8). With medium supplemented at 48 h, TEER measured at 72 h was 595±24 Ω cm2 in mono-cultured cells, and 779±19 Ω cm2 in cells co-cultured with astrocytes in the bottom of the well (Fig. 9). The apparent permeability (Papp) to [14C]mannitol measured across the same inserts was in the range 0.1–2.6×10−5 cm/s ( Fig. 10), and showed an inverse relation to the TEER. The careful removal of meninges, including its invaginating cAMP inhibitor folds into sulci, was designed to remove the large surface vessels, including many of the penetrating arterioles which run perpendicularly into the brain cortex ( Dacey and Duling, 1982). This will not only remove most of the potential contamination by leptomeningeal

cells with fibroblast-like properties, but also by arterial and arteriolar smooth muscle cells, which

tend to grow more rapidly than endothelial cells in Selleck Z-VAD-FMK culture. The two-stage filtration is designed to retain vessel fragments, allowing isolated cells including most glial cells to pass through. Examination of the material collected from the coarser and finer filters (150 µm and 60 µm mesh respectively) shows that the 150 µm filters retain a less pure (and generally larger diameter) vessel fraction than the 60 µm filters; the latter generate a more homogeneous and higher TEER monolayer consistent with it being derived from relatively pure capillary endothelium. Isolation of predominantly capillary rather than arteriolar or venular microvessels is important as there are several phenotypic and functional differences between the endothelial cells of these different segments of the microvasculature. In particular, compared with arteriolar or venular endothelium, cerebral capillary endothelium has more a more complex and complete pattern of tight junction strands in freeze-fracture images ( Nagy et al., 1984) consistent with tighter tight junctions ( Wolburg and Lippoldt, 2002), high expression of solute transporters including efflux transporters ( Ge et al., 2005, Macdonald

Thalidomide et al., 2010 and Saubamea et al., 2012), and of certain receptors involved in transcytosis such as transferrin receptor ( Ge et al., 2005). Arteriolar endothelium shows higher expression of certain enzymes including 5′-nucleotidase, Mg2+-ATPase and Na+-K+-ATPase than capillary or venular endothelium ( Vorbrodt et al., 1982 and Vorbrodt, 1988), and significant absence of P-glycoprotein ( Saubamea et al., 2012); bidirectional transcytosis of horseradish peroxidase (creating a local ‘leak’) has been reported in certain brain arterioles but not in capillaries or venules ( Westergaard and Brightman, 1973 and van Deurs, 1977). The post-capillary venule segment is specialised as a site regulating adhe-sion and traffic of leucocytes into the perivascular space ( Bechmann et al., 2007, Owens et al., 2008 and Muldoon et al., 2013), shows higher expression of genes involved in inflam-mation-related tasks ( Macdonald et al.