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Aggregating Brain Cell Cultures for Neurotoxicological Studies

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In Vitro Neurotoxicology

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Because of the limited accessibility of the brain for experimentation, but also for ethical and economical reasons, there is considerable interest in culture models suitable for neurotoxicological research. Although it is generally accepted that in vitro models cannot cover the entire spectrum of brain functions, they have proven to be indispensable for investigations in the life sciences since the early work of Harrison (1). To date, many in vitro models of various complexity are available, ranging from monolayer cultures of immortalized cell lines to organotypic cultures. Each of these culture systems has its particularities, therefore, it is of great importance to select the model that is most appropriate for the question to be solved.

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References

  1. Harrison, R.G. (1907) Observations on the living developing nerve fiber. Anat. Rec. 1, 116–118.

    Article  Google Scholar 

  2. Moscona, A. A. (1960) Patterns and mechanisms of tissue reconstruction from dissociated cells, in Developing Cell Systems and their Control (Rudnick, D., ed.), Ronald, New York, pp. 45–70.

    Google Scholar 

  3. Honegger, P., Lenoir, D., and Favrod, P. (1979) Growth and differentiation of aggregating fetal brain cells in a serum-free defined medium. Nature 282, 305–308.

    Article  PubMed  CAS  Google Scholar 

  4. Honegger, P. and Monnet-Tschudi, F. (1997) Aggregating neural cell cultures, in Protocols for Neural Cell Culture (Fedoroff, S. and Richardson, A., eds.), Humana, Totowa, NJ, pp. 25–49.

    Google Scholar 

  5. Honegger, P. and Werffeli, P. (1988) Use of aggregating cell cultures for toxicological studies. Experientia 44, 817–822.

    Article  PubMed  CAS  Google Scholar 

  6. Loughlin, A. J., Honegger, P., Woodroofe, M. N., Comte, V., Matthieu, J.-M., and Cuzner, M. L. (1994) Myelination and cytokine-induced demyelination in aggregating rat brain cell cultures: effects of macrophage-enrichment. Neurosci. Res. 37, 647–653.

    Article  CAS  Google Scholar 

  7. Honegger, P. (1985) Biochemical differentiation in serum-free aggregating brain cell cultures, in Cell Culture in the Neurosciences (Bottenstein, J. E. and Sato, G., eds.), Plenum, New York, pp. 223–243.

    Google Scholar 

  8. Riederer, B. M., Monnet-Tschudi, F., and Honegger, P. (1992) Development and maintenance of the neuronal cytoskeleton in aggregated cell cultures of fetal rat telencephalon and influence of elevated K+ concentrations. J. Neurochem. 58, 649–658.

    Article  PubMed  CAS  Google Scholar 

  9. Corthésy-Theulaz, I., Mérillaz, A. M., Honegger, P., and Rossier, B. C. (1990) Na+-K+-ATPase gene expression during in vitro development of rat fetal forebrain. Am. J. Physiol. 258, C1062–C1069.

    PubMed  Google Scholar 

  10. Honegger, P. and Pardo, B. (1999) Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium. J. Cereb. Blood Flow Metab. 19, 1051–1059.

    Article  PubMed  CAS  Google Scholar 

  11. Honegger, P. and Matthieu, J.-M. (1990) Aggregating brain cell cultures: a model to study myelination and demyelination, in Cellular and Molecular Biology of Myelination (Jeserich, G., Althaus, H. H., and Waehneldt, T. V., eds.), Springer-Verlag, Berlin, pp. 155–170.

    Google Scholar 

  12. Honegger, P. and Richelson, E. (1979) Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain. Brain Res. 62, 89–101.

    Article  Google Scholar 

  13. Mata, M., Honegger, P., and Fink, D. J. (1997) Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization. Cell. Mol. Neurobiol. 17, 129–140.

    Article  PubMed  CAS  Google Scholar 

  14. Norton, W. T., Aquino, D. A., Hozumi, I., Chiu, F.-C., and Brosnan, C. F. (1992) Quantitative aspects of reactive gliosis: a review. Neurochem. Res. 17(9), 877–885.

    Article  PubMed  CAS  Google Scholar 

  15. O’Callaghan, J. P. (1991) Assessment of neurotoxicity: use of glial fibrillary acidic protein as a biomarker. Biomed. Environ. Sci. 4, 197–206.

    Google Scholar 

  16. McMillian, M. K., Thai, L., Hong, J.-S., O’Callaghan, J. P., and Pennypacker, K. R. (1994) Brain injury in a dish: a model for reactive gliosis. TINS 17, 138–142.

    PubMed  CAS  Google Scholar 

  17. Wu, V. W. and Schwartz, J. P. (1998) Cell culture models for reactive gliosis: new perspectives. J. Neurosci. Res. 51, 675–681.

    Article  PubMed  CAS  Google Scholar 

  18. Rozovsky, I., Laping, N. J., Krohn, K., Teter, B., O’Callaghan, J. P., and Finch, C. E. (1995) Transcriptional regulation of glial fibrillary acidic protein by corticosterone in rat astrocytes in vitro is influenced by the duration of time in culture and by astrocyte-neuron interactions. Endocrinology 136, 2066–2073.

    Article  PubMed  CAS  Google Scholar 

  19. Monnet-Tschudi, F., Zurich, M.-G., Riederer, B. M., and Honegger, P. (1995) Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage. NeuroToxicology 16, 97–104.

    PubMed  CAS  Google Scholar 

  20. Monnet-Tschudi, F., Zurich, M.-G., and Honegger, P. (1996) Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res. 741, 52–59.

    Article  PubMed  CAS  Google Scholar 

  21. Zurich, M.-G., Honegger, P., Schilter, B., Costa, L. G., and Monnet-Tschudi, F. (2000) Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides. NeuroToxicology 21, 599–606.

    PubMed  CAS  Google Scholar 

  22. Aquino, D.A., Chiu, F.-C., Brosnan, C. F., and Norton, W. T. (1988) Glial fibrillary acidic protein increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis. J. Neurochem. 51, 1085–1095.

    Article  PubMed  CAS  Google Scholar 

  23. Streit, W. J. and Kreutzberg, G. W. (1987) Lectin binding by resting and reactive microglia. J. Neurocytol. 16, 249–260.

    Article  PubMed  CAS  Google Scholar 

  24. Giulian, D. (1987) Ameboid microglia as effectors of inflammation in the central nervous system. J. Neurosci. Res. 18, 155–171.

    Article  PubMed  CAS  Google Scholar 

  25. Perry, V. H. and Gordon, S. (1991) Macrophage and the nervous system. Int. Rev. Cytol. 125, 203–244.

    Article  PubMed  CAS  Google Scholar 

  26. Morioka, T. and Streit, W. J. (1991) Expression of immunomolecules on microglial cells following neonatal sciatic nerve axotomy. J. Neuroimmunol. 35, 21–30.

    Article  PubMed  CAS  Google Scholar 

  27. Marty, S., Dusart, I., and Peschanski, M. (1991) Glial changes following an excitotoxic lesion in the CNS-I. Microglia/macrophages. Neuroscience 45, 529–539.

    Article  PubMed  CAS  Google Scholar 

  28. Stoll, G. and Jander, S. (1999) The role of microglia and macrophages in the pathophysiology of the CNS. Prog. Neurobiol. 58, 233–247.

    Article  PubMed  CAS  Google Scholar 

  29. Streit, W. J., Walter, S. A., and Pennell, N. A. (1999) Reactive microgliosis. Prog. Neurobiol. 57, 563–581.

    Article  PubMed  CAS  Google Scholar 

  30. Gehrmann, J. and Kreutzberg, G. W. (1995) Microglia in experimental neuro-pathology, in Neuroglia (Kettenmann, H. and Ransom, B. R., eds.), Oxford University Press, New York, pp. 883–904.

    Google Scholar 

  31. Monnet-Tschudi, F., Zurich, M.-G., Pithon, E., van Melle, G., and Honegger, P. (1995) Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity. Brain Res. 690, 8–14.

    Article  PubMed  CAS  Google Scholar 

  32. McCann, M. J., O’Callaghan, J. P., Martin, P. M., Bertram, T., and Streit, W. J. (1996) Differential activation of microglia and astrocytes following trimethyl tin-induced neurodegeneration. Neuroscience 72, 273–281.

    Article  PubMed  CAS  Google Scholar 

  33. Monnet-Tschudi, F., Sorg, O., Honegger, P., Zurich, M.-G., Huggett, A. C., and Schilter, B. (1997) Effects of naturally occurring food mycotoxin ochratoxin A on brain cells in culture. NeuroToxicology 18, 831–840.

    PubMed  CAS  Google Scholar 

  34. Charleston, J.S., Bolender, R.P., Mottet, N.K., Body, R.L., Vahter, M.E. and Burbacher, T.M. (1994) Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. Toxicol. Appl. Pharmacol. 129, 196–206.

    Article  PubMed  CAS  Google Scholar 

  35. Monnet-Tschudi, F., Zurich, M.-G., and Honegger, P. (1993) Evaluation of the toxicity of different metal compounds in the developing brain using aggregating cell cultures as a model. Toxic. In Vitro 7, 335–339.

    Article  CAS  Google Scholar 

  36. Honegger, P. and Schilter, B. (1992) Serum-free aggregate cultures of fetal rat brain and liver cells: methodology and some practical application in neurotoxicology, in The Brain in Bits and Pieces. In Vitro Techniques in Neurobiology, Neuropharmacology and Neurotoxicology (Zbinden, G., ed.), MTC Verlag, Zollikon, Switzerland, pp. 51–79.

    Google Scholar 

  37. Benke, G. M. and Murphy, S. D. (1975) The influence of age on the toxicity and metabolism of methyl parathion and parathion in male and female rats. Toxicol. Appl. Pharmacol. 31, 254–269.

    Article  PubMed  CAS  Google Scholar 

  38. Mortensen, S. R., Chanda, S. M., Hooper, M. J., and Padilla, S. (1996) Maturational differences in chlorpyrifos-oxonase activity may contribute to age-related sensitivity to chlorpyrifos. J. Biochem. Toxicol. 11, 279–287.

    Article  PubMed  CAS  Google Scholar 

  39. Lassiter, T. L., Padilla, S., Mortensen, S. R., Chanda, S. M., Moser, V. C., and Barone, S., Jr. (1998) Gestational exposure to chlorpyrifos: apparent protection of the fetus? Toxicol. Appl. Pharmacol. 152, 56–65.

    Article  PubMed  CAS  Google Scholar 

  40. Monnet-Tschudi, F., Zurich, M.-G., Schilter, B., Costa, L. G., and Honegger, P. (2000) Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures. Toxicol. Appl. Pharmacol. 165, 175–183.

    Article  PubMed  CAS  Google Scholar 

  41. Liu, J., Olivier, K., and Pope, C. N. (1999) Comparative neurochemical effects of repeated methyl parathion or chlorpyrifos exposures in neonatal and adult rats. Toxicol. Appl. Pharmacol. 158, 186–196.

    Article  PubMed  CAS  Google Scholar 

  42. Honegger, P. and Schilter, B. (1995) The use of serum-free aggregating brain cell cultures in neurotoxicology, in Neurotoxicology: Approaches & Methods (Chang, L. W., ed.), Academic, New York, pp. 507–516.

    Google Scholar 

  43. Zurich, M.-G., Monnet-Tschudi, F., and Honegger, P. (1994) Long-term treatment of aggregating brain cell cultures with low concentrations of lead acetate. NeuroToxicology 15(3), 715–720.

    PubMed  CAS  Google Scholar 

  44. Tiffany-Castiglioni, E., Zmudzki, J., and Bratton, G. R. (1986) Cellular targets of lead neurotoxicity: in vitro models. Toxicology 42, 303–315.

    Article  PubMed  CAS  Google Scholar 

  45. Tiffany-Castiglioni, E., Zmudzki, J., Wu, J. N., and Bratton, G. R. (1987) Effects of lead treatment on intracellular iron and copper concentrations in cultured astroglia. Metab. Brain Dis. 2(1), 61–79.

    Article  PubMed  CAS  Google Scholar 

  46. Guentert-Lauber, B., Monnet-Tschudi, F., Omlin, F. X., Favrod, P., and Honegger, P. (1985) Serum-free aggregate cultures of rat CNS glial cells: biochemical, immunocytochemical and morphological characterization. Dev. Neurosci. 7, 33–44.

    Article  PubMed  CAS  Google Scholar 

  47. He, M., Howe, D. G., and McCarthy, K. D. (1996) Oligodendroglial signal transduction systems are regulated by neuronal contact. J. Neurochem. 67, 1491–1499.

    Article  PubMed  CAS  Google Scholar 

  48. Pouly, S., Storch, M. K., Matthieu, J.-M., Lassman, H., Monnet-Tschudi, F., and Honegger, P. (1997) Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures. J. Neurosci. Res. 49, 121–132.

    Article  PubMed  CAS  Google Scholar 

  49. Monnet-Tschudi, F., Zurich, M.-G., Sorg, O., Matthieu, J.-M., Honegger, P., and Schilter, B. (1999) The naturally occuring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture. NeuroToxicology 20, 41–48.

    PubMed  CAS  Google Scholar 

  50. Arenender, A. T. and De Vellis, J. (1989) Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, Raven, New York, pp. 479–506.

    Google Scholar 

  51. Hunt, R. K. (1987) Neural Development, Part IV: Cellular and Molecular Differentiation, Current Topics in Developmental Biology, Vol. 21, Academic, New York.

    Google Scholar 

  52. Steindler, D. A., Faissner, A. and Schachner, M. (1989) Brain “cordones”: transient boundaries of glia and adhesion molecules that define developing functional units. Comments on Developmental Neurobiology 1, 29–60.

    Google Scholar 

  53. Thomas, W. E. (1992) Brain macrophages: evaluation of microglia and their functions. Brain Res. Rev. 17, 61–74.

    Article  PubMed  CAS  Google Scholar 

  54. Kreutzberg, G. W. (1996) Microglia: a sensor for pathological events in the CNS. TINS 19, 312–318.

    PubMed  CAS  Google Scholar 

  55. Sawada, M., Kondo, N., and Marunouchi, T. (1989) Production of tumor necrosis factor-alpha by microglia and astrocytes in culture. Brain Res. 491, 394–397.

    Article  PubMed  CAS  Google Scholar 

  56. Finsen, B. R., Jorgensen, M. B., Diemer, N. H., and Zimmer, J. (1993) Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus. Glia 7, 41–49.

    Article  PubMed  CAS  Google Scholar 

  57. Beyer, M., Gimsa, U., Eyüpoglu, I. Y., Hailer, N. P., and Nitsch, R. (2000) Phagocytosis of neuronal or glial debris by microglial cells: upregulation of MHC class II expression and multinuclear giant cell formation in vitro. Glia 31, 262–266.

    Article  PubMed  CAS  Google Scholar 

  58. Ashwell, K. (1990) Microglia and cell death in the developing mouse cerebellum. Dev. Brain Res. 55, 219–230.

    Article  CAS  Google Scholar 

  59. Lassmann, H., Schmied, M., Vass, K., and Hickey, W. F. (1993) Bone marrow derived elements and resident microglia in brain inflammation. Glia 7, 19–24.

    Article  PubMed  CAS  Google Scholar 

  60. Banati, R. B., Gehrmann, J., Schubert, P., and Kreutzberg, G. W. (1993) Cytotoxicity of microglia. Glia 7, 111–118.

    Article  PubMed  CAS  Google Scholar 

  61. Mizuno, T., Sawada, M., Suzumura, A., and Marunouchi, T. (1994) Expression of cytokines during glial differentiation. Brain Res. 656, 141–146.

    Article  PubMed  CAS  Google Scholar 

  62. Mallat, M., Houlgatte, R., Brachet, P., and Prochiantz, A. (1989) Lipopolysac-charide-stimulated rat brain macrophages release NGF in vitro. Dev. Biol. 133, 309–311.

    Article  PubMed  CAS  Google Scholar 

  63. Miwa, T., Furukawa, S., Nakajima, K., Furukawa, Y., and Kohsaka, S. (1997) Lipopolysaccharide enhances synthesis of brain-derived neurotrophic factor in cultured rat microglia. J. Neurosci. Res. 50, 1023–1029.

    Article  PubMed  CAS  Google Scholar 

  64. Elkabes, S., diCicco-Bloom, E. M., and Black, I. B. (1996) Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J. Neurosci. 16, 2508–2521.

    PubMed  CAS  Google Scholar 

  65. Eskes, C. (2000) Implication of microglial cells in the cascade of cell-cell interactions following subchronic neurotoxicant treatments: a multiple in vitro study. Thesis dissertation, University of Lausanne.

    Google Scholar 

  66. Eskes, C., Honegger, P., Juillerat-Jeanneret, L., and Monnet-Tschudi, F. Microglial reaction induced by non-cytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37, 43–52.

    Google Scholar 

  67. Walton, K., Walker, R., van de Sandt, J. J. M., et al. (1999) The application of in vitro data in the derivation of the Acceptable Daily Intake (ADI) of food additives. Food Chem. Toxicol. 37, 1175–1197.

    Article  PubMed  CAS  Google Scholar 

  68. Schilter, B., Holzhäuser, D., Cavin, C., and Huggett, A. C. (1996) An integrated in vivo/in vitro strategy to improve food safety evaluation. Trends Food Sci. Technol. 7, 327–332.

    Article  Google Scholar 

  69. Huggett, A. C., Schilter, B., Roberfroid, M., Antignac, E., and Koemen, J. H. (1996) Comparative methods of toxicity testing. Food Chem. Toxicol. 34, 183–192.

    Article  PubMed  CAS  Google Scholar 

  70. Funk, K. A., Liu, C.-H., Higgins, R. J., and Wilson, B. W. (1994) Avian embryonic brain reaggregate culture system II. NTE activity discriminates between effects of a single neuropathic or nonneuropathic organophosphorus compound exposure. Toxicol. Appl. Pharmacol. 124, 159–163.

    Article  PubMed  CAS  Google Scholar 

  71. Atterwill, C. K., Hillier, G., Johnston, H., and Thomas, S. M. (1992) A tiered system for in vitro neurotoxicity testing: a place for neural cell line and organotypic cultures? in The Brain in Bits and Pieces (Zbinden, G., ed.), MTC Verlag, Zollikon, Switzerland, pp. 81–114.

    Google Scholar 

  72. Fox, R. M., Davenport Jones, J. E., and Atterwill, C. K. (1995) Oxidative stress induces neurotoxicity in rat brain reaggregate cultures that can be prevented by β-tocopherol. NeuroToxicology 16, 558.

    Google Scholar 

  73. Fox, R. M., Davenport Jones, J. E., and Atterwill, C. K. (1995) Excitatory amino acids produce toxic glial responses in whole rat brain reaggregate cultures. NeuroToxicology 16, 559.

    Google Scholar 

  74. Hayes, G. M., Fox, R. M., Cuzner, M. L., and Griffin, G E. (2000) Human rotation-mediated fetal mixed brain cell aggregate culture: characterization and N-methyl-d-aspartate. Neurosci. Lett. 287, 146–150.

    Article  PubMed  CAS  Google Scholar 

  75. Pulliam, L., Gascon, R., Stubblebine, M., McGuire, D., and McGrath, M. S. (1997) Unique monocyte subset in patients with AIDS dementia. Lancet 349, 692–695.

    Article  PubMed  CAS  Google Scholar 

  76. Pulliam, L., Stubblebine, M., and Hyun, W. (1998) Quantification of neurotoxicity and identification of cellular subsets in a three-dimensional brain model. Cytometry 32, 66–69.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute of Physiology, University of Lausanne, Lausanne, Switzerland

    Marie-Gabrielle Zurich (Premiere Assistante), Florianne Monnet-Tschudi (Maitre Assistant) & Paul Honegger (Professeur Associe)

  2. Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA

    Lucio G. Costa (Professor)

  3. Nestlé Research Centre, Vers-chez-les Blanc, Lausanne, Switzerland

    Benoît Schilter (Nestlé Research Centre)

Authors
  1. Marie-Gabrielle Zurich
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  2. Florianne Monnet-Tschudi
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  3. Lucio G. Costa
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  4. Benoît Schilter
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  5. Paul Honegger
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Editors and Affiliations

  1. Department of Veterinary Anatomy and Public Health, Texas A&M University College of Veterinary Medicine, College Station, TX

    Evelyn Tiffany-Castiglioni

  2. Center for Environmental and Rural Health, College Station, TX

    Evelyn Tiffany-Castiglioni

  3. University of California, Davis, CA

    Mannfred A. Hollinger

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Zurich, MG., Monnet-Tschudi, F., Costa, L.G., Schilter, B., Honegger, P. (2004). Aggregating Brain Cell Cultures for Neurotoxicological Studies. In: Tiffany-Castiglioni, E., Hollinger, M.A. (eds) In Vitro Neurotoxicology. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-651-7:243

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  • DOI: https://doi.org/10.1385/1-59259-651-7:243

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-047-2

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