Short communication Cortical changes in cholecystokinin mRNA are related to spontaneous pain behaviors following excitotoxic spinal cord injury in the rat Kori L. Brewer * , David McMillan, Todd Nolan, Kerry Shum Department of Emergency Medicine, The Brody School of Medicine at East Carolina University, Division of Research, Physician’s Quadrangle, Building M, Greenville NC 27858, USA Accepted 13 August 2003 Abstract Cholecystokinin (CCK) in the CNS antagonizes the opioid system and has been implicated post-spinal cord injury (SCI) pain. The current study found that excitotoxic SCI alters levels of CCK mRNA levels in the cortex, diencepahlon, and mesencephalon of rats. Animals that developed pain post-SCI had significantly higher levels than animals that did not develop pain. Upregulation of CCK mRNA in the cortex may be related to post-SCI pain in rats. D 2003 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Neuropathic pain; Excitotoxicity; Quisqualic acid; Slot blot Spinal cord injuries (SCI) are known to cause anatomi- cal, physiological, and molecular changes within the cord [24,36,39,40] that may contribute to the development of pain-related behavioral syndromes that appear following injury [5,6,14,38,40]. Excitotoxic SCI results in the appear- ance of overgrooming in approximately 70–80% of injured animals, a behavior believed to represent the presence of at- level neuropathic pain [10,16,37,40]. Previous macroarray analysis demonstrated a 3.5 Â increase in cholecystokinin (CCK) mRNA levels in the brains of mice that developed pain after SCI compared to those that did not develop pain [4]. CCK has been implicated in many models of neuro- pathic pain including post-SCI pain [34,35], and blocking the actions of CCK is effective in treating many of these conditions [7,15,32]. The current study examines the effects of excitotoxic SCI on CCK mRNA levels in the brains of rats after injury and relates these changes overgrooming behavior. Each animal received 1.2 Al of PBS or quisqualic acid (QUIS) by intraspinal injection as described previously [2,40]. Animals were assessed daily for the presence of excessive grooming behavior. At 3 (n = 4 QUIS, n =3 sham), 7 (n = 4 QUIS, n =3 sham) or 14 (n = 7 QUIS, n = 3 sham) days postinjection, brains were removed and grossly dissected into cerebral cortex, diencephalon, and mesencephalon. Total RNA was extracted and purified according to manufacturer’s protocols (Gibco, Gaithersburg, MD). RNA was also extracted from intestinal tissue for positive control, liver for negative control, and brains of two naive animals. Three micrograms of total RNA from each sample was loaded onto one of three nylon membranes (Amersham, Buckinghamshire, England). Each membrane contained (+) and ( À ) controls, plus samples from one brain region of all experimental, sham, and naive animals. A digoxigenin- labeled ribobrobe was hybridized to each membrane, and detection of the probe was performed using BrightStar BioDetct Kit (Ambion). Each membrane was exposed to film and developed after 2 (cortex), 5 (diencephalons) or 10 (mesencephalon) min. Radiographic images were scanned into Kodak 1D Image Analysis Software Program for quantification of signal using the signal from the naive animals as a reference. All analyses represent comparisons of survival times and behaviors within a single region of the brain. Comparisons 0169-328X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.08.006 * Corresponding author. Tel.: +1-252-744-2158; fax: +1-252-744- 2655. E-mail address: brewerk@mail.ecu.edu (K.L. Brewer). www.elsevier.com/locate/molbrainres Molecular Brain Research 118 (2003) 171 – 174