in vivo 28: 21-32 (2014) Minocycline Modulates Cytokine and Gene Expression Profiles in the Brain After Whole-body Exposure to Radiation SHALINI MEHROTRA2, MICHAEL J. PECAUT1,2 and DAILA S. GRIDLEY1,2 Department of Basic Sciences, 1Division of Radiation Research and 2Division of Biochemistry and Microbiology, Loma Linda University School of Medicine, Loma Linda, CA, U.S.A. Abstract. An effective countermeasure against radiation radiation on normal tissues in the context of radiological/ damage to normal tissues is urgently needed. The major goal of nuclear events. As astronauts may be exposed to doses as high the present study was to determine if minocycline could modify as 1-3 Gray (Gy) during a solar particle event (SPE) (1), the the immunomodulatory effects of radiation on the brain. development of effective radioprotectants is also important to C57BL/6 mice were treated with minocycline intraperitoneally agencies such as the National Aeronautics and Space for 5 days beginning immediately before total-body exposure Administration (NASA). Due to the highly radiosensitive to 0, 1, 2 and 3 Gray (Gy) 60Co γ-rays. Brains were collected nature of the hematopoietic system and gastrointestinal tract, on days 4 and 32 post-irradiation for cytokine and gene most studies to date have focused on pharmacological agents analyses. Minocycline treatment significantly increased the that mitigate the damage associated with acute radiation levels of interleukin (IL)-10, IL-15 and vascular endothelial syndrome (ARS) (2). Although various cytokines and growth growth factor (VEGF) in the brain on day 4 in one or more factors appear promising, minimal efficacy and side-effects irradiated groups compared to radiation-alone (p<0.05). IL- remain problematic (3). Currently, although there are several 10 is anti-inflammatory, IL-15 can prevent apoptosis and agents that have Investigational New Drug (IND) status, VEGF is nuroprotective. On day 32, the drug decreased IL-1β granulocyte colony-stimulating factor (G-CSF) is the only drug in the 2- Gy group (p<0.05 vs. 2-Gy alone); this cytokine is approved by the Food and Drug Administration (FDA) of the implicated in immune-related central nervous system United States as an Emergency Use IND. In contrast, research pathologies. Microarray analysis of brains on day 32 showed on agents with potential to protect the central nervous system that while radiation increased expression of inflammatory (CNS) against radiation damage has been minimal and needs to genes such as Il1f10, Il17, Tnfrsf11b, Tnfsf12, Il12b and Il1f8, be further explored. these were no longer up-regulated in the minocycline-treated Although the brain is considered to be relatively groups. Similarly, the pro-apoptotic gene Bik and nitric oxide radioresistant, irradiation of neural tissues is often associated synthase producer (Nostrin) were no longer up-regulated in the with localized inflammation (4, 5). The neuroinflammatory drug-treated groups. Pathway analysis based on gene data milieu generated by resident and infiltrating populations in suggested that catenin-β1 and tumor suppressor-related response to radiation includes pro-inflammatory cytokines and transcription regulation were significantly activated by other factors indicative of increased and prolonged oxidative radiation and/or minocycline (activation z-score >2.0). stress. For example, the acute response to radiation in the brain Overall, the data warrant further testing of minocycline as a involves an increase in inflammatory cytokines and mediators potential neuroprotectant against radiation-induced damage. such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL- 1β), intercellular adhesion molecule-1 (ICAM-1) and There continues to be great urgency to develop safe and cyclooxygenase-2 (COX-2), as well as activation of effective agents that protect against the damaging effects of transcription factors such as nuclear factor kappa-light-chain- enhancer of activated B cells (NF-kB) and activator protein-1 (AP-1) (6-8). Prolonged oxidative stress, in turn, is responsible for the long-term pathogenesis of radiation-induced brain injury Correspondence to: Daila S. Gridley, Ph.D., Chan Shun Pavilion, (9, 10). Indeed, irradiation of the brain has already been shown Room A-1010, 11175 Campus Street, Loma Linda University, to increase apoptosis in the neural stem cell pools of the Loma Linda, CA 92354, U.S.A. Tel: +1 9095588361, Fax: hippocampus, a structure critical to memory and other +1 9095580825, e-mail: [email protected] behaviors (11, 12). Furthermore, radiation-induced decrement Key Words: Gamma radiation, radioprotectant, apoptosis, in stem cell populations has been shown to impact behavior in inflammation, mouse model. animal models (13, 14). 0258-851X/2014 $2.00+.40 21 in vivo 28: 21-32 (2014) Numerous antioxidants are in various stages of development (n=10/cage) under standard vivarium conditions. Animals were for protection against or mitigation of oxidative stress triggered assigned to 8 groups (10 mice/group): a) deionized water (dH2O) + by radiation exposure (15). These include superoxide dismutase 0 Gy; b) dH2O + 1 Gy; c) dH2O +2 Gy; d) dH2O + 3 Gy; e) minocycline + 0 Gy; f) minocycline + 1 Gy; g) minocycline + 2 Gy; (SOD) mimetics (16), resveratrol (17), melatonin (18) and and h) minocycline +3 Gy. Animals were rapidly euthanized on days others. Currently, however, only amifostine is approved by the 4 and 32 post-irradiation using 100% CO2 in compliance with the FDA for use in a clinical setting. Although amifostine, a free recommendations of the National Institutes of Health and the Panel radical scavenger, can be useful as a radioprotectant of healthy of Euthanasia of the American Veterinary Medical Association. tissues, it has numerous side-effects, e.g. nausea/vomiting, acute Brains were collected following euthanasia, cut into right and left hypersensitivity reaction, pruritis, urticaria, seizures and hemispheres, and immediately snap-frozen in liquid nitrogen. All reduction in blood pressure (http://www.drugs.com/sfx/ procedures were approved by the Institutional Animal Care and Use Committee of Loma Linda University. amifostine-side-effects.html). In some cases, the side-effects are serious and require discontinuation of the drug. Drug treatment and irradiation of mice. Minocycline hydrochloride Minocycline is a semi-synthetic tetracycline derivative that was purchased from Triax Pharmaceuticals, LLC, Cranford, NJ, USA. is commonly prescribed for a variety of bacterial infections. Animals in the respective treatment groups were injected However, the drug has a number of actions unrelated to its intraperitonealy (i.p.) with the drug (45 mg/kg in 0.1 ml) or dH2O anti-microbial properties that may be useful for therapy. immediately before irradiation. A Co-60 source (Eldorado machine, Although the mechanisms of action are still being worked Atomic Energy of Canada Ltd, Commercial Products Division, out, minocycline can efficiently cross the blood-brain barrier Ottawa, Canada) was used to administer 1, 2 or 3 Gy whole-body radiation at 1.58 Gy/min to mice placed individually into rectangular (BBB) and exert neuroprotective effects in animal models of plastic aerated boxes (30×30×60 mm3). A second injection of cerebral ischemia, traumatic brain injury and several other minocycline (45 mg/kg) or dH2O was administered to the appropriate pathologies related to the CNS (19). The drug is being groups immediately after irradiation. Three consecutive injections of evaluated as a possible adjunct treatment for early-stage minocycline (22.5 mg/kg) or dH2O were then administered on the schizophrenia and other psychiatric disorders (20-22). following three days post-irradiation. Sham-irradiated groups were Minocycline reportedly also has anti-apoptotic, anti- given similar treatment, but without the radiation. inflammatory and anti-tumorigenic effects that include reduction of glioma cell invasiveness (23). Perhaps most Analysis of cytokines. For cytokine analysis, left hemispheres obtained from mice on days 4 and 32 were thawed. Homogenates important to the present study is that minocycline has free were prepared using phosphate buffered saline (PBS without radical scavenging properties (24) and potential to protect calcium and magnesium) containing a cocktail of protease inhibitor neurons against radiation-induced apoptotic death (25). In tablets from Hoffman-La Roche, Inc. (Pleasanton, CA, USA) and addition, minocycline can be administered orally and has a 0.05% Tween-20. One ml of buffer solution was used for every relatively long half-life, characteristics that are favorable in 0.2 g tissue for homogenization, followed by centrifugation. The case of a nuclear disaster or during deep space travel where supernatants were stored at –80˚C until analysis. Supernatants from medical support is likely to be minimal. brain homogenates were thawed and analyzed for 22 different cytokines and chemokines using the Mouse Cytokine/Chemokine The data presented here are part of a larger study that Milliplex MAP Kit (Millipore, MA, USA) and Luminex100 (Linco involved whole-body irradiation of mice, with the intent to Research, Inc., St. Charles, MO, USA) as per the manufacturer’s assess the effects of minocycline on the hematopoietic instructions. The cytokines/chemokines evaluated were as follows: system in the context of ARS followed by recovery (26, 27). IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p70), IL- However, brains were also harvested to evaluate the potential