Neurotoxicity Research

, Volume 32, Issue 1, pp 71–81 | Cite as

Complex Changes in the Innate and Adaptive Immunity Accompany Progressive Degeneration of the Nigrostriatal Pathway Induced by Intrastriatal Injection of 6-Hydroxydopamine in the Rat

  • Giulia Ambrosi
  • Natasa Kustrimovic
  • Francesca Siani
  • Emanuela Rasini
  • Silvia Cerri
  • Cristina Ghezzi
  • Giuseppe Dicorato
  • Sofia Caputo
  • Franca Marino
  • Marco Cosentino
  • Fabio Blandini


We investigated changes in innate and adaptive immunity paralleling the progressive nigrostriatal damage occurring in a neurotoxic model of Parkinson’s disease (PD) based on unilateral infusion of 6-hydroxydopamine (6-OHDA) into the rat striatum. A time-course analysis was conducted to assess changes in morphology (activation) and cell density of microglia and astrocytes, microglia polarization (M1 vs. M2 phenotype), lymphocyte infiltration in the lesioned substantia nigra pars compacta (SNc), and modifications of CD8+ and subsets of CD4+ T cell in peripheral blood accompanying nigrostriatal degeneration. Confirming previous results, we observed slightly different profiles of activation for astrocytes and microglia paralleling nigral neuronal loss. For astrocytes, morphological changes and cell density increases were mostly evident at the latest time points (14 and 28 days post-surgery), while moderate microglia activation was present since the earliest time point. For the first time, in this model, we described the time-dependent profile of microglia polarization. Activated microglia clearly expressed the M2 phenotype in the earlier phase of the experiment, before cell death became manifest, gradually shifting to the M1 phenotype as SNc cell death started. In parallel, a reduction in the percentage of circulating CD4+ T regulatory (Treg) cells, starting as early as day 3 post-6-OHDA injection, was detected in 6-OHDA-injected rats. Our data show that nigrostriatal degeneration is associated with complex changes in central and peripheral immunity. Microglia activation and polarization, Treg cells, and the factors involved in their cross-talk should be further investigated as targets for the development of therapeutic strategies for disease modification in PD.


Parkinson’s disease 6-OHDA Rat Microglia Astrocytes T regulatory cells 



This study was supported by a grant from Fondazione CARIPLO to Marco Cosentino and Fabio Blandini (Project 2011-0504: Dopaminergic modulation of CD4+ T lymphocytes: relevance for neurodegeneration and neuroprotection in Parkinson’s disease—the dopaminergic neuro-immune connection). Natasa Kustrimovic (postdoc fellow) and Cristina Ghezzi (lab technician) appointments were supported by the grant. We would like to acknowledge Dr. Marco Gnesi for performing the statistical analysis of data and correlations. The skillful technical assistance of Dr. Emanuela Rasini (Center for Research in Medical Pharmacology, University of Insubria) in the development and validation of flow cytometric assays as well as in data analysis is also gratefully acknowledged.

Author Contribution

FB, MC, and FM conceived and designed the study. GA, NK, FS, CG, SC, SC, GD and ER acquired data. GA, NK, FS, MC, and FB analyzed and interpreted data. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved and declare to have confidence in the integrity of the contributions of their co-authors.

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing financial interests.

Supplementary material

12640_2017_9712_Fig7_ESM.gif (501 kb)
Supplementary Fig. 1

Representative image of microglia polarization at different time points (24 h, 7 and 14 days post-6-OHDA injection) in the lesioned SNc of a 6-OHDA-treated rat. Blue signal: DAPI (nuclei); green signal: CD11b+ cells; red signal: CD32+/CD206+ cells, respectively, M1 and M2 phenotype. (GIF 501 kb)

12640_2017_9712_MOESM1_ESM.tif (13.3 mb)
High resolution (TIFF 13570 kb)
12640_2017_9712_MOESM2_ESM.docx (16 kb)
ESM 2 (DOCX 15 kb)


  1. Ambrosi G, Armentero MT, Levandis G, Bramanti P, Nappi GBF (2010) Effects of early and delayed treatment with an mGluR5 antagonist on motor impairment, nigrostriatal damage and neuroinflammation in a rodent model of Parkinson’s disease. Brain Res Bull 82:29–38. doi: 10.1016/j.brainresbull.2010.01.011 CrossRefPubMedGoogle Scholar
  2. Ambrosi G, Ghezzi C, Sepe S, Milanese C, Payan-Gomez C, Bombardieri CR, Armentero MT, Zangaglia R, Pacchetti C, Mastroberardino PGBF (2014) Bioenergetic and proteolytic defects in fibroblasts from patients with sporadic Parkinson’s disease. Biochim Biophys Acta 1842:1385–1394. doi: 10.1016/j.bbadis.2014.05.008 CrossRefPubMedGoogle Scholar
  3. Appel SH (2009) CD4+ T cells mediate cytotoxicity in neurodegenerative diseases. J Clin Invest 119:13–15. doi: 10.1172/JCI38096 PubMedGoogle Scholar
  4. Armentero MT, Levandis G, Nappi G, Bazzini EBF (2006) Peripheral inflammation and neuroprotection: systemic pretreatment with complete Freund’s adjuvant reduces 6-hydroxydopamine toxicity in a rodent model of Parkinson's disease. Neurobiol Dis 24:492–505. doi: 10.1016/j.nbd.2006.08.016 CrossRefPubMedGoogle Scholar
  5. Armentero MT, Levandis G, Bazzini E, Cerri S, Ghezzi CBF (2011) Adhesion molecules as potential targets for neuroprotection in a rodent model of Parkinson’s disease. Neurobiol Dis 43:663–668. doi: 10.1016/j.nbd.2011.05.017 CrossRefPubMedGoogle Scholar
  6. Baba Y, Kuroiwa A, Uitti RJ, Wszolek ZKYT (2005) Alterations of T-lymphocyte populations in Parkinson disease. Park Relat Disord 11:493–498. doi: 10.1016/j.parkreldis.2005.07.005 CrossRefGoogle Scholar
  7. Barcia C, Ros CM, Annese V et al (2011) IFN-γ signaling, with the synergistic contribution of TNF-α, mediates cell specific microglial and astroglial activation in experimental models of Parkinson’s disease. Cell Death Dis 2:e142. doi: 10.1038/cddis.2011.17 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Blandini F (2013) Neural and immune mechanisms in the pathogenesis of Parkinson’s disease. J NeuroImmune Pharmacol 8:189–201. doi: 10.1007/s11481-013-9435-y CrossRefPubMedGoogle Scholar
  9. Blandini F, Levandis G, Bazzini E et al (2007) Time-course of nigrostriatal damage, basal ganglia metabolic changes and behavioural alterations following intrastriatal injection of 6-hydroxydopamine in the rat: new clues from an old model. Eur J Neurosci 25:397–405. doi: 10.1111/j.1460-9568.2006.05285.x CrossRefPubMedGoogle Scholar
  10. Brochard V, Combadière B, Prigent A et al (2009) Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J Clin Invest 119:182–192. doi: 10.1172/JCI36470 PubMedGoogle Scholar
  11. Bruchelt G, Grygar G, Treuner J, Esterbauer H, Niethammer D (1989) Cytotoxic effects of 6-hydroxydopamine, merocyanine-540 and related compounds on human neuroblastoma and hematopoietic stem cells. Free Radic Res Commun 7:205–212CrossRefPubMedGoogle Scholar
  12. Carvey PM, Zhao CH, Hendey B et al (2005) 6-Hydroxydopamine-induced alterations in blood-brain barrier permeability. Eur J Neurosci 22:1158–1168. doi: 10.1111/j.1460-9568.2005.04281.x CrossRefPubMedGoogle Scholar
  13. Chaturvedi RK, Beal M (2013) Mitochondria targeted therapeutic approaches in Parkinson’s and Huntington's diseases. Mol Cell Neurosci 55:101–114. doi: 10.1016/j.mcn.2012.11.011 CrossRefPubMedGoogle Scholar
  14. Chaudhuri KR, Odin P, Antonini A-MP (2011) Parkinson’s disease: the non-motor issues. Park Relat Disord 17:717–723CrossRefGoogle Scholar
  15. Colburn RW, DeLeo JA, Rickman AJ, Yeager MP, Kwon PHW (1997) Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J Neuroimmunol 79:163–175CrossRefPubMedGoogle Scholar
  16. Doorn KJ, Lucassen PJ, Boddeke HW, Prins M, Berendse HW, Drukarch B, van Dam AM (2012) Emerging roles of microglial activation and non-motor symptoms in Parkinson’s disease. Prog Neurobiol 98:222–238. doi: 10.1016/j.pneurobio.2012.06.005 CrossRefPubMedGoogle Scholar
  17. Double KL, Reyes S, Werry EL HG (2010) Selective cell death in neurodegeneration: why are some neurons spared in vulnerable regions? Progr Neurobiol 92:316–329. doi: 10.1016/j.pneurobio.2010.06.001 CrossRefGoogle Scholar
  18. Espinosa-Oliva AM, de Pablos RM, Sarmiento M, Villarán RF, Carrillo-Jiménez A, Santiago M, Venero JL, Herrera AJ, Cano JMA (2014) Role of dopamine in the recruitment of immune cells to the nigro-striatal dopaminergic structures. Neurotoxicology 41:89–101. doi: 10.1016/j.neuro.2014.01.006 CrossRefPubMedGoogle Scholar
  19. González H, Pacheco R (2014) T-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases. J Neuroinflammation 11:201. doi: 10.1186/s12974-014-0201-8 CrossRefPubMedPubMedCentralGoogle Scholar
  20. González-Hernández T, Cruz-Muros I, Afonso-Oramas D et al (2010) Vulnerability of mesostriatal dopaminergic neurons in Parkinson’s disease. Front Neuroanat 4:140. doi: 10.3389/fnana.2010.00140 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Greenamyre JT, Hastings TG (2004) Biomedicine. Parkinson’s—divergent causes, convergent mechanisms. Science 304:1120–1122. doi: 10.1126/science.1098966 CrossRefPubMedGoogle Scholar
  22. Hauser DN, Hastings TG (2013) Mitochondrial dysfunction and oxidative stress in Parkinson’s disease and monogenic parkinsonism. Neurobiol Dis 51:35–42. doi: 10.1016/j.nbd.2012.10.011 CrossRefPubMedGoogle Scholar
  23. He F, Balling R (2013) The role of regulatory T cells in neurodegenerative diseases. Wiley Interdiscip Rev Syst Biol Med 5:153–180. doi: 10.1002/wsbm.1187 CrossRefPubMedGoogle Scholar
  24. Herrero M-T, Estrada C, Maatouk L, Vyas S (2015) Inflammation in Parkinson’s disease: role of glucocorticoids. Front Neuroanat 9:32. doi: 10.3389/fnana.2015.00032 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hirsch EC, Hunot S (2009) Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol 8:382–397CrossRefPubMedGoogle Scholar
  26. Hirsch EC, Vyas SHS (2012) Neuroinflammation in Parkinson’s disease. Park Relat Disord 18:S210–S212. doi: 10.1016/S1353-8020(11)70065-7 CrossRefGoogle Scholar
  27. Hu X, Li P, Guo Y et al (2012) Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke 43:3063–3070. doi: 10.1161/STROKEAHA.112.659656 CrossRefPubMedGoogle Scholar
  28. Kannarkat GT, Boss JM, Tansey MG (2013) The role of innate and adaptive immunity in Parkinson’s disease. J Parkinsons Dis 3:493–514. doi: 10.3233/JPD-130250 PubMedPubMedCentralGoogle Scholar
  29. Kipnis J, Cardon M, Avidan H et al (2004) Dopamine, through the extracellular signal-regulated kinase pathway, downregulates CD4+CD25+ regulatory T-cell activity: implications for neurodegeneration. J Neurosci 24:6133–6143. doi: 10.1523/JNEUROSCI.0600-04.2004 CrossRefPubMedGoogle Scholar
  30. Kitamura Y, Inden M, Minamino H, Abe M, Takata K, Taniguchi T (2010) The 6-hydroxydopamine-induced nigrostriatal neurodegeneration produces microglia-like NG2 glial cells in the rat substantia nigra. Glia 58(14):1686–1700. doi: 10.1002/glia.21040 CrossRefPubMedGoogle Scholar
  31. Kustrimovic N, Rasini E, Legnaro M et al (2014) Expression of dopaminergic receptors on human CD4+ T lymphocytes: flow cytometric analysis of naive and memory subsets and relevance for the neuroimmunology of neurodegenerative disease. J NeuroImmune Pharmacol 9:302–312. doi: 10.1007/s11481-014-9541-5 CrossRefPubMedGoogle Scholar
  32. Kustrimovic N, Rasini E, Legnaro M et al (2016) Dopaminergic receptors on CD4+ T naive and memory lymphocytes correlate with motor impairment in patients with Parkinson's disease. Sci Rep 6:33738. doi: 10.1038/srep33738 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Levite M (2016) Dopamine and T cells: dopamine receptors and potent effects on T cells, dopamine production in T cells, and abnormalities in the dopaminergic system in T cells in autoimmune, neurological and psychiatric diseases. Acta Physiol (Oxf) 216:42–89. doi: 10.1111/apha.12476 CrossRefGoogle Scholar
  34. Lowther DE, Hafler DA (2012) Regulatory T cells in the central nervous system. Immunol Rev 248:156–169. doi: 10.1111/j.1600-065X.2012.01130.x CrossRefPubMedGoogle Scholar
  35. Lucin KM, Wyss-Coray T (2009) Immune activation in brain aging and neurodegeneration: too much or too little? Neuron 64:110–122. doi: 10.1016/j.neuron.2009.08.039 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lull ME, Block ML (2010) Microglial activation and chronic neurodegeneration. Neurotherapeutics 7:354–365Google Scholar
  37. Mak SK, McCormack AL, Manning-Bog AB et al (2010) Lysosomal degradation of alpha-synuclein in vivo. J Biol Chem 285:13621–13629. doi: 10.1074/jbc.M109.074617 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Martinez-Pasamar S, Abad E, Moreno B et al (2013) Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity. BMC Syst Biol 7:34. doi: 10.1186/1752-0509-7-34 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Massano J, Bhatia KP (2012) Clinical approach to Parkinson’s disease: features, diagnosis, and principles of management. Cold Spring Harb Perspect Med 2:a008870. doi: 10.1101/cshperspect.a008870 CrossRefPubMedPubMedCentralGoogle Scholar
  40. McGeer PL, McGeer EG (2008) Glial reactions in Parkinson’s disease. Mov Disord 23:474–483. doi: 10.1002/mds.21751 CrossRefPubMedGoogle Scholar
  41. McNaught KSP, Jackson T, JnoBaptiste R et al (2006) Proteasomal dysfunction in sporadic Parkinson’s disease. Neurology 66:S37–S49CrossRefPubMedGoogle Scholar
  42. Miklossy J, Doudet DD, Schwab C, Yu S, McGeer EGMP (2006) Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys. Exp Neurol 192:275–283. doi: 10.1016/j.expneurol.2005.10.034 CrossRefGoogle Scholar
  43. Miller KR, Streit WJ (2007) The effects of aging, injury and disease on microglial function: a case for cellular senescence. Neuron Glia Biol 3:245–253. doi: 10.1017/S1740925X08000136 CrossRefPubMedGoogle Scholar
  44. Perego C, Fumagalli S, De Simoni M-G (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8:174. doi: 10.1186/1742-2094-8-174 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Ramesh G, MacLean AG, Philipp MT (2013) Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain. Mediat Inflamm 2013:480739. doi: 10.1155/2013/480739 Google Scholar
  46. Reynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL (2007) Neuroprotective activities of CD4þCD25þ regulatory T cells in an animal model of Parkinson’s disease. J Leuk Biol 82:1083–1094CrossRefGoogle Scholar
  47. Reynolds AD, Stone DK, Mosley RL, Gendelman HE (2009) Proteomic studies of nitrated alpha-synuclein microglia regulation by CD4+CD25+ T cells. J Proteome Res 8:3497–3511. doi: 10.1021/pr9001614 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Reynolds AD, Stone DK, Hutter JAL et al (2010) Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson’s disease. J Immunol 184:2261–2271. doi: 10.4049/jimmunol.0901852 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Shechter R, London A, Schwartz M (2013) Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol 13:206–218. doi: 10.1038/nri3391 CrossRefPubMedGoogle Scholar
  50. Spillantini MG, Schmidt ML, Lee VM et al (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840. doi: 10.1038/42166 CrossRefPubMedGoogle Scholar
  51. Stephens LA, Malpass KH, Anderton SM (2009) Curing CNS autoimmune disease with myelin-reactive Foxp3+ Treg. Eur J Immunol 39:1108–1117. doi: 10.1002/eji.200839073 CrossRefPubMedGoogle Scholar
  52. Su X, Federoff HJ (2014) Immune responses in Parkinson’s disease: interplay between central and peripheral immune systems. Biomed Res Int 2014:275178. doi: 10.1155/2014/275178 PubMedPubMedCentralGoogle Scholar
  53. Theodore S, Maragos W (2015) 6-Hydroxydopamine as a tool to understand adaptive immune system-induced dopamine neurodegeneration in Parkinson’s disease. Immunopharmacol Immunotoxicol 37:393–399. doi: 10.3109/08923973.2015.1070172 CrossRefPubMedGoogle Scholar
  54. Vairetti M, Ferrigno A, Rizzo V, Ambrosi G, Bianchi A, Richelmi P, Blandini F, Armentero MT (2012) Impaired hepatic function and central dopaminergic denervation in a rodent model of Parkinson's disease: a self-perpetuating crosstalk? Biochim Biophys Acta 1822:176–184CrossRefPubMedGoogle Scholar
  55. Wheeler CJ, Seksenyan A, Koronyo Y et al (2014) T-lymphocyte deficiency exacerbates behavioral deficits in the 6-OHDA unilateral lesion rat model for Parkinson’s disease. J Neurol Neurophysiol. doi: 10.4172/2155-9562.1000209 PubMedPubMedCentralGoogle Scholar
  56. Xie L, Choudhury GR, Winters A et al (2015) Cerebral regulatory T cells restrain microglia/macrophage-mediated inflammatory responses via IL-10. Eur J Immunol 45:180–191. doi: 10.1002/eji.201444823 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Giulia Ambrosi
    • 1
  • Natasa Kustrimovic
    • 2
  • Francesca Siani
    • 1
  • Emanuela Rasini
    • 2
  • Silvia Cerri
    • 1
  • Cristina Ghezzi
    • 1
  • Giuseppe Dicorato
    • 1
  • Sofia Caputo
    • 1
  • Franca Marino
    • 2
  • Marco Cosentino
    • 2
  • Fabio Blandini
    • 1
  1. 1.Laboratory of Functional Neurochemistry, Center for Research in Neurodegenerative DiseasesC. Mondino National Neurological InstitutePaviaItaly
  2. 2.Center of Research in Medical PharmacologyUniversity of InsubriaVareseItaly

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