitro model of BBB, based on a three-cell-type co-culture system, in which rat neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological BBB. Herein we report that the serum from patients with secondary progressive multiple sclerosis (SPMS) has a damaging effect on isolated neurons. This finding suggests that neuronal damaging in MS could be a primary event and not only secondary to myelin damage, as generally assumed. SPMS serum affects the permeability of the BBB model, as indicated by the decrease of the transendothelial electrical resistance (TEER). Moreover, as shown by both immunofluorescence and Western blot analyses, BBB breaking is accompanied by a decrease of the synthesis as well as the peripheral localization of occludin, a structural protein of the tight junctions that are responsible for BBB properties.

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 cm 2 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 ( R 2 = 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.

Assays to Predict Drug Permeation Across the Blood-Brain Barrier, and Distribution to Brain

Drug discovery programmes to target or avoid the brain need to take into account the properties of the blood-brain barrier (BBB). The importance to CNS PK of the free drug concentration in brain is increasingly recognised, and assays for drug discovery programmes are being adjusted accordingly. In vitro models of the BBB continue to play an important role in this process. Good cell-based models using brain endothelium have been developed and validated for mechanistic studies, and some are suitable for medium to high throughput permeability screening and toxicology. Brain homogenate and brain slice methods allow estimation of drug partition into brain. In combination with in silico and in vivo models, the portfolio of methods establishing and predicting CNS drug PK is now very powerful, allowing much more accurate iterative feedback to chemists to optimise compound profiles through the drug discovery and development programme. The advantage of using models based on real BBB cellular anatomy and physiology is that they have the power to reveal and incorporate previously undiscovered properties, such as new transporters, metabolic enzymes and modulation, to form the basis for models mimicking neurological disorders as well as normal function, and to allow physiologically-based pharmacokinetic (PBPK) extrapolation from animal models to humans.

Cancer and the blood–brain barrier: ‘Trojan horses’ for courses?

The blood-brain barrier (BBB) limits the bioavailability of most bioactive molecules and drugs in the CNS, leaving clinicians with only a few options for pharmacotherapy. In this issue Regina et al. demonstrate that a 'Trojan horse' drug conjugate, acting as a substrate of a physiological BBB receptor that facilitates transcytosis, significantly improves drug transport into the CNS. Specifically, the low-density lipoprotein receptor-related protein (LRP) is used to carry a conjugate of paclitaxel and Angiopep-2, an aprotinin-derived peptide, across the BBB. Interestingly, in its conjugated form paclitaxel circumvents the efflux pumps at the BBB but still retains its activity against microtubules. Importantly, the authors were able to demonstrate improved therapeutic efficacy of this approach in orthotopic models of primary and metastatic brain cancer. This proof-of-principle study thus represents a milestone for drug delivery across the BBB but also a starting point for studies exploring wider applicability and potential limitations of the approach.

Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function

Basolateral condition of the brain microvascular endothelium is believed to influence blood–brain barrier (BBB) phenotype, although the precise transcriptional and post-translational mechanisms involved are poorly defined. In vivo, the basolateral surface of the blood–brain endothelium is bathed in serum-free interstitial fluid and encompassed by astrocytic end-feet. We hypothesized that these conditions impact on BBB function by directly modulating expression and biochemical properties of tight junctions. To investigate this, an in vitro transwell culture model was employed to selectively modify the basolateral environment of bovine brain microvascular endothelial cells (BBMvECs). In the absence of basolateral (but not apical) serum, we observed higher levels of expression, association and plasma membrane localization for the tight junction proteins, occludin and zonula occludens-1 (ZO-1), in parallel with elevated transendothelial electrical resistance (TEER) and reduced 14[C]-sucrose permeability of BBMvEC monolayers. We further examined the effects of non-contact co-culture with basolateral astrocytes (C6 glioma) on indices of BBMvEC barrier function in both the presence and absence of serum. Astrocyte co-culture with serum led to enhanced occludin protein expression, occludin/ZO-1 association, and ZO-1 membrane localization, in parallel with increased TEER of BBMvEC monolayers. Astrocyte co-culture in the absence of serum (i.e. basolateral conditions most consistent with in vivo BBB physiology) however, gave the highest increases in BBMvEC barrier indices. Thus, we can conclude that factors influencing condition of the basolateral environment of the brain microvasculature can directly, and independently, modify BBB properties by regulating the expression and biochemical properties of the tight junction proteins, occludin and ZO-1.

Neutrophils and the blood–brain barrier dysfunction after trauma

Despite the fact that neutrophils are essential for the protection from invading pathogens, hyperactive neutrophils may elicit detrimental cerebral damage after severe trauma. The neutrophil interactions with the neurovascular unit entail endothelial dysfunction involving endothelial leakage, formation of edema, coagulation abnormalities, disturbed hemodynamics, tissue infiltration etc. These elements of the "whole body inflammation," designated systemic inflammatory response syndrome (SIRS) in conjunction with intracerebral proinflammatory activities, are important triggers of post-traumatic cerebral damage and mortality according to the "second hit" concept. From the immunologic point of view, the brain is an immune privileged site, known to resist autodestructive inflammatory activity much more efficiently than other organs because of the highly efficient diverse functions of the blood-brain barrier (BBB). However, both the underlying strategy of the BBB to maintain cerebral protecting functions against the post-traumatic neutrophil-mediated "second hit" and how activated neutrophils may overcome the BBB are currently unknown. Therefore, this review summarizes the current understanding of the "second hit," the BBB physiology, and its role in the maintenance of cerebral immune privilege, and discusses recent findings that may explain the pathophysiologic neutrophil-BBB interactions occurring after severe trauma, thus offering novel therapeutic options to protect from post-traumatic brain damage.

Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice.

Tight junctions are well-developed between adjacent endothelial cells of blood vessels in the central nervous system, and play a central role in establishing the blood-brain barrier (BBB). Claudin-5 is a major cell adhesion molecule of tight junctions in brain endothelial cells. To examine its possible involvement in the BBB, claudin-5–deficient mice were generated. In the brains of these mice, the development and morphology of blood vessels were not altered, showing no bleeding or edema. However, tracer experiments and magnetic resonance imaging revealed that in these mice, the BBB against small molecules (<800 D), but not larger molecules, was selectively affected. This unexpected finding (i.e., the size-selective loosening of the BBB) not only provides new insight into the basic molecular physiology of BBB but also opens a new way to deliver potential drugs across the BBB into the central nervous system.

Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro

A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.