Elsevier

Neuroscience

Volume 105, Issue 2, 27 July 2001, Pages 287-297
Neuroscience

Intracellular generation of free radicals and modifications of detoxifying enzymes in cultured neurons from the developing rat forebrain in response to transient hypoxia

https://doi.org/10.1016/S0306-4522(01)00189-0Get rights and content

Abstract

To address the influence of oxidative stress and defense capacities in the effects of transient hypoxia in the immature brain, the time course of reactive oxygen species generation was monitored by flow cytometry using dihydrorhodamine 123 and 2′,7′-dichlorofluorescein-diacetate in cultured neurons issued from the fetal rat forebrain and subjected to hypoxia/reoxygenation (6 h/96 h). Parallel transcriptional and activity changes of superoxide dismutases, glutathione peroxidase and catalase were analyzed, in line with cell outcome. The study confirmed hypoxia-induced delayed apoptotic death, and depicted increased mitochondrial and cytosolic productions of free radicals (+30%) occurring over the 48-h period after the restoration of oxygen supply, with sequential stimulations of superoxide dismutases. Whereas catalase mRNA levels and activity were augmented by cell reoxygenation, glutathione peroxidase activity was transiently repressed (−24%), along with reduced glutathione reductase activity (−27%) and intracellular glutathione depletion (−19%).

Coupled with the neuroprotective effects of the glutathione precursor N-acetyl-cysteine (50 μM), these data suggest that hypoxia/reoxygenation-induced production of reactive oxygen species can overwhelm glutathione-dependent antioxidant capacity, and thus may contribute to the resulting neuronal apoptosis.

Section snippets

Neuronal cell culture and exposure to hypoxia

Animal experimentation was carried out with the highest standards of animal care and housing, according to the N.I.H. Guide for the Care and Use of Laboratory Animals. Primary cultured neurons were obtained from 14-day-old rat embryo forebrains as previously described (Chihab et al., 1998a, Chihab et al., 1998b, Bossenmeyer-Pourié et al., 1999b, Bossenmeyer-Pourié et al., 2000b, Lièvre et al., 2000). When they were in the proestrus period, as shown by the observation of daily vaginal smears,

Neuronal injury

By using antibodies raised against neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), immunohistochemical characterization of culture preparations grown for 6 days in chemically defined medium confirmed the high proportion of differentiated neuronal cells, showing 92.5±2.8% of NSE-positive cells, with 7.6±1.6% of GFAP-positive cells (data not shown).

Transient exposure to the anaerobic gas mixture usually reduced the partial pressure of oxygen (PO2) in the culture medium

Discussion

Due to its metabolic properties, oxidative capacities and specific composition, the brain is particularly sensitive to oxidative damage. Within the CNS, the response to oxidative stress has been shown to vary according to the various cellular phenotypes, and the antioxidant capacity of neurons is lower than that of glia (Makar et al., 1994). Furthermore, it has been reported that the immature brain exhibits reduced defense mechanisms against oxidative events (Mishra and Delivoria-Papadopoulos,

Acknowledgements

V.L. wishes to express her gratitude to Mrs. Catherine Charoy for her generous support.

References (85)

  • V Castagne et al.

    Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system

    Prog. Neurobiol.

    (1999)
  • R Cathcart et al.

    Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay

    Anal. Chem.

    (1983)
  • C.K Chang et al.

    Changes of superoxide dismutase (SOD) mRNA and activity in response to hypoxic stress in cultured Wistar rat glioma cells

    Neurosci. Lett.

    (1997)
  • R Chihab et al.

    Sequential activation of activator protein-1-related transcription factors and JNK protein kinases may contribute to apoptotic death induced by transient hypoxia in developing brain neurons

    Mol. Brain Res.

    (1998)
  • D.W Choi

    Ischemia-induced neuronal apoptosis

    Curr. Opin. Neurobiol.

    (1996)
  • P Chomczynski et al.

    Single-step method of RNA isolation by acid guanidium-thiocyanate-phenol-chloroform extraction

    Anal. Biochem.

    (1987)
  • R Del Maestro et al.

    Subcellular localization of superoxide dismutases, glutathione peroxidase and catalase in developing rat cerebral cortex

    Mech. Ageing Dev.

    (1989)
  • R Dringen et al.

    N-acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain

    Neurosci. Lett.

    (1999)
  • E.D Hall et al.

    Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation

    Free Radic. Biol. Med.

    (1989)
  • M.B Hansen et al.

    Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill

    J. Immunol. Methods

    (1989)
  • H.M Hassan

    Biosynthesis and regulation of superoxide dismutases

    Free Radic. Biol. Med.

    (1988)
  • S.L Hempel et al.

    Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123

    Free Radic. Biol. Med.

    (1999)
  • Y.S Ho et al.

    Cloning and characterization of the rat glutathione peroxidase gene

    FEBS Lett.

    (1992)
  • V Lièvre et al.

    Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture

    Free Radic. Biol. Med.

    (2000)
  • R Liu et al.

    Oxygen free radicals mediate the induction of manganese superoxide dismutase gene expression by TNF-α

    Free Radic. Biol. Med.

    (2000)
  • D Maulik et al.

    Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

    Brain Res.

    (1998)
  • O.P Mishra et al.

    Anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia

    Dev. Brain Res.

    (1988)
  • O.P Mishra et al.

    Lipid peroxidation in developing fetal guinea pig during normoxia and hypoxia

    Dev. Brain Res.

    (1989)
  • O.P Mishra et al.

    Cellular mechanisms of hypoxic injury in the developing brain

    Brain Res. Bull.

    (1999)
  • P Moldeus et al.

    N-acetylcysteine

    Methods Enzymol.

    (1994)
  • H Mover et al.

    Antioxidant enzymatic activity in embryos and placenta of rats chronically exposed to hypoxia and hyperoxia

    Comp. Biochem. Physiol.

    (1997)
  • C.S Niu et al.

    Modification of superoxide dismutase (SOD) mRNA and activity by a transient hypoxic stress in cultured glial cells

    Neurosci. Lett.

    (1998)
  • T Ohtsuki et al.

    Effect of transient forebrain ischemia on superoxide dismutases in gerbil hippocampus

    Brain Res.

    (1993)
  • G Rothe et al.

    Flow cytometric measurement of the respiratory burst activity of phagocytes using dihydrorhodamine 123

    J. Immunol. Methods

    (1991)
  • J.A Royall et al.

    Evaluation of 2′,7′-dichlorofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells

    Arch. Biochem. Biophys.

    (1993)
  • D.C Salo et al.

    Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

    Free Radic. Biol. Med.

    (1988)
  • S Schoonbroodt et al.

    Oxidative stress interference with the nuclear factor-κB activation pathway

    Biochem. Pharmacol.

    (2000)
  • R.S Sidhu et al.

    Nuclear condensation and fragmentation following cerebral hypoxia-ischemia occurs more frequently in immature than older rats

    Neurosci. Lett.

    (1997)
  • P.M Sinet et al.

    Inactivation of the human CuZn superoxide dismutase during exposure to O2radical dot and H2O2

    Arch. Biochem. Biophys.

    (1981)
  • A.K Singh et al.

    A novel glutathione peroxidase in bovine eye

    J. Biol. Chem.

    (1998)
  • J.A Smith et al.

    Further characterization of the neutrophil oxidative burst by flow cytometry

    J. Immunol. Methods

    (1993)
  • I Stoian et al.

    Apoptosis and free radicals

    Biochem. Mol. Med.

    (1996)
  • Cited by (59)

    • NO-dependent protective effect of VEGF against excitotoxicity on layer VI of the developing cerebral cortex

      2012, Neurobiology of Disease
      Citation Excerpt :

      In term infants, brain lesions more frequently affect gray matter structures and an important neuronal death is currently described (Mishra et al., 2001). Several mechanisms such as depletion of adenosine triphosphate (Garnier et al., 2002), reduction of neurotrophin levels (Riikonen et al., 1999), induction of reactive oxygen species (Lievre et al., 2001) and inflammation (Aly et al., 2006) are involved in hypoxia–ischemia-induced cell death. Among them, the massive release of glutamate by presynaptic neurons is thought to play a key role in the development of neonatal brain lesions through induction of an excitotoxic process (Jensen, 2002; Johnston, 2005; Laudenbach et al., 2001).

    • Modes of Action of Taurine and Granulocyte Colony-stimulating Factor in Neuroprotection

      2012, Journal of Experimental and Clinical Medicine
      Citation Excerpt :

      Based on the current literature, it is apparent that taurine protects against hypoxia-induced apoptosis by preventing mitochondrial dysfunction.60 Calcium overload and ionic imbalances in neurons induce mitochondria to produce free radicals.61,62 Elevated levels of ROS is a hallmark of neurodegenerative diseases, especially Parkinson’s disease.63

    • Preventive effect of Piracetam and Vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture

      2011, Food and Chemical Toxicology
      Citation Excerpt :

      Hypoxia has been implicated in neurodegenerative disorders and is a key mediator for neuronal energy depletion, excitotoxic cellular injury and enhanced free radical production, that subjugates the antioxidant scavenging capacity, causing oxidative damage of DNA, lipids and proteins, and leads to development of cellular damage (Acker and Acker, 2004; Lievre et al., 2001; Maiti et al., 2006).

    • Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: Findings from a human, in-vitro hypoxia model

      2010, European Journal of Pharmacology
      Citation Excerpt :

      Cell cultures grown for 2 h under hypoxic conditions also contained numerous rounded cells displaying signs of detachment from the growth surface (Fig. 1B, C; 24 h post hypoxia). Moreover, semiquantitative RT-PCR suggested that the relative gene expression levels of several typical hypoxia associated genes (Irwin et al., 2009; Lievre et al., 2001; Rauchova et al., 2005; Said et al., 2007) were regulated under hypoxic conditions in IMR-32 cells (VEGF: normoxia: 0.06 ± 0.02 a.u, hypoxia: 0.15 ± 0.09 a.u, P > 0.05. CAT: normoxia: 0.05 ± 0.01 a.u, hypoxia: 0.20 ± 0.10 a.u, P < 0.05.

    View all citing articles on Scopus
    View full text