CHAPTER 8 Blood-Brain Barrier
The blood-brain barrier (BBB) is a “neurovascular unit” composed of microvascular endothelium, basement membrane, neurons, and neuroglial structures: astrocytes, pericytes, and microglia. More recently, it has become apparent that in human brain pathology, the BBB also interacts substantially with intravascular signals and circulating blood cells. In this respect, it becomes clear that despite its location at the blood-brain interface, the potential impact and topography of BBB cells are more widespread than initially believed. For example, the interaction of circulating white blood cells infected by human immunodeficiency virus (HIV) has been shown to have an impact on BBB function; in contrast, the BBB in patients with acquired immunodeficiency syndrome seems to act as a reservoir for the virus, thus further extending the reach of this cellular interface.1,2 The BBB is an active and dynamic organ that ensures adequate concentration of essential compounds such as oxygen and glucose and at the same time protects the brain from deleterious substances in the peripheral circulation. The BBB selectively prevents transportation of substances into brain via tight junctions (TJs), enzymatic reactions, and neurotransmitter signaling and selectively transports small and large molecules by passive and facilitated diffusion and active transport. The synergistic integration of all molecular and structural components gives rise to this functional complex called the BBB. Disruption of the BBB is seen in numerous pathologic processes. However, the discriminatory nature of the neurovascular unit also prevents the delivery of therapies to the brain, including chemotherapy agents, antiviral drugs, and beneficial neuromodulators. There are novel methods of circumventing the BBB that may provide novel therapies to treat a variety of neurological disorders. Scientific investigation of the BBB continues to provide insight into this complex and dynamic system and may generate much needed therapies to treat numerous neurological diseases.
History
Scientific investigation in identifying the BBB dates back to the 19th century. In 1885, Paul Ehrlich, a bacteriologist, observed that aniline dyes intravenously injected into animals colored all organs with the exception of the brain and spinal cord.3 His interpretation at the time was that there was poor uptake of the dye by the brain. Later, one of Ehrlich’s protégés, Edwin Goldmann, injected trypan blue intravenously and was able to visualize the dye in the choroid plexus and meninges but not the brain itself.4 However, when he injected trypan blue into cerebrospinal fluid (CSF), the dye was present throughout the brain, although it was absent in the rest of the body.5
In the 1920s, experiments performed by Stern and Gautier led to greater understanding of the blood-CSF barrier. They studied the transport of substances from blood into CSF. Chemicals such as bromide, bile salts, and morphine injected into the bloodstream were found in CSF, whereas fluorescein and epinephrine were absent, even though they were administered in the same fashion.6 Moreover, substances that entered the brain affected its activity and substances that were unable to penetrate the brain had no functional consequence.7 They coined this semipermeable protection of agents entering the brain “barrière hématoencéphalique.”
A series of investigations in the 1960s led to the identification of properties of molecular compounds that facilitated transport across the BBB, such as lipid solubility.8 Moreover, a gradient existed between extracellular fluid in the brain and CSF. This allows substances to be filtered out of the brain through CSF, termed the sink effect.9 More recently, it was shown that molecular transport of substances across the BBB could be determined from their log octanol-water partition coefficients (Fig. 8-1).10 A plot of log BBB permeability (cm/sec) versus log octanol-water partition coefficient shows that increased lipophilicity directly correlates with increased membrane permeability in that the more lipid soluble a molecule is, the more readily it moves from the aqueous environment of blood across the lipid environment of the endothelial cell (EC) membrane and enters the brain. Compounds subject to active transport will exceed their predicted permeability based on membrane lipophilicity. Counteracting influences that may slow diffusion across the BBB include pH, temperature, and retention in blood because of protein binding. As a general rule, lipid-soluble molecules with a molecular size less than 400 daltons can cross the BBB. Unfortunately, few central nervous system (CNS) diseases respond to small-molecule drugs.
Finally, between 1965 and 1967, a number of scientists identified the structure of the BBB as consisting of a network of capillaries and ECs known as TJs.11–13
Anatomy of the Blood-Brain Barrier
The anatomic BBB is formed by a monolayer of microvascular ECs that line the intraluminal space of brain capillaries. The BBB consists of ECs packed close together and forming TJs. The EC layer has a luminal (inside) and abluminal (outside) compartment separated by 300 to 500 nm of cytoplasm between the blood and brain. The EC layer is composed of TJs, which consist of occludin and claudin; adherent junctions, including cadherin, catenins, vinculin, and actinin; and junctional adhesion molecules (Fig. 8-2).
In addition to the structural integrity of the BBB, there exists an enzymatic surveillance system that metabolizes drugs and other compounds bypassing the structural barrier. Three main catalytic agents regulate transportation across the BBB: γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase, and aromatic acid decarboxylase. All are highly concentrated in cerebral vessels.14
There is charge polarity between the abluminal and luminal surface of ECs. This polarity influences permeability of the barrier. Alkaline phosphatase and γ-GTP are concentrated on the luminal compartment, whereas sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) and other transporters are clustered on the abluminal side. Other shuttling proteins that contribute to transport polarity include glucose transporter-1 (GLUT-1), which is concentrated at the abluminal membrane,15 and the drug transporter P-glycoprotein (Pgp), which is concentrated at the luminal membrane.16
The tissue microenvironment is necessary for continued regulation of barrier function. The BBB, also known as the neurovascular unit, consists of astrocytes, pericytes, microglia, neurons, and the extracellular matrix (ECM), all of which play a supportive role in maintaining integrity of the BBB.17,18 Astrocytes have end-feet that border the basement membrane of vessels of the parenchyma. More than 90% of astrocyte foot processes surround ECs.19 They are associated with adrenergic and cholinergic nerve terminals, as well as those that respond to peptides (Fig. 8-3).
Astrocytes also densely surround TJs and augment ECs by reducing the size of the gap of TJs.20 In vitro experiments imply that without astrocytes, the integrity of the BBB is significantly compromised.20,21 In contrast, other studies indicate that BBB integrity is retained amid degradation of astrocytes,22 thus suggesting that astrocytes regulate BBB activity indirectly rather than through physical obstruction. Astrocytes are considered to be inducers of both the barrier and permeability properties of the endothelium. Stewart and Wiley in 1981 first demonstrated that newly formed vessels originating from the coelomic cavity display BBB characteristics when placed in contact with grafts of neural tissue.23 Later, Janzer and Raff first demonstrated that a functional BBB was induced in nonbrain ECs in the anterior chamber of the eye in the presence of astrocytic aggregates.24
Pericytes are undifferentiated contractile connective tissue cells that localize to capillary walls and share a common basement membrane with brain ECs. They may not be involved in vessel contraction because they lack a contractile actin subtype.25 In vitro studies have revealed communication between ECs and pericytes. The proposed mechanism of this communication is through cellular projections, which penetrate the basal lamina and cover 20% to 30% of the microvascular circumference.25 Pericytes express macrophage functions and are actively involved in the immune response, where they operate as a second line of defense at the BBB. Pericytes are the most abundant on venules, for which they provide mechanical support and also 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 has been 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. Patients treated with imatinib and hydroxyurea had a 20% response rate, and the drug combination was reasonably well tolerated in phase II studies.26,27 Pathologic conditions that increase BBB permeability, such as trauma or hypoxia, result in a significantly decreased pericyte concentration as they migrate away from the site of injury.28
Neurons are the building blocks of the CNS. The role of neuronal modulation at the BBB is principally enzymatic (Table 8-1). Functional brain imaging studies, such as positron emission tomography (PET) and functional magnetic resonance imaging (MRI), are based on regional increases in cerebral blood flow and glucose and oxygen consumption, which are associated with regional increases in neuronal activity.29 Neurons upregulate catalytic factors specific to ECs.30 Astrocytes and their associated ECs are innervated by noradrenergic,31 serotoninergic,32 cholinergic,33 and GABAergic (transmitting or secreting γ-aminobutyric acid [GABA]) neurons.34 Lesions of the norepinephrine-producing locus caeruleus sensitize the BBB to hypertension. In Alzheimer’s disease, cholinergic inhibition impairs cerebrovascular blood flow.33,35
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Decreased Blood-Brain Barrier Permeability |
Adapted from Abbott NJ. Dynamics of CNS barriers: Evolution, differentiation, and modulation. Cell Mol Neurobiol. 2005;25:5-23.
Microglia serve as surveillance cells for the BBB. They identify foreign compounds that have bypassed the BBB and act as antigen-presenting cells by engulfing these substances and presenting them to activated T cells for destruction. Microglia also secrete cytokines, or proinflammatory molecules, and rapidly proliferate to contain the offending agent.15
The ECM 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, which in turn compromises its activity. Structural integrity of the BBB is achieved through interaction with several structural proteins, including laminin, collagen type IV, and integrins.36 Matrix proteins also upregulate TJ protein expression.37
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 an electron microscope, Majno and colleagues found that carbon particles injected into blood entered the parenchyma after brain tissue had been exposed to histamine.38 In addition, these authors were able to see gaps between ECs. The concept of osmotic control of the BBB was also based on electron microscope studies, in which it was shown that the nuclei of ECs seemed to have a contracted, raisin-like appearance after exposure to histamine.39 This method of osmotic regulation of the BBB has since been further described.40
The precise vascular localization of the functional term BBB might be extended beyond the capillary segments to CNS microvessels (Fig. 8-4). The average total surface area of the brain microvasculature is 20 m2, whereas the surface area of cerebral capillary endothelium is 100 cm2/g tissue.41 The total length is 650 km, the inner capillary lumen is 6 µm, and capillaries are 20 µm apart from one another. The BBB occupies more than 99% of brain capillaries, with the exception of the circumventricular organs, which have a blood-CSF barrier. Circumventricular organs include the median eminence, pituitary gland, choroid plexus, subfornical organ, lamina terminalis, and area postrema. Although not as stringent as the BBB, the blood-CSF barrier prevents blood-borne substances from entering the brain. Other mechanisms of controlling traffic across the BBB besides the structural support include ion channels and transport carriers, which control the traffic of hydrophilic nutrients, metabolites, vitamins and hormones, and ions across the BBB.
Transport Across the Blood-Brain Barrier
The biochemical BBB is established by transport systems of the BBB, which can be grouped into four types (Fig. 8-5):
Glucose
The glucose transporter at the BBB is of special importance because glucose is the primary source of energy for the brain and is required for normal brain activity and function. Transportation across the BBB occurs via a glucose transporter. There are five members of the sodium-independent glucose transporters in the brain, including GLUT-1 (ECs), GLUT-3 (neurons),43 and GLUT-5 (microglia).44 Each transports 2-deoxyglucose, 3-O-methylglucose, mannose, galactose, and glucose across the membrane.44 GLUT-1 is a 45- to 55-kD protein, depending on its glycosylation state. It is present in high concentration in the ECs of arterioles, venules, and capillaries and facilitates movement of d-glucose enantiomers from the peripheral circulation to the brain.45 Although the BBB is small, glucose transport is the rate-limiting step of primary energy acquisition. GLUT-1 expression is three to four times higher on the abluminal membrane and is altered with processes such as diabetes, epilepsy, trauma, and tumors.
Amino Acids
Delivery of amino acids across the BBB is achieved by carrier-mediated transport across the abluminal and luminal membranes in both a concentration-dependent and stereospecific manner.46 Factors affecting amino acid uptake include plasma concentration, affinity of the transport system for a particular amino acid, and competition among amino acids for a particular transport system. Amino acid transport is classified into four systems based on seminal experiments conducted on the BBB and amino acid transport.47 The first group is the large, neutral-charged amino acid transporter (L type), which has a preference for leucine residues. These transporters are sodium independent, saturable, and stereospecific. The rate of transfer across the BBB is high because of the high requirement for this subtype of amino acids by the brain. The second group of transporters consists of small, neutral-charged amino acids (A type). These transporters are dependent on sodium for movement across the BBB. Independent transporters for acidic and basic amino acids make up the third transport system. The rate of transfer of basic amino acids is high because of a high requirement for this subtype by the brain. Finally, alanine, serine, cysteine, and threonine residues use a sodium-dependent transport system that shuttles small neutral amino acids across the BBB. Investigation of this transport system has suggested that its primary role is to transport amino acids out of the brain.48
Glutamate is the most abundant amino acid in the CNS and is stored intracellularly against its concentration gradient. It is an excitatory neurotransmitter that serves a number of functions in the brain. It is involved in energy metabolism, molecular synthesis of glutathione and GABA, and breakdown and removal of ammonia. In addition to transport systems, the BBB also plays a role in glutamate metabolism.49
Ions and Water
Nitric oxide (NO) is well known for its properties as a vasodilatory agent and also plays an important role in BBB signal regulation and autocrine activity.50,51 It mediates the transport of ions and nutrients essential for brain function and is regulated by cyclic guanosine monophosphate and cyclic adenosine monophosphate.
All movement of ions across the BBB is also associated with the movement of water. Water passes through the plasma membranes via facilitated diffusion through water channels called aquaporins, by cotransport with organic or inorganic ions, and by diffusion across the lipid bilayer. The water channel expressed most in the CNS is aquaporin-4, principally by astrocytes. Immunolocalization studies with double staining for aquaporin-4 and glial fibrillary acidic protein (GFAP), which stains astrocytes, show strong colocalization at the level of the BBB.52,53 Involvement of aquaporin-4 in brain edema and water homeostasis has been well established.
Lipoproteins
The mechanism underlying lipoprotein transport across the BBB has garnered considerable interest among researchers for potential exploitation of this channel for delivering drug therapy. Low-density lipoprotein (LDL) receptors are expressed on the BBB, and LDL is transported across the BBB by endocytosis. Scientists are now attempting to use this receptor as a vehicle for delivering therapeutic drugs into the brain.54
Multidrug Resistance
Multidrug resistance (MDR) protein has also been intensely studied as a possible vehicle for drug delivery. Pgp is an efflux transporter protein found in ECs, astrocytes, and microglia.55 It is expressed on the luminal surface of the endothelial membrane and glia55 and prevents toxins from entering the brain.56 Many drugs are substrates for Pgp, which limits their accumulation in the brain. Vinca alkaloids, anthracyclines, and taxanes are among the anticancer agents known to be transported by Pgp. Preclinical models have revealed that patients with deletion of Pgp have 100-fold increased sensitivity to chemotherapy agents and antiviral compounds in comparison to control subjects.57–60 Pgp is also protective in disease states such as Alzheimer’s, in which there is decreased deposition of β amyloid.61 However, overexpression of Pgp is also found in patients with epilepsy, although it is unclear whether upregulation of the transporter is a pathologic process of epilepsy or secondary to resistance to antiepileptic drugs (AEDs).62 In vivo studies have shown depletion of Pgp in patients with Parkinson’s disease.63
Leukocytes
It was an early notion that leukocytes are rare within the brain and that the architecture of brain microvessels maintains its immune-privileged status. There is now evidence that leukocytes traverse microvessels via a transcellular route. In addition, activated T lymphocytes can cross the endothelial wall in the normal state. Immunologic response to foreign pathogens, surveillance, and inflammation are regulated by leukocyte transportation across the BBB. Transportation occurs by means of integrins, intercellular adhesion molecules (ICAMs), and direct intercellular activity.64,65 In HIV encephalitis, actin cytoskeletal proteins such as Rho guanosine triphosphatase (GTPase) facilitate leukocyte migration, and blocking these compounds maintains the integrity of the BBB against leukocyte transport.66
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 that reside intracellularly. 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 transportation of solutes into the brain (Fig. 8-6).
Membrane-Associated Guanylate Kinase Homologue
This class of proteins forms multimers that anchor TJs to the cell membrane. There are three main adhesion proteins: zona occludin-1 (ZO-1), ZO-2, and ZO-3. The first TJ-associated protein identified was ZO-1, a 220-kD membrane-associated protein expressed on both EC and epithelial cell surfaces.67 It serves as a scaffold for TJ formation by binding to the C-terminal of the cytoplasmic tail of occludin and the cytoskeletal protein spectrin.68 The importance of ZO-1 in TJ stability is illustrated by research showing that barrier stringency is compromised when ZO-1 expression is reduced or dissociated from membrane proteins.69 ZO-2 is a 160-kD protein with homologous regions to ZO-1 and has been identified as a ZO-1–associated protein.70 It binds to a transcription factor and transmembrane domains of the TJ. During proliferation of the TJ, ZO-2 migrates to the nucleus.71,72 Although ZO-1 and ZO-2 are seen in both brain and peripheral ECs, ZO-1 is continuous in brain but discontinuous in peripheral ECs.73 ZO-3 is seen solely in epithelial cells, unlike ZO-1 and ZO-2, which are intricate components of ECs.74
Occludin
Occludin is a 65-kD transmembrane protein located at TJ margins19,75 and is associated with cytoskeletal signaling proteins, including ZO-1, and ZO-2.70 Antibodies against occludin cluster around cell-cell contacts in brain ECs.76 Occludin is composed of four transmembrane domains. The terminal ends of the protein face the cytoplasm, and two extracellular loops span the intracellular cleft.77 The concentration of occludin in peripheral ECs is much lower than in brain ECs. Occludin is thought to be transcriptionally regulated, as measured by significant differences in mRNA levels between EC types.73 Occludin contains numerous phosphorylation sites, which are directly related to substance permeability. The cytoplasmic C-terminal provides association among occludin, ZO-1, and ZO-2. Occludin dysfunction is seen in numerous diseases such as HIV.66,78 Furthermore, downregulation of 55-kD occludin expression is seen in high-grade (III and IV) brain tumors.79
Claudins
Claudins are a third group of membrane proteins. These proteins function to selectively permit entry of cations through TJs.80 There is sequence conservation in the first and fourth transmembrane domains in the approximately 24 claudins identified in mammals.81 Both homophilic and heterophilic binding of extracellular domains facilitates close interaction of cell layers.82 In elucidating the roles of claudins and occludin in TJ formation, overexpression of claudin generated cell layers resembling TJs, which was not the case with occludin.83 Based on staining experiments, claudins are arranged continuously along ECs, in contrast to occludins.83 These results imply that claudins form the fundamental scaffold of TJs and occludins secure TJs. ECs express at least four claudin subtypes, claudin-1, claudin-3, claudin-5, and claudin-12. In vitro studies have revealed that claudin expression is significantly downregulated in anaplastic astrocytoma and GBM cell cultures.84,85
Junctional Adhesion Molecules
Members of the immunoglobulin supergene family are localized within TJs. Junctional adhesion molecule-1 (JAM-1) is a 40-kD IgG protein that regulates cell membrane attachment between large-domain and smaller chains.86,87 In animal models, JAMs regulate transendothelial cell migration, but their role in the BBB in vivo remains unclear.86
Endothelial TJs can rapidly and reversibly alter their conformation to permit plasma constituents to pass. The mechanism of control of this system is the degree of tyrosine phosphorylation of ZO-1 or other contact proteins; with increased phosphorylation, there is decreased electrical resistance and TJ permeability increases.88 Other mechanisms of control of cell-cell junctions are regulated by factors secreted by astroglial cells. These factors alter the microenvironment of the junction, thereby modifying permeability of the TJ.
Tight Junction Regulation
Ion Signaling
Phosphorylation indirectly controls TJ permeability by acting on associated proteins.89 Various pathologic states affect the function of phosphorylation on TJs. For example, there is significant phosphorylation of TJs in the presence of vascular endothelial growth factor (VEGF), which compromises the integrity of the BBB.90 In contrast, in bacterial infections there is significant dephosphorylation of TJs, which increases BBB permeability.91,92
Calcium
Calcium (Ca2+) acts as second messenger for regulation of BBB activity. Low levels of extracellular Ca2+ decrease TJ barrier stability,93 possibly dispersing ZO-1 and occludin away from TJ sites.94 Intracellular Ca2+ levels also affect BBB integrity by activating signaling cascades, regulating TJ transcription, and modulating the distribution of TJs.95 Release of intracellular Ca2+ compromises the BBB by inducing phosphorylation of TJs.96
G Protein
G proteins regulate scaffold proteins responsible for the cytoarchitecture of the TJ and membrane-associated proteins regulating TJ activity.97 They also facilitate leukocyte transport into brain via Rho GTPases.66 Protein kinase C (PKC) regulation also modulates BBB function. Control of PKC-α and PKC-ζ influences BBB permeability.98 In HIV disease, the cell surface protein gp120 activates PKC. Inhibitors of gp120 activity block PKC activation and prevent breakdown of the BBB.99 AF-6 is a Ras-binding protein that also binds to ZO-1 to stabilize the BBB. Overexpression of Ras results in disruption of ZO-1 and increased permeability of the BBB.100
Pathologic Changes in the Blood-Brain Barrier
Many of the same factors that regulate permeability under normal conditions are altered during pathologic conditions and result in enhanced vascular permeability and edema formation. Many neurologic conditions, including trauma, inflammatory and autoimmune disease, infection, cerebrovascular disease, neurodegenerative disease, epilepsy, and neoplasia, result in disruption of the BBB. Head trauma alters transporter activity, which may inhibit essential compounds from crossing the BBB while permitting pathogens and toxins to do so. Lymphoid surveillance of the CNS occurs via lymphatic vessels such as olfactory nerves or arachnoid granulations. BBB permeability is increased in autoimmune inflammatory diseases such as multiple sclerosis. Studies have also revealed that extravasation of lymphoid cells mediated by the vascular and intracellular adhesion molecules VCAM-1 and ICAM-1 is a seminal event in the pathophysiology of multiple sclerosis.101 Extravasation of leukocytes is achieved by three fundamental processes:
In addition, leukocytes secrete matrix metalloproteinases (MMPs), which degrade the ECM.
Infectious agents cross the BBB by transcytosis. HIV-1 enters the brain by adsorptive endocytosis.66,102 Because brain ECs lack CD4 and galactosylceramide receptors, they are protected from direct infection. HIV enters the CNS via infected white blood cells. The virus continues to proliferate in glial cells and is protected from therapy by the BBB.2
Clostridium perfringens has high affinity for TJ claudin proteins, which when bound, increases BBB permeability.103 Pneumococci bind to ECs through receptors for platelet-activating factor, thereby resulting in increased paracellular transport.104 Subsequent degradation of TJs results in the release of cytokines, including interleukin-1 (IL-1), tumor necrosis factor (TNF), and MMPs.105 In contrast, Neisseria meningitidis adheres to ECs, induces phosphorylation of EC binding proteins, inhibits leukocyte transportation, and in turn, inhibits inflammation.106 In patients with bacterial meningitis, steroids are given in conjunction with antibiotics to minimize inflammation in the brain. Dexamethasone, a synthetic glucocorticoid, may impede antibiotic permeability by tightening the BBB in the setting of meningitis. Herpes enters the CNS through olfactory nerves, whereas rabies enters through spinal nerves. In general, viruses perturb the BBB less than bacterial infection does.
Cerebrovascular disease and ischemia have deleterious effects on the BBB by depleting the brain of nutrients, inducing inflammation, and activating the cytokine cascade. This results in release of MMP, which causes vasogenic edema and degradation of the ECM. Like the edema seen in trauma, no significant improvement is provided with steroids. Stroke causes disruption of TJs and destruction of basal lamina proteins, including collagen type IV, laminin, and fibronectin.107 The severity of infarction is correlated with the degree of BBB dysfunction,10 which may place the patient at greater risk for hemorrhagic conversion after reperfusion. Klatzo first characterized brain edema as a cytotoxic versus a vasogenic process.108 In cytotoxic edema, the brain cells swell at the expense of the extracellular space, whereas the BBB remains intact. In vasogenic edema, permeability is increased because of disruption of the BBB, which allows an influx of plasma constituents and expansion of the extracellular space. During stroke, features of both cytotoxic edema and vasogenic edema occur simultaneously.
Breakdown of the BBB is implicated in the pathophysiology of Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. In Alzheimer’s disease, Aβ amyloid deposition is a histopathologic hallmark of the disease. Amyloid is transported across the BBB by the receptor for advanced glycation end products (RAGE). This receptor transports Aβ amyloid into the brain, whereas LDL receptor–related protein-1 transports Aβ amyloid out.109 Alzheimer’s patients have changes in RAGE and LDL receptor–related protein-1 concentrations on the hippocampus and cerebral cortex, thus indicating that Aβ amyloid deposition secondary to BBB dysfunction is involved in the early pathogenesis of the disease.110 Anti-RAGE therapies are currently being developed to test this hypothesis.
Seizures induce extravasation of intravascular markers, which results in a transient increase in BBB permeability. The BBB characteristics seen in epilepsy include downregulation of GLUT-1, inflammation, and MDR to AEDs, findings implicating both MDR receptors and efflux transporters.111
Brain tumors represent a disease process in which the dynamic characteristics of the BBB present significant challenges for therapy. ECs in tumor vessels are characterized by frequent membrane fenestrations, prominent pinocytotic vesicles, and lack of perivascular glial end-feet, and they display abnormal TJ morphology. The blood-tumor barrier is characterized by higher permeability to small molecules, although a large majority of drug therapies are still unable to penetrate the CNS in adequate concentration because of their large size and transport mechanisms, such as Pgp, that shuttle drug away from the brain.112 The center of a tumor’s BBB is often vastly compromised, thus making it ideal for drug delivery. However, surrounding normal parenchyma has an intact BBB, which prevents clinically significant doses of drugs from reaching the lesion. Corticosteroid therapy after brain tumor surgery has reduced mortality because it helps reestablish BBB integrity.113
Central Nervous System Drug Administration and the Blood-Brain Barrier
Some speculate that strong selective pressure must have existed to allow such a complex structure as the BBB to evolve.114,115 The CNS has no lymphatic system or other method of parenchymal drainage and is enclosed within the cranium, a rigid, nonexpandable structure. A net influx of molecules into the CNS would increase osmolarity and allow water from the vasculature to enter the brain, thereby leading to an increase in intracranial pressure. The evolution of the BBB fortunately makes large increases in intracranial pressure rare occurrences.116,117 Additionally, the BBB serves to prevent potentially harmful toxins from reaching the brain.
Despite these important functional roles, the BBB’s unique selectivity has created a strong challenge for medicine by hindering the ability of CNS medications to pass through it. Many drugs that have potentially useful action in vitro are found on in vivo evaluation to be unable to enter the CNS. New techniques to make the BBB more permeable would allow a number of potentially useful drugs currently unable to traverse the barrier to reach the CNS. Considerable research is currently under way to accomplish this task via a variety of approaches. The difficulty in administering drugs for the CNS can be seen in surveys of currently available medications. One study analyzing 6304 medications, excluding diagnostic dyes, revealed that only 6% of the drugs were used for treatment of the CNS118 despite heavy research in CNS pharmaceutical development. Other groups are focusing on strengthening the barrier, which would help prevent damage from exposure to neurotoxins and limit CNS side effects caused by drugs acting on other organ systems. In a related area, studies are searching to find more effective methods of analyzing BBB permeability, which is often disrupted in many disease states. All these new areas of research have developed rapidly in recent years, and some interesting and unique strategies to both “read” and “write” the BBB have been formulated.
Importance of Adequate Central Nervous System Drug Penetration
Improved regulation of BBB permeability would result in increased efficacy in treating the vast majority of CNS disorders. Nevertheless, most research in this area has focused on a handful of specific diseases, including brain tumors, HIV disease, epilepsy, Parkinson’s disease, Alzheimer’s disease, and infections. Both primary and metastatic CNS neoplasms, the most common of which in adults are astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas,119 are difficult to treat because most chemotherapeutic agents are unable to adequately traverse the BBB as a result of their low lipophilicity.40,119 Partly as a consequence of poor drug delivery to the CNS, many tumors are currently treated by surgery and radiation therapy. However, difficulties often arise in surgery because precise boundaries of some tumors are difficult to locate. Other tumors may lie in inoperable locations or have already metastasized to multiple sites. Although radiotherapy is often beneficial as well, it may lead to secondary, more aggressive tumors.120,121 Chemotherapy is the best option in these cases; however, the drugs must be able to enter the CNS to be effective.
AEDs are often excluded from the CNS by the BBB and are therefore clinically unusable despite demonstrating potent and selective in vitro action.122 Pgp has been implicated as an important transport protein for some of these AEDs,123 including phenytoin124 and carbamazepine.125 However, other reports question the role of Pgp in AED extrusion.126,127 Approximately 30% of epileptic patients do not respond to the common pharmaceutical treatments, thereby resulting in increased morbidity and mortality in these resistant patients.128 In an immunohistochemical study, Volk and Loscher129 demonstrated that rats resistant to AED therapy had higher levels of Pgp expression in brain capillary ECs than did control animals. Similar variations in Pgp in human epileptics would help explain the existence of patients with medically intractable epilepsy who do not respond to AEDs.
In the treatment of Parkinson’s disease, it is estimated that only 5% of orally administered levodopa reaches the circulation after first pass through the liver. Less than 1% of the oral dose enters the brain because of blockage at the BBB.130 Recent studies are beginning to elucidate the potential mechanisms for exclusion of levodopa from the CNS. Levodopa enters the brain via the luminal neutral amino acid transporter L1 on ECs, but it may be excreted by the sodium-dependent large neutral amino acid (LNAA) transporter on the abluminal side of the membrane.131,132 Other molecules of levodopa are degraded by the endothelial enzyme monoamine oxidase B in the periphery,133 although drugs such as carbidopa are now available to inhibit this enzyme.
Medications for Alzheimer’s disease are excluded by the BBB as well.134 Given the rising proportion of elderly individuals in whom this disease is diagnosed in the United States, effective treatment of Alzheimer’s disease will be critical. Certain modifications of the BBB may eventually be used to provide better treatment of this debilitating illness.
Infectious disease could also be treated more successfully by opening the BBB, which would allow CNS penetration of more effective antibacterial, antifungal, and antiviral agents for the treatment of meningitis, encephalitis, abscesses, and other infectious diseases of the CNS. Once bacteria are killed, the BBB must be permeable to the remnants of their cell walls, which could otherwise irritate sensitive nervous tissue.135
HIV may harbor itself in the brain where antiviral drugs cannot penetrate effectively and then re-emerge later.136,137 Several studies have shown that zidovudine (azidothymidine [AZT]) does not effectively penetrate the BBB in rodents,138–140 although this does not appear to be the result of Pgp export.141 Strazielle and colleagues142 suggested that the organic anion transporters, a different class of protein pumps at the BBB, may be responsible for transport of AZT. Other anti-HIV medications are exported by similar means. The nucleoside reverse transcriptase inhibitor (NRTI) stavudine (d4T) may be removed from the BBB by the same transporter as AZT.143 Another NRTI, lamivudine (3TC), does not significantly penetrate the CNS either.140 Some protease inhibitors used for the treatment of HIV also appear to be susceptible to BBB exporters, primarily Pgp.144 However, there are some anti-HIV drugs that do enter the CNS in significant amounts. Some non-NRTIs, including nevirapine, appear to cross the BBB,145 whereas other anti-HIV drugs are believed to cross the BBB as a result of their CNS side effects.146
Drug Modifications
Expanding on the initial experiments of Brodie and associates8 in the 1960s, recent studies have shown that the permeability of most molecules can be predicted by determining their octanol-water coefficients based on their respective nonpolar and polar solubilites.147,148 Specifically, substances with the greatest ability to pass through the BBB generally have a log octanol-water coefficient between −0.5 and 6.0149 and a molecular mass of less than 400 to 500 daltons and do not form hydrogen bonds with water.150
