Vascular delivery of intraperitoneal Evans blue dye into the blood–brain barrier-intact and disrupted rat brains Hwai-Lee Wang a , Eva Yuhua Kuo a and Ted Weita Lai a,b,c,d Blood–brain barrier (BBB) integrity can be determined by tracer infusion into the circulation followed by measurements of its penetration into the brain parenchyma. Tracer injection through the intraperitoneal (i.p.) route (rather than intravascular injection) avoids confounding effects of animal anesthesia or immobilization/surgical stress. Evans blue dye (EBD) can be administered by i.p. injection, and once in circulation, it binds to plasma albumin to become an endogenous protein tracer. Here, we investigated whether a similar level of EBD is extravasated into the brain following i.p. versus intravenous (i.v.) injection in rats. In comparison with i.v. EBD injection, i.p. EBD injection resulted in much of the tracer residing in the peritoneal cavity. Accordingly, comparatively less EBD was found in the blood, liver, or brain of BBB-intact rat. In addition, following unilateral osmotic BBB disruption, i.v. but not i.p. EBD stained the ipsilateral hemisphere blue. Nevertheless, following either route of tracer administration in these rats, spectrophotometric quantification detected more EBD in the ipsilateral (BBB-disrupted) than in the contralateral hemisphere. Taken together, in contrast to a recent report, we found that i.p. EBD resulted in less tracer in circulation and in peripheral/central organs than EBD delivered i.v. We nevertheless conclude that i.p. EBD delivered sufficient tracer for the detection of regional BBB disruption. NeuroReport 00:000–000 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. NeuroReport 2018, 00:000–000 Keywords: blood–brain barrier, Evans blue dye, intraperitoneal injection, rat, route of administration a Graduate Institute of Clinical Medical Science, b Graduate Institute of Biomedical Sciences, c Drug Development Center, China Medical University and d Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan Correspondence to Ted Weita Lai, PhD, Graduate Institute of Biomedical Sciences, China Medical University, 40402 Taichung, Taiwan Tel: + 886 422 052 121 x7338; fax: + 886 422 052 121 x7837; e-mail: ted.weita@me.com Received 11 April 2018 accepted 27 April 2018 Introduction The most direct method for gauging functional blood– brain barrier (BBB) integrity in laboratory animals is by infusion of a tracer into the circulation for a fixed period of time, and then removing the tracer from the circulation by means of perfusion before collecting the brain par- enchyma for qualitative and quantitative analyses of tracer location and concentration. Evans blue dye (EBD) is one of the most commonly used tracers for this purpose owing to its low cost, ease of handling and preparation, and wide margin for signal detection [1,2]. Following intravenous (i.v.) injection, EBD binds to serum protein to become a high-molecular-weight (HMW) protein tracer. As such, its pattern of cerebral extravasation has been shown to be distinct from that of a low-molecular- weight (LMW) tracer that does not bind to serum pro- teins [3]. Nevertheless, because of its LMW, EBD can be administered by intraperitoneal (i.p.) injection [4–7]. This is a major advantage given that intravascular injec- tions in laboratory animals most often require subjecting the animal to anesthesia [8–11] and/or stress-bearing restraint or surgical implantation of a cannula [12,13], both of which have been reported to either directly alter BBB permeability [10,13,14] and/or indirectly affect BBB integrity against other insults [7,15–19]. In our experience working with EBD, i.v. injections typically stain the entire animal and all of its internal organs (except for the brain and the spinal cord) dark blue. In contrast, i.p. injections at the same dose had little noticeable effect on the coloration of the organs. To us, this marked difference in rat appearance following i.v. and i.p. EBD injections casted doubt on the use of i.p. EBD in studies investigating the BBB. Surprisingly, a recent study reported that i.v. and i.p. injections of EBD resulted in similar concentrations of this tracer in the brains of mice subjected to BBB disruption caused by intracerebral hemorrhage [4]. This raised the intriguing possibility that i.p. and i.v. EBD, perhaps because of its high affinity for serum proteins, results in similar uptake into circulation. Another possibility is that a threshold level of EBD limits further cerebral extravasation regardless of increased levels of circulating EBD. Given the prominent use of EBD in many laboratories including our own, we sought to compare the EBD concentrations residing in the blood and in central/per- ipheral tissues following either i.v. or i.p. injections and the pattern and regional-specificity of EBD distribution in the brain following these two routes of administration. Materials and methods Ethics statement All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the China Medical University (Protocol no: 102-27-N) and were Pathology 1 0959-4965 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/WNR.0000000000001052 Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.