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Open Access 01-12-2007 | Review

Microglial cells in astroglial cultures: a cautionary note

Author: Josep Saura

Published in: Journal of Neuroinflammation | Issue 1/2007

Abstract

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions.

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Pellerin L: How astrocytes feed hungry neurons. Mol Neurobiol. 2005, 32: 59-72. 10.1385/MN:32:1:059.CrossRefPubMed

Araque A, Perea G: Glial modulation of synaptic transmission in culture. Glia. 2004, 47: 241-248. 10.1002/glia.20026.CrossRefPubMed

Abbott NJ, Ronnback L, Hansson E: Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006, 7: 41-53. 10.1038/nrn1824.CrossRefPubMed

Simard M, Nedergaard M: The neurobiology of glia in the context of water and ion homeostasis. Neuroscience. 2004, 129: 877-896. 10.1016/j.neuroscience.2004.09.053.CrossRefPubMed

Koehler RC, Gebremedhin D, Harder DR: Role of astrocytes in cerebrovascular regulation. J Appl Physiol. 2006, 100: 307-317. 10.1152/japplphysiol.00938.2005.PubMedCentralCrossRefPubMed

Darlington CL: Astrocytes as targets for neuroprotective drugs. Curr Opin Investig Drugs. 2005, 6: 700-703.PubMed

Sofroniew MV: Reactive astrocytes in neural repair and protection. Neuroscientist. 2005, 11: 400-407. 10.1177/1073858405278321.CrossRefPubMed

Lim R, Bosch EP: Isolation of Astrocytes and Schwann Cells for Culture. Methods in Neurosciences, Volume 2, Cell Culture. Edited by: Coon PM. 1990, San Diego, Academic Press, 47-55.

Hertz L, Juurlink BHJ, Szuchet S: Cell cultures. Handbook of Neurochemistry, vol 8. Edited by: A L. 1985, New York, Plenum, 603-661. 2nd

10.

Booher J, Sensenbrenner M: Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology. 1972, 2: 97-105.PubMed

11.

McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980, 85: 890-902. 10.1083/jcb.85.3.890.CrossRefPubMed

12.

Kimelberg HK, Schools GP, Cai Z, Zhou M: Freshly isolated astrocyte (FIA) preparations: a useful single cell system for studying astrocyte properties. J Neurosci Res. 2000, 61: 577-587. 10.1002/1097-4547(20000915)61:6<577::AID-JNR1>3.0.CO;2-T.CrossRefPubMed

13.

Hansson E, Thorlin T: Brain primary cultures and vibrodissociated cells as tools for the study of astroglial properties and functions. Dev Neurosci. 1999, 21: 1-11. 10.1159/000017360.CrossRefPubMed

14.

Wiesinger H, Schuricht B, Hamprecht B: Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain. Brain Res. 1991, 550: 69-76. 10.1016/0006-8993(91)90406-L.CrossRefPubMed

15.

Hildebrand B, Olenik C, Meyer DK: Neurons are generated in confluent astroglial cultures of rat neonatal neocortex. Neuroscience. 1997, 78: 957-966. 10.1016/S0306-4522(96)00676-8.CrossRefPubMed

16.

Crang AJ, Blakemore WF: Attempts to produce astrocyte cultures devoid of oligodendrocyte generating potential by the use of antimitotic treatment reveal the presence of quiescent oligodendrocyte precursors. J Neurosci Res. 1997, 49: 64-71. 10.1002/(SICI)1097-4547(19970701)49:1<64::AID-JNR7>3.0.CO;2-B.CrossRefPubMed

17.

Hansson E: Cellular composition of a cerebral hemisphere primary culture. Neurochem Res. 1984, 9: 153-172. 10.1007/BF00964164.CrossRefPubMed

18.

McNaught KS, Jenner P: Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures. J Neurochem. 1999, 73: 2469-2476. 10.1046/j.1471-4159.1999.0732469.x.CrossRefPubMed

19.

Theis M, Speidel D, Willecke K: Astrocyte cultures from conditional connexin43-deficient mice. Glia. 2004, 46: 130-141. 10.1002/glia.10350.CrossRefPubMed

20.

Falsig J, Porzgen P, Lund S, Schrattenholz A, Leist M: The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006, 96: 893-907. 10.1111/j.1471-4159.2005.03622.x.CrossRefPubMed

21.

Castel H, Diallo M, Chatenet D, Leprince J, Desrues L, Schouft MT, Fontaine M, Dubessy C, Lihrmann I, Scalbert E, Malagon M, Vaudry H, Tonon MC, Gandolfo P: Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes. J Neurochem. 2006, 99: 582-595. 10.1111/j.1471-4159.2006.04130.x.CrossRefPubMed

22.

Giulian D, Baker TJ: Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci. 1986, 6: 2163-2178.PubMed

23.

Murphy S: Production of nitric oxide by glial cells: regulation and potential roles in the CNS. Glia. 2000, 29: 1-13. 10.1002/(SICI)1098-1136(20000101)29:1<1::AID-GLIA1>3.0.CO;2-N.CrossRefPubMed

24.

Saura J, Angulo E, Ejarque A, Casado V, Tusell JM, Moratalla R, Chen JF, Schwarzschild MA, Lluis C, Franco R, Serratosa J: Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. J Neurochem. 2005, 95: 919-929. 10.1111/j.1471-4159.2005.03395.x.CrossRefPubMed

25.

Hamby ME, Hewett JA, Hewett SJ: TGF-beta1 potentiates astrocytic nitric oxide production by expanding the population of astrocytes that express NOS-2. Glia. 2006, 54: 566-577. 10.1002/glia.20411.CrossRefPubMed

26.

Hamby ME, Uliasz TF, Hewett SJ, Hewett JA: Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes. J Neurosci Methods. 2006, 150: 128-137. 10.1016/j.jneumeth.2005.06.016.CrossRefPubMed

27.

von Bernhardi R, Eugenin J: Microglial reactivity to beta-amyloid is modulated by astrocytes and proinflammatory factors. Brain Res. 2004, 1025: 186-193. 10.1016/j.brainres.2004.07.084.CrossRefPubMed

28.

Cassina P, Peluffo H, Pehar M, Martinez-Palma L, Ressia A, Beckman JS, Estevez AG, Barbeito L: Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J Neurosci Res. 2002, 67: 21-29. 10.1002/jnr.10107.CrossRefPubMed

29.

Kong LY, McMillian MK, Maronpot R, Hong JS: Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures. Brain Res. 1996, 729: 102-109. 10.1016/0006-8993(96)00417-9.CrossRefPubMed

30.

Korcok J, Wu F, Tyml K, Hammond RR, Wilson JX: Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition. J Neurochem. 2002, 81: 185-193. 10.1046/j.1471-4159.2002.00814.x.CrossRefPubMed

31.

Akama KT, Van Eldik LJ: Beta-amyloid stimulation of inducible nitric-oxide synthase in astrocytes is interleukin-1beta- and tumor necrosis factor-alpha (TNFalpha)-dependent, and involves a TNFalpha receptor-associated factor- and NFkappaB-inducing kinase-dependent signaling mechanism. J Biol Chem. 2000, 275: 7918-7924. 10.1074/jbc.275.11.7918.CrossRefPubMed

32.

Buskila Y, Farkash S, Hershfinkel M, Amitai Y: Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices. Glia. 2005, 52: 169-176. 10.1002/glia.20217.CrossRefPubMed

33.

Salvemini D, Mollace V, Pistelli A, Anggard E, Vane J: Cultured astrocytoma cells generate a nitric oxide-like factor from endogenous L-arginine and glyceryl trinitrate: effect of E. coli lipopolysaccharide. Br J Pharmacol. 1992, 106: 931-936.PubMedCentralCrossRefPubMed

34.

Lee SC, Dickson DW, Liu W, Brosnan CF: Induction of nitric oxide synthase activity in human astrocytes by interleukin-1 beta and interferon-gamma. J Neuroimmunol. 1993, 46: 19-24. 10.1016/0165-5728(93)90229-R.CrossRefPubMed

35.

Boje KM, Arora PK: Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res. 1992, 587: 250-256. 10.1016/0006-8993(92)91004-X.CrossRefPubMed

36.

Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK: Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol. 1992, 149: 2736-2741.PubMed

37.

Hewett SJ, Corbett JA, McDaniel ML, Choi DW: Interferon-gamma and interleukin-1 beta induce nitric oxide formation from primary mouse astrocytes. Neurosci Lett. 1993, 164: 229-232. 10.1016/0304-3940(93)90898-U.CrossRefPubMed

38.

Xiong H, Yamada K, Jourdi H, Kawamura M, Takei N, Han D, Nabeshima T, Nawa H: Regulation of nerve growth factor release by nitric oxide through cyclic GMP pathway in cortical glial cells. Mol Pharmacol. 1999, 56: 339-347.PubMedCentralPubMed

39.

Sola C, Casal C, Tusell JM, Serratosa J: Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells. Eur J Neurosci. 2002, 16: 1275-1283. 10.1046/j.1460-9568.2002.02199.x.CrossRefPubMed

40.

Saura J, Petegnief V, Wu X, Liang Y, Paul SM: Microglial apolipoprotein E and astroglial apolipoprotein J expression in vitro: opposite effects of lipopolysaccharide. J Neurochem. 2003, 85: 1455-1467. 10.1046/j.1471-4159.2003.01788.x.CrossRefPubMed

41.

Falsig J, Latta M, Leist M: Defined inflammatory states in astrocyte cultures: correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004, 88: 181-193.CrossRefPubMed

42.

Oropeza RL, Wekerle H, Werb Z: Expression of apolipoprotein E by mouse brain astrocytes and its modulation by interferon-gamma. Brain Res. 1987, 410: 45-51. 10.1016/S0006-8993(87)80018-5.CrossRefPubMed

43.

Pitas RE, Boyles JK, Lee SH, Foss D, Mahley RW: Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta. 1987, 917: 148-161.CrossRefPubMed

44.

Stone DJ, Rozovsky I, Morgan TE, Anderson CP, Hajian H, Finch CE: Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol. 1997, 143: 313-318. 10.1006/exnr.1996.6360.CrossRefPubMed

45.

Aleong R, Aumont N, Dea D, Poirier J: Non-steroidal anti-inflammatory drugs mediate increased in vitro glial expression of apolipoprotein E protein. Eur J Neurosci. 2003, 18: 1428-1438. 10.1046/j.1460-9568.2003.02869.x.CrossRefPubMed

46.

Mori K, Yokoyama A, Yang L, Yang L, Maeda N, Mitsuda N, Tanaka J: L-serine-mediated release of apolipoprotein E and lipids from microglial cells. Exp Neurol. 2004, 185: 220-231. 10.1016/j.expneurol.2003.10.010.CrossRefPubMed

47.

Gebicke-Haerter PJ, Bauer J, Schobert A, Northoff H: Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells. J Neurosci. 1989, 9: 183-194.PubMed

48.

Dalmau I, Castellano B, Pedersen EB, Finsen B, Zimmer J, Gonzalez B: Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate. J Neuroimmunol. 1996, 70: 123-129. 10.1016/S0165-5728(96)00074-4.CrossRefPubMed

49.

Kloss CU, Bohatschek M, Kreutzberg GW, Raivich G: Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture. Exp Neurol. 2001, 168: 32-46. 10.1006/exnr.2000.7575.CrossRefPubMed

50.

Gorfien S, Spector A, DeLuca D, Weiss S: Growth and physiological functions of vascular endothelial cells in a new serum-free medium (SFM). Exp Cell Res. 1993, 206: 291-301. 10.1006/excr.1993.1149.CrossRefPubMed

51.

Saura J, Tusell JM, Serratosa J: High-yield isolation of murine microglia by mild trypsinization. Glia. 2003, 44: 183-189. 10.1002/glia.10274.CrossRefPubMed

52.

Tanaka J, Toku K, Sakanaka M, Maeda N: Morphological differentiation of microglial cells in culture: involvement of insoluble factors derived from astrocytes. Neurosci Res. 1999, 34: 207-215. 10.1016/S0168-0102(99)00041-3.CrossRefPubMed

53.

Jauneau AC, Ischenko A, Chatagner A, Benard M, Chan P, Schouft MT, Patte C, Vaudry H, Fontaine M: Interleukin-1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes. J Neuroinflammation. 2006, 3: 8-10.1186/1742-2094-3-8.PubMedCentralCrossRefPubMed

54.

Hertz L, Peng L, Lai JC: Functional studies in cultured astrocytes. Methods. 1998, 16: 293-310. 10.1006/meth.1998.0686.CrossRefPubMed

55.

Linnemann D, Skarsfelt T: Regional changes in expression of NCAM, GFAP, and S100 in aging rat brain. Neurobiol Aging. 1994, 15: 651-655. 10.1016/0197-4580(94)00060-3.CrossRefPubMed

56.

Ogura K, Ogawa M, Yoshida M: Effects of ageing on microglia in the normal rat brain: immunohistochemical observations. Neuroreport. 1994, 5: 1224-1226.CrossRefPubMed

57.

Abe K, Saito H: L-glutamate suppresses amyloid beta-protein-induced stellation of cultured rat cortical astrocytes. J Neurochem. 2000, 74: 280-286. 10.1046/j.1471-4159.2000.0740280.x.CrossRefPubMed

58.

Yanni PA, Rising LJ, Ingraham CA, Lindsley TA: Astrocyte-derived factors modulate the inhibitory effect of ethanol on dendritic development. Glia. 2002, 38: 292-302. 10.1002/glia.10071.CrossRefPubMed

59.

Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS: Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci. 2000, 20: 6309-6316.PubMed

60.

Hao C, Richardson A, Fedoroff S: Macrophage-like cells originate from neuroepithelium in culture: characterization and properties of the macrophage-like cells. Int J Dev Neurosci. 1991, 9: 1-14. 10.1016/0736-5748(91)90067-V.CrossRefPubMed

61.

Milner R, Campbell IL: Cytokines regulate microglial adhesion to laminin and astrocyte extracellular matrix via protein kinase C-dependent activation of the alpha6beta1 integrin. J Neurosci. 2002, 22: 1562-1572.PubMed

62.

Olsen ML, Higashimori H, Campbell SL, Hablitz JJ, Sontheimer H: Functional expression of Kir4.1 channels in spinal cord astrocytes. Glia. 2006, 53: 516-528. 10.1002/glia.20312.PubMedCentralCrossRefPubMed

63.

Imura T, Nakano I, Kornblum HI, Sofroniew MV: Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes. Glia. 2006, 53: 277-293. 10.1002/glia.20281.CrossRefPubMed

64.

Frith JC, Monkkonen J, Blackburn GM, Russell RG, Rogers MJ: Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res. 1997, 12: 1358-1367. 10.1359/jbmr.1997.12.9.1358.CrossRefPubMed

65.

Kohl A, Dehghani F, Korf HW, Hailer NP: The bisphosphonate clodronate depletes microglial cells in excitotoxically injured organotypic hippocampal slice cultures. Exp Neurol. 2003, 181: 1-11. 10.1016/S0014-4886(02)00049-3.CrossRefPubMed

66.

Brown DR, Schmidt B, Kretzschmar HA: A neurotoxic prion protein fragment enhances proliferation of microglia but not astrocytes in culture. Glia. 1996, 18: 59-67. 10.1002/(SICI)1098-1136(199609)18:1<59::AID-GLIA6>3.0.CO;2-Z.CrossRefPubMed

67.

Ciccarelli R, Di Iorio P, D'Alimonte I, Giuliani P, Florio T, Caciagli F, Middlemiss PJ, Rathbone MP: Cultured astrocyte proliferation induced by extracellular guanosine involves endogenous adenosine and is raised by the co-presence of microglia. Glia. 2000, 29: 202-211. 10.1002/(SICI)1098-1136(20000201)29:3<202::AID-GLIA2>3.0.CO;2-C.CrossRefPubMed

68.

Galea E, Feinstein DL, Reis DJ: Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. Proc Natl Acad Sci U S A. 1992, 89: 10945-10949. 10.1073/pnas.89.22.10945.PubMedCentralCrossRefPubMed

69.

Murphy S: Reply to "Histochemical localization of nitric oxide synthase in the CNS". Trends Neurosci. 1994, 17: 106-10.1016/0166-2236(94)90116-3.CrossRef

70.

Vincent VA, Van Dam AM, Persoons JH, Schotanus K, Steinbusch HW, Schoffelmeer AN, Berkenbosch F: Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1 beta production in rat microglial cells of endotoxin-treated mixed glial cell cultures. Glia. 1996, 17: 94-102. 10.1002/(SICI)1098-1136(199606)17:2<94::AID-GLIA2>3.0.CO;2-6.CrossRefPubMed

71.

Vincent VA, Tilders FJ, Van Dam AM: Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: a role for transforming growth factor beta. Glia. 1997, 19: 190-198. 10.1002/(SICI)1098-1136(199703)19:3<190::AID-GLIA2>3.0.CO;2-3.CrossRefPubMed

72.

Yang L, Tanaka J, Zhang B, Sakanaka M, Maeda N: Astrocytes modulate nitric oxide production by microglial cells through secretion of serine and glycine. Biochem Biophys Res Commun. 1998, 251: 277-282. 10.1006/bbrc.1998.9457.CrossRefPubMed

73.

Noack H, Possel H, Chatterjee S, Keilhoff G, Wolf G: Nitrosative stress in primary glial cultures after induction of the inducible isoform of nitric oxide synthase (i-NOS). Toxicology. 2000, 148: 133-142. 10.1016/S0300-483X(00)00204-3.CrossRefPubMed

74.

Possel H, Noack H, Putzke J, Wolf G, Sies H: Selective upregulation of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) and cytokines in microglia: in vitro and in vivo studies. Glia. 2000, 32: 51-59. 10.1002/1098-1136(200010)32:1<51::AID-GLIA50>3.0.CO;2-4.CrossRefPubMed

75.

Roychowdhury S, Wolf G, Keilhoff G, Bagchi D, Horn T: Protection of primary glial cells by grape seed proanthocyanidin extract against nitrosative/oxidative stress. Nitric Oxide. 2001, 5: 137-149. 10.1006/niox.2001.0335.CrossRefPubMed

76.

Possel H, Noack H, Keilhoff G, Wolf G: Life imaging of peroxynitrite in rat microglial and astroglial cells: Role of superoxide and antioxidants. Glia. 2002, 38: 339-350. 10.1002/glia.10066.CrossRefPubMed

77.

Choi SH, Choi DH, Song KS, Shin KH, Chun BG: Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells. J Neurosci Res. 2002, 67: 411-421. 10.1002/jnr.10102.CrossRefPubMed

78.

Pyo H, Yang MS, Jou I, Joe EH: Wortmannin enhances lipopolysaccharide-induced inducible nitric oxide synthase expression in microglia in the presence of astrocytes in rats. Neurosci Lett. 2003, 346: 141-144. 10.1016/S0304-3940(03)00505-6.CrossRefPubMed

Title: Microglial cells in astroglial cultures: a cautionary note
Author: Josep Saura
Publication date: 01-12-2007
Publisher: BioMed Central
Published in: Journal of Neuroinflammation / Issue 1/2007
Electronic ISSN: 1742-2094
DOI: https://doi.org/10.1186/1742-2094-4-26

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Microglial cells in astroglial cultures: a cautionary note

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