MEMBRANE AND CELLULAR BIOPHYSICS AND BIOCHEMISTRY NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 12 No 5 17 April 2001 1045 The antioxidant enzyme quinone reductase is up-regulated in vivo following cerebral ischemia Adrian W. Laxton, Ming-Chieh Sun, 1 Hao Shen, Timothy H. Murphy 2 and Christopher R. Honey CA Divisions of Neurosurgery and 1 Neurological Sciences, Kinsmen Lab, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Detwiller Pavilion Rm. 4N1, Vancouver, BC., Canada, V6T 1Z3; 2 Cardinal Tien Hospital and Fu-Jen Catholic University, Taiwan, R.O.C. CA Corresponding Author Received 21 December 2000; accepted 30 January 2001 An astrocyte antioxidant enzyme, quinone reductase (QR), was studied in vivo to assess whether its activity was up-regulated following cerebral ischemia. Rats were given a unilateral focal cerebral infarct and regions of interest within the ischemic penumbra compared to the non-ischemic side for QR activity. At 7 days post-ischemia, QR activity was signi®cantly up- regulated within cells of astrocyte morphology in the cortex ( p  0.007) and subcortical ( p  0.005) areas adjacent to the infarct. This enzyme activity peaked at 7 days but was still signi®cantly up-regulated at 14 days. Up-regulation of QR activity occurs within the ischemic penumbra of a stroke in this animal model and may contribute to factors that limit ischemic damage to neurons in this area. NeuroReport 12:1045±1048 & 2001 Lippincott Williams & Wilkins. Key words: Antioxidant enzyme; Astrocyte; Ischemia; Quinone reductase; Rat; Stroke INTRODUCTION Up-regulation of astrocyte antioxidant enzymes in vivo could be a new and powerful method of protecting neurons in the ischemic penumbra of a stroke. Astrocytes are the most abundant non-neuronal cell type in the brain. They are intimately linked with neurons and increased activity in either cell type can activate the other [1,2]. They help buffer the microenvironment around the neurons and proliferate in response to brain injury [3,4]. Astrocytes can protect neurons in vitro from excitotoxicity [5,6] and from hypoxic injury [7±9]. They produce more antioxidants such as glutathione and have a greater resistance to free radicals than neurons [10,11]. Astrocytes may therefore protect neurons by metabolizing extracellular free radicals (e.g. H 2 O 2 ) or by producing antioxidants (e.g. cysteine or glutathione) that can be used by the neurons [12]. Given the documented in vitro role of astrocytes in aiding neurons during oxidative stress, we proposed to study if astrocytes could up-regulate the antioxidant enzyme qui- none reductase (QR) in vivo in a focal model of ischemia. This enzyme is one of the antioxidant enzymes produced by astrocytes and has been shown to be up-regulated in vitro in response to electrophilic small molecule inducers [13]. MATERIALS AND METHODS Animals: All experiments were approved by the Animal Care Committee at the University of British Columbia. Four groups of six adult male Wistar rats (200±250 g) received unilateral, focal brain ischemia produced by a modi®cation of the technique described by Chen et al. [14]. Under general anesthesia with sodium pentobarbital (62 mg/kg, Sigma, Mississagua, ON), their right common (CCA) and middle cerebral arteries (MCA) were ligated and the left CCA temporarily occluded with an aneurysm clip for 30 min. During surgery, animals had their tempera- ture monitored with a rectal thermometer and controlled by a warming blanket. Animals were then caged in pairs and fed freely until sacri®ce at 30 min (Group 1), day 3 (Group 2), day 7 (Group 3), and day 14 (Group 4). Histochemistry: Under deep anesthesia (chloral hydrate), animals were perfused via a transcardiac route with 4% paraformaldehyde and their brains removed and left over- night in paraformaldehyde. The brains were then im- mersed in 20% sucrose for 48 h. Frozen sections were cut coronally (40 ìm) and two adjacent sections were selected for staining every 400 ìm through the brain. One section was stained for brain histology with cresyl violet (CV) and the other for QR activity [15]. Quinone reductase activity was determined by exposing the brain sections to a quinone compound, LY 83583, in the presence of NAD(P)H. The endogenous QR reduced the LY 83583 and produced NAD(P)  allowing a simultaneous reduction of nitroblue tetrazolium into the opaque, insoluble, blue formazan compound. Since under these conditions most of the blue staining was produced by QR [15], the amount of blue staining was used as a measure of QR activity.