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PubMed2005

[Ultrastructural study of epiretinal membrane stained by trypan blue: 15 case reports].

INTRODUCTION: Idiopathic epiretinal membrane results from detachment of the posterior hyaloid and is believed to be related to naturally occurring defects in the internal limiting membrane (ILM) of the retina. Vitrectomy and peeling are the treatment of choice. Trypan blue 0.15% (TB) stains epiretinal membrane and internal limiting membrane. It allows selective and complete removal, facilitating surgery, with less retinal damage. An ultrastructural study was conducted showing ultrastructural features of idiopathic epiretinal membranes (ERM) and those of the internal limiting membrane and its connections with the retinal side. MATERIAL AND METHODS: After pars plana vitrectomy and induction of posterior vitreous detachment, 0.2 ml TB 0.15% was injected over the ERM in an air-filled eye. The stained tissue was peeled with intraocular forceps. Specimens were at once collected in 4% glutaraldehyde for a transmission electron microscopy study. RESULTS: TB may allow complete and easier ERM and ILM peeling. The staining does not present toxic effects. The major cellular contingent is represented by glial cells, participating actively in neocollagen synthesis. Their presence supports the hypothesis of a migratory movement of retinal cells toward the vitreoretinal side. CONCLUSION: The presence of an intact internal limiting membrane, the absence of optical fibers belonging to the under retina, and the absence of any sign of apoptosis make TB a useful staining agent for ERM and ILM peeling.

Aged↗
PubMed1990

Maternal age, fertility and failure of teratogenicity by trypan blue in Jcl:SD rats.

The study compared the fertility of nulliparous Jcl:SD rats, young mature (12-week-old) versus middle aged (47-week-old) or old animals (64-week-old), both groups near the termination of their reproductive capability. Trypan blue (TB) was given subcutaneously at 0 (control), 10 or 50 mg/kg on days 7 through 9 of gestation to increase the malformation rate. However, no clear teratogenic action of TB was observed. The dose of 10 mg/kg TB was well tolerated, while 50 mg/kg was of severe maternal toxicity and 14 of 53 females died. In comparison with the young control group, the average per litter from middle aged or old dams was significantly decreased for implantation sites, live fetuses and fetal weight, while the percentages of pre-implantation loss and dead implants were extremely high. No increase of the malformation rate occurred with maternal aging. Histologically, middle aged and old rats exhibited age-related lesions of the pituitary, adrenals and reproductive organs with pituitary adenomas, cystic endometrium and sclerotic atrophy of uterine glands being significantly increased in non-pregnant, old rats.

Abnormalities, Drug-Induced↗
PubMed2000

HPC viability measurement: trypan blue versus acridine orange and propidium iodide.

BACKGROUND: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering. STUDY DESIGN AND METHODS: A fluorometric cell viability assay using acridine orange and propidium iodide (AO/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO/PI fluoresce green under darkfield fluorescence microscopy, while nonviable cells fluoresce orange. Mixtures of fresh and heat-killed bone marrow were prepared and used as viability standards for evaluation of both assays. The frequency of CFU-GM was determined for each specimen. RESULTS: Cell viability measured by AO/PI was extremely linear, with measured and predicted viability in agreement from 0 to 100 percent of the viable cells and a coefficient of regression (r(2)) of 0.9921. The predicted-viability regression line fell within the 95% CI for AO/PI-measured viability. The coefficient of regression for TB-measured viability was 0.9584, with the predicted-viability regression line almost entirely outside the 95% CI. TB overestimated the percentage of viable cells, particularly below the 50-percent level. CFU-GM frequency correlated better with cell viability measured by AO/PI (r(2) = 0.979) than with that measured by TB (r(2) = 0.930). CONCLUSIONS: The AO/PI viability assay is a rapid, highly linear, functionally correlated assay that is superior to conventional viability measurement by TB exclusion.

Acridine Orange↗
PubMed2000

Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA.

The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca(2+), internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res. 853 (2000) 32-40). A short incubation time (10 min) with 30 microM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10(-5) M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes.

Adenosine Triphosphate↗
PubMed1978

[Vital staining of the endothelium of preserved corneae with Trypan Blue (author's transl)].

As a development of Capella's and Kaufman's procedure we examined on four series each with 20 pig corneae the four antifreezing solutions described by the authors singly with regard to their protective effect against freezing. After thawing at +60 degrees within 50 seconds and rehydration with 25% Serum albumin (in the fourth series 10 corneae were rehydrated with polyvinylpyrrolidone) the endothelium was stained with Trypan blue. Microphotographs were made of the endothelial surface, stained nuclei counted and the results were analysed statistically and compared one with other. Hence we could show that with increasing concentration of the antifreeze solution the vitality of the cornea increases.

Animals↗
PubMed1976

[Studies on the prenatally toxic effect of parathionemethyl in Wistar rats compared to cyclophosphamide and trypan blue].

Higher yield and performance in plant and animal production will depend on reinforced use of pesticides, particularly alkyl phosphate esters and insecticidal carbamates. Prenatal toxic action of the pesticides used today may be detrimental to human and animal health. Prenatal-toxicological tests, using parathion-methyl, were conducted on the basis of known but modified experimental methods. The vulnerability of a rat strain was tested by applying to it teratogenic model substances (trypan blue and cyclophosphamide). Several oral applications of parathion-methyl (3 ppm) during the sensitive phases of germ formation and growth (fifth to ninth and eleventh to fifteenth or eleventh to nineteenth days from conception) were followed by clear embryotoxic effects, such as retardation and increased resorption. The average number of resorption sites grew from nine in the control group to 20 in the experimental group. Postnatal malformations were not observed.

Abnormalities, Drug-Induced↗
PubMed1993

[The effect of oxygen supply in continuous cold perfusion of the rat liver using perfluorochemical emulsion--parenchymal and nonparenchymal cell injuries evaluated with trypan blue perfusion/fixation techniques].

We investigated the effect of hypothermic aerobic perfusion of the rat liver using perfluoro-N-methyl-decahydroisoquinoline (FMIQ) on the cell viabilities of hepatic parenchyma and nonparenchyma in comparison with University of Wisconsin (UW) solution. Rat liver were perfused at 4 degrees C up to 24hr at a flow rate of 0.2 ml/g liver tissue/min with either UW or a perfusate (m-FMIQ) containing 20% FMIQ with or without oxygen saturation. After storage, livers were perfused with Krebs-Henseleit buffer containing trypan blue (200 microM). Nuclear dye uptake was measured as an indicator of cell death. Dye uptake of parenchymal cells remained less than 2% for up to 24hr, regardless of perfusate or O2-saturation. In UW perfused livers, nonparenchymal cells showed time related increases of dye positive nuclei, which were not affected by O2-saturation (39.9 +/- 4.7% and 36.5 +/- 4.2% at 24hr in O2-saturated and nonsaturated groups, respectively). In contrast, we found excellent protective effects of oxygen supply with m-EMIQ on the nonparenchymal cell viability, the dye uptake values being 16.9 +/- 5.7% and 39.4 +/- 9.1% at 24hr in O2-saturated and nonsaturated groups, respectively (p < 0.001). In conclusion, oxygen supply during hypothermic perfusion may be useful in preventing nonparenchymal cell injury of the liver, thereby leading to dramatic improvement of the hepatic microcirculation.

Adenosine↗
PubMed1999

[Observation of the endothelial healing in rabbit corneal alkali wounds by alizarin red S-trypan blue staining method].

PURPOSE: To study the healing of the corneal endothelial cells in rabbit corneal alkali wounds. METHODS: 26 New Zealand white rabbits were used. A alkali wound was created with a round filter paper, 8 mm in diameter, which was soaked in 0.5 N NaOH for 5 seconds and was placed centrally on the cornea for 60 seconds. The rabbits were killed in 20 minutes; 1, 2, 6, 12 h; 1, 2, 3, 5, 7, 10, 14, 30 days after injury. The corneal endothelium was examined in the light microscope using a lizarin red S-trypan blue staining method. RESULTS: Twenty minutes after the wound, the corneal endothelial cells were destroyed. 72 hours following the wound, the endothelial cells surrounding the burns became larger, irregular and started to migrate onto the denuded descemet's membrane. The endothelial cells covered the burn with the spindle-shaped cells 7 days after wound. CONCLUSION: The endothelial damage is healed by the division and migration of remaining endothelial cells at the margin of the wound, which has some characters of fibroblast.

Alkalies↗
PubMed2005

[Evaluation of trypan blue toxicity in idiopathic epiretinal membrane surgery with macular function test using multifocal electroretinography: seven prospective case studies].

INTRODUCTION: Idiopathic epiretinal membranes are considered the consequence of glial proliferation through a defect of the internal limiting membrane and can induce a functional macular syndrome associating mainly metamorphopsia and visual acuteness. Visual disturbance is not bound to the area and the degree of transparency of the membrane, but to distortion of external layers of the retina. Multifocal electroretinography (ERGm) can assess the macular area and retina extending 20 degrees. We used this tool to study macular function pre- and post-operatively with seven patients presenting idiopathic epiretinal membrane. PATIENT AND METHOD: All patients had successful vitrectomy, with 0.2 ml of 0.15% trypan blue (TB) staining to facilitate peeling. This stain is not toxic for the pigment epithelium but it becomes so for photoreceptors at doses exceeding 0.2%. We tried to estimate the echo of TB 0.15% on photoreceptors through this exam. An ERGm was done 1 week before the operation as well as 1 month and 4 months after on 14 eyes of seven patients with ERMs. RESULTS: Results show a pre-operative decrease in the electrical retinal response densities in the foveal, perifoveal and parafoveal areas. One month after operation, no significant difference was found compared to pre-operative results. However, at 4 months, an improvement of the retinal response density was observed. Retinal response densities in the macular area increased progressively after ERM surgery. CONCLUSION: The 0.15% TB facilitated ERM peeling and does not seem to present any retinal toxicity.

Electroretinography↗
PubMed1971

The effect of Trypan Blue, suramin and aurothiomalate on the breakdown of 125 I-labelled albumin within rat liver lysosomes.

1. A fraction enriched in lysosomes was prepared by centrifugation from the livers of rats that had been injected 0.5h before death with (125)I-labelled albumin. When suspended in sucrose-protected buffer, pH7.4, and incubated at 22 degrees C for 2h, the particles progressively released iodotyrosine into the medium. Albumin digestion did not occur if the particles were subjected to treatments known to break lysosomes or if particles from uninjected rats were incubated in medium containing (125)I-labelled albumin. It is concluded that the observed production of iodotyrosine results from protein hydrolysis within intact heterolysosomes. 2. Particles from rats pre-treated with Trypan Blue, suramin or aurothiomalate released iodotyrosine more slowly than controls. Since these compounds are enzyme inhibitors that concentrate in liver lysosomes after administration in vivo, their effect is ascribed to intralysosomal inhibition of proteolysis. The doses used did not decrease endocytosis of albumin into liver or cause increased lysosome breakage during incubation, thus allowing some alternative explanations of the decreased proteolysis to be eliminated. Particulate carbon, a non-inhibitor that also concentrates in lysosomes, did not affect albumin hydrolysis.

Animals↗
PubMed1978

Effects of vinyl chloride exposure alone and in combination with trypan blue--applied systematically during all thirds of pregnancy on the fetuses of CFY rats.

Vinyl chloride (VC) has been shown to be present in the fetal and maternal blood as well as in the amniotic fluid after the exposition of pregnant CFY rats to VC at an atmospheric concentration of 5500, 18 000 or 33 000 mg/m3 (approximately 2000, 7000 or 12 000 ppm) for 2.5 h on the 18th day of pregnancy, indicating the permeability of the placenta to the agent. Teratological investigation of the offspring of pregnant rats exposed continuously to VC at an atmospheric concentration of 4000 mg/m3 air (1500 ppm) during the first, second or last third of pregnancy has shown that VC has no teratological effect in the rat and has no embryotoxic effects either, when applied during the second or last third of pregnancy in the above concentration. Exposition to VC during the first third of pregnancy resulted in an increased fetal mortality and in the manifestation of embryotoxic effects. Fetal losses and induction of central nervous system malformation due to trypan blue administration were not potentiated by a combined exposure of pregnant rats to VC and the dye.

Amniotic Fluid↗
PubMed1981

Vascular hypervolemia and hematoma formation in chick embryos treated with trypan blue.

The relationship between cardiovascular disturbances and the formation of hematomas in trypan blue-treated chick embryos was investigated. Hematocrits from Hamburger-Hamilton stage 15 and 16 embryos (8-10 hours after treatment at stage 13) were obtained. The hematocrit value from the treated embryos was significantly lower (P less than 0.001) than the values from controls. Unopened and saline-injected control values were not significantly different (P greater than 0.05). We interpret this to mean that there is excess circulating fluid volume in the treated embryos. The results of direct injection of measured volumes of fluid into the cardiovascular system of normally developing embryos (stages 15 and 16) provided evidence to suggest that the caudal dorsal aortae are susceptible to fluid-induced distension and rupture.

Animals↗