Unfavorable effect of levetiracetam on cultured hippocampal neurons after hyperthermic injury.
BACKGROUND
The aim of this study was to examine the viability of neurons and the putative neuroprotective effects of second-generation antiepileptic drug, levetiracetam (LEV), on cultured hippocampal neurons injured by hyperthermia.


METHODS
Primary cultures of rat's hippocampal neurons at 7day in vitro (DIV) were incubated in the presence or absence of LEV in varied concentrations under hyperthermic conditions. Cultures were heated in a temperature of 40°C for 24h or in a temperature of 41°C for 6h. Flow cytometry with Annexin V/PI staining as well as fluorescent microscopy assay were used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, the release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Assessment was performed after 9DIV and 10 DIV.


RESULTS
Incubation of hippocampal cultures in hyperthermic conditions resulted in statistically significant increase in the number of injured neurons when compared with non-heated control cultures. Intensity of neuronal destruction was dependent on temperature-value. When incubation temperature 40°C was used, over 80% of the population of neurons remained viable after 10 DIV. Under higher temperature 41°C, only less than 60% of neurons were viable after 10 DIV. Both apoptotic and necrotic pathways of neuronal death induced by hyperthermia were confirmed by Annexin V/PI staining.


CONCLUSIONS
LEV showed no neuroprotective effects in the current model of hyperthermia in vitro. Moreover, drug, especially when used in higher concentrations, exerted unfavorable intensification of aponecrosis of cultured hippocampal neurons.
Department of Pediatric Neurology and Rehabilitation, Medical University of Bialystok, Białystok, Poland. Electronic address: krsen@wp.pl.
Krzysztof
K
Sendrowski
Department of Pediatric Neurology and Rehabilitation, Medical University of Bialystok, Białystok, Poland.
Piotr
P
Sobaniec
Department of Pediatric Laboratory Diagnostic, Medical University of Bialystok, Białystok, Poland.
Elżbieta
E
Poskrobko
Department of Hematological Diagnostics, Medical University of Bialystok, Białystok, Poland.
Małgorzata
M
Rusak
Department of Pediatric Neurology and Rehabilitation, Medical University of Bialystok, Białystok, Poland.
Wojciech
W
Sobaniec
D000818:Animals; D000927:Anticonvulsants / Q000008:administration & dosage* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005334:Fever / Q000473:pathology*; D006624:Hippocampus / Q000187:drug effects / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D000077287:Levetiracetam; D009336:Necrosis / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D018696:Neuroprotective Agents / Q000008:administration & dosage* / Q000633:toxicity; D010889:Piracetam / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley
D000927:Anticonvulsants; D018696:Neuroprotective Agents; D000077287:Levetiracetam; D007770:L-Lactate Dehydrogenase; D010889:Piracetam
Aponecrosis; Hippocampal culture; Hyperthermia; Levetiracetam