The BBB a Neurovascular Physiological Unit The Concept

The BBB, which separates the blood from the brain, and vice versa, was first described in 1885 by Paul Ehrlich and confirmed later in 1909 by Edwin Goldman. Both investigators showed that, following intravenous injection, trypan blue (an albumin-binding dye) was dispersed throughout the whole body, except for the brain. Conversely, injection of this dye into the brain subarachnoidal space selectively stained only the brain. Reese and Karnovsky [161], Brightman and Reese [162] and others, demonstrated that movement of the markers from the bloodstream to the brain, and from the brain to the bloodstream, was stopped at the level of the endothelial junctions [163].

The anatomic structure of the BBB is composed of the cerebral microvascular endothelium which, together with astrocytes, pericytes, neurons, and the extracellular matrix, constitutes a "neurovascular physiological entity" (Fig. 1.7A) that is essential for the health and function of the CNS [164].

Astrocytic projections [165] and neurons almost completely cover the basolateral surface of the brain microvessel ECs, which form capillaries with tight junctions. The tight junction is an intricate complex of transmembrane (junctional adhesion molecule-1, occludin, claudin, etc.) and cytoplasmic proteins (zonula occludens-1,2, cingulin, AF-6, 7H6, etc.) linked to the cytoskeleton. These tight junctions represent the physical barrier which limits the penetration of most chemical compounds into the brain [166].

The BBB functions primarily as a physical barrier to the passage of small molecules by a paracellular route (between the cells) (Table 1.3).

Although astrocytes via their "end-feet" - which are in direct contact with the ECs - also participate in the BBB structure, they take no part in the physical barrier. However, it is this interaction of the astrocytes that induces the unique BBB endothelial phenotype in the brain. The major characteristics of this barrier are: (1) small intercellular space between adjacent ECs (< 10 A) [162]; (2) high electrical resistance (> 2000 Q, X cm- ) [167]; and (3) low paracellular permeability [168]. The tight junctions between ECs of the BBB restrict the paracellular diffusion

Fig. 1.7 The principle and technology of the in-vitro blood-brain barrier model.

(A) A single neurovascular barrier unit modeled in vitro using transwell inserts.

(B) SEM micrograph of confluent monolayer insert membrane. (C) SEM micrograph of glial cell projection entering a pore in the basolateral (glia) side; the insert shows glial projections transversing the transwell insert membrane and extending to the apical

Fig. 1.7 The principle and technology of the in-vitro blood-brain barrier model.

(A) A single neurovascular barrier unit modeled in vitro using transwell inserts.

(B) SEM micrograph of confluent monolayer insert membrane. (C) SEM micrograph of glial cell projection entering a pore in the basolateral (glia) side; the insert shows glial projections transversing the transwell insert membrane and extending to the apical of endothelial cells on the top of the transwell (endothelial) side.

of water-soluble chemicals. Passive permeation of the BBB is generally related to the degree of the solute's lipophilicity.

The microvascular endothelium in the BBB serves as an active, energy-dependent barrier, with unique transport properties. In line with their high enzymatic and metabolic activities, brain ECs possess a large number of mitochondria, fuelling an elaborate system of transport proteins (influx and efflux carriers and pumps). The asymmetric distribution of the transporters on the luminal and abluminal EC membrane allows for the vectorial exchange of selected chemicals into or out of the brain. This biochemical-transport barrier ensures a selective permeability of nutrients, neurotransmitter precursors, and xenobiotics. As an enzymatic-barrier, BBB microvascular ECs express a large variety of metabolizing enzymes which serve as biotransformation and detoxification systems, metabolizing and excreting lipophilic endogenous and exogenous chemicals which may have invaded the brain environment.

The ECM of the BBB is mainly composed of fibronectin, laminin, and collagen type IV [169]. Developmentally, the matrix constitutes a pivotal biological platform for the growth of brain microvascular ECs. The cells interact with the ECM proteins through specific integrin receptors [170] which, upon activation, induce important

Table 1.3 The evolution of in-vitro blood-brain barrier (BBB) models.

BBB model (generation)

Endothelium (abbreviation)

Astroglia

Neurons/ Pericytes

Major finding

Ref.

HUVEC-304 MBCEC

-

-

Polycarbonate membranes are highly suitable for in-vitro BBB models

Elevated intracellular cAMP levels - increases TEER Addition of hydrocortisone - increases TEER

[245]

2

PBCEC

C6-glioma

-

Co-culture - increases y-GT and Na+/K+ ATPase activity

[246]

PBCEC

Rat astrocytes and C6

-

Co-culture - increases y-GT and ALP activities

[247]

HCEC

-

-

Astrocytes conditioned media - increases TEER

[248]

RBE4

C6-glioma

-

C6-glioma conditioned media or matrix - increases P-gp activity and levels

[249]

b. End5

C6-glioma

-

Co-culture - increases TEER

[170]

BAEC

C6-glioma

-

Co-culture - increases TEER

[250]

RBMEC

Rat astrocytes

-

Co-culture - increases TEER and y-GT activity

[189]

b. End3

C6-glioma

-

Co-culture - increases TEER and ALP activity

[251]

PBCEC

Porcine astrocytes

-

Co-culture - increases TEER and decreases inulin permeability

[252]

RBE4

U-373 MG

-

Co-culture - increases TEER

[253]

BAEC

C6-glioma

-

Addition of dexamethasone - increases TEER and decreases sucrose permeability

[254]

BBB model

Endothelium

Astroglia

Neurons/

Major finding

Ref.

(generation)

(abbreviation)

Pericytes

3

PBEC

-

Murine cortical neurons

Co-culture - increases y-GT and Na+/K+-ATPase activities

[246]

RBMEC

Rat astrocytes

B14

Tri-culture decreases sucrose permeability

[255]

RBE4.B

-

Rat neurons

Neurons regulate occludin localization

[256]

RBE4.B

-

Rat neurons

Neurons induce selective exclusion of dopamine, but allow l-tryptophan and l-DOPA permeability

[190]

RBE4.B

Rat astrocytes

Rat neurons

Neurons and astrocytes synergistically induce localization of occludin in endothelial plasma membrane

[257]

RBE4.B

Rat astrocytes

Rat neurons

Tri-culture decreases sucrose permeability

[198]

= bovine brain microvascular endothelial cells = rat brain microvascular endothelial cells = porcine brain capillary endothelial cells = bovine aortic endothelial cells = mouse brain capillary endothelial cells = human capillary endothelial cells = mouse brain endothelial cells = rat brain endothelial cells = trans-endothelial electrical resistance

Abbreviations:

BBMEC

RBMEC

PBCEC

BAEC

MBCEC

HCEC

bEnd3 and 5

RBE4 and4B

TEER

genetic and phenotypic changes [171]. The ECM proteins have a crucial role in inducing and maintaining the barrier properties of ECs by contributing to their differentiation processes.

Astrocytic projections, also known as "end-feet", are instrumental for maintaining the barrier characteristics of cerebral ECs [165]. The barrier-generating effect of astrocytes can be separated into: (1) the secretion of a set of humoral growth factors that encourage barrier generation by acting on ECs [172]; and (2) an extension of"end-feet" bulbs to make direct contact with the ECs. Both these factors synergistically affect formation of the BBB-specific barrier function [173].

Passive paracellular permeability relies on simple diffusion. Only small molecules with a molecular weight of< 400-500 Da [174] and a molecular diameter < 20 A [175] can cross the barrier through the paracellular route. In contrast to most other tissues, the mean distance between cerebral capillaries is rather short, on the average ~40 }im [176]. For small gaseous molecules (e.g., oxygen) and volatile anesthetics, which penetrate the BBB passively by the paracellular route, the high capillary density facilitates almost immediate equilibration throughout the brain parenchyma. Normally, the paracellular pathway has little therapeutic relevance for neurologically active small molecular weight drugs, such as L-dopa, due to an inability to accumulate therapeutically effective drug levels inside the cerebral milieu. By contrast, for most drugs the transcellular pathway (permeability through the cells) is the major route of entry into the brain. Transcellular transport can be divided into two general subgroups: (1) passive transcellular permeability, which depends mainly on the lipophilic properties of a given drug; and (2) active transcellular permeability, which utilizes a variety of transporters with bidirectional activity into and out of the brain [177].

The BBB is the bottleneck in the development of neurotherapeutics, as more than 98% of all potential CNS-targeted drugs do not cross the BBB [178]. The recognition that "poor permeability into the brain is one ofthe reasons for the failure of CNS-targeted drug candidates" has revolutionized neurological drug discovery research. Thus, given the large number of drug candidates generated by modern combinatorial chemistry-based approaches, poor brain-penetrating candidates need to be identified and eliminated at the early stages of drug discovery [179]. Due to high costs, time-consuming assays and ethical considerations, previously accepted in-vivo methods for measuring BBB permeability are no longer applicable for such enormous numbers of candidate drugs. Permeability evaluation through the BBB should be relatively simple, widely applicable, robust, and able to comply with HTS methods [180]. This calls for the generation ofnovel, in-vitro BBB models, which can provide the pharmacologically validated multitude of HTS screening tests required throughout the lead compound selection process.

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