Comparison of neurotoxicity induced by some glutathione depletors in mouse cortical cell cultures

We examined the neurotoxic effects of 3 glutathione (GSH) depletors, buthionine sulfoximine (BSO), diethyl maleate (DEM) and phorone, under the presence of trolox, cycloheximide (CHX), pyrrolidine dithiocarbamate (PDTC) or MK-801 in primary mouse cortical cell cultures. All three depletors induced neuronal death in dose and exposure time dependent manner, and decreased total cellular GSH contents. The patterns of the neuronal death and the GSH decrements were dependent on the individual agents. DEM (200 micrometer) induced rapid and irreversible decrement of the GSH. BSO (1 mM) also decreased the GSH irreversibly but the rate of decrement was more progressive than that of DEM. Phorone (1 mM) reduced the GSH content to 40% by 4 hr exposure, that is comparable to the decrement of BSO, but the GSH recovered and reached over the control value by 36 hr exposure. BSO showed a minimal neurotoxicity (0-10%) at the end of 24 hr exposure, but marked neuronal cell death at the end of 48 hr exposure. The BSO (1 mM)-induced neurotoxicity was markedly inhibited by trolox or CHX and partially attenuated by MK-801. DEM induced dose-dependent cytotoxicity at the end of 24 hr exposure. Over the doses of 400 micrometer, glial toxicity also appeared. DEM (200 micrometer)-induced neurotoxicity was markedly inhibited by trolox or PDTC. Phorone (1 mM) induced moderate neurotoxicity (40%) at the end of 48 hr exposure. Only CHX showed significant inhibitory effect on the phorone-induced neurotoxicity. These results suggest that the GSH depletors induce neuronal injury via different mechanisms and that GSH depletors should be carefully employed in the researches of neuronal oxidative injuries.

Effect of Vasoactive Intestinal Peptide on the Necrotic or Apoptotic Neuronal Death in Murine Cortical Cell Cultures

OBJECTIVE: The purpose of this study was to examine the effects of VIP on the apoptotic neuronal death induced by serum deprivation and the necrotic(excitotoxic) neuronal death by the exposure of NMDA in primary murine cortical cell cultures. MATERIALS AND METHODS: Near-pure neuronal cell cultures were prepared by plating fetal mice cortical cells onto polyethyleneimine-coated 24 well vessels. At 7 days in vitro(DIV), serum was removed from culture media for 24 hrs in near-pure neuronal cultures. Mixed cortical cell cultures containing both neurons and glia were prepared by plating fetal mice cortical cells onto an established monolayer of glia. At 12-14 DIV, excitotoxic neuronal death was induced by the addition of NMDA into the mixed cortical cultures. The neuronal cell death was assessed by either MTT or LDH assay after microscopic examination. RESULTS: Near-pure neuronal cell cultures deprived of serum undergo neuronal apoptosis marked by cell body shrinkage, chromatin condensation and DNA ladders. The apoptotic neuronal death following serum deprivation was significantly attenuated by the inclusion of a membrane-permeable cAMP analogue(8-bromo-cAMP; 100nM), an adenyl cyclase stimulator(forskolin; 10nM), a protein synthesis inhibitor(cycloheximide; 0.1ng/ml) or cell membrane depolarization(25mM KCl) during serum deprivation, but was not affected by the addition of an NMDA antagonist (MK-801; 10nM) or an antioxidant(trolox; 100nM). The inclusion of VIP(1, 3, 10nM) during deprivation also significantly prevented the neuronal death without dose-dependency. The neuroprotective effect of VIP(1nM) was not reversed by concomitant treatment with a VIP receptor antagonist([D-p-Cl-Phe6, Leu17]-VIP; 10, 30nM). Neuronglia co-cultures exposed to 300nM NMDA for 5 min produced neuronal death marked by cell body swelling. The neuronal death induced by the exposure of NMDA was markedly attenuated by MK-801 but not affected by 8-bromo-cAMP, forskolin, cycloheximide, high potassium or VIP. CONCLUSION: These results provide an evidence that VIP prevent apoptotic neuronal death induced by serum deprivation and suggest that VIP may have therapeutic potential for diseases in central nervous system linked to apoptotic neuronal death.

Effect of Trolox, Cycloheximide or MK-801 on the Neuronal Cell Death Induced by FeCl2, Buthionine Sulfoximine or KCN in Primary Murine Mixed Cortical Cell Culture

Oxidative stress is known to be a major neuropathologic mechanism in chronic neurodegenerative disorders as well as in stroke, trauma and epilepsy, and many kinds of oxidative insults induce neuronal injury. The purpose of this study was to examine the temporal effects of trolox(TLX; water and lipid soluble vitamin E analog), cycloheximide(CHX; protein synthesis inhibitor) and MK-801(NMDA receptor antagonist) on neuronal death indu-ced by different kinds of oxidative insults in primary murine mixed cortical cell culture(14-16 days in vitro), and to gain information on the mechanisms underlying oxidative cell death. As oxidative insults, the authors used iron(FeCl2), buth-ionine sulfoximine(BSO; glutathione depletor) and potassium cyanide(KCN; ATP depletor). Cell death was assessed by measurement of LDH efflux to bathing media at the end of exposure. All three agents induced neuronal cell death associated with cell body swelling. FeCl2(30nM-1mM) induced conce-ntration- and exposure time-dependent neurotoxicity, while BSO(10nM-3mM) showed little neurotoxicity at the end of 24 hrs exposure, but marked neuronal cell death at the end of 48 hrs; at concentrations of over 100uM of BSO neurotoxicity reached a plateau. KCN(0.1mM-1mM) also showed dose-dependent neurotoxicity. TLX(100nM) did not affect the neurotoxicity induced by KCN(1mM) but almost completely block BSO(1mM)- or FeCl2(100, 300nM)- induced neuronal cell death. CHX(1ng/ml) significantly attenuated BSO-induced cell death but did not protect against KCN(1mM)-induced cell death. CHX treatment, on the other hand, significantly potentiated FeCl2(100 or 300nM)-induced death. MK-801(10nM) markedly inhibited KCN-induced cell death but had no effect on FeCl2-induced death. MK-801 also significantly attenuated BSO-induced neurotoxicty after exposure for 48hrs but this protective effect disappeared at the end of 72hr. These results suggest that protein synthesis as well as lipid peroxidation of cell membrane may involve oxidative neuronal injury and that one oxidative agent may induce various cell death processes.