A total of 4% Evan’s Blue (Sigma Aldrich, St. Louis, MO, USA) solution made in 0.9% NaCl (saline; Baxter, Deerfield, IL, USA) was infused into the jugular vein of an anesthetized rat and circulated for 30 min followed by a saline flush. The brain was dissected into the following four regions: frontal cortex (region above the anterior of the circle of Willis), posterior cortex, brainstem, and cerebellum. These four regions were selected as patients with CKD had evidence of impairment in these regions [42 (link),43 (link),44 (link)]. The brain regions were weighed and homogenized in trichloroacetic acid (TCA; Fisher Scientific, Fair Jawn, NJ, USA) buffer solution (1:1 20% w/v TCA and 0.9% saline) at a 1:3 dilution (w/v) as previously described [45 (link)]. The homogenized samples were centrifuged for 20 min at 10,000× g. The supernatant was assayed in triplicates, and samples and standards received 95% ethanol. The Evan’s Blue concentration was measured immediately at an excitation of 620 nm and an emission of 720 nm using Synergy LX (BioTek, Santa Clara, CA, USA) and Gen5 3.05 software (Biotek, Santa Clara, CA, USA). Data are presented as concentration of Evan’s Blue (pg)/weight of tissue (g).
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