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PubMed1990

Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.

Acrosome↗
PubMed1994

Identification of two human sperm populations using flow and image cytometry.

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder ("marginal population"). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide.

Cell Separation↗
PubMed2006

Effects of 17beta-estradiol on intracellular calcium changes and neuronal survival after mechanical strain injury in neuronal-glial cultures.

The neuroprotective effects of 17beta-estradiol (E2) were investigated using an in vitro model of traumatic brain injury in which cortical neuronal cultures were subjected to mechanical strain-injury. The rise in intracellular calcium ([Ca(2+)](i)) following neuronal injury was reduced by addition of 10 or 100 nM E2 to the cultures immediately following injury. Neuronal damage was measured 24 h after injury by propidium iodide uptake and cell viability by carboxyfluorescein diacetate uptake. Addition of 1, 10, or 100 nM E2 to cell cultures immediately following injury decreased neuronal damage and increased neuronal viability compared to vehicle-treated neurons. These results demonstrate the neuroprotective activity of E2 in an in vitro model of neuronal injury, and suggest that such effects may be related to the ability of E2 to modulate [Ca(2+)](i).

Animals↗
PubMed1992

Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts.

Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.

Autolysis↗
PubMed1999

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.

Apoptosis↗
PubMed2001

Dissociation of DNA fragmentation from other hallmarks of apoptosis in nitric oxide-treated neutrophils: differences between individual nitric oxide donor drugs.

The events of apoptotic cell death can be experimentally dissociated from each other in certain cell types. Here we demonstrate the ability of structurally diverse nitric oxide (NO) donating compounds to delay or enhance neutrophil apoptosis and to differentially influence distinct parameters of programmed cell death. We provide evidence that high concentrations of the NO donors GEA 3162, SPER/NO, and DEA/NO induce morphological and biochemical markers of neutrophil apoptosis, but that only DEA/NO causes a concomitant increase in DNA fragmentation as evidenced by nuclear propidium iodide intercalation and the classical laddering pattern of electrophoresed DNA. In contrast, both GEA 3162 and SPER/NO inhibit DNA cleavage in a time- and concentration-dependent manner. We are the first to show that DNA fragmentation can be dissociated from other changes of apoptosis in NO-treated neutrophils and that it may therefore be inappropriate to assess NO-induced apoptosis solely by measuring DNA fragmentation in this cell type.

Annexin A5↗
PubMed1994

Cryopreservation of the common carotid artery of the rabbit.

We describe experiments on the cryopreservation of the rabbit common carotid artery aimed at improving upon previous results. We describe the design of a double clamp which holds the artery during transportation and storage, preventing twisting, shortening, and collapse of the vessel. The device allowed perfusion with solutions as desired and markedly reduced the extent of endothelial loss during procurement and processing. We also studied the effects of three vehicle solutions; a modified Hanks' solution, a solution originally developed for the cryopreservation of smooth muscle (K-Pipes), and a solution designed for corneal endothelium (CP-Tes). The criteria used to make the assessments were smooth muscle contractility and the structure and function of the vascular endothelium. A new staining method for vascular endothelium (combining propidium iodide with silver nitrate) is described. We found that there was significantly more endothelial cell damage in rabbit carotid arteries frozen in Hanks' solution than in the other solutions, and the recovery of smooth muscle contractility was lowest in the Hanks' group. Arteries cryopreserved using CP-Tes as the vehicle solution showed less endothelial cell damage than arteries preserved with either K-Pipes or Hanks' solution, and these arteries also exhibited the greatest relaxation response to acetylcholine. We conclude that careful handling of the vessels is important; of the solutions studied, CP-Tes is preferred for the cryopreservation of rabbit carotid artery with Me2SO.

Animals↗
PubMed1999

Interferon alpha augments activation-induced T cell death by upregulation of Fas (CD95/APO-1) and Fas ligand expression.

Interferon alpha (IFN-alpha) plays a prominent role in the therapy of a variety of diseases. The Fas/FasL system is crucial for the cytotoxic function and the peripheral elimination of activated T lymphocytes (ATC) by a mechanism referred to as activation-induced cell death (AICD). Recent studies suggest a link between IFN-alpha, the 2', 5'- oligoadenylate system and apoptosis. We therefore asked whether IFN-alpha is able to regulate the Fas/FasL pathway and thereby affects AICD. Peripheral blood mononuclear cells (PBMC), purified T cells and ATC of healthy volunteers were stimulated with various agents and the influence of IFN-alpha on Fas/FasL was assessed by mRNA and protein studies. The proportion of ATC undergoing AICD or anti-Fas-induced apoptosis was determined by FITC-annexin V staining and propidium iodide uptake. IFN-alpha upregulated mRNA expression of Fas and FasL in activated PBMC. Furthermore the concentration of the soluble form of FasL (sFasL) was increased in PBMC and T cells co-stimulated with IFN-alpha and various agents, whereas Fas surface expression was enhanced by IFN-alpha alone. IFN-alpha enhanced apoptosis induced by anti-Fas antibody and augmented AICD via the Fas/FasL pathway. IFN-alpha-regulated AICD may contribute to lymphopenia observed during IFN-alpha therapy. Our data further support that IFN-alpha is a multifunctional cytokine with profound effects on the immune cascades.

Annexin A5↗
PubMed1994

Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations.

We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Höechst 33342, and Höechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone or tributyltin. In the nuclear technique, apoptotic cells appeared as a single population containing reduced DNA content, while in the cellular techniques, apoptotic cells appeared as two or more subpopulations exhibiting increased fluorescence. Of these subpopulations, early apoptotic cells [which excluded propidium iodide (PI), indicating maintenance of membrane integrity] exhibited higher fluorescence but the same level of axial light loss (i.e., size/refractive index) as viable cells; late apoptotic and dead cells (which incorporated PI) exhibited decreased axial light loss. However, while the Höechst dyes allowed discrimination of late apoptotic from dead cells, CF did not. In comparing sensitivity to staining conditions, Höechst 33258 fluorescence was the most stable over time, Höechst 33342 the least, and CF fluorescence not only varied with time, but with tri-n-butyltin methoxide concentration. Comparison of single-parameter analyses revealed that axial light loss was sensitive only to late apoptotic changes; nuclear fluorescence was a better indicator of apoptotic subpopulations, but still underestimated the total percentage of affected cells, and Höechst 33342 distinguished early apoptotic cells as those with elevated fluorescence. Early apoptotic cells stained with Höechst 33258 also exhibited increased fluorescence, but could not be distinguished from late apoptotic and dead cells without a second parameter. These findings indicate that of the methods investigated, the method of choice for detecting apoptosis depends on the goal of analysis: Höechst 33258 was best for discriminating apoptotic subpopulations, and CF was best for assessing alterations of membrane fluidity. For single-parameter analyses, Höechst 33258 was best for determining the total percentage of affected cells, while Höechst 33342 could be used to determine the percentage in early apoptosis.

Animals↗
PubMed1995

Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy.

FDCP-Mix, a pluripotent murine hemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis by either drugs or withdrawal of growth factor (interleukin-3) was studied after treatment with the topoisomerase II inhibitor etoposide (0.5-4 microM). An increase in autolytic activity was the major early morphological change within the cytoplasm, with mitochondria as the main target for autolytic digestion. Despite this macroautophagy, thin sections showed a high number of mitochondria, suggesting mitochondrial proliferation as a result of drug treatment. This observation of an increase in the number of mitochondria was confirmed by flow cytometric studies of mitochondrial overall mass. Multiparameter flow cytometry of cells double stained with propidium iodide and nonyl-acridine orange gave an accurate assay for mitochondrial mass in relation to cell cycle stages. The increase in mitochondrial mass was found in all cell cycle stages. The results suggest a drug-induced proliferation of mitochondria separate from the processes involved in the doubling of mitochondrial mass during the cell cycle and a decline of mitochondria in the later stages of apoptosis.

Acridine Orange↗
PubMed1996

Differential effects of 3,5,3'-triiodothyronine on control and mdx myoblasts and fibroblasts: analysis by flow cytometry.

The possibility of differential effects of triiodothyronine (T3) treatment in vivo on myoblast and fibroblast cell proliferation was examined in control and mdx muscle cultures. Cell isolates were purified in a Percoll gradient, sorted by flow cytometry (light scatter), and characterized as myoblasts and fibroblasts using antiskeletal muscle myosin fluorescence. The two cell types were grown separately or remixed (1:1). Cultures were incubated with or without T3 (10(-9) M) for 19 h. Cells were either exposed to [3H]thymidine for 1 h and DNA prepared for scintillation counts or stained with propidium iodide for cell cycle analysis by flow cytometry. Overall [3H]thymidine uptake per cell was greater in mdx than control cells (mainly fibroblasts and mixed cells) and was decreased by T3 only in myoblast and mixed cultures. Cell cycle data showed that the effects of T3 originated primarily at the G0/G1 phase. There were more mdx than control myoblasts at G0/G1 without T3. After T3 treatment, more control fibroblasts than myoblasts were at G0/G1, but more mdx myoblasts than fibroblasts were at G0/G1. In the absence of T3, there were also fewer mdx than control myoblasts in S. After T3, only the proportion of mdx myoblasts in S phase was reduced. Results are consistent with distinct T3 effects on muscle regeneration in vivo and support the hypothesis that cycling and proliferation of mdx and control myoblasts are differentially modulated by T3. As control and mdx fibroblasts also showed distinct responses to T3, muscle regeneration likely occurs by a complex regulation of gene expression endogenous to specific cell types as well as interactions between cells of different lineage.

Animals↗
PubMed1998

Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells.

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants.

Acids↗
PubMed1998

Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility.

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.

Adult↗
PubMed2000

Reactive oxidizing species produced near the plasma membrane induce apoptosis in bovine aorta endothelial cells.

Many cytotoxic agents initiate apoptosis by generating reactive oxidizing species (ROS). The goal of this study was to determine whether apoptosis could be induced by initial reactions of ROS near the plasma membrane. Bovine aorta endothelial cells (BAEC) were illuminated with evanescent wave visible radiation, which has limited penetration into the basal surface of cells, or by trans-radiation. Imaging of fluorescent dyes localizing in the plasma membrane, mitochondria, or nucleus confirmed that evanescent wave radiation excited only dyes in and near the plasma membrane. Singlet oxygen, an ROS generated by photosensitization, has a very short lifetime, ensuring that it oxidizes molecules residing in or very close to the plasma membrane when evanescent wave radiation is used. Cells with condensed nuclei were considered apoptotic and were quantified after treatment with varying doses of light. Annexin V staining without propidium iodide staining confirmed that these cells were apoptotic. The doses required to induce apoptosis using evanescent wave radiation were 10-fold greater than those needed for trans-irradiation. Quantitative analysis of the evanescent wave penetration into cells supports a mechanism in which the singlet oxygen created near the plasma membrane, rather than at intracellular sites, was responsible for initiation of apoptosis.

Animals↗
PubMed2002

3-Nitropropionic acid neurotoxicity in hippocampal slice cultures: developmental and regional vulnerability and dependency on glucose.

We investigated whether neurotoxic effects of the mitochondrial toxin 3-nitropropionic acid (3-NP) in hippocampal slice cultures are dependent on glucose levels in the culture medium and whether such effects occur via apoptosis or necrosis. In addition, 3-NP toxicity was investigated at two developmental stages of the cultures, prepared from rat brain at postnatal day 5-7 and grown in Neurobasal medium for 1 or 3 weeks. Cultures were exposed to 3-NP in the presence of high (25 mM), normal (5 mM), or low (3 mM) glucose for 48 h, followed by 48 h incubation in medium without 3-NP. Cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) efflux into the medium revealed time- and dose-dependent cell death by 3-NP, with EC(50) values of about 60 microM in high or normal glucose. Regional vulnerability, as assessed by PI uptake and MAP2 immunostaining, in 3-week-old cultures was as follows: CA1 > CA3 > fascia dentata. In low glucose much lower concentrations of 3-NP (25 microM) triggered neurotoxicity. One-week-old cultures were less susceptible to 3-NP toxicity than 3-week-old cultures, but the dentate granule cells were relatively more affected in the immature cultures. We found no evidence for apoptotic cell death by 3-NP in 3-week-old cultures, but in 1-week-old cultures the putative apoptotic marker c-JUN/AP1 and nuclear fragmentation (Hoechst) were significantly increased in the dentate granule cells.

Animals↗
PubMed1997

Leishmania infantum promastigotes: flow cytometry as a possible tool for assessing the effects of drugs on cellular functions.

The capacity of flow-cytometric techniques to detect drug-specific biochemical targets and side effects in Leishmania infantum promastigotes was estimated by assessing the effects of three antileishmanial drugs (pentamidine, allopurinol, and amphotericin B) on parasite metabolism. Cell cycle and total protein content were estimated by staining cells with propidium iodide and fluorescein isothiocyanate, nonprotein thiols were stained by mercury orange, and membrane potential was measured by the accumulation of 3,3'-dipenthyloxacarbocyanine iodide inside the cell. Results showed that dynamic studies in parasites treated with subtoxic concentrations of drugs allowed the detection of drug-specific targets: pentamidine primarily affected nonprotein thiol contents and DNA synthesis, allopurinol primarily affected intracellular protein contents, and amphotericin B primarily affected membrane potential. Moreover, the assessment of cellular functions in parasites treated with increasing concentrations of drugs certified the capacity of these techniques to establish dose-response curves and to permit the detection of side effects.

Allopurinol↗
PubMed1993

An in vitro model of cyclosporine-induced nephrotoxicity.

The predominant hypothesis for cyclosporine-induced acute renal failure is postulated to be prerenal vasoconstriction with concomitant hemodynamic changes; an alternate hypothesis, however, may be that cyclosporine (CsA) affects intrarenal processes, i.e., direct renal parenchymal cell injury. However, reports on this direct effect of CsA on renal parenchymal cells are contradictory. Therefore, the purpose of this study was to address whether CsA is directly toxic to renal parenchymal cells in a primary culture system of rat renal cortical epithelial cells. The cytotoxicity of Sandimmune, the commercial form of CsA in a polyoxyethylated castor oil vehicle (Cremophor), CsA without vehicle, and the Cremophor vehicle was assessed by plasma membrane integrity (lactate dehydrogenase leakage), mitochondrial metabolic activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and gross morphology (phase-contrast microscopy). The cytotoxicity of Sandimmune was also assessed by lysosomal activity (neutral red uptake), by proximal tubular enzyme activity (alkaline phosphatase), and by three fluorescent probes using a multiwell scanner. The three fluorescent probes were propidium iodide which stains nuclei of nonviable cells; bis-carboxyethyl-carboxyfluorescein which is retained by viable cells; and rhodamine 123, which assesses mitochondrial membrane potential. The results of this study demonstrated that Sandimmune caused dose- (10, 25, and 50 microM) and time- (12, 24, and 48 hr) dependent cytotoxicity, while Cremophor caused cytotoxicity only at high concentrations and long incubations. We conclude that (1) CsA is directly toxic to renal parenchymal cells in vitro and this system potentially represents a sensitive model for further mechanistic studies; (2) CsA plus vehicle (Sandimmune) was more cytotoxic to renal cells than CsA alone (without the polyoxyethylated castor oil vehicle).

Alkaline Phosphatase↗
PubMed1999

Heat shock attenuates oxidation and accelerates apoptosis in human neutrophils.

BACKGROUND: The heat shock response entails the increased expression of heat shock proteins (hsp) which are capable of protecting cells from subsequent metabolic insults. Here we are interested in determining whether activation of the heat shock response might affect polymorphonuclear leukocyte (PMN) function and/or longevity. METHODS: Freshly isolated human PMN were either left at 37 degrees C or subjected to a 43 degrees C heat shock treatment (60 min) and subsequently returned to 37 degrees C. During the course of the recovery period a number of parameters were examined for the control and heat shock-treated neutrophils: the relative expression of the highly stress-inducible hsp72; respiratory burst activity as measured by intracellular peroxidation in response to phorbol ester addition; cell-surface expression of CD16; and finally, the extent of apoptosis as determined by both annexin V staining and nuclear propidium iodide staining. RESULTS: Heat shock treatment resulted in a progressive increase in hsp72 production, peaking at 8 h following return of the cells to 37 degrees C. Net intracellular oxidant production was diminished by 46% immediately following the heat shock treatment and deteriorated even further over the next 4 h. Finally, a significant early increase in the rate of apoptosis was observed in the cells subjected to the hyperthermic treatment. This increase in the heat-induced rate of apoptosis was associated with a marked reduction in cell-surface CD16 levels. CONCLUSIONS: By decreasing PMN oxidative functions and by accelerating their apoptotic demise, it would appear that heat shock is anti-inflammatory and not cytoprotective for PMN.

Apoptosis↗