Journal of Histochemistry and Cytochemistry, Vol. 47, 661-672, May 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Direct Temporal Analysis of Apoptosis Induction in Living Adherent Neurons

Andrea M. Vincenta and Kenneth Maiesea
a Laboratory of Cellular and Molecular Cerebral Ischemia, Departments of Neurology and Anatomy and Cell Biology, Center for Molecular and Cellular Toxicology and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan

Correspondence to: Kenneth Maiese, Neurology and Anatomy & Cell Biology, 6E-19 UHC, Wayne State U. School of Medicine, 4201 St. Antoine, Detroit, MI 48201.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 47:661–671, 1999)

Key Words: annexin V, apoptosis, fluorescence microscopy, neurodegeneration, nitric oxide, phosphatidylserine residues, primary hippocampal neurons, rat


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Wyllie et al. 1980 ). A cell in PCD can be identified by nuclear chromatin condensation, nuclear shrinkage, ordered cleavage of the nuclear DNA, compactness of cytoplasmic organelles, and the appearance of an irregular plasma membrane (Kerr et al. 1972 ). A discrete change that occurs early is the redistribution of phosphatidylserine from the plasma membrane inner leaflet to become exposed at the cell surface. The process of PCD is essential in the control of tissue shape and size (Alison and Sarraf 1992 ) and therefore plays an important role during development of the nervous system (Deckwerth and Johnson 1993 ).

PCD is a necessary component not only in the development of the nervous system (Lo et al. 1995 ) but also in several pathophysiological conditions that lead to neurodegeneration. Neuronal PCD can be induced by a variety of toxic insults to the nervous system, such as cerebral ischemia (Hara et al. 1995 ), excitotoxicity (Didier et al. 1996 ), and nitric oxide (NO) exposure (Palluy and Rigaud 1996 ; Maiese 1998b ; Vincent and Maiese in press ). In some experimental systems, free radical NO-induced PCD induction is both rapid and robust, occurring within hours in at least 70% of the neuronal population (Vincent et al. 1997 ). Because of its ability to modulate neuronal survival, NO has become aggressively employed as a valuable tool to dissect the downstream cellular pathways that mediate PCD (Maiese et al. 1994 ; Vincent et al. 1997 ; Maiese 1997 ; Maiese 1998b ).

The externalization of phosphatidylserine is known to occur very early during PCD. This phenomenon was first described in lymphocytes (Koopman et al. 1994 ) but has since been attributed to many murine and human cell types (Martin et al. 1995 ). A recent report has demonstrated that increased surface phosphatidylserine is an early marker for PCD in neuronal cell lines (Rimon et al. 1997 ). Membrane asymmetry is believed to be maintained through the activity of aminophospholipid translocase (Vanags et al. 1996 ). Phosphatidylserine externalization during PCD could result from reversal of aminophospholipid translocase activity or loss of this enzyme coupled with random externalization of membrane phospholipids (Bratton et al. 1997 ). The presence of an unidentified "inside-outside phosphatidylserine translocase" also has been proposed (Allen et al. 1997 ). Factors known to inhibit PCD, such as trophic factors (Maiese and Boccone 1995 ; Maiese et al. 1993a ), metabotropic glutamate receptor activation (Maiese et al. 1996 ; Vincent et al. 1997 ; Maiese 1998a ; Vincent et al. in press ), or benzothiazole administration (Maiese et al. 1997 ), also can prevent phosphatidylserine exposure, suggesting that this event may be an integral part of the PCD pathway that occurs only after the cell has committed to die.

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 (Hill et al. 1995 ; Vincent et al. 1997 ; Maiese 1998b ). Other assays have incorporated the use of annexin V, a member of a family of calcium-dependent phospholipid-binding proteins that possesses strong affinity for phosphatidylserine (Andree et al. 1990 ). Because phosphatidylserine translocation occurs early in PCD when cell membrane integrity is still intact, flow cytometric analysis using fluorescein isothiocyanate-labeled annexin V is now widely used as a quantitative measure of early PCD (Koopman et al. 1994 ; Homburg et al. 1995 ; Vanags et al. 1996 ). However, flow cytometric techniques have limited application to adherent cultures because harvesting of the sample generates a certain amount of mechanical membrane damage and the use of trypsin can artificially induce phosphatidylserine exposure (van Engeland et al. 1996 ). Assays that eliminate cell harvesting continue to require cell fixation (Rimon et al. 1997 ).

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 (Tait and Gibson 1992 ), we have developed a novel technique for continuous assessment of the initiation and progression of PCD over time in adherent, monolayer individual neurons. Our technique offers a unique investigative advantage to identify the earliest stages of PCD in viable cells that can further the understanding of the mechanisms that modulate PCD.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Primary Hippocampal Neuronal Cultures
The hippocampi were obtained from 1-day-old Sprague–Dawley 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 (10–20 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 10–20% 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 70–80% of neurons over a 24-hr period (Maiese et al. 1993a , Maiese et al. 1993b ). More than one NO generator is used as a control to demonstrate that the neurons were responding to NO rather than to other byproducts of these agents. After treatment with the NO donors, the culture medium was replaced with fresh growth medium and the cultures were placed in a normoxic, humidified incubator at 37C with 5% CO2 for periods determined by the specific experimental protocol.

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 10–20 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 Epon–Araldite and PO for 1 hr, with 3:1 mixture for 3 hr, with straight Epon–Araldite for 1–4 days, and then with Epon–Araldite-added accelerator for 24–48 hr. Neurons were embedded in fresh Epon–Araldite, 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.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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).



View larger version (72K):
[in this window]
[in a new window]
 
Figure 1. PCD after free radical injury is identified by reversible annexin V-PE labeling. The figure illustrates a series of images of the same microscope field. (A,C,E,G) Images generated using the transmitted light illustrating the neurons. (B,D,F,H) Corresponding images were obtained using 490-nm excitation and 585-nm emission wavelengths to locate the PE label on the surface of neurons undergoing PCD. Five hours after exposure to SNP (300 µM), the neurons were examined before addition of the label to demonstrate that there was no background fluorescence (B). In C and D, the neurons were stained with annexin V-phycoerythrin conjugate. Stained fluorescent neurons undergoing PCD are shown in D. The label was then removed by washing three times in calcium-free buffer. The efficiency of removal is demonstrated while the neurons were still present (E,F). Neurons could then be re-stained and imaged. (G,H) The neurons that were re-stained 2 hr later demonstrate increased annexin V labeling. Bar = 30 µm.

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.



View larger version (113K):
[in this window]
[in a new window]
 
Figure 2. Tracking of individual neurons over time illustrates progressive increase in annexin V labeling after free radical injury. Neurons were stained, imaged, and washed repeatedly over 24 hr. Representative fields of stained neurons are shown indicating minimal induction of PCD in control cultures (left column: T, transmitted light; F, fluorescence) and induction of PCD in cultures exposed to SNP (300 µM) (right column: T, transmitted light; F, fluorescence). Over a 24-hr course, a statistically insignificant induction in annexin V labeling of control cultures may be secondary to repeated mechanical manipulation. In contrast to the control cultures, NO exposure resulted in a marked, progressive increase in the labeling of phosphatidylserine residues over a 24-hr course, illustrating the initial onset and progression of PCD. Bars = 20 µm.

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.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 3. Quantitation of neurons undergoing PCD. At each indicated time point after exposure to either SNP (300 µM) or NOC-9 (300 µM), the percentage of neurons labeled with annexin V was determined. By 3 hr, a significant increase in annexin V label was present after NO exposure. Over a 24-hr period, annexin V label progressively increased to a maximum of approximately 60%. Data represent the mean and SEM from seven individual experimental cultures. In each experiment, the percentage of stained neurons was counted in three to seven discrete fields with 5–25 neurons in each field. Results of neuronal cultures exposed to NO (*) were significantly different from control (untreated) cultures not exposed to NO. p<0.05 by ANOVA.

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 (Rimon et al. 1997 ). If this is the case, then positive labeling of externalized phosphatydylserine residues should occur before the onset of cell membrane disruption. We therefore characterized the ability of annexin V to identify early PCD induction before cell membrane leakage with a dye exclusion system in sister cultures (Figure 4). The loss of membrane integrity was assessed by counting the proportion of trypan blue-positive neurons as described for the survival assays. In this experiment, the NO generators NOC-9 (300 µM) and SNP (300 µM) were employed. To simplify the analysis, results for the two NO donors were combined. Loss of membrane integrity increased by insignificant increments from approximately 2% at 1 hr to 12% at 7 hr after NO exposure (Figure 4). After the 7-hr time period, trypan blue staining markedly increased to 72 ± 2%. In contrast, annexin V labeling rapidly and significantly outpaced the induction of cellular membrane disruption at the time points of 3, 5, and 7 hr after NO exposure (Figure 4). Our work illustrates that annexin V binding identifies neuronal injury and the dislruption of PCD before the loss of cellular membrane integrity.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 4. Phosphatidylserine exposure precedes the loss of membrane integrity. At each indicated time point after exposure to either SNP (300 µM) or NOC-9 (300 µM), the percentage of neurons labeled with annexin V was determined. In sister cultures, the staining with trypan blue was counted at the same times after identical NO exposure. To simplify the figure, the results for the two NO donors were combined. In each experiment, the percentage of annexin V-labeled neurons was counted in three to seven discrete fields with 5–25 neurons in each field. The trypan blue data represent the mean and SEM from two separate experimental cultures, counting nine randomly selected, nonoverlapping fields with each containing approximately 10–20 neurons in each culture dish. * The annexin V labeling at 3, 5, and 7 hr after NO exposure was significantly higher than the induction of cellular membrane leakage of trypan blue (p<0.001, ANOVA).

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 (Maiese et al. 1993a ; Maiese and Boccone 1995 ; Vincent et al. 1997 ). Our studies also illustrate that some neurons that lose membrane integrity, as evidenced by trypan blue staining, no longer label with annexin V (Figure 5). These results are consistent with the findings in other cellular systems (Rimon et al. 1997 ) that loss of annexin V labeling after severe membrane disruption may be secondary to shedding of phosphatidylserine residues or the loss of membrane fluidity. Therefore, our method is sensitive for assessment of the initial stages of PCD induction before more "global" cellular injury.



View larger version (44K):
[in this window]
[in a new window]
 
Figure 5. Annexin V-labeled neurons do not stain with trypan blue during early cell injury. The images are representative and were obtained from the same field of neurons 5 hr after NOC-9 (300 µM) exposure. The left panel (T) illustrates the transmitted light image after addition of trypan blue dye. The black arrow identifies a neuron unable to exclude trypan blue. In the corresponding fluorescent image (F), the annexin V-labeled neurons are indicated with white arrows and the neuron unable to exclude trypan blue is identified by the black arrow. The majority of neurons, including the annexin V-labeled neurons, did not take up the trypan blue dye, indicating that their cell membranes were intact. Bar = 20 µm.

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 (Takei et al. 1994 ). Neurotrophic effects have been reported with a continuous 5-day exposure within a low concentration range (1–30 ng/ml). Although we employ a brief application of annexin V for 10 min, our concentration of annexin V (3 µg/ml) is greater than concentrations that have been demonstrated to yield neurotrophic effects. For these reasons, we investigated the ability of annexin V to alter neuronal survival in our primary hippocampal cultures. Assessing neuronal survival over a 24-hr period, repeated application of annexin V at 1 hr, 3 hr, 5 hr, 7 hr, and 24 hr slightly decreased neuronal survival from 72 ± 2% (untreated control) to 61 ± 2% (repeated annexin application) (Figure 6). However, application of annexin V in conjunction with an NO generator did not significantly alter neuronal survival compared to application of an NO generator alone. In light of these results, annexin V application appears not to be directly toxic to the neurons, but a 10% decrease in neuronal survival may be secondary to mechanical cellular disruption after repeated washing and staining applications over a 24-hr period. The neuronal injury that follows this mechanical damage may occur through necrosis or PCD.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 6. Repeated staining with annexin V does not alter neuronal survival. Neuronal survival was assessed over a 24-hr period after repeated application of annexin V at 1 hr, 3 hr, 5 hr, 7 hr, and 24 hr. Although neuronal survival slightly decreased from 72 ± 2% (untreated control) to 61 ± 2% [annexin V only; p<0.05 by ANOVA (*)], application of annexin V in conjunction with an NO generator did not significantly alter neuronal survival compared to application of an NO generator alone. The results demonstrate that annexin V application is not directly toxic to the neurons, but a 10% decrease in neuronal survival may be secondary to mechanical cellular disruption after repeated washing and staining applications over a 24-hr period. Data represent the mean and SEM from two separate experimental cultures.

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 (10–20%) 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).



View larger version (134K):
[in this window]
[in a new window]
 
Figure 7. Nuclear morphological changes correlate with annexin V labeling. Transmission electron microscopy was employed to examine subcellular manifestations of PCD after NO exposure. Each image is representative of a neuronal nucleus at 1 hr (A), 3 hr (B), 5 hr (C), and 7 hr (D) after exposure to NOC-9 (300 µM). The figure demonstrates the progressive appearance of condensed chromatin with margination at the nuclear membrane. Nuclear cleavage began to appear at 5 hr after NO (C) and became more evident at 7 hr after NO (D). Bar = 2 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Maiese et al. 1997 ; Vincent et al. 1997 ; Vincent and Maise in press; Vincent et al. in press ). This article describes a novel use of a labeling technique that detects both the early induction of PCD and the actual progression of PCD in living cells.

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 phycoerythrin–annexin 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 1–3 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 (Koopman et al. 1994 ). This simultaneous appearance of chromatin condensation and phosphatidylserine exposure also suggests that these cellular processes may be downstream of other signal transduction events that initiated PCD.

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.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Alison MR, Sarraf CE (1992) Apoptosis: a gene-directed programme of cell death. J R Coll Physicians (London) 26:25-35[Medline]

Allen RT, Hunter WR, Agrawal DK (1997) Morphological and biochemical characterization and analysis of apoptosis. J Pharmacol Toxicol Methods 37:215-228[Medline]

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM (1990) Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem 265:4923-4928[Abstract/Free Full Text]

Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM (1997) Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase. J Biol Chem 272:26159-26165[Abstract/Free Full Text]

Deckwerth TL, Johnson E, Jr (1993) Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J Cell Biol 123:1207-1222[Abstract]

Didier M, Bursztajn S, Adamec E, Passani L, Nixon RA, Coyle JT, Wei JY, Berman S (1996) DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures. J Neurosci 16:2238-2250[Abstract]

Hara A, Yoshimi N, Hirose Y, Ino N, Tanaka T, Mori H (1995) DNA fragmentation in granular cells of human cerebellum following global ischemia. Brain Res 697:247-250[Medline]

Hill IE, MacManus JP, Rasquinha I, Tuor UI (1995) DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat. Brain Res 676:398-403[Medline]

Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger CP, Roos D (1995) Human neutrophils lose their surface Fc gamma RIII and acquire annexin V binding sites during apoptosis in vitro. Blood 85:532-540[Abstract/Free Full Text]

Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257[Medline]

Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415-1420[Abstract/Free Full Text]

Lo AC, Houenou LJ, Oppenheim RW (1995) Apoptosis in the nervous system: morphological features, methods, pathology, and prevention. Arch Histol Cytol 58:139-149[Medline]

Maiese K (1997) Neuronal survival: cellular and molecular pathways of protection. In Frontiers of Neurology: A symposium in honor of Fred Plum. Ann NY Acad Sci 835:255-273[Medline]

Maiese K (1998a) Cellular mechanisms of neuronal protection by metabotropic glutamate receptors. In Robertson JT, Nowak TS, eds. Frontiers in Cerebrovascular Disease: Mechanisms, Diagnosis, and Treatment. Armonk, NY, Futura Publishing, 281-297

Maiese K (1998b) From the bench to the bedside: the molecular management of cerebral ischemia. Clin Neuropharmacol 21:1-7[Medline]

Maiese K, Boccone L (1995) Neuroprotection by peptide growth factors against anoxia and nitric oxide toxicity requires modulation of protein kinase C. J Cereb Blood Flow Metab 15:440-449[Medline]

Maiese K, Boniece I, DeMeo D, Wagner JA (1993a) Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity. J Neurosci 13:3034-3040[Abstract]

Maiese K, Boniece IR, Skurat K, Wagner JA (1993b) Protein kinases modulate the sensitivity of hippocampal neurons to nitric oxide toxicity and anoxia. J Neurosci Res 36:77-87[Medline]

Maiese K, Swiriduk M, TenBroeke M (1996) Cellular mechanisms of protection by metabotropic glutamate receptors during anoxia and nitric oxide toxicity. J Neurochem 66:2419-2428[Medline]

Maiese K, TenBroeke M, Kue I (1997) Neuroprotection of lubeluzole is mediated through the signal transduction pathways of nitric oxide. J Neurochem 68:710-714[Medline]

Maiese K, Wagner J, Boccone L (1994) Nitric oxide: a downstream mediator of calcium toxicity in the ischemic cascade. Neurosci Lett 166:43-47[Medline]

Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182:1545-1556[Abstract]

Palluy O, Rigaud M (1996) Nitric oxide induces cultured cortical neuron apoptosis. Neurosci Lett 208:1-4[Medline]

Rimon G, Bazenet CE, Philpott KL, Rubin LL (1997) Increased surface phosphatidylserine is an early marker of neuronal apoptosis. J Neurosci Res 48:563-570[Medline]

Tait JF, Gibson D (1992) Phospholipid binding of annexin V: effects of calcium and membrane phosphatidylserine content. Arch Biochem Biophys 298:187-191[Medline]

Takei N, Ohsawa K, Imai Y, Nakao H, Iwasaki A, Kohsaka S (1994) Neurotrophic effects of annexin V on cultured neurons from embryonic rat brain. Neurosci Lett 171:59-62[Medline]

Vanags DM, Porn-Ares MI, Coppola S, Burgess DH, Orrenius S (1996) Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J Biol Chem 271:31075-31085[Abstract/Free Full Text]

van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP (1996) A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24:131-139[Medline]

Vincent AM, Maiese K (in press) Nitric oxide induction of neuronal endonuclease activity in programmed cell death. Exp Cell Res

Vincent AM, Mohammad Y, Ahmad I, Greenberg R, Maiese K (1997) Metabotropic glutamate receptors prevent nitric oxide induced programmed cell death. J Neurosci Res 50:549-564[Medline]

Vincent AM, TenBroeke M, Maiese K (in press) Metabotropic glutamate receptors prevent programmed cell death through the modulation of neuronal endonuclease activity and intracellular pH. Exp Neurol

Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251-306[Medline]