Cell Death In Primary Cultures Research Articles

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor.

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3alpha-ol-5beta-pregnan-20-one hemisuccinate (3alpha5betaHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3alpha5betaHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3alpha5betaHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3alpha5betaHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

Potentiation by K+ of anoxic release of newly synthesized neuronal glutamate.

Glutamate synthesis from [14C]glutamine, release of newly synthesized, labelled glutamate and cell death in primary cultures of the glutamatergic cerebellar granule cell neurones under anoxic conditions were increased by an elevation of the extracellular potassium concentration. Phenylsuccinate, an inhibitor of transmitochondrial transport and hence of glutamate synthesis from glutamine, decreased the potassium-enhanced glutamate synthesis and the release of newly synthesized glutamate and reduced cell death. Since the extracellular concentration of potassium is elevated during brain anoxia and glutamate neurotoxicity is thought to contribute to neuronal cell death under this condition, these observations may be of functional and potentially therapeutic relevance.

Vinpocetine is a highly potent neuroprotectant against veratridine-induced cell death in primary cultures of rat cerebral cortex

The effects of vinpocetine and phenytoin against veratridine-induced cell death were investigated in primary cultures of rat cerebral cortex. Toxicity was evaluated by phase contrast microscopy and quantified by measuring lactic dehydrogenase leakage from damaged cells. Vinpocetine was highly potent in inhibiting the cell death evoked by veratridine. The concentrations of the drug evoking 50% protection (IC 50 values) against 100μM (maximal response) and 50μM (half-maximal response) veratridine were 490 nM and 63 nM, respectively. The protective efficacy of vinpocetine exceeded about 100-fold that of phenytoin (IC 50 = 44.2 μM against 100μM veratridine), a prototype sodium-channel blocker. These data suggest that the blockade of voltage-gated sodium channels is a possible mechanism of action for the well-known neuroprotective and anticonvulsant properties of vinpocetine.

Serum amyloid P component-induced cell death in primary cultures of rat cerebral cortex

The influence of serum amyloid P component (SAP) on the survival of rat cerebrocortical cultures was tested. Cytotoxic cell death was examined on 8–9-day-old cell cultures by phase contrast microscopy and quantified by the measurement of lactate dehydrogenase (LDH) leakage. SAP (16–48 nM) evoked a concentration-dependent cell death within 24 h exposure. Our results suggest that SAP, as contituent of cerebral amyloid deposits, may play a role in the pathomechanism of Alzheimer's disease.

Cell death in primary cultures of mouse neurons and astrocytes during exposure to and ‘recovery’ from hypoxia, substrate deprivation and simulated ischemia

Effects of hypoxia, substrate deprivationand simulated ischemia (combined hypoxia and substrate deprivation) on cell survival during the insult itself and during a 24 h ‘recovery’ period were studied in primary cultures of mouse astrocytes and in cerebral cortical neuronal-astrocytic co-cultures. Cell death was determined by release of the cytosolic high molecular enzyme lactate dehydrogenase (LDH) as well as morphologically (retention of staining with rhodamine 123 and lack of staining with propidium iodide as an indicator of live cells). Glutamate concentrations were measured in the incubation media at the end of the metabolic insults. Astrocytes were very resistant to hypoxia, but less so to simulated ischemia; under both conditions the glutamate concentrations in the media remained low. Cerebral cortical neurons were almost equally susceptible to damage by hypoxia and by stimulated ischemia, although hypoxia had a faster deleterious effects on some of the neurons and simulated ischemia during a long-term insult (9 h) killed all neurons, whereas a non-negligible neuronal subpopulation survived 9 h of hypoxia. Neuronal cell death after long-term hypoxia (but not after simulated ischemia) was correlated with high concentrations of glutamate in the incubation media. After certain insults, most notably relatively short lasting simulated ischemia (3 h) in neurons (which caused no increased cell death during the insult), there was a large release of LDH during the ‘recovery’ period.

Vimentin-crossreactive antibodies induce cell death in primary cultures of embryonic spinal cord

Sera from guinea pigs inoculated with spinal cord gray matter were cytotoxic for embryonic day 14 (ED14) primary spinal cord cell cultures. All cell types including astroglia and neurons were affected. Immunoglobulin (IgG) from inoculated animals and biologically active complement proteins were both identified as necessary components for the observed cytotoxicity. Adsorption with cytoskeletal constituents removed the cytotoxic effect. Adsorption with purified vimentin substantially reduced cytotoxicity, while adsorption with glial fibrillary acid protein (GFAP) had no cytoprotective effects. Antiserum from vimentin-immunized guinea pigs was also toxic to spinal cord cell cultures. Antibody and complement-mediated toxicity appeared to result from interaction with vimentin bound to the surface of embryonic cultured glia and neurons, although interaction with a cross-reacting epitope could not be definitely excluded. Cytotoxic vimentin-directed antibody was not noted in control sera, and the presence of this antibody in guinea pig and human sera did not correlate with the clinicopathological state.

Vinpocetin protects against excitotoxic cell death in primary cultures of rat cerebral cortex

The protective effect of vinpocetin, a drug clinically useful in brain hypoxia/ischemia, was examined in vitro on cerebrocortical cultures treated with glutamate and related excitotoxins. The extent of cell death was quantified by measuring lactic dehydrogenase activity released from damaged cells into the culture medium. Vinpocetin partially protected the cortical cells against cell death induced by N-methyl-D-aspartate, quisqualate and kainate, indicating that the drug exerts a direct protective action on cerebrocortical cells bearing excitatory amino acid receptors.

Strychnine protection against excitotoxic cell death in primary cultures of rat cerebral cortex

Strychnine-insensitive glycine receptors are known to modulate the toxicity of excitatory amino acids via an allosteric action at the N-methyl-D-aspartate receptor complex. To elucidate whether strychnine-sensitive glycine receptors may also influence excitotoxicity, the effect of strychnine on the excitotoxic cell death was examined in primary cultures of the rat cerebral cortex. To exclude any interference at the N-methyl-D-aspartate receptor complex, cell death was evoked by kainic acid. The release of lactic dehydrogenase (LDH) into the culture medium was taken as a quantitative measure of cell death. Strychnine reduced the excitotoxic cell death in a concentration-dependent fashion. This finding indicates that glycine may modulate the vulnerability of cortical cells to excitotoxic insults not only via the strychnine-insensitive population of glycine receptors within the N-methyl-D-aspartate receptor-complex, but also via strychnine-sensitive receptor channels.