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

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.

Novel peptides secreted from human neuroblastoma: useful clinical tools?

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Treatment with nimodipine or mannitol reduces programmed cell death and infarct size following focal cerebral ischemia

“…Oxidative stress is one trigger of apoptotic cell death and treatment of cells with a variety of different exogenous antioxidants blocks apoptosis [5,7,8,44,45,[54][55][56]. However, it was demonstrated that hypertonic stress and mannitol could induce programmed cell death in vascular endothelial cells [36].…”

Lactosylceramide is required in apoptosis induced by N-Smase

“…Pro-oxidants and redox cycling agents [18][19][20][21] can induce apoptosis. Other apoptotic stimuli, such as treatment with TNF-α [22] lipopolysaccharide [23], growth factor withdrawal [24], and human immunodeficiency virus infection [25], can stimulate ROS production. Studies with TNF-α have shown that these agents activate ROS generation by mitochondria [22].…”