Representative immunohistochemical staining of albumin in the adult and juvenile brains (scale bars, 5 μm).
A) Stereotaxic implantation of the cannulae was checked using intracerebroventricular (ICV) methylene blue-injected rats which permitted visualization of the stereotaxic implantation of the injection cannulae and the injection site into the brain lateral ventricles. Cannula implantation reached the lateral ventricle as shown by the blue color and cannula scare (blood trace) (as referred to in Paxinos and Watson’s stereotaxic atlas). Moreover, methylene blue diffused to the hippocampus. B) Blood–brain barrier (BBB) permeability after endothelin-1 (ET-1) injection was assessed using albumin immunohistochemical (IHC) in IV Evans blue injected rats. ET-1 was administered intracerebroventricularly. Twenty-two hours later a solution of 3% Evans Blue dissolved in 0.9% saline was administered intravenously (4 ml/kg). No albumin labeling or blue trace was detected in the cortex or in the hippocampus of adult or juvenile rat brains, suggesting a lack of BBB breakdown after icv injection of ET-1. C) BBB permeability after ET-1 injection was also assessed by measuring (using real-time PCR) mRNA relative expression of a tight-junction component, the ZO-1 gene, in brain microvessels isolated 24 h after ET-1 treatment. Results represent mean ± S.E.M from three batches of brain endothelial microvessels. Statistical comparisons: *P <0.05, **P <0.01 and ***P <0.0001.