SPERMINE REDUCES INFARCTION AND NEUROLOGICAL DEFICIT FOLLOWING A RAT MODEL OF MIDDLE CEREBRAL ARTERY OCCLUSION: A MAGNETIC RESONANCE IMAGING STUDY MD. SHIRHAN, a S. M. MOOCHHALA, b * P.-Y. NG, c J. LU, b K. C. NG, b A. L. TEO, b E. YAP, c I. NG, c P. HWANG, c T. LIM, c Y. Y. SITOH, c H. RUMPEL, d R. JOSE d AND E. LING e a Department of Pharmacology, National University of Singapore, Sin- gapore b Defence Medical & Environmental Research Institute, DSO National Laboratories (Kent Ridge), 27 Medical Drive 09-01, Singapore 117510 c National Neuroscience Institute, Singapore d Department of Radiology, Singapore General Hospital, Singapore e Department of Anatomy, National University of Singapore, Singapore Abstract—The role of nitric oxide (NO) in post-ischemic ce- rebral infarction has been extensively examined, but few studies have investigated its role on the neurological deficit. In the present study, we investigated the effect of spermine on the temporal evolution of infarct volume, NO production and neurological deficit using magnetic resonance imaging in a model of permanent focal cerebral ischemia in rats. Spermine given at 10 mg/kg 2 h after ischemia reduced the infarct volume by 40% and abolished brain NO production and improved the neurological score 24 h, 48 h and 72 h after ischemia. Spermine also reduced the neurological deficit as evaluated by rotamex, grip strength and neurological severity score tests. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: polyamine, nitric oxide, MRI, TTC staining. Natural polyamines, e.g. spermidine and spermine and their precursor putrescine are of considerable importance for the development and maturation of nervous system. They exhibit a number of neurophysiological and metabolic effects in the nervous system, including control of nuclei acid and protein synthesis, modulation of ionic channels, calcium-dependent transmitter release and regulation of nitric oxide synthase (NOS) and free radical scavenging (Coert et al., 2000). The polyamine system is also known to be involved in various brain pathological events such as seizure, stroke, Alzheimer’s disease and others (Berstein and Muller, 1999). One of the biochemical processes involved in brain trauma is the synthesis and release of the natural polyamines and their subsequent effect on several targets such as the N-methyl-D-aspartate (NMDA; Ferchmin et al., 2000). Current data on the neurotoxicity of polyamines have been conflict- ing. Spermine, the most abundant of the polyamines, ap- pears to be released in ischemia (Gilad et al., 1993). It has been suggested that pathological conditions are likely to en- hance the neuroprotective effect of spermine (Ferchmin et al., 2000). Spermine has been reported to be neuroprotective in a gerbil model of forebrain ischemia as i.p. administration of spermine significantly decreased hippocampus and striatal cell loss (Gilad and Gilad, 1991; Gilad et al., 1993; Harada and Sugimoto, 1997). However, others have reported poly- amines to be neurotoxic (Sparapani et al., 1997). Nitric oxide (NO) has been associated with both posi- tive and negative physiological roles. The NO generated from endothelial NOS (eNOS) is critical in maintaining cerebral blood flow and reducing infarct volume. However, NO produced by both neuronal and inducible NOS has been associated with a detrimental effect (Verrecchia et al., 1995; Balkan et al., 1997; Samdani et al., 1997; Lecanu et al., 1998; Salom et al., 2000; Ding-Zhou et al., 2002). Inducible NOS (iNOS) which is not present under physio- logical condition can be induced shortly after ischemia and contributes to secondary late-phase damage (Samdani et al., 1997). However, most of these studies have focused on the infarct volumes while only few have looked into the neurological deficit aspect. In the present study, the neurological effects of sperm- ine on the brain pathogenesis and neurological perfor- mance in a rat model of permanent middle cerebral artery occlusion (pMCAO) were evaluated using physiological variables, serial magnetic resonance imaging (MRI), 2,3,5,-triphenyltetrazolium chloride (TTC) staining, brain nitrate/nitrite content and neurobehavioral tests. EXPERIMENTAL PROCEDURES Animals and reagents Male Sprague-Dawley rats (250 –350 g), n=20 for each group, were deprived of food 24 h prior to the experiment but were allowed free access to water. Group 1: sham-operated. Group 2: saline-treated with pMCAO. Group 3: spermine-treated with pM- CAO. A Clinical Research Center (CRC) cocktail consisting of one part hypnorm (Jansen Pharmaceutica, Beerse, Belbica), one part dormicum (Roche, Basel, Switzerland), and two parts water was used for anesthesia. Spermine tetrahydrochloride-N,N 1 -Bis(3- aminopropyl)-1,4-butanediamine was obtained from Sigma Chemicals (St. Louis, MO, USA). The drug was dissolved in 0.9% *Corresponding author. Tel: +65-64857201; fax: +65-64857226. E-mail address: nmiv19@nus.edu.sg (S. M. Moochhala). Abbreviations: ANOVA, analysis of variance; BP, blood pressure; CRC, Clinical Research Center; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; MABP, mean arterial blood pressure; MRI, magnetic resonance imaging; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; NSS, neurological severity score; pMCAO, permanent middle cerebral artery occlusion; TTC, 2,3,5,-triphenyltetrazolium chloride. Neuroscience 124 (2004) 299 –304 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.10.050 299