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PubMed1985

The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus.

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [3H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [3H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.

Animals↗
PubMed2002

[Effects of nitric oxide on pleural lymphatic stomata and lymphatic drainage in the rat].

To investigate the effects of nitric oxide (NO) on the pleural lymphatic stomata and lymph absorption from the pleural cavity, the NOS (nitric oxide synthase) inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME) and the NO donor isosorbide dinitrate (ISDN) were injected into the peritoneal cavity of the rats respectively. Trypan blue was used as a tracer. Then the concentrations of NO and trypan blue in the blood serum were measured, and the ultrastructural changes in pleural lymphatic stomata were observed under a scanning electron microscope (SEM) and studied by a computer image processing system attached to SEM. It turned out that the concentration of NO in the serum was 49.34+/-18.47 micromol/L, and the area and density of the pleural lymphatic stomata were 6.80+/-1.13 microm(2) and 170.24+/-66.60 /0.1 mm(2) respectively in the NO donor group. The concentration of NO reduced to 17.72+/-6.58 micromol/L, and the area and density of the pleural lymphatic stomata were 5.72+/-1.54 microm(2) and 61.71+/-12.73/0.1 mm(2) in the NOS inhibitor group. We found that the area and density of the pleural lymphatic stomata were positively correlated with the NO quantity. After the tracer was injected into the pleural cavity, the NO donor group exhibited a higher trypan blue concentration than the control group. The ability of the pleura to absorb trypan blue was enhanced because of the large opening of the stomata. It is suggested that NO can increase lymph absorption of the pleura by relaxing pleural lymphatic stomata.

Animals↗
PubMed1977

Electron microscope studies on the relationship between mitochondria and inclusion body in spheroid alveolar epithelial cell after vital staining with acidic and basic dyes.

Electron microscope observations were conducted on the relationship between mitochondria and inclusion body in mice spheroid alveolar epithelial cells after injection of trypan blue, an acidic dye and Alcian blue 8GS, a basic dye, by vital staining procedures. When both dyes were injected, the mitochondria of the spheroid alveolar epithelial cell became degenerated; however, in injection of only trypan blue, the cristae showed an increase in electron density. In injection on only Alcian blue 8GS, the cristae showed negative contrast. In most cases the trypan blue particles did not enter into mitochondria, whereas particles of Alcian blue 8GS sometimes entered into the mitochondria. When trypan blue particles entered mitochondria, deposits were not evident in the inclusion body, whereas when Alcian blue particles entered mitochondria deposits were seen in the inclusion body. In both of these cases only a few inclusion bodies were formed so that only traces or no inclusion bodies with vacuolar appearance were observed. From these findings it is suggested that mitochondria maybe convert to inclusion bodies.

Alcian Blue↗
PubMed1993

Caco-2 cell metabolism of oxygen-derived radicals.

Reactive oxygen metabolites have been associated with gastrointestinal injury and may play a role as mediators of inflammation. The effect of oxygen metabolites on Caco-2 cell viability (trypan blue exclusion and 51Cr release), hexose monophosphate shunt activity, and glutathione was assessed. Caco-2 cells were incubated with amino acid oxidase, xanthine oxidase, menadione, and t-butylhydroperoxide in the presence and absence of superoxide dismutase, catalase, mannitol, and butylated hydroxytoluene. With amino acid oxidase, trypan blue exclusion decreased (P < 0.01) and 51Cr release, oxidized glutathione, and shunt activity increased (P < 0.05). The addition of catalase attenuated these changes. Trypan blue exclusion decreased (P < 0.005) and 51Cr release, oxidized glutathione, and shunt activity increased (P < 0.01) with xanthine oxidase. The addition of superoxide dismutase caused a further increase in 51Cr release, oxidized glutathione, and shunt activity (P < 0.01), which was prevented by the addition of catalase or mannitol. t-Butylhydroperoxide did not effect 51Cr release or trypan blue exclusion, but oxidized glutathione and shunt activity increased (P < 0.01). The increase in shunt activity was prevented by preincubation with butylated hydroxytoluene (P < 0.01). Menadione did not alter trypan blue exclusion or 51Cr release, but caused an increase in oxidized glutathione and shunt activity (P < 0.001). The increase in shunt activity was attenuated by preincubation with butylated hydroxytoluene (P < 0.001). Menadione also caused a depletion of total glutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromium Radioisotopes↗
PubMed1981

An experimental study on the morphogenesis of spina bifida.

This investigation was performed to study the morphogenesis of caudal meningomyeloschisis or spina bifida aperta during neurulation. Pregnant rats were treated wih trypan blue to induce these defects in the closure of the neural walls. Maternal serum was studied to determine trypan blue levels. Both control and treated embryos were examined grossly, at cellular and subcellular level on days 10 through 13 of gestation. Trypan blue was distributed throughout the maternal tissues. There existed a maternal blood-brain barrier and a barrier at the level of the visceral yolk sac and embryonic gut preventing passage of the dye through these entodermal structures. The interstitial space in the paraxial mesenchyme was considerably increased in day 10 treated embryos (6--7 somites) but cellular and ultrastructural changes were not seen. This accumulation of intercellular fluid was only seen in the restricted area of the mesoderm above the invaginating intra-embryonic entoderm. On day 11 the closure of the neural walls was completed in control embryos. In the treated embryos of the similar stages a blister which contained embryonic blood cells had replaced the interstitial mesenchymal fluid. This structural disturbance in the non-segmented mesenchyme interfered primarily with the neurulation and resulted in caudal meningomyeloschisis (spina bifida aperta). Trypan blue was thought to interfere with the function of the extra-embryonic membranes and the primitive gut. The results of this study supported the hypothesis of indirect teratogenic effect upon the embryo.

Abnormalities, Drug-Induced↗
PubMed1982

Cell structure and percent viability by a slide centrifuge technique.

It was found that a slide centrifuge (Cytospin) preparation of a cell suspension allowed a reliable assessment of not only cell structure but also the percentage of non-viable cells. The non-viable cells appeared as "smear" cells and paralleled in number the cells taking up trypan blue. Direct experiment showed the unstained viable cells in a trypan blue cell suspension remained intact in a Cytospin preparation while the cells taking up trypan blue were the "smear" cells. The non-viability of the "smear" cells was confirmed by their inability to survive in culture.

Animals↗
PubMed1998

Effects of cell concentration on viability and metabolic activity during cryopreservation.

Increasing the cell concentration during the cryopreservation of red blood cells (RBC) increased hemolysis. Similarly, increasing the cell concentration during the cryopreservation of hepatocytes reduced both the viability of the cells as assessed by trypan blue exclusion and the metabolic activity of the trypan blue-excluding cells. In both cell types, significant damage appeared at cell concentration levels exceeding 60%. With tightly packed RBC, hemolysis reached 63%, while for hepatocytes after thawing and dilution, trypan blue viability was reduced to 41% of that of the cells initially isolated. The viability of cryopreserved hepatocytes, estimated by trypan blue exclusion, was considerably reduced following incubation for 1 h at 37 degrees C and the extent of this reduction was also a function of cell concentration during freezing and thawing. The trypan blue viability of hepatocytes that were tightly packed during cryopreservation and then incubated at a concentration of 5 x 10(6) viable cells/ml fell dramatically to only 12.5%. The protein-synthesizing activity and the membrane transport activity of these cells, expressed in terms of cells that excluded trypan blue immediately following thawing and removal of the cryoprotectant, were reduced to 38.7 and 33.5%, respectively, of the activity in cells that were packed at 10% cell concentration. The fall in metabolic activity may have been due to complete loss of activity in some of the cells or reduced activity in most or all of the cells or any combination of these factors. It is concluded that there may be many mechanisms involved, but the most important factor is probably stresses produced by unphysiological cell-cell contacts occurring during the freeze-thaw cycle.

Animals↗
PubMed1982

The release of 4,4'-diaminobiphenyls from azodyes in the rat.

Six azodyes derived from benzidine, o-tolidine or o-dianisidine were separately administered orally by gavage to rats. Urine was collected over a 24 h period. Following dichloromethane extraction, urines were analysed by h.p.l.c. for the presence of the respective parent amine and its N-acetylated and N,N'-diacetylated derivatives. After alkaline hydrolysis, urines were analysed for the amines resulting from the cleavage of N-conjugates. All six dyes, direct black 38, direct brown 95, direct blue 6, Congo red, trypan blue and Chicago sky blue were found to be reduced, N-acetylated and N-conjugated. However, no N,N'-diacetylated metabolites were detected. After administration of the same dyes via injection into the hepatic portal vein, bile was collected over a 3 h period by cannulation of the bile duct. Urine was withdrawn from the bladder by syringe at the end of the three hours. Both body fluids were analysed for reduction products which were found only in the case of direct black 38, direct brown 95 and direct blue 6. Of the six dyes examined only the three direct dyes were mutagenic to S. typhimurium strains TA98 and TA1538 in the absence of flavin mononucleotide. The same three dyes were also substrates for rat liver microsomal azoreductase enzymes whereas Congo red, trypan blue and Chicago sky blue were shown to be inactive in a previous publication. The possible relationship between these results and the potent carcinogenicity exhibited by direct black 38, direct blue 6 and direct brown 95 is discussed.

Aminobiphenyl Compounds↗
PubMed1977

Antitumor activity of macrophages induced by Corynebacterium liquefaciens.

Corynebacterium liquefaciens can induce a marked resistance to tumor cell (AH-7974) challenge in Donryu rats. This effect was also induced in T-cell-deficient athymic nude mice. The macrophages harvested from the peritoneal cavity of a rat injected with C. liquefaciens showed a marked inhibition of tumor growth in vitro. Therefore, the effector cells were assumed to be peritoneal macrophages. The time needed for activation of macrophages in vivo was within 3 to 5 days after injection of C. liquefaciens. Before or after this period, the peritoneal macrophages did not inhibit tumor cell proliferation. Furthermore, normal peritoneal macrophages were activated in vitro by C. liquefaciens and displayed remarkable antitumor activity. These results suggest that C. liquefaciens rendered the macrophages inhibitory to tumor cells even under T-cell depleted condition. On the other hand, in vitro antitumor activity of the activated macrophages disappeared completely after in vivo treatment of the peritoneal cavity of a rat with Trypan Blue, a lysosomal enzyme blocker. Resistance of the rat induced by C. liquefaciens to tumor cells was also abrogated by Trypan Blue treatment just 1 day before tumor cell inoculation. Trypan Blue blocked the acid-phosphatase (a marker enzyme of lysosomes) activity of the activated macrophages but it seemed not to affect other macrophage activities, at least in the following two points; normal macrophages treated with Trypan Blue had almost normal phogocytic function of India ink particles and, moreover, they could be rendered tumoricidal, same as normal macrophages, in vitro by C. liquefaciens.

Animals↗
PubMed2006

Capsule staining as an adjunct to cataract surgery: a report from the American Academy of Ophthalmology.

OBJECTIVE: This document evaluates currently available data in the published literature to answer the question of whether the use of dye such as indocyanine green or trypan blue to stain the lens capsule to improve visualization is safe and effective as an adjunct to cataract surgery. METHODS: Literature searches conducted in March 2003 and May 2004 retrieved 139 citations. The panel members reviewed the abstracts and selected 47 of possible clinical relevance for review. An additional 14 articles were identified for evaluation. Of the 61 articles reviewed, the panel members selected 36 for the panel methodologist to review and rate according to the strength of the evidence. A level I rating was assigned to properly conducted, well-designed, randomized clinical trials; a level II rating was assigned to well-designed cohort and case-control studies; and a level III rating was assigned to case series and case reports. RESULTS: There is level III evidence that indocyanine green, trypan blue, and fluorescein are each effective in staining the lens capsule and that indocyanine green and trypan blue provide better ease of use and visualization of the capsule than fluoroscein. There is level II evidence that staining the capsule is helpful in completing capsulorrhexis and that it is helpful for pediatric patients under age 5 years and in cases of white cataract. The overall surgical advantage of a completed continuous curvilinear capsulorrhexis using dye has not been demonstrated, but this may be related to the outcome measures chosen rather than a failure to confer advantage. There are substantial data indicating that trypan blue 0.1% is not toxic to the cornea. There are limited data suggesting that indocyanine green 0.125% to 0.5% is not toxic to anterior segment structures. CONCLUSIONS: There are data confirming that dye is safe and effective as an adjunct for capsule visualization in cataract surgery. It is reasonable to use dye when inadequate capsule visualization may compromise the outcome in cataract surgery. More studies are needed to confirm a lack of toxicity of indocyanine green and trypan blue, particularly in the event of posterior segment or longer duration exposure.

Academies and Institutes↗
PubMed1977

Detection of cytotoxic antibody to erythroblasts.

A two-stage test of cytotoxicity for erythroblasts has been developed that is faster and more sensitive than a previous method. Release of 59Fe from erythroid precursors was used as an index of cytotoxic injury. Optimal release was obtained by pretreating the labeled marrow cells with 0.46M reduced glutathione (GSH) at pH 8.0 for 1 hour at 37 degrees C. The first-stage test plasmas or IgG globulins were incubated with the treated cells at 22 degrees C. for 1 hour and the second-stage source of complement was incubated with the cells for 1 hour at 37 degrees C. These changes permitted cytotoxic plasmas to be detected when they previously would not have been identified. Substitution of the GSH by trypsin or neuraminidase did not yield comparable results. Measurement of the cytotoxicity for GSH-treated cells by the trypan blue exclusion technique showed that the loss of exclusion of trypan blue by marrow erythroblasts increased with an increased release of 50Fe. No cytotoxicity was detected if complement was inactivated at 56 degrees C. The results observed with trypan blue and the occurence of cytotoxicity with the addition of IgG globulins and complement indicate that this system can be used to detect cytotoxic antibody to erythroblasts.

Animals↗
PubMed1995

Role of calcium in thromboxane B2-mediated injury to rabbit gastric mucosal cells.

A sustained increase in cytosolic Ca2+ can damage gastric mucosal cells. The present study has examined the role of Ca2+ in thromboxane B2 (TXB2)-mediated damage of rabbit isolated gastric mucosal cells. Cells were isolated from rabbit oxyntic mucosa by collagenase-EDTA digestion. Cell metabolic activity and cell damage were estimated by alamar blue dye absorbance and trypan blue uptake, respectively. Cellular Ca2+ was monitored by indo-1 dye fluorescence. Addition of TXB2 (10(-6) and 10(-8) M) to the cell suspension resulted in a decrease in metabolic activity, and this effect was reduced when Ca2+ was removed from the incubation medium. TXB2 addition to the incubation medium resulted in an increase in cytosolic Ca2+ and incubation of cells with the intracellular Ca2+ chelator, BAPTA-AM (20 microM), reduced cell injury in response to TXB2. Incubation of cells with the Ca2+ ionophore A23187 (1-25 microM) resulted in a dose-dependent increase in trypan blue uptake and a reduction in cell metabolism. Cell injury in response to A23187 were exacerbated by addition of TXB2 (10(-8) M) to the cell suspension. TXB2 treatment reduced cellular content of reduced glutathione (GSH), while exogenous GSH addition (10 mM) reduced TXB2-mediated cell injury. These data demonstrate that TXB2 can directly injure gastric mucosal cells. Gastric mucosal cellular damage in response to TXB2 is mediated in part by a disruption of Ca2+ homeostasis as well as a reduction in cellular GSH content.

Animals↗
PubMed2005

Retinal tolerance to dyes.

BACKGROUND: Dye solutions for intraoperative staining of epiretinal membranes and the internal limiting membrane improve the visualisation of these thin structures and facilitate their removal. In the present study the authors investigated the effects of indocyanine green 0.05%, trypan blue 0.15%, and patent blue 0.48% on bovine retinal function. METHODS: Bovine retina preparations were perfused with a standard solution and the electroretinogram (ERG) was recorded repeatedly. After recording of stable ERG amplitudes the nutrient solution was substituted by one of the dye solutions. The duration of retinal exposure to a dye solution was varied between 10 seconds and 2 minutes. Thereupon, the preparation was reperfused with standard solution for at least 115 minutes. The percentage of b-wave reduction after exposition was calculated. RESULTS: Reductions of the b-wave amplitude were found for each dye solution tested. The effects after application of patent blue and indocyanine green were completely reversible within the recovery time for an exposure period of 60 and 30 seconds, respectively. The application of trypan blue lead to a loss of the b-wave when the retina was exposed for 15 seconds or longer. This effect was only partly reversible within the recovery time. CONCLUSION: The ERG showed toxic effects of trypan blue after a short period of retinal exposure. The intraocular application of trypan blue should be limited to selected cases. However, intraocular application of indocyanine green and patent blue in a sufficient concentration and taking account of a short period of retinal exposure seems possible.

Animals↗
PubMed1985

Eradication of a disseminated syngeneic mouse lymphoma by systemic adoptive transfer of immune lymphocytes and its dependence upon a host component(s).

We have studied the in vivo effects of carrageenan and trypan blue on the adoptive immunotherapy of an established local and disseminated syngeneic mouse FBL-3 lymphoma. Mice receiving 500 rads total-body irradiation before injection of FBL-3 tumor into the footpad were treated 4 to 5 days later when a palpable local tumor and disseminated metastases were present. Injection of in vivo immune lymphocytes i.v. caused complete regression of footpad tumor and cured 96% of all mice (greater than 60 days mean survival; p less than 0.0005). Carrageenan or trypan blue treatment of the tumor-bearing host abrogated the therapeutic effect of adoptively transferred cells. Cure rates were significantly reduced to 27% (p less than 0.004) and 0% (p less than 0.0001) and mean survival times to 40.2 days (p less than 0.0005) and 15.2 (p less than 0.005) days for mice treated with carrageenan and trypan blue, respectively, in addition to immune cells. In vivo treatment of the immune spleen cell donors with carrageenan or trypan blue had no significant effect on the ability of those splenocytes to mediate cure when adoptively transferred into tumor-bearing hosts, indicating that the inhibitory activity of these agents cannot be attributed to direct toxicity to immune lymphoid cells. These results demonstrate that a recipient component(s), possibly macrophages, sensitive to carrageenan and to trypan blue but relatively resistant to radiation (500 rads), plays a vital role in the cure of tumor-bearing mice that receive the adoptive transfer of immune splenocytes.

Animals↗
PubMed1976

Human spinal arachnoid villi and granulations.

Human spinal arachnoid villi and granulations were studied after distension by a subarachnoid perfusion of Berlin blue and trypan blue. These proliferations were distributed on almost every nerve root in the thoracic and lumbar region. Human spinal arachnoid villi and granulations were divided into those located entirely internal to the dura, those that extended into the dura, and those that penetrated the dura completely. Venous sinuses were closely related to most arachnoid proliferations.

Arachnoid↗
PubMed1996

Human immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes.

Cationic liposomes may be valuable for the delivery of anti-sense oligonucleotides, ribozymes, and therapeutic genes into human immunodeficiency virus type 1 (HIV-1)-infected and uninfected cells. We evaluated the toxicity of three cationic liposomal preparations, Lipofectamine, Lipofectin, and 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) reagent, to HIV-infected and uninfected cells. Monocyte/macrophages were infected with HIV-1BaL and treated with liposomes in medium containing 20% fetal bovine serum (FBS) for 4 h or 24 h at 37 degree C. Uninfected monocytic THP-1 cells and chronically infected THP-1/HIV-1IIIB cells were treated with phorbol 12-myristate 13-acetate (PMA) and exposed to liposomes in the presence of 10% FBS. Toxicity was evaluated by the Alamar Blue assay and viral p24 production. The toxic effect of cationic liposomes was very limited with uninfected cells, although concentrations of liposomes that were not toxic within a few days of treatment could cause toxicity at later times. In HIV-1BaL-infected macrophages, Lipofectamine (up to 8 microM) and Lipofectin (up to 40 microM) were not toxic after a 4-h treatment, while DMRIE reagent at 40 microM was toxic. While a 4-h treatment of THP-1/HIV-1IIIB cells with the cationic liposomes was not toxic, even up to 14 days post-treatment, all three cationic liposomes were toxic to cells at the highest concentration tested after a 24-h treatment. Similar results were obtained with the Alamar Blue assay, Trypan Blue exclusion and a method that enumerates nuclei. Infected cells with relatively high overall viability could be impaired in their ability to produce virions, indicating that virus production appears to be more sensitive to treatment with the cationic liposomes than cell viability. Our results indicate that HIV-infected cells are more susceptible than uninfected cells to killing by cationic liposomes. The molecular basis of this differential effect is unknown; it is proposed that alterations in cellular membranes during virus budding cause enhanced interactions between cationic liposomes and cellular membranes.

Animals↗
PubMed1998

Supramolecular ligands: monomer structure and protein ligation capability.

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.

Congo Red↗