In many cell types, apoptosis is characterized by a specific sequence of nuclear changes culminating in chromatin condensation and nuclear fragmentation (Wyllie et al., 1980). To examine nuclear alterations during apoptosis in BV-2 cells, we used DAPI staining and electron microscopy. Unless specifically noted, apoptosis was induced by UV-irradiation.#

The localization of heterochromatin and condensed chromatin, respectively, was assessed with DAPI labeling and visualized by confocal microscopy. Although DAPI-staining may not be identical with the entire volume of the nucleus (see below), we use the expression “nucleus” to describe extension and shape of DAPI-labeled chromatin. Nuclei of cells not exposed to UV-irradiation were characterized by punctuate staining of chromatin, indicating a highly compartmentalized nucleus. These nuclei had an ellipsoid shape with a diameter along their long axis of about 15.1±1.3 μm (n=30) (Fig. 1a). Upon UV-irradiation, chromatin structure collapsed. In the early stages of apoptosis, condensed chromatin accumulated along the periphery of the nucleus leaving chromatin-free areas in the center of the nucleus. Furthermore, BV-2 nuclei became disfigured showing irregularly shaped nuclei ranging from ellipsoid to lobulated (Fig. 1b) and decreased to 11.9±2.2 μm (n=30). In the late stage of apoptosis, DAPI staining appeared homogenous in the center of the nucleus, but faded gradually at the periphery (Fig. 1c). The nucleus further decreased to 9.8±1.4 μm in diameter (n=20). In the terminal apoptotic stage, the nuclei appeared pycnotic and showed an intense homogenous labeling by DAPI (Fig. 1d). These cells had a diameter of 7.9±1.3 μm (n=10). Unlike lymphocytes, which show clear nuclear fragmentation in their final apoptotic stages (Wyllie et al., 1980), microglial nuclei did not show an obvious fragmentation.#

The absence of classical nuclear fragmentation in BV-2 cells differs radically from most reports addressing the morphology of nuclei during the course of apoptosis. We, therefore, evaluated nuclear changes during apoptosis using transmission electron microscopy to obtain more information on this critical step during apoptosis. Similar to our light-microscopical observations, our electron-microscopical studies showed that the nuclear shape changed continuously from ellipsoid and lobulated in control cells and early apoptotic cells to more spherical at late apoptotic stages, while the nuclear volume decreased markedly (Figs. 2 and 5). In addition, formation of condensed ribonucleoproteins in the central area of the nucleus represented one of the earliest visible indications of apoptosis, which persisted until late stages of apoptosis (Fig. 2b). Gradual marginalization of chromatin also appeared during the early phase of apoptosis (Fig. 2b). In later apoptotic stages, the nucleus was filled by condensed chromatin to a high degree (Figs. 2c 5b). Parallel to condensation of chromatin, the nuclear envelope showed characteristic alterations, including dilation, ultrastructural changes resembling vesicle formation at the nuclear membrane, and accumulation of electron lucent vesicles close to the nuclear envelope (Fig. 2c). Surprisingly, chromatin was localized in the nucleo—as well as in the cytoplasm (Figs. 2b and d). Like nuclear chromatin, cytoplasmic chromatin was attached to the nuclear envelope and displayed similar electron dense and granular structures as nuclear chromatin. Because nuclear and cytoplasmic chromatin were located opposite to each other on the same dilated areas of the nuclear envelope, we assume that chromatin is transported by an unknown mechanism across the nuclear envelope. Cytoplasmic chromatin was identified 3 and 5 h (59 out of 89 cells), but not 1 h following UV-irradiation.#

Although all cells are doomed following UV-irradiation (250 mJ/cm²), the onset of apoptosis differs in each cell. The histogram in Fig. 3 illustrates the distribution of nuclei at specific apoptotic stages after UV-irradiation. Prior to UV-irradiation, we detected about 1.6% apoptotic cells (early and late apoptotic stages), which increased to 94.9% 6 h, and to 90,5% 12 h following UV-irradiation (Fig. 3). The number of pycnotic nuclei increased to 0.7% within 6 h, and to 3% within 12 h after irradiation.#

To evaluate whether the lack of fragmentation of nuclei in terminal stages of apoptosis is restricted to UV-irradiation or is a more general feature in BV-2 cells, we induced apoptosis via exposure to TNF-α (50 ng/ml) and TNF-α with subsequent ligation of CD 95, respectively. Cells pre-exposed to TNF-α (50 ng/ml) for 24 h, followed by ligation of CD 95 showed an increase of apoptosis by 6% after 3 h, by 14% after 6 h, and by 44% after 24 h. Neither stimulation with TNF-α nor ligation of CD 95 induced nuclear fragmentation in BV-2 cells.#

In a series of experiments, we compared the phenotypic characteristics of BV-2 cells before and after UV-irradiation using electron microscopy and FACS analysis. Although control BV-2 cells show a variety of different cell shapes, ranging from rounded to elongated phenotypes (Figs. 4a and b), almost all of them had numerous delicate filopodia and the cell surface was either smooth or covered by microridges. During an early phase of apoptosis, however, their phenotype changed significantly, partly because length and number of filopodia decreased, mainly because the cell body became covered with blebs (zeiosis) (Figs. 4c and d). During terminal stages of apoptosis, the cell body fragmentized. Remarkably, only one pole of the cell was engaged in fragmentation, whereas the other pole was still paunchy, because it housed the nucleus (Figs. 4e and f; see also Fig. 5). Three and five hours after UV-irradiation 57% (51 out of 89 cells) of the cells showed this type of polarization in their formation of apoptotic bodies.#

Furthermore, induction of apoptosis established a clear asymmetric zonation of cell organelles. Nucleus and endoplasmic reticulum were found on opposite poles of the apoptotic cells, whereas mitochondria were localized between these organelles (Fig. 5). Apoptotic bodies contained mainly ER cisternae or mitochondria, whereas the remaining nucleus was surrounded by a thin cytoplasmic layer at this stage (Fig. 5). The segregation of cell organelles during apoptosis may indicate that an intracellular gradient of signaling factors governs transition of intact cells into apoptotic bodies.#

Since apoptotic volume decrease is a classical feature of apoptosis, we evaluated cell volume in BV-2 cells following UV-irradiation by flow cytometry (Fig. 6). Control cells showed two peaks. The first indicates small particles, probably severed filopodia (see also Fig. 4), and the second peak indicates larger particles, presumably cell bodies. One hour after UV-irradiation the distribution did not differ from that found in control cells, but significantly changed over 2 and 3 h following irradiation: the first peak significantly increased, whereas the second peak significantly decreased. In UV-irradiated cells, the first peak presumably represents apoptotic bodies and cell debris (see also Figs. 4e and f; Fig. 5b).#

Because apoptotic cells expose phosphatidylserine on their surface, we studied the phosphatidylserine asymmetry in the plasma membrane in BV-2 cells before and after UV-irradiation using annexin V labeling and confocal microscopy (Fig. 7). Accordingly, a cell was considered apoptotic, when it showed at least one intense accumulation of annexin V labeling (Fig. 7b). When a cell showed annexin V labeling and propidium iodide staining, it was considered necrotic. Prior to UV-irradiation, the relative number of annexin V labeled cells was low (14%), but significantly increased following UV-irradiation. Furthermore, we detected that the number of annexin V accumulating areas increased and, finally, the entire plasma membrane was labeled (Fig. 7c). In terminal apoptotic stages, nuclei were stained by propidium iodide (Fig. 7d).#

To explore whether a caspase cascade is involved in UV-induced apoptosis in BV-2 cells, we quantified caspase-3/7 activity (Fig. 8). Caspase activity increased and was already saturated 3 and 6 h following UV-irradiation. Notably, the temporal increase of caspase-3/7 activity after UV-irradiation was similar to that of chromatin condensation (see also Fig. 2).#

Unless otherwise stated, all reagents were obtained from Sigma-Aldrich (Sigma, St. Louis, Missouri, USA).#

BV-2 cells were cultivated in 25 cm² culture flasks in DMEM supplemented with 2.2 g glucose/l and 10% FCS under standard culture conditions (37 °C, 5% CO2, 95% rel. humidity). For UV-irradiation experiments, 3×10⁴–3×10⁶ cells were plated on poly-d-lysine-coated glass dishes, covered with DMEM (10% FCS), and cultured for 12 h. After UV-irradiation (250 mJ/cm²; λmax=254 nm; UV Stratagene's Stratalinker 2400, Amsterdam, Netherlands), cells were assayed for apoptosis using electron microscopy, DAPI staining, annexin V labeling, and caspase-3/7 activity, respectively. For Fas-induced apoptosis cells were pretreated with 50 ng/ml TNF-α for 24 h and incubated for additional 3, 6, and 24 h, respectively, with an antibody to CD95 combined with a catch antibody. Fas-induced apoptosis was quantified via DAPI staining.#

Confocal microscopy images were collected using a laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany). We used an UV laser with λEX=364 nm extinction to visualize DAPI-stained nuclei, an argon laser with λEX=488 nm extinction for FITC-labeled Annexin V, and a helium-neon laser (λEX=543 nm) for visualization of PI staining. Images were acquired using a plan-apochromat 63×/1.4 oil and a plan-neofluar 40×/1.3 oil and differential interference contrast III (Zeiss, Jena, Germany). Images were digitized and postacquisition processing was performed with Zeiss LSM Image Examiner.#

Cells were washed with phosphate-buffered saline (PBS) once and fixed with 1 ml methanol/glacial acetic acid (100%) [3:1] for 15 min in the dark. Thereafter the cells were stained with DAPI (0.2 μg DAPI/ml) in Mc Ilvaine buffer (0.2 M Na2HPO4/0.1 M citric acid, pH 7) for 30 min in the dark. Cells were briefly rinsed with PBS and washed twice with distilled water and mounted with glycergel (DAKO).#

BV-2 cells were grown on glass coverslips for at least 12 h. Following UV-irradiation (250 mJ/cm²), cells were cultured for additional 3, 6, and 12 h, respectively. Then, cells were immersed in 2.5% glutaraldehyde–PBS (phosphate-buffered saline) (pH 7.4) at room temperature for 1 h. After fixation, samples were washed in PBS for 1 h, changing PBS for at least three times, and dehydrated in a graded series of ethanol (50%, 70%, 80%; 20 min per change, and 90%, 96%, 100%; 15 min per change; 100% ethanol was changed three times). Subsequently to dehydration, samples were critical-point dried with a Baltec Critical Point Dryer LPD030 (eleven times for 2 min), glued on pins, and sputter coated with a 50 nm layer of gold using an Agar Sputter Coater (15 s). Specimens were inspected and photographed with a CAMBRIDGE stereoscan 250 scanning electron microscope.#

Cells exposed to UV-irradiation (250 mJ UV) and untreated controls were trypsinated from their culture dishes, centrifuged at 50×g for 3 min and then fixed in 1% glutaraldehyde in 50 mM cacodylate buffer for 15 min. After fixation and between the three washing steps (5 min) with 50 mM cacodylate buffer the samples were centrifuged again. Thereafter, the cells were postfixed in buffered OsO4 (2%) at 4 °C over night. The cells were then washed again three times in cacodylate buffer, transferred into 50% and 70% ethanol before en-bloc staining for 1 h in 2% uranylacetate (in 70% ethanol). Thereafter, the cells were dehydrated in 70%, 90% and 100% ethanol (each four times 5 min) and then transferred into propylenoxide mixed with ethanol (1:1) for 10 min and, finally, into pure propylenoxide (10 min). Subsequently, the preparations were embedded in a 1:1 mixture of propylenoxide and EMbed 812 resin. After evaporation of propylenoxide the preparations were polymerized at 60 °C for 72 h. Ultrathin sections were cut on a Reichert Ultracut and mounted on copper grids coated with Formvar film. Sections were viewed in a LEO 912AB TEM (Zeiss, Oberkochen) operated at 80 kV.#

Caspase-3/7 activity was quantified either via a modified CaspACE^TM assay or the Apo-ONE^TM homogenous Caspase 3/7 Assay. In the modified CaspACE^TM assay, we quantified caspase activity by determination of cleavage of the fluorogenic peptide substrate Ac-DEVD-AMC (Promega, Madison, USA). Adherent and non-adherent cells were harvested using Accutase (PAA-laboratories). Following centrifugation at 840×g for 2 min at room temperature to pellet cells, apoptotic bodies, and cell debris, the pellet was resuspended in 100 μl PBS, and subjected to further centrifugation at 840×g for 5 min at room temperature. 10 μl of each sample was removed for protein determination (see below). After centrifugation, cells were lysed in 20 μl of caspase lysis buffer (Promega, Madison, USA) and subjected to three freeze and thaw cycles. 10 μl of the cell lysate was transferred to an ELISA well (96-well plate) in the presence of 90 μl of the modified caspase assay, including 2 μl caspase-3/7 substrate (Promega, Madison, USA), 1 μl DTT (1 M), 55 μl distilled water, and 32 μl caspase assay buffer (Promega, Madison, USA), and incubated for 1 h at 30 °C. Changes in fluorescence intensity were quantified by use of a Spectrafluor microplate reader (Tecan, Salzburg, Austria) with an excitation wavelength of 405 nm and an emission wavelength of 505 nm. Caspase activity was estimated as the difference in arbitrary fluorescence units between blank and samples and normalized to the protein content. Protein content of cell extracts was quantified by the BCA Protein assay, which is based on the biuret reaction and the bicinchoninic acid detection of cuprous cation. Cells were permeabilized with 40 μl 3% Triton X100 at room temperature for 10 min followed by incubation in 150 μl dyeing solution (BCA-Reagenz [Pierce Biotechnology, IL] and 4% CuSO4) at 37 °C for 30 min. The absorbance was measured at 565 nm using a Spectrofluor microplate reader (Tecan, Salzburg, Austria). All measurements were performed in duplicate.#

Detection of phosphatidylserine, translocated to the outer surface of the cell membrane on apoptotic cells, was accomplished by use of a commercially available FITC-coupled Annexin V Apoptosis Detection Kit (Bio Vision, Mountain View, CA). Cells were incubated in the dark in 500 μl binding buffer, supplemented with 5 μl FITC-coupled annexin V and 5 μl propidium iodide, at room temperature for 5 min. Then, the cells were washed once with PBS and immersed in 2% paraformaldehyde for 30 min in the dark. Subsequently, the cells were washed once in PBS and twice in ddH2O and, finally, mounted with Glycergel (Dako, Cytomation, USA) and examined using confocal microscopy.#

Twelve  hours prior to experiments, cells were seeded at a density of 1×10⁵ on uncoated petri dishes. One, two, three, and six hours after UV-irradiation, cells were trypsinized, washed once in PBS, and relative cell volume was estimated by flow cytometry using forward and sideward light scattering (FACS-Calibur form Becton Dickinson, Franklin Lakes, NJ, USA).#

All data were presented as mean ± SD from at least three independent experiments. Student's double-sided t-test for independent samples was applied to calculate the levels of significance for the effects of UV-irradiation on BV-2 cells (Caspase Assay). Statistical comparison between different incubation times following UV-irradiation was done by either Student's t-test (Caspase Assay) or χ²-test to compare distributions (DAPI-staining, Annexin V labeling) (Zierler and Kerschbaum, 2005). Differences with probability value α less than 0.02 were considered as statistically significant.#