Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: Protection with antioxidants and glutamate receptor antagonists

Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and... Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr after serum removal) we found significant impairment of mitochondrial functioning (3‐(4,5‐dimethyl thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay) and an increase in the percentage of neurons showing signs of DNA fragmentation (in situ terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants (1 mM: 1,4‐diazobicyclo(2.2.2)octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01–1 μM ascorbic acid). We also measured neuronal oxidation utilizing the oxidation‐sensitive fluorescent dye 2′,7′‐dichlorofluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min after serum deprivation. The blockade of glutamate receptors by (+)‐5‐methyl‐10,11‐dihydroxy‐5H‐dibenzo(a,d)cyclohepten‐5,10‐imine (MK‐801) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK‐801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress‐induced apoptosis. © 1996 Wiley‐Liss, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuroscience Research Wiley

Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: Protection with antioxidants and glutamate receptor antagonists

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Publisher
Wiley
Copyright
Copyright © 1996 Wiley‐Liss, Inc.
ISSN
0360-4012
eISSN
1097-4547
D.O.I.
10.1002/(SICI)1097-4547(19960215)43:4<465::AID-JNR7>3.0.CO;2-D
Publisher site
See Article on Publisher Site

Abstract

Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr after serum removal) we found significant impairment of mitochondrial functioning (3‐(4,5‐dimethyl thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay) and an increase in the percentage of neurons showing signs of DNA fragmentation (in situ terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants (1 mM: 1,4‐diazobicyclo(2.2.2)octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01–1 μM ascorbic acid). We also measured neuronal oxidation utilizing the oxidation‐sensitive fluorescent dye 2′,7′‐dichlorofluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min after serum deprivation. The blockade of glutamate receptors by (+)‐5‐methyl‐10,11‐dihydroxy‐5H‐dibenzo(a,d)cyclohepten‐5,10‐imine (MK‐801) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK‐801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress‐induced apoptosis. © 1996 Wiley‐Liss, Inc.

Journal

Journal of Neuroscience ResearchWiley

Published: Feb 15, 1996

References

  • Melatonin protects neurons from singlet oxygen‐induced apoptosis
    Cagnoli, Cagnoli; Atabay, Atabay; Kharlamova, Kharlamova; Manev, Manev
  • Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor
    Deckwerth, Deckwerth; Johnson, Johnson
  • Cloned glutamate receptors
    Hollmann, Hollmann; Heinemann, Heinemann
  • Oxidative stress induces apoptosis in embryonic cortical neurons
    Ratan, Ratan; Murphy, Murphy; Baraban, Baraban
  • Sodium, calcium and late potassium currents are reduced in cerebellar granule cells cultured in the presence of a protein complex conferring resistance to excitatory amino acids
    Zona, Zona; Ragozzino, Ragozzino; Cictti, Cictti; Mercanti, Mercanti; Avoli, Avoli; Brancati, Brancati; Calissano, Calissano

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