Advertisement

Neurotoxicity Research

, Volume 29, Issue 4, pp 447–459 | Cite as

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways

  • Flávia V. Santa-Cecília
  • Benjamin Socias
  • Mohand O. Ouidja
  • Julia E. Sepulveda-Diaz
  • Leonardo Acuña
  • Rangel L. Silva
  • Patrick P. Michel
  • Elaine Del-Bel
  • Thiago M. CunhaEmail author
  • Rita Raisman-VozariEmail author
Original Article

Abstract

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson’s disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1β). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

Keywords

Parkinson’s disease Doxycycline Microglia Cytokines 

Notes

Acknowledgments

Our study received funding from the MINCyT-ECOS Program, A12S02; from the São Paulo Research Foundation (FAPESP) under Grant Agreements No. 2011/19670-0 (Thematic Project); 2013/08216-2 (Center for Research in Inflammatory Disease); from CNPq; and from the Program Investissements d’avenir “ANR-10-IAIHU-06” and the Translational Research Infrastructure for Biotherapies in Neurosciences ANR-11-INBS-0011-NeurATRIS. EDB and RRV are coordinators of Science without Borders-Special Visitor Research (CNPQ). FVSC received a fellowship from the Science Without Borders CNPq Program. BS and LA are recipients of a fellowship from the Bernardo Houssay Program, MINCyT-CONICET-CAMPUS FRANCE.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Abd-El-Basset E, Fedoroff S (1995) Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures. J Neurosci Res 41:222–237CrossRefPubMedGoogle Scholar
  2. Ahler E, Sullivan WJ, Cass A, Braas D, York AG, Bensinger SJ, Graeber TG, Christofk HR (2013) Doxycycline alters metabolism and proliferation of human cell lines. PLoS One 8:e64561CrossRefPubMedPubMedCentralGoogle Scholar
  3. Austin PJ, Moalem-Taylor G (2010) The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines. J Neuroimmunol 229:26–50CrossRefPubMedGoogle Scholar
  4. Bachstetter AD, Eldik LJV (2010) The p38 MAP kinase family as regulators of proinflammatory cytokine production in degenerative diseases of the CNS. Aging dis 3:199–211Google Scholar
  5. Barnum CJ, Eskow KL, Dupre K, Blandino P Jr, Deak T, Bishop C (2008) Exogenous corticosterone reduces L-DOPA-induced dyskinesia in the hemi-parkinsonian rat: role for interleukin-1beta. Neuroscience 156:30–41CrossRefPubMedPubMedCentralGoogle Scholar
  6. Blackwell TS, Christman JW (1997) The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 17:3–9CrossRefPubMedGoogle Scholar
  7. Block ML, Hong JS (2005) Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol 76:77–98CrossRefPubMedGoogle Scholar
  8. Block ML, Zecca L, Hong JS (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69CrossRefPubMedGoogle Scholar
  9. Bortolanza M, Cavalcanti-Kiwiatkoski R, Padovan-Neto FE, da-Silva CA, Mitkovski M, Raisman-Vozari R, Del-Bel E (2014) Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson’s disease. Neurobiol Dis. doi: 10.1016/j.nbd.2014.10.017
  10. Bosscher KD, Beck IM, Dejager L, Bougarne N, Gaigneaux A, Chateauvieux S et al (2014) Selective modulation of the glucocorticoid receptor can distinguish between transrepression of NF-κB and AP-1. Cell Mol Life Sci 71:143–163CrossRefPubMedPubMedCentralGoogle Scholar
  11. Butterfield DA, Kanski J (2001) Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mech Ageing Dev 15:945–962CrossRefGoogle Scholar
  12. Caldeira C, Oliveira AF, Cunha C, Vaz AR, Falcão AS, Fernandes A, Brites D (2014) Microglia change from a reactive to an age-like phenotype with the time in culture. Front Cell Neurosci. doi: 10.3389/fncel.2014.00152 PubMedPubMedCentralGoogle Scholar
  13. Cho Y, Son HJ, Kim EM, Choi JH, Kim ST, Ji IJ, Choi DH, Joh TH, Kim YS, Hwang O (2009) Doxycycline is neuroprotective against nigral dopaminergic degeneration by a dual mechanism involving MMP-3. Neurotox Res 16:361–371CrossRefPubMedGoogle Scholar
  14. Clark WM, Calcagno FA, Gabler WL, Smith JR, Coull BM (1994) Reduction of central nervous system reperfusion injury in rabbits using doxycycline treatment. Stroke 25:1411–1415CrossRefPubMedGoogle Scholar
  15. Clark WM, Lessov N, Lauten JD, Hazel K (1997) Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci 9:103–108CrossRefPubMedGoogle Scholar
  16. Cunha BA, Comer JB, Jonas M (1982) The tetracyclines. Med Clin North Am 66:293–302PubMedGoogle Scholar
  17. Cunningham C (2013) Microglia and neurodegeneration: the role of systemic inflammation. Glia 61:71–90CrossRefPubMedGoogle Scholar
  18. Dendorfer U, Oettgen P, Libermann TA (1994) Multiple regulatory elements in the interleukin-6 gene mediate induction by prostaglandins, cyclic AMP, and lipopolysaccharide. Mol Cell Biol 14:4443–4454CrossRefPubMedPubMedCentralGoogle Scholar
  19. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489CrossRefPubMedPubMedCentralGoogle Scholar
  20. Domercq M, Matute C (2004) Neuroprotection by tetracyclines. Trends Pharmacol Sci 25:609–612CrossRefPubMedGoogle Scholar
  21. Dutta G, Zhang P, Liu B (2008) The lipopolysaccharide Parkinson’s disease animal model: mechanistic studies and drug discovery. Fundam Clin Pharmacol 22:453–464CrossRefPubMedPubMedCentralGoogle Scholar
  22. Edan RA, Luqmani YA, Masocha W (2013) COL-3, a chemically modified tetracycline, inhibits lipopolysaccharide-induced microglia activation and cytokine expression in the brain. PLoS One 8:e57827CrossRefPubMedPubMedCentralGoogle Scholar
  23. Emerit J, Edeas M, Bricaire F (2004) Neurodegenerative diseases and oxidative stress. Biomed Pharmacother 58:39–46CrossRefPubMedGoogle Scholar
  24. Fan LW, Pang Y, Lin S, Rhodes PG, Cai Z (2005) Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain. Neuroscience 133:159–168CrossRefPubMedGoogle Scholar
  25. Forno LS (1996) Neuropathology of Parkinson’s disease. J Neuropathol Exp Neurol 55:259–272CrossRefPubMedGoogle Scholar
  26. Gandhi S, Wood NW (2005) Molecular pathogenesis of Parkinson’s disease. Hum Mol Genet 2:2749–2755CrossRefGoogle Scholar
  27. Gerhard A, Pavese N, Hotton G, Turkheimer F, Es M, Hammers A, Eggert K, Oertel W, Banati RB, Brooks DJ (2006) In vivo imaging of microglial activation with [11C](R)-PK11195 PET in idiopathic Parkinson’s disease. Neurobiol Aging 21:404–412Google Scholar
  28. Giasson BI, Ischiropoulos H, Lee VM, Trojanowski JQ (2002) The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer’s and Parkinson’s diseases. Free Radic Biol Med 32:1264–1275CrossRefPubMedGoogle Scholar
  29. Gordon PH, Moore DH, Gelinas DF, Qualls C, Meister ME, Werner J, Mendoza M, Mass J, Kushner G, Miller RG (2004) Placebo-controlled phase I/II studies of minocycline in amyotrophic lateral sclerosis. Neurology 62:1845–1847CrossRefPubMedGoogle Scholar
  30. Henry CJ, Huang Y, Wynne A, Hanke M, Himler J, Bailey MT, Sheridan JF, Godbout JP (2008) Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia. J Neuroinflamm 5:15CrossRefGoogle Scholar
  31. Henry CJ, Huang Y, Wynne AM, Godbout JP (2009) Peripheral lipopolysaccharide (LPS) challenge promotes microglial hyperactivity in aged mice that is associated with exaggerated induction of both pro-inflammatory IL-1beta and anti-inflammatory IL-10 cytokines. Brain Behav Immun 23:309–317CrossRefPubMedPubMedCentralGoogle Scholar
  32. Hirsch EC, Hunot S, Damier P, Faucheux B (1998) Glial cells and inflammation in Parkinson’s disease: a role in neurodegeneration? Ann Neurol 44:115–120CrossRefGoogle Scholar
  33. Horvath RJ, Nutile-McMenemy N, Alkaitis MS, Deleo JA (2008) Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortal- ized BV-2 and HAPI cell lines and primary microglial cultures. J Neurochem 107:557–569CrossRefPubMedPubMedCentralGoogle Scholar
  34. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S (1999) Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product. J Immunol 162:3749–3752PubMedGoogle Scholar
  35. Huang Y, Li R, Chen X, Zhuo Y, Jin R, Qian XP, Jiang YQ, Zeng ZH, Zhang Y, Shao QX (2011) Doxycycline up-regulates the expression of IL-6 and GM-CSF via MAPK/ERK and NF-kappaB pathways in mouse thymic epithelial cells. Int Immunopharmacol 111:1143–1149Google Scholar
  36. Ito D, Tanaka K, Suzuki S, Dembo T, Fukuuchi Y (2001) Transient focal cerebral ischemia in rat brain enhanced expression of iba 1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32:1208–1215CrossRefPubMedGoogle Scholar
  37. Kaneko YS, Mori K, Nakashima A, Sawada M, Nagatsu I, Ota A (2005) Peripheral injection of lipopolysaccharide enhances expression of inflammatory cytokines in murine locus coeruleus: possible role of increased norepinephrine turnover. J Neurochem 94:393–404CrossRefPubMedGoogle Scholar
  38. Kang YJ, Chen J, Otsuka M, Mols J, Ren S, Wang Y, Han J (2008) Macrophage deletion of p38α partially impairs lipopolysaccharide-induced cellular activation. J Immunol 180:5075–5082CrossRefPubMedGoogle Scholar
  39. Kang SM, More SV, Park JY, Kim BW, In PJ, Yoon SH, Choi DK (2014) A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated inflammatory response by suppressing the p38 MAPK/JNK and NF-kB activation pathways. Pharmacol Rep 66:471–479CrossRefPubMedGoogle Scholar
  40. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev. doi: 10.1152/physrev.00011.2010 PubMedGoogle Scholar
  41. Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci 20:6309–6316PubMedGoogle Scholar
  42. Kim SS, Kong PJ, Kim BS, Sheen DH, Nam SY, Chun W (2004) Inhibitory action of minocycline on lipopolysaccharide-induced release of nitric oxide and prostaglandin E2 in BV2 microglial cells. Arch Pharm Res 27:314–318CrossRefPubMedGoogle Scholar
  43. Langston JW, Forno LS, Tetrud J, Reeves AG, Kaplan JA, Karluk D (1999) Evidence of active nerve cell degeneration in the substantia nigra of humans years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure. Ann Neurol 46:598–605CrossRefPubMedGoogle Scholar
  44. Lawson LJ, Perry VH, Dri P, Gordon S (1990) Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience 39:151–170CrossRefPubMedGoogle Scholar
  45. Lazzarini M, Martin S, Mitkovski M, Vozari RR, Stuhmer W, Bel ED (2013) Doxycycline restrains glia and confers neuroprotection in a 6-OHDA Parkinson model. Glia 61:1084–1100CrossRefPubMedGoogle Scholar
  46. Lehnardt S, Massillon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, Volpe JJ, Vartanian T (2003) Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci USA 100:8514–8519CrossRefPubMedPubMedCentralGoogle Scholar
  47. Liu B, Hong JS (2003) Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304:1–7CrossRefPubMedGoogle Scholar
  48. Lund S, Christensen KV, Hedtjarn M, Mortensen AL, Hagberg H, Falsig J, HasseldamH Schrattenholz A, Pörzgen P, Leist M (2006) The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol 180:71–87CrossRefPubMedGoogle Scholar
  49. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S (1993) Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 90:193–197CrossRefGoogle Scholar
  50. McGeer PL, Itagaki S, Boyes BE, McGeer EG (1988) Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology. doi: 10.1212/WNL.38.8.1285 PubMedGoogle Scholar
  51. Metz LM, Zhang Y, Yeung M, Patry DG, Bell RB, Stoian CA, Yong VW, Patten SB, Duquette P, Antel JP, Mitchell JR (2013) Minocycline reduces gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis. Ann Neurol 55:756CrossRefGoogle Scholar
  52. More SV, Kumar H, Kim IS, Song SY, Choi DK (2013) Cellular and molecular mediators of neuroinflammation in the pathogenesis of Parkinson’s disease. Mediators Inflamm. doi: 10.1155/2013/952375 PubMedPubMedCentralGoogle Scholar
  53. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefPubMedGoogle Scholar
  54. Nikodemova M, Duncan ID, Watters JJ (2006) Minocycline exerts inhibitory effects on multiplemitogen-activated protein kinases and IΚBα degradation in a stimulus-specific manner in microglia. J Neurochem 96:314–323CrossRefPubMedGoogle Scholar
  55. Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B (2000) The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J Neurosci 20:6048–6054PubMedGoogle Scholar
  56. Page TH, Brown A, Timms EM, Foxwell BM, Ray KP (2010) Inhibitors of p38 suppress cytokine production in rheumatoid arthritis synovial membranes: does variable inhibition of interleukin-6 production limit effectiveness in vivo? Arthritis Rheum 62:3221–3231CrossRefPubMedGoogle Scholar
  57. Reasoner DK, Hindman BJ, Dexter F, Subieta A, Cutkomp J, Smith T (1997) Doxycycline reduces early neurologic impairment after cerebral arterial air embolism in the rabbit. Anesthesiology 87:569–576CrossRefPubMedGoogle Scholar
  58. Roberts MC (2003) Tetracycline therapy: update. Clin Infect Dis 36:462–467CrossRefPubMedGoogle Scholar
  59. Roy A, Jana A, Yatish K, Freidt MB, Fung YK, Martinson JA, Pahan K (2008) Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: implications for neurodegenerative diseases. Free Radic Biol Med 45:686–699CrossRefPubMedPubMedCentralGoogle Scholar
  60. Russo I, Bubacco L, Greggio E (2014) LRRK2 and neuroinflammation: partners in crime in Parkinson’s disease? J Neuroinflamm. doi: 10.1186/1742-2094-11-52 Google Scholar
  61. Sanchez-Guajardo V, Barnum CJ, Tansey MG, Romero-Ramos M (2013) Neuroimmunological processes in Parkinson’s disease and their relation to α-synuclein: microglia as the referee between neuronal processes and peripheral immunity. ASN Neuro 5:113–139CrossRefPubMedGoogle Scholar
  62. Shi Q, Cheng L, Liu Z et al (2015) The p38 MAPK inhibitor SB203580 differentially modulates LPS-induced interleukin 6 expression in macrophages. Cent Eur J Immunol 40:276–282CrossRefPubMedPubMedCentralGoogle Scholar
  63. Singh V, Mitra S, Sharma AK, Gera R, Ghosh D (2014) Isolation and characterization of microglia from adult mouse brain: selected applications for ex vivo evaluation of immunotoxicological alterations following in vivo xenobiotic exposure. Chem Res Toxicol 27:895–903CrossRefPubMedGoogle Scholar
  64. Skaper SD, Giusti P, Facci L (2012) Microglia and mast cells: two tracks on the road to neuroinflammation. FASEB J 26:3103–3117CrossRefPubMedGoogle Scholar
  65. Skaper SD, Facci L, Giusti P (2014) Mast cells, glia and neuroinflammation: partners in crime? Immunology 141:314–327CrossRefPubMedPubMedCentralGoogle Scholar
  66. Smith K, Leyden JJ (2005) Safety of doxycycline and minocycline: a systematic review. Clin Ther 27:1329–1342CrossRefPubMedGoogle Scholar
  67. Smith JA, Das A, Ray SK, Banik NL (2012) Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull 87:10–20CrossRefPubMedGoogle Scholar
  68. Stolp HB, Dziegielewska KM (2009) Role of developmental inflammation and blood-brain barrier dysfunction in neurodevelopmental and neurodegenerative diseases. Neuropathol Appl Neurobiol 35:132–146CrossRefPubMedGoogle Scholar
  69. Tarpey MM, Wink DA, Grisham MB (2004) Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol 286:R431–R444CrossRefPubMedGoogle Scholar
  70. Teismann P, Tieu K, Cohen O, Choi DK, Wu DC, Marks D, Vila M, Jackson-Lewis V, Przedborski S (2003) Pathogenic role of glial cells in Parkinson’s disease. Mov Disord 18:121–129CrossRefPubMedGoogle Scholar
  71. Thomas M, Ashizawa T, Jankovic J (2004) Minocycline in Huntington’s disease: a pilot study. Mov Disord 19:692–695CrossRefPubMedGoogle Scholar
  72. Wang PQ, Sun SG, Qiao X (2009) Protective effects of doxycycline upon dopaminergic neuron in LPS-induced rat model of Parkinson’s disease. Zhonghua Yi Xue Za Zhi 89:1346–1350PubMedGoogle Scholar
  73. Wojtera M, Sikorska B, Sobow T, Liberski PP (2005) Microglial cells in neurodegenerative disorders. Folia Neuropathol 43:311–332PubMedGoogle Scholar
  74. Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 22:1763–1771PubMedGoogle Scholar
  75. Yim CW, Flynn NM, Fitzgerald FT (1985) Penetration of oral doxycycline into the cerebrospinal fluid of patients with latent or neurosyphilis. Antimicrob Agents Chemother 28:347–348CrossRefPubMedPubMedCentralGoogle Scholar
  76. Yrjänheikki J, Keinänen R, Pellikka M, Hökfelt T, Koistinaho J (1998) Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA 95:15769–15774CrossRefPubMedPubMedCentralGoogle Scholar
  77. Zecca L, Casella L, Albertini A, Bellei C, Zucca FA, Engelen M, Zadlo A, Szewczyk G, Zareba M, Sarna T (2008) Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson’s disease. J Neurochem. doi: 10.1111/j.1471-4159.2008.05541.x Google Scholar
  78. Zhang Y, Dawson VL, Dawson TM (2000) Oxidative stress and genetics in the pathogenesis of Parkinson’s disease. Neurobiol Dis 7:240–250CrossRefPubMedGoogle Scholar
  79. Zhang R, Zhao M, Ji HJ, Yuan YH, Chen NH (2013) Study on the dynamic changes in synaptic vesicle-associated protein and axonal transport protein combined with LPS neuroinflammation model. ISRN Neurol. doi: 10.1155/2013/496079 Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, UM75, U1127, UMR 7225, Institut du Cerveau et de la Moelle EpinièreParisFrance
  2. 2.Department of Pharmacology, Ribeirão Preto Medical SchoolUniversity of São Paulo (USP)Ribeirão PretoBrazil
  3. 3.Instituto Superior de Investigaciones Biológicas, INSIBIO (CONICET-UNT) and Instituto de Química Biológica “Dr Bernabé Bloj”, Facultad de Bioquímica, Química y Farmacia (UNT)San Miguel de TucumánArgentina
  4. 4.Department of Morphology, Physiology and Pathology, School of Odontology of Ribeirão Preto (FORP)University of São Paulo (USP)Ribeirão PretoBrazil

Personalised recommendations