A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture

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Abstract

An automated fluorescence method for the detection of neuronal cell death by necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide (EB) staining using confocal microscopy is described. Since cell nuclei during apoptosis become acidic, AO staining was utilized to distinguish live neurons from neurons undergoing apoptosis, using the AO property to shift its fluorescence from green at normal pH toward brilliant orange-red in the process of acidification. Further EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB can be employed as an express vitality test to count fractions of live and dead cell via apoptosis and necrosis, respectively. An algorithm of automatic quantification of cell types is based on the image correlation analysis. Our conclusion is validated by experiments with the vital dye trypan blue and the pharmacological study of receptor subtypes involved in the excitotoxicity. The approach described here, therefore, offers an express, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Because this assay does not require any preliminary tissue treatment, fixation or dissociation in a cell suspension its utility is likely to be extended for measuring cell viability and cytotoxicity on a variety of living preparations (tissues, brain slices and cell cultures).

Introduction

Neurodegeneration or neuronal cell death is critically involved in both physiological processes of developing vertebrate central nervous system (CNS) and diseased states induced by many pathological factors such as hypoxia, hypoglycemia, energy deprivation, oxidative stress, or a combination of those mimicked by ischemia (Choi, 1988, Beal, 1992, Lipton, 1999, Khodorov, 2004). The most common feature of neuronal cell loss is an accumulation of glutamate (Glu), an excitatory neurotransmitter in the CNS, resulting in hyperactivation of glutamate receptors (GluR) and sustained cell depolarization (Antonov and Magazanik, 1988, Szatkowski et al., 1990, Rossi et al., 2000, Antonov, 2001). Glu excitotoxicity, which destroys neurons following excessive Glu exposure, can be revealed experimentally. When injected in the CNS, applied to brain slices or neuron cultures, GluR agonists trigger necrotic membrane injuries of neurons (Choi et al., 1987) that usually appear during hours of treatment and the delayed apoptotic death that begins from DNA digestion and nucleus fragmentation (Wyllie et al., 1980).

Primary cell cultures are routinely used to determine the mechanism by which GluR agonists mediate neuronal cell death. Quantification of neuronal cell death in experimental systems can be achieved using a variety of in vitro assays with different technical advantages (Bergmeyer and Bernt, 1974, Koh and Choi, 1987, Uliasz and Hewett, 2000). In many studies, the cellular uptake of the fluorescence dye propidium iodide (PI, or similar stain ethidium bromide, EB) has been used as a marker of dead or dying cells, since this dye is unable to penetrate the plasma membrane of live cells, and can enter cells exclusively through necrotic membrane damages (Monette et al., 1998, Noraberg et al., 1999). Apoptosis can be recognized by a set of morphological features (Wyllie et al., 1980). At the biochemical level apoptosis is characterized by DNA digestion (Compton, 1992) and is accompanied by expression of many specific proteins and molecules that can be visualized by immunostaining on fixed preparations (Nagata, 1997, Wolf and Green, 1999).

In principle, acridine orange (AO) staining without PI or EB may monitor apoptosis. In addition to the property to label double-stranded nucleic acids in green (Darzynkiewicz, 1990), AO in living cells serves as a pH indicator, being trapped by acidic compartments, which then fluoresce as brilliant orange-red (Zelenin, 1966). As nuclei during apoptosis undergo morphological changes and acidification, AO has been used to identify apoptotic cells in live Drosophila embryos (Abrams et al., 1993, White et al., 1994), Tetrahymena and chicken chondrocytes (Mpoke and Wolfe, 1997), and cell aggregates from human fetal brain (Pulliam et al., 1998). Based on these unique properties, we demonstrate that AO itself as a membrane permeable vital stain allows to discriminate live neurons from neurons undergoing apoptosis. Further, EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB, therefore, can be employed as an express test to count proportions of live cells and those dead by apoptosis and necrosis, respectively. Verification of our conclusion was accomplished by parallel experiments with the vital dye, trypan blue, and the pharmacological analysis of receptor subtypes that mediate Glu-induced neurodegeneration. Combined with confocal microscopy and an automatic quantification of neuron states, this method is fast and reliable and eliminated manual counting thus minimizing sample and systematic measurement errors.

Section snippets

Cell culture

Experiments were performed at room temperature (20–22 °C) on primary cultures of neurons from embryonic rat brain cortex. All procedures using animals were in accordance with the European Communities Council Directive (24th November 1986; 86/609/EEC) and were approved by the local Institutional Animal Care and Use Committee. Cultures were prepared using a modification of the method as previously described (Dichter, 1978, Antonov et al., 1998). Pregnant Wistar rats were sacrificed by CO2

Double sequential AO/EB staining as a marker for apoptosis and necrosis

In order to determine the feasibility, sensitivity and reproducibility of the double sequential AO/EB staining as a reliable confocal microscopy assay for the discrimination of neuronal necrosis and apoptosis, experiments on excitotoxic neuronal cell death were performed. The data were compared to those obtained by the TB light microscopy assay previously characterized for the detection of neuronal cell membrane injuries in mixed cortical cell cultures following Glu exposure (Patterson, 1979).

Discussion

Understanding the nature and extent of changes in cell structure and tissue organization under various physiological conditions is an essential step toward elucidating the cellular substrates of CNS development, function, injury and repair. A variety of stains (Noraberg et al., 1999), biochemical criteria (Bergmeyer and Bernt, 1974, Koh and Choi, 1987, Uliasz and Hewett, 2000) and their combinations for tracing live and dead cell dynamics have recently become available. Here we described the

Acknowledgements

The authors thank Dr. N.N. Nalivaeva for comments on the manuscript. This work was supported by RFBR grant 05-04-49789 and grant SPb SC RAS to S.M.A.

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