Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels
| Autor: | Giuseppe Campiani, Roger Griffiths, Clive Meredith, Annamarie Rogers, Gabriele Scholz, D. Clive Williams, Gabriele Schmuck, Arne Schousboe |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Cell Survival
Excitatory Amino Acids Health Toxicology and Mutagenesis Excitotoxicity Mice Inbred Strains Kainate receptor AMPA receptor Biology Toxicology medicine.disease_cause Receptors N-Methyl-D-Aspartate c-Fos Mice Cerebellum medicine Animals Excitatory Amino Acid Agents RNA Messenger Receptor 6-Cyano-7-nitroquinoxaline-2 3-dione Reverse Transcriptase Polymerase Chain Reaction Glutamate receptor General Medicine Molecular biology Reverse transcription polymerase chain reaction Gene Expression Regulation NIH 3T3 Cells biology.protein NMDA receptor Biological Assay Excitatory Amino Acid Antagonists Proto-Oncogene Proteins c-fos |
| Zdroj: | Archives of Toxicology. 79:129-139 |
| ISSN: | 1432-0738 0340-5761 |
| DOI: | 10.1007/s00204-004-0617-5 |
| Popis: | Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15-30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies. |
| Databáze: | OpenAIRE |
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