Inhibition of GSK-3 reduces infarct volume and improves neurobehavioral functions Seong-Ho Koh a , A Rum Yoo a , Dae-Il Chang b , Se J. Hwang c, * , Seung H. Kim a, * a Department of Neurology, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea b Department of Neurology, College of Medicine, Kyung-Hee University, Seoul, Republic of Korea c Department of Anatomy, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea article info Article history: Received 22 April 2008 Available online 12 May 2008 Keywords: Glycogen synthase kinase-3 Ischemia Stroke Inflammation Neuronal cell death abstract In the present study, we have investigated the effects of glycogen synthase kinase-3 (GSK-3) inhibition on infarct volume and neurobehavioral functions in a focal cerebral ischemia model. To achieve our goals, GSK-3 inhibitor II or VIII was injected at several time points and in varing dosages. GSK-3 inhibitor VIII was more effective than inhibitor II, and infarct volume and water content in the VIII group were signif- icantly decreased 24 h after the onset of ischemic stroke, as compared with the control group. These pro- tective effects were associated with reductions of TUNEL-positive cells, neutrophil infiltration, glucose levels after ischemia, and GSK-3 enzyme activity. In addition, expressions of death and inflammation- related signals decreased and those of survival-related signals increased. Lastly, neurobehavioral func- tions were restored to a greater extent in the VIII group than in the control group. Together, these results suggest that GSK-3 inhibition reduces infarct volume and restores neurobehavioral functions. Ó 2008 Elsevier Inc. All rights reserved. Glycogen synthase kinase-3 (GSK-3) has recently been empha- sized in the mechanism of neuronal cell death [1–3]. Therefore, GSK-3 may also be important in stroke-related cell death. Although it has been reported that several kinds of compounds inducing GSK-3 inhibitory effects can reduce infarct volume in ischemic stroke model rats [4–6], the precise effects of direct GSK-3 inhibi- tion on infarct volume and neurobehavioral functions have not yet been fully elucidated. In the present study, the effects and protective mechanisms of direct GSK-3 inhibition in ischemic stroke were investigated using the ischemia/reperfusion injury rat model. Materials and methods Animal preparation and ischemic surgery. All animal procedures were performed in accordance with the guidelines for the care and use of laboratory animals, approved by the Institutional Ani- mal Care and Use Committee (IACUC) of Hanyang University. A to- tal of 305 Sprague–Dawley (SD) rats, whose weights ranged from 281 to 308 g, were purchased from Biogenomics Incorporated (Seoul, Korea). To inhibit GSK-3, GSK-3b inhibitor II (2-thio(3-iodobenzyl)-5- (1-pyridyl)-[1,3,4]-oxidiazole) and GSK-3 inhibitor VIII [AR- A014418: N-(4-Methoxybenzyl)-N 0 -(5-nitro-1,3-thiazol-2-yl)urea] were purchased from Santa Cruz Biotech (Delaware, CA, USA) and Calbiochem (Cambridge, MA, USA), respectively. GSK-3 inhibitor II, a 2-thio-[1,3,4]-oxadiazole-pyridyl derivative that potently inhibits GSK-3 (IC 50 = 390 nM, C 14 H 10 IN 3 OS; molecu- lar weight: 395.2) [7] (See the Supplementary Fig. 1A), and GSK-3 inhibitor VIII (C 12 H 12 N 4 O 4 S, molecular weight: 308.3, IC 50 = 104 nM), which potently, selectively, and specifically inhibits GSK-3 and can cross the brain–blood barrier (BBB, albeit poorly [8] (See the Supplementary Fig. 1B), were dissolved in 0.9% sterile NaCl. In order to determine the most effective dose, the inhibitor II was administered at levels of 2, 4, 8, 16, and 32 mg/kg B.W. 10 min before reperfusion (n =5 for each concentration, to- tal = 25); and the inhibitor VIII at levels of 1, 2, 4, and 8 mg/kg B.W. (n = 5 for each concentration, total = 20). To determine the therapeutic window, the most effective dose of GSK-3 inhibitor II (16 mg/kg) or VIII (4 mg/kg) (Fig. 1A and B), was administered through the left external jugular vein either 1 h before, 10 min be- fore, or 2, 4, or 8 h after left middle cerebral artery (MCA) occlusion (n = 5 for each interval and control, 25 rats with GSK-3 inhibitor II and 25 rats with GSK-3 inhibitor VIII). When compared with the control group, administration of both inhibitors 10 min before the occlusion was determined as the most effective time point (Fig. 1C). Thus, 4 mg/kg of GSK-3 inhibitor VIII, which was more effective than 16 mg/kg of GSK-3 inhibitor II, was administered to 80 animals 10 min before left MCA occlusion for the remainder of the study. As a control, an equivalent volume of vehicle was 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.006 * Corresponding authors. Fax: +82 2 2296 8370. E-mail addresses: hwangsj@hanyang.ac.kr (S.J. Hwang), kimsh1@hanyang.ac.kr (S.H. Kim). Biochemical and Biophysical Research Communications 371 (2008) 894–899 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc