Brain Endothelial permeability

Abbott, N.J. 1998. Role of intracellular calcium in regulation of brain endothelial permeability. In... 

Freshly isolated or subcultured brain microvascular endothelial cells offer a notable in vitro tool to study drug transport across the blood-brain barrier. Cells can be grown to monolayers on culture plates or permeable membrane supports. The cells retain the major characteristics of brain endothelial cells in vivo, such as the morphology, specific biochemical markers of the blood-brain barrier, and the intercellular tight junctional network. Examples of these markers are y-glutamyl transpeptidase, alkaline phosphatase, von-Willebrandt factor-related antigen, and ZO-1 tight junctional protein. The methods of... 

The rather time- and cost-expensive preparation of primary brain microvessel endothelial cells, as well as the limited number of experiments which can be performed with intact brain capillaries, has led to an attempt to predict the blood-brain barrier permeability of new chemical entities in silico. Artificial neural networks have been developed to predict the ratios of the steady-state concentrations of drugs in the brain to those of the blood from their structural parameters [117, 118]. A summary of the current efforts is given in Chap. 25. Quantitative structure-property relationship models based on in vivo blood-brain permeation data and systematic variable selection methods led to success rates of prediction of over 80% for barrier permeant and nonper-meant compounds, thus offering a tool for virtual screening of substances of interest [119]. 

Figure 2.5. Setup for in vitro measurement of blood-brain barrier permeability with a co-culture of bovine brain microvascular endothelial cells (BBMEC) and an astro ioma cell line, C6. The BBMEC are grown on top of a filter insert. The C6 cells are either grown on the opposite side of the filter or on the bottom of the wells. Transport across the BBMEC monolayer is measured by adding the test substance to the upper chamber and sampling from the lower chamber. The tightness of the monolayer is also characterized by the transendothelial electrical resistance (TEER). Courtesy of T. Abbruscato.

Abbott, N.J. 2002. Astrocyte-endothelial cell interactions and blood-brain barrier permeability. J Anat 200 629. 

In addition to the permeability barrier of the capillary endothelium, highly active enzymes present in the brain endothelial cells, pericytes and astrocytes, represent a further metabolic component of the BBB that also restricts the entry of substances to the brain. This is further compounded by the presence of the p-glycoprotein active efflux system in the astrocyte membranes (see below) these various components work in parallel with the permeability barrier of the capillary endothelium, to form a multicomponent BBB. 

Bornstein MB (1958) Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes. Lab Invest 7 134-137 Bowman PD, Ennis SR, Rarey KE et al. (1983) Brain microvessel endothelial cells in tissue culture a model for study of blood-brain barrier permeability. Ann Neurol 14 396-402 Eisenblatter T, Galla HJ (2002) A new multidrug resistance protein at the blood-brain barrier. Biochem Biophys Res Com-mun 293 1273-1278... 

Abbott NJ (2002) Astrocyte endothelial interaction and blood brain barrier. Permeability. A Anat 200 629-638. 

The following method could be used to either monitor the integrity of the in vitro BBB by calculating the permeability of a non-permeant marker molecule or measure the BBB permeability of a test compound. The most important criterion for a compound to be used as an integrity marker is that it should not be a ligand for uptake or efflux transporters and brain endothelial receptors or a substrate for an endothelial enzyme. Most tracers are labelled by a fluorescent dye or isotope that helps the quantification of the molecule. 

The currently available human brain endothelial cell lines do not provide enough paracellular restriction to be useful in permeability screens, even if their high throughput and ease of culture are of advantage. Another possibility would be to use human pluripotent stem cells, because it might constitute a new way of obtaining a reliable human in vitro BBB model if they can be made to differentiate into endothelial cells displaying the BBB phenotype in vivo. 

Figure 3.1. Schematic representation of the cellular components of the blood-hrain (Panel A) and blood-cerehral spinal fluid (Panel B) barriers. The blood-brain barrier consists of continuous type endothelial cells with complex tight junctions to limit paracellular diffusion. The astrocytes and pericytes located in close proximity to the brain endothelial cells release various endogenous factors that modulate endothelial cell permeability. In contrast, the choroid endothelial cells are fenestrated and the blood-cerebral spinal fluid barrier properties are provided by the tight junctions formed between the choroid epithelial cells.

Roux F, Couraud PO. Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions. Cell Mol Neurobiol 2005 25(l) 41-58. 

This correlation is useful for rapid estimation of the blood-brain barrier permeability. More accurate estimation can be obtained by examining other physical characteristics of the compound, such as cross-sectional area and critical micelle concentration [27] (Figure 5.28). Because of the extremely low permeability of brain capillaries to most molecules, brain endothelial cells have a variety of specialized transport systems—including transporters for glucose, amino acids, insulin, and transferrin—that enable essential molecules to move from the blood into the brain extracellular space. Useful reviews of the blood-brain barrier are available [28, 29]. 

Several strategies for increasing the permeability of the brain capillaries to proteins have been developed. The permeability of the BBB can be transiently increased by intra-arterial injection of the solutions with high osmolarity, which disrupts inter-endothelial tight junctions [11]. Certain protein modifications, such as cationization by hexamethyldiamine [12] and anionization by succinylation [13], produce enhanced uptake in the brain. Modification of drugs [14] and proteins [15] by linkage to an anti-transferrin receptor antibody also appears to enhance transport into the brain. This approach depends on receptor-mediated transcytosis of transferrin-receptor complexes by brain endothelial cells substantial uptake also occurs in the liver. 

Cellular models of blood-brain barrier permeability are also important, both for drugs meant to penetrate the CNS and those meant to stay out of it. Various Caco-2-like assays have been used for this, such as one using MDCK cells engineered to overexpress P-gp, a major barrier to CNS drug penetration. Other cell lines such as brain microvessel endothelial... 

In vitro reconstituted models of the BBB from different mammalian species have been used since the late 1970s. However, their comparison is difficult because of the different species and methods used for isolation, culture, coculture, and characterization of the models. Lundquist et al. (2002) confirmed that the epithelial cells might not represent a valid and reliable in vitro BBB model, because results obtained on epithelial monolayers correlated poorly with in vivo BBB permeability values. Bowman et al. (1983) introduced the first in vitro BBB filter model. The insert was made of nylon mesh and polycarbonate tubing, and bovine brain endothelial cells were seated on it for stud5ung the effect of calcium-free medium and osmotic shock on sucrose flux. Since then, a variety of... 

In in vitro studies, although no permeability change was foxmd in the case of basolateral application on a bovine coculture (Gaillard et al., 1996), apical glutamate treatment increased the flux of 70kDa FITC-dextran (Collard et al., 2002) and decreased TEER (Sharp et al., 2003) in human brain endothelial monolayers. These findings support that brain endothelial cells express fxmctional glutamate receptors (Krizbai et al., 1998 Sharp et al., 2003). 

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