Administration of N-Acetylcysteine after Focal Cerebral Ischemia Protects Brain and Reduces Inflammation in a Rat Model of Experimental Stroke Mushfiquddin Khan, 1 * Bipanjeet Sekhon, 1 Manu Jatana, 1 Shailendra Giri, 1 Anne G. Gilg, 1 Charanpal Sekhon, 1 Inderjit Singh, 1 and Avtar K. Singh 2,3 1 Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 2 Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 3 Ralph H. Johnson V.A. Medical Center, Charleston, South Carolina Free radicals and inflammatory mediators are involved in transient focal cerebral ischemia (FCI). Preadministration of N-acetylcysteine (NAC) has been found to attenuate the cerebral ischemia-reperfusion injury in a rat model of ex- perimental stroke. This study was undertaken to investigate the neuroprotective potential of NAC administered after ischemic events in experimental stroke. FCI was induced for 30 min by occluding the middle cerebral artery (MCA). NAC (150 mg/kg) was administered intraperitoneally at the time of reperfusion followed by another dose 6 hr later. Animals were sacrificed after 24 hr of reperfusion. The cerebral infarct consistently involved the cortex and stria- tum. Infarction was assessed by staining the brain sections with 2,3,5-triphenyltetrazolium chloride. Animals treated with NAC showed a significant reduction in infarct area and infarct volume and an improvement in neurologic scores and glutathione level. Reduction in infarction was significant even when a single dose of NAC was administered at 6 hr of reperfusion. Immunohistochemical and quantitative real- time PCR studies demonstrated a reduction in the expres- sion of proinflammatory cytokines such as tumor necrosis factor  (TNF) and interleukin 1 (IL-1) and inducible nitric oxide synthase (iNOS) in NAC compared to that in vehicle-treated animals. The expression of activated macrophage/microglia (ED1) and apoptotic cell death in ischemic brain was also reduced by NAC treatment. These results indicate that in a rat model of experimental stroke, administration of NAC even after ischemia onset protected the brain from free radical injury, apoptosis, and inflamma- tion, with a wide treatment window. © 2004 Wiley-Liss, Inc. Key words: stroke; inflammation; apoptosis; antioxidant; N-acetylcysteine Stroke is a significant cause of adult disability and the major cause of death in the older human population worldwide. It commonly results from thrombosis or em- bolism. Ischemic or hypoxic brain injury often causes irreversible brain damage and is the third leading cause of death in the USA (Brown et al., 1996; Menotti et al., 1996; Tuomilehto et al., 1996). Cerebral ischemia (par- ticularly in the period after reperfusion) is accompanied by the enhanced formation of reactive oxygen species (ROS) in brain tissue. Excessive production of ROS such as superoxide anion (O 2 - ), hydroxyl radical (HO  ), hydro- gen peroxide (H 2 O 2 ), and nitric oxide (NO) has been shown to play a critical role in the development of ischemia/reperfusion injury (Tominaga et al., 1993). These oxidant radicals contribute to increased neuronal death by oxidizing proteins, damaging DNA, and induc- ing lipid peroxidation (Barber et al., 2003). Cerebral ischemia initiates a cascade of detrimental events including release of glutamate, accumulation of intracellular calcium, formation of free radicals, degrada- tion of membrane lipids, and induction of inflammation including expression of tumor necrosis factor  (TNF) and interleukin 1 (IL-1). These detrimental events lead to disruption of cellular homeostasis and structural damage of ischemic brain tissue (Kochanek and Hallenbeck, 1992; Feuerstein et al., 1994). The expression of proinflamma- tory cytokines during ischemia/reperfusion and related oxidative stress results in upregulation of inducible nitric oxide synthase (iNOS), thereby producing large amounts of NO. NO reacts with O 2 - to generate peroxynitrite (ONOO - ), which is capable of nitrating tyrosine residues of protein and enzymes, leading to tissue injury (Brezis et al., 1984; Shanley et al., 1986; Takada et al., 1997). Some Contract grant sponsor: National Institute of Health; Contract grant num- ber: NS-40144, NS-22576, NS-34741, NS-37766, NS-40810. *Correspondence to: Mushfiquddin Khan, PhD, Medical University of South Carolina, Department of Pediatrics, 316 CSB, 96 Jonathan Lucas Street, Charleston, SC 29425. E-mail: khanm@musc.edu Received 9 May 2003; Revised 23 January 2004; Accepted 26 January 2004 Published online 1 April 2004 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.20087 Journal of Neuroscience Research 76:519 –527 (2004) © 2004 Wiley-Liss, Inc.