Journal of Molecular Neuroscience

, Volume 49, Issue 1, pp 68–79 | Cite as

Protective Effects of Ascorbic Acid on Behavior and Oxidative Status of Restraint-Stressed Mice

  • Morgana Moretti
  • Josiane Budni
  • Danubia Bonfanti dos Santos
  • Alessandra Antunes
  • Juliana Felipe Daufenbach
  • Luana Meller Manosso
  • Marcelo Farina
  • Ana Lúcia S. RodriguesEmail author


Studies have demonstrated an association between stressful conditions and the onset of clinical depression. Considering the antidepressant-like properties of ascorbic acid in both experimental and clinical approaches, we evaluated the beneficial effect of this vitamin on restraint stress-induced behavioral and neurochemical alterations. Acute restraint stress caused a depressive-like behavior in the forced swimming test, accompanied by increased lipid peroxidation (cerebral cortex and hippocampus); increased superoxide dismutase (cerebral cortex and hippocampus), glutathione reductase (cerebral cortex), and glutathione peroxidase (cerebral cortex and hippocampus) activities; and elevated expression of Bcl-2 (hippocampus). Oral administration of ascorbic acid (1 mg/kg) or fluoxetine (10 mg/kg) 1 h before restraint stress prevented the stress-induced increase on immobility time in the forced swimming test. Moreover, this vitamin reduced lipid peroxidation to control levels and restored the activity of superoxide dismutase, glutathione reductase, and glutathione peroxidase. Ascorbic acid had no effect on the increased level of Bcl-2 induced by stress. Glutathione levels, glycogen synthase kinase-3β phosphorylation, and Bax expression were not altered by stress or ascorbic acid administration. Besides reinforcing the antioxidant potential of ascorbic acid, our results support the notion that oxidative stress plays a role in the pathogenesis and treatment of stress-induced depression.


Ascorbic acid Acute restraint stress Forced swimming test Oxidative stress Antioxidant 


  1. Adams JM, Cory S (2007) The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26:1324–1337PubMedCrossRefGoogle Scholar
  2. Ahmad A, Rasheed N, Ashraf GM et al (2012) Brain region specific monoamine and oxidative changes during restraint stress. Can J Neurol Sci 39:311–318PubMedGoogle Scholar
  3. Andreazza AC, Kauer-Sant’Anna M, Frey BN et al (2008) Oxidative stress markers in bipolar disorder: a meta-analysis. J Affect Disord 111:135–144PubMedCrossRefGoogle Scholar
  4. Atif F, Yousuf S, Agrawal SK (2008) Restraint stress-induced oxidative damage and its amelioration with selenium. Eur J Pharmacol 600:59–63PubMedCrossRefGoogle Scholar
  5. Bai F, Li X, Clay M, Lindstrom T, Skolnick P (2001) Intra- and interstrain differences in models of “behavioral despair”. Pharmacol Biochem Behav 70:187–192PubMedCrossRefGoogle Scholar
  6. Balk RDS, Bridi JC, Portella Rde L et al (2010) Clomipramine treatment and repeated restraint stress alter parameters of oxidative stress in brain regions of male rats. Neurochem Res 35:1761–1770CrossRefGoogle Scholar
  7. Beaulieu JM, Gainetdinov RR, Caron MG (2009) Akt/GSK3 signaling in the action of psychotropic drugs. Annu Rev Pharmacol Toxicol 49:327–347PubMedCrossRefGoogle Scholar
  8. Beaulieu JM, Zhang X, Rodriguiz RM et al (2008) Role of GSK3beta in behavioral abnormalities induced by serotonin deficiency. Proc Natl Acad Sci USA 105:1333–1338PubMedCrossRefGoogle Scholar
  9. Benkovic SA, Connor JR (1993) Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain. Neurology 338:97–113Google Scholar
  10. Bilici M, Efe H, Koroglu MA, Uydu HA, Bekaroglu M, Deger O (2001) Antioxidative enzyme activities and lipid peroxidation in major depression: alterations by antidepressant treatments. J Affect Disord 64:43–51PubMedCrossRefGoogle Scholar
  11. Binfaré RW, Rosa AO, Lobato KR, Santos ARS, Rodrigues ALS (2009) Ascorbic acid administration produces an antidepressant-like effect: evidence for the involvement of monoaminergic neurotransmission. Prog Neuropsychopharmacol Biol Psychiatry 33:530–540PubMedCrossRefGoogle Scholar
  12. Brocardo PS, Assini F, Franco JL et al (2007) Zinc attenuates malathion-induced depressant-like behavior and confers neuroprotection in the rat brain. Toxicol Sci 97:140–148PubMedCrossRefGoogle Scholar
  13. Brody S (2002) High-dose ascorbic acid increases intercourse frequency and improves mood: a randomized controlled clinical trial. Biol Psychiatry 52:371–374PubMedCrossRefGoogle Scholar
  14. Budni J, Gadotti VM, Kaster MP, Santos AR, Rodrigues ALS (2007) Role of different types of potassium channels in the antidepressant-like effect of agmatine in the mouse forced swimming test. Eur J Pharmacol 575:87–93PubMedCrossRefGoogle Scholar
  15. Buynitsky T, Mostofsky DI (2009) Restraint stress in biobehavioral research: recent developments. Neurosci Biobehav Rev 33:1089–1098PubMedCrossRefGoogle Scholar
  16. Capra JC, Cunha MP, Machado DG et al (2010) Antidepressant-like effect of scopoletin, a coumarin isolated from Polygala sabulosa (Polygalaceae) in mice: evidence for the involvement of monoaminergic systems. Eur J Pharmacol 643:232–238PubMedCrossRefGoogle Scholar
  17. Carlberg I, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490PubMedCrossRefGoogle Scholar
  18. Checkley S (1996) The neuroendocrinology of depression and chronic stress. Br Med Bull 52:597–617PubMedCrossRefGoogle Scholar
  19. Cocchi P, Silenzi M, Calabri G, Salvi G (1980) Antidepressant effect of vitamin C. Pediatrics 65:862–863PubMedGoogle Scholar
  20. Cordova FM, Rodrigues ALS, Giacomelli MB et al (2004) Lead stimulates ERK1/2 and p38MAPK phosphorylation in the hippocampus of immature rats. Brain Res 998:65–72PubMedCrossRefGoogle Scholar
  21. Doble BW, Woodgett JR (2003) GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116(Part 7):1175–1186PubMedCrossRefGoogle Scholar
  22. de Vasconcellos AP, Nieto FB, Crema LM et al (2006) Chronic lithium treatment has antioxidant properties but does not prevent oxidative damage induced by chronic variate stress. Neurochem Res 31:1141–1151PubMedCrossRefGoogle Scholar
  23. Dringen R, Pawlowski PG, Hirrlinger J (2005) Peroxide detoxification by brain cells. J Neurosci Res 79:157–165PubMedCrossRefGoogle Scholar
  24. Elhwuegi AS (2004) Central monoamines and their role in major depression. Prog Neuropsychopharmacol Biol Psychiatry 28:435–451PubMedCrossRefGoogle Scholar
  25. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77PubMedCrossRefGoogle Scholar
  26. Fontella FU, Siqueira IR, Vasconcellos APS, Tabajara AS, Netto CA, Dalmaz C (2005) Repeated restraint stress induces oxidative damage in rat hippocampus. Neurochem Res 30:105–111PubMedCrossRefGoogle Scholar
  27. Frame S, Cohen P (2001) GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359(Part 1):1–16PubMedCrossRefGoogle Scholar
  28. Galecki P, Szemraj J, Bienkiewicz M, Florkowski A, Galecka E (2009a) Lipid peroxidation and antioxidant protection in patients during acute depressive episodes and in remission after fluoxetine treatment. Pharmacol Rep 61:436–447PubMedGoogle Scholar
  29. Galecki P, Szemraj J, Bienkiewicz M, Zboralski K, Galecka E (2009b) Oxidative stress parameters after combined fluoxetine and acetylsalicylic acid therapy in depressive patients. Hum Psychopharmacol 24:277–286PubMedCrossRefGoogle Scholar
  30. Gamaro GD, Manoli LP, Torres IL, Silveira R, Dalmaz C (2003) Effects of chronic variate stress on feeding behavior and on monoamine levels in different rat brain structures. Neurochem Int 42:107–114PubMedCrossRefGoogle Scholar
  31. García-Fernández M, Castilla-Ortega E, Pedraza C et al (2012) Chronic immobilization in the malpar1 knockout mice increases oxidative stress in the hippocampus. Int J Neurosci 122:583–589PubMedCrossRefGoogle Scholar
  32. Gould TD, Manji HK (2005) Glycogen synthase kinase-3: a putative molecular target for lithium mimetic drugs. Neuropsychopharmacology 30:1223–1237PubMedGoogle Scholar
  33. Gould TD, Einat H, Bhat R, Manji HK (2004) AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int J Neuropsychopharmacol 7:387–390PubMedCrossRefGoogle Scholar
  34. Graumann R, Paris I, Martinez-Alvarado P et al (2002) Oxidation of dopamine to aminochrome as a mechanism for neurodegeneration of dopaminergic systems in Parkinson’s disease. Possible neuroprotective role of DT-diaphorase. Pol J Pharmacol 54:573–579PubMedGoogle Scholar
  35. Harkin AJ, Bruce KH, Craft B, Paul IA (1999) Nitric oxide synthase inhibitors have antidepressant-like properties in mice. 1. Acute treatments are active in the forced swim test. Eur J Pharmacol 372:207–213PubMedCrossRefGoogle Scholar
  36. He B, Meng YH, Mivechi NF (1998) Glycogen synthase kinase 3β and extracellular signal-regulated kinase inactivate heat shock transcription factor 1 by facilitating the disappearance of transcriptionally active granules after heat shock. Mol Cell Biol 18:6624–6633PubMedGoogle Scholar
  37. Hernández-Martínez JM, Domínguez G, Blancas S, Morán J (2011) Oxidation of biomolecules in the apoptotic death of cerebellar granule neurons induced by potassium deprivation. Neurochem Res 36:677–685PubMedCrossRefGoogle Scholar
  38. Holmes PV (2003) Rodent models of depression: reexamining validity without anthropomorphic inference. Crit Rev Neurobiol 15:143–174PubMedCrossRefGoogle Scholar
  39. Jope RS, Johnson GVW (2004) The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 29:95–102PubMedCrossRefGoogle Scholar
  40. Khanzode SD, Dakhale GN, Khanzode SS, Saoji A, Palasodkar R (2003) Oxidative damage and major depression: the potential antioxidant action of selective serotonin re-uptake inhibitors. Redox Rep 8:365–370PubMedCrossRefGoogle Scholar
  41. Kioukia-Fougia N, Antoniou K, Bekris S, Liapi C, Christofidis I, Papadopoulou-Daifoti Z (2002) The effects of stress exposure on the hypothalamic–pituitary–adrenal axis, thymus, thyroid hormones and glucose levels. Prog Neuropsychopharmacol Biol Psychiatry 26:823–830PubMedCrossRefGoogle Scholar
  42. Kovacheva-Ivanova S, Bakalova R, Ribavov SR (1994) Immobilization stress enhances lipid peroxidation in the rat lungs. Materials and methods. Gen Physiol Biophys 13:469–482Google Scholar
  43. Kovachich GB, Mishra OP (1983) The effect of ascorbic acid on malonaldehyde formation, K+, Na+ and water content of brain slices. Exp Brain Res 50:62–68PubMedCrossRefGoogle Scholar
  44. Kowaltowski AJ, Fiskum G (2005) Redox mechanisms of cytoprotection by Bcl-2. Antioxid Redox Signal 7:508–514PubMedCrossRefGoogle Scholar
  45. Kowaltowski AJ, Fenton RG, Fiskum G (2004) Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress. Free Radic Biol Med 37:1845–1853PubMedCrossRefGoogle Scholar
  46. Kozlovsky N, Belmaker RH, Agam G (2002) Lack of effect of acute, subchronic, or chronic stress on glycogen synthase kinase-3beta protein levels in rat frontal cortex. Prog Neuropsychopharmacol Biol Psychiatry 26:1309–1312PubMedCrossRefGoogle Scholar
  47. Kumar A, Goyal R (2008) Quercetin protects against acute immobilization stress-induced behaviors and biochemical alterations in mice. J Med Food 11:469–473PubMedCrossRefGoogle Scholar
  48. Kumari B, Kumar A, Dhir A (2007) Protective effect of non-selective and selective COX-2-inhibitors in acute immobilization stress-induced behavioral and biochemical alterations. Pharmacol Rep 59:699–707Google Scholar
  49. Li X, Jope RS (2010) Is glycogen synthase kinase-3 a central modulator in mood regulation? Neuropsychopharmacology 35:2143–2154PubMedCrossRefGoogle Scholar
  50. Li X, Zhu W, Roh MS, Friedman AB, Rosborough K, Jope RS (2004) In vivo regulation of glycogen synthase kinase-3beta (GSK3beta) by serotonergic activity in mouse brain. Neuropsychopharmacology 29:1426–1431PubMedCrossRefGoogle Scholar
  51. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  52. Maes M, Fišar Z, Medina M, Scapagnini G, Nowak G, Berk M (2012) New drug targets in depression: inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors. Inflammopharmacology 20:127–150PubMedCrossRefGoogle Scholar
  53. Manoli LP, Gamaro GD, Silveira PP, Dalmaz C (2000) Effect of chronic variate stress on thiobarbituric-acid reactive species and on total radical-trapping potential in distinct regions of rat brain. Neurochem Res 25:915–921PubMedCrossRefGoogle Scholar
  54. McCord JM, Edeas MA (2005) SOD, oxidative stress and human pathologies: a brief history and a future vision. Biomed Pharmacother 59:139–142PubMedCrossRefGoogle Scholar
  55. Michel TM, Frangou S, Thiemeyer D et al (2007) Evidence for oxidative stress in the frontal cortex in patients with recurrent depressive disorder—a postmortem study. Psychiatry Res 151:145–150PubMedCrossRefGoogle Scholar
  56. Mills GC (1957) Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J Biol Chem 229:189–197PubMedGoogle Scholar
  57. Misra HP, Fridovich I (1972) The purification and properties of superoxide dismutase from Neurospora crassa. J Biol Chem 247:3410–3414PubMedGoogle Scholar
  58. Moretti M, Budni J, Ribeiro CM, Rodrigues ALS (2012a) Involvement of different types of potassium channels in the antidepressant-like effect of ascorbic acid in the mouse tail suspension test. Eur J Pharmacol 687:21–27PubMedCrossRefGoogle Scholar
  59. Moretti M, Colla A, de Oliveira Balen G et al (2012b) Ascorbic acid treatment, similarly to fluoxetine, reverses depressive-like behavior and brain oxidative damage induced by chronic unpredictable stress. J Psychiatr Res 46:331–340PubMedCrossRefGoogle Scholar
  60. Moretti M, Freitas AE, Budni J, Fernandes SC, Balen GD, Rodrigues ALS (2011) Involvement of nitric oxide–cGMP pathway in the antidepressant-like effect of ascorbic acid in the tail suspension test. Behav Brain Res 225:328–333PubMedCrossRefGoogle Scholar
  61. Naert G, Ixart G, Maurice T, Tapia-Arancibia L, Givalois L (2011) Brain-derived neurotrophic factor and hypothalamic–pituitary–adrenal axis adaptation processes in a depressive-like state induced by chronic restraint stress. Mol Cell Neurosci 46:55–66PubMedCrossRefGoogle Scholar
  62. Ng F, Berk M, Dean O, Bush AI (2008) Oxidative stress in psychiatric disorders: evidence base and therapeutic implications. Int J Neuropsychopharmacol 11:851–876PubMedCrossRefGoogle Scholar
  63. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358PubMedCrossRefGoogle Scholar
  64. Oishi K, Yokoi M, Maekawa S et al (1999) Oxidative stress and haematological changes in immobilized rats. Acta Physiol Scand 165:65–69Google Scholar
  65. O'Mahony CM, Sweeney FF, Daly E, Dinan TG, Cryan JF (2010) Restraint stress-induced brain activation patterns in two strains of mice differing in their anxiety behaviour. Behav Brain Res 213:148–154PubMedCrossRefGoogle Scholar
  66. Osburn WO, Kensler TW (2008) Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults. Mutat Res 659:31–39PubMedCrossRefGoogle Scholar
  67. Pajović SB, Pejic S, Stojiljkovic V, Gavrilovic L, Dronjak S, Kanazir DT (2006) Alterations in hippocampal antioxidant enzyme activities and sympatho-adrenomedullary system of rats in response to different stress models. Physiol Res 55:453–460PubMedGoogle Scholar
  68. Park SH, Sim YB, Han PL, Lee JK, Suh HW (2010) Antidepressant-like effect of kaempferol and quercitirin, isolated from Opuntia ficus-indica var. saboten. Exp Neurobiol 19:30–38PubMedCrossRefGoogle Scholar
  69. Park SW, Phuong VT, Lee CH et al (2011) Effects of antipsychotic drugs on BDNF, GSK-3β, and β-catenin expression in rats subjected to immobilization stress. Neurosci Res 71:335–340PubMedCrossRefGoogle Scholar
  70. Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356PubMedCrossRefGoogle Scholar
  71. Poleszak E, Wlaz P, Kedzierska E et al (2006) Immobility stress induces depression-like behavior in the forced swim test in mice: effect of magnesium and imipramine. Pharmacol Rep 58:746–752PubMedGoogle Scholar
  72. Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327–336Google Scholar
  73. Prediger ME, Gamaro GD, Crema LM, Fontella FU, Dalmaz C (2004) Estradiol protects against oxidative stress induced by chronic variate stress. Neurochem Res 29:1923–1930PubMedCrossRefGoogle Scholar
  74. Radak Z, Sasvari M, Nyakas C et al (2001) Single bout of exercise eliminates the immobilization-induced oxidative stress in rat brain. Neurochem Int 39:33–38Google Scholar
  75. Rice ME (2000) Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci 23:209–216PubMedCrossRefGoogle Scholar
  76. Rudin CM, Yang Z, Schumaker LM et al (2003) Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res 63:312–318PubMedGoogle Scholar
  77. Sahin E, Gumuslu S (2007) Immobilization stress in rat tissues: alterations in protein oxidation, lipid peroxidation and antioxidant defense system. Comp Biochem Physiol C Toxicol Pharmacol 144:342–347PubMedCrossRefGoogle Scholar
  78. Santos IM, Tomé AR, Saldanha GB, Ferreira PM, Militão GC, Freitas RM (2009) Oxidative stress in the hippocampus during experimental seizures can be ameliorated with the antioxidant ascorbic acid. Oxid Med Cell Longev 2:214–221PubMedCrossRefGoogle Scholar
  79. Selek S, Savas HA, Gergerlioglu HS, Bulbul F, Uz E, Yumru M (2008) The course of nitric oxide and superoxide dismutase during treatment of bipolar depressive episode. J Affect Disord 107:89–94PubMedCrossRefGoogle Scholar
  80. Seregi A, Schaefer A, Komlós M (1978) Protective role of brain ascorbic acid content against lipid peroxidation. Experientia 34:1056–1057PubMedCrossRefGoogle Scholar
  81. Shoji H, Mizoguchi K (2010) Acute and repeated stress differentially regulates behavioral, endocrine, neural parameters relevant to emotional and stress response in young and aged rats. Behav Brain Res 211:169–177PubMedCrossRefGoogle Scholar
  82. Steckert AV, Valvassori SS, Moretti M, Dal-Pizzol F, Quevedo J (2010) Role of oxidative stress in the pathophysiology of bipolar disorder. Neurochem Res 35:1295–1301PubMedCrossRefGoogle Scholar
  83. Trevisan R, Uliano-Silva M, Pandolfo P et al (2008) Antioxidant and acetylcholinesterase response to repeated malathion exposure in rat cerebral cortex and hippocampus. Basic Clin Pharmacol Toxicol 102:365–369PubMedCrossRefGoogle Scholar
  84. Trivedi MH, Rush AJ, Wisniewski SR et al (2006) Evaluation of outcomes with citalopram for depression using measurement-based care in STAR*D: implications for clinical practice. Am J Psychiatry 163:28–40PubMedCrossRefGoogle Scholar
  85. Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:325–333PubMedCrossRefGoogle Scholar
  86. Wilkinson MB, Dias C, Magida J et al (2011) A novel role of the WNT-disheveled–GSK3β signaling cascade in the mouse nucleus accumbens in a social defeat model of depression. J Neurosci 31:9084–9092PubMedCrossRefGoogle Scholar
  87. Zafir A, Ara A, Banu N (2009) In vivo antioxidant status: a putative target of antidepressant action. Prog Neuropsychopharmacol Biol Psychiatry 33:220–228PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Morgana Moretti
    • 1
  • Josiane Budni
    • 1
  • Danubia Bonfanti dos Santos
    • 1
  • Alessandra Antunes
    • 1
  • Juliana Felipe Daufenbach
    • 1
  • Luana Meller Manosso
    • 1
  • Marcelo Farina
    • 1
  • Ana Lúcia S. Rodrigues
    • 1
    Email author
  1. 1.Departamento de Bioquímica, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianópolisBrazil

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