Fig. 4

Cortistatin-deficient brain endothelial cells (BECs) show increased leakage and exacerbated immune activation. a BECs were isolated from wild type (Cort^+/+) and cortistatin-deficient (partial: Cort^+/−; complete: Cort^−/−) mice and exposed to 4 h oxygen–glucose deprivation followed by 20 h of reoxygenation (OGD-R). Barrier integrity was evaluated and represented as the index (%) of EBA permeability vs an empty-coated insert. N = 5–8 cultures/group. b–d Representative immunofluorescence images of cellular junctions distribution in BECs incubated in basal conditions (NX/NG) and after OGD-R. b Immunofluorescence of tight-junctions ZO-1 and claudin-5 (both in red, arrowheads point out membrane disruption) and claudin-1 (green, arrowheads indicate augmented cytosolic expression). Delocalization of ZO-1 was evaluated through the ratio of ZO-1 staining intensity in the membrane vs the cytosol. Claudin-1 and claudin-5 cellular expression was quantified by fluorescence intensity as described below. c, d Adherens-junction protein VE-cadherin (c, green, arrowheads show discontinuous expression) and stress fibers (d, red rhodamine–phalloidin F-actin labelling, arrowheads reveal cytosolic reorganization of F-actin thick bundles) were analysed by quantifying fluorescence intensity. 25–50 selected ROIs in 4 independent fields were analysed (expressed as arbitrary units, a.u). N = 6 cultures/group. Scale bar: 20 µm. e Levels of inflammatory factors were determined in culture supernatants after OGD-R. N = 5–10 cultures/group. Data are the mean ± SEM with dots representing individual values of independent cultures. Cells for each culture were derived from 4 pooled brains. *vs Cort^+/+ either in NX/NG and OGD-R; ^&vs corresponding genotype (Cort^+/+, Cort^+/−, Cort^−/−) in NX/NG. *^/&p ≤ 0.05, **^/&&p ≤ 0.01, ***^/&&&p ≤ 0.001, ****^/&&&&p ≤ 0.0001