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PubMed2003

Trypan blue-assisted peeling of the internal limiting membrane during macular hole surgery.

PURPOSE: To evaluate the outcome of surgeries for macular holes using trypan blue to facilitate delamination of the internal limiting membrane. DESIGN: A retrospective series of 18 patients. METHOD: All patients underwent a three-port pars plana vitrectomy with internal limiting membrane delamination using trypan blue 0.06% to assist visualization. The main outcome measures were postoperative visual acuity and clinically evident retinal pigment epithelial changes. RESULTS: The median preoperative visual acuity was 20/200, and the median postoperative visual acuity was 20/70. All but one macular hole were closed after surgery (94%). At the last recorded follow-up, 10 patients (56%) had improved visual acuity of at least two chart lines. No adverse reaction related to trypan blue was observed up to 1 year postsurgery. CONCLUSIONS: Trypan blue staining of internal limiting membrane may accomplish more complete dissections of the internal limiting membrane and better surgical and visual outcomes.

Adolescent↗
PubMed1981

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail.

It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induce by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5 x 10(-3) mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured. It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.

Animals↗
PubMed1984

Evidence for repair in trypan blue-treated mouse egg cylinders: an electron microscopic study.

The question of whether mammalian fetuses can correct damage induced by teratogens during early stages of embryogenesis was reinvestigated. Toward this purpose female mice were injected on day 6 of gestation with the teratogenic dye trypan blue. Within 6, 24, and 48 hr of exposure to the teratogen, egg cylinders were removed, sectioned, and prepared for electron microscopic analysis. The following organelles were examined: 1) mitochondria, 2) Golgi apparatus, 3) endoplasmic reticulum, and 4) free polysomes. On the ultrastructural level, exposure to trypan blue lead within 6 hr to fragmentation of the endoplasmic reticulum and a depletion of free polysomes in the endoderm of the egg cylinders. In ectodermal cells only the distribution of polysomes was disturbed following exposure to trypan blue. Egg cylinders harvested 24 hr after injection of trypan blue had partially recovered. Their endoplasmic reticulum and polysomes appeared closer to controls. The cells of both germ layers of most egg cylinders obtained 48 hr after injection were indistinguishable from controls when viewed with the electron microscope. No consistent changes were found in mitochondria or Golgi apparatus following trypan blue treatment. It is concluded that mouse embryos appear to be able to correct damage sustained during the egg cylinder stage, and that in spite of earlier injury affecting both germ layers such egg cylinders can develop normally as revealed by microscopic examination.

Abnormalities, Drug-Induced↗
PubMed1984

Ultrastructure of trypan blue-induced ocular defects II. Cornea and mesenchyme.

Using scanning and transmission electron microscopy, the histological and cytological alterations induced in the developing eye by trypan blue were examined. Microphthalmic and anophthalmic eyes were analyzed from 16-day rat fetuses whose dams had received a teratogenic dose of trypan blue. Cell and tissue damage within cornea and mesenchyme were assessed. Corneas from the treated groups were undulating and uneven owing to abnormal lens placement and corneal cell injury. Extensive extracellular spacing, few cell-to-cell contacts, patchy glycocalyces, and abnormal surface morphology characterized the corneal cells of treated tissues. Mesenchyme of severely malformed eyes typically filled the eye field in regions normally occupied by retina and lens. As a tissue, mesenchyme lacked organization and those surface features normally present in component cells were altered. Mesenchymal cells were packed so closely that extracellular spaces were nearly obliterated. Microvilli, filopodia, and lamellipodia, cell processes observed in normal mesenchyme, were altered in both number and structure. The cytoplasm of mesenchymal cells was condensed and decreased in volume. These data suggested that 1) in this trypan blue ocular model, virtually all major tissue types were morphologically altered; 2) because healthy mesenchyme and cornea are required for normal development of lens, optic vesicle and ocular adnexa it follows that abnormal cornea and mesenchyme also will compromise these tissues; and 3) since necrotic reminants were not present, this trypan blue model probably causes developmental arrest. The most likely teratogenic pathway is presented with emphasis on the cell surface as a probable site of dye action.

Abnormalities, Drug-Induced↗
PubMed1981

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence.

Shortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1979) obtained by autoradiography after 3H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged "retarding" effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.

Animals↗
PubMed1982

The characterization of trypan blue-induced tumors in Wistar rats.

Trypan blue is an azo dye widely used for testing cell viability. The dye has been identified as a mutagen and a carcinogen. In some strains of rats, particular Wistar rats, chronic exposure induces a reticuloendothelial neoplasm, predominantly in the liver. These tumors were studied with the use of immunologic cell membrane markers, electron microscopy, and histochemistry to characterize tumor cell type. The authors have studied this tumor in two inbred lines of Wistar rats to compare the efficacy of two previously described dye regimens on tumor incidence and to ascertain whether a short, intense exposure was as effective as chronic protracted exposure. No significant difference in tumor incidence was observed between the two regimens. These studies suggest that the tumor is composed of a macrophage-like cell that retains some characteristics of normal macrophages and that is a reproducible model for carcinogen-induced lymphoreticular human lymphomas.

Animals↗
PubMed1999

Early alterations in cerebral hemodynamics, brain metabolism, and blood-brain barrier permeability in experimental intracerebral hemorrhage.

OBJECT: The authors sought to ascertain the nature of the hemodynamic and metabolic derangement underlying acute pathophysiological events that occur after intracerebral hemorrhage (ICH). METHODS: Cerebral perfusion pressure (CPP), flow velocity (FV) of the middle cerebral artery, and the arteriovenous contents of oxygen and lactate were investigated in 24 dogs subjected to sham operations (Group A, four animals) or intracerebral injections of 3 ml (Group B, 11 animals) or 5 ml (Group C, nine animals) autologous arterial blood. Twelve additional dogs received intravenous injections of 2% Evans blue or trypan blue dye to evaluate blood-brain barrier (BBB) changes. Within 1 hour, animals with ICH exhibited a rise in FV associated with significant reductions (p<0.05) in CPP and the arteriovenous content difference (AVDO2). In Group C animals significant increases in lactate concentration were found in arterial and superior sagittal sinus (SSS) samples compared with those in the other two groups (p<0.05). Additionally, perihematomal dye extravasation was observed in animals subjected to ICH and trypan blue dye injections, with profound and mild leakages in Group C and Group B animals, respectively, but not in Group A and Evans blue dye-injected animals. During the subsequent 4 hours, the FV and AVDO2 returned to normal in Group B animals, indicating a balanced cerebral metabolic rate for oxygen (CMRO2) compared with a deranged CMRO2 in Group C animals due to their lowered FV and AVDO2. However, no coupling increase in brain lactate clearance in Group C animals accounted for either the early lactate elevation in SSS or the decrease in CMRO2. CONCLUSIONS: Profound reductions in CPP and brain oxygenation after ICH may rapidly exhaust hemodynamic compensation and, thus, impede cerebral homeostasis; however, these reductions only modestly enhance anaerobic glycolysis. Furthermore, the data suggest that a selective increase in permeability, rather than anatomical disruption, of the BBB is involved in the acute pathophysiological events that occur after ICH, which may provide a possible gateway for systemic arterial lactate entering the SSS.

Animals↗
PubMed2002

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

Brazil↗
PubMed1998

Uptake of L-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups.

The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.

Animals↗
PubMed1979

A mammary gland whole mount technique that preserves cell fine structure for electron miscroscopy.

A mammary gland whole mount technique has been developed that preserves cell fine structure and makes it possible to also examine the preparations by electron microscopy. The glands are placed on glass microscope slides, fixed in a paraformaldehyde-glutaraldehyde mixture, defatted in acetone, stained with 0.5% methylene blue (or trypan blue) in saline, and dehydrated in ethanol. They are evaluated and photographed in 100% ethanol. Then specific areas (i.e. containing small growths, tumors, or other lesions) are selected, excised and prepared for electroscopy. The ultrastructural preservation is good, organelles are evident and there is no observable dye precipitate. The only unusual finding is that cell membranes display a "negative" image.

Animals↗
PubMed1985

Further evidence for the procidin function of C3.

It was found that heat-inactivated serum supported phagocytosis, but did not promote oxygen consumption or killing of Candida, by human polymorphonuclear leucocytes (PMNs). It was also established that C3 is required for activation of the PMN candidacidal related mechanisms of superoxide anion generation and iodination. Addition of purified C3 to C3 deficient serum restored phagocytic killing. Further evidence for C3 involvement in Candida killing was obtained from studies of PMN ingestion and killing in the presence of trypan blue or heparin. Trypan blue, which antagonises C3 receptors, prevented efficient killing, as did heparin, which affects C3 conversion in normal serum. From these results it is evident that phagocytic killing is not an obligatory consequence of ingestion. The findings also indicate that killing by PMNs is dependent upon the interaction of humoral factors, termed procidins, with receptors responsible for initiation of microbicidal mechanisms. Activation products of C3 may stimulate phagocytic killing by engagement of their counterpart receptors on PMNs.

Candida albicans↗
PubMed2004

Evans Blue and other dyes as protein tyrosine phosphatase inhibitors.

Commonly used dyes including Evans Blue and Trypan Blue were examined for their inhibitory activities against protein tyrosine phosphatases (PTPases), all of them showed inhibition of PTPases with different potencies. Of the 13 dyes tested, four exhibited IC(50) value of less than 10 microM, Evans Blue lowest IC(50) of 1.3 microM against PTP1B. Care must be taken in the use of dyes for clinical or biochemical experiments to avoid unwanted side effects. Some of the low molecular weight dyes might be useful as lead compounds for the development of potent and selective PTPase inhibitors.

Coloring Agents↗
PubMed2004

In vivo vascular leakage assay.

This chapter describes the methods for measuring the increase in vascular permeability induced by tumor necrosis factor alpha (TNFalpha, or other mediators, in vivo in animal models. Mouse liver or kidney are perfused through the portal vein or the renal artery, respectively, by intravascular injection of a blue dye (trypan blue or Evans blue) combined with albumin. When endothelial permeability is increased, the dye extravasates and reaches the subendothelial spaces. Thus, subsequent washing of dye-perfused organs with saline cannot remove the dye. The perfusates are drawn from the suprahepatic inferior vena cava (for the liver) and from the renal vein (for the kidney). After perfusion, the livers and kidneys are removed, homogenized, and centrifuged. Spectrophotometric analysis of supernatants, at 540 nm, is then performed. The increase in optical density values is indicative of the increase in dye retention, hence of vascular leakage.

Albumins↗
PubMed2003

Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme.

The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was below 2.0 for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K(m)=38 microM), 4.0 for 2,6-dimethoxyphenol (K(m)=48 microM), 4.5 for guaiacol (K(m)=93 microM) and 7.0 for syringaldazine (K(m)=131 microM). The temperature optimum was between 60 and 70 degrees C depending on the pH and buffer used. The enzyme was stable up to 45 degrees C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu(2+) and inhibited by Mn(2+), sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.

Anthraquinones↗
PubMed1993

Cerebrovascular permeability to azo dyes and plasma proteins in rodents of different ages.

The often quoted investigation by Behnsen [4] provides some evidence for an increased permeability of the neonatal mouse blood-brain barrier (BBB) to trypan blue compared to the adult. Trypan blue, which circulates in plasma mainly bound to albumin, is commonly used as a macromolecular tracer, although Behnsen probably injected dyes in amounts exceeding the dye-binding capacity of plasma proteins. The cerebrovascular permeability in neonatal and adult mice and rats was investigated using the visual tracers trypan blue and Evans blue and immunocytochemical staining for endogenous plasma proteins. By administering different concentrations of the dyes, the permeability of the BBB was assessed. The presence of the dyes in plasma as either dye-protein complexes or as free dye was measured by a plasma protein binding assay. When dyes occurred in plasma as macromolecular dye-protein complexes, dyes and plasma proteins were restricted to CNS regions normally devoid of a BBB in both neonates and adults. When unbound (free) dyes occurred in plasma, dyes were observed intraneuronally in regions without projections beyond the BBB in both neonates and adults corresponding to that observed by Behnsen; intraneuronal accumulation of plasma proteins also occurred in regions with projections confined to the BBB but only in neonates. It is concluded that the BBB to macromolecular tracers is fully developed at birth in mouse and rats. However, when free dye is present in plasma, there is a differential permeability to plasma proteins between neonates and adults.

Aging↗
PubMed2004

Effect of blue staining of expandable hydrophilic intraocular lenses on contrast sensitivity and glare vision.

PURPOSE: To evaluate the effect of trypan blue staining of expandable hydrophilic acrylic intraocular lenses (IOLs) on contrast sensitivity and glare. SETTING: Department of Ophthalmology, Federal University, and Oculistas Associados, Rio de Janeiro, Brazil. METHODS: Phacoemulsification with expandable hydrophilic IOL implantation was performed in 19 eyes. Group 1 (stained group) consisted of 12 eyes with the IOL unintentionally stained by trypan blue 0.1% solution during surgery; Group 2 (unstained group) consisted of 7 eyes with IOLs that were not stained because trypan blue was not used during surgery. The eyes were examined postoperatively for Snellen best corrected visual acuity (BCVA), contrast sensitivity BCVA, and glare vision. The examiners were masked to whether the IOL was stained by trypan blue. Inclusion criteria included a BCVA of 20/30 or better 1 month postoperatively. Patients who had other ocular pathology or previous ocular surgery were excluded from the study. RESULTS: The mean postoperative BCVA was 20/24 in the stained group and 20/23 in the unstained group (P =.73). The mean contrast sensitivity visual acuity was 20/31 in the stained group and 20/26 in unstained group (P =.10). The mean glare visual acuity was 20/75 and 20/33, respectively (P =.03). CONCLUSIONS: Patients with expandable hydrophilic acrylic IOLs stained with trypan blue performed significantly worse on a glare vision test than patients whose IOLs were not stained. Cataract surgeons should avoid using trypan blue with this IOL type.

Acrylic Resins↗
PubMed1979

Combination of a viability test and a quantification method for Schistosoma haematobium eggs (filtration--trypan blue-staining-technique).

The efficiency of the polycarbamate membrane filtration technique for detecting Schistosoma haematobium eggs in urine has been increased by using a pore size of 14 micrometers diameter and the suction of a water jet pump. The technique permits filtration of several 1000 ml of urine in one test. Eggs concentrations of 1 egg in more than 1000 ml of urine were detected. Viability was assess after filtration by staining with trypan blue. Preliminary data of 118 Schistosoma haematobium-positive urine samples of 32 patients suggest that the filtration trypan blue-staining-technique is of high validity in light infection with scanty egg-output.

Filtration↗
PubMed2006

Trypan blue-assisted neodymium:YAG laser treatment for overfiltering bleb.

Overfiltering blebs can lead to postoperative problems including hypotony maculopathy, decreased vision, and increased ocular discomfort. Many methods have been used in the past with varying results. Various lasers have been used in an attempt to cause localized scarring and fibrosis. We report a treatment technique to noninvasively treat a patient with an overfiltering bleb using trypan blue and a frequency-doubled continuous-wave neodymium:YAG laser emitting green light.

Aged↗