Short communication Regional distribution of cyclooxygenase-3 mRNA in the rat central nervous system Bela Kis a, * , Andy Snipes a , Ferenc Bari a,b , David W. Busija a a Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157, USA b Department of Physiology, Faculty of Medicine, University of Szeged, Hungary Accepted 18 March 2004 Available online 6 May 2004 Abstract We determined COX-3 mRNA expression in regions of the rat central nervous system (CNS). On a regional basis, levels were the highest in choroid plexus and spinal chord followed by pituitary gland, hypothalamus, hippocampus, medulla, cerebellum, and cortex. COX-3 mRNA levels were higher in major brain arteries, and dramatically higher in brain microvessels. Our results suggest that the expression pattern of COX-3 mRNA in the rat CNS primarily relates to the vascular density of a given region. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Signal transduction: gene expression Keywords: Brain; Choroid plexus; Cyclooxygenase-3; Microvessel; RT-PCR; Spinal cord A new variant of the cyclooxygenase family, COX-3, was identified in canine as an acetaminophen sensitive isoform which is strongly expressed in brains of dogs and humans [4]. This finding offers a possible explanation for the analgesic and antipyretic effects of acetaminophen in the central nervous system (CNS) [2]. However, whether the COX-3 mRNA leads to the production of a protein capable of converting arachidonic acid to prostaglandins is contro- versial [5]. Very recently we showed that this new COX isoform also exists in primary cultures of cells from rat brain [6]. Whether COX-3 mRNA is expressed in intact brain and whether the in vitro expression pattern is reflected in COX-3 mRNA levels in different brain regions in vivo has not been examined previously. There were two purposes of our present study. First, we determined whether COX-3 mRNA was present in the rat brain in vivo. Second, if present, we compared the expression levels of COX-3 mRNA in eight regions of the rat CNS and also in the choroid plexus, the superficial major cerebral arteries and in freshly isolated brain microvessels. We also compared levels of COX-3 and COX-1 mRNA to indirectly assess the relative importance of this newly described isoform. Male Wistar rats (body weight 200 F 20 g) were pur- chased from Harlan (Indianapolis, IN). Two animals per cage were housed in a temperature (22–24 jC) and light controlled room (lights on at 7 am and off at 7 pm) with free access to food and tap water for at least 3 days prior the experiments. All animal experiments were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences. Eighteen rats were sacrificed by left ventricular transcardiac perfusion with chilled saline con- taining 1000 U/l heparin under halothane (5% in oxygen) anesthesia. Tissue samples were collected and immediately placed on dry ice and kept there until total RNA isolation. To isolate microvessels, cerebral cortices of saline perfused brains were freed from larger vessels, pial membranes and myelin, and then were finely minced and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Grand Island, NY, USA) containing collagenase (200 U/ml, Worthington, Lakewood, NJ) and DNase (30 U/ml, Sigma, St. Louis, MO, USA) at 37 jC for 2 h. After this incubation the digested tissue was triturated, mixed with 20% bovine serum albumin in DMEM and centrifuged at 1000 Â g for 20 min. The pellet containing the microvessels 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.03.015 * Corresponding author. Tel.: +1-336-716-4367; fax: +1-336-716- 0237. E-mail address: bkis@wfubmc.edu (B. Kis). www.elsevier.com/locate/molbrainres Molecular Brain Research 126 (2004) 78 – 80