The blood-brain barrier (BBB) is a critical biological interface that maintains brain homeostasis by regulating the entry of substances, ions, and xenobiotics into the brain’s extracellular space. While it protects the brain, it also poses a significant challenge for drug development targeting central nervous system (CNS) disorders and brain cancers. Various biological and analytical methods have been developed to study the BBB’s role in drug delivery and CNS-PNS (Peripheral Nervous System) communication.

Key Components and Functions of the BBB

• Brain Microvessel Endothelial Cells (BMVECs): These cells form tight junctions (TJs) that restrict the passive transport of small hydrophilic molecules. The tightness of these junctions is measured by transendothelial electrical resistance (TEER).
• Physical and Electrostatic Barriers: The BBB’s surface is highly anionic, creating an electrostatic barrier for negatively charged compounds. It also includes metabolic barriers with enzymes that convert substrates into less permeable or toxic forms.
• Immunological Barrier: The BBB prevents bacteria and viruses from entering the brain.

Roles of the BBB

1. Maintaining Homeostasis: Prevents uncontrolled influx of molecules that could disrupt neuronal activity or cause brain damage.
2. Nutrient Supply: Uses specific transport systems for essential molecules like glucose, insulin, and amino acids.
3. Protection Against Toxins: Employs transport proteins to efflux undesirable compounds.
4. Immune Response: Directs inflammatory cells to respond to changes in the neurovascular space.
5. Chemical Messaging: Facilitates communication between the CNS and PNS, involving substances like cytokines and neuropeptides.

Transport Mechanisms Across the BBB

1. Transcellular Passive Diffusion: For small lipophilic compounds.
2. Carrier-Mediated Transport: For essential polar molecules like amino acids and glucose.
3. Receptor-Mediated Endocytosis: For large peptides and proteins.
4. Adsorptive-Mediated Endocytosis: For cationic molecules.
5. TJ Modulation: Allows nonspecific passage of molecules when TJs are disrupted.

Importance of the BBB

Understanding the BBB is crucial for developing effective treatments for neurological and psychiatric disorders, and brain tumors, and for maintaining the brain’s chemical messaging system. Compromised BBB integrity can lead to immune or inflammatory responses, while effective drug delivery requires overcoming the BBB’s restrictive properties.

Methods for studying the BBB

Two primary methods for studying the BBB are:

1. In Vivo Methods for Studying the Transport of Substances Across the Blood-Brain Barrier
2. In Vitro Models of the Blood-Brain Barrier

In Vivo methods

In response to the growing interest in the blood-brain barrier (BBB) for understanding and treating neurological diseases, various methods have been developed to investigate BBB transport. These methods range from in silico and cell-culture models to live animal studies and positron emission tomography (PET). Key Methods for Studying BBB Transport are as following:

Intravenous Injection Methods:

The most common approach involves administering a compound intravenously to an animal and measuring its concentration in the brain, plasma, and cerebrospinal fluid over time. This method keeps physiological and metabolic systems intact but requires a separate animal for each data point, leading to high variability and the need for large sample sizes.

Brain Perfusion Techniques:

This method involves directly infusing a compound dissolved in artificial blood, plasma, or saline into the heart or a major vessel leading to the brain. It allows for controlled experiments and the use of radiolabelled compounds for detection. However, it is labor-intensive and requires a new animal for each experiment.

Tomographic Methods:

Positron emission tomography (PET) and single-photon emission-computed tomography (SPECT) are used to study brain uptake kinetics, cerebral blood flow, BBB integrity, and efflux mechanisms. These non-invasive techniques provide high-resolution images and can be used on both humans and animals. However, they require expensive instrumentation and radiolabelled compounds, which can have short half-lives and different transport properties.

Microdialysis Sampling:

Developed in the 1970s, this minimally invasive method monitors neurotransmitters and other substances in the brain. It involves implanting a probe in the brain or other tissue to sample the chemical makeup of the interstitial fluid. This method allows for long-term sampling on a single animal, reducing the number of animals needed for statistically significant results. It can measure concentrations of drugs, neurotransmitters, and metabolic markers simultaneously.

In Vitro Models

In vitro models, particularly cell-culture systems, are essential tools for screening BBB permeability to drugs and other compounds. These models help investigate the metabolic components of the BBB and their effects on transport.

Primary Brain Microvessel Endothelial Cells (BMVECs)

• Bovine and Porcine Models: Bovine (BBMECs) and porcine (PBMECs) brain microvessel endothelial cells are commonly used. These cells are grown to confluency on polycarbonate membranes and used in Side-by-Side™ diffusion chambers to study bidirectional permeation.
• Advantages: This setup allows for temperature control, constant stirring, and manipulation of variables like temperature and compound concentration to distinguish between passive and active transport processes.
• Efflux Transporters: BBMECs express efflux transporters like P-glycoprotein (P-gp), which are crucial for studying drug delivery obstacles.

Immortalized Cell Lines

• Development: Immortalized brain endothelial cell lines have been developed to reduce the time to reach confluency and lessen the workload. These lines form monolayers but not complete tight junctions, making them useful for studying endothelial uptake but not transport.
• Alternative Models: The Mardin-Darby canine kidney cell line transfected with the MDR1 gene is a notable non-cerebral epithelial model for BBB studies.
• Co-Culture Systems: These systems involve endothelial cells and astrocytes to study the role of astrocytes in tight junction formation.

Methods for Measuring Transport and Metabolism

• Analytical Techniques: Sensitive analytical methods are required to monitor transport across cell monolayers. Factors to consider include drug concentrations, labelling capabilities, sample volume requirements, and matrix compatibility.

• Permeability Markers: Control compounds like radiolabelled sucrose or fluorescein assess cell monolayer integrity.

• Scintillation Counting: Used for competitive studies to determine carrier-mediated processes. It is simple but costly due to the need for labeled compounds.

• Capillary Electrophoresis (CE): Suitable for small sample volumes and sensitive detection, though it requires sample derivatization.

• Liquid Chromatography/Mass Spectrometry (LC-MS): Effective for analyzing compounds in high-salt matrices, with methods to protect MS equipment from damage.

• Microfluidic applications: Microfluidic devices have recently been used to study the transport and metabolism of substances by endothelial cells and the release of signaling compounds. These devices, made from biocompatible materials, can integrate multiple functions such as cell culture, stimulation, and analysis into a single platform. They mimic physiological conditions by controlling channel size and fluid flow, allowing for better sensitivity in detecting secreted molecules. Nitric oxide (NO), a vasodilator that increases BBB permeability, can be directly measured using microfluidic devices. These devices allow for the detection of NO release from cells with high sensitivity and temporal resolution. Researchers have constructed devices that model vasculature and monitor NO release from erythrocytes, providing realistic in vitro representations of blood vessel circulation. Microfluidic platforms can also be used for high-throughput analysis, with multiple detection wells fabricated onto a single chip. These devices are useful for BBB transport and metabolism studies. For example, a microchip with cell-culture chambers and fluidic networks can study substance permeation across cell layers. Another device monitors the release of dopamine and norepinephrine from PC-12 cells, using pneumatic valves to isolate cell perfusion channels and separate analytes electrophoretically. Overall, microfluidic devices offer significant advantages in studying BBB transport and metabolism, providing precise control and high sensitivity in a compact, integrated format.

Why Use FITC-Dextran and TRITC-Dextran?

FITC-dextran and TRITC-dextran are fluorescently labeled dextrans used to study the permeability of the BBB. FITC-dextran is ideal for experiments where pH sensitivity is crucial. It is often used to monitor dynamic changes in pH within cells or tissues, which can be important in various physiological and pathological processes. On the other hand, TRITC-dextran is preferred when pH stability is needed. It is particularly useful in studies where the pH remains constant, such as in microcirculation and permeability studies.

Combining FITC-dextran and TRITC-dextran allows researchers to leverage the strengths of both dyes. This dual labeling can provide comprehensive data on the blood-brain barrier’s (BBB) permeability under varying pH conditions. For instance, FITC-dextran can indicate pH changes, while TRITC-dextran provides a stable reference. This combination is particularly useful in intravital microscopy, where real-time monitoring of pH and permeability is required.

Another significant reason for using both FITC-dextran and TRITC-dextran simultaneously is to distinguish between the two colors. This distinction is particularly useful in experiments where different aspects of vascular integrity and permeability need to be assessed. High molecular weight FITC-dextran (e.g., 70 kDa) can be used to demonstrate that the blood vessels are intact and not damaged. Its larger size means it should not pass through an intact BBB, thus serving as a marker for vessel integrity. Smaller-sized TRITC-dextran can be used to show permeability. If the BBB is compromised, TRITC-dextran will pass through, indicating areas of increased permeability.

By using both dyes, researchers can simultaneously assess the integrity of the blood vessels and the permeability of the BBB, providing a more comprehensive understanding of the barrier’s function and any pathological changes.

Selecting Molecular Weights

The molecular weight of dextrans is crucial for studying the blood-brain barrier (BBB) permeability. Low molecular weight dextrans, such as 4 kDa, can pass through the BBB more easily, making them suitable for studying the barrier’s tight junction integrity. However, there are considerations about the selected molecular weight as dextrans with a molecular weight below 15 kDa are filtered unrestricted by the kidneys, resulting in a short elimination half-life. The elimination of dextrans depends on the carbohydrate chain length, administration route, and molecular weight.  For example in humans, dextran 1 has a half-life of about 2 hours, dextran 40 about 10 hours, and dextran 60 about 42 hours. Therefore, the quick removal of low molecular weight dextrans by the kidneys should be considered, as they may not be visible for long in monitoring studies. Low molecular weight dextrans, such as 3-5 kDa, are rapidly filtered by the glomerulus and are used to study the filtration capacity and permeability of the glomerular barrier. Medium molecular weight dextrans, such as 40 kDa, are used to assess the selective permeability of the BBB. High molecular weight dextrans, like 70 kDa, are used to test the barrier’s ability to restrict the passage of large substances, which is critical for maintaining the brain’s protected environment.

References

1. Sun, H., Hu, H., Liu, C. et al. Methods used for the measurement of blood-brain barrier integrity. Metab Brain Dis 36, 723–735 (2021). https://doi.org/10.1007/s11011-021-00694-8
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