In contrast, the second phase of paw licking (1160 min) that involves a period of sensitization was not affected by the vitamin combination, nor by Vit C or Vit E given alone (Fig. pain behavior induced by the vitamin C and E combination was paralleled by a reduced AG 957 p38 phosphorylation in the spinal cord and AG 957 in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states. == Introduction == Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and hydroxyl radicals, are derived from the metabolism of molecular oxygen[1],[2]. Due to their highly reactive nature, AG 957 ROS can damage nucleic acids, proteins and lipids, especially at high concentrations. Accordingly, excessive production of ROS in the central nervous system (CNS) contributes to the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis[3]. Recent studies suggest that ROS at physiological concentrations can mediate reversible regulatory processes and AG 957 serve as functional messenger molecules, possibly fulfilling a large range of physiologic and pathophysiologic functions[4]. Notably, accumulating evidence indicates that ROS are involved in the sensitization of pain pathways during persistent pain. For example, an increased ROS production has been detected in the spinal cord and in peripheral tissues after noxious hindpaw stimulation or nerve injury[5][7]. Mice lacking the superoxide-generating NADPH oxidases Nox1 or Nox2 demonstrated a reduced nociceptive behavior in animal models of inflammatory or neuropathic pain, respectively, indicating a contribution of NADPH oxidase-mediated superoxide production to pain sensitization[8],[9]. Moreover, inflammatory and neuropathic pain was effectively inhibited in animal models after administration of free radical scavengers or superoxide dismutase mimetics[10][14], further suggesting that ROS contribute to nociceptive processing. Vitamin C (Vit C) and vitamin E (Vit E) are essential nutrients that function as antioxidants in the human body. Vit C (L-ascorbic acid) is a water-soluble sugar acid that exists at physiological pH as a monovalent anion, ascorbate, which is capable to scavenge ROS[15]. Vit E (alpha-tocopherol), a fat-soluble vitamin, is the major chain-breaking antioxidant in body tissues and is the first line of defense against lipid peroxidation, protecting cell membranes from free radical attack[16]. Notably, Vit C is able to recycle Vit E by reduction of the tocopheroxyl radical of Vit E, thereby permitting Vit E to function again as a free radical chain-breaking antioxidant[17]. Based on the observation that (i) ROS contribute to nociceptive signaling and that (ii) the natural compounds Vit C and Vit E may exert additive antioxidant effects, we hypothesized that a combination of both vitamins might attenuate persistent pain. Hence, the goal of this study was to evaluate whether or not exogenous Vit C and Vit E are able to inhibit nociceptive behavior in animal models of inflammatory or neuropathic pain. == Methods == == Ethics Statement == All experiments were approved by the federal authority for animal research (Regierungsprsidium Darmstadt, Hessen, Germany) and were carried out in strict accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering. == Animals == Male C57BL/6 mice (89 weeks old, 251 g) were obtained from Harlan Laboratories. Animals were maintained under a 12/12-h light/dark cycle with free access to water and standard chow (ssniff R/M-H; ssniff, Soest, Germany). == Drugs == Commercially available liquid parenteral formulations of Vit C (Pascorbin, Pascoe, Giessen, Germany and Vitamin C-Rotexmedica, Rotexmedica, Trittau, Germany; containing 150 and 100 mg/ml L-ascorbic acid, respectively) and Vit E (Vitamin E Sanum, Sanum-Kehlbeck, Hoya, Germany and Ephynal, Bayer, Barcelona, Spain; containing 75 and 50 mg/ml alpha-tocopherol acetate, respectively), or 0.9% saline (B. Braun, Melsungen, Germany) were used. For AG 957 multiple daily intraperitoneal (i.p.) injections and oral administrations of low vitamin doses, the vitamin formulations were diluted with 0.9% saline. For intrathecal (i.t.) injections (injection volume 2.5 l), L-ascorbic acid (Sigma-Aldrich, Munich, Germany) was added to the Vit C formulation (Pascorbin) to obtain a final concentration of 300 mg/ml and NaHCO3was used to adjust pH to 7.0, whereas alpha-tocopherol acetate (Sigma-Aldrich) was added to the Vit E formulation (Vitamin E Sanum) to obtain a final concentration of 150 mg/ml. Vitamins C and E in combination were administered by consecutive single doses of Vit C and Vit E, respectively. All drug preparations were performed immediately before administration to minimize air oxidation of the vitamin solutions. == Behavioral Testing == Prior to behavioral testing, animals were habituated to the experimental room for at least 1 h. Experiments were performed by an observer blinded for the treatment of the animals. == Formalin test == Rabbit polyclonal to HNRNPH2 Twenty or two minutes after i.p. drug injection, 15 l.