Advertisement

Rosmarinic acid improves oxidative stress parameters and mitochondrial respiratory chain activity following 4-aminopyridine and picrotoxin-induced seizure in mice

  • Jordana Griebler Luft
  • Luiza Steffens
  • Ana Moira Morás
  • Mateus Strucker da Rosa
  • Guilhian Leipnitz
  • Gabriela Gregory Regner
  • Pricila Fernandes Pflüger
  • Débora Gonçalves
  • Dinara Jaqueline Moura
  • Patrícia PereiraEmail author
Original Article

Abstract

Studies have indicated that epilepsy, an important neurological disease, can generate oxidative stress and mitochondrial dysfunction, among other damages to the brain. In this context, the use of antioxidant compounds could provide neuroprotection and help to reduce the damage caused by epileptic seizures and thereby the use of anticonvulsant drugs. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylactic acid that prevents cell damage caused by free radicals, acting as an antioxidant. It also presents anti-inflammatory, antimutagenic, and antiapoptotic properties. In this work, we used two models of acute seizure, 4-aminopyridine (4-AP) and picrotoxin (PTX)-induced seizures in mice, to investigate the anticonvulsant, antioxidant, and neuroprotective profile of RA. Diazepam and valproic acid, antiepileptic drugs already used in the treatment of epilepsy, were used as positive controls. Although RA could not prevent seizures in the models used in this study, neither enhance the latency time to first seizure at the tested doses, it exhibited an antioxidant and neuroprotective effect. RA (8 and 16 mg/kg) decreased reactive oxygen species production, superoxide dismutase activity, and DNA damage, measured in hippocampus, after seizures induced by PTX and 4-AP. Catalase activity was decreased by RA only after seizures induced by 4-AP. The activity of the mitochondrial complex II was increased by RA in hippocampus samples after both seizure models. The results obtained in this study suggest that RA is able to reduce cell damage generated by the 4-AP and PTX seizures and therefore could represent a potential candidate in reducing pathophysiological processes involved in epilepsy.

Keywords

4-aminopyridine Mitochondrial respiratory chain Oxidative stress Picrotoxin Rosmarinic acid Seizure 

Notes

Acknowledgments

The authors were supported by the Brazilian’s agencies: National Council for Scientific and Technological Development (CNPq) (Dr. Guilhian Leipnitz and Dr. P Pereira), and Federal University of Rio Grande do Sul (UFRGS).

Authors’ contributions

JGL, LS, AMM, MSR, and DG conducted the behavioral and biochemical experiments. GRG and PFP analyzed data. GL, DJM, JGL, and PP developed the experimental design, analyzed the data, and wrote the paper. All authors read and approved the manuscript.

Compliance with ethical standards

Experimental protocol adhered to the Guidelines of Brazilian Council of Animal Experimentation—CONCEA—and EU Directive 2010/63/EU for animal experiments.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Aebi H (1984) Catalase in Vitro. Methods Enzymol 105:121–126.  https://doi.org/10.1016/S0076-6879(84)05016-3 CrossRefGoogle Scholar
  2. Avoli M, de Curtis M (2011) GABAergic synchronization in the limbic system and its role in the generation of epileptiform activity. Prog Neurobiol 95:104–132.  https://doi.org/10.1016/j.pneurobio.2011.07.003 CrossRefGoogle Scholar
  3. Awad R, Muhammad A, Durst T, Trudeau VL, Arnason JT (2009) Bioassay-guided fractionation of lemon balm (Melissa officinalis L .) using an In Vitro measure of GABA transaminase activity. Phytother Res 23:1075–1081.  https://doi.org/10.1002/ptr CrossRefGoogle Scholar
  4. Bhat AH, Dar KB, Anees S, Zargar MA, Masood A, Sofi MA, Ganie SA (2015) Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight. Biomed Pharmacother 74:101–110.  https://doi.org/10.1016/j.biopha.2015.07.025 CrossRefGoogle Scholar
  5. Coelho VR, Vieira CG, de Souza LP, Moysés F, Basso C, Papke DKM, Pires TR, Siqueira IR, Picada JN, Pereira P (2015) Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice. Life Sci 122:65–71.  https://doi.org/10.1016/j.lfs.2014.11.009 CrossRefGoogle Scholar
  6. Coelho VR, Vieira CG, de Souza LP, da Silva LL, Pfluger P, Regner GG, Papke DKM, Picada JN, Pereira P (2016) Behavioral and genotoxic evaluation of rosmarinic and caffeic acid in acute seizure models induced by pentylenetetrazole and pilocarpine in mice. Naunyn Schmiedeberg's Arch Pharmacol 389:1195–1203.  https://doi.org/10.1007/s00210-016-1281-z CrossRefGoogle Scholar
  7. Coelho VR, Viau CM, Staub RB, de Souza MS, Pfluger P, Regner GG, Pereira P, Saffi J (2017) Rosmarinic acid attenuates the activation of murine microglial N9 cells through the downregulation of inflammatory cytokines and cleaved Caspase-3. Neuroimmunomodulation 24:171–181.  https://doi.org/10.1159/000481095 CrossRefGoogle Scholar
  8. da Silva CG, Ribeiro CAJ, Leipnitz G, Dutra CS, Wyse ATS, Wannmacher CMD, Sarkis JJF, Jakobs C, Wajner M (2002) Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro. Biochim Biophys Acta 1586:81–91.  https://doi.org/10.1016/S09254439(01)00088-6 CrossRefGoogle Scholar
  9. De Oliveira NCD, Sarmento MS, Nunes EA, Porto CM, Rosa DP, Bona SR, Rodrigues G, Marroni NP, Pereira P, Picada JN, Ferraz ABF, Thiesen FV, da Silva J (2012) Rosmarinic acid as a protective agent against genotoxicity of ethanol in mice. Food Chem Toxicol 50:1208–1214.  https://doi.org/10.1016/j.fct.2012.01.028 CrossRefGoogle Scholar
  10. Devinsky O, Vezzani A, De Lanerolle NC, Rogawski MA (2013) Glia and epilepsy: excitability and inflammation. Trends Neurosci 36:174–184.  https://doi.org/10.1016/j.tins.2012.11.008
  11. Dóczi J, Banczerowski-Pelyhe I, Barna B, Világi I (1999) Effect of a glutamate receptor antagonist (GYKI 52466) on 4-aminopyridine-induced seizure activity developed in rat cortical slices. Brain Res Bull 49(6):435–440.  https://doi.org/10.1016/S0361-9230(99)00079-9 CrossRefGoogle Scholar
  12. Fachel FNS, Schuh RS, Veras KS, Bassani VL, Koester LS, Henriques AT, Braganhol E, Teixeira HF (2019) An overview of the neuroprotective potential of rosmarinic acid and its association with nanotechnology-based delivery systems: a novel approach to treating neurodegenerative disorders. Neurochem Int 122:47–58.  https://doi.org/10.1016/j.neuint.2018.11.003 CrossRefGoogle Scholar
  13. Fallarini S, Miglio G, Paoletti T, Minassi A, Amoruso A, Bardelli C, Brunelleschi S, Lombardi G (2009) Clovamide and rosmarinic acid induce neuroprotective effects in in vitro models of neuronal death. Br J Pharmacol 157:1072–1084.  https://doi.org/10.1111/j.1476-5381.2009.00213.x CrossRefGoogle Scholar
  14. Fischer JC, Ruitenbeek W, Berden JA, Trijbels JMF, Veerkamp JH, Stadhouders AM, Sengers RCA, Janssen AJM (1985) Differential investigation of the capacity of succinate oxidation in human skeletal muscle. Clin Chim Acta 153:23–36.  https://doi.org/10.1016/0009-8981(85)90135-4 CrossRefGoogle Scholar
  15. Fisher RS, Boas WE, Blume W, Elger C, Genton P, Lee P, Engel J (2005) Epileptic seizures and epilepsy: definitions proposed by the international league against epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 46(4):470–472.  https://doi.org/10.1111/j.0013-9580.2005.66104.x CrossRefGoogle Scholar
  16. Fisher RS, Acevedo C, Arzimanoglou A, Bogacz A, Cross JH, Elger CE, Engel J, Forsgren L, French JA, Glynn M, Hesdorffer DC, Lee BI, Mathern GW, Moshé SL, Perucca E, Scheffer IE, Tomson T, Watanabe M, Wiebe S (2014) A practical clinical definition of epilepsy. Epilepsia 55(4):475–482.  https://doi.org/10.1111/epi.12550 CrossRefGoogle Scholar
  17. Freitas RM, Nascimento VS, Vasconcelos SMM, Sousa FCF, Viana GSB, Fonteles MMF (2004) Catalase activity in cerebellum, hippocampus, frontal cortex and striatum after status epilepticus induced by pilocarpine in Wistar rats. Neurosci Lett 365:102–105.  https://doi.org/10.1016/j.neulet.2004.04.060 CrossRefGoogle Scholar
  18. Gao LP, Wei HL, Zhao HS, Xiao SY, Zheng RL (2005) Antiapoptotic and antioxidant effects of rosmarinic acid in astrocytes. Pharmazie 60:62–65Google Scholar
  19. Gok DK, Hidisoglu E, Ocak GA, Er H, Acun AD, Yargıcoglu P (2018) Protective role of rosmarinic acid on amyloid beta 42-induced echoic memory decline: implication of oxidative stress and cholinergic impairment. Neurochem Int 118:1–13.  https://doi.org/10.1016/j.neuint.2018.04.008 CrossRefGoogle Scholar
  20. Grigoletto J, de Oliveira CV, Grauncke ACB, de Sousa TL, Souto NS, de Freitas ML, Furian AF, Santos ARS, Oliveira MS (2016) Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice. Epilepsy Behav 62:27–34.  https://doi.org/10.1016/j.yebeh.2016.06.037 CrossRefGoogle Scholar
  21. Grings M, Moura AP, Parmeggiani B, Pletsch JT, Cardoso GMF, August PM, Matté C, Wyse ATS, Wajner M, Leipnitz G (2017) Bezafibrate prevents mitochondrial dysfunction, antioxidant system disturbance, glial reactivity and neuronal damage induced by sulfite administration in striatum of rats: implications for a possible therapeutic strategy for sulfite oxidase deficiency. Biochim Biophys Acta Mol basis Dis 1863(9):2135–2148.  https://doi.org/10.1016/j.bbadis.2017.05.019 CrossRefGoogle Scholar
  22. Guzy RD, Sharma B, Bell E, Chandel NS, Schumacker PT (2008) Loss of the SdhB, but not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Mol Cell Biol 28(2):718–731.  https://doi.org/10.1128/MCB.01338-07 CrossRefGoogle Scholar
  23. Hartmann A, Speit G (1997) The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay). Toxicol Lett 90:183–188.  https://doi.org/10.1016/S0378-4274(96)03847-7 CrossRefGoogle Scholar
  24. Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR (2003) Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 18(1):45–51.  https://doi.org/10.1093/mutage/18.1.45 CrossRefGoogle Scholar
  25. Hasan ZA, Razzak RLA, Alzoubi KH (2014) Comparison between the effect of propofol and midazolam on picrotoxin-induced convulsions in rat. Physiol Behav 128:114–118.  https://doi.org/10.1016/j.physbeh.2014.01.027 CrossRefGoogle Scholar
  26. Jacob S, Nair AB (2016) An updated overview on therapeutic drug monitoring of recent antiepileptic drugs. Drugs RD 16:303–316.  https://doi.org/10.1007/s40268-016-0148-6
  27. Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ, Ischiropoulos H (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52:1–6.  https://doi.org/10.1016/j.freeradbiomed.2011.09.030 CrossRefGoogle Scholar
  28. Kang M, Yun S, Won J (2003) Rosmarinic acid inhibits Ca2+-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-Gamma1 and Itk activity. Blood 101(9):3534–3542.  https://doi.org/10.1182/blood-2002-07-1992 CrossRefGoogle Scholar
  29. Khamse S, Sadr SS, Roghani M, Hasanzadeh G, Mohammadian M (2015) Rosmarinic acid exerts a neuroprotective effect in the kainate rat model of temporal lobe epilepsy: underlying mechanisms. Pharm Biol 53(12):1818–1825.  https://doi.org/10.3109/13880209.2015.1010738 CrossRefGoogle Scholar
  30. Kudin AP, Kudina TA, Seyfried J, Vielhaber S, Beck H, Elger CE, Kunz WS (2002) Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus. Eur J Neurosci 15:1105–1114.  https://doi.org/10.1152/jn.01053.2003 CrossRefGoogle Scholar
  31. Kwon YO, Hong JT, Oh KW (2017) Rosmarinic acid potentiates pentobarbital-induced sleep behaviors and non-rapid eye movement (NREM) sleep through the activation of GABAa-ergic systems. Biomol Ther 25(2):105–111.  https://doi.org/10.4062/biomolther.2016.035 CrossRefGoogle Scholar
  32. Li X, Fang P, Mai J, Choi ET, Wang H, Yang X (2013) Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J Hematol Oncol 6(1):19.  https://doi.org/10.1186/1756-8722-6-19 CrossRefGoogle Scholar
  33. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  34. Martinc B, Grabnar I, Vovk T (2014) Antioxidants as a preventive treatment for epileptic process: a review of the current status. Curr Neuropharmacol 12:527–550.  https://doi.org/10.2174/1570159X12666140923205715 CrossRefGoogle Scholar
  35. Michiels C, Raes M, Toussaint O, Remacle J (1994) Importance of SE-glutathione peroxidase, catalase, and CU/ZN-SOD for cell survival against oxidative stress. Free Radic Biol Med 17(3):235–248.  https://doi.org/10.1016/0891-5849(94)90079-5 CrossRefGoogle Scholar
  36. Misra HP, Fridovich I (1972) The role of superoxide anion in the epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247(10):3170–3175Google Scholar
  37. Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1–13.  https://doi.org/10.1042/BJ20081386 CrossRefGoogle Scholar
  38. Najjar S, Pearlman D, Miller DC, Devinsky O (2011) Refractory epilepsy associated with microglial activation. Neurologist 17(5):249–254.  https://doi.org/10.1097/NRL.0b013e31822aad04 CrossRefGoogle Scholar
  39. Ngugi AK, Bottomley C, Kleinschmidt I, Sander J, Newton CR (2010) Estimation of the burden of active and life-time epilepsy: A meta-analytic approach. Epilepsia 51:883–889.  https://doi.org/10.1111/j.1528-1167.2009.02481.x
  40. Orr AL, Quinlan CL, Perevoshchikova IV, Brand MD (2012) A refined analysis of superoxide production by mitochondrial sn-glycerol 3-phosphate dehydrogenase. J Biol Chem 287(51):42921–42935.  https://doi.org/10.1074/jbc.M112.397828 CrossRefGoogle Scholar
  41. Paranagama MP, Sakamoto K, Amino H, Awano M, Miyoshi H, Kita K (2010) Contribution of the FAD and quinone binding sites to the production of reactive oxygen species from Ascaris suum mitochondrial complex II. Mitochondrion 10:158–165.  https://doi.org/10.1016/j.mito.2009.12.145 CrossRefGoogle Scholar
  42. Patel M (2004) Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures. Free Radic Biol Med 37:1951–1962.  https://doi.org/10.1016/j.freeradbiomed.2004.08.021 CrossRefGoogle Scholar
  43. Peña F, Tapia R (2000) Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GAMA-mediated neurotransmission and of ion channels. Neuroscience 101(3):547–561CrossRefGoogle Scholar
  44. Pereira P, De Oliveira PA, Ardenghi P, Rotta L, Henriques JAP, Picada JN (2006) Neuropharmacological analysis of Caffeic acid in rats. Basic Clin Pharmacol Toxicol 99:374–378.  https://doi.org/10.1016/j.phrs.2005.03.003 CrossRefGoogle Scholar
  45. Pflüger P, Regner GG, Coelho VR, da Silva LL, Nascimento L, Viau CM, Zanette RA, Hoffmann C, Picada JN, Saffi J, Pereira P (2018) Gamma-Decanolactone improves biochemical parameters associated with pilocarpine-induced seizures in male mice. Curr Mol Pharmacol 11(2):162–169.  https://doi.org/10.2174/1874467210666171002114954 CrossRefGoogle Scholar
  46. Quinlan CL, Orr AL, Perevoshchikova IV, Treberg JR, Ackrell BA, Brand MD (2012) Mitochondrial complex II can generate reactive oxygen species at high rates in both the forward and reverse. J Biol Chem 287(32):27255–27264.  https://doi.org/10.1074/jbc.M112.374629 CrossRefGoogle Scholar
  47. Rahbardar MG, Amin B, Mehri S, Mirnajafi-Zadeh SJ, Hosseinzadeh H (2018) Rosmarinic acid attenuates development and existing pain in a rat model of neuropathic pain: an evidence of anti-oxidative and anti-inflammatory effects. Phytomedicine 40:59–67.  https://doi.org/10.1016/j.phymed.2018.01.001 CrossRefGoogle Scholar
  48. Rizk HA, Masoud MA, Maher OW (2017) Prophylactic effects of ellagic acid and rosmarinic acid on doxorubicin-induced neurotoxicity in rats. J Biochem Mol Toxicol 31(12).  https://doi.org/10.1002/jbt.21977
  49. Rocha J, Eduardo-Figueira M, Barateiro A, Fernandes A, Brites D, Bronze R, Duarte CMM, Serra AT, Pinto R, Freitas M, Fernandes E, Silva-Lima B, Mota-Filipe H, Sepodes B (2015) Anti-inflammatory effect of Rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation. Basic Clin Pharmacol Toxicol 116:398–413.  https://doi.org/10.1111/bcpt.12335 CrossRefGoogle Scholar
  50. Rowles J, Olsen M (2012) Perspectives on the Development of Antioxidant Antiepileptogenic Agents. Mini-Reviews Med Chem 12:1015–1027.  https://doi.org/10.2753/RES1060-939322036 CrossRefGoogle Scholar
  51. Rowley S, Patel M (2013) Mitochondrial involvement and oxidative stress in temporal lobe epilepsy. Free Radic Biol Med 62:121–131.  https://doi.org/10.1016/j.freeradbiomed.2013.02.002
  52. Salami P, Lévesque M, Gotman J, Avoli M (2015) Distinct EEG seizure patterns reflect different seizure generation mechanisms. J Neurophysiol 113:2840–2844.  https://doi.org/10.1152/jn.00031.2015 CrossRefGoogle Scholar
  53. Salgo MG, Pryor WA (1996) Trolox inhibits Peroxynitrite-mediated oxidative stress and apoptosis in rat Thymocytes. Arch Biochem Biophys 333(2):482–488.  https://doi.org/10.1006/abbi.1996.0418 CrossRefGoogle Scholar
  54. Schapira AHV, Mann VM, Cooper JM, Dexter D, Daniel SE, Jenner P, Clark JB, Marsden CD (1990) Anatomic and disease specificity of NADH CoQ1 reductase (complex I) deficiency in Parkinson’s disease. J Neurochem 55:2142–2145.  https://doi.org/10.1111/j.1471-4159.1990.tb05809.x CrossRefGoogle Scholar
  55. Selivanov VA, Votyakova TV, Pivtoraiko VN, Zeak J, Sukhomlin T, Trucco M, Roca J, Cascante M (2011) Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain. PLoS Comput Biol 7(3):1–17.  https://doi.org/10.1371/journal.pcbi.1001115 CrossRefGoogle Scholar
  56. Shiha AA, de la Rosa RF, Delgado M, Pozo MA, García-García L (2017) Subacute fluoxetine reduces signs of hippocampal damage induced by a single Convulsant dose of 4-Aminopyridine in rats. CNS Neurol Disord Drug Targets 16(6):694–704.  https://doi.org/10.2174/1871527315666160720121723 CrossRefGoogle Scholar
  57. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191CrossRefGoogle Scholar
  58. Singh A, Kukreti R, Saso L, Kukreti S (2019) Oxidative stress: a key modulator in neurodegenerative diseases. Molecules 24(8):1–20.  https://doi.org/10.3390/molecules24081583 CrossRefGoogle Scholar
  59. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344.  https://doi.org/10.1113/jphysiol.2003.049478 CrossRefGoogle Scholar
  60. Van Houten B, Woshner V, Santos JH (2006) Role of mitochondrial DNA in toxic responses to oxidative stress. DNA Repair (Amst) 5:145–152.  https://doi.org/10.1016/j.dnarep.2005.03.002 CrossRefGoogle Scholar
  61. Venditti P, Di Stefano L, Di Meo S (2013) Mitochondrial metabolism of reactive oxygen species. Mitochondrion 13:71–82.  https://doi.org/10.1016/j.mito.2013.01.008 CrossRefGoogle Scholar
  62. Wahab A, Albus K, Gabriel S, Heinemann U (2010) In search of models of pharmacoresistant epilepsy. Epilepsia 51:154–159.  https://doi.org/10.1111/j.1528-1167.2010.02632.x CrossRefGoogle Scholar
  63. Wang J, Xu H, Jiang H, Du X, Sun P, Xie J (2012) Neurorescue effect of rosmarinic acid on 6-hydroxydopamine-lesioned nigral domapine neurons in rat model of Parkinson´s disease. J Mol Neurosci 47:113–119.  https://doi.org/10.1007/s12031-011-9693-1

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jordana Griebler Luft
    • 1
  • Luiza Steffens
    • 2
  • Ana Moira Morás
    • 2
  • Mateus Strucker da Rosa
    • 3
  • Guilhian Leipnitz
    • 3
    • 4
  • Gabriela Gregory Regner
    • 1
  • Pricila Fernandes Pflüger
    • 1
  • Débora Gonçalves
    • 1
  • Dinara Jaqueline Moura
    • 2
  • Patrícia Pereira
    • 1
    Email author
  1. 1.Neuropharmacology and Preclinical Toxicology Laboratory, Institute of Basic Health SciencesFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  2. 2.Laboratory of Genetic ToxicologyFederal University of Health Sciences of Porto Alegre (UFCSPA)Porto AlegreBrazil
  3. 3.Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Institute of Basic Health SciencesFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  4. 4.Programa de Pós-Graduação em Fisiologia, Institute of Basic Health SciencesFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil

Personalised recommendations