Experimental Brain Research

, Volume 188, Issue 1, pp 33–43 | Cite as

Acute anoxia stimulates proliferation in adult neural stem cells from the rat brain

  • Heinrich F. Bürgers
  • Dominik W. Schelshorn
  • Wolfgang Wagner
  • Wolfgang Kuschinsky
  • Martin H. Maurer
Research Article


Hypoxic-ischemic damage is a major challenge for neuronal tissue. In the present study, we investigated the effects of anoxia and glucose deprivation on adult neural stem cells (NSCs) in vitro. We assessed glucose deprivation, anoxia and the combination of the latter separately. After 24 h of anoxia, cell numbers increased up to 60% compared to normoxic controls. Whereas nearly all normoxic cells incorporated the mitotic marker BrdU (99%), only 85% of the anoxic cells were BrdU-positive. Counting of interphase chromosomes showed 8-fold higher cell division activity after anoxia. The number of necrotic cells doubled after anoxia (14% compared to 7% after normoxia). Apoptosis was measured by two distinct caspases assays. Whereas the total caspase activity was reduced after anoxia, caspase 3/7 showed no alterations. Glucose deprivation and oxygen glucose deprivation both reduced cell viability by 56 and 53%, respectively. Under these conditions, total caspases activity doubled, but caspase 3/7 activity remained unchanged. Erythropoietin, which was reported as neuroprotective, did not increase cell viability in normoxia, but moderately under oxygen glucose deprivation by up to 6%. Erythropoietin reduced total caspase activity by nearly 30% under all the conditions, whereas caspase 3/7 activity was not affected. Our results show that anoxia increases proliferation and viability of adult NSCs, although a fraction of NSCs does not divide during anoxia. In conclusion, anoxia increased cell viability, cell number and proliferation in NSCs from the rat brain. Anoxia turned out to be a highly stimulating environmental for NSCs and NSCs died only when deprived of glucose. We conclude that the availability of glucose but not of oxygen is a crucial factor for NSC survival, regulating apoptotic pathways via caspases activity other than the caspases 3/7 pathway. Therefore, we conclude that NSCs are dying from glucose deprivation, not from hypoxic-ischemic damage.


Erythropoietin Oxygen glucose deprivation Neural stem cell Cell viability Caspases 









Ethidium bromide


Fluorescein diacetate


Glucose deprivation


Neural stem cell


Oxygen glucose deprivation


Phosphate buffered saline


  1. Abdelkarim GE, Gertz K, Harms C, Katchanov J, Dirnagl U, Szabo C, Endres M (2001) Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke. Int J Mol Med 7:255–260PubMedGoogle Scholar
  2. Buttke TM, McCubrey JA, Owen TC (1993) Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. J Immunol Methods 157:233–240PubMedCrossRefGoogle Scholar
  3. Clark LC Jr, Wolf R, Granger D, Taylor Z (1953) Continuous recording of blood oxygen tensions by polarography. J Appl Physiol 6:189–193PubMedGoogle Scholar
  4. Csete M (2005) Oxygen in the cultivation of stem cells. Ann N Y Acad Sci 1049:1–8PubMedCrossRefGoogle Scholar
  5. Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391–397PubMedCrossRefGoogle Scholar
  6. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci USA 96:11619–11624PubMedCrossRefGoogle Scholar
  7. Hofmann J, Sernetz M (1983) A kinetic study on the enzymatic hydrolysis of fluorescein diacetate and fluorescein-di-beta-D-galactopyranoside. Anal Biochem 131:180–186PubMedCrossRefGoogle Scholar
  8. Holst G, Glud RN, Kuhl M, Klimant I (1997) A microoptode array for fine-scale measurement of oxygen distribution. Sensors and actuators B: chemical, 3rd European conference on optical chemical sensors and biosensors 38:122–129Google Scholar
  9. Klimant I, Kuhl M, Glud RN, Holst G (1997) Optical measurement of oxygen and temperature in microscale: strategies and biological applications. Sensors and actuators B: chemical, 3rd European conference on optical chemical sensors and biosensors 38:29–37Google Scholar
  10. Lewczuk P, Hasselblatt M, Kamrowski-Kruck H, Heyer A, Unzicker C, Siren AL, Ehrenreich H (2000) Survival of hippocampal neurons in culture upon hypoxia: effect of erythropoietin. Neuroreport 11:3485–3488PubMedCrossRefGoogle Scholar
  11. Li X, Zhu L, Chen X, Fan M (2007) Effects of hypoxia on proliferation and differentiation of myoblasts. Med Hypotheses 69:629–636PubMedCrossRefGoogle Scholar
  12. Macas J, Nern C, Plate KH, Momma S (2006) Increased generation of neuronal progenitors after ischemic injury in the aged adult human forebrain. J Neurosci 26:13114–13119PubMedCrossRefGoogle Scholar
  13. Maurer MH, Feldmann RE Jr, Fütterer CD, Butlin J, Kuschinsky W (2004) Comprehensive proteome expression profiling of undifferentiated versus differentiated neural stem cells from adult rat hippocampus. Neurochem Res 29:1129–1144PubMedCrossRefGoogle Scholar
  14. Maurer MH, Geomor HK, Bürgers HF, Schelshorn DW, Kuschinsky W (2006) Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression. FEBS Lett 580:4430–4434PubMedCrossRefGoogle Scholar
  15. Plane JM, Liu R, Wang TW, Silverstein FS, Parent JM (2004) Neonatal hypoxic-ischemic injury increases forebrain subventricular zone neurogenesis in the mouse. Neurobiol Dis 16:585–595PubMedCrossRefGoogle Scholar
  16. Potrovita I, Zhang W, Burkly L, Hahm K, Lincecum J, Wang MZ, Maurer MH, Rossner M, Schneider A, Schwaninger M (2004) Tumor Necrosis Factor-Like Weak inducer of apoptosis-induced neurodegeneration. J Neurosci 24:8237–8244PubMedCrossRefGoogle Scholar
  17. Ruscher K, Freyer D, Karsch M, Isaev N, Megow D, Sawitzki B, Priller J, Dirnagl U, Meisel A (2002) Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model. J Neurosci 22:10291–10301PubMedGoogle Scholar
  18. Santra M, Katakowski M, Zhang RL, Zhang ZG, Meng H, Jiang F, Chopp M (2006) Protection of adult mouse progenitor cells and human glioma cells by de novo decorin expression in an oxygen- and glucose-deprived cell culture model system. J Cereb Blood Flow Metab 26:1311–1322PubMedCrossRefGoogle Scholar
  19. Shingo T, Sorokan ST, Shimazaki T, Weiss S (2001) Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci 21:9733–9743PubMedGoogle Scholar
  20. Sontag W (1977) A comparative kinetic study on the conversion of fluoresceindiacetate to fluorescein in living cells and in vitro. Radiat Environ Biophys 14:1–12PubMedCrossRefGoogle Scholar
  21. Studer L, Csete M, Lee SH, Kabbani N, Walikonis J, Wold B, McKay R (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 20:7377–7383PubMedGoogle Scholar
  22. Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T, Narasimhan P, Maier CM, Chan PH (2004) Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx 1:17–25PubMedCrossRefGoogle Scholar
  23. Vannucci SJ (1994) Developmental expression of GLUT1 and GLUT3 glucose transporters in rat brain. J Neurochem 62:240–246PubMedCrossRefGoogle Scholar
  24. Wagner W, Ansorge A, Wirkner U, Eckstein V, Schwager C, Blake J, Miesala K, Selig J, Saffrich R, Ansorge W, Ho AD (2004) Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis. Blood 104:675–686PubMedCrossRefGoogle Scholar
  25. Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov KV, Tarasova Y, Wersto RP, Boheler KR, Wobus AM (2004) Nestin expression—a property of multi-lineage progenitor cells? Cell Mol Life Sci 61:2510–2522PubMedCrossRefGoogle Scholar
  26. Youssoufian H, Longmore G, Neumann D, Yoshimura A, Lodish HF (1993) Structure, function, and activation of the erythropoietin receptor. Blood 81:2223–2236PubMedGoogle Scholar
  27. Yu AC, Gregory GA, Chan PH (1989) Hypoxia-induced dysfunctions and injury of astrocytes in primary cell cultures. J Cereb Blood Flow Metab 9:20–28PubMedGoogle Scholar
  28. Zhang RL, Zhang ZG, Lu M, Wang Y, Yang JJ, Chopp M (2006) Reduction of the cell cycle length by decreasing G1 phase and cell cycle reentry expand neuronal progenitor cells in the subventricular zone of adult rat after stroke. J Cereb Blood Flow Metab 26:857–863PubMedCrossRefGoogle Scholar
  29. Zhou L, Miller CA (2006) Mitogen-activated protein kinase signaling, oxygen sensors and hypoxic induction of neurogenesis. Neurodegener Dis 3:50–55PubMedCrossRefGoogle Scholar
  30. Zhu LL, Wu LY, Yew DT, Fan M (2005) Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol 31:231–242PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Heinrich F. Bürgers
    • 1
  • Dominik W. Schelshorn
    • 1
  • Wolfgang Wagner
    • 1
    • 2
  • Wolfgang Kuschinsky
    • 1
  • Martin H. Maurer
    • 1
    • 3
  1. 1.Department of Physiology and PathophysiologyUniversity of HeidelbergHeidelbergGermany
  2. 2.Department of MedicineUniversity of HeidelbergHeidelbergGermany
  3. 3.SYGNIS Bioscience GmbH & Co KGHeidelbergGermany

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