Lack of Correlation Between the Effects of Transient Exposure to Glutamate and Those of Hypoxia/Reoxygenation in Immature Neurons In Vitro

Abstract

To assess the influence of brain immaturity on the effects of oxygen deprivation and the participation of excitotoxicity, the consequences of a 6‐h exposure to either hypoxia (95% N2/5% CO2) or 100 µM glutamate were studied in cultured fetal rat forebrain neurons taken at two maturational stages, i.e., 6 and 13 days in vitro. Cells were examined for their morphology, viability, energy metabolism reflected by 2‐d‐[3H]deoxyglucose uptake, and protein synthesis assessed by [3H]leucine incorporation. Apoptosis and necrosis were scored using the fluorescent dye 4,6‐diamidino‐2‐phenylindole. Whereas 6‐day‐old neurons responded to a 6‐h hypoxia by transient hypermetabolism, biphasic increase in protein synthesis, and cycloheximide‐sensitive apoptotic death within 72 h postexposure, glutamate did not affect cell characteristics by the same time. In 13‐day‐old neurons, hypoxia induced both apoptosis (8.2%) and necrosis (22.3%). At this age, glutamate definitely reduced energy metabolism (26%) and protein synthesis (17%) by the end of exposure. The percentage of necrotic neurons reached 40.7%, but the rate of apoptosis was unchanged compared with controls. Therefore, excitotoxicity cannot account for hypoxia‐induced injury in immature neurons, but its participation is suggested in older cells by the suppression of the necrotic component of hypoxia by glutamate receptor antagonists at 13 days.

Bilirubin Exerts Additional Toxic Effects in Hypoxic Cultured Neurons from the Developing Rat Brain by the Recruitment of Glutamate Neurotoxicity

“…These findings are consistent with earlier studies that demonstrated a lack of correlation between the effects of exposure to glutamate and those of transient hypoxia in immature neurons (53,54). Although the presence of functional receptors for glutamate, including NMDA receptors, has been demonstrated in cultured neurons at the corresponding maturational stage (55), these neurons, in contrast to more mature cells, are not sensitive to toxic concentrations of either glutamate or one of its derivatives, such as NMDA, kainate, or AMPA (␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate) (54,56). Pharmacological studies have revealed that a loss of protein kinase C activity is necessary for glutamate-mediated induction of neurotoxicity.…”

“…This phenomenon appears to be an essential element of the excitotoxic death of neuronal cells, and cellular events responsible for linking glutamate-associated Ca 2ϩ influx to protein kinase C inactivation are undeveloped in such immature neurons in vitro (53,54,57). Specifically in our model, we have previously shown that neurons became vulnerable to the application of glutamate when they were co-exposed to 30 nM staurosporine, a protein kinase C inhibitor, and that cells can be rescued by glutamate receptor antagonists (54,56). On the other hand, accumulative evidence has been provided to demonstrate that bilirubin inhibits protein kinase C activity with subsequent blockade of protein kinase C-mediated phosphorylation of endogenous substrates in various tissue systems.…”

NMDA Channel Antagonist MK-801 Does Not Protect against Bilirubin Neurotoxicity

“…Cells partly compensate by uptake of calcium into intracellular organelles, disrupting the electrochemical potential across the inner mitochondrial membrane and inhibiting ATP production leading to apoptosis [44] . In some models (ischemia, epilepsy and traumatic brain injury), blocking calcium entry with the NMDA channel antagonist MK-801 prevents excitotoxicity [45,46] .…”

“…Since we grow the glial feeder layer for two weeks prior to plating the neurons, it is unlikely that we have significant glial cell loss, to contribute to the neuronal damage. Additionally, in this cell culture model system a 10-min exposure to glutamate kills almost all the cells and NMDA antagonists are typically protective [33,46,54,55] .…”

Transforming Growth Factor-α Attenuates N-Methyl-D-aspartic Acid Toxicity in Cortical Cultures by Preventing Protein Synthesis Inhibition through an Erk1/2-dependent Mechanism

“…Mnk1, a kinase activated by Erk1/2 and p38 (19), phosphorylates eIF4E. Cellular stresses such as excitotoxicity or hydrogen peroxide in neurons (12,20,21), oxygen and glucose deprivation (22), and serum deprivation (23) in PC12 cells down-regulate protein synthesis. On the other hand, growth factors and neurotrophins enhance translation in cultured neurons, but different agents regulate specific translation factors through activation of Akt and/or Erk1/2 signaling pathways (24 -26).…”