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Long-Term Primary Culture of Highly-Pure Rat Embryonic Hippocampal Neurons of Low-Density

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Abstract

In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (103 cells/cm2), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the β-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.

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References

  1. Sasaki Y, Fukushima N, Yoshida A, Ueda H (1998) Low-density induced apoptosis of cortical neurons is inhibited by serum factors. Cell Mol Neurobiol 18:487–496

    Article  CAS  PubMed  Google Scholar 

  2. Fujita R, Yoshida A, Mizuno K, Ueda H (2001) Cell density-dependent death mode switch of cultured cortical neurons under serum-free starvation stress. Cell Mol Neurobiol 21:317–324

    Article  CAS  PubMed  Google Scholar 

  3. Martin DL (1992) Synthesis and release of neuroactive substances by glial cells. Glia 5:81–94

    Article  CAS  PubMed  Google Scholar 

  4. Pellitteri R, Zicca A, Mancardi GL, Savio T, Cadoni A (2001) Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth. Eur J Histochem 45:367–376

    CAS  PubMed  Google Scholar 

  5. Walsh E, Ueda Y, Nakanishi H, Yoshida K (1992) Neuronal survival and neurite extension supported by astrocytes co-cultured in transwells. Neurosci Lett 138:103–106

    Article  CAS  PubMed  Google Scholar 

  6. Price PJ (1975) Preparation and use of rat-tail collagen. Methods in Cell Sci 1:43–44

    CAS  Google Scholar 

  7. Phifer CB, Terry LM (1986) Use of hypothermia for general anesthesia in preweanling rodents. Physiol Behav 38:887–890

    Article  CAS  PubMed  Google Scholar 

  8. Goslin K, Asmussen H, Banker G (1998) Rat hippocampal neurons in low density culture. Culturing nerve cells. MIT Press, Cambridge, pp 339–370

    Google Scholar 

  9. James CD, Davis R, Meyer M, Turner A, Turner S, Withers G, Kam L, Banker G, Craighead H, Isaacson M, Turner J, Shain W (2000) Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays. IEEE Trans Biomed Eng 47:17–21

    Article  CAS  PubMed  Google Scholar 

  10. Woerly S, Maghami G, Duncan R, Subr V, Ulbrich K (1993) Synthetic polymer derivatives as substrata for neuronal adhesion and growth. Brain Res Bull 30:423–432

    Article  CAS  PubMed  Google Scholar 

  11. Ruegg UT, Hefti F (1984) Growth of dissociated neurons in culture dishes coated with synthetic polymeric amines. Neurosci Lett 49:319–324

    Article  CAS  PubMed  Google Scholar 

  12. Maxwell GD (1976) Substrate dependence of cell migration from explanted neural tubes in vitro. Cell Tiss Res 172:325–330

    Article  CAS  Google Scholar 

  13. Bunge RP, Bunge MB (1978) Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers. J Cell Biol 78:943–950

    Article  CAS  PubMed  Google Scholar 

  14. Elsdall T, Bard J (1972) Collagen substrate for studies on cell behavior. J Cell Biol 54:626–637

    Article  Google Scholar 

  15. Lee HY, Greene LA, Mason CA, Manzini MC (2009) Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons. J Vis Exp 16:990

    Google Scholar 

  16. Tafti M, Ghyselinck NB (2007) Functional implication of the vitamin A signaling pathway in the brain. Arch Neurol 64:1706–1711

    Article  PubMed  Google Scholar 

  17. Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobio 62:649–671

    Article  CAS  Google Scholar 

  18. Butterfield DA, Koppal T, Subramaniam R, Yatin S (1999) Vitamin E as an antioxidant/free radical scavenger against amyloid beta-peptide-induced oxidative stress in neocortical synaptosomal membranes and hippocampal neurons in culture: insights into Alzheimer’s disease. Rev Neurosci 10:141–149

    CAS  PubMed  Google Scholar 

  19. Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M (2004) Vitamin A exhibits potent antiamyloidogenic and fibril-destabilizing effects in vitro. Exp Neurol 189:380–392

    Article  CAS  PubMed  Google Scholar 

  20. Diaz-Hernandez JI, Almeida A, Delgado-Esteban M, Fernandez E, Bolanos JP (2005) Knockdown of glutamate-cysteine ligase by small hairpin RNA reveals that both catalytic and modulatory subunits are essential for the survival of primary neurons. J Biol Chem 280:38992–39001

    Article  CAS  PubMed  Google Scholar 

  21. Mizui T, Kinouchi H, Chan PH (1992) Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats. Am J Physiol 262:H313–H317

    CAS  PubMed  Google Scholar 

  22. Sagara JI, Fujiwara K, Sakakura Y, Sato H, Bannai S, Makino N (2007) Beneficial effect of antioxidants in purified neurons derived from rat cortical culture. Brain Res 1131:11–16

    Article  CAS  PubMed  Google Scholar 

  23. Sagara JI, Miura K, Bannai S (1993) Maintenance of neuronal glutathione by glial cells. J Neurochem 61:1672–1676

    Article  CAS  PubMed  Google Scholar 

  24. Slivka A, Spina MB, Cohen G (1987) Reduced and oxidized glutathione in human and monkey brain. Neurosci Lett 74:112–118

    Article  CAS  PubMed  Google Scholar 

  25. Dringen R, Hirrlinger J (2003) Glutathione pathways in the brain. Bio Chem 384:505–516

    Article  CAS  Google Scholar 

  26. Martinez-Cruz F, Pozo D, Osuna C, Espinar A, Marchante C, Guerrero JM (2002) Oxidative stress induced by phenylketonuria in the rat: prevention by melatonin, vitamin E and vitamin C. J Neurosci Res 69:550–558

    Article  CAS  PubMed  Google Scholar 

  27. Ioudina M, Uemura E, Greenlee HW (2004) Glucose insufficiency alters neuronal viability and increases susceptibility to glutamate toxicity. Brain Res 1004(1–2):188–192

    Article  CAS  PubMed  Google Scholar 

  28. Liu D, Chan SL, de Souza-Pinto NC, Slevin JR, Wersto RP, Zhan M, Mustafa K, de Cabo R, Mattson MP (2006) Mitochondrial UCP4 mediates an adaptive shift in energy metabolism and increases the resistance of neurons to metabolic and oxidative stress. Neuromol Med 8:389–414

    Article  CAS  Google Scholar 

  29. Albrecht J, Sonnewald U, Waagepetersen HS, Schousboe A (2007) Glutamine in the central nervous system: function and dysfunction. Front Biosci 12:332–343

    Article  CAS  PubMed  Google Scholar 

  30. Yang H, Liang Z, Li J, Cheng X, Luo N, Ju G (2006) Optimized and efficient preparation of astrocyte cultures from rat spinal cord. Cytotechnology 52:87–97

    Article  PubMed  Google Scholar 

  31. Geshi M, Takenouchi N, Yamauchi N, Nagai T (2000) Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod 63:1730–1734

    Article  CAS  PubMed  Google Scholar 

  32. Suh SW, Aoyama K, Matsumori Y, Liu J, Swanson RA (2005) Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54:1452–1458

    Article  CAS  PubMed  Google Scholar 

  33. Andrae U, Singh J, Ziegler-Skylakakis K (1985) Pyruvate and related alphaketoacids protect mammalian cells in culture against hydrogen peroxideinduced cytotoxicity. Toxicol Lett 28:93–98

    Article  CAS  PubMed  Google Scholar 

  34. Needels DL, Nieto-Sampedro M, Cotman CW (1986) Induction of a neurite-promoting factor in rat brain following injury or deafferentation. Neurosci 18:517–526

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by Natural Science Foundation of China (Nos. 30973088, 30872829 and 30571998).

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Correspondence to Gong Ju or Si-Wei You.

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Hao Yang and Rui Cong contributed equally to this work.

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Yang, H., Cong, R., Na, L. et al. Long-Term Primary Culture of Highly-Pure Rat Embryonic Hippocampal Neurons of Low-Density. Neurochem Res 35, 1333–1342 (2010). https://doi.org/10.1007/s11064-010-0189-0

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