Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures.
Generation of free radicals may have a key role in the nerve cell damage induced by prolonged or frequently recurring convulsions (status epilepticus). Mitochondrial function may also be altered due to production of free radicals during seizures. We therefore studied changes in field potentials (fp) together with measurements of extracellular, intracellular, and intramitochondrial calcium concentration ([Ca(2+)]e, [Ca(2+)]i, and [Ca(2+)]m, respectively), mitochondrial membrane potential (deltapsi), NAD(P)H auto-fluorescence, and dihydroethidium (HEt) fluorescence in hippocampal slice cultures by means of simultaneous electrophysiological and microfluorimetric measurements. As reported previously, each seizure-like event (SLE) resulted in mitochondrial depolarization associated with a delayed rise in oxidation of HEt to ethidum, presumably indicating ROS production. We show here that repeated SLEs led to a decline in intracellular and intramitochondrial Ca(2+) signals despite unaltered Ca(2+) influx. Also, mitochondrial depolarization and the NAD(P)H signal became smaller during recurring SLEs. By contrast, the ethidium fluorescence rises remained constant or even increased from SLE to SLE. After about 15 SLEs, activity changed to continuous afterdischarges with steady depolarization of mitochondrial membranes. Staining with a cell death marker, propidium iodide, indicated widespread cell damage after 2 h of recurring SLEs. The free radical scavenger, alpha-tocopherol, protected the slice cultures against this damage and also reduced the ongoing impairment of NAD(P)H production. These findings suggest involvement of reactive oxygen species (ROS) of mitochondrial origin in the epileptic cell damage and that free radical scavenging may prevent status epilepticus-induced cell loss.
Kovács
Richard
R
Department of Neurochemistry, Chemical Institute, Chemical Research Center, Hungarian Academy of Sciences, Budapest 1025, Hungary.
Schuchmann
Sebastian
S
Gabriel
Siegrun
S
Kann
Oliver
O
Kardos
Julianna
J
Heinemann
Uwe
U
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D046508:Culture Techniques; D004996:Ethidium / Q000493:pharmacokinetics; D005110:Extracellular Space / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005609:Free Radicals / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D007425:Intracellular Membranes; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D009249:NADP / Q000502:physiology; D009994:Osmolar Concentration; D051381:Rats; D013226:Status Epilepticus / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D024502:alpha-Tocopherol / Q000494:pharmacology
D005456:Fluorescent Dyes; D005609:Free Radicals; D009249:NADP; D004996:Ethidium; D024502:alpha-Tocopherol; D002118:Calcium