Metab Brain Dis DOI 10.1007/s11011-014-9602-8
RESEARCH ARTICLE
Minocycline protects against oxidative damage and alters energy metabolism parameters in the brain of rats subjected to chronic mild stress Gislaine Z. Réus & Helena M. Abelaira & Amanda L. Maciel & Maria Augusta B. dos Santos & Anelise S. Carlessi & Amanda V. Steckert & Gabriela K. Ferreira & Samira D. De Prá & Emilio L. Streck & Danielle S. Macêdo & João Quevedo
Received: 4 June 2014 / Accepted: 6 August 2014 # Springer Science+Business Media New York 2014
Abstract Studies have been suggested that minocycline can be a potential new agent for the treatment of depression. In addition, both oxidative stress and energy metabolism present an important role in pathophysiology of depression. So, the present study was aimed to evaluate the effects of minocycline on stress oxidative parameters and energy metabolism in the brain of adult rats submitted to the chronic mild stress protocol (CMS). After CMS Wistar, both stressed animals as controls received twice ICV injection of minocycline (160 μg) or vehicle. The oxidative stress and energy metabolism parameters were assessed in the prefrontal cortex (PF), hippocampus (HIP), amygdala (AMY) and nucleus accumbens (Nac). Our G. Z. Réus : H. M. Abelaira : A. L. Maciel : M. A. B. dos Santos : A. S. Carlessi : A. V. Steckert : J. Quevedo Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil G. Z. Réus : J. Quevedo Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral Sciences, Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA G. K. Ferreira : S. D. De Prá : E. L. Streck Laboratório de Bioenergética, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil D. S. Macêdo Neuropharmacology Laboratory, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil G. Z. Réus (*) Department of Psychiatry and Behavioral Sciences, Center for Experimental Models in Psychiatry,Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA e-mail:
[email protected]
findings showed that stress induced an increase on protein carbonyl in the PF, AMY and NAc, and mynocicline injection reversed this alteration. The TBARS was increased by stress in the PF, HIP and NAc, however, minocycline reversed the alteration in the PF and HIP. The Complex I was incrased in AMY by stress, and minocycline reversed this effect, however reduced Complex I activity in the NAc; Complex II reduced in PF and AMY by stress or minocycline; the Complex II-III increased in the HIP in stress plus minocycline treatment and in the NAc with minocycline; in the PF and HIP there were a reduced in Complex IV with stress and minocycline. The creatine kinase was reduced in AMY and NAc with stress and minocycline. In conclusion, minocycline presented neuroprotector effects by reducing oxidative damage and regulating energy metabolism in specific brain areas. Keywords Minocycline . Oxidative stress . Energy metabolism . Depression
Introduction Depression is a serious disorder that has enormous consequences for the quality of life and can cause severe impairment in occupational, social, or other important areas of functioning (American Psychiatric Association, 2013). Still, depression is a clinically and biologically heterogeneous disorder, with 10–30 % of women and 7–15 % of men susceptible to depression in their lifetime (Kessler and Wang, 2008). For many decades, the accepted pathogenesis of major depression has involved the dysfunction of the monoaminergic system. In fact, the drugs which enhance monoamine function show efficacy for depression promoting serotonergic and norepinephrinergic neurotransmission (Pae et al., 2008).
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Some evidence have reported that major depression, as well as the drugs which are used to treat this disorder can produce several adverse outcomes, among which impairment on mitochondrial function, increased production of free radicals and oxidative stress, leading to cellular changes and significantly affecting the patient’s quality of life (Duncan and Thompson, 2003). In this way, the present study was aimed to evaluate the effects of minocycline on stress oxidative parameters and energy metabolism in the brain of adult rats submitted to the chronic mild stress protocol.
However, such drugs are associated with some limitations, for example, only one-third of patients undergoing monotherapy with an antidepressant achieve complete remission of depressive symptoms and gain functional recovery (Skolnick, 2002).In this regard, the minocycline, a second-generation tetracycline, which presents powerful anti-inflammatory and neuroprotective effects, has been investigated as a potential new agent for the treatment of major depression (Pae et al., 2008). Minocycline effectively crosses the bloodebrain barrier leading to the inhibition of cytochrome c release from the mitochondria, inhibition of caspase expression, and the suppression of microglial activation (Domercq and Matute, 2004; Kim and Suh, 2009). Moreover, minocycline has a regulatory effect on pro-inflammatory agents, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-a), and interleukin- 1β (Lai and Todd, 2006), which are consistently reported to be increased in patients with major depression, and in other hand are normalized after antidepressant treatment (Maes, 1995; Herken et al., 2007). Although many has been known about the neurobiology of depression in recent years, its aetiology is still largely unknown. However, recent theoretical focus has been on biological factors, including a wealth of information supporting stress as a causal factor in depression, largely concerning chronic stress-related hypothalamic–pituitary–adrenal (HPA) axis dysregulation and toxicity from excessive glucocorticoid release (Lupien et al., 2009). Though other theories posit that a downregulation of hippocampal neurogenesis underlies the disorder (Kempermann and Kronenberg, 2003), or suggest genetic or epigenetic factors for developing depression (Karg et al., 2011; Menke et al., 2012). In this way, many preclinical studies attempting to model aspects of depression have focused on behaviors thought to represent anhedonia, reduced locomotor activity or behavior despair by exposure to different kinds of mild stress every day (Lu et al., 2006; Rygula et al., 2005; Willner, 1997). Also, unpredictable stressors have greater negative impact in humans than predictable ones, perhaps due to temporal uncertainty and inability to anticipate the event (Anisman and Matheson, 2005; Willner and Mitchell, 2002).
For this study, male rats used were divided into 4 experimental groups (n = 5–8): 1) Control + saline; 2) Control + minocycline; 3) Stress + saline; and 4) Stress + minociclyne. Firstly, the animals were subjected to the chronic mild stress protocol for 40 days and in the the forty-first day a surgical procedure was performed in the stereotaxic apparatus (Fig. 1).
Fig. 1 Schematic drawing of the stress protocol and treatment with minocycline. CMS procedures will be performed for 40 days. Surgery will be realized day 41st. In the fiftieth day minocycline (160 or 20 μg/
side) or saline (control) will be infused bilaterally into the right cerebral ventricle and in the fifty-first minocycline (160 or 20 μg/side) or saline will be infused bilaterally into the left cerebral ventricle
Materials and methods Animals Male Adult Wistar rats (60 days old) were obtained from the breeding colony at UNESC (Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil). They were housed five per cage with food and water available ad libitum (except on the day that the stressor used in the stress protocol were deprived of water or food) and were maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental procedures involving animals were performed in accordance with the NIH Guide for the Care and Usage of Laboratory Animals and under the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care, and with approval by the local Ethics Committee under protocol number 45/2012. Experimental design and treatment
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After surgery, the animals were left undisturbed for nine days until the start of treatment. Minocycline hydrochloride purchased from (Sigma-Aldrich Corp., St Louis, USA) was dissolved in physiological saline. In the first day of treatment, rats received bilateral microinjection of different amounts of minocycline (160 or 20 μg/side) or saline (control) into the right cerebral ventricle; and in the second day of treatment, the bilateral microinjection of minocycline (160 or 20 μg/side) or saline was into the left cerebral ventricle (Arakawa et al., 2012). A total volume of 4.0 μl was infused into each side over 10 min, and the injection syringe was left in place for an additional 5 min to allow for diffusion. After treatment, the animals were killed by decapitation and the prefrontal cortex, hippocampus, amygdala and nucleus accumbens were removed for biochemical analyzes.
of 1 mm were placed through the following coordinates: 0.9 mm behind bregma, 1.5 mm to the right of bregma; cannula being located 2.6 mm deep to the ventricle. The fixation of the tubes was made with acrylic cement. The animals were left undisturbed for 9 days until the start of treatment. Tissue and homogenate preparation The rat prefrontal cortex, hippocampus, amygdala and nucleus accumbens for thiobarbituric acid reactive species (TBARS) was homogenized in 30 mM Na2PO4, 14 mM KCl, pH=7.4 and for carbonyl protein was homogenized in (12 mM KCl, 0.038 mM KH2PO4, pH=7.4. Protein determination
Chronic mild stress protocol The chronic mild stress (CMS) protocol was adapted from the procedure described by Gamaro et al. (2003). The animals were divided in two groups: control and stressed. The control groups were kept undisturbed in their home cages during the 40 days of treatment receiving only ordinary daily care with daily supports of food and water. The 40-days chronic mild stress paradigm was used for the animals in the stressed group. Individual stressors and length of time applied each day were as follows. The following stressors were used: (i) 24 h food deprivation was applied on days 2, 7, 15, 21 and 30; (ii) 24 h water deprivation on days 1, 10, 20 and 33; (iii) 1–3 h restraint on days 9, 23 and 31, (iv) 1.5–2 h restraint at 4 °C on days 13, 26 and 34; (v) forced swimming for a duration of 10 or 15 min on days 8, 16, 27, 35 and 40; (vi) flashing light over a duration of 120–210 min on days 6, 14, 22, 28, 32 and 39; (vii) isolationon days 3, 4, 5, 17, 18, 19, 24, 25, 36 and 37. Stressor stimuli were applied at different times every day, in order to minimize its predictability. The restraint test was carried out by placing the animal in a 25 × 7 cm plastic tube and adjusting it with plaster tape on the outside, so that the animal was unable to move. There was a 1 cm hole at the far end for breathing. The Forced swimming was carried out by placing the animal in a glass tank measuring 50 × 47 cm filled with 30 cm of water at 23±2 °C in which the animal cannot touch the bottom. The exposure to flashing light test was undertaken by placing the animal in a 60 × 60 x 25 cm plywood box divided into 16 cells of 15 × 15 × 25 cm with a frontal glass wall. A 40 w lamp, flashing at frequency of 60 flashes/min, was used. Surgical procedure The animals were anesthetized with ketamine 30 mg/kg and xylazine 30 mg/kg intraperitoneally. In stereotaxic apparatus the skin and scalp rat in the skull region were removed. 2 tubes
All oxidative stress measures were normalized to the protein content with bovine albumin as standard (Lowry et al., 1951). Oxidative stress parameters MDA equivalents To determine oxidative damage in lipid, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as previously described by Draper and Hadley (1990). The samples were mixed with 1 ml of trichloroacetic acid 10 % and 1 ml of thiobarbituric acid 0.67 %, and then heated in a boiling water bath for 30 min. Malondialdehyde equivalents were determined in tissue and in submitochondrial particles of the rat brain spectrophotometrically by the absorbance at 532 nm. Carbonyls protein formation The oxidative damage to proteins was assessed by the determination of carbonyl groups content based on the reaction with dinitrophenylhidrazine (DNPH), as previously described by Levine et al. (1994). Proteins were precipitated by the addition of 20 % trichloroacetic acid and were redissolved in DNPH. The absorbance was monitored spectrophotometrically at 370 nm. Respiratory chain enzyme activities NADH dehydrogenase (complex I) was evaluated using the method described by Cassina and Radi (1996) relating to the rate of NADH-dependent ferricyanide reduction at 420 nm. The activity of succinate: Cytochrome c oxidoreductase (complexes II and II–III) were determined according to the method of Fischer et al. (1995), measured by Cytochrome c reduction from succinate. The activity of Cytochrome c oxidase
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(complex IV) was assayed according to the method described by Rustin et al. (2002), measured by following the decrease in absorbance due to the oxidation of previously reduced Cytochrome c at 550 nm with 580 nm as reference wavelength ( = 19.1 mM–1 × cm–1). The reaction buffer contained 10 mM potassium phosphate, pH 7.0, 0.6 mM n-dodecyl-d maltoside, 2–4 lg homogenate protein and the reaction was initiated with addition of 0.7 lg reduced cytochrome c. The activity of complex IV was measured at 25 °C for 10 min. The activities of the mitochondrial respiratory chain complexes were described as nmol. min-1. mg protein-1. Creatine kinase activity The creatine kinase activity was measured in the brain homogenates pretreated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 μg protein in a final volume of 100 μL. After 15 min of pre-incubation at 37 °C, the reaction was started by the addition of 3.2 mmol of ADP plus 0.8 mmol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 μmol of p-hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes (1962). The color was developed by the
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Statistical analysis Evaluation of study variables showed that normal distribution parametric tests would be most appropriate All data are presented as mean ± S.E.M. Differences among experimental groups in the assessment of oxidative stress parameters and energy metabolism were determined by one ANOVA, followed by Tukey post-hoc test when ANOVA was significant; P values
