2.12. Blood-brain barrier (BBB) permeability assay

Evans Blue is a dye that has a high affinity for serum albumin [10]. An intact BBB is impermeable to serum albumin and thus the injected Evans Blue remains bound to the serum albumin and does not stain the neuronal tissue. When the BBB has been compromised, albumin-bound Evans Blue dye enters the CNS and stains it blue, allowing visual qualitative confirmation in addition to the quantitative immunofluorescence assay Drug- and vehicle-treated mice (n = 3) were weighed and injected intraperitoneally (i.p.) with Evans Blue dye (Sigma Aldrich, #E2129), 50 μg (in 100 µl PBS) per gram weight of the mouse [10,42]. Three hours after injection, the mice were terminally anesthetized with ketamine (100 mg/kg i.p.; Narketan, Vetoquinol) and xylazine (10 mg/kg i.p.; Rompun, Bayer) and transcardially perfused with PBS for 5 min at a 5 ml/minute flow rate [10]. After removing the cerebellum and olfactory bulbs, the brains were weighed. The Evans blue dye was extracted as follows: one mL of 50% trichloroacetic acid was added to the brain and the samples were Dounce homogenized by pulling the plunger up and down 10 times. The homogenates were centrifuged at 19000 g (approx. 13,000 rpm) for 10 min and the supernatant diluted 1:4 with 100% ethanol. The optical density of the supernatant was read with an ELISA plate reader (Spectra Max 190; Molecular Devices, Sunnyvale, CA) at 620 nm [42]. The readings were divided by the weight of the brain and the data were statistically analyzed with unpaired t-tests.