MINOCYCLINE ALLEVIATES HYPOXIC–ISCHEMIC INJURY TO DEVELOPING OLIGODENDROCYTES IN THE NEONATAL RAT BRAIN Z. CAI,* S. LIN, L.-W. FAN, Y. PANG AND P. G. RHODES Department of Pediatrics, Division of Newborn Medicine, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA Abstract—The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia–ischemia. Hypoxia–ischemia was achieved through bilateral carotid ar- tery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague–Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/ kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusionhypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white mat- ter area of the rat brain 1 and 5 days after hypoxia–ischemia. Hypoxia–ischemia insult also resulted in apoptotic oligoden- drocyte cell death, loss of O4 and O1 oligodendrocyte immunoreactivity, and hypomyelination as indicated by de- creased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline signif- icantly attenuated hypoxia–ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia–ischemia-induced microglial activa- tion as indicated by the decreased number of activated mi- croglia, which were also interleukin-1 and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia– ischemia-induced oxidative and nitrosative stress as indi- cated by 4-hydroxynonenal and nitrotyrosine positive oli- godendrocytes, respectively. The reduction in hypoxia– ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the mino- cycline-treated hypoxia–ischemia rat brain as compared with that in the vehicle-treated hypoxia–ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia–ischemia injury. © 2005 Pub- lished by Elsevier Ltd on behalf of IBRO. Key words: hypoxia–ischemia, microglial activation, minocy- cline, myelination, oligodendrocyte, oxidative stress. White matter damage (WMD) in preterm neonates is as- sociated with adverse neurological outcomes such as ce- rebral palsy and mental retardation (Volpe, 2001). Hypoxia– ischemia (HI) is considered to be a common cause of WMD. Considerable clinical and experimental data have demonstrated that immature oligodendrocytes (OLs) are highly susceptible to a HI insult (Back et al., 2001, 2002). Excitotoxicity, oxidative stress, inflammation and apoptosis are some of the mechanisms involved in the vulnerability of OLs to HI (Volpe, 2001). Treatments aiming at the above mechanisms might provide protection to OLs. But up to now, there is still no promising therapy for WMD. Minocycline is a semi-synthetic second-generation de- rivative of tetracycline and has been used in humans to treat a variety of infectious diseases with relatively few adverse effects. Tetracyclines are bacteriostatic agents with broad-spectrum anti-microbial activity. Minocycline has a superior tissue penetration into the brain and is absorbed rapidly and completely (Aronson, 1980). Re- cently, minocycline has been shown to exert biological effects that are completely separate and distinct from its anti-microbial action (Amin et al., 1996; Yrjanheikki et al., 1998, 1999). Minocycline has been shown to provide pro- tection against brain injury due to global brain ischemia in adult gerbils (Yrjanheikki et al., 1998), focal cerebral isch- emia in adult rats (Wang et al., 2003; Xu et al., 2004; Yrjanheikki et al., 1999), and neonatal HI in rats (Arvin et al., 2002). Minocycline has anti-inflammatory effects. Our recent studies have shown that this drug provides protec- tion against preferential white matter injury induced by intracerebral administration of lipopolysaccharide (LPS) in the neonatal rat brain, presumably through inhibition of microglial activation (Fan et al., 2005). Secondary inflam- matory responses contribute significantly to neonatal HI brain injury (Johnston et al., 2000). Therefore, the anti- inflammatory effects of minocycline may contribute, at least partially, to its protection against HI brain injury. However, the initial report by Arvin et al. (2002) about the protection of minocycline in a neonatal rat model of HI has not been substantiated in many other laboratories. While this agent has been found to ameliorate HI brain injury in developing rats, it has also been found to worsen HI brain injury in a neonatal mouse model (Tsuji et al., 2004). Recently, minocycline has been reported to impair OL progenitor cell responses and remyelination in a non-im- mune adult rat model of demyelination (Li et al., 2005). Effects of minocycline on neonatal HI brain injury and the underlying mechanisms remain to be elucidated. The objective of the present study is to evaluate effects of minocycline in our previously developed neonatal rat *Corresponding author. Tel: +1-601-984-2786; fax: +1-601-815-3666. E-mail address: zcai@ped.umsmed.edu (Z. Cai). Abbreviations: APC-CC1, adenomatous polyposis coli (clone CC1); BCAO, bilateral carotid artery occlusion; GFAP, glial fibrillary acidic protein; HI, hypoxia–ischemia; IL-1, interleukin-1beta; iNOS, induc- ible nitric oxide synthase; LPS, lipopolysaccharide; MBP, myelin basic protein; NeuN, neuron-specific nuclear protein; NO, nitric oxide; NT, nitrotyrosine; OL, oligodendrocyte; P, postnatal day; PBS, phosphate- buffered saline; PVL, periventricular leukomalacia; ROS, reactive ox- ygen species; SH, sham operation; TNF, tumor necrosis factor- alpha; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated uridine 5=-triphosphate-biotin nick end labeling; WMD, white matter damage; 4-HNE, 4-hydroxynonenal; 8-ISO, 8-isoprostane. Neuroscience 137 (2006) 425– 435 0306-4522/06$30.00+0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.09.023 425