Huang et al. BMC Neurosci (2015) 16:61 DOI 10.1186/s12868-015-0199-6 RESEARCH ARTICLE Open Access Effects of melatonin on the nitric oxide system and protein nitration in the hypobaric hypoxic rat hippocampus Chih‑Chia Huang1,2,3†, Chia‑Jou Lai4, Mang‑Hung Tsai5†, Ya‑Chieh Wu6, Kuang‑Ti Chen4†, Ming‑Jia Jou7, Pin‑I Fu5, Ching‑Hsiang Wu8* and I‑Hua Wei5* Abstract Background: It is well documented that the nitric oxide (NO) might be directly involved in brain response to hypo‑ baric hypoxia, and could contribute to memory deficiencies. Recent studies have shown that melatonin could attenu‑ ate hypoxia or ischemia-induced nerve injuries by decreasing the production of free radicals. The present study, using immunohistochemical and immunoblot methods, aimed to explore whether melatonin treatment may affect the expression of nitric oxide system and protein nitration, and provide neuroprotection in the rat hippocampus injured by hypobaric hypoxia. Prior to hypoxic treatment, adult rats were pretreated with melatonin (100 mg/kg, i.p.) before they were exposed to the altitude chamber with 48 Torr of the partial oxygen concentration (pO2) for 7 h to mimic the ambience of being at 9000 m in height. They were then sacrificed after 0 h, 1, and 3 days of reoxygenation. Results: The results obtained from the immunohistochemical and immunoblotting analyses showed that the expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine (Ntyr) and Caspase 3 in the hypoxic hippocampus were increased from 0 h to 3 days of reoxygenation. Interestingly, the hypoxia-induced increase of nNOS, eNOS, iNOS, Ntyr and Caspase 3 protein expres‑ sion was significantly depressed in the hypoxic rats treated with melatonin. Conclusions: Activation of the nitric oxide system and protein nitration constitutes a hippocampal response to hypobaric hypoxia and administration of melatonin could provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaric hypoxia. Keywords: Hippocampus, Hypobaric hypoxia, Nitric oxide, Protein nitration, Melatonin Background of the decreased partial pressure of oxygen (pO2) availa- Sudden exposure to high altitude (HA) (i.e., rapid ascent ble to the central nervous system (CNS) [1, 2]. It has been without acclimatization, as in mountain climbing) results established that CNS is highly sensitive to hypoxia and in the development of hypobaric hypoxia (HBH). Hypo- that some areas, such as the hippocampus, are especially baric hypoxia leads to appearance of neuropsychological vulnerable to hypoxic damage [3]. Several studies have disorders and mental dysfunctions such as insomnia, diz- determined that hypobaric hypoxia in the CA1 region of ziness, and memory deficiencies which are consequences the hippocampus provokes metabolic, electrophysiologi- cal, and morphological modifications related to neuronal *Correspondence: [email protected]; [email protected] death [4–7]. The obvious cell damage in the hippocam- †Chih-Chia Huang, Mang-Hung Tsai and Kuang-Ti Chen contributed pus and learning/memory deficits were evidenced after equally to this work exposure to HBH [8]. The exact mechanisms of neuronal 5 Department of Anatomy, China Medical University, No. 91 Hsueh‑Shih Road, Taichung, Taiwan damage in hypobaric hypoxia remain to be elucidated. 8 Department of Anatomy and Cell Biology, College of Medicine, Taipei Growing evidence showed that nitric oxide (NO) system Medical University, 250 Wuxing Street, Taipei, Taiwan involved in certain neuronal modifications and could Full list of author information is available at the end of the article © 2015 Huang et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Neurosci (2015) 16:61 Page 2 of 11 contribute to memory deficiencies related to ischemic prevent and/or treat the symptoms associated with hypo- hypoxia, normobaric and hypobaric hypoxia [9–11]. NO baric disease. is a short-lived bioactive molecule that participates in the physiology and pathophysiology of various systems Methods in mammals. NO is produced by nitric oxide synthases Experimental animals (NOS) that constitute a family of enzymes that catalyze Healthy adult male Wistar rats weighing 150–250 g the oxidation of l-arginine and nicotinamide adenine obtained from the Laboratory Animal Center of the dinucleotide phosphate by oxygen to yield L-citrulline National Taiwan University were used in this study. and NO [12, 13]. Three distinct NOS isoforms have been These animals were housed in conditions with con- identified: neuronal NOS (nNOS), endothelial NOS trolled temperature (22 °C) and exposed to an automati- (eNOS), and inducible NOS (iNOS). It has been sug- cally regulated light/dark cycle of 12:12 h (light on at gested that the formation of NO is directly linked to the 07:00–19:00 h), with ad libitum access to food and water glutamate, which can cause post-synaptic calcium influx throughout the study period. For the care and handling and trigger a cascade of events leading to cell damage fol- of all experimental animals, the guidelines as stated in lowing N-methyl-d-aspartate (NMDA) receptors activa- the Guide for the Care and Use of Laboratory Animals tion during hypoxia insults [14–16]. Part of the released (1985) as stated in the United States NIH guidelines (NIH NO after hypoxic injury can rapidly react with superox- publication No. 86-23), were followed. All the experi- ide produced in excess during reoxygenation, forming ments were approved by our Laboratory Animal Center, peroxynitrite, a potent oxidizing agent with neurotoxic China Medical University, Taiwan (No. 96-181-B). All actions [17, 18]. Peroxynitrite can act on tyrosine resi- efforts were made to minimize animal suffering and the dues in proteins to form the stable end product 3-nitro- smallest numbers of animal were used for the experi- l-tyrosine (nitrotyrosine; Ntyr). This compound can thus ments presented. The experimental animals were carried be used as a marker for the potentially cytotoxic effect of out to evaluate the post-treatment effect of melatonin in NO production in the presence of superoxide [18, 19]. hypoxic exposure. The animals (n = 180) were divided Pharmacological agents can reduce NO production or into four groups (I–IV) with 45 rats each. Groups I and prevent its biological effects by a variety of mechanisms, II served as controls, the rats were subjected to normoxic including the inhibition of l-arginine uptake into the cell, breathing by receiving intraperitoneal administrations of the reduction of cellular availability of necessary cofac- vehicle (normal saline) and melatonin (100 mg/kg body tors by preventing their formation or promoting their weight in saline), respectively. Rats of groups III and IV breakdown, or inhibition of the cellular mechanisms were pretreated with intraperitoneal injections of nor- leading to induction of different NOS isoforms [12]. Of mal saline and melatonin, respectively, 30 min before the the many substances indentified, recent studies suggest hypobaric hypoxic insult. Melatonin (Sigma, St Louis, that the melatonin and its metabolites are highly effective MO, USA) was dissolved freshly in pure absolute etha- physiological antioxidants and free radical scavengers nol and later liquidized with isotonic sodium chloride [20, 21]. Many biochemical and histopathological find- (0.9 % NaCl) amounting to final concentration of 1:10 ings have revealed that melatonin exerts neuroprotec- in a freshly prepared solution form, under sterile condi- tive effects in suppressing NO production and enhancing tions. Pretreatment of both melatonin and normal saline superoxide dismutase (SOD) activity following numer- was carried out at 10 am. Hypoxia was achieved by keep- ous experimental and clinical oxidative injury [22–24]. ing the rats in an altitude chamber at 9000 m with the Lowering circulating levels of melatonin also exagger- partial pressure of oxygen set at the level of 0.303 atm ates the oxidative damage to tissues that are subjected to (pO2 = 48 Torr) for 7 h. Following hypobaric hypoxic increased oxidative stress [25]. Thus, melatonin serving exposure, each of the experimental groups was further as a powerful agent in the treatment of various neuro- divided into three subgroups (n = 15 each) sacrificed at toxicities is anticipated. To our knowledge, the potential 0 h, 1, and 3 days, respectively. effects of melatonin on nitric oxide system and protein nitration following hypobaric hypoxic insults in the hip- Perfusion and tissue preparation pocampus have not yet been explored. Therefore, this At each of the respective time points, both the hypoxic study was using immunohistochemical and immunoblot treated and control rats were deeply anesthetized with methods, aimed to explore the time course alteration of an intramuscular injection of mixtures of zoletil (30 mg/ NOS and nitrotyrosine expression in the hippocampus kg) and xylazine (10 mg/kg) and perfused transcardially of HBH rats. We also sought to elucidate whether mel- with 100 ml of normal saline followed by 300 ml of 4 % atonin treatment would have any beneficial effects to paraformaldehyde in 0.1 M phosphate buffer (PB), pH Huang et al. BMC Neurosci (2015) 16:61 Page 3 of 11 7.4. After perfusion, the hippocampus were removed and Caspase 3 (1:1000; Millipore) overnight at 4 °C, respec- postfixed in the same fixative for 2 h. Tissue samples were tively. The nitrocellulose sheet was further processed for then rinsed in 0.1 M PB and placed overnight in sucrose chemiluminescence detection (Santa Cruz) using horse- buffer (10–30 %) for cryoprotection at 4 °C.