Research report Nicotine increases in vivo blood–brain barrier permeability and alters cerebral microvascular tight junction protein distribution Brian T. Hawkins a , Thomas J. Abbruscato b , Richard D. Egleton c , Rachel C. Brown d , Jason D. Huber e , Christopher R. Campos c , Thomas P. Davis a,c, * a Program in Neuroscience, College of Medicine, The University of Arizona, Tucson, AZ 85724, United States b Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, United States c Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, AZ 85724, United States d Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, 77030, United States e Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, United States Accepted 19 August 2004 Available online 18 September 2004 Abstract The blood–brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free based kg 1 d day 1 ) for 1 and 7 days led to increased permeability of the BBB to [ 14 C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins. D 2004 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Blood–brain barrier Keywords: Blood–brain barrier; Nicotine; Tight junction; ZO-1; ZO-2; Claudin 1. Introduction The blood–brain barrier (BBB) is a vascular system that regulates the passage of materials between the peripheral circulation and the central nervous system (CNS). The BBB is essential for maintaining brain homeostasis and enabling proper neuronal function [32,53] but makes the non- invasive delivery of therapeutics to the brain problematic [48]. Situated at the level of the cerebral microvascular endothelium [44], the BBB presents a diffusion barrier to most non-lipophillic molecules [33]. The barrier is estab- lished by a lack of fenestrations [13] and the presence of tight junctions (TJ) in the apical region of the interendo- thelial cleft. TJ are elaborate, interconnected membrane- spanning structures composed of transmembrane proteins linked via accessory proteins to the actin cytoskeleton [59] found in epithelial and endothelial barrier tissues through- out the body. Once thought to be static structures, TJ are in fact regulated in both physiological and pathological states [25], and changes in TJ protein expression and/or organ- ization have been associated with altered permeability [4,15,27,37,46,62]. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.043 * Corresponding author. 1501 North Campbell Avenue, P.O. Box 245050, Tucson, AZ, 85724-5050, United States. Tel.: +1 520 626 7643; fax: +1 520 626 4053. E-mail address: davistp@u.arizona.edu (T.P. Davis). Brain Research 1027 (2004) 48 – 58 www.elsevier.com/locate/brainres