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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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Influence of Aβ on MTT reduction of single cell cultures.A) Influence of Aβ on MTT reduction of neuron and microglia single cell cultures. When applied to cell cultures for 3 days, at 1 µM all Aβ species diminished the MTT reduction significantly in both cell types. The dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group B) Concentration dependent influence of Aβ on MTT reduction activity of astroglia single cell culture. When applied to cell culture for 3 days, any Aβ species diminished the MTT reduction significantly, compared to control. Congo red (2 µM) completely reverses the Aβ effect; Aβ (25-35) diminished the MTT reduction in NB medium, normally used for cultivation of OHC; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group C) Electron microscopic images (EMI) revealed that freshly dissolved Aβ (25-35) did not form aggregates. Moreover, EMI conformed the needle like structure of fibrillar Aβ (1-40) and the smaller, spherical shape of oligomeric Aβ (1-40) and oligomeric Aβ (1-42).
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pone-0003236-g001: Influence of Aβ on MTT reduction of single cell cultures.A) Influence of Aβ on MTT reduction of neuron and microglia single cell cultures. When applied to cell cultures for 3 days, at 1 µM all Aβ species diminished the MTT reduction significantly in both cell types. The dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group B) Concentration dependent influence of Aβ on MTT reduction activity of astroglia single cell culture. When applied to cell culture for 3 days, any Aβ species diminished the MTT reduction significantly, compared to control. Congo red (2 µM) completely reverses the Aβ effect; Aβ (25-35) diminished the MTT reduction in NB medium, normally used for cultivation of OHC; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group C) Electron microscopic images (EMI) revealed that freshly dissolved Aβ (25-35) did not form aggregates. Moreover, EMI conformed the needle like structure of fibrillar Aβ (1-40) and the smaller, spherical shape of oligomeric Aβ (1-40) and oligomeric Aβ (1-42).

Mentions: We extensively investigated different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-40) and oligomeric Aβ (1-42) for their effects on MTT reduction in neuronal, astroglia and microglia single cell cultures, representing the majority of cell types within the brain. In accordance with the literature [2], [10], each Aβ species led to a pronounced diminution of MTT reduction in all cell types tested (Neurons: control 100±4.4%, Aβ (25-35) 84.4±3.9%, fibrillar Aβ (1-40) 61.1±3.5%, oligomeric Aβ (1-40): 46.0±2.4%, Aβ (1-42) 77.7±5.1%; Microglia: control: 100±5.6%, Aβ (25-35) 25.9±6.2%, fibrillar Aβ (1-40) 42.3±6.5%, oligomeric Aβ (1-40): 49.1±2.5%, Aβ (1-42) 72.7±3.1% Figure 1A). As we intended to investigate the effect of Aβ on MTT reduction in OHCs, where the most abundant cell type is astroglia, we determined the Aβ effect in detail in astroglia single cell cultures. Because OHCs and astroglial cultures are cultivated in different culture media we elucidated whether or not the Aβ mediated disruption of MTT reduction is influenced by the culture medium.


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 of single cell cultures.A) Influence of Aβ on MTT reduction of neuron and microglia single cell cultures. When applied to cell cultures for 3 days, at 1 µM all Aβ species diminished the MTT reduction significantly in both cell types. The dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group B) Concentration dependent influence of Aβ on MTT reduction activity of astroglia single cell culture. When applied to cell culture for 3 days, any Aβ species diminished the MTT reduction significantly, compared to control. Congo red (2 µM) completely reverses the Aβ effect; Aβ (25-35) diminished the MTT reduction in NB medium, normally used for cultivation of OHC; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group C) Electron microscopic images (EMI) revealed that freshly dissolved Aβ (25-35) did not form aggregates. Moreover, EMI conformed the needle like structure of fibrillar Aβ (1-40) and the smaller, spherical shape of oligomeric Aβ (1-40) and oligomeric Aβ (1-42).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2529401&req=5

pone-0003236-g001: Influence of Aβ on MTT reduction of single cell cultures.A) Influence of Aβ on MTT reduction of neuron and microglia single cell cultures. When applied to cell cultures for 3 days, at 1 µM all Aβ species diminished the MTT reduction significantly in both cell types. The dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group B) Concentration dependent influence of Aβ on MTT reduction activity of astroglia single cell culture. When applied to cell culture for 3 days, any Aβ species diminished the MTT reduction significantly, compared to control. Congo red (2 µM) completely reverses the Aβ effect; Aβ (25-35) diminished the MTT reduction in NB medium, normally used for cultivation of OHC; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group C) Electron microscopic images (EMI) revealed that freshly dissolved Aβ (25-35) did not form aggregates. Moreover, EMI conformed the needle like structure of fibrillar Aβ (1-40) and the smaller, spherical shape of oligomeric Aβ (1-40) and oligomeric Aβ (1-42).
Mentions: We extensively investigated different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-40) and oligomeric Aβ (1-42) for their effects on MTT reduction in neuronal, astroglia and microglia single cell cultures, representing the majority of cell types within the brain. In accordance with the literature [2], [10], each Aβ species led to a pronounced diminution of MTT reduction in all cell types tested (Neurons: control 100±4.4%, Aβ (25-35) 84.4±3.9%, fibrillar Aβ (1-40) 61.1±3.5%, oligomeric Aβ (1-40): 46.0±2.4%, Aβ (1-42) 77.7±5.1%; Microglia: control: 100±5.6%, Aβ (25-35) 25.9±6.2%, fibrillar Aβ (1-40) 42.3±6.5%, oligomeric Aβ (1-40): 49.1±2.5%, Aβ (1-42) 72.7±3.1% Figure 1A). As we intended to investigate the effect of Aβ on MTT reduction in OHCs, where the most abundant cell type is astroglia, we determined the Aβ effect in detail in astroglia single cell cultures. Because OHCs and astroglial cultures are cultivated in different culture media we elucidated whether or not the Aβ mediated disruption of MTT reduction is influenced by the culture medium.

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