CAV1 expression is necessary for ETX induced BBB permeability but not ETX binding to brain microvasculature.
(A) To determine if CAV1 was necessary for ETX binding to brain microvasculature, cerebellum cryosections from Cav1+/+ and Cav1-/- were probed with epsilon protoxin (proETX). ProETX binding was detected by and anti-ETX antibody (red). FITC-BSL1 was used to identify vasculature (green). Note that proETX is observed binding to the microvasculature in the molecular layer (ML) as well as myelin in the granular layer (GL) in both Cav1+/+ and Cav1-/- mice. (B) Higher magnification images of areas in white boxes in image A. (C) Quantification of proETX binding to vasculature in cerebellum gray matter in Cav1+/+ and Cav1-/- mice. proETX fluorescence was normalized to BSL1 fluorescence. Results expressed as Mean ± STDEV, *p value determined by T-Test, n = 4–7. D) To evaluate CAV1’s role in ETX induced BBB permeability, Cav1+/+ Cav1-/- were treated with or without 5ng of ETX per gram of body weight for up to 180 minutes. Mice were perfused with PBS, brains harvested, and cryosectioned. BBB permeability was accessed by immunohistochemistry staining for endogenous mouse IgG in the pericollosal white matter of sagittal sections. Results are normalized to individual genotype controls and expressed as IgG Extravasation (% CT). Results expressed as Mean ± STDEV, * p < 0.05 determined by ANOVA, n = 2–3 mice per group. (E) Representative images from ETX treated Cav1+/+ and Cav1-/- treated mice. Note in ETX treated Cav1+/+ mice, endogenous IgG (red) can be observed leaking for large medullary veins (white arrows) and smaller capillaries (white arrow heads). FITC-BSL1 (green) was used to identify vasculature.