the blood – brain barrier

M E T H O D S I N M O L E C U L A R M E D I C I N E TM Edited by Sukriti Nag The Blood–Brain Barrier Biology and Research Protocols Edited by Sukriti Nag The Blood–Brain Barrier Biology and Research Protocols 3 1 Morphology and Molecular Properties of Cellular Components of Normal Cerebral Vessels Sukriti Nag 1. Introduction The blood–brain barrier (BBB) includes anatomical, physicochemical, and bio- chemical mechanisms that control the exchange of materials between blood and brain and cerebrospinal fluid (CSF). Thus two distinct systems, the BBB and the blood–CSF barrier systems, control cerebral homeostasis. However, both systems are unique, the BBB having a 5000-fold greater surface area than the blood–CSF barrier (1,2). The con- centrations of substances in brain interstitium, which is determined by transport through the BBB, can differ markedly from concentrations in CSF, the composition of which is determined by secretory processes in the choroid plexus epithelia (3). This review will focus on cellular components of cerebral vessels with emphasis on endothelium, base- ment membrane, and pericytes as well as the perivascular macrophage (Figs. 1 and 2A), which in light of new information is distinct from pericytes. This review deals less with pathogenesis and more with some of the molecules that have been discovered in these cell types in the past decade. Although astrocytes invest 99% of the brain surface of the capillary basement membrane and are important in induction and maintenance of the BBB, this topic will not be discussed and readers are referred to reviews in the literature (4–11). 2. Cerebral Endothelial Cells Cerebral capillaries are continuous capillaries, their wall being composed of one or more endothelial cells. The endothelial surface of 1 g of cerebral tissue has been cal- culated to be approx 240 cm 2 (12). Cerebral endothelial cell surface properties such as charge and lectin binding are discussed in Chapter 9. Some of the markers specific for localization of cerebral endothelium and others that are ubiquitous, being present in endothelia of non-neural vessels, are given in Table 1 (13–28). 2.1. Cerebral Endothelial Cells in Vasculogenesis and Angiogenesis Vasculogenesis is the process whereby a primitive network is established during embryogenesis from multipotential mesenchymal progenitors. This occurs in the rat by embryonic d 10 after which the intraparenchymal network develops by sprouting from preexisting vessels, a process termed angiogenesis. Research in the past decade has led From: Methods in Molecular Medicine, vol. 89: The Blood–Brain Barrier: Biology and Research Protocols Edited by: S. Nag © Humana Press Inc., Totowa, NJ to a greater understanding of cerebral angiogenesis during brain development (28,29) and in pathological states, including neoplastic (28) and non-neoplastic conditions (30). During angiogenesis, endothelial cells participate in proteolytic degradation of the base- ment membrane and extracellular matrix and migrate with concomitant proliferation and tube formation. Subsequent stages of angiogenesis involve increases in the length of indi- vidual sprouts, the formation of lumens, and the anastomosis of adjacent sprouts to form vascular loops and networks. An integral component of angiogenesis is microvascular hyperpermeability, which results in the deposition of plasma proteins in the extracellu- lar space forming a matrix that supports the ingrowth of new vessels (31,32). Endothelial proliferation is tightly regulated during brain development (33). In the mouse brain, for example, endothelial turnover and sprouting are maximal at postna- tal d 6–8 (34). Proliferation then slows and the turnover is very low in the adult brain (35). However, endothelial cells in the adult are not terminally differentiated and post- mitotic cells and when stimulated such as occurs during wound healing or tumor growth, they can rapidly resume cell proliferation giving rise to new capillaries. 4 Nag Fig. 1. Segment of normal cerebral cortical capillary wall consists of endothelium (e) and a pericyte (p) separated by basement membrane. This rat was injected with ionic lanthanum, which has penetrated the interendothelial space upto the tight junction (arrowhead). ×70,000. Fig. 2. (see facing page) (A) A cryostat section shows perivascular macrophages using anti- ED2 antibody. The inset shows these cells at higher magnification. Note that these cells are asso- ciated with vessels, which have the caliber of veins and not capillaries. (C,D) Merged confocal images of normal rat brain dual labeled for Ang-1 and Ang-2 proteins. Normal vessels show endothelial localization of Ang-1 (green) only in rat brain (B) and choroid plexuses (C) and there is no detectable localization of Ang-2. Note the granular immunostaining in choroid plexus epithelial cells indicating colocalization of Ang-1 and Ang-2 (yellow). (D) Cultured cells derived from cerebral microvessels show adherance of antibody-coated ox red blood cells forming rosettes indicating presence of Fc receptors. Note that many of these cells contain Factor VIII indicating that they are endothelial cells (arrowheads). (E) Electron micrograph demonstrating that the cells to which antibody-coated ox red cells have adhered also show cytoplasmic Factor VIII immunostaining indicating its endothelial nature. Scale bar A–C = 50 µm; Inset = 25 µm; D × 100; E × 8000. Morphology and Molecular Properties 5 Morphologic studies have shown that brain capillaries are derived from endothelial cells from outside the brain that invade the neuroectoderm and differentiate in response to the neural environment (28). Using chick-quail transplantation experiments, con- vincing evidence was presented that BBB characteristics could be induced in endothe- lial cells, which invade brain transplants (36). Conversely, brain capillaries become permeable after invasion of somite transplants (36). These results indicate that organ- specific characteristics of endothelial cells may be induced and maintained by the local environment. Janzer and Raff (5) provided direct evidence that when purified type I astrocytes are transplanted into the rat anterior eye chamber or the chick chorioallan- toic membrane, the astrocytes induce a permeability barrier in invading endothelial cells. The specific mechanisms regulating angiogenesis are not fully understood but sev- eral potential regulators of this process include fibroblast growth factor, epidermal growth factor, transforming growth factors α and β, platelet-derived growth factor, ephrins, the family of the vascular endothelial growth factors (VEGF) and the angiopoi- etins (Ang). The VEGF family includes several members; VEGF or VEGF-A is best characterized, and numerous studies indicate the importance of VEGF-A in vasculo- genesis and angiogenesis during brain development (28,33,37). During embryonic brain angiogenesis, VEGF-A is expressed in neuroectodermal cells of the subependy- mal layer correlating with the invasion of endothelial cells from the perineural plexus (33). The high affinity tyrosine kinase receptors that bind VEGF-A, VEGFR-1 (flt-1; 38) and VEGFR-2 (flk-1; 39,40) are highly expressed in invading and proliferating 6 Nag Table 1 Markers for Localization of Normal Cerebral Endothelium Markers Specific for Cerebral Endothelium Glucose transporter-1 Kalaria et al. (13) γ-glutamyl transpeptidase Albert et al. (14) Neurothelin/HT7 protein (chick) Risau et al. (15); Schlosshauer and Herzog (16) (human) Prat et al. (17) OX-47 (Rat homologue of HT7) Fossum et al. (18) Endothelial barrier antigen (only in rat) Sternberger & Sternberger (19); Cassella et al. (20) Jefferies et al. (21) Transferrin receptor Dermietzel and Krause (22) Tight junction proteins: Liebner et al. (23) ZO-1 Occludin Markers Common to all Endothelia Endoglin (CD 105) Personal Observation Factor VIII Weber et al. (24) Growth Factors: VEGF-B Nag et al. (25) Angiopoietin-1 Nourhaghighi et al. (26) Lectin binding: Ulex europaeus agglutinin I Weber et al. (24) Enzymes: Endothelial nitric oxide synthase Nag et al. (27) Platelet/endothelial cell adhesion Plate (28) molecule-1, (PECAM-1, CD31) endothelial cells during brain development. This suggests that VEGF-A may act as a paracrine angiogenic factor. Both VEGF-A and its receptors are largely switched off in the adult (37,39). The importance of VEGF-A in cerebral angiogenesis has also been demonstrated in central nervous system (CNS) neoplasia (28) and non-neoplastic conditions, such as brain infarction (41–43) and brain injury (25,32). Recent studies demonstrate that VEGF-B, another member of the VEGF family, is constitutively expressed in cerebral endothelial cells (25). Angiogenesis following injury is also associated with increased expression of VEGF-B at both the gene and protein level at the injury site during angiogenesis (25). Angiopoietin-1 and -2 constitute a novel family of endothelial growth factors that function as ligands for the endothelial-specific receptor tyrosine kinase, Tie-2 (44). Angiopoietins are involved in later stages of angiogenesis when vessel remodeling and maturation takes place and have a role in the interaction of endothelial cells with smooth muscle cells/pericytes (45). Recent studies show constitutive expression of angiopoietin-1 protein in endothelium of normal cerebral vessels (Fig. 2B; 26,46). This protein is not specific for cerebral endothelium because it is also present in endothe- lium of choroid plexus and pituitary vessels (Fig. 2C). Increased angiopoietin-2 expres- sion at both the gene and protein level occurs during angiogenesis after brain injury (26,46),in cerebral tumors (47,48), and after infarction (43,49,50). 2.2. Properties of Cerebral Endothelial Cells Features that distinguish cerebral endothelial cells from those of non-neural vessels and form the structural basis of the BBB include the presence of tight junctions between cerebral endothelial cells, reduced endothelial plasmalemmal vesicles or caveolae, and increased numbers of mitochondria. 2.2.1. Endothelial Junctions 2.2.1.1 M ORPHOLOGY Transmission electron microscopy (TEM) shows that the junctions between adjacent cerebral endothelial cells are characterized by fusion of the outer leaflets of adjacent plasma membranes at intervals along the interendothelial space producing a penta- laminar apperarance and forming tight or occluding junctions that prevent paracellular diffusion of solutes via the intercellular route (see Figs. 3 and 4; 51–53). These tight junctions form the most apical element of the junctional complex, which includes both tight and adherens junctions. Subsequent studies using horseradish peroxidase as a tracer suggested that tight junctions extend circumferentially around cerebral endothelial cells; hence, their name zonula occludens (54,55). Permeability of these junctions to protein and protein tracers is further discussed in Chapter 6. Tight junctions are also present between arachnoidal cells located at the outer layers of the dura (56,57), and at the apical ends of choroid plexus epithelial cells (52,58–60) and ependymal cells (61). Certain areas of the brain, most of which are situated close to the ventricle and are therefore called circumventricular organs, have endothelial cells that do not form tight junctions. These areas include the hypothalamic median eminence, pituitary gland, choroid plexus, pineal gland, subfornicial organs, the area postrema, and the organum vasculosum of the lamina terminalis (60,62–64); together they comprise less than 1% Morphology and Molecular Properties 7 of the brain. Endothelium in these areas is fenestrated with circular pores having a diam- eter of 40–60 nm that are covered by diaphragms that are thinner than a plasma mem- brane and of unknown composition. Fenestrations allow free exchange of molecules between the blood and adjacent neurons. The epithelial cells, which delimit the cir- cumventricular organs, however, impede diffusion into the rest of the brain and the CSF (65). Therefore, substances that have entered these areas do not have unrestricted access to the rest of the brain. Freeze-fracture studies show that the tight junctions of cerebral endothelium of mammalian species are characterized by the highest complexity of any other body ves- sels (66). Eight to 12 parallel junctional strands having no discontinuities run in the lon- gitudinal axis of the vessel, with numerous lateral anastomotic strands. This pattern extends into the postcapillary venules, although in a less complex fashion (66). In cere- bral arteries, tight junctions consist of simple networks of junctional strands, with occa- sional discontinuities, whereas collecting veins, of which there are a few, have tight junctional strands that are free-ending and widely discontinuous (66). Another feature of cerebral endothelial tight junctions is the high association with the protoplasmic (P)-face of the membrane leaflet, which is 55% as compared with endothelial cells of non-neural blood vessels, which have a P-face association of only 10% (67). The tight junctions of choroidal epithelium consist of four or more strands or fibrils, arranged in parallel with few interconnections (68). In addition, focal discontinuities have been noted in the junctional strands, which may represent hydrated channels, thus explain- ing the leakiness of choroid plexus epithelium (69). Further details of freeze fracture studies of endothelial tight junctions are discussed in Chapter 3 and in studies in the literature (22,23,67,70–72). 2.2.1.2. TRANSENDOTHELIAL RESISTANCE The physiologic correlate of tightness in epithelial membranes is transepithelial resis- tance. Leaky epithelia generally exhibit electrical resistances between 100–200 Ω/cm 2 . 8 Nag Fig. 3. (A) Segment of cortical arteriolar endothelium from a control rat injected intra- venously with HRP showing tight junctions (arrowheads) and a zonula adherens (za) junction along the intercellular space between two endothelial cells. Also present are cross sections of actin filaments (ac) and microtubules (m) and two plasmalemmal vesicles (v). The vesicle at the luminal plasma membrane contains HRP. × 132,000. Cultured brain endothelial cells grown in the absence of astrocytes have an electrical resistance of approximately 90 Ω/cm 2 (73). The latter is 100-fold less than the electri- cal resistance across the BBB in vivo, which is estimated to be approx 4–8000 Ω/cm 2 (74,75). The electrical resistance of cultured endothelial cells can be increased to 400–1000 by using special substrata such as type IV collagen and fibronectin (76). Co-culture of brain microvascular endothelial with astrocytes increases transendothe- lial electrical resistance by 71% (77) and treatment with glial-derived neurotrophic factor and cAMP increases transendothelial electrical resistance by approx 250%. (78). The resistance of the isolated arachnoid membrane with its tight junctions is less than that of intracerebral vessels being approx 2000 Ω/cm 2 (79),while the junctions of Morphology and Molecular Properties 9 Fig. 4. Proposed locations of the major proteins associated with tight junctions (TJs) at the BBB are shown. The tight junction is embedded in a cholesterol-enriched region of the plasma membrane (shaded). Three integral proteins—claudin 1 and 2, occludin and junctional adhe- sion molecule (JAM)—form the tight junction. Claudins make up the backbone of the TJ strands forming dimers and bind homotypically to claudins on adjacent cells to produce the primary seal of the TJ. Occludin functions as a dynamic regulatory protein, whose presence in the mem- brane is correlated with increased electrical resistance across the membrane and decreased para- cellular permeability. The tight junction also consists of several accessary proteins, which contribute to its structural support. The zonula occludens proteins (ZO-1 to 3) serve as recog- nition proteins for tight junctional placement and as a support structure for signal transduction proteins. AF6 is a Ras effector molecule associated with ZO-1. 7H6 antigen is a phosphopro- tein found at tight junctions impermeable to ions and molecules. Cingulin is a double-stranded myosin-like protein that binds preferentially to ZO proteins at the globular head and to other cingulin molecules at the globular tail. The primary cytoskeletal protein, actin, has known bind- ing sites on all of the ZO proteins. (Modified from ref. 93.) choroid plexus epithelial cells have a resistance of 73 Ω/cm 2 and hence are considered to be leaky epithelia (80). 2.2.1.3. MOLECULAR STRUCTURE OF TIGHT JUNCTIONS Research in the past decade has provided new information on the proteins composing tight junctions using Madin Darby canine kidney epithelial cells, endothe- lial cells of non-neural vessels, cerebral endothelial cells, and other cell types. Tight junctions are composed of an intricate combination of transmembrane and cytoplasmic proteins linked to an actin-based cytoskeleton that allows these junctions to form a seal while remaining capable of rapid modulation and regulation (Fig. 4). Three integral pro- teins—claudin 1 and 2 (81), occludin (82) and junction adhesion molecule (JAM) (83)—form the tight junction. Claudins form dimers and bind homotypically to claudins on adjacent endothelial cells to form the primary seal of the tight junction (84). Occludin is a regulatory protein, whose presence at the BBB is correlated with increased elec- trical resistance across the barrier and decreased paracellular permeability (85). Occludin is not present in non-neural vessels thus differentiating the tight junctions of cerebral and non-neural vessels (85). Junctional adhesion molecules are localized at the tight junction and are members of the immunoglobulin superfamily, which can func- tion in association with platelet endothelial cellular adhesion molecule 1 (PECAM) to regulate leukocyte migration (83). Overexpression of JAM in cells that do not normally form tight junctions increases their resistance to the diffusion of soluble tracers, sug- gesting that JAM functionally contributes to permeability control (83). Tight junctions are also made up of several accessory proteins that are necessary for structural support such as ZO-1 to 3, AF-6, 7H6 and cingulin. The zonula occludens (ZO) proteins 1–3 (86,87) belong to a family of proteins known as membrane- associated guanylate kinase-like proteins (88),afamily of multidomain cytoplasmic molecules involved in the coupling of transmembrane proteins to the cytoskeleton. ZO-1 is a component of the human and rat BBB (89). The ALL-1 fusion partner from chromosome 6 (AF-6) is associated with ZO-1 and serves as a scaffolding component of tight junctional complexes by participating in regulation of cell–cell contacts via interaction with ZO-1 at the N terminus Ras-binding domain (90). 7H6 antigen is a phos- phoprotein found at tight junctions that are impermeable to ions and macromolecules (91). A recent review suggests that 7H6 is sensitive to the functional state of the tight junction (92). In response to cellular adenosine triphosphate (ATP) depletion, 7H6 reversibly disassociates from the tight junction while ZO-1 remains attached and there is concurrent increase in paracellular permeability (93). Cingulin is a double-stranded myosin-like protein localized at the tight junction and found in endothelial cells as well. Recent in vitro studies have shown that ZO-1, ZO-2, ZO-3, myosin, JAM and A6 inter- act with cingulin at the N-terminus, while myosin and ZO-3 bind at the C-terminus (94). Thus, cingulin appears to serve as a scaffolding protein that links tight junction acces- sory proteins to the cytoskeleton. Availability of antibodies to some of the tight junction proteins has allowed local- ization of these proteins in cerebral endothelium by immunohistochemistry. ZO-1 immunoreactivity occurs along the entire perimeter of cultured cerebral endothelial cells (73) and endothelial cells in cryostat sections of human brain (95). Cryostat sections 10 Nag of adult brain also show anti-ZO-1 immunoreactivity as fibrillar fluorescence along the lateral aspects of brain microvessels (22) which constitute interendothelial tight junc- tion domains. The ZO-1 protein occurs in approximately the same quantities (molecules per micron) as the intramembranous particles that constitute the junctional fibrils in freeze-fracture preparations (96,97). ZO-1 immunoreactivity is also observed in brain endothelial cells of chick and rat along with immunoreactivity for occludin, claudin-1 and claudin-5 (23). ZO-1 immunoreactivity is also present at the other known sites of tight junction locations within the CNS such as in the leptomeningeal layer, choroid plexus epithelium, and the ependyma (22). The primary cytoskeletal protein, actin, has known binding sites on all ZO proteins, and on claudin and occludin (98). Electron microscopy shows microfilaments having the dimensions of actin grouped near the cytoplasmic margins in proximity to cell junc- tions in cerebral endothelium (see Fig 3; 10,99,100) and actin has been localized to the plasma membrane by molecular techniques (101). ZO-1 binds to actin filaments and the C-terminus of occludin (98), which couples the structural and dynamic properties of perijunctional actin to the paracellular barrier. Tight junctions are localized at cholesterol-enriched regions along the plasma mem- brane associated with caveolin-1 (102). Caveolin-1 interacts with and regulates the activ- ity of several signal transduction pathways and downstream targets (103). Several cytoplasmic signaling molecules are concentrated at tight junction complexes and are involved in signaling cascades that control assembly and disassembly of tight junctions (104). Regulation of tight junctions is discussed in previous reviews (67,93,104–106). Adherens junctions are located near the basolateral side of endothelial cells (Fig. 3). Adherens junction proteins include the E, P, and N cadherins, which are single-pass transmembrane glycoproteins that interact homotypically in the presence of Ca 2+ (107). These cadherins are not specific for cerebral endothelial junctions being present in endothelium of non-neural blood vessels as well (108). Cadherins are linked intracel- lularly to a group of proteins termed catenins (109). α-catenin is a vinculin homolog that binds to β-catenin and probably links cadherins to the actin-based cytoskeleton and to other signaling components. γ-catenin is related to β-catenin and can substitute for it in the cadherin-catenin complex. Catenins are, thereby, part of the system by which adherens and tight junctions communicate. All these molecules are expressed at junc- tions in brain endothelial cells (110,111). The newer cadherin-associated protein, p120 and a related protein p100 are associated with the cadherin/catenin complex in both epithelial and endothelial cells (112). The interaction of these proteins in junctional per- meability has been recently reviewed (67,106). 2.2.2. Endothelial Plasmalemmal Vesicles or Caveolae 2.2.2.1. M ORPHOLOGY TEM studies show membrane-bound vesicles open to both the luminal and ablumi- nal plasmalemma through a neck 10–40 nm in diameter and also free in the endothe- lial cytoplasm of vessels of most organs (Fig. 3; Chapter 6, Fig. 1). These non-coated structures referred to in the previous literature as pinocytotic vesicles are now gener- ally referred to as plasmalemmal vesicles or caveolae. These vesicles are distinct from clathrin-coated vesicles, which have an electron-dense coat and are involved in Morphology and Molecular Properties 11 [...]... + – + + + – – – + ± – – – + + + + – + + + + + + + + – – – – – NK NK NK + – – – NK NK + NK – NK + + + + + SM = smooth muscle; NM = nonmuscle; GSA = Griffonia Simplicifolia Agglutinin; NK = not known References: Alliott et al (231); Balabanov and Dore-Duffy (232); Bandopadhyay et al (233) filaments (238,239) have been demonstrated in pericytes in vitro with a portion of this actin corresponding to the. .. cerebral endothelium during normal and hyperosmotic conditions Lab Invest 50, 31 3–3 22 67 Kniesel, U., and Wolburg, H (2000) Tight junctions of the blood- brain barrier Cell Mol Neurobiol 20, 5 7–7 4 68 Brightman, M W., and Tao-Cheng, J H (1993) Tight junctions of brain endothelium and epithelium In The Blood- Brain Barrier Cellular and Molecular Biology (Pardridge, W M., ed.), Raven, New York, pp 10 7–1 25 69... control at the blood- brain barrier Brain Res Rev 36, 25 8–2 64 106 Rubin, L L., and Staddon, J M (1999) The cell biology of the blood- brain barrier Annu Rev Neurosci 22, 1 1–2 8 107 Takeichi, M (1995) Morphogenetic roles of classic cadherins Curr Biol 7, 61 9–6 27 108 Lampugnani, M G., and Dejana, E (1997) Interendothelial junctions: structure, signalling and functional roles Curr Opin Cell Biol 9, 67 4–6 82 109... nitrotyrosine during blood- brain barrier breakdown and repair after cold injury Lab Invest 81, 4 1–4 9 28 Plate, K H (1999) Mechanisms of angiogenesis in the brain J Neuropathol Exp Neurol 58, 31 3–3 20 29 Risau, W (1997) Mechanisms of angiogenesis Nature 386, 67 1–6 74 30 Nag, S (2002) The blood- brain barrier and cerebral angiogenesis: lessons from the coldinjury model Trends Mol Med 8, 3 8–4 4 31 Dvorak, H F.,... distribution of an endothelial barrier antigen between the pial and cortical microvessels of the rat Brain Res 744, 33 5–3 38 21 Jefferies, W A., Brandon, M R., Hunt, S V., Williams, A F., Gatter, K C., and Mason, D Y (1984) Transferrin receptor on endothelium of brain capillaries Nature 312, 16 2–1 63 22 Dermietzel, R., and Krause, D (1991) Molecular anatomy of the blood- brain barrier as defined by immunocytochemistry... in the choroid plexus epithelium, a freeze fracture study including complementary replicas J Cell Biol 80, 66 2–6 73 70 Connell, C J., and Mercer, K L (1974) Freeze-fracture appearance of the capillary endothelium in the cerebral cortex of mouse brain Am J Anat 140, 59 5–5 99 71 Farrell, C L., and Shivers, R R (1984) Capillary junctions of the rat are not affected by osmotic opening of the blood- brain barrier. .. 181, 42 7–4 41 59 van Deurs, B (1980) Structural aspects of brain barriers, with special reference to the permeability of the cerebral endothelium and choroidal epithelium Int Rev Cytol 65, 11 7–1 91 24 Nag 60 Nag, S (1991) Effect of atrial natriuretic factor on permeability of the blood- cerebrospinal fluid barrier Acta Neuropathol (Berl.) 82, 27 4–2 79 61 Brightman, M W., and Palay, S L (1963) The fine... small proteins in the continuous endothelium Am J Physiol 272, H937–H949 130 Simionescu, N., Simionescu, M., and Palade, G E (1975) Permeability of muscle capillaries to small hemepeptides Evidence for the existence of patent transendothelial channels J Cell Biol 64, 58 5–6 07 131 Nag, S (1998) Blood- brain barrier permeability measured with histochemistry In Introduction to the Blood- Brain Barrier Methodology,... 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Endothelium in these. molecules between the blood and adjacent neurons. The epithelial cells, which delimit the cir- cumventricular organs, however, impede diffusion into the rest of the brain and the CSF (65). Therefore,. systems, the BBB and the blood–CSF barrier systems, control cerebral homeostasis. However, both systems are unique, the BBB having a 5000-fold greater surface area than the blood–CSF barrier (1,2). The