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PubMed2000

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

BACKGROUND: Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. METHODS: We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. RESULTS AND CONCLUSIONS: With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Animals↗
PubMed2003

Timed sequential therapy of acute leukemia with flavopiridol: in vitro model for a phase I clinical trial.

PURPOSE: The survival of adults with acute leukemias remains unsatisfactory and requires new treatment approaches. Flavopiridol modulates cell cycle progression, inhibits transcription, and induces apoptosis. We designed an in vitro model of timed sequential therapy for acute leukemia to determine whether flavopiridol can: (a). trigger apoptosis in fresh acute leukemia; and (b). recruit surviving leukemic cells to a proliferative state, thereby priming such cells for the S-phase-related cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). EXPERIMENTAL DESIGN: Bone marrow cells from 20 adults with relapsed and refractory acute leukemias were enriched for blasts by Ficoll Hypaque sedimentation. Blasts were cultured on day 0 in flavopiridol 250 nM for 24 h, removed from flavopiridol for 24 h, and then cultured in ara-C 1 microM for an additional 72 h (F(250)A(1)). Apoptosis and cell cycle phase distribution were estimated from cells stained with propidium iodide. Cell survival was determined after the 72 h ara-C exposure by double cytofluorescence assay with fluorescein diacetate and propidium iodide. RESULTS: Flavopiridol induced a 4.3-fold increase in apoptosis in human leukemia samples within the first 24 h of culture. Subsequent removal of flavopiridol led to a 1.7-fold increase in the proportion of cells in S phase by day 2. Mean survival in F(250)A(1) cultures after 72 h exposure to ara-C was 35.6% compared with flavopiridol alone (F(250)A(0), 56.1%; P = 0.0003) and ara-C alone (F(0)A(1), 65.2%; P < 0.00001). CONCLUSIONS: Flavopiridol induces apoptosis in marrow blasts from patients with refractory acute leukemias. Furthermore, flavopiridol pretreatment increases the proapoptotic and cytotoxic effects of ara-C. The advantage of sequential FP(250)A(1) over either agent alone is seen for both acute myelogenous leukemia and acute lymphoblastic leukemia. These findings support a clinical trial of timed sequential therapy where flavopiridol is given for cytoreduction and subsequent priming of remaining leukemic cells for enhanced cycle-dependent drug cytotoxicity.

Adult↗
PubMed1990

A fluorometric viability assay for single human and rat islets.

A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluorescein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (454 nm). The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively. All measurements were corrected for background fluorescence. The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, P less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells. This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.

Animals↗
PubMed1985

On the heterogeneity of Purkinje neurons in vertebrates. Cytochemical and morphological studies of chromatin during eel (Anguilla anguilla L.) life cycle.

The relationship between the heterogeneity of Purkinje cell chromatin and the functional involvement of cerebellum has been analyzed comparing two stages of eel life cycle (yellow eel and silver eel) which are characterized by a different degree of swimming activity. The DNA content and the chromatin condensation have been studied by means of microdensitometry (Feulgen-DNA, Feulgen-DNA/area), microfluorometry (DNA after intercalation with Propidium Iodide at low and high concentration) and electron microscopy. In the transition from the yellow eel to the silver eel, the Purkinje cell heterogeneity undergoes variations. In the silver eel, which shows a higher swimming activity, high Feulgen-DNA and Propidium Iodide-DNA (H2c to 4c) were more frequent and a greater heterogeneity in the chromatin condensation was observed. In particular, the changes were more evident in the lobus vestibulolateralis; mainly in this area, which receives inputs from the lateral line, the Purkinje cells are scattered in the molecular layer. On the contrary, in the yellow eel these large cells occupy the zone near or within the granular layer. The data suggest a relationship between the degree of heterogeneity of Purkinje cells and the afferent systems of the cerebellar areas examined. The changes of cerebellar cytoarchitectonics may be expression of the higher degree of the swimming activity of silver eel.

Anguilla↗
PubMed1985

Stromal and nuclear markers for rapid identification of pituitary adenomas at biopsy.

We tested fluorescent and light microscopic markers to improve recognition of pituitary adenomas at biopsy. The optimal reagent was 100 mg/L of fluoresceinated Ricinus communis agglutinin 120 (RCA 120) lectin plus 3 mg/L of propidium iodide. The refrigerated solution was immediately available for use on routine frozen sections. The sections were stained for one minute and viewed immediately after they were rinsed with saline and coverslips applied. Fluorescein-labeled RCA 120, which binds galactose, localized vascular stroma. Propidium iodide, which binds nucleic acids, stained nuclei. Stromal configuration, nuclear morphology, and cell to stroma ratio were illuminated and used to distinguish adenoma from adenohypophysis. We also describe a method utilizing peroxidase-conjugated RCA 120 that demonstrates the same features by light microscopy. Fluoresceinated RCA 120-stained vessels and stroma of routinely processed material more reliably than hematoxylin-eosin or labeled antibody to fibronectin and faster than peroxidase-conjugated RCA 120 or the rapid method for reticulin.

Adenoma↗
PubMed1986

Automated fluorescent analysis for drug-induced cytotoxicity assays.

The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug-exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P less than 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency.

Adult↗
PubMed1982

Flow cytometry of mouse bone marrow cells cultured in vivo or in vitro.

We have developed new methods for counting cyto-(fluoro)-metrically cells retrieved from diffusion chamber and methylcellulose cultures of mouse bone marrow. Total cell counting and DNA distribution measurements were based on detection of the fluorescence from propidium iodide stained nuclei. Staining of cell nuclei with propidium iodide further allowed the separate enumeration of avid phagocytes that had endocytosed fluorescent latex beads. Finally, we quantified peroxidase positive cells by using o-Dianisidine as a substrate for the enzyme, the formation of brown reaction product being enhanced by the presence of lidocaine and dichlorophenol. We found the new techniques precise, reliable and suitable for analysis of cell growth and maturation in culture.

Animals↗
PubMed1995

Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study.

The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (delta psi) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but delta psi decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.

Animals↗
PubMed1997

Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors.

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

Animals↗
PubMed1997

Confocal fluorescence analysis of the depth and focus of cytogenetic preparations.

OBJECTIVE: To characterize differences in the depth of fluorescent probes, to observe estimated depth levels (focal planes) on fluorescent in situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase. STUDY DESIGN: We used labeling of the targets by the probes (sequences labeled by fluoroscein isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. The investigation was performed on this model by three-dimensional (3-D) sequences of images obtained on a single photomultiplier detector of a confocal microscope by selection of emission between 510 and 550 nm and by (z) displacement. The investigation was also performed by obtaining sequences of images from spherical fluorescent beads to verify (z) focusing, to visualize depth differences and to analyze estimated factor images. RESULTS: Estimated images showed depth differences in FITC-stained targets as well as in nuclei, stained with propidium iodide, in interphase and in fluorescent beads, that could not be visualized by conventional 3-D reconstruction. CONCLUSION: It is possible to study 3-D architecture of flattened preparations and penetration of fluorophores inside the beads and to evaluate depth differences.

Cell Nucleus↗
PubMed1998

An automated method for discriminating aneugen- vs. clastogen-induced micronuclei.

A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between aneugen- and clastogen-induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC-conjugated anti-CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single-parameter histogram. The histogram statistic "median channel" was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or aneugenic activity, and 2) MN resulting from aneugens can be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content.

Animals↗
PubMed1998

Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration.

BACKGROUND: Choroidal neovascular membranes (CNVMs) in age-related macular degeneration show progressive histologic changes from active, cellular, highly vascularized membranes to inactive paucicellular scars. The purpose of this study was to determine whether apoptosis was involved in the evolution of these changes, what cell types are involved, and whether there was an association with the Fas antigen (Fas or CD95) and Fas ligand (FasL). METHODS: Serial frozen sections from 10 surgically excised CNVMs were stained by the TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine 5-triphosphate nick-end labeling) method for detection of DNA strand breaks and by propidium iodide staining for morphologic detection of apoptosis. Immunoperoxidase staining was used for detection of Fas, FasL, and cell-type specific antigens. RESULTS: Highly vascularized membranes contained cells with TUNEL-positive nuclei, particularly in the regions of neovascularization, while fibrotic membranes showed few, if any, TUNEL-positive cells. Many of the TUNEL-positive cells were stromal retinal pigment epithelial cells, although smaller numbers were identified as endothelial cells and macrophages. Confocal microscopy of propidium idoide-stained sections confirmed the presence of apoptotic nuclei. The extent of Fas antigen expression correlated with extent of apoptosis. FasL expression was found in all specimens but was most intense in the highly vascularized membranes. CONCLUSIONS: Highly vascularized CNVMs related to age-related macular degeneration show apoptosis in stromal retinal pigment epithelial cells, endothelial cells, and occasional macrophages. Apoptosis is associated with prominent Fas and FasL expression.

Aged↗
PubMed1996

Apoptotic inhibition of head and neck squamous cell carcinoma cells by tumor necrosis factor alpha.

OBJECTIVES: To evaluate the effect and mechanism of action of tumor necrosis factor alpha (TNF-alpha) on head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, to find out whether TNF-alpha induces apoptotic (programmed) cell death. DESIGN: Cytotoxicity and kinetics were evaluated through the use of a methylene blue colorimetric assay. The mechanism of cell death was evaluated through gel electrophoresis of extracted DNA, double-stranded DNA fragmentation, and Hoechst 33342-propidium iodide double staining. TARGET CELLS: Four HNSCC cell lines were investigated: UMSCC-1, UMSCC-8, UMSCC-19, and CAL-27. RESULTS: Tumor necrosis factor alpha at a concentration of 10000 U/mL induced cytotoxic effects in all four cell lines with varying sensitivities. Significant cytotoxic effects required 3 to 4 days of exposure to TNF-alpha. Further experiments demonstrated that TNF-alpha induced double-stranded DNA fragmentation of targets, which preceded the detection of actual cytotoxic effects. Treated HNSCC cell lines had their DNA fragmented into a ladder pattern of endonucleolytic cleavage of multiples of 180 to 200 base pairs, consistent with apoptosis. Propidium iodide-Hoechst 33342 double staining confirmed that TNF-alpha-induced nuclear alterations precede permeabilization of the plasma membrane, supporting cell death in these tumor cells by apoptosis. CONCLUSION: Tumor necrosis factor alpha exposure results in variable cytotoxic reactions in HNSCC cell lines, with DNA fragmentation preceding significant cell death. The mechanism of cell death is apoptosis, with typical morphological features. Further studies are needed to better elucidate the role of TNF-alpha in the treatment of in vivo HNSCC.

Apoptosis↗
PubMed1996

Sequestration of mitotic (M-phase) chromosomes in autophagosomes: mitotic programmed cell death in human Chang liver cells induced by an OH* burst from vanadyl(4).

BACKGROUND: Fragmentation of genomic DNA in apoptosis/programmed cell death (PCD) is a characteristic hallmark in which both 2N and 4N DNA from G1, S, and G2/M cell cycle phases were seen degraded to the sub-2N Ao level in PCD such as from serum deprivation, glucocorticoid treatment, and gamma-radiation. However M-phase (mitotic) cells are said to perish only via non-programmed or necrotic cell death unless they were allowed to complete cytokinesis and re-enter interphase. The morphological criteria of PCD refer only to interphase cells with intact nuclear membranes, none seems applied to mitotic cells. We show here autophagic sequestration of mitotic chromosomes in a typical PCD response where G1, S, and G2/M DNA were replaced by a sub-2N Ao peak, suggesting that mitotic cells may yet have the option of PCD or suicide. Autophagy is absent in necrosis. METHODS: Mitotic human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts initiated by a burst of hydroxyl free radicals (OH*) generated from vanadium internalized by an NH4Cl prepulse containing vanadyl(4) ions. Total (free and bound) vanadium uptake was quantitated by elemental spectral analysis of single cells using a) Particle-Induced X-ray Emission (PIXE) profiling, and b) Scanning Transmission Ion Microscopy (STIM) in the nuclear microscope. The Coulter EPICS PROFILE II flow cytometer was used for a) the cell cycle analysis using propidium iodide-DNA binding, b) intracellular pH (pHi) evaluation in the acidification-and-recovery cycle, using ratiometric 2',7'-bis(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF) fluorescence quantitation. Transmission electron microscopy examined the morphological changes. Vanadyl(4)- generated hydroxyl free radicals (OH*) were evaluated by measuring OH*-benzoic adduct fluorescence at 304/413 nm using the SPEX Fluoromax photon counting spectrofluorometer. RESULTS: Nuclear microscopy showed that a 30 min acidification prepulse containing 4mM vanadyl(4) ions, V(4), had increased the total (free and bound) vanadium concentration of human Chang liver cells from normal ultratrace levels to 56,992 ppm of dry wt (1.1174 Eq per kg dry wt). After the prepulse, cells realkalinized in DMEM growth medium, recovering to the physiological pHi level in 30 min. At the physiological pH 7 level, V(4) generated a burst of OH* free radicals in the order of 15,000 folds above the prepulse (pH 4.5) level. In these conditions, spectrofluorometric evaluation showed loss of DNA intercalation with propidium iodide (PI-DNA binding) indicating DNA degradation. Cell-by-cell evaluation of the PI-DNA binding by flow cytometry showed abolition of G1, S, and G2/M phases and their replacement by a sub-2N Ao peak of fragmented DNA, emulating serum deprivation PCD in these cells. Immediately upon initiating an OH* burst ultrastructural profiles showed mitotic chromosomes (M-phase chromatin) being surrounded by rough endoplasmic reticulum (RER) and small vesicles, indicating their sequestration in autophagosomes. Autophagy was also seen to be a prominent feature in serum deprivation PCD. CONCLUSION: Sequestration of mitotic chromosomes by autophagosomes in a typical PCD response showed a well-defined morphological pathway for direct degradation of M-phase chromatin without first completing cytokinesis. Mitotic cells could commit suicide via autophagy directed at its own chromatin. Autophagic sequestration of chromatin in PCD is novel.

Apoptosis↗
PubMed1997

Apoptotic condensations in M-phase cells.

BACKGROUND: Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion. METHODS: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease. RESULTS: The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells. CONCLUSION: Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.

Apoptosis↗
PubMed2000

Cell death in denervated skeletal muscle is distinct from classical apoptosis.

Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. The purpose of the present study was to investigate the occurrence of cell death and its mechanism in rat skeletal muscle undergoing post-denervation atrophy. We studied the expression of specific markers of apoptosis and necrosis in experimentally denervated tibialis anterior, extensor digitorum longus and soleus muscles of adult rats. Fluorescent staining of nuclear DNA with propidium iodide revealed the presence of nuclei with hypercondensed chromatin and fragmented nuclei typical of apoptotic cells in the muscle tissue 2, 4 and to a lesser extent 7 months after denervation. This finding was supported by electron microscopy of the denervated muscle. We found clear morphological manifestations of muscle cell death, with ultrastructural characteristics very similar if not identical to those considered as nuclear and cytoplasmic markers of apoptosis. With increasing time of denervation, progressive destabilization of the differentiated phenotype of muscle cells was observed. It included disalignment and spatial disorganization of myofibrils as well as their resorption and formation of myofibril-free zones. These changes initially appeared in subsarcolemmal areas around myonuclei, and by 4 months following nerve transection they were spread throughout the sarcoplasm. Despite an increased number of residual bodies and secondary lysosomes in denervated muscle, we did not find any evidence of involvement of autophagocytosis in the resorption of the contractile system. Dead muscle fibers were usually surrounded by a folded intact basal lamina; they had an intact sarcolemma and highly condensed chromatin and sarcoplasm. Folds of the basal lamina around the dead cells resulted from significant shrinkage of cell volume. Macrophages were occasionally found in close proximity to dead myocytes. We detected no manifestations of inflammation in the denervated tissue. Single myocytes expressing traits of the necrotic phenotype were very rare. A search for another marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, revealed the presence of multiple DNA fragments in cell nuclei in only a very small number of cell nuclei in 2 and 4 month denervated muscle and to less extent in 7 month denervated muscle. Virtually no TUNEL reactivity was found in normal muscle. Double labeling of tissue denervated for 2 and 4 months for genome fragmentation with the TUNEL method and for total nuclear DNA with propidium iodide demonstrated co-localization of the TUNEL-positive fragmented DNA in some of the nuclei containing condensed chromatin and in fragmented nuclei. However, the numbers of nuclei of abnormal morphology containing condensed and/or irregular patterns of chromatin distribution, as revealed by DNA staining and electron microscopy, exceeded by 33-38 times the numbers of nuclei positive for the TUNEL reaction. Thus, we found a discrepancy between the frequences of expression of morphological markers of apoptosis and DNA fragmentation in denervated muscle. This provides evidence that fragmentation of the genomic DNA is not an obligatory event during atrophy and death of muscle cells, or, alternatively, it may occur only for a short period of time during this process. Unlike classical apoptosis described in mammalian thymocytes and lymphoid cells, non-inflammatory death of muscle fibers in denervated muscle occurs a long time after the removal of myotrophic influence of the nerve and is preceded by the progressive imbalance of the state of terminal differentiation. Our results indicate that apoptosis appears to be represented by a number of distinct isotypes in animals belonging to different taxonomic groups and in different cell lineages of the same organism.

Animals↗
PubMed1996

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Aneuploidy↗
PubMed1997

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

Animals↗