PHYSIOLOGICAL RESEARCH  ISSN 0862-8408 (print)  ISSN 1802-9973 (online)  2008 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres Physiol. Res. 57: 283-288, 2008 Influence of Melatonin Pretreatment and Preconditioning by Hypobaric Hypoxia on the Development of Cortical Photothrombotic Ischemic Lesion I. MATĚJOVSKÁ 1 , K. BERNÁŠKOVÁ 1 , D. KRÝSL 1,2 , J. MAREŠ 1 1 Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University, Prague and 2 Department of Neurology, University Hospital Motol, Prague, Czech Republic Received October 25, 2006 Accepted February 6, 2007 On-line available February 8, 2007 Summary Photothrombotic model of ischemia (PT) is based on free radical- mediated endothelial dysfunction followed by thrombosis. Free radicals are also involved in hypoxic preconditioning. We tested the sensitivity of PT to preconditioning with hypobaric hypoxia and to pretreatment with melatonin. In adult Wistar rats, after intravenous application of Rose Bengal, a stereo-tactically defined spot on the denuded skull was irradiated by a laser for 9 min. The first experimental group underwent hypobaric hypoxia three days before irradiation. In the second experimental group, melatonin was applied intraperitoneally one hour before irradiation. Three days after irradiation, animals were sacrificed, the brains perfused, and stained with TTC. Ischemic lesions were divided into grades (I, II, III). In the control group (where no manipulation preceded photothrombosis), most animals displayed deep damage involving the striatum (grade III). The group pre- exposed to hypoxia showed similar results. Only 28.57 % of the melatonin pretreated animals exhibited grade III lesions, and in 57.14 % no signs of lesions were detected. Pre-exposure to hypoxia was not protective in our model. Pretreatment with melatonin lead to a significant reduction of the number of large ischemic lesions. This result is probably caused by protection of endothelial cells by melatonin. Key words Melatonin • Hypoxia • Ischemia • Free Radicals • Models • Animal Corresponding author J. Mareš, Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University, Ke Karlovu 4, 120 00 Prague 2, Czech Republic. Fax: +420 224 923 827, + 420 224 916 896. E-mail: jan.mares@lf3.cuni.cz Introduction Several experimental models have been devised for the study of the pathophysiology of focal ischemia of the central nervous system (Xi et al. 2004, Johnston et al. 2007, Tejkalová et al. 2007). Among these, the model of photothrombosis (PT) (Watson et al. 1985) is frequently used, because it is non-invasive and reliable. It involves photodynamic generation of free radicals (mainly singlet oxygen 1 O 2 ) triggered by the exposition of intravenously injected Rose Bengal (disodium tetraiodotetrachloro- fluorescein) to green light. By mechanisms not yet completely understood, newly generated 1 O 2 mediates endothelial dysfunction, which leads to thrombotization and vascular occlusion (Dietrich et al. 1987, Ishikawa et al. 2002). PT is pathophysiologically similar to naturally occurring ischemic stroke in man – not only due to common pathological mechanisms (including free radical mediated damage), but also due to the subsequent possibility of spontaneous reperfusion. Free radicals lead to cellular injury when generated in excess. However, their physiological role as signal molecules is now increasingly recognized (Kamsler and Segal 2004, Brune 2005, Mareš et al. 2006). Importantly, hypoxic preconditioning (i.e. tolerance to severe hypoxia/ischemia induced by previous exposure to mild hypoxia) is considered to be a free radical-mediated phenomenon (Liu et al. 2005), at least because it is effectively abolished by pretreatment with reactive oxygen species (ROS) scavengers (such as dimethylthiourea or ebselen) (Puisieux et al. 2004). It