Fig. 2

Cortistatin reverses murine brain endothelium disruption after ischemic-like conditions. a Graphical illustration of the experimental design. Primary brain endothelial cells (BECs) were isolated from 8-week-old Cort^+/+ mice and enriched after 60 h puromycin treatment. One week later, cells were plated in collagen/fibronectin-coated transwells as described before. BECs were exposed to OGD-R with or without cortistatin (CST, 100 nM) and gene profile expression analysis or permeability assays were performed. b mRNA expression levels of cortistatin system receptors (left, Sstr and Ghsr1a) and ligands (right, somatostatin, Sst and cortistatin, Cort) in the brain endothelium under NX/NG. Data represent the mean of mRNA expression levels quantified by real-time qPCR analyses and normalized to Rplp0. N = 5–7 cultures/group. Mouse brain (n = 3) was used as an internal reference for endogenous Cort expression. c Endothelium barrier functionality was assessed by evaluating permeability to EBA and NaF and tight-junctions integrity. Index (%) of the tracer permeability vs an empty-coated insert. N = 5 cultures/group. d Representative immunofluorescence images of the cellular distribution of ZO-1 (red), claudin-1 (green) and claudin-5 (red) in BECs after OGD-R in the absence or presence of cortistatin. Arrowheads indicate ZO-1 and claudin-5 disruption throughout the cell membrane and augmented cytosolic expression of claudin-1. After cortistatin treatment, asterisks point out the recovery of ZO-1 and claudin-5 continuous pattern in the membrane and the reduced claudin-1 intracellular location. Scale bar: 20 µm. e Levels of immune factors were determined in culture supernatants after OGD-R in the presence or absence of cortistatin. N = 7 cultures/group. Data are the mean ± SEM with dots representing individual values of independent cultures. Cells for each culture derived from 4 pooled brains. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001