Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis.

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H(2)O(2). Cell viability was quantified by spectrophotometry, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 microM) or H(2)O(2) (50-800 microM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H(2)O(2) treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H(2)O(2) - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo.

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro, KA (100-500 microM) induced a concentration-dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo, twenty-four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.