Transendothelial electrical resistance

With disruption of this barrier, molecules such as albumin freely enter the brain and ions and water follow. Because the brain lacks a well-developed lymphatic system, clearance of plasma constituents is slow, edema occurs, and intracranial pressure rises. At lower levels of exposure, subtle dysfunction of the blood-brain barrier may contribute to neurobehavioral deficits in children (Bressler and Goldstein 1991 Goldstein 1993). The particular vulnerability of the fetus and infant to the neurotoxicity of lead may be due in part to immaturity of the blood-brain barrier and to the lack of the high-affinity leadbinding protein in astroglia, which is discussed later in this section. Results of measurements of transendothelial electrical resistance across the blood-brain barrier from mice of various ages showed that lead potentiates cytokines-induced increase in ion permeability of the blood-brain barrier (Dyatlov et al. 

Numerous modifications of in vitro culture systems have been developed for the estimation of BBB transfer [52]. Culture systems in use are either primary cultures of brain microvessel endothelial cells (BMEC) or immortalized endothelial cell hues. BMEC may be grown in co-culture with astrocytes or in astrocyte-conditioned medium. Astrocyte-derived factors increase the tightness of the barrier as measured by transendothelial electrical resistance (TEER) and by the permeability of hydrophUic markers such as sucrose. They also up-regulate the expression of BBB-enriched enzymes such as y-glutamyl transpeptidase (y-GTP) and alkaline phosphatase. A setup of the in vitro technique in a transwell system for transport studies is depicted in Figure 2.5. 

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.

Directly prior the experiment the quality, that is the tightness, of the PBCEC monolayer is assessed by determination of the transendothelial electrical resistance (TEER) of each filter. TEER measurements can be easily performed with the ENDOHM-24 chamber and the EVOHM voltometer (World Precision Instruments, Inc,). The resistance data, expressed in Q, are multiplied with the filter surface (4.52 cm2), corrected for blank filter resistance and expressed in L2 x cm2. 

Drags are tested at 10 p,M concentration and in two directions (apical to basolateral (a-b) and basolateral to apical (b-a)). Monolayer efflux studies are conducted at 37 °C in a humidified incubator with shaking (90 rpm) for 60 min. Transendothelial electrical resistance is measured with an Endohm Meter (World Precision Instruments, New Haven, CT). Reference drags for paracellular transport (14C-mannitol), tran-scellular transport (3H-propranolol), and Pgp efflux (amprenavir) should be included in each experiment. Concentrations of 14C-mannitol and 3H-propranolol are measured by liquid scintiallation counting. Amprenavir is analyzed by cassette LC/MS/MS analysis along with the test drags. 

There are complex interactions among the different cellular components of the neurovascular unit and the extracellular matrix, determining its permeability properties during both physiological and pathological conditions. This highlights the severe limitations of cell culture-based models to mimic neurological diseases associated with BBB disruption. Transwell culture systems of endothelial cells alone rarely achieve adequate transendothelial electrical resistance (TEER). Cocultures of... 

Fig. 8.1 Schematic drawing of the in vitro BBB model. Astrocytes are seeded on the bottom of the collagen-coated filter at a density of 45.000 cells per filter, allowed to adhere for 8 min, and cultured for 2 or 3 days. BBB endothelial cells (BCECs) are seeded at a density of 30.000 cells per filter. BCEC-astrocyte co-cultures are cultured to tight monolayers in growth medium for the first 2 or 3 days and on differentiation medium for the last 2 or 3 days. Transport or transendothelial electrical resistance (TEER), drug transport or receptor characterization experiments are performed after a total of 9 or 10 days after the brain capUlaries are seeded...

The main component of the blood-brain barrier is the brain endothelium, which exhibits a physical, an efflux and a metabolic barrier for the transport of drugs into the CNS. The physical barrier, an efflux, is a result of the tight junctions between adjacent endothelial cells, which are around 50-100 times tighter than in the peripheral endothelium, so that penetration across the endothelium is effectively confined to transcellular mechanisms [26, 27]. These junctions significantly restrict even the movement of small ions such as Na" " and Cl , so that the transendothelial electrical resistance (TEER), which is typically 2-20 2 cm in peripheral capillaries, can be over 1000 1 cm in brain endothelium [28]. 

To characterize the transport properties of in vitro BBB models, the solute permeability P of the in vitro BBB was determined by measuring the flux of the selected tracer. The most commonly used cell culture substrate consists of a porous membrane support submerged in the culture medium (Transwell apparatus). The Transwell system is characterized by a horizontal side-by-side or vertical diffusion system. During the experiment, the flux of tracers into the abluminal compartment of the Transwell system is recorded as a function of the time and the solute permeability P is calculated from the slope of the flux. The tracers used in the transport experiments are labeled by a fluorescent dye or isotope whose intensity can be measured quantitatively. Another index, transendothelial electrical resistance (TEER), or the ionic conductance of the monolayer, is also a measurement of the tightness of the in vitro BBB models. 

D. M. Shasby and S. S. Shasby. Effects of calcium on transendothelial albumin transfer and electrical resistance. J. Appl. Physiol. 60(l) 7l (1986). 

Deli et al. (2005) presented permeability data from various in vitro BBB models by measuring transendothe-lial electrical resistance (TEER) and by calculation of permeability coefficients for paracellular or transendothelial tracers. These authors summarized the results of primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin. This also described the effect of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as the physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the BBB fimction. 

Because microvessels are isolated directly from the brain, they express all transporters displayed in vivo. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity [13]. Their disadvantage is that the preparation is laborious, requires animals, and has a shorter life span in vitro [21]. Permeability of freshly isolated microvessels can be assessed by measuring the uptake of FITC-labeled dextran and transendothelial cell electrical resistance, and paracellular transport in cell culture models can be evaluated as well [13]. 

---