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Cellular and Molecular Neurobiology

, Volume 35, Issue 7, pp 961–975 | Cite as

Microglia in Glia–Neuron Co-cultures Exhibit Robust Phagocytic Activity Without Concomitant Inflammation or Cytotoxicity

  • Alexandra C. Adams
  • Michele Kyle
  • Carol M. Beaman-Hall
  • Edward A. MonacoIII
  • Matthew Cullen
  • Mary Lou VallanoEmail author
Original Research

Abstract

A simple method to co-culture granule neurons and glia from a single brain region is described, and microglia activation profiles are assessed in response to naturally occurring neuronal apoptosis, excitotoxin-induced neuronal death, and lipopolysaccharide (LPS) addition. Using neonatal rat cerebellar cortex as a tissue source, glial proliferation is regulated by omission or addition of the mitotic inhibitor cytosine arabinoside (AraC). After 7–8 days in vitro, microglia in AraC cultures are abundant and activated based on their amoeboid morphology, expressions of ED1 and Iba1, and ability to phagocytose polystyrene beads and the majority of neurons undergoing spontaneous apoptosis. Microglia and phagocytic activities are sparse in AraC+ cultures. Following exposure to excitotoxic kainate concentrations, microglia in AraC cultures phagocytose most dead neurons within 24 h without exacerbating neuronal loss or mounting a strong or sustained inflammatory response. LPS addition induces a robust inflammatory response, based on microglial expressions of TNF-α, COX-2 and iNOS proteins, and mRNAs, whereas these markers are essentially undetectable in control cultures. Thus, the functional effector state of microglia is primed for phagocytosis but not inflammation or cytotoxicity even after kainate exposure that triggers death in the majority of neurons. This model should prove useful in studying the progressive activation states of microglia and factors that promote their conversion to inflammatory and cytotoxic phenotypes.

Keywords

Microglia Co-cultures Phagocytosis Excitotoxicity Inflammation 

Notes

Conflict of interest

The authors, Alexandra C. Adams, Michele Kyle, Carol M. Beaman-Hall, Edward A. Monaco III, Matthew Cullen, Mary Lou Vallano, do not have any conflicts of interest to report.

References

  1. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15:961–973CrossRefPubMedGoogle Scholar
  2. Araki E, Forster C, Dubinsky JM, Ross ME, Iadecola C (2001) Cyclooxygenase-2 inhibitor ns-398 protects neuronal cultures from lipopolysaccharide-induced neurotoxicity. Stroke 32:2370–2375CrossRefPubMedGoogle Scholar
  3. Bachstetter AD, Rowe RK, Kaneko M, Goulding D, Lifshitz J, Van Eldik LJ (2013) The p38α MAPK regulates microglial responsiveness to diffuse traumatic brain injury. J Neurosci 33:6143–6153. doi: 10.1523/JNEUROSCI.5399-12.2013 PubMedCentralCrossRefPubMedGoogle Scholar
  4. Bal-Price A, Brown GC (2001) Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci 21:6480–6491PubMedGoogle Scholar
  5. Beaman-Hall CM, Leahy JC, Benmansour S, Vallano ML (1998) Glia modulate NMDA-mediated signaling in primary cultures of cerebellar granule cells. J Neurochem 71:1993–2005CrossRefPubMedGoogle Scholar
  6. Bechmann I, Nitsch R (1997) Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique. Glia 20:145–154CrossRefPubMedGoogle Scholar
  7. Blaylock RL (2013) Immunoexcitatory mechanisms in glioma proliferation, invasion and occasional metastasis. Surg Neurol Int 4:15–24. doi: 10.4103/2152-7806.106577 PubMedCentralCrossRefPubMedGoogle Scholar
  8. Bocchini V, Rebel G, Massarelli R, Schuber F, Muller CD (1988a) Latex beads phagocytosis capacity and ecto-nad glycohydrolase activity of rat brain microglia cells in vitro. Int J Dev Neurosci 6:525–534CrossRefPubMedGoogle Scholar
  9. Bocchini V, Artault JC, Rebel G, Dreyfus H, Massarelli R (1988b) Phagocytosis of polystyrene latex beads by rat brain microglia cell cultures is increased by treatment with gangliosides. Dev Neurosci 10:270–276CrossRefPubMedGoogle Scholar
  10. Cebers G, Zhivotovsky B, Ankarcrona M, Liljequist S (1997) AMPA neurotoxicity in cultured cerebellar granule neurons: mode of cell death. Brain Res Bull 43:393–403CrossRefPubMedGoogle Scholar
  11. Chen Y, Won SJ, Xu Y, Swanson RA (2014) Targeting microglial activation in stroke therapy: pharmacological tools and gender effects. Curr Med Chem 21:2146–2155PubMedCentralCrossRefPubMedGoogle Scholar
  12. Cho IH, Hong J, Suh EC, Kim JH, Lee H, Lee JE, Lee S, Kim CH, Kim DW, Jo EK, Lee KE, Karin M, Lee SJ (2008) Role of microglial IKKbeta in kainic acid-induced hippocampal neuronal cell death. Brain 131(Pt 11):3019–3033. doi: 10.1093/brain/awn230 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Choi SH, Joe EH, Kim SU, Jin BK (2003) Thrombin-induced microglia activation produces degeneration of nigral dopaminergic neurons in vivo. J Neurosci 23:5877–5886PubMedGoogle Scholar
  14. Christensen RN, Ha BK, Sun F, Bresnahan JC, Beattie MS (2006) Kainate induces redistribution of the actin cytoskeleton in ameboid microglia. J Neurosci Res 84:170–181CrossRefPubMedGoogle Scholar
  15. Claycomb KI, Winokur PN, Johnson KM, Nicaise AM, Giampetruzzi AW, Sacino AV, Snyder EY, Barbarese E, Bongarzone ER, Crocker SJ (2014) Aberrant production of tenascin-C in globoid cell leukodystrophy alters psychosine-induced microglial functions. J Neuropathol Exp Neurol 73:964–974CrossRefPubMedGoogle Scholar
  16. Dambach H, Hinkerohe D, Prochnow N, Stienen MN, Moinfar Z, Haase CG, Hufnagel A, Faustmann PM (2014) Glia and epilepsy: experimental investigation of antiepileptic drugs in an astroglia/microglia co-culture model of inflammation. Epilepsia 55:184–192CrossRefPubMedGoogle Scholar
  17. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson CC, Dijkstra CD (1994) Rat macrophage lysosomal membrane antigen recognize by monoclonal antibody ED1. Immunology 83:140–147PubMedCentralPubMedGoogle Scholar
  18. de Haas AH, Boddeke HW, Biber K (2008) Region-specific expression of immunoregulatory proteins on microglia in the healthy CNS. Glia 56:888–894. doi: 10.1002/glia.20663 CrossRefPubMedGoogle Scholar
  19. Dessi F, Pollard H, Moreau J, Ben-Ari Y, Charriaut-Marlangue C (1995) Cytosine arabinoside induces apoptosis in cerebellar neurons in culture. J Neurochem 64:1980–1987CrossRefPubMedGoogle Scholar
  20. Dinkins MB, Dasgupta S, Wang G, Zhu G, Bieberich E (2014) Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer’s disease. Neurobiol Aging 35:1792–1800PubMedCentralCrossRefPubMedGoogle Scholar
  21. Duan L, Chen BY, Sun XL, Luo ZJ, Rao ZR, Wang JJ, Chen LW (2013) LPS-induced proNGF synthesis and release in the N9 and BV2 microglial cells: a new pathway underling microglia toxicity in neuroinflammation. PLoS One 8:e73768. doi: 10.1371/journal.pone.0073768 PubMedCentralCrossRefPubMedGoogle Scholar
  22. Elkabes S, DiCicco-Bloom EM, Black IB (1996) Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci 16:2508–2521PubMedGoogle Scholar
  23. Faustmann PM, Haase CG, Romberg S, Hinkerohe D, Szlachta D, Smikalla D, Krause D, Dermietzel R (2003) Microglia activation influences dye coupling and Cx43 expression of the astrocytic network. Glia 42:101–108CrossRefPubMedGoogle Scholar
  24. Favaron M, Manev H, Alho H, Bertolino M, Ferret B, Guidotti A, Costa E (1988) Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex. Proc Natl Acad Sci USA 85:7351–7355PubMedCentralCrossRefPubMedGoogle Scholar
  25. Gallo V, Kingsbury A, Balazs R, Jorgensen OS (1987) The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J Neurosci 7:2203–2213PubMedGoogle Scholar
  26. Gerber AM, Beaman-Hall CM, Mathur A, Vallano ML (2010) Reduced blockade by extracellular Mg(2+) is permissive to NMDA receptor activation in cerebellar granule neurons that model a migratory phenotype. J Neurochem 114:191–202. doi: 10.1111/j.1471-4159.2010.06746.x PubMedGoogle Scholar
  27. Giardina SF, Beart PM (2001) Excitotoxic profiles of novel, low-affinity kainate receptor agonists in primary cultures of murine cerebellar granule cells. Neuropharmacology 41:421–432CrossRefPubMedGoogle Scholar
  28. Gregory CD, Devitt A (2004) The Macrophage and the apoptotic cell: an innate immune interaction viewed simplistically? Immunology 113:1–14PubMedCentralCrossRefPubMedGoogle Scholar
  29. Griffiths MR, Gasque P, Neal JW (2009) The multiple roles of the innate immune system in the regulation of apoptosis and inflammation in the brain. J Neuropathol Exp Neurol 68:217–226. doi: 10.1097/NEN.0b013e3181996688 CrossRefPubMedGoogle Scholar
  30. Hewlett LJ, Prescott AR, Watts C (1994) The coated pit and macropinocytic pathways serve distinct endosome populations. J Cell Biol 124:689–703CrossRefPubMedGoogle Scholar
  31. Hong J, Cho IH, Kwak KI, Suh EC, Seo J, Min HJ, Choi SY, Kim CH, Park SH, Jo EK, Lee S, Lee KE, Lee SJ (2010) Microglia toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death. J Biol Chem 285:39447–39457. doi: 10.1074/jbc.M110.132522 PubMedCentralCrossRefPubMedGoogle Scholar
  32. Huang LQ, Zhu GF, Deng YY, Jiang WQ, Fang M, Chen CB, Cao W, Wen MY, Han YL, Zeng HK (2014) Hypertonic saline alleviates cerebral edema by inhibiting microglia-derived TNF-α and IL-1β-induced Na-K-Cl cotransporter up-regulation. J Neuroinflammation 11:102. doi: 10.1186/1742-2094-11-102 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Ito U, Tanaka K, Suzuki S, Dembo T, Fukuuchi Y (2001) Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32:1208–1215CrossRefPubMedGoogle Scholar
  34. Ito U, Nagasao J, Kawakami E, Oyanagi K (2007) Fate of disseminated dead neurons in the cortical ischemic penumbra: ultrastructure indicating a novel scavenger mechanism of microglia and astrocytes. Stroke 38:2577–2583CrossRefPubMedGoogle Scholar
  35. Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem 33:77–79CrossRefPubMedGoogle Scholar
  36. Kabadi SV, Stoica BA, Loane DJ, Luo T, Faden AI (2014) CR8, a novel inhibitor of CDK, limits microglial activation, astrocytosis, neuronal loss, and neurologic dysfunction after experimental traumatic brain injury. J Cereb Blood Flow Metab 34:502–513. doi: 10.1038/jcbfm.2013.228 PubMedCentralCrossRefPubMedGoogle Scholar
  37. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91:461–553. doi: 10.1152/physrev.00011.2010 CrossRefPubMedGoogle Scholar
  38. 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
  39. Kingsbury AE, Gallo V, Woodhams PL, Balazs R (1985) Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium. Brain Res 349:17–25CrossRefPubMedGoogle Scholar
  40. Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kosaka S, Inoue K (2007) UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature 446:1091–1095PubMedCentralCrossRefPubMedGoogle Scholar
  41. Kolodny JM, Leonard JL, Larsen PR, Silva JE (1985) Studies of nuclear 3,5,3′-triiodothyronine binding in primary cultures of rat brain. Endocrinology 117:1848–1857CrossRefPubMedGoogle Scholar
  42. Koval M, Preiter K, Adles C, Stahl PD, Steinberg TH (1998) Size of IgG-opsonized particles determines macrophage response during internalization. Exp Cell Res 242:265–273CrossRefPubMedGoogle Scholar
  43. 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
  44. Leahy JC, Chen Q, Vallano ML (1994) Chronic mild acidosis specifically reduces functional expression of N-methyl-d-aspartate receptors and increases long-term survival in primary cultures of cerebellar granule cells. Neuroscience 63:457–470CrossRefPubMedGoogle Scholar
  45. Lee H, Kim YO, Kim H, Kim SY, Noh HS, Kang SS, Cho GJ, Choi WS, Suk K (2003) Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia. FASEB J 17:1943–1954PubMedGoogle Scholar
  46. Martin DP, Wallace TL, Johnson EM Jr (1990) Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction. J Neurosci 10:184–193PubMedGoogle Scholar
  47. Min KJ, Yang MS, Kim SU, Jou I, Joe EH (2006) Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci 26:1880–1887CrossRefPubMedGoogle Scholar
  48. Neher JJ, Neniskyte U, Zhao JW, Bal-Price A, Tolkovsky AM, Brown GC (2011) Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 186:4973–4983. doi: 10.4049/jimmunol.1003600 CrossRefPubMedGoogle Scholar
  49. Neher JJ, Emmrich JV, Fricker MPK, Thery C, Brown GC (2013) Phagocytosis executes delayed neuronal death after focal brain ischemia. Proc Natl Acad Sci USA 110:E4098–E4107. doi: 10.1073/pnas.1308679110 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglia cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318CrossRefPubMedGoogle Scholar
  51. Ousman SS, Kubes P (2012) Immune surveillance in the central nervous system. Nat Neurosci 15:1096–1101CrossRefPubMedGoogle Scholar
  52. Paludan SR (2000) Synergistic action of pro-inflammatory agents: cellular and molecular aspects. J Leukoc Biol 67:18–25PubMedGoogle Scholar
  53. Perry VH, Holmes C (2014) Microglial priming in neurodegenerative disease. Nat Rev Neurol 10:217–224. doi: 10.1038/nrneurol.2014.38 CrossRefPubMedGoogle Scholar
  54. Pratten MK, Lloyd JB (1986) Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro. Biochim Biophys Acta 881:307–313CrossRefPubMedGoogle Scholar
  55. Raivich G, Jones LL, Werner A, Bluthmann H, Doetschmann T, Kreutzberg GW (1999) Molecular signals for glial activation: pro- and anti-inflammatory cytokines in the injured brain. Acta Neurochir Suppl 73:21–30PubMedGoogle Scholar
  56. Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 27:119–145. doi: 10.1146/annurev.immunol.021908.132528 CrossRefPubMedGoogle Scholar
  57. Ren L, Lubrich B, Biber K, Gebicke-Haerter PJ (1999) Differential expression of inflammatory mediators in rat microglia cultured from different brain regions. Brain Res 65:198–205CrossRefGoogle Scholar
  58. Rosenstiel P, Lucius R, Deuschi G, Sievers J, Wilms H (2001) From theory to therapy: implications from an in vitro model of ramified microglia. Microsc Res Tech 54:18–25CrossRefPubMedGoogle Scholar
  59. Salter MW, Beggs S (2014) Sublime microglia: expanding roles for the guardians of the CNS. Cell 158:15–24. doi: 10.1016/j.cell.2014.06.008 CrossRefPubMedGoogle Scholar
  60. Schramm M, Eimerl S, Costa E (1990) Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of the glutamate receptor responsive to N-methyl-d-aspartate. Proc Natl Acad Sci USA 87:1193–1197PubMedCentralCrossRefPubMedGoogle Scholar
  61. Sloka S, Metz LM, Hader W, Starreveld Y, Yong VW (2013) Reduction of microglial activity in a model of multiple sclerosis by dipyridamole. J Neuroinflammation 10:89. doi: 10.1186/1742-2094-10-89 PubMedCentralCrossRefPubMedGoogle Scholar
  62. Streit WJ, Xue Q-S, Tischer J, Bechmann I (2014) Microglia pathology. Acta Neuropathol Commun 2:142–158PubMedCentralCrossRefPubMedGoogle Scholar
  63. Sudo S, Tanaka J, Toku K, Desaki J, Matsuda S, Arai T, Sakanaka M, Maeda N (1998) Neurons induce the activation of microglia cells in vitro. Exp Neurol 154:499–510CrossRefPubMedGoogle Scholar
  64. Thangnipon W, Kingsbury A, Webb M, Balazs R (1983) Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurons and astrocytes. Brain Res 313:177–189CrossRefPubMedGoogle Scholar
  65. Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J (2001) Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21:2580–2588PubMedGoogle Scholar
  66. Vallano ML, Lambolez B, Audinat E, Rossier J (1996) Neuronal activity differentially regulates NMDA receptor subunit expression in cerebellar granule cells. J Neurosci 15:631–639Google Scholar
  67. van Eldik LJ, Thompson WL, Ralay Ranaivo H, Behanna HA, Martin Watterson D (2007) Glia proinflammatory cytokine upregulation as a therapeutic target for neurodegenerative diseases: function-based and target-based discovery approaches. Int Rev Neurobiol 82:277–296CrossRefPubMedGoogle Scholar
  68. Voll RE, Herrmann M, Roth EA, Stach C, Kalden JR, Girkontaite I (1997) Immunosuppressive effects of apoptotic cells. Nature 390:350–351CrossRefPubMedGoogle Scholar
  69. Von Bernhardi R, Ramirez G, Toro R, Eugenin J (2007) Pro-inflammatory conditions promote neuronal damage mediated by amyloid precursor protein and decrease its phagocytosis and degradation by microglial cells in culture. Neurbiol Dis 26:153–164CrossRefGoogle Scholar
  70. Xie L, Sun F, Wang J, Mao X, Xie L, Yang SH, Su DM, Simpkins JW, Greenberg DA, Jin K (2014) mTOR signaling inhibition modulates macrophage/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia. J Immunol 192:6009–6019. doi: 10.4049/jimmunol.1303492 PubMedCentralCrossRefPubMedGoogle Scholar
  71. Xing B, Bachstetter AD, Van Eldik LJ (2015) Inhibition of neuronal p38α, but not p38β MAPK, provides neuroprotection against three different neurotoxic insults. J Mol Neurosci 55:509–518. doi: 10.1007/s12031-014-0372-x PubMedCentralCrossRefPubMedGoogle Scholar
  72. Yang MS, Min KJ, Joe E (2007) Multiple mechanisms that prevent excessive brain inflammation. J Neuro Res 85:2298–2305. doi: 10.1002/jnr.21254 CrossRefGoogle Scholar
  73. Zheng H, Zhu W, Zhao H, Wang X, Wang W, Li Z (2010) Kainic acid-activated microglia mediate increased excitability of rat hippocampal neurons in vitro and in vivo: crucial role of interleukin-1beta. Neuroimmunomodulation 17:1–8. doi: 10.1159/000243083 CrossRefGoogle Scholar
  74. Zhu W, Zheng H, Shao X, Wang W, Yao Q, Li Z (2010) Excitotoxicity of TNFalpha derived from KA activated microglia on hippocampal neurons in vitro and in vivo. J Neurochem 114:386–396. doi: 10.1111/j.1471-4159.2010.06763.x CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Alexandra C. Adams
    • 1
    • 3
  • Michele Kyle
    • 2
  • Carol M. Beaman-Hall
    • 1
  • Edward A. MonacoIII
    • 1
    • 4
  • Matthew Cullen
    • 1
    • 5
  • Mary Lou Vallano
    • 1
    Email author
  1. 1.Department of Neuroscience & PhysiologySUNY Upstate Medical UniversitySyracuseUSA
  2. 2.Department of NeurosurgerySUNY Upstate Medical UniversitySyracuseUSA
  3. 3.Department of Pulmonary and Critical CareMount Sinai Beth Israel Medical CenterNew YorkUSA
  4. 4.Department of Neurological SurgeryUniversity of Pittsburgh Medical CenterPittsburghUSA
  5. 5.Department of AnesthesiologyPhelps Memorial Hospital CenterSleepy HollowUSA

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