ETX treatment causes an increase in caveolae dependent BBB permeability and internalization.

(A) Wild type mice were treated with 5ng ETX per gram of body weight for 1 hour. CAV1 staining was evaluated by IHC. White asterisks denote normal CAV1 staining in various sized blood vessels. In ETX treated mice, aberrant CAV1 staining was observed in some small capillaries as punctate staining (white arrow). In some medium sized vessels, CAV1 staining appeared basally located (white arrow head). In some large vessels, a dramatic increase in CAV1 staining was observed (red asterisk). (B) After ETX treatment, mice were intravenously injected with BSA-594 and then perfused. Coronal sections in control treated mice revealed BSA-594 (red) confined to vasculature lumen (white arrow head). In ETX treated mice, BSA-594 extravasation was observed accumulating in the corpus collosum (CC, thickness denoted by white dotted line) and as halos surrounding smaller vessels (white arrows). PDGF receptor-beta (green) was used to identify pericytes/vasculature. (C) Retinal whole mounts of control and ETX treated mice reveal increased internalization of BSA-594 by retinal endothelial cells after ETX treatment (white arrows). FITC-BSL1 (green) was used to identify vasculature. (D) Primary BEC were treated with our without 50nM ETX for 2 hours in the presence BSA-594 and CAV1 staining was evaluated by ICC. Internalized BSA-594 was observed in perinuclear organelles in ETX treatment cells (red arrows). In control treated cells, CAV1 staining can be observed extending all the way to cell-to-cell contacts (white arrows). After ETX treatment, CAV1 is absent from cell-to-cell contacts (white arrow heads). (E) and (F) Fluorescent quantification of CAV1 and BSA-594, respectively. Results expressed as Mean ± STDEV, p values determined by T-Test, n = 4.