Tight junctions of brain endothelium in vitro are enhanced by astroglia

Abstract

The belts of endothelial tight junctions, which impede diffusion between blood and brain, were reduced to fragmentary, small junctions in subcultured brain endothelium. When cocultured with the capillaries' nearest neighbor, the astrocytes, these endothelial tight junctions were enhanced in length, width, and complexity, as seen by en face views of the cell membranes with freeze-fracture electron microscopy. Gap junctions, common in brain endothelium in vitro but absent in mature brain capillaries in wiwo, were markedly diminished in area from among the enhanced tight junctions of the cocultures. Thus, astrocytes in vitro play a role in the formation, extent, and configuration of the junctional complexes in brain endothelium, whose diffusion barrier may likewise be influenced by astrocytes in viva.Brain capillaries have zonular tight junctions (TJs) impermeable to hydrophilic solutes (Reese and Kamovsky, 1967;Brightman and Reese, 1969). In freeze-fracture replicas of TJs, the constituent strands are continuous, anastomose extensively without free ends, and are devoid of gap junctions (Connel and Mercer, 1974;Dermietzel, 1975;Shivers, 1979;Nagy et al., 1984). The ability of brain endothelial cells to form this barrier depends on the environment in which they grow (Stewart and Wiley, 198 1). The astrocyte, as the cell immediately ensheathing brain capillaries, is the most likely candidate to affect the capillaries' passive permeability by modulating the formation and maintenance of the TJs between their endothelial cells. To test this notion, we have cultured beef brain microvessel endothelium alone or together with rat astroglial cultures, and examined the endothelial TJ with freeze-fracture electron microscopy. In thin plastic sections, tight junctions appear as fused spots between 2 cell membranes, and it is very difficult to derive any quantitative information from these cross-sectional views of the TJs. The freeze-fracture technique displays the en face vista of the interior of the cell membrane (Branton, 1966), where the TJs appear as strands of particles in a 2-dimensional plane. Furthermore, the length and width of the TJ strands can be measured morphometrically. Preliminary results of the present work have been published as an abstract (Tao-Cheng et al., 1986).Received Dec. 9, 1986; revised Feb. 24, 1987; accepted Apr. 1, 1987. We Bowman et al. (1983). The endothelial culture medium consisted of equal volumes of minimum essential medium and nutrient F12 (Gibco) with 5% plasma-derived horse serum (P. D. Bowman et al., personal communication), 10 mM HEPES, 13 mM sodium bicarbonate, 100 ILLg/ml heparin, 20 &ml endothelial cell growth supplement (Sigma), 100 pg/ml penicillin, 100 &ml streptomycin, and 2.5 &ml amphotericin B. After the cells were confluent at 7-20 d in vitro, 0.05% trypsin and 0.02% EDTA in Puck's saline solution without Ca*+ or Mg*+ were used to dissociate the cells for subculturing. In the present study, only the subcultures were examined because (1) primary cultures require laborious pro...

Anin vitroblood–brain barrier model: Cocultures between endothelial cells and organotypic brain slice cultures

“…With this technique, TJs appear as long parallel linear fibrils that circumscribe the cell, with short fibril fragments interconnecting the main parallel array (51). TJs observed in freeze-fracture replica of cocultures displayed similar structures and organization to those described in in vitro BBB models (16,(52)(53)(54). Moreover, in our cocultures, TJs exhibit the morphological appearance found in brain microvessels with strands forming complex networks (55,56).…”

Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

“…Cells surrounding brain capillaries, such as astrocytes, pericytes, perivascular microglia, and neurons contribute to the formation and maintenance of a functional BBB in the central nervous system. Among these cells, astrocytes were the first to be recognized as regulators of brain endothelial characteristics and functions (for reviews see Abbott 2005;Abbott et al 2006;Haseloff et al 2005) including the induction of tight junctions (TJs) (Tao-Cheng et al 1987), a fundamental characteristic of the BBB. Brain pericytes, the cells sharing the basal membrane with capillary endothelial cells, were recently found to be also able to induce BBB functions in cultured brain endothelial cells (Hayashi et al 2004;Dohgu et al 2005).…”

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

“…One of the major problems of in vitro studies is the loss of blood-brain barrier properties such as low ion permeability and the activity of certain enzymes in culture (Mischeck et al, 1989;Laterra and Goldstein, 1993). Based on the observation that astrocytes can induce tight junction formation in endothelial cells (Janzer and Raff, 1987;Tao-Cheng et al, 1987), both co-cultures with astrocytes (e.g., Dehouck et a!., 1990;Meyer et al, 1991) and astroglia-conditioned media (e.g., Rubin et al, 1991;Wolburg et a!., 1994) have been applied to induce barrier properties in cultured BCECs. One aspect rarely recognized until now is the question of whether the basement membrane located between BCECs and astrocytes in vivo may be involved in the differentiation of brain capillary endothelium leading to the formation of a functional barrier.…”