Blood Brain Barrier

Darryl Ronnie Peterson, Chicago Medical School, Chicago, Illinois, USA

The blood brain barrier serves to restrict and control the movement of substances between the general circulation and brain extracellular fluid. It participates in regulating the volume and composition of fluid surrounding the brain through specific transport processes, and thus contributes to homeostasis of the central nervous system.

Introductory article
Article Contents
. Entry of Substances from Blood into Brain . Brain Capillaries are Impermeant Except in Specialized Areas . Tight Junctions and High Electrical Resistance Between Capillary Endothelial Cells . Contributions of Astrocytes to a Well-sealed Blood Brain Barrier . Getting Past the Barrier: Small Lipophilic Molecules and Molecules with Special Transport Systems . Delivering Drugs to the Brain . Summary

Entry of Substances from Blood into Brain

The bloodbrain barrier serves to restrict and control the movement of substances between the general circulation and brain extracellular uid. It participates in regulating the volume and composition of uid surrounding the brain through specic transport processes, and thus contributes to homeostasis of the central nervous system. Nutrients such as glucose and certain amino acids are allowed to enter the brain, but blood-to-brain passage of neuroexcitatory substances, which could otherwise alter brain function, is essentially prevented. Because of its restrictive properties, drugs intended to treat neurological disorders at the level of the brain must be designed to penetrate the barrier. The bloodbrain barrier consists of endothelial cells which line cerebral capillaries (Figure 1). Unlike endothelium elsewhere in the body, these cells possess tight junctions which separate the plasmalemma into luminal
Abluminal membrane Tight junction Astrocyte Luminal membrane Brain capillary Astrocyte Blood Endothelial cell

(blood facing) and abluminal (brain facing) membrane domains, each with a dierent set of properties. Thus, the endothelial cells forming the bloodbrain barrier are polarized, and function in many respects like an epithelium. This allows for net unidirectional movement of certain electrolytes and water across the barrier, and selective transport of other substances. Because the tight junctions greatly limit paracellular transport (i.e. between cells), substances must cross the bloodbrain barrier by penetrating two plasma membrane domains arranged in series. The organization of transport proteins in these two membrane domains determines the direction and extent of transport. The volume of extracellular uid surrounding the brain is quite small, averaging only about 250 mL in an average human adult. The total surface area of the bloodbrain barrier is approximately 12 m2 (100 ft2), however, suggesting a relationship sucient for adequate regulation. Because the size of the brain extracellular uid compartment is so small, it is implied that even modest changes in its volume or composition could impact signicantly on brain function. For instance, a pathological accumulation of brain extracellular uid could potentially increase damaging pressure to the brain in a compartment restricted by the skull.

Brain Capillaries are Impermeant Except in Specialized Areas

Basement membrane

Neuron Pericyte

Figure 1 The blood brain barrier consists of endothelial cells lining brain capillaries. These cells are connected by tight junctions, which separate the plasmalemma into luminal (blood facing) and abluminal (brain facing) plasma membrane domains. The capillaries are surrounded by a basement membrane, and are closely associated with astrocytes, neurons and pericytes.

Cerebral capillary endothelial cells provide a tight barrier between the blood and brain tissue in most regions of the brain, with a few exceptions. Relatively small regions associated with the brain ventricles are termed circumventricular organs (CVOs), which include the area postrema, median eminence, neurohypophysis, organum vasculosum lamina terminalis, pineal gland and subfornical organ. Capillaries perfusing these structures are highly porous, and allow substances in the blood to come in contact with
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Blood Brain Barrier

neurons in the central nervous system. In some instances, this may provide a means for circulating hormones to inuence brain function, or for neurohormones to enter the blood. It has been estimated, however, that the surface area of the bloodbrain barrier is about 5000 times that of the CVOs, indicating that the barrier is essentially intact.

Tight Junctions and High Electrical Resistance Between Capillary Endothelial Cells
Closely associated with cerebral capillaries forming the bloodbrain barrier are pericytes, astrocytes and neurons (see Figure 1). These cells are present at the abluminal pole of the endothelial cells, separated by a basement membrane. Because of the intimate relationship of these cells with the capillaries, it was originally questioned whether they formed part of the barrier. To address this issue, early studies using horseradish peroxidase showed that this macromolecular marker could pass across the basement membrane and between the pericytes, astrocytes and neurons, but was stopped at the level of the endothelial tight junctions. This conrmed that the capillary endothelial cells represent the true bloodbrain barrier. The presence of tight junctions in the bloodbrain barrier greatly increases electrical resistance across the endothelium. This indicates that passage of electrolytes (i.e. Na 1 , Cl 2 ) between the cells is largely restricted, and that movement is dependent upon specic transport proteins present in the luminal and abluminal plasma membranes. Thus, as indicated above, a largely transcellular rather than a paracellular route of movement is implied. The specialized endothelial cells of the blood brain barrier develop a resistance as high as 8000Ocm 2 2, which is typical of some tight epithelia. The high electrical resistance also implies that solutes other than electrolytes must cross the bloodbrain barrier transcellularly.

cells are grown in tissue culture without astrocytes, they develop a typical nonpolar endothelium without tight junctions, and a low electrical resistance. If these same cells are grown in the presence of astrocytes, however, they become polarized and dierentiate into a high resistance bloodbrain barrier in vitro. Similar results can be achieved by culturing the cells in a medium that has been conditioned by previous exposure to astrocytes. In both cases, it is believed that a growth factor released by astrocytes stimulates dierentiation of growing endothelial cells destined to become the bloodbrain barrier. Specically, dierentiation includes the development of tight junctions and polarization of the cells. Furthermore, astrocytes appear to induce the expression of certain membrane-associated proteins, including the glucose transporter GLUT-1 (see below), potassium transporters, and the enzyme g-glutamyltranspeptidase. Upregulation of the glucose carrier is especially noteworthy, since transport of glucose from blood to brain is necessary for energy production in the central nervous system. Recently, this discussion has included the possibility that neurons and pericytes may also participate in the development of brain capillary endothelial cells.

Getting Past the Barrier: Small Lipophilic Molecules and Molecules with Special Transport Systems
Substances that are able to pass across the bloodbrain barrier include small lipophilic molecules. A small size and lipophilic disposition allow these molecules to penetrate the lipid components of the limiting plasma membranes. Lipid-soluble substances with a molecular weight of less than 600 Da readily permeate the bloodbrain barrier by simple diusion. Thus, alcohol, certain steroid hormones such as progesterone, as well as small lipophilic drugs such as codeine, can reach the brain. The lipophilic nature of a naturally occurring substance or drug may be determined by measuring its permeability coecient. This is done by quantifying its distribution between nonpolar and polar phases, such as 1-octanol and water. The permeability coecient varies directly with the ratio of distribution in the nonpolar over the polar solution. As mentioned above, nutrients such as glucose and certain amino acids are able to enter the brain by crossing the bloodbrain barrier. Entry of glucose is expected, since it is poorly stored by the brain, and is required for energy metabolism. Movement of glucose from blood to brain across the barrier is carrier mediated (Figure 2). The process is passive, with glucose moving down its concentration gradient from blood to brain. Carriers on both the luminal and abluminal plasma membranes facilitate its passage into the brain extracellular uid. Several isoforms of

Contributions of Astrocytes to a Well-sealed Blood Brain Barrier

The dierentiation of a specialized bloodbrain barrier is dependent upon the presence of astrocytes. Astrocytes are nonneuronal, multifunctional cells in the brain which apparently secrete a paracrine substance that stimulates the endothelial cells of the bloodbrain barrier to form tight junctions and develop a polar conguration. Normally astrocytes are closely aligned with the bloodbrain barrier, and project cellular extensions (end-feet) that terminate near the endothelium. Thus, they are in an ideal position to inuence its dierentiation. If the endothelial
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Blood Brain Barrier

Blood G AA AA Luminal membrane L y+ Gln AA Glu AA G Na+ Glu H+ Tight junction Brain capillary endothelial cell Abluminal AA membrane L Na+ AA y+ A Na+ AA Bo,+ N Glu Na+ AA G G Glu K+ Na+

Na+ AA Na+ AA Brain

Figure 2 Brain capillary endothelial cells forming the blood brain barrier are polarized and possess distinct transport proteins in the luminal and abluminal plasma membranes. This illustration depicts a current model describing some of these transporters. Organic nutrients such as amino acids (AA) and glucose (G) move passively from blood to brain extracellular fluid, with the assistance of carrier systems (i.e. L, y 1 , G) in both plasma membranes. Sodium-dependent amino acid cotransporters positioned at the abluminal membrane (i.e. A, Bo, 1 ) apparently serve to limit the influx of certain amino acids. Transporters for glutamate (Glu) are arranged to remove this neuroexcitatory amino acid from the brain. Together, the transport systems for glutamate and glutamine (Gln, N) in the blood brain barrier may contribute to regulating the presence of these nitrogen-containing amino acids in the brain, and thus participate in balancing brain nitrogen. Finally, the arrangement of sodium transporters (Na 1 /H 1 antiporter, Na channel, Na 1 /K 1 adenosine triphosphatase) in the luminal and abluminal plasma membranes favours net unidirectional blood to brain movement of fluid.

glucose transporters have been identied in a variety of tissues. As indicated above, the facilitative glucose carrier of the bloodbrain barrier is called GLUT-1. Several amino acid transport systems have been identied in the bloodbrain barrier (Figure 2). These are carrier-mediated pathways, and may be classied generally into sodium-independent and sodium-dependent processes. Large neutral amino acids, such as leucine and phenylalanine, are transported from blood to brain by system L (leucine preferring), which is typical of the sodium-independent category. Movement across the barrier is passive, with the amino acids moving down their concentration gradients, and transport is facilitated by L carriers on both plasma membrane domains. Although these carrier proteins have a greater anity for large neutral amino acids, other amino acids may also be transported to varying degrees. Basic amino acids such as arginine, lysine and ornithine are passively transported across the bloodbrain barrier by system y 1 , with these sodium-independent carriers presumably on both the luminal and abluminal membranes. Various sodium-dependent amino acid transporters have been described in the bloodbrain barrier, including systems A, ASC, Bo, 1 , N, and a glutamate carrier. System A has a high anity for small neutral amino acids such as alanine, and preferentially transports N-(methylamino)isobutyric acid (MeAIB), which has become its dening substrate. This carrier protein is characterized by secondary active transport of amino acids from the brain

extracellular uid into the endothelial cells forming the bloodbrain barrier, driven by cotransport of sodium down its electrochemical gradient. For this reason, the transporter functions to reduce entry of amino acids into the brain. Recently it has been shown that the system A transport protein is upregulated by oxoproline, an intracellular metabolite that is probably formed during enhanced blood to brain transport of amino acids. Thus it appears that a negative feedback mechanism is in place that serves to limit brain uptake of certain amino acids, when the blood to brain gradient would otherwise enhance their passive movement across the bloodbrain barrier. Sodium-dependent transport of neutral amino acids by the bloodbrain barrier can be accounted for by other carriers, in addition to system A. A component of sodiumdependent neutral amino acid transport has been observed in isolated rat cerebral capillaries that is not inhibited by MeAIB, presumably occurring at the abluminal membrane of the endothelial cells. This transport has been attributed to system ASC, because of its preference for small neutral amino acids. However, recent studies have challenged the presence of system ASC in the bloodbrain barrier, and have suggested that a carrier typical of system Bo, 1 is present. This is a sodium-dependent transporter positioned at the abluminal membrane, which is characterized by a high anity for neutral amino acids. Furthermore, there is recent evidence for the presence of system N at the abluminal membrane, which has a high anity for the nitrogen-containing amino acid glutamine. Since a facil3

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Blood Brain Barrier

itative carrier for glutamine is also present at the luminal membrane, it appears that the bloodbrain barrier has the capability to transport glutamine from brain to blood, and thus may serve as a means of removing nitrogen from the brain. Conceivably, this could be important in certain pathological disorders (e.g. liver disease) associated with increased levels of nitrogen-containing compounds, including ammonia, in brain extracellular uid. Finally, there appears to be a sodium-dependent amino acid carrier in the abluminal membrane that is capable of transporting glutamate from brain extracellular uid into the endothelium of the bloodbrain barrier. Under normal circumstances, the barrier is largely impermeable to glutamate entering the brain from the blood. This is expected, since glutamate is a common neurotransmitter in the central nervous system, and its inux could alter nerve impulses and brain function. Like glutamine, glutamate may passively cross the luminal membrane of the barrier by a facilitative carrier. However, a facilitative transporter does not appear to be present at the abluminal membrane. This arrangement of carriers is consistent with blood to brain movement of glutamate, and suggests that the bloodbrain barrier may serve to remove glutamate from the brain extracellular uid. In addition to the amino acid transporters mentioned above, there is evidence for systems T, b and X 2 in the bloodbrain barrier. These are low-capacity carriermediated systems that transport thyroid hormones (T3, T4), b amino acids (e.g. taurine) and anionic amino acids (e.g. aspartate) respectively. Furthermore, there are reports describing transport of monocarboxylic acids, amines, purine bases and nucleosides. The bloodbrain barrier appears to be generally impermeable to peptides and proteins, although some exceptions have been described. There are reports that the following peptides may enter the brain by crossing the barrier: enkephalin, thyroid-releasing hormone, d sleepinducing peptide, vasopressin and glutathione. The apparent uptake of several peptides is characterized by saturation kinetics, suggesting the presence of carrier-mediated transport systems. There is evidence that insulin may be transcytosed across the bloodbrain barrier. In addition, much attention has been given recently to the circulating protein transferrin, which appears to cross the blood brain barrier transcytotically. Both insulin and transferrin bind to specic receptor sites located on the luminal plasma membrane. After binding, endocytosis of the substrate occurs, after which the endocytic vesicle moves across the endothelial cell and exocytically releases the substrate at the abluminal membrane. It is currently believed that the bloodbrain barrier serves to regulate extracellular uid homeostasis in the brain. The emerging picture is that the barrier drives net unidirectional movement of electrolytes and water from blood to brain. Thus, uid is secreted into the brain. This is thought to be accomplished by a polar arrangement of
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transport proteins in the luminal and abluminal plasma membrane domains of the endothelial cells that form the barrier. Furthermore, the processes responsible for uid and electrolyte transport appear to be under the control of peptide hormones released within the brain. Studies suggest that a Na 1 /H 1 antiporter is present in the luminal membrane, along with a nonspecic cationic channel, and a Na 1 /K 1 adenosine triphosphatase is located at the abluminal membrane (Figure 2). Such an arrangement of sodium transporters would allow for net unidirectional transport of uid from blood to brain. Thus, sodium would enter the endothelial cells from the blood, utilizing the transport proteins in the luminal membrane. It would then be actively extruded into the brain extracellular uid by the sodium pump positioned at the abluminal membrane. As net unidirectional sodium transport occurs from blood to brain, water follows passively. It appears that alterations in brain extracellular uid homeostasis may bring about the release of peptide hormones within the brain, which can modulate the rate of net uid transport across the blood brain barrier. Centrally released peptides that have been implicated in this process include atrial natriuretic factor, angiotensin II and vasopressin. Normally, uid secretion and drainage are balanced in the central nervous system, preventing uid accumulation.

Delivering Drugs to the Brain

Drugs may potentially cross the bloodbrain barrier by a variety of mechanisms. These include: simple diusion, carrier-mediated transport and transcytosis. Simple diusion occurs by unassisted movement of solute down its electrochemical gradient, from blood to brain. For a drug to diuse across the bloodbrain barrier, it must either pass between the endothelial cells via a paracellular pathway, or traverse the endothelium by penetrating the respective luminal and abluminal plasma membranes. As discussed above, since the endothelial cells are joined by tight junctions, relatively little paracellular transport occurs, and solutes enter the brain primarily by crossing the limiting plasma membranes. Thus, a hydrophilic drug must be small enough to enter water-lled channels in the membranes to get across by simple diusion. A lipophilic drug, however, could penetrate the lipid components of the membranes and traverse the cell. For this reason, a common strategy for designing drugs to cross the blood brain barrier is to make them more lipophilic. The disadvantages of this approach, however, are that: (1) the specicity of drug delivery may be compromised, since these drugs would likely enter cells throughout the body, and (2) the action of the drug may be aected by altering its chemical composition to make it more lipophilic. An additional strategy that has been used is to perfuse the brain with a hyperosmotic solution, temporarily disrupt-

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Blood Brain Barrier

ing the tight junctions of endothelial cells forming the bloodbrain barrier, and thus allowing for simple diusion of administered drugs through a paracellular pathway into the brain. A second mode of transport across the bloodbrain barrier involves membrane carriers. If a drug is recognized by carrier proteins in both membranes, and the conditions favour blood to brain movement, then passage of this drug across the bloodbrain barrier is carrier mediated. Carriermediated transport is either active or passive, depending on whether transfer occurs against an electrochemical gradient, or not. Theoretically, primary active transport would involve the direct application of energy in the form of adenosine triphosphate to one of the carriers. Secondary active transport would require co- or counter-transport of a solute passing down its electrochemical gradient, which in turn drives movement of the drug against its electrochemical gradient. Passive carrier-mediated transport would involve passage of drugs across the bloodbrain barrier in the direction of an electrochemical gradient, but movement is facilitated by the presence of carriers. Since substances such as glucose and amino acids normally enter the brain by utilizing carriers in the bloodbrain barrier, a possible strategy for drug delivery is to fashion therapeutics that are recognized by these or other transporters. Transcytosis represents a third mechanism by which drugs may be delivered to the brain. For this process, a solute binds to the luminal plasma membrane of the bloodbrain barrier, and is carried across the endothelial cell in an endocytic vesicle. Exocytosis occurs at the abluminal membrane, releasing the solute into the brain extracellular uid. A current strategy utilizing this mechanism is to conjugate a drug to a vehicle solute which is transcytosed by the bloodbrain barrier. Transferrin, which is recognized by a specic receptor on the luminal membrane, has been used in this way. Cationic albumin has also been suggested as a possible vehicle for transcytosis of drugs across the bloodbrain barrier.

allowing certain ones to pass and preventing the passage of others. Small lipophilic molecules (Mr 5 600 Da) such as alcohol or progesterone are able to penetrate the blood brain barrier by permeating the lipid components of the limiting plasma membranes, and diusing across the endothelium. Glucose and certain amino acids are transported by specic carrier proteins present in the membranes. These organic nutrients move passively from blood to brain extracellular uid down their electrochemical gradients, utilizing their respective carriers to facilitate transport. In addition, there is evidence describing transport of monocarboxylic acids, amines, purine bases and nucleosides by the bloodbrain barrier. Fluid and electrolyte transport is mediated by channels and carriers. It is currently believed that net unidirectional uid movement occurs from blood to brain across the bloodbrain barrier. This is accomplished by a polar arrangement of sodium transporters in the respective luminal and abluminal plasma membranes, with water passively following transport of sodium. The process appears to be regulated by hormones produced in the brain. With few exceptions, circulating peptides and proteins generally do not cross the bloodbrain barrier. There is evidence for carrier-mediated transport of enkephalin, thyroid-releasing hormone, d sleep-inducing peptide, vasopressin and glutathione. Furthermore, insulin and transferrin are able to cross by the process of receptormediated transcytosis. When designing systemically administered drugs for the treatment of central nervous system disorders, the molecules must be fashioned to cross the bloodbrain barrier in order to reach their site of action in the brain. This may involve making the molecules lipophilic, or designing them to interact with known transport mechanisms.

Further Reading
Betz AL and Goldstein GW (1986) Specialized properties and solute transport in brain capillaries. Annual Reviews of Physiology 48: 241 250. Davson H, Zlokovic B, Rakic L and Segal MB (1993) An Introduction to the Blood Brain Barrier. Boca Raton, FL: CRC Press. Doczi T (1993) Volume regulation of the brain tissue a survey. Acta Neurochirurgica 121: 18. Drewes LR (1998) Biology of the bloodbrain glucose transporter. In: Pardridge WM (ed.) Introduction to the Blood Brain Barrier, pp. 165 174. Cambridge: Cambridge University Press. Frelin C and Vigne P (1998) Ion channels in endothelial cells. In: Pardridge WM (ed.) Introduction to the Blood Brain Barrier, pp. 214 220. Cambridge: Cambridge University Press. Pardridge WM (1991) Peptide Drug Delivery to the Brain. New York: Raven Press. Peterson DR and Hawkins RA (1998) Isolation and behavior of plasma membrane vesicles made from cerebral capillary endothelial cells. In: Pardridge WM (ed.) Introduction to the Blood Brain Barrier, pp. 62 70. Cambridge: Cambridge University Press.

Summary
The bloodbrain barrier is formed by endothelial cells which line capillaries in the brain. Unlike endothelium elsewhere in the body, these cells possess tight junctions which form cell-to-cell attachments and separate the limiting plasmalemma into luminal (blood facing) and abluminal (brain facing) plasma membrane domains. The junctional complexes display a high electrical resistance, indicating that solute transport across the bloodbrain barrier is predominantly transcellular, rather than paracellular. Thus, for substances to pass from the blood to brain extracellular uid, they must cross both the luminal and abluminal plasma membranes. These membranes are selectively permeable to solutes carried in the blood,

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Smith QR and Stoll J (1998) Bloodbrain barrier amino acid transport. In: Pardridge WM (ed.) Introduction to the Blood Brain Barrier, pp. 188197. Cambridge: Cambridge University Press. Strange K (1992) Regulation of solute and water balance and cell volume in the central nervous system. Journal of the American Society of Nephrology 3: 1227.

Tsuji A and Tamai I (1998) Bloodbrain barrier transport of drugs. In: Pardridge WM (ed.) Introduction to the Blood Brain Barrier, pp. 238 247. Cambridge: Cambridge University Press.

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