ARTICLE |
Correspondence to: Kenneth Maiese, Neurology and Anatomy & Cell Biology, 6E-19 UHC, Wayne State U. School of Medicine, 4201 St. Antoine, Detroit, MI 48201.
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Summary |
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Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells. (J Histochem Cytochem 47:661671, 1999)
Key Words: annexin V, apoptosis, fluorescence microscopy, neurodegeneration, nitric oxide, phosphatidylserine residues, primary hippocampal neurons, rat
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Introduction |
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Programmed cell death (PCD) is a selective, genetically controlled process of cell deletion. This contrasts with cellular necrosis, a passive cellular injury that results in loss of membrane integrity and cell lysis (
PCD is a necessary component not only in the development of the nervous system (
The externalization of phosphatidylserine is known to occur very early during PCD. This phenomenon was first described in lymphocytes (
Because neuronal injury can occur through the induction of PCD, the ability to rapidly identify the onset and progression of PCD has become crucial to elucidate the multiple mechanisms that modulate PCD. Current studies that characterize PCD rely on a variety of "fixed" assays to identify the end stages of PCD, which include gel electrophoresis DNA fragmentation assays, DNA 3'-OH end-labeling, electron microscopy, and hematoxylin and eosin staining (
We describe a significant advancement for the use of annexin V labeling of phosphatidylserine residue translocation during PCD in viable neurons. Employing the reversible calcium-dependent nature of annexin V binding to phosphatidylserine (
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Materials and Methods |
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Primary Hippocampal Neuronal Cultures
The hippocampi were obtained from 1-day-old SpragueDawley rat pups, following institutional guidelines, and were incubated in dissociation medium (90 mM Na2SO4, 30 mM K2SO4, 5.8 mM MgCl2, 0.25 mM CaCl2, 10 mM kynurenic acid, and 1 mM HEPES with the pH adjusted to 7.4) containing papain (10 U/ml) and cysteine (3 mmol/liter) for two 20-min periods. The hippocampi were then rinsed in dissociation medium and incubated in dissociation medium containing trypsin inhibitor (1020 U/ml) for three 5-min periods. The neurons were washed in growth medium (Leibovitz's L-15 medium; Gibco BRL, Gaithersburg, MD) containing 6% sterile rat serum (Bioproducts for Science; Indianapolis, IN), 150 mM NaHCO3, 2.25 mg/ml transferrin, 2.5 µg/ml insulin, 10 nM progesterone, 90 µM putrescine, 15 nM selenium, 35 mM glucose, 1 mM L-glutamine, penicillin, and streptomycin (50 µg/ml), and vitamins. The dissociated neurons were plated at a density of ~1.5 x 103 cells/mm2 in 35-mm polylysine/laminin-coated plates (Falcon Labware; Lincoln Park, NJ). Neurons were maintained in growth medium at 37C in a humidified atmosphere of 5% CO2 and 95% room air. All experiments were performed with neurons that had been in culture for 2 weeks. Non-neuronal cells accounted for 1020% of the total cell population.
Experimental Treatments
NO administration was performed by replacing the culture medium with media containing either sodium nitroprusside (SNP, 300 µM) (Sigma; St Louis, MO) or 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (NOC-9, 300 µM) (Calbiochem; San Diego, CA) for 5 min. We have previously demonstrated that each of these agents yields neuronal injury through a mechanism that involves the direct release of NO, with a 5-min application of 300 µM resulting in the death of approximately 7080% of neurons over a 24-hr period (
Staining for Externalization of Phosphatidylserine Residues
Annexin V conjugated to phycoerythrin (PE) was purchased from R&D Systems (Minneapolis, MN). The stock solution was 30 µg/ml concentration. This was diluted directly before use 1:10 in warmed (37C) binding buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2). The growth medium was removed from culture plates, and annexin V conjugate was applied at a final concentration of 3 µg/ml, and then incubated at 37C in a humidified atmosphere in the dark for 10 min. Plates were then rinsed twice with fresh binding buffer and neurons were examined using a Leitz DMIRB microscope (Leica; McHenry, IL) and Oncor Image 2.0 imaging software (Oncor; Gaithersburg, MD). Images were acquired using a cooled charge-coupled device with both transmitted light and fluorescent single-excitation light at 490 nm and detected emission at 585 nm.
After examination, the annexin V label was detached by washing three times in dissociation buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM KCl, 2.8 mM MgCl2), which differed from binding buffer in that the calcium was replaced with magnesium. Plates could then be reexamined to confirm that the annexin V was completely removed, then returned to the incubator for a further specified period. Plates could then be re-stained using the same method. By drawing a grid on the bottom of the culture dish, the same fields of neurons could be relocated for sequential imaging.
Neuronal Survival Assays
Hippocampal neuronal injury was determined by brightfield microscopy using a 0.4% trypan blue dye exclusion method at specified times after treatment with the NO donors. Neurons were identified by morphology. The mean survival was determined by counting nine randomly selected, nonoverlapping fields with each containing approximately 1020 neurons (viable + nonviable) in each 35-mm Petri dish. The mean survival from each culture dish represents an n = 1 determination.
Transmission Electron Microscopy of Hippocampal Neurons
Electron microscopy was used to visualize the neuronal subcellular structure to assess changes in the cell nucleus at times relevant to the externalization of phosphatidylserine. Neurons were grown on 35-mm glass coverslips coated with laminin and poly-L-lysine. After treatments as indicated, the hippocampal neurons were fixed at 4C for 1.5 h with a fixative consisting of 1:1:1 of 2% aqueous OsO4, 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and 0.2 M phosphate buffer (pH 7.4). Neurons were then rinsed with phosphate buffer and dehydrated with graded ethanol in the following sequence: 50% for 5 min, 70% for 10 min, 85% for 10 min, 95% for 10 min, 100% for 10 min, and an additional 30 min in 100% ethanol. Neurons were removed from the coverslips and placed in a glass scintillation vial containing propylene oxide (PO). Dehydration was performed with three changes of PO, each for 10 min, and one change for 30 min. Neurons were infiltrated with mixture of 1:1 EponAraldite and PO for 1 hr, with 3:1 mixture for 3 hr, with straight EponAraldite for 14 days, and then with EponAraldite-added accelerator for 2448 hr. Neurons were embedded in fresh EponAraldite, and ultrathin sections were mounted on copper grids and stained with 3% aqueous uranyl acetate and Reynolds' lead citrate. These were viewed using a JEM 1010 transmission electron microscope (Jeol; Tokyo, Japan).
Statistical Analysis
For each experiment, the mean and SEM were determined. The sample size is defined in each individual experiment. Statistical significance was assessed using the Student's paired t-test and ANOVA with 95% confidence intervals.
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Results |
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Annexin V Binding to Injured Neurons Is Reversible and Reproducible
Using the reversible annexin V assay, we could determine the time when cultured neurons externalized phosphatidylserine at the plasma membrane. Figure 1 illustrates the reversibility of the annexin V and phosphatidylserine interaction after removal of calcium. Each panel in the figure illustrates the same field of neurons in a culture dish after a representative exposure to the NO generator SNP (300 µM). The neurons were imaged before staining with annexin V-PE (Figure 1A and Figure 1B), during staining with annexin V-PE 5 hr after NO exposure (Figure 1C and Figure 1D), then after rinsing three times in dissociation buffer (Figure 1E and Figure 1F). Three washes with dissociation buffer consistently removed all of the staining in the culture plate. Neurons were then restained 2 hr later with annexin V-PE in the same manner (Figure 1G and Figure 1H).
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NO Exposure Induces a Progressive Increase in Phosphatidylserine Residue Translocation
Figure 2 illustrates a series of representative images obtained to characterize changes in annexin V labeling over a 24-hr period. In untreated control cultures, there appeared to be a slight increase in annexin V staining by the 24-hr time point. In neurons exposed to NO, a progressive and significant increase in annexin V labeling was evident over a 24-hr period. Figure 2 illustrates a representative set of images for the NO generator SNP (300 µM). In this series of images, the staining at 3 hr after NO exposure was no different from that at 1 hr. At 5 hr after NO injury, two of the three neurons in this field are annexin V-positive. The intensity of staining in these neurons continued to increase at 7 and 24 hr after NO exposure. The third neuron also became positive for annexin V labeling 7 hr after NO exposure.
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We further assessed the degree of annexin V staining by determining the proportion of labeled neurons in each microscope field. Initial percentage of annexin V staining in untreated control cultures at the 1-hr time point was 6 ± 4% (Figure 3). This amount of annexin V staining approached a trend to gradually increase in the same neuronal cells to 17 ± 4% (24 hr) but was not statistically significant (Figure 3). Within 3 hr after the exposure to NO, annexin V labeling is significantly increased from approximately 5 to 20% in the identical neuronal cells (Figure 3), suggesting a rapid induction of PCD. These neurons continue to significantly progress with phosphatidylserine membrane translocation to a maximum of approximately 60% over a 24-hr time period (Figure 3). Therefore, our ability to follow the temporal course of phosphatidylserine membrane translocation in individual neurons after free radical exposure with NO demonstrates that the resultant induction of PCD is both an early (within 3 hr) and a robust process that serially progresses in individual neurons over a 24-hr course.
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Annexin V Binding Identifies Neurons Early in PCD Before the Loss of Membrane Integrity
Translocation of membrane phosphatidylserine residues has been reported to be an early marker of PCD induction (
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As shown in Figure 5, we further assessed the ability of neurons labeled with annexin V to simultaneously stain with trypan blue. After identification of neurons that label for annexin V at each time period, warmed (37C) 0.4% trypan blue was added in a 1:1 volume:volume dilution for 1 min, removed, and then neurons were re-imaged. At the time points of 3 hr, 5 hr, and 7 hr, the majority of neurons labeling positive for annexin V did not stain for trypan blue. Figure 5 is a representative image at the 5-hr time point, which illustrates that annexin V labeling of phosphatidylserine residue translocation precedes cell membrane disruption that is detected with trypan blue staining. In contrast, over a 24-hr period, labeling with annexin V coincides with trypan blue staining in the same neuronal population (Figure 4), suggesting that PCD has progressed to a level that now involves cell membrane disruption. These results correlate with our prior studies that demonstrated PCD expression and trypan blue staining in 70% of primary hippocampal neurons 24 hr after NO exposure (
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Annexin V Staining Does Not Independently Alter Neuronal Survival
Depending on the mode and concentration of application, annexin V application can have either toxic or neurotrophic effects on neurons (
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Annexin V Staining Correlates Closely with Changes in Nuclear Morphology
Our present results illustrate that our technique offers a sensitive approach for the early detection of the induction of PCD. However, it also is vital to assess the specificity of our assay to detect PCD. Electron microscopy provides a secondary means of visualizing changes in the neuronal subcellular structure that are consistent with PCD. Primary hippocampal neurons were exposed to an NO generator and then processed 1 hr, 3 hr, 5 hr, 7 hr, and 24 hr later for transmission electron microscopy. Evidence for apoptotic neuronal cell death was characterized by the preservation of membrane integrity and internal organelle structure and by the presence of chromatin condensation with nuclear fragmentation (Figure 7). Assessment by electron microscopy for PCD closely paralleled PCD characterization through the annexin V labeling. Approximately 10% or fewer neurons displayed evidence of PCD after 1 hr, with the majority of neurons showing no evidence of PCD (Figure 7A). A slightly greater proportion (1020%) of neurons displayed some chromatin condensation after 3 hr (Figure 7B). At 5 hr, approximately 35% of the neurons were identified with chromatin condensation (Figure 7C). The percentage of neurons on electron microscopy that were consistent with PCD increased to approximately 50% by 7 hr (Figure 7D) and to almost 70% by 24 hr (not shown).
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Discussion |
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Early detection of PCD is crucial for dissecting the molecular mechanisms that mediate both cellular development and cellular injury. We have previously defined PCD sequentially in hippocampal neuronal cultures, using techniques that do not permit the later reassessment of the neuronal population (
We incorporated several modifications to prior techniques that label fixed, nonviable cells for PCD. The majority of studies employ fluorescein isothiocyanate-conjugated annexin V for use in flow cytometry. For microscopy, we found that this fluorescent marker lacked sufficient intensity with a 40 x 0.5 flat objective lens to adequately detect annexin V labeling. With the fluorescent conjugate phycoerythrinannexin V, detection became efficient and prior fixation with mounting for microscopy became unnecessary. The present technique also permits the tracking of individual neurons over time. Through the construction of a grid on the bottom of a culture plate, individual neurons and field could be made available for repeated examination over time. Access to a mechanically controlled microscope stage would further ease the reassessment of individual cells. To maintain cell adherence to the culture plates, extreme care was required during dissociation of the label because cell-to-cell and cell-to-matrix adhesion processes are calcium-dependent. The magnesium ions that we have chosen to replace the calcium in the dissociation buffer should assist in maintaining cell adherence to the culture surface. Removal of annexin V in calcium-free conditions also was performed gently, with minimal shear forces at the culture surface. A longer incubation in a calcium-free buffer or undue agitation of the neuronal layer could result in detachment of the neurons.
Our technique to identify the initial induction of PCD and to follow the course of PCD progression has proved to be specific, sensitive, and nontoxic for the detection of PCD. Within 13 hr after a free radical insult with an NO generator, we could identify the onset of phosphatidylserine residue membrane translocation. This exposure of the phosphatidylserine residues was consistent with the induction of PCD and was independent of global cellular injury as detected with the trypan blue dye exclusion method. The use of an exclusion dye is an important aspect of this assay to ensure that annexin V staining is facilitated through loss of membrane asymmetry and not through loss of membrane integrity. Once we had established that early annexin V staining was caused by phosphatidylserine exposure, we were then able to investigate the same population of neurons repeatedly without exposing them to exclusion dyes that are toxic and can compromise viability in later assessments.
Co-assessment of phosphatidylserine labeling with electron microscopic imaging further supported the specificity and sensitivity of our assay. Appearance of the nuclear morphological alterations documented by electron microscopy paralleled our ability to identify phosphatidylserine residue membrane translocation. Our results are consistent with prior studies that demonstrate a close correlation between chromatin condensation identified by flow cytometry and annexin V labeling (
Our work is novel in that it offers a procedure for critical assessment of both the onset of PCD and the subsequent progression of PCD. The technique is able to identify the initial stages of PCD and can continue to follow the course of PCD in individual living cells. Staining with fluorescent annexin V for microscopic examination was rapid, reliable, and reversible, permitting minimal disruption to the neurons under examination. The technique should be readily applicable to other adherent monolayer cell types.
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Acknowledgments |
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Supported by the following grants (to KM): Alzheimer's Association, American Heart Association, Boehringer Ingelheim Award, Janssen Neuroscience Award, Johnson and Johnson Focused Investigator Award, NIH/NINDS, and United Cerebral Palsy Foundation.
Received for publication August 25, 1998; accepted November 25, 1998.
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