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Most in vitro models are only used to assess the short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2–3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100μM L-AP3, with PI (2μM) in the culture medium. Exposure to 100μM L-AP3 induced severe toxicity after 4–6 days, as shown by massive PI uptake, LDH leakage, and changes in MAP2 and GFAP immunostaining and in Nissl and Timm staining. In contrast, 10μM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist, NBQX, together with 100μM L-AP3, reduced neurodegeneration to close to control values. It is concluded that the continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies on inducible neuronal degeneration over time.
Alternatives to Laboratory Animals – SAGE
Published: May 1, 2007
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