The Blood-Brain Barrier

Functional Anatomy of the Blood-Brain Barrier

The BBB consists of astrocytes, pericytes, microglia, neurons, and the extracellular matrix (ECM), all of which play a supportive role in maintaining integrity of this structure. ^, The concept of the neurovascular unit has recently been married to the BBB concept to illustrate the fact that an intimate contact exists between BBB cells (endothelial cells and perivascular glia) and the brain parenchyma (principal neurons, interneurons, microglia, oligodendroglia, and astroglia). More recently, it has become clear that cells involved in the production and reabsorption of CSF also need to be considered as members of the neurovascular unit. This is discussed in more detail later.

This deceptively simple anatomic structure conceals a complex, multifaceted network of pathways and interactions that create a far more dynamic unit than the usual barrier function descriptor implies. Increasing characterization of BBB structure and function has been facilitated by the identification of BBB pathology in neurological disorders such as stroke, epilepsy, and neuroinflammation, among others. The anatomic BBB is formed by a monolayer of microvascular endothelial cells (ECs) that line the intraluminal space of brain capillaries and are supported by astrocyte end-feet. The low permeability that characterizes the BBB is conferred in part by tight packing of ECs and formation of TJs and adherens junctions (AJs). The EC layer has a luminal (inside the brain) and abluminal (outside the brain) membrane separated by 300 to 500 nm of cytoplasm between the blood and brain. The luminal surface is in contact with the blood supply, whereas on the abluminal surface, ECs are enveloped in a basement membrane and separated into regions associated with a pericyte. Neighboring cells are connected by TJs and AJs. TJs regulate paracellular transport and consist of occludin, claudin, and junctional adhesion molecules. AJs mediate cell spacing and transmit cytoskeletal signals and include cadherin, catenins, vinculin, and actinin ( Fig. 68.1 ).

There are two fundamental morphologic characteristics that differentiate brain ECs from peripheral ECs. The cytoplasm of brain microvascular ECs has rare pinocyte vesicles—fluid-filled cell membrane invaginations that allow compounds to cross the BBB. These ECs also contain a greater concentration of mitochondria, presumably to generate the greater energy needed to actively transport nutrients.

In addition to the structural integrity of the BBB, there is an enzymatic surveillance system that metabolizes drugs and other compounds that manage to bypass the structural barrier. Three main catalytic agents regulate transportation across the BBB: γ-glutamyl transpeptidase, alkaline phosphatase, and aromatic l -amino acid decarboxylase. These are concentrated in cerebral vessels. The role of cytochrome P-450 (CYP) enzymes at the BBB has taken on a greater significance in local drug metabolism. Heterogeneity in the activity and expression of these enzymes may impact variability in drug response or have other pathologic implications ( Box 68.1 ).

BOX 68. 1

Metabolic Activity at the Blood-Brain Barrier

Cytochrome P-450 (CYP) enzymes are essential for the metabolism of many drugs. Although this molecular family comprises more than 50 enzymes, a handful of CYP enzymes are responsible for the metabolism of 90% of drugs. These include CYP3A4, CYP2D6, CYP2E1, CYP2C9, and CYP2C19. CYPs are abundant in the liver, but appreciable amounts are also expressed in many extrahepatic tissues, including the brain. Although the total amount of CYP is less in the brain than in the liver, the localized expression of these drug-metabolizing enzymes at the blood-brain barrier and the neurovascular unit provides a potent mechanism of biotransformation in proximity to neurons. ^{, ,} CYP enzymes catalyze the formation of neurosteroids and eicosanoids, and metabolize substrates as diverse as vitamins A and D, centrally acting drugs, anesthetics, and environmental neurotoxins. These unique characteristics allow this family of proteins to control vital functions such as neurotrophic support, neuroprotection, control of cerebral blood flow, and maintenance of brain cholesterol homeostasis.

CYP enzymes can be inhibited or induced by drugs, causing clinically significant drug-drug interactions leading to unanticipated adverse reactions or unexpected therapeutic benefits. Thereby, changes in the function of brain CYP metabolism can influence drug response, drug toxicity, and drug-induced behavioral changes. As an example, metabolism of drugs by CYP enzymes causes biotransformation of an antiepileptic drug, carbamazepine, into a proconvulsant toxic metabolite. Finally, central nervous system drug metabolism may contribute to interindividual variation in response because brain CYP enzymes have unique regional induction patterns that are regulated independently of their hepatic isoforms. Understanding these different aspects of CYP enzymes in brain function and physiology is essential for improved future drug therapy and reduced neurotoxic side effects.

There is charge polarity between the abluminal and luminal surfaces of ECs. This polarity influences permeability of the barrier and is regulated in part by TJs. Differential receptor expression and density on the luminal versus abluminal surfaces allow for directional transport of solutes such as glucose and ions such as sodium, potassium, and calcium. EC polarity plays a significant role in generating interstitial fluid and maintaining homeostatic gradients within the interstitial fluid. Perturbation of transporter activity has been implicated as a potential cause of edema following ischemic stroke. Differential expression of luminal versus abluminal ion transporters is still being characterized; however, transporters such as Na ⁺ ,K ⁺ -ATPase, the Na ⁺ /H ⁺ exchange transporter, and the Na ⁺ amino acid transporter are found predominantly on the abluminal membrane. Other shuttling proteins that contribute to transport polarity include glucose transporter protein 1 (GLUT1), which is concentrated at the abluminal membrane, and drug efflux pumps, which are differentially distributed between luminal and abluminal membranes. P-glycoprotein (aka multidrug resistance protein 1 [MDR1]), which is the product of the human multidrug resistance 1 gene ( MDR1 ), is concentrated at the luminal membrane. Enzyme polarity is also present, with examples including alkaline phosphatase and γ-glutamyl transpeptidase, which are concentrated in the luminal compartment. Redistribution of multidrug resistance transporters occurs during pathologic events involving BBB breakdown, presumably to enhance clearance of toxins, and is relevant for delivery of drugs to the central nervous system (CNS). BBB polarity is additionally maintained by astrocytes, in which distribution of aquaporin-4 plays a significant role in water absorption and potentially in edema formation.

The neurovascular unit is made up of several key cell types in addition to the brain ECs, which include astrocytes, pericytes, neurons, and microglia. Astrocytes reinforce barrier function and contact ECs on the abluminal side of the BBB endothelium. Astrocytes have end-feet that border the basement membrane of parenchymal vessels and densely surround TJs, reducing the size of the gap between ECs. More than 90% of astrocyte foot processes surround ECs. They are associated with adrenergic and cholinergic nerve terminals, as well as those that respond to peptides ( Fig. 68.2 ).

Astrocytes appear to be critical in regulating BBB permeability, and the majority of in vitro experiments have demonstrated BBB compromise in their absence. ^, Several studies, however, have indicated that BBB integrity is retained amid degradation of astrocytes, thus suggesting that astrocytes regulate BBB activity indirectly rather than through physical interaction. Astrocytes are able to induce both the barrier and permeability properties of the endothelium. This was first demonstrated in 1981 by Stewart and Wiley, who found that newly formed vessels originating from the coelomic cavity display BBB characteristics when placed in contact with grafts of neural tissue. Later, Janzer and Raff demonstrated that astrocytic aggregates induced a functional BBB in nonbrain ECs of the anterior chamber of the eye. Astrocyte media can also be used to induce BBB-like properties in ECs in vitro. Thus the mechanism of astrocyte-mediated induction, maintenance, and obliteration of the BBB is only partially understood. To address this issue, researchers have described a “BBB transcriptome” as a means to better understand the complexity and dynamic changes that occur at the brain-blood interface.

Efforts to develop a microfluidic model of the BBB—a “BBB on a chip”—have attempted to recapitulate barrier function by incorporating sheers stress into a variety of culture systems, for example, EC-astrocyte co-culture with cell types separated by a porous membrane ( Box 68.2 ). Models incorporating fluid flow and shear stress have achieved higher transendothelial electrical resistance than static models, suggesting more effective barrier function. These systems have been used to test drug permeability and may eventually be effective tools to study pharmaceutical approaches to modulating BBB integrity.

BOX 68.2

In Vivo and In Vitro Modeling of the Blood-Brain Barrier

As the blood-brain barrier (BBB) rises to prominence in a wide variety of neurological research studies, efforts to model both the intact and dysfunctional BBB have led to development of many animal and in vitro models. Efficient and accurate BBB modeling would be a significant step forward in drug screening for compounds able to permeate the BBB as well as in investigation of BBB dysfunction in pathologic conditions such as multiple sclerosis, stroke, and epilepsy.

In vivo modeling of the BBB has utilized rat and murine transgenics to assess the importance of various BBB components and signaling pathways to barrier function and integrity. Transgenic models, particularly knockouts, have been used to characterize the role of efflux and influx transporters (e.g., ATP-binding cassette and glucose transporter protein 1), aquaporin-4, matrix metalloproteinases, calveolin, and angiogenic factors such as vascular endothelial growth factor in BBB function. Other models have assessed the role of pericytes, tight junction proteins, integrins, selectins, ^, and chemokines in BBB integrity and leukocyte extravasation. ^, One novel model to investigate BBB permeability during development and selected interventions utilized a zebrafish with a blood-borne endogenous fluorescent tracer.

In vitro models have emphasized the use of endothelial cell–astrocyte co-cultures, or more recently tricultures with pericytes and have primarily utilized porcine, rat, murine, and bovine cells. The role of astrocytes in conferring BBB properties on endothelial cells in conjunction with the importance of flow and shear stress in BBB function has clearly demonstrated the need for integrated modeling systems incorporating these components. Microfluidic systems have allowed for the development of compact BBB models that include fluid flow. These systems have the potential for facilitating high-throughput screening of central nervous system–targeted drugs. However, significant characterization of transendothelial electrical resistance measurement, permeability, and other parameters is required to determine the validity of each model. The fundamental role of intraluminal shear stress has been emphasized, and recent modeling efforts have shown that BBB function is modulated by shearing forces acting on the endothelial membrane. ^, Models have also been developed to allow for leukocyte adhesion studies under flow or altered flow conditions.

With increasing improvements in protocols for differentiation of induced pluripotent stem cells (iPSCs), BBB models using human iPSCs may hold promise for the future. This approach would not only address the challenges of using nonhuman cells to model the human BBB, but would also open up new avenues for research addressing the genetic components of diseases such as multiple sclerosis or drug-resistant epilepsy. However, widespread application of this method will require further improvement in differentiation protocols and further characterization of the resulting cells.

Pericytes are undifferentiated contractile connective tissue cells that localize to capillary walls, aid in regulating the growth of ECs, and modulate the integrity of capillary cells. They share a common basement membrane with brain ECs and act as a secondary BBB in a manner similar to macrophages by monitoring the perivascular space and phagocytosing unwanted substances. Although pericytes lack contractile actin, they have been implicated in regulation of cerebral blood flow (CBF) and in pathologic states such as the no-reflow phenomenon of impaired blood flow following an ischemic stroke.

In vitro studies have demonstrated communication between ECs and pericytes. The proposed mechanism of this communication is via cellular projections, which penetrate the basal lamina and cover 20% to 30% of the microvascular circumference. Pericytes are most abundant on venules, where they provide mechanical support and synthesize ECM proteins such as laminin and fibronectin. Platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase receptor expressed on the surface of pericytes that was targeted for the treatment of malignant brain tumors. Clinical trials were conducted with imatinib, a PDGFR inhibitor, on patients with glioblastoma multiforme (GBM) who were refractory to chemotherapy and radiation therapy. However, while initially promising in early phase 1 and 2 trials, later phase 2 and 3 trials failed to demonstrate superior efficacy compared to standard therapy. Pericytes migrate away from the site of injury in pathologic conditions that increase BBB permeability, such as trauma or seizures. Increasing recognition of the role of pericytes in homeostatic regulation at the BBB should prompt investigation into pericytes as possible therapeutic targets, particularly in seizures.

Neurons are the building blocks of the CNS. The role of neuronal modulation at the BBB is primarily enzymatic ( Table 68.1 ). Dynamic brain imaging studies, such as positron emission tomography (PET) and functional magnetic resonance imaging (MRI), detect regional increases in CBF and glucose and oxygen consumption, which are associated with local increases in neuronal activity. Neurons upregulate catalytic factors specific to ECs and provide noradrenergic, serotoninergic, cholinergic, and GABAergic (transmitting or secreting γ-aminobutyric acid [GABA]) innervation to astrocytes and their associated ECs.

Microglia are the resident macrophages of the CNS and provide immunosurveillance of the CNS and the perivascular space. They act as antigen-presenting cells, identifying, engulfing, and presenting foreign compounds that have “bypassed” the BBB to T cells. Microglia secrete proinflammatory cytokines and rapidly proliferate to contain offending agents. Microglia play important roles in the sequelae of stroke and trauma and are relevant in conditions characterized by pathologic inflammation, such as MS and, as shown more recently, other neurodegenerative diseases. Although microglial overactivity may reflect or promote harmful neuroinflammation, recently the role of microglia in clearance of debris has received significant attention regarding amyloid deposition in Alzheimer disease (AD). Documented microglial clearance of plaques suggests that in certain pathologic conditions, stimulation of microglial activity may be beneficial. ^, The ideal balance between the negative consequences of neuroinflammation and the positive role of inflammatory cells in clearance must be better defined in the context of individual neurological diseases.

The extracellular matrix provides physical stability to the BBB. It is a critical anchoring site that mediates polarity at the EC-astrocyte interface. Disruption of the ECM predictably impairs the structural integrity of the BBB, thus compromising its activity. Structural integrity of the BBB is maintained through interaction with several structural proteins, including laminin, collagen type IV, and integrins. Matrix proteins also upregulate TJ protein expression.

The permeability of the BBB to macromolecules is determined by both TJ-controlled paracellular permeability (through cell-cell junctions) and caveolae-mediated transcellular permeability. Caveolae are sites of endothelial transcytosis, endocytosis, and signal transduction. The relationship between paracellular and transcellular permeability is of crucial importance for the regulation of transendothelial permeability. Using electron microscopy, Majno and colleagues found that carbon particles injected into blood entered the parenchyma after brain tissue had been exposed to histamine, which promotes vasodilation. In addition, the authors reported gaps between ECs. Osmotic control of the BBB has been supported by electron microscopy studies, in which it was shown that the nuclei of ECs seemed to have a contracted, raisin-like appearance after exposure to histamine. This method of osmotic regulation of the BBB has since been further described. Clinical work has utilized this concept to induce osmotic disruption of the BBB and improve drug delivery to the CNS (see Table 68.1 ).

The precise vascular localization of the functional term blood-brain barrier might be extended beyond the capillary segments to CNS microvessels ( Fig. 68.3 ). The average total surface area of the brain microvasculature is 20 m ² , whereas the surface area of cerebral capillary endothelium is 100 cm ² /g tissue. The total length is 650 km, the inner capillary lumen is 6 μm, and in the cortex capillaries are 20 μm apart from one another. More than 99% of brain capillaries have a BBB, with the exception of the circumventricular organs. Circumventricular organs are important for peripheral chemosensing and include the median eminence, pituitary gland, choroid plexus, subfornical organ, lamina terminalis, and area postrema. Other nonstructural mechanisms of controlling traffic across the BBB include ion channels and transport carriers, which regulate influx and efflux of hydrophilic nutrients, metabolites, vitamins, hormones, and ions.

In summary, the brain microvascular endothelium differs from peripheral endothelium in three primary ways:

• 1.

  The BBB lacks fenestrations and is characterized by low pinocytotic activity, both of which markedly impair fluid uptake.

• 2.

  TJs are ubiquitous in brain ECs and impede paracellular transport of large or hydrophobic molecules across the BBB into the brain parenchyma.

• 3.

  Mitochondria are present at a much higher concentration, which provides the energy needed for active transport of various proteins and factors required by the brain.

Transport Across the Blood-Brain Barrier

Tight regulation of solute gradients and molecular transport is mediated by four primary transport mechanisms ( Fig. 68.4 ):

• 1.

  Paracellular aqueous diffusion—small water-soluble molecules are able to pass through this extracellular pathway, which is regulated by TJs.

• 2.

  Transcellular lipophilic diffusion—a transmembrane process by which substances cross the BBB at a rate proportional to their molecular weight and lipid solubility.

• 3.

  Adsorptive transcytosis—an endocytotic process mediated by clathrin-coated pits and, to a lesser extent, caveolae. It is generally selective for cationic molecules and is the predominant mechanism for passage of human immunodeficiency virus type 1 (HIV-1) into the brain. This process is being investigated as a possible pathway for therapeutic drug delivery to the CNS. ^,

• 4.

  Saturable transport

  • a.

    Receptor-mediated transcytosis—transport of solutes via receptor binding and subsequent endocytosis. This can occur against a concentration gradient, but requires energy.

  • b.

    Channel-mediated transport—a saturable mode of transit that mediates influx and efflux via transport proteins.

BBB transporters ensure delivery of critical compounds that are essential for brain function. Several carrier systems exist at the BBB for transport of hexoses (glucose, galactose); neutral, basic, and acidic amino acids; monocarboxylic acids (lactate, pyruvate); purines (adenine, guanine); nucleosides (adenosine); amines; and ions. Uptake of larger molecules, including insulin and transferrin, occurs via receptor-mediated endocytosis.

Tight Junctions

The most prominent feature of the BBB is the presence of complex TJs between CNS ECs, which establish high transendothelial electrical resistance and determine the permeability of the BBB to hydrophilic molecules. TJs form a protective layer around ECs and are composed of various adhesive molecules linked to cytoskeletal signaling proteins. The proteins that make up the cellular architecture of the TJ and secure its connection to the actin cytoskeleton provide the structural integrity for the junctional complex to regulate transport of solutes into the brain ( Fig. 68.5 ).

An interesting twist is the recent proposition that the gut microbiota influences BBB permeability through altered expression of claudin-5 and occludin. In these experiments, mice with a germ-free gut demonstrated greater BBB permeability than those with normal gut flora. These preliminary results, although intriguing, were obtained in rodents under tightly controlled experimental conditions; given the impact that diet has on the microbiome and the differences between human and rodent dietary patterns, the clinical relevance remains unknown.