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Cipolla MJ. The Cerebral Circulation. San Rafael (CA): Morgan & Claypool Life Sciences; 2009.

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The Cerebral Circulation.

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Chapter 6Barriers of the CNS

The first studies to demonstrate the existence of a selective barrier between the blood and the brain were done by Ehrlich in 1885 [173]. In a classic series of experiments, Ehrlich infused Evan’s blue dye intravenously into a rat and found that all organs in the body stained except for the brain. However, he incorrectly surmised that the brain was made of tissue for which the dye could not adhere to. It was his graduate student, Goldmann, in 1913 that did the decisive experiment and injected dye into the CSF, finding that in this case, only the brain tissue stained [174]. He correctly surmised that there was a barrier between the brain and the blood. These experiments also determined that although there was a barrier between the blood and the brain, there was free access from CSF to brain and, therefore, there was no CSF–brain barrier (Figure 18). There are three main interfaces in the brain that protect neurons from blood-borne substances and help to maintain water homeostasis and an appropriate milieu for neuronal function: the blood–CSF interface, the blood–brain interface (BBB), and the CSF–blood interface [175,176] (Figure 19). The cerebral endothelium forms the largest barrier in the brain, the BBB, while epithelial cells of the choroid plexus form the blood–CSF barrier, and the avascular arachnoid epithelium lies under the dura and completely encases the brain, forming the CSF–blood barrier. Other interfaces with blood and neural tissue include the blood–retinal barrier and the blood–spinal cord barrier. These barriers within the central nervous system provide several protective functions for the brain. They are protective against unwanted pathogens and control the immunologic status of the brain [177]. The tight junctions at the BBB do not allow ions to move passively into the brain and thus prevent fluctuations in electrolytes that occur in the blood [178]. They also prevent proteins (albumin) and circulating blood cells (erythrocytes, leukocytes) from passing into the brain, which can damage neuronal tissue and interfere with tightly controlled water homeostasis [175178].

FIGURE 18. The blood–brain barrier, or BBB, to trypan blue and its diffusion from the cerebrospinal fluid, or CSF, into the brain.


The blood–brain barrier, or BBB, to trypan blue and its diffusion from the cerebrospinal fluid, or CSF, into the brain. Used with permission from Neuron 2008;57:178–201.

FIGURE 19. Schematics of the sites of the barrier interfaces (indicated in orange) in the adult and developing brain.


Schematics of the sites of the barrier interfaces (indicated in orange) in the adult and developing brain. (a) The blood–brain barrier is a barrier between the lumen of cerebral blood vessels and brain parenchyma. The endothelial cells (Endo) (more...)

The Blood–CSF Barrier

CSF is formed in the lateral third and fourth ventricles mainly by the choroid plexus and cerebral capillaries [179]. CSF functions as a cushion for the brain and spinal cord and provides important nutrients. CSF has the same composition as interstitial fluid (ISF) and mixes freely together across pial surfaces [179181]. The capillaries of the choroid plexus do not have BBB properties, but are fenestrated and leaky [180]. However, the tight junctions of the ependymal cells of the choroid plexuses form the blood–CSF barrier [180]. Ependymal cells of the choroid plexus are epithelial-like. Ionic pumps, most importantly, the Na+–K+ ATPase on the apical surface of the ependymal cells produces the chemiosmotic energy for the osmotic gradient that contributes to fluid formation by the cells of the choroid plexus [182]. Water flow follows the osmotic gradient set up by the removal of three Na+ ions for 2 K+ ions. Formation of CSF by the choroid plexus is facilitated by the very high rates of blood flow to the choroid plexus [183].

CSF is formed at a rate of ~600 ml/day [184]. This rapid production results in turnover of CSF several times during the day. ISF formed by cerebral capillaries joins CSF formed by the choroid plexus in the cerebral ventricles and is the starting point of circulating CSF [181]. From the ventricles, CSF exits into the cisterna magna and then over the cerebral covexities. CSF is absorbed at the arachnoid granulations into the blood before flowing up over the cerebral hemispheres.

The Blood–Brain Barrier

In the brain and spinal cord, the BBB is formed by cerebral endothelial cells that have highly specialized structural and functional properties [185,186]. Brain endothelial cells are phenotypically unique compared to endothelium in the periphery in that they have apical tight junction complexes that more closely resemble epithelium than endothelium [12,185,186]. In addition, while specialized tight junctions of the BBB limit passive diffusion of blood-borne solutes, brain endothelium also contains transporters that actively control transport of nutrients into the brain from the blood [12,185187]. Like peripheral endothelium, cerebral endothelial cells are polarized and express specific transporters apically to actively transport nutrients from the blood into the brain and basolaterally to inactivate toxic substances and remove (efflux) them from the brain into the blood [12]. Thus, the cerebral endothelium provides a highly restricted, but controlled barrier to plasma constituents. Other unique features of the cerebral endothelium are a lack of fenestrations, a very low rate of pinocytosis that limits transcellular transport, and a high number of mitochondria associated with its high metabolic activity [6,7,12,88].

Although often overlooked, the BBB is present in cerebral endothelium throughout the brain, including pial arteries and arterioles and veins, but is absent from the circumventricular organs (CVO) [6,7,12,188]. The CVO are highly specialized areas in the brain and include area postrema and median eminence, neurohypophysis, pineal gland, sub-fornica organ, and lamina terminalis, which require significant cross-talk between the brain and peripheral blood, e.g., release and transport of hormones [188,189]. Therefore, the cerebral endothelium of the CVO are fenestrated and do not have BBB properties. It is often thought that the CVO are an area in the brain without barrier properties, but this is not the case. The barrier for the CVO lies in the epithelial cells known as tanycytes and ependymal cells. Thus, circulating substances can diffuse into the CVO but not beyond.

Ultrastructure of the BBB

Ultrastructural studies by Reese and Karnovsky characterized brain endothelium as the morphologic site of the BBB [185]. Cerebral endothelial cells are connected paracellularly at junctional complexes by tight junctions and adherens junctions [12,188]. The molecular organization of the BBB tight junction and its adapter proteins that link to the actin cytoskeleton forms a continuous membrane that confers the high electrical resistance of the BBB (~1500–2000 Ω-cm2) and retention of ions in the vascular lumen [12,99,178,188] (Figure 20). Many disease states including chronic disease, such as multiple sclerosis, experimental autoimmune encephalomyelitis, and Alzheimer’s disease, and acute conditions, such as ischemic stroke, hypertension, and seizure, have been associated with dysregulation of tight junction proteins [12,99].

FIGURE 20. A simplified molecular atlas of the BBB.


A simplified molecular atlas of the BBB. (A) Tight junctions. Claudins (claudin-3, claudin-5, and claudin-12) and occludin have four transmembrane domains with two extracellular loops. Zonula occludens proteins (ZO -1, ZO-2, and ZO-3) and the calcium-dependent (more...)

Tight Junctions

Tight junctions consist of three integral membrane proteins (claudin, occludin, and junction adhesion molecules (JAM)) and several accessory proteins including zona occludens (ZO) (ZO-1, ZO-2, ZO-3), cingulin, and others (190–193). Claudins are 22-kDa phosphoproteins that comprise the major component of tight junctions (194). Greater than 20 members of the claudin family have been identified [195]. They are located at the tight junction strand and bind other claudins homotypically on adjacent endothelial cells to form the primary seal of the tight junction [194]. The carboxy terminus of the claudins bind to cytoplasmic proteins including ZO-1, ZO-2, and ZO-3 (194). The zona occludens proteins (ZO-1, ZO-2, and ZO-3) together with cingulin and several others are cytoplasmic proteins involved in tight junction formation [196,197]. ZO-1 and ZO-2 bind to the actin cytoskeleton at their carboxy terminus [194]. This critical link provides for structural stability of the endothelial cell and is an important means of regulating paracellular permeability [99]. Occludin is a 65-kDa phosphoprotein with four transmembrane domains, a long carboxy-terminal cytoplasmic domain, and a short amino-terminal cytoplasmic domain [188,198,199]. Two extracellular loops of occludin and claudin originating in neighboring cells form the paracellular barrier of the tight junction [188]. Occludin is directly linked to the zona occludens proteins and thereby regulates permeability through their association with the actin cytoskeleton [200]. JAM are 40-kDa membrane proteins within the tight junction that also bind ZO-1 [201]. Of the three JAM molecules identified, only JAM-1 and JAM-3, but not JAM-2, are expressed in brain endothelium [202]. JAM-1 localizes with actin and is involved in cell-to-cell adhesion [202].

Adherens Junctions

Adherens junctions form adhesive contacts between cells and consist of the membrane protein cadherin that joins the actin cytoskeleton via intermediary proteins, the catenins [188,203]. Adherens junctions form from homophilic interactions between extracellular domains of cadherins on the surface of adjacent cells [204]. The cytoplasmic domains of cadherins bind to β- or γ-catenin, which are linked to the cytoskeleton via α-catenin [204]. Adherens junctions interact with tight junctions via ZO-1 and catenins to influence tight junction assembly [205].

Regulation of Paracellular Permeability

The contractile activity of endothelial cells via actin stress fibers within the cytoplasm is of central importance to regulating tight junction permeability [99,206]. Agonists that promote relaxation of stress fibers (e.g., cyclic-AMP) decrease permeability by cell spreading, which strengthens cell–cell contact and reduces paracellular transport [207]. Alternatively, agonists that promote stress fiber contraction (e.g., PKC, VEGF) promote increased permeability by causing cell rounding, which decreases cell–cell contact [208]. Studies have demonstrated that inhibition of myosin light-chain (MLC) phosphorylation, which inhibits actin stress fiber contraction, decreases agonist-induced permeability [209,210]. Stress fiber activity as an underlying mechanism of paracellular transport is an important consideration to the numerous mediators of cerebral edema formation that are produced by the injured brain, e.g., histamine, bradykinin, arachidonic acid, etc. [99,206].

BBB Transporters

The BBB freely passes oxygen, carbon dioxide, and small lipophilic substances, but is impermeable to hydrophilic molecules such as glucose, amino acids, and other nutrients essential to life [12]. Thus, a major physiological function of the BBB is the tight regulation of transport of nutrients and other molecules into and out of the brain. In addition, BBB transporters are involved in inactivation and reuptake of neurotransmitters [12,188]. The high electrical-resistant tight junctions only allow small lipid-soluble molecules (<400 Da) to cross the BBB [211]. All other substances must cross the BBB through specific transporters on either the apical or basolateral endothelial membrane [12,188]. Specific carrier-mediated transport (facilitated diffusion) systems facilitate transport of nutrients such as glucose and galactose, amino acids, nucleosides, purines, amines, and vitamins down their concentration gradient from the blood to the brain [12,188]. Transport of these nutrients is in general regulated by brain metabolic needs and the concentration of substrates in plasma. There are also receptor-mediated transport systems for proteins and peptides to transport neuroactive peptides, chemokines, and cytokines into the brain [2,188]. Large proteins such as transferrin, low-density lipoprotein (LDL), leptin, insulin, and insulin-like growth factor also use specific receptor-mediated transport systems to cross the BBB [264,265]. Active efflux transporters are located on both apical and basolateral endothelial membranes and are important for removal of molecules from the brain into the blood. A number of active efflux transporters have been identified, most belonging to a large family of proteins called the ATP-binding cassette (ABC) transporter superfamily [12,188]. Transporters such as these use ATP-bound energy for the transport of molecules across the cell membrane and include the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) that mediates removal of toxic lipophilic metabolites and cationic drugs, multidrug resistance-associated proteins (MRP), the breast cancer resistance protein (BCRP), and other transporters of anionic compounds [12,188].

Transcellular Transport

While tight junction proteins control paracellular movement of ions and solutes, a transcellular route also exists in cerebral endothelium for passage of lipophobic molecules through three distinct routes. Fluid-phase endocytosis is a constitutive process for passing macromolecules into the brain and for recycling the plasma membrane [212,213]. Molecules are internalized indiscriminantly without binding to the cell surface. In general, fluid-phase endocytosis is low in the cerebral endothelium, but is induced under pathologic conditions such as ischemic stroke and acute hypertension [206]. Absorptive endocytosis occurs when molecules such as lectins bind to carbohydrate moieties or to the negatively charged glycocaylyx causing endocytosis [12,188]. Receptor-mediated endocytosis occurs when a specific molecule (ligand) binds to a receptor on the endothelial cell surface, triggering internalization of the receptor–ligand complex [12,188]. This process can be clathrin-mediated and involves cavaolae or clathrin-44-independent.

Water Homeostasis in the Brain

The brain is unique in how it deals with water flow from the blood [178]. For all other tissues, there is convective water flow into the tissue with solute between endothelial cells such that only plasma proteins are retained in the vascular lumen [214]. Protein osmotic pressure offsets the efflux of fluid due to blood hydrostatic pressure and gives rise to Starling’s forces. However, at the BBB, there is limited molecular transport due to a low rate of fluid-phase endocytosis, which limits transcellular flux, and coupling by high electrical resistance tight junctions, which limits paracellular flux [215,216). These morphologic features prevent the extravasation of large and small solutes and, importantly, ions [178,214,215]. The tight junctions of the brain effectively prevent the movement of hydrophilic substances, including plasma proteins and univalent cations such as Na+ and K+ [178]. These unique barrier properties modifies Starling’s forces such that any movement of water into the brain by normal blood hydrostatic pressure (CPP) is immediately opposed by the osmotic pressure gradient set up by the ions retained in the vascular lumen [178]. This unique situation prevents vasogenic edema formation and is considered to be a protective role of the BBB.

Hydraulic Conductivity

Another unique feature of cerebral endothelium is that is has an unusually high resistance to water filtration in response to hydrostatic pressure, a parameter known as hydraulic conductivity (Lp). Unlike measures of solute or tracer permeability, Lp is the critical transport parameter that relates water flux to hydrostatic pressure [178]. Together with transvascular filtration (Jv), Lp is an important

determinant of the movement of water into the brain [217,218]. Lp is also a characteristic parameter of convective fluid motion and can influence the mass transport of solutes and other molecules through the endothelium [219]. Therefore, this parameter encompasses both transcellular and paracellular routes of permeability.

Role of Astrocytes in BBB Function

While it is the structural properties of the cerebral endothelium that make up the BBB (e.g., tight junctions), associated cells within the brain parenchyma contribute to its barrier properties, most notably astrocytes. Astrocytes are specific brain cells located among endothelium, pericytes, and neurons. An early concepts of the BBB was that astrocytic end-feet, which are in close proximity to cerebral endothelial cells, provided a structural barrier and thus contributed to BBB properties. However, studies by Reese and Karnovsky showed that the site of barrier function was at the level of the cerebral endothelial cells and not astrocytes [185]. A role for astrocytes in inducing BBB properties of the cerebral endothelium, as opposed to being a physical barrier, became apparent [220]. In vitro cell culture studies confirm an important role of glial cells in inducing BBB phenotype, including upregulation of tight junction proteins [221,222]. The interaction of astrocytes and cerebral endothelium is not one way. There is considerable evidence that the cerebral endothelium signals astrocytes. For example, the water channel aquaporin-4 (AQP-4) is primarily expressed only in astrocytic end-feet surrounding vessels in the brain parenchyma, but not in astrocytes just interacting with neurons [223] (Figure 21). Thus, the maintenance of BBB properties and function likely depends on cross-talk between the endothelium and astrocytes. In addition, astrocytes have a large number of K+ channels (Kir4.1 and rSloKCa) and spatially buffer K+ in the perivascular space [224]. Astrocytes have other important functions in regulating water and ionic homeostasis in the brain and are important contributors to cytotoxic edema in the brain during injury [206] (see Vasogenic Edema).

FIGURE 21. Double immunolabeling of AQP-4 (red) and GFAP (green).


Double immunolabeling of AQP-4 (red) and GFAP (green). AQP-4 immunolabeling reveals that the entire network of vessels, including capillaries, is covered by astrocytic processes, albeit GFAP-negative. Smaller vessels and capillaries are mostly GFAP-negative (more...)

Cerebral Edema Formation

Cytotoxic vs. Vasogenic Edema

Klatzo first characterized brain edema as cytotoxic vs. vasogenic depending on whether or not the BBB is disrupted [225]. Cytotoxic edema occurs when brain cells swell at the expense of the extracellular space, but BBB properties are present. Vasogenic edema occurs when there is increased permeability of the BBB, which allows an influx of plasma constituents, and water, which expand the extracellular space. Several pathological conditions cause breakdown of the BBB and vasogenic edema, including ischemic stroke, acute hypertension, seizure, and traumatic brain injury. Cell swelling associated with cytotoxic edema may be compensatory or even protective. Precapillary astrocyte end-feet are the first cellular elements to swell during ischemia [226], a process thought to normalize the composition of the extracellular environment for normal neuronal activity [227]. Glial cells can also inactivate neurotransmitter [228], take up excess potassium ions produced during neuronal activity [229], and scavenge reactive oxygen species [230] (see Role of Astrocytes).

Under normal conditions, the barrier property of the cerebral endothelium has low hydraulic conductivity, and prevents bulk flow of water, ions, and proteins into the brain from hydrostatic forces such as blood pressure [231]. When the BBB is disrupted (e.g., during ischemia or acute hypertension), hydrostatic forces become significant enough that the rate of protein entry into the brain is directly related to the pressure gradient between the blood and the brain. However, while the passage of protein from the plasma into the brain is a measure of BBB disruption, it does not significantly contribute to edema formation. Albumin and other proteins passing into the brain have been used as a measure of BBB disruption; however, the concentration of a large protein is several orders of magnitude smaller than that of ions. Therefore, the increase in osmolality that occurs when albumin enters the brain is small compared to that of ions. Albumin and protein entry into the brain may be an indicator of BBB disruption, but its contribution to edema formation is small compared to that of ions.

Copyright © 2010 by Morgan & Claypool Life Sciences.
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