Laser scanning cytometry in human brain slices

Mosch, Birgit; Mittag, Anja; Lenz, Dominik; Arendt, Thomas; Tárnok, Attila

doi:10.1002/cyto.a.20228

Cited by 37 publications

(33 citation statements)

References 17 publications

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“…Since LSC has previously been used successfully to analyze intracellular markers of single cells within subpopulation of neurons (22,23), an automated LSC cytome approach was developed in our study to analyze single cells from various cell types in the buccal mucosa. Their DNA content and neutral lipid content and their ratios were quantified to identify which parameters, if any, were most strongly associated with those subjects who were diagnosed with MCI or classified as AD.…”

Section: Introductionmentioning

confidence: 99%

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease

et al. 2014

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Previous studies have shown that mild cognitive impairment (MCI) may be reflective of the early stages of more pronounced neurodegenerative disorders such as Alzheimer's disease (AD). There is a need for a minimally invasive and inexpensive diagnostic to identify those who exhibit cellular pathology indicative of MCI and AD risk so that they can be prioritized for primary preventative measures. The hypothesis was that a minimally invasive approach using cytological markers in isolated buccal mucosa cells can be used to identify individuals of both MCI and AD. An automated buccal cell assay was developed using laser scanning cytometry (LSC) to measure buccal cell type ratios, nuclear DNA content and shape, and neutral lipid content of buccal cells from clinically diagnosed AD (n 5 13) and MCI (n 5 13) patients prior to treatment compared to age-and gender-matched controls (n 5 26). DNA content was significantly higher in all cell types in both MCI (P < 0.01) and AD (P < 0.05) compared with controls mainly due to an increase in >2N nuclei. Abnormal nuclear shape (circularity) was significantly increased in transitional cells in MCI (P < 0.001) and AD (P < 0.01) when compared to controls. In contrast, neutral lipid content (as measured by Oil red O "ORO" staining) of buccal cells was significantly lower in the MCI group (P < 0.05) compared with the control group. The ratio of DNA content/ORO in buccal basal cells for both MCI and AD was significantly higher compared to the control group, with ratios for MCI being approximately 2.8-fold greater (P < 0.01) and AD approximately 2.3-fold greater (P < 0.05) than the control group. Furthermore, there was a strong negative correlation between buccal cell DNA content and ORO content in the AD group (r 2 5 0.75, P < 0.0001) but not in MCI or controls. The changes in the buccal cell cytome observed in this study could prove useful as potential biomarkers in identifying individuals with an increased risk of developing MCI and eventually AD. V C 2014 International Society for Advancement of Cytometry

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Section: Introductionmentioning

confidence: 99%

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease

et al. 2014

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“…(Adams et al, 2004;Binder, 2006;Bowen and Wylie, 2006;Green, 1990;Harnett, 2007;Kamentsky, 2001;Levenson and Mansfield, 2006;Lidke et al, 2003;Roman et al, 2002;Tarnok and Gerstner, 2002;Whimster et al, 1995). There is a relatively large body of work that has focused on scanning cytometry applications to neurons, such as the identification of specific neuronal subtypes via biomarkers and antibodies, gene expression, functional characterization of ion channels and anatomical identification (Aguila et al, 2006;Bingham et al, 2006;Blass-Kampmann et al, 1997;Crang et al, 2004;Dahlstrom et al, 1982;Ma and Cui, 2004;Mosch et al, 2006;Schild et al, 1995;Shepherd et al, 2005;Sureda et al, 1997;Verdaguer et al, 2002). However, these analyses are very different both methodologically and in their applications to the spatiotemporal detection of a second messenger during functional signaling events as, we describe here.…”

Section: Discussionmentioning

confidence: 99%

“…However, these analyses are very different both methodologically and in their applications to the spatiotemporal detection of a second messenger during functional signaling events as, we describe here. There has been even less work done on glia in general or astrocytes in particular, which have only been described within the context of neurons (Mosch et al, 2006). Although some work has focused on the identification and quantitative spatial analysis of intercellular calcium waves at the cellular level in cardiac myocytes (Bray et al, 2007) and developing zebra fish (Ashworth and Bolsover, 2002;Gilland et al, 1999;Van der Linden et al, 2002;Webb and Miller, 2003), to the best of our knowledge there is no report describing the automated identification and quantitative spatiotemporal characterization of calcium transients or intercellular calcium waves in either glia or neurons.…”

Section: Discussionmentioning

confidence: 99%

Automated detection of intercellular signaling in astrocyte networks using the converging squares algorithm

Hashemi¹,

Buibas²,

Silva³

2008

Journal of Neuroscience Methods

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Intercellular calcium waves in central nervous system astrocyte networks underline the principle mechanism of cell signaling in astrocyte syncsytiums, which putatively contribute to the modulation of neuronal signaling and metabolic regulation. In support of carrying out systems level analyses of astrocyte networks, we have optimized and validated the converging squares image segmentation algorithm to automatically detect the relative spatial locations of all cells in a visible network as a preliminary step towards analyzing the dynamics of astrocyte intracellular calcium transients, which are the signals that mediate intercellular calcium waves. We used the temporal derivatives of pixel intensities as the data source for the algorithm. The method works by converging progressively smaller squares until the signal peak is reached. It is robust to noise and performs comparably to manual cell signal identification, but is much faster and efficient. This is the first reported application of this algorithm to glial networks that we are aware of.

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“…LSC, a method that has been applied to such diverse research interests as cell cycle analysis (17), oncology (18), gene therapy (19), chemotaxis (20), immunophenotyping of peripheral blood leucocytes (21,22), histological analysis of tissue sections (23,24), and G-protein coupled receptor activation (25), offers the noninvasive capture of multiple fluorescence parameters from adherent cells (13). As such, it is a technology well suited to measuring the morphological correlates of neuronal apoptosis.…”

Section: Lsc Scoring Of An Exogenous Proapoptotic Injury To Cgnsmentioning

confidence: 99%

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons

Bingham¹,

Kotnis²,

McHendry-Rinde³

et al. 2006

Cytometry Pt A

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BackgroundLow transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants.MethodsCerebellar granule neurons (CGNs) were transfected with EGFP‐fusion constructs of Caspase‐3 and Caspase‐9 using a DNA‐calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs.ResultsIn Caspase‐3 and Caspase‐9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture‐mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase‐9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision.ConclusionLSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects. © 2006 International Society for Analytical Cytology

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Laser scanning cytometry in human brain slices

Cited by 37 publications

References 17 publications

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease

Automated detection of intercellular signaling in astrocyte networks using the converging squares algorithm

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons

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