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Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

Rönicke R, Klemm A, Meinhardt J, Schröder UH, Fändrich M, Reymann KG - PLoS ONE (2008)

Bottom Line: Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue.Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay.This may indicate that the Abeta 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 Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

View Article: PubMed Central - PubMed

Affiliation: Project Group Neuropharmacology, Leibniz Institute for Neurobiology, Magdeburg, Germany. roenicke@zenit-magdeburg.de

ABSTRACT
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 (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta 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 Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta 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 Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta 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 Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

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Related in: MedlinePlus

Influence of Aβ on MTT reduction activity of OHC and single cells, generated from OHC.A) Aβ (25-35) 1 µM was applied to the slice for 3 days. The MTT assay was done 2 hours after the preparation of the single cells out of the slice. In this case, 1 µM Aβ did not diminish the MTT reduction of OHC and single cells; B) 1 µM Aβ was applied to the slices and single cells after the preparation for 2 days. In this case, Aβ (25-35) 1 µM significantly reduced the MTT reduction of single cells, compared to control; * = p≤0.05, Mann–Whitney U-test, n = 10 per group.
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pone-0003236-g003: Influence of Aβ on MTT reduction activity of OHC and single cells, generated from OHC.A) Aβ (25-35) 1 µM was applied to the slice for 3 days. The MTT assay was done 2 hours after the preparation of the single cells out of the slice. In this case, 1 µM Aβ did not diminish the MTT reduction of OHC and single cells; B) 1 µM Aβ was applied to the slices and single cells after the preparation for 2 days. In this case, Aβ (25-35) 1 µM significantly reduced the MTT reduction of single cells, compared to control; * = p≤0.05, Mann–Whitney U-test, n = 10 per group.

Mentions: Considering our conflicting findings in single cells and OHCs it appeared likely that the susceptibility of cells to Aβ mediated diminution of MTT reduction activity depends on their environment. To address this matter, we split one OHC preparation into two groups. One group was cultivated further and the other group was separated into single cells. For the first time we prepared single cells from OHCs. Because of the matured state of the isolated cells only few neurons survived the isolation procedure and thus the cultures consisted largely of astrocytes (Figure 3).


Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

Rönicke R, Klemm A, Meinhardt J, Schröder UH, Fändrich M, Reymann KG - PLoS ONE (2008)

Influence of Aβ on MTT reduction activity of OHC and single cells, generated from OHC.A) Aβ (25-35) 1 µM was applied to the slice for 3 days. The MTT assay was done 2 hours after the preparation of the single cells out of the slice. In this case, 1 µM Aβ did not diminish the MTT reduction of OHC and single cells; B) 1 µM Aβ was applied to the slices and single cells after the preparation for 2 days. In this case, Aβ (25-35) 1 µM significantly reduced the MTT reduction of single cells, compared to control; * = p≤0.05, Mann–Whitney U-test, n = 10 per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2529401&req=5

pone-0003236-g003: Influence of Aβ on MTT reduction activity of OHC and single cells, generated from OHC.A) Aβ (25-35) 1 µM was applied to the slice for 3 days. The MTT assay was done 2 hours after the preparation of the single cells out of the slice. In this case, 1 µM Aβ did not diminish the MTT reduction of OHC and single cells; B) 1 µM Aβ was applied to the slices and single cells after the preparation for 2 days. In this case, Aβ (25-35) 1 µM significantly reduced the MTT reduction of single cells, compared to control; * = p≤0.05, Mann–Whitney U-test, n = 10 per group.
Mentions: Considering our conflicting findings in single cells and OHCs it appeared likely that the susceptibility of cells to Aβ mediated diminution of MTT reduction activity depends on their environment. To address this matter, we split one OHC preparation into two groups. One group was cultivated further and the other group was separated into single cells. For the first time we prepared single cells from OHCs. Because of the matured state of the isolated cells only few neurons survived the isolation procedure and thus the cultures consisted largely of astrocytes (Figure 3).

Bottom Line: Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue.Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay.This may indicate that the Abeta 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 Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

View Article: PubMed Central - PubMed

Affiliation: Project Group Neuropharmacology, Leibniz Institute for Neurobiology, Magdeburg, Germany. roenicke@zenit-magdeburg.de

ABSTRACT
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 (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta 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 Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta 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 Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta 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 Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

Show MeSH
Related in: MedlinePlus