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PubMed2000

Epinephrine, but not dexamethasone, induces apoptosis in retinal pigment epithelium cells in vitro: possible implications on the pathogenesis of central serous chorioretinopathy.

BACKGROUND: The pathogenesis of central serous chorioretinopathy is poorly understood. It is believed to be due to dysfunction of the retinal pigment epithelium and/or choroid and has been associated with elevated levels of epinephrine and administration of corticosteroids. Epinephrine and corticosteroids have previously been shown to induce apoptosis (programmed cell death) in various types of cells. The objective of this study was to investigate whether these agents can induce apoptosis in cultured retinal pigment epithelium cells. This may help elucidate the pathogenesis of central serous chorioretinopathy. METHODS: Third-passage porcine retinal pigment epithelium cells were grown to confluence and incubated for 1-7 days in culture medium containing epinephrine (10(2)-10(9) pg/ml) or a corticosteroid, dexamethasone (4-4x10(4) ng/ml). The cultures were evaluated for apoptosis by phase-contrast microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. RESULTS: Epinephrine (7x10(7)-10(9) pg/ml) induced apoptosis in a dose- and time-dependent manner. Exposure to lower concentrations of epinephrine (10(2)-6x10(7) pg/ml) and all tested levels of dexamethasone did not result in apoptosis. CONCLUSION: Retinal pigment epithelium cells may undergo apoptosis following exposure to elevated levels of epinephrine. These findings suggest a possible pathophysiologic mechanism for the development of central serous chorioretinopathy.

Animals↗

PubMed1975

Endothelial injury induced by thrombin or thrombi.

A variety of blood constituents was injected into an isolated segment of rabbit aorta to determine which elements might be involved in early endothelial injury. Test materials consisted of platelet-rich plasma (PRP) alone; PRP plus adenosine diphosphate (ADP); PRP plus tendon extract; PRP plus thrombin; ultrasonicated PRP alone; platelet-poor plasma alone; and thrombin in saline. Each experimental mixture was left in the aorta for 15 minutes, followed by reflow for 20 minutes. The vessel was then fixed by glutaraldehyde perfusion. Thick sections of the entire circumference of the aorta were taken for phase contrast microscopy and representative arease were selected for electron microscopy. In control PRP alone, platelet-poor plasma alone and with PRP plus ADP there were occasional subendothelial vesicles. When PRP plus thrombin and platelet-poor plasma plus thrombin were injected separately to form a thrombus or when thrombin in saline was used, there was extensive subendothelial vesiculation with focal ulceration and adherence of thrombus to endothelium. Severe injury was associated with the presence of thrombin initiating the polymerization of fibrinogen to fibrin. Electron micrographs demonstrate the earliest lesion as a disruption of the superficial fibrilliary elastica with separation of overlying endothelium.

Adenosine Diphosphate↗

PubMed1996

The bundling of actin with polyethylene glycol 8000 in the presence and absence of gelsolin.

Actin filament and bundle formation occur in the cytosol under conditions of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a means of simulating crowded conditions in vitro. Column-purified Ca-actin was polymerized in the absence and presence of gelsolin (to regulate mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCl and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by fluorescence and phase-contrast microscopy. The minimum concentration of KCl required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cation, bundling is reversible on dilution, as determined by intensity levels, diffusion coefficients, and microscopy. However, with either 2 mM Mg2+ or Ca2+ added, bundling is irreversible under conditions of higher PEG concentrations or longer filaments, indicating that osmotic pressure effects cannot fully explain actin bundling with PEG. Weaker divalent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.

Actins↗

PubMed1986

A method for standardizing cytologic sampling for the estimation of nasal ciliary activity.

A method for sampling cytological brushings of human nasal mucosa using phase-contrast microscopy is detailed and the modes for counting the ciliary beat frequency (CBF) are reviewed. To test the reproducibility of our method, we performed two sampling procedures on subsequent days on each of 19 volunteers and patients. The total volume of harvested cells, the number of beating ciliated cells, and the CBF were assessed. Since there are some differences present in subsequent cell samples, our findings indicate that it may be better to critically evaluate a single sampling procedure in order to establish a correct clinical diagnosis, especially if the number of harvested cells is small. The need to standardize methods for counting the CBF to achieve comparable results is stressed.

Cilia↗

PubMed2005

Fidelity of micropatterned cell cultures.

Methods that enable the culture of micropatterned cells may help advance our fundamental understanding of cell-cell and cell-surface interactions, while facilitating the development and implementation of cell-based biological assays. However, the long-term stability of the cell patterns can limit the time scales over which such methods can be informative. Here we used self-assembling monolayers (SAMs) to localize the adsorption of baby hamster kidney (BHK-21) cells as well as cells from a murine astrocytoma-derived cell line (delayed brain tumor) in linear arrays. We tested the effects of surface chemistries, fibronectin pre-treatments, array dimensions, and cell types on pattern fidelity. Changes in patterns were monitored by phase-contrast microscopy up to 96 h post-plating, followed by digital imaging, and these changes were quantified by measuring an "intrusion distance" or the average distance cells extend beyond the initial adhesive/non-adhesive boundary. Loss of pattern boundaries involved different mechanisms for different cells. Treatment of patterned surfaces with fibronectin prior to plating of cells tended to promote earlier loss of pattern fidelity, and the extent of pattern loss was further augmented for SAMs formed using hydrophobic monolayers. Finally, reduction of gap spacing between adjacent cell arrays promoted pattern loss.

Adsorption↗

PubMed1978

Early effects of vinblastine and vincristine on the rat spermatogenesis: analyses by a new transillumination-phase contrast microscopic method.

Male sterility belongs to the recently recognized complications of cancer chemotherapy and has an increasing importance. Therefore, more information about the mode of action of anticancer drugs on mammalian spermatogenesis is needed. We have developed a technique based on transillumination of living, freshly isolated unstained rat seminiferous tubules, where the cells specifically killed by the drugs are recognized as dull zones. Early stages of cell degeneration can be rapidly analyzed by phase contrast microscopy of living cells. Because the transillumination technique, in addition, permits an accurate recognition of the segments of the seminiferous epithelial wave, the cells representing various stages of the mitotic and meiotic cell cycles during spermatogenesis can be isolated in living state for morphological analysis. Vinblastine and vincristine cause an arrest of mitotic and meiotic divisions to metaphase followed by cell death, which was more rapid after vincristine administration. Both alkaloids had a slight damaging effect on the pachytene spermatocytes. Large doses of both drugs primarily affected the Sertoli cells by destroying their microtubules and mitochondria. Vincristine specifically damaged the acrosomic system and the cytoplasmic bridges of the young spermatids.

Animals↗

PubMed1989

[Use of native microscopic examination of the vaginal flora in the diagnosis of trichomoniasis].

Microscopic diagnosis of vaginal discharge established so far by means of microbial vaginal pictures (MVP) has proved to be insufficient. Phase contrast microscopy has an irreplaceable value in diagnosing vaginal infections transmitted by sexual intercourse. It yields quick and exact diagnosis of the disease, thus providing the possibility to administer treatment addressly and immediately. In equivocal findings and relapses vaginal cultivation is indispensable. In a series of 600 examined women vaginal trichomoniasis was diagnosed in 28.5% of cases. Simultaneous occurrence of other sexually transmitted diseases was recorded in 42% of 171 women infected with Trichomonas vaginalis. In these cases, the course of the disease was markedly altered by the associated infection. The result of MVP was incomplete or false in 26% of the examined women, trichomoniasis failed to be diagnosed in 80 cases.

Animals↗

PubMedDate not supplied

Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture.

We report here the in vitro isolation of Cajal-like interstitial cells from human inactive mammary-gland stroma. Primary cell cultures examined in phase-contrast microscopy or after vital methylene-blue staining revealed a cell population with characteristic morphological phenotype: fusiform, triangular or polygonal cell body and the corresponding (very) long, slender, moniliform cytoplasmic processes. Giemsa staining pointed out the typical knobbed aspect of cell prolongations. Immunofluorescence (IF) showed, like in situ immunohistochemistry, that Cajal-type cells in vitro (primary cultures), expressed c-kit/CD117 and vimentin. In conclusion, the images presented here reinforce our previous hypothesis that human mammary glands have a distinct population of Cajal-like cells in non-epithelial tissue compartments.

Breast↗

PubMed1987

A predictive model for determining asbestos concentrations for fibers less than five micrometers in length.

The controversy of whether small asbestos fibers are biologically significant has not been resolved. The present standard method for evaluating asbestos fiber concentrations in workroom air excludes fibers less than 5 micron long even though it has been shown that small fiber concentrations dominate in a dust cloud. This research project was conducted to develop a mathematical model whereby one could predict small (less than 5 micron length) asbestos fiber concentration based on the fiber count concentration determined by phase contrast microscope analysis. Dry chrysotile asbestos was aerosolized into a chamber and sampled by membrane filtration. Segments from each filter were analyzed by both the NIOSH technique using phase contrast microscopy (PCM) and scanning electron microscopy (SEM) at 2000 X for fiber concentrations. A linear relationship was found to exist between the natural logarithm of the SEM-determined concentration and the natural logarithm of the PCM-determined concentration (r = 0.852). Using these data, a mathematical model was developed to predict SEM concentrations based on PCM counts. This model may have application in retrospective epidemiological studies for estimating small fiber exposure levels to determine if small fibers play a role in disease production. The greatest utility would be in those retrospective studies where the only exposure information available is based on PCM counts.

Air Pollutants↗

PubMed2002

Use of vascular explants for ex vivo neovascularization of biomaterials.

Biomaterial polymers have been proposed as scaffolds for cell assembly in vascular bioengineering. We describe here a new method for the neovascularization of polyurethane meshes from explants of rat aorta. Aortic rings embedded in collagen-permeated polyurethane meshes and cultured in medium supplemented with fetal bovine serum and vascular endothelial growth factor generated florid microvascular outgrowths that efficiently vascularized the available spaces between polyurethane fibers. The neovessels could be identified in the live cultures by phase-contrast microscopy, and in formalin-fixed preparations by the ABC peroxidase procedure, using the endothelial-specific Griffonia isolectin B4. The aortic outgrowths were successfully labeled with the intravital fluorescent dyes Calcein AM or SPDiOC(18), which are nontoxic and can be used for tracking studies. This study shows that artificial biomaterial meshes can be colonized ex vivo with histotypic microvascular networks, and provides the proof of concept for the future development of stably vascularized devices for in vivo implantation.

Animals↗

PubMed1995

Anabolic-androgenic steroid-induced toxicity in primary neonatal rat myocardial cell cultures.

Recent literature reports of myocardial infarction in athletes who self-administer anabolic-androgenic steroid (AAS) and previous animal studies of the effects of AASs on the heart suggest that these drugs may be directly injurious to the myocardium. We have previously demonstrated that 100 microM testosterone cypionate (TC) inhibits all beating activity of primary neonatal rat myocardial cell cultures within 1 hr of exposure and causes significant LDH release by 4 hr of exposure, indicating a direct toxic effect of TC. The purpose of this investigation was to evaluate the effects of commonly abused AASs on primary neonatal rat myocardial cell cultures and to provide insight into early cellular changes that may lead to TC-induced toxicity. Significant LDH release was observed in 5-day-old primary myocardial cell cultures (obtained from 3-to-5-day-old Sprague-Dawley rats) exposed to 100 microM testosterone enanthate (TE), testosterone propionate (TP), and oxymetholone (O) for 4 and 24 hr and in cultures exposed to 100 microM testosterone (T) for 24 hr. Neutral red retention and MTT formazan production were significantly decreased in cell cultures exposed to 100 microM TE, TP, and O after only 4 hr of exposure, indicating a loss of viability and mitochondrial activity. However, there was no effect on viability of cell cultures exposed for 24 hr to 100 microM of a variety of other commonly abused AASs. Phase-contrast microscopy revealed complete disruption of the monolayer in cell cultures treated with 100 microM TE, TP, and O for 4 hr. Treatment of fura-2-loaded myocardial cell cultures with 100 microM TC produced no significant changes in calcium transients or baseline calcium levels for up to 13 min of exposure. These results indicate that O, T, TC, TE, and TP produce a direct toxic effect in heart cell cultures and that early (< 13 min) changes in calcium homeostasis are unlikely to participate in the mechanism of toxicity.

Anabolic Agents↗

PubMed1998

Regional influences of the fallopian tubes on the rate of boar sperm capacitation in surgically inseminated gilts.

Aliquots of ejaculated boar semen containing known numbers of spermatozoa were deposited into the caudal isthmus or rostral ampulla of the Fallopian tubes of gilts at, or immediately after, ovulation to assess regional influences on the rate of capacitation. Eggs were recovered during a second intervention 4, 5, 6 or 7 h after surgical insemination and were examined by phase-contrast microscopy. Results were obtained from ten animals in each of the 4-, 5- and 6-h groups and from eight animals in the 7-h group. With two exceptions, fertilized eggs were not recovered until 6 h after insemination into the isthmus, the proportion (45.6%) being significantly greater than the corresponding figure (1.4%) for ampullary insemination (P < 0.001). Similarly, the proportion of fertilized eggs recovered 7 h after insemination into the isthmus (58.7%) was significantly greater than after ampullary insemination (21.9%; P < 0.01). Numbers of spermatozoa associated with the zona pellucida remained low in all these instances, with mean figures per egg ranging from 0.3 to 3.8. Insemination into the isthmus gave a 1-2 h advantage in fertilization compared with insemination into the ampulla. Although relative rates of sperm cell progression to the site of fertilization may have contributed to this, there is strong evidence that rates of capacitation differ significantly in the respective portions of the Fallopian tube. Therefore, attention was focused on: (1) the viscous glycoprotein secretion in the caudal isthmus acting to remove seminal plasma from the sperm surface; and (2) the phase of sperm head binding to the isthmus epithelium. Gradients in local endocrine modulation by the adjacent ovary offer one explanation for the functional specialization of different regions of the Fallopian tubes.

Animals↗

PubMed1997

[Differentiation between glomerular and non-glomerular erythrocyturia: what is the value of differential microhematuria diagnosis?].

A few years ago, a new and simple method has been proposed to help guiding the investigation of microhematuria. This method which consists in quantifying the percentage of deformed polymorphous erythrocytes in the urinary sediment using phase contrast microscopy allows to distinguish glomerular from non-glomerular erythrocytes. In this paper, we have reviewed the recent literature concerning this approach and have discussed the conclusions according to our own experience based on the analysis of 147 patients presenting with microhematuria. Our results demonstrate that this technique is still limited by the difficulty to obtain well-defined cut-off values which effectively differentiate renal from urologic diseases. Thus, only extreme results showing either the total absence or the presence of a very high percentage of dismorphic erythrocytes appear to be helpful for the physician. Despite the introduction of this new method, many patients with microhematuria are insufficiently investigated.

Diagnosis, Differential↗

PubMed1997

Three-dimensional endothelial-tumor epithelial cell interactions in human cervical cancers.

The purpose of this study is to understand the multicellular interaction between tumor epithelial (TEC) and human umbilical vein endothelial cells (HUVEC). The development of in vitro systems in which to coculture these cells as multicellular aggregates is very critical. Cell lines were established from cervical tumor cells (n = 6) and two from HUVEC (n = 2) and they were cultured as three-dimensional (3-D) multicellular-cultures using Cytodex-3 microcarrier beads in the rotating wall vessel (RWV). After a 240-h incubation, TEC and HUVEC proliferated exponentially to 4.2 x 10(7) and 2.2 x 10(7) cells/ml, respectively, without requiring a feeder layer; in contrast to the two-dimensional (2-D) cultures that average about 8 x 10(5) cells/ml. Phase contrast microscopy indicated formation of 3-D aggregates that varied in size from 0.5 to 5 mm. The size of the aggregates (1-5 mm, 6-14 microcarriers) increased over time; however, the number of aggregates (0.5-1 mm, 2-5 microcarriers) decreased over a long-term incubation (240 h) because the cells merged to form large clumps. Maximum aggregation was observed with TEC at 120 h and HUVEC at 96 h. The culture of TEC in the absence of HUVEC produced minimal differentiation in contrast to cocultures. The TEC and HUVEC as cocultures in RWV proliferated at an accelerated rate (1.3 x 10(7) cells/ml, 96 h). The TEC-HUVEC coculture presented tubular structures penetrating the tumor cell masses, forming aggregates larger in size than the monocultures and typically with greater cell mass and number. The cells were viable (trypan blue exclusion) and metabolically active (glucose utilization) until 240 h. These data suggest that RWV provides a new model that allows us to investigate the regulatory factors that govern tumor angiogenesis.

Adenocarcinoma↗

PubMed1988

Purification of meiotic spindles and cytoplasmic asters from mouse oocytes.

The unfertilized mouse oocyte is arrested at second metaphase of meiosis with microtubules existing exclusively in the meiotic spindle. Multiple inactive cytoplasmic microtubule organizing centers (MTOCs) are also present. These MTOCs can be identified immunocytochemically with an autoimmune serum (No. 5051) directed against pericentriolar material (PCM) and also by their nucleating capacity in the presence of taxol which effectively lowers the critical concentration for tubulin polymerization. Taxol induces the formation of cytoplasmic microtubule asters around the PCM foci, a process which also occurs in untreated eggs after fertilization. The molecular characterization of these structures has not been undertaken previously, probably due to the very small amount of material available. We have developed a single-step purification procedure by which very clean preparations of meiotic spindles and cytoplasmic asters can be obtained, as judged by phase-contrast microscopy and transmission electron microscopy. The purified structures were shown to correspond to those observed in vivo: positive staining of the spindles was observed with anti-tubulin and anti-phosphoprotein (MPM2) antibodies, and positive staining of the MTOCs was observed with MPM2, No. 5051, and anti-calmodulin antibodies. As expected, tubulin was the major protein present in the preparations. Silver staining of SDS-PAGE also revealed the presence of a small number of other polypeptides (Mr of around 47, 35, and 25K). Amongst newly synthesized polypeptides associated with the preparation, two prominent high molecular weight proteins (greater than 200K) were enriched in addition to tubulin and polypeptides with Mr of around 52, 41, and 35K.

Animals↗

PubMed1995

In vitro studies of human choroidal endothelial cells.

Vascular endothelial cells play an important role in progression or healing of various retinal or choroidal diseases and they have a broad organ specificity. We have grown and studied human choroidal endothelial cells (CECs) cultured in a collagen gel, where they form tube-like structures. CECs were differentially isolated from choroidal tissues and cultured to near homogeneity. The cells were embedded in a type I collagen gel in 24-well culture dishes and incubated with M199 medium containing 10% fetal calf serum. Gels were observed using phase contrast microscopy and were evaluated histologically by light and electron microscopy. The CECs were strongly positive for factor VIII-related antigen and actively ingested diI-acetylated LDL, indicating their endothelial nature. Ultrastructural analysis of the tube-like structures revealed a central lumen surrounded by cells joined apically by junctions and showing prominent pinocytotic activity, fenestrations and basement membrane formation; these features are typical of CECs in vivo. This preparation should be a useful tool to study the development of physiology and pathology of choroidal endothelium.

Carbocyanines↗

PubMed1988

[Assessment of the intensity of lymphocyte blast transformation in the crypts of the palatal tonsils by using DNA cytophotometry].

Cell fractions isolated from lacunae of palatine tonsils with different functional activities were taken from 40 children 6-15 years of age. The cell fractions were examined using vital phase-contrast microscopy, light microscopy of Pappenheim-stained smears and the Feulgen cytophotometry. Lymphocytes with signs of immunological involvement were shown to constitute 95 per cent of the whole cell lacunar population of the active functional tonsils. 35 per cent of lymphocytes, being in different stages of blast transformation, were found. The most intense blast transformation was revealed in patients with tonsil inflammation. 5-13 per cent of lacunar nuclei with DNA content above the diploid level were found in these patients. Some multicellular islets accumulating small lymphocytes, lymphocytes at different stages of blast transformation, and macrophages were revealed to imitate the cooperation of immunocompetent cells. The data obtained can be helpful in the practical surgery of palatine tonsils.

Adolescent↗

PubMed2006

Apoptotic activity of a novel synthetic cantharidin analogue on hepatoma cell lines.

Cantharidin isolated from Mylabris caraganae and other insects is used traditionally as an anti-cancer drug. However, its toxicity on the renal system and suppression effect on bone marrow limits its clinical usage. Recently, we have synthesized two cantharidin analogues, CAN 029 (compound 2) and CAN 030 (compound 3). Although both showed an apoptotic induction ability on cancer cells, they were still relatively toxic towards non-malignant haematological disordered bone marrow. Based on the principle structure of cantharidin, we have further chemically synthesized another analogue, CAN 032. The cytotoxic activity of this analogue was screened on both Hep3B hepatocellular carcinoma and SK-Hep-1 liver adenocarcinoma cell lines by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. Morphological changes of hepatoma cell lines were recorded under an inverted microscope. The possible tolerance of these analogues was further investigated using non-malignant haematological bone marrow primary culture. CAN 032 showed a significant cytotoxic response on both hepatoma cell lines in which the potencies were comparable to that of cantharidin. Further screening on the bone marrow tolerance revealed that compound CAN 032 showed a relatively less toxic effect. Phase contrast microscopy demonstrated that cell shrinkage, rounding, loss of adherent property and loss of colony-formation ability were induced. The dose-dependence of the response of CAN 032 on Hep3B was further assayed by DNA fragmentation gel electrophoresis. The G1 peak of Hep3B cells was reduced. Chemically synthesized CAN 032 may provide an improved therapeutic advantage over traditional cantharidin.

Adult↗