A beta Mediated Diminution of MTT Reduction-An Artefact of Single Cell Culture?

作者：Roenicke Raik; Klemm Anja; Meinhardt Jessica; Schroeder Ulrich H; Faendrich Marcus; Reymann Klaus G

来源：PLos One, 2008, 3(9): e3236.

DOI：10.1371/journal.pone.0003236

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Ab) toxicity in different types of single cell culture. To our knowledge, the influence of Ab on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Ab species, namely freshly dissolved Ab (25-35), fibrillar Ab (1-40), oligomeric Ab (1-42) and oligomeric Ab (1-40). In contrast to the findings in single cell cultures, none of these Ab species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Ab to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Ab also did not influence the MTT reduction in the respective tissue. Failure of Ab penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Ab (1-40), but not by freshly dissolved Ab (25-35) or fibrillar Ab (140). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Ab species on MTT reduction. Particularly, the differential effect of oligomeric versus other Ab forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Ab oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Ab, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

• 出版日期2008-9-18