Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies.
This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.
Noraberg
Jens
J
NeuroScreen ApS, Anatomy and Neurobiology, University of Southern Denmark, Winslowparken 21, 5000 Odense, Denmark. jnoraberg@health.sdu.uk
D000826:Animal Testing Alternatives; D000818:Animals; D001921:Brain / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects; D007608:Kainic Acid / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009410:Nerve Degeneration; D009474:Neurons / Q000187:drug effects; D009498:Neurotoxins / Q000633:toxicity*; D009924:Organ Culture Techniques / Q000379:methods*; D011419:Propidium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018675:Toxicity Tests / Q000379:methods*
D009498:Neurotoxins; D011419:Propidium; D018698:Glutamic Acid; D007608:Kainic Acid