Assessment of hepatotoxicity and nephrotoxicity in Cirrhinus mrigala induced by trypan blue - An azo dye.

Cytotoxicity in freshwater fishes induced by industrial effluents and dyes is a global issue. Trypan blue dye has many applications in different sectors, including laboratories and industries. This study determines to detect the cytotoxic effects of trypan blue dye in vivo. The objective of this study was to estimate the sub-lethal effects of azodye in fish. Cirrhinus mrigala, a freshwater fish, was exposed to three different grading concentrations of dye 5 mg/L, 10 mg/L, and 20 mg/L in a glass aquarium. Significant (p < 0.05) decrease in the weight of fish was observed as 0.728 ± 0.14 g and 2.232 ± 0.24 g, respectively, in the trial groups exposed to 10 and 20 mg/L of dye in a week. After exposure to trypan blue dye, fishes were dissected to remove liver and kidney tissues. Histopathological assessments determined hepatotoxicity and nephrotoxicity induced by trypan blue through the paraffin wax method. This dye induces mild alterations in the liver such as congestion, hemolysis, dilated sinusoids, ruptured hepatocytes, vacuolization, edema of hepatocytes, necrosis, degeneration, aggregation, and inflammation. This dye not only alters liver tissue, also induces an acute level of tissue alterations in the kidneys, such as degeneration of epithelial cells of renal tubules, shrinkage of the glomerulus, congestion, reduced lumen, degeneration of glomerulus, absence of space of bowmen, glomerulonephritis, necrosis in hematopoietic interstitial tissues and glomerulus, reduced lumen, vacuolar degeneration of renal tubules, increased per tubular space. The current study concludes that trypan blue dye released even in small amounts is found to be associated with a high incidence of cytotoxicity. Such tissue alterations in this species could be used as biomarkers for azo dyes.

Ex vivo evaluation of retinal cytotoxicity after the use of multiple medical devices in pars plana vitrectomy in porcine eyes.

This study aimed to evaluate viability of retinal cells after the use of multiple intraoperative devices, namely a vitreal dye (triamcinolone acetonide,TA), a ERM/ILM dye (solution of trypan blue 0.15% and brilliant blue 0.025%), and two intraocular tamponades, namely perfluoro-n-octane, (PFO) and silicone oil (SO 1000 cSt), with minimal and maximal removal of their residues, during a simulated pars plana vitrectomy (PPV) in porcine eyes ex-vivo. The in vitro cytotoxicity of each of these compounds was verified on ARPE-19 cells by direct tests according to the ISO 10993-5 (2009). Pars plana vitrectomy was performed on 25 enucleated porcine eyes divided in five groups according to the following conditions: Group A) No surgery control: eye bulbs were kept at room temperature for 40 min; Group B) Sham surgery: PPV with the sole use of BSS for 40 min; Group C) Cytotoxic control: PPV with BSS infusion (20 min) followed by intravitreal injection of 1H-PFO (contact time: 20 min); Group D) Surgery with residues: PPV with BSS infusion and sequential intravitreal injection of TA, ERM/ILM dye, PFO and SO, with minimal removal of each compound after a specified contact-time (overall duration: 40 min); Group E) Surgery with minimal residues: PPV performed as in group D, but with maximal removal of each compound (overall duration: 40 min). All the experimental procedures were performed at room temperature. Immediately after surgery, the retina was extracted from each eye bulb and samples of 3-mm diameter were prepared. Retinal viability was determined for each sample by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. A cell viability <70% was considered the cytotoxicity threshold. Kruskal-Wallis test was used to evaluate the differences in retinal viability between groups. No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The tested conditions indicated that the combined use of TA, ERM/ILM dye, PFO and SO during PPV does not affect retinal cells viability if all the devices are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability due to a cumulative and/or synergistic cytotoxic effect between them, supporting the crucial role of an optimal removal of the intraoperative medical devices to ensure a safe vitrectomy to the patient.

Comparative effects of commonly used intraocular dyes on the viability of human retina Müller cells.

This study investigated the in vitro effect of various vital dyes in common clinical use on human Müller cell viability, and it compared the toxicity of these dyes using a cell culture model. Müller cells were exposed to a series of concentrations (1 %, 0.5 %, 0.25 %, and 0.125 % or 12.9 mM, 6.45 mM, 3.22 mM and 1.61 mM) of Indocyanine green (ICG) for 2, 24, 48, and 72 h. Similarly, groups of Müller cells were stained with "Heavy" brilliant blue G (HBBG), Trypan blue (TB) (0.15 %, or 1.56 mM), Membrane-blue-dual (MBD), and ICG (0.25 %, or 3.22 mM) or BBG (0.025 %, or 0.3 mM) with glucose (GS) (50 %, 66 % and 75 % or 2.78 M, 3.67 M and 4.17 M) for 30, 60, and 120 s. Cell viability was measured with the Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) release assays. We found that high stain concentration and long exposure time resulted in increased toxicity to Müller cells. Nevertheless, ICG seemed to be safe at the clinically relevant concentration of 0.25 % (3.22 mM) in the short time of exposure. TB was safer than both HBBG and MBD, especially HBBG. Hypertonic GS as a dilution was not safe for Müller cells, and the negative effect was more obvious in 0.025 % (0.3 mM) BBG than that in 0.25 % (3.22 mM) ICG. This is the first report to observe the cytotoxicity of commonly used stains in clinical on human Müller cells in vitro, and to provide some basis for further studies, including in vivo investigation.

Evaluation of Patent Blue as the Vital Dye in an Animal Model of Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate the safety and feasibility of patent blue (PB) as the vital dye in Descemet membrane endothelial keratoplasty (DMEK). METHODS: Bovine corneal endothelial cells were incubated with different concentrations (0.02%-2.5%) of PB. The cell viability, which was assessed by Cell Counting Kit-8 assay, was compared with that of untreated control and 0.06% to 0.4% trypan blue. The dyes were also used for graft preparation and implantation in the porcine eye model to evaluate stain quality, dye retention, and the feasibility of using PB in DMEK surgery. RESULTS: No obvious increase in cytotoxicity was detected for 0.06% to 0.4% trypan blue and PB at concentrations up to 1.0%, but the cell viability after incubating with 1.5% to 2.5% PB was significantly reduced. PB at 0.5% to 1.0% generated good staining quality that can be used to facilitate graft implantation. Although the staining quality of 0.5% to 1.0% PB faded to an intermediate level after a 30-minute wash in phosphate-buffered saline, dye retention persisted for up to 24 hours. CONCLUSIONS: PB at 0.5% to 1.0% is biocompatible and can stain the graft sufficiently, making it an alternative for DMEK surgery.

Foetal bovine serum can reduce toxicity of indocyanine green, brilliant blue G and trypan blue in ARPE-19 cellular model that suggests new surgical staining protocols for internal limiting membrane peeling procedure.

BACKGROUND: To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). METHODS: The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. RESULTS: Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. CONCLUSIONS: As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

A low-cost wheat bran medium for biodegradation of the benzidine-based carcinogenic dye Trypan Blue using a microbial consortium.

Environmental release of benzidine-based dyes is a matter of health concern. Here, a microbial consortium was enriched from textile dye contaminated soils and investigated for biodegradation of the carcinogenic benzidine-based dye Trypan Blue using wheat bran (WB) as growth medium. The PCR-DGGE analysis of enriched microbial consortium revealed the presence of 15 different bacteria. Decolorization studies suggested that the microbial consortium has high metabolic activity towards Trypan Blue as complete removal of 50 mg∙L-1 dye was observed within 24 h at 30 ± 0.2 °C and pH 7. Significant reduction in TOC (64%) and COD (88%) of dye decolorized broths confirmed mineralization. Induction in azoreductase (500%), NADH-DCIP reductase (264%) and laccase (275%) proved enzymatic decolorization of dye. HPLC analysis of dye decolorized products showed the formation of six metabolites while the FTIR spectrum indicated removal of diazo bonds at 1612.30 and 1581.34 cm-1. The proposed dye degradation pathway based on GC-MS and enzyme analysis suggested the formation of two low molecular weight intermediates. Phytotoxicity and acute toxicity studies revealed the less toxic nature of the dye degradation products. These results provide experimental evidence for the utilization of agricultural waste as a novel low-cost growth medium for biodegradation of benzidine-based dyes, and suggested the potential of the microbial consortium in detoxification.

Dye solutions based on lutein and zeaxanthin: in vitro and in vivo analysis of ocular toxicity profiles.

PURPOSE: To study the safety profile of Lutein/Zeaxanthin(L/Z)-based natural dye solutions in in vitro and in vivo models. MATERIAL AND METHODS: In vitro cytotoxicity and cellular growth experiments were carried out on ARPE-19 and human corneal epithelial (HCE) cell lines using different L/Z-based dye solutions, either alone or in association with brilliant blue (BB) or trypan blue (TB). Light and transmission electron microscopy studies were performed seven days after intravitreal injection of dye solutions in rabbits. Electroretinogram (ERG) recordings were taken at baseline and before histopathology. RESULTS: In vitro cytotoxicity assays demonstrated that the different L/Z-based solutions (from 0.3 to 2%), either alone or in association with BB (0.025%) or TB (0.04%), did not significantly alter mitochondrial activity (≤15%) in the cell lines tested. In addition, in vitro cell growth was inhibited by up to 60% depending on the dye solution, and in direct proportion to the concentration assayed. There was no evidence of structural alterations in the neurosensory retina, retinal pigment epithelium (RPE), or choriocapillaris-choroidal complex. b-Wave ERG records showed no significant differences (±15.2%) in comparison with baseline. CONCLUSIONS: L/Z-based dye solutions demonstrated a safe profile in in vitro and in vivo models, and may be a useful tool for staining intraocular structures.

The effects of vital dyes on retinal pigment epithelium cells in oxidative stress.

PURPOSE: To determine the effect of the most commonly used vital dyes in vitrectomy [trypan blue at 0.15% concentration and indocyanine green (ICG) at 0.5% concentration] on the viability of human retinal pigment epithelium (RPE) cell lines (ARPE-19) exposed to oxidative stress. METHODS: ARPE-19 cells unexposed or exposed to oxidative stress (hypoxic chamber) were treated for 1 min with one of the dyes. RPE proliferation was measured by (3)H-thymidine incorporation, adhesion by ability to adhere to fibronectin, and safety by annexin V staining. RESULTS: Proliferation: The dyes affected the proliferation of RPE cells differently under non-hypoxic and hypoxic conditions (p = 0.001). In non-hypoxic conditions, there was no statistically significant difference between the proliferation of the treated (both dyes) and untreated RPE cells (p = 0.279). Under hypoxia, both dyes significantly suppressed proliferation, more so with ICG (p = 0.001). Adhesion: The dyes affected adhesion differently under non-hypoxic and hypoxic conditions (p = 0.04). In non-hypoxic conditions, both increased the adhesive properties of RPE cells to fibronectin, ICG more than trypan blue (p = 0.001). Under hypoxia, both dyes suppressed adhesion, with no statistically significant difference between treated and non-treated RPE cells. Apoptosis: Both dyes increased early apoptosis of RPE cells compared with no treatment (p = 0.001), ICG more than trypan blue. Hypoxia increased the apoptosis of both dyes compared to non-hypoxic conditions (p = 0.02). CONCLUSIONS: In hypoxic conditions, both dyes showed an inhibition of RPE adhesion to fibronectin and proliferation capacity and an increase in early apoptosis compared with non-hypoxic conditions. Apoptosis was greater in ICG-treated RPE cells than in trypan blue-treated cells.

Increase in lens capsule stiffness caused by vital dyes.

PURPOSE: To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue. SETTING: Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Fifteen unstained lens capsules were dissected into 7 wedge-shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 µm × 10 µm were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz. RESULTS: Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2-fold after illumination. CONCLUSION: Staining significantly increased the mechanical properties of the human lens capsule. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

The retinal biocompatibility of dyes in the ex vivo model of the isolated superfused vertebrate retina.

BACKGROUND: Peeling of the internal limiting membrane or epiretinal membranes is a successful principle in macular surgery to achieve a functional benefit. Different dyes are used to facilitate the identification of intraocular tissues. The aim of our work was to investigate the retinal tolerance to the different dyes and their solvent carriers to provide valuable data for surgeons in handling for an optimal intraoperative use. METHODS: Using the ex vivo model of the isolated superfused vertebrate retina technique, the effects of the dyes were tested on human and bovine retinal function. The retinas were perfused with an oxygen preequilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl electrodes. After recording stable ERG amplitudes, the dyes brilliant blue G, indocyanine green, trypan blue, patent blue, triamcinolone and their solvent carriers were investigated. RESULTS: Reductions of the ERG amplitudes were found for each tested dye. The effects after application of the dyes were dependent on time and concentration of the applied dyes, which were different for each dye. CONCLUSION: In part, the ERG has shown strong effects already after a short period of dye application. Surgeons who rely on the intraocular use of the dyes should keep in mind our findings, and the use of some dyes should be limited to selected cases. The well-considered use of the dyes by the surgeons could lead to a better functional outcome and avoid a possible harmful outcome of the surgery after mishandling.

Brilliant Blue G as protective agent against trypan blue toxicity in human retinal pigment epithelial cells in vitro.

BACKGROUND: Combinations of trypan blue (TB), Brilliant Blue G (BBG) and polyethyleneglycol had been shown before to be less toxic to ARPE retinal pigment epithelial cells than TB alone. We studied systematically the influence of combinations of dyes on cell damage. METHODS: ARPE cells were exposed to TB (concentration range 0.025 to 1 %), BBG (0.0025 to 0.5 %), and combinations of the two dyes, dissolved in phosphate buffered saline (PBS), for periods between 5 and 60 min. Cell damage was monitored with the WST-1 assay. The effect of different salt concentration was measured in the same way. RESULTS: TB in concentrations of 0.075 % and higher was toxic to the cells already after 30 min incubation. BBG was toxic after 30 min in concentration of 0.1 % and higher, but had a protective effect on cells with incubation time of 5 min and concentrations up to 0.1 %. BBG at concentrations of 0.025 % protected against TB-induced damage at 5 min and 30 min incubation. Salt concentrations between 113 and 225 mM did not influence cell survival even after 30 min. In the presence of TB, propidium iodide bound strongly to the cells. CONCLUSIONS: BBG acts as a protecting agent against TB toxicity.

Time-dependent morphological alterations and viability of cultured human trabecular cells after exposure to Trypan blue.

BACKGROUND: To determine the time-dependent toxicity of Trypan blue at 0.06% concentration in cultured human trabecular meshwork cells. METHODS: Human trabecular meshwork cells cultured in vitro were exposed to Trypan blue and acute toxicity was evaluated. Cells were exposed for 5, 30, 60 s and for 5, 15, 30, 60 min to a commercially available Trypan blue preparation (Vision Blue; DORC International Zuidland, The Netherlands). Morphology was observed by phase-contrast microscopy and cell viability was measured using Hoechst 33342 (Intergen; Purchase, NY, USA) and propidium iodide assays to determine the percentage of living and dead cells. RESULTS: Morphological changes occurred mainly after 5 min of exposure to Trypan blue. Viability was 96.0% ± 3.6%, 94.8% ± 3.5% and 92.5% ± 4.4% after 5, 30 and 60 s of exposure, respectively; a significant toxic effect of Trypan blue was observed after 5, 15, 30 and 60 min of exposure with a viability of 85.0% ± 3.7%, 77.0% ± 6.7%, 70.8% ± 5.9% and 68.3% ± 8.7%, respectively. CONCLUSIONS: Trypan blue is not toxic, in terms of cell viability, over an exposure time of up to 60 s; however, further exposure results in a gradual increase in damage of cultured human trabecular meshwork cells.

Investigating the biocompatibility of two new heavy intraocular dyes for vitreoretinal surgery with an isolated perfused vertebrate retina organ culture model and a retinal ganglion cell line.

BACKGROUND: During vitreoretinal surgery, vital dyes are used to visualize anatomical structures. Substances with a density higher than water are added to facilitate sedimentation and staining. BBG with 4% PEG (ILM Blue) and BBG with TB and 4% PEG (MBB Dual) are two new dyes. This study evaluates biocompatibility of the new dye solutions, using cell cultures and electrophysiological evaluation. METHODS: To determine cytotoxicity of ILM Blue and MBB Dual for 30, 60, 120 and 320 seconds, monolayer cultures of retinal ganglion cells (RGC5) were used. For functionality testing, bovine retinas were isolated and superfused with an oxygen-saturated nutrient solution, and the electroretinogram (ERG) was recorded. The two dye solutions were applied epiretinally for 30, 60 and 120 seconds. ERG recovery was monitored. RESULTS: After staining with ILM Blue, no statistical significant reduction of a- or b-wave amplitudes at the end of the wash-out was recorded. For MBB Dual, only a significant reduction of the a-wave amplitudes after 30 seconds of application at the end of the wash-out was noticed, while no statistically different changes for a- and b-wave amplitudes up to 120 seconds were noted. During the MTT assay, we noted no significant difference in cell viability after 30, 60, 120 and 320 seconds of staining with ILM blue, MBB Dual or 4% PEG in comparison to the control group (DMEM, Triton X-100 0.9% as positive control) after formazan extraction. CONCLUSIONS: ILM Blue and MBB Dual seem to be safe for clinical use for a staining period of up to 120 seconds, probably even up to 320 seconds.

Methyl blue and aniline blue versus patent blue and trypan blue as vital dyes in cataract surgery: capsule staining properties and cytotoxicity to human cultured corneal endothelial cells.

PURPOSE: To evaluate capsule-staining properties and biocompatibility of the triarylmethane dyes methyl blue and aniline blue compared with patent blue and trypan blue on cultured human corneal endothelial cells. SETTING: Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany. DESIGN: Experimental study. METHODS: Human corneal endothelial cell cultures were harvested from human donor cells and exposed to various concentrations (0.025 to 5.0 mg/mL) of methyl blue, aniline blue, patent blue, and trypan blue. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test after 24 hours of incubation. Calcein live cell staining was performed at the same time point. The dyes were also used to stain pig lens capsules in vitro by incubating the lenses for 1 minute with 3 concentrations (0.5, 1.5, and 2.5 mg/mL) of dye, after which the staining properties were evaluated. RESULTS: No significant cytotoxicity was detected for patent blue and methyl blue at any tested concentration. However, aniline blue exerted significant cytotoxicity at concentrations of 1.5 mg/mL or higher and trypan blue at 2.5 mg/mL or higher. Capsule staining of the tested triarylmethane dyes was suitable for performing capsulorhexis, but only at higher concentrations than with trypan blue. CONCLUSIONS: High concentrations and long incubation times of trypan blue and aniline blue showed significant cytotoxicity to human cultured endothelial cells in contrast to patent blue and methyl blue. All tested dyes were able to stain lens capsules sufficiently for capsulorhexis creation. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Comparative toxicology of trypan blue, brilliant blue G, and their combination together with polyethylene glycol on human pigment epithelial cells.

PURPOSE: To determine the toxicity in ARPE-19 human retinal pigment epithelium cells of trypan blue (TB) at 0.15% and 0.25% concentration, brilliant blue G (BBG) at 0.025% and 0.05%, their combination, and the effect of the addition of 4% polyethyleneglycol (PEG), as an additive for increasing the density and thus improving the staining in internal limiting membrane removal, on the individual dyes and their combinations, and compare the toxicity of the dyes to that of clinically used preparations. METHODS: Cells were exposed for 5 and for 30 minutes to the different preparations. Cell viability was measured with the WST-1 assay measuring intracellular dehydrogenase activity. RESULTS: Solutions containing PEG with BBG (0.025%), TB (0.15%), and mixtures of BBG (0.025%) with TB (0.15% and 0.25%) were the least toxic of the preparations as well as preparations of BBG at 0.025% in phosphate-buffered saline solution, while TB at 0.25% in phosphate-buffered saline solution was the most toxic. The addition of PEG reduced the toxicity of preparations containing TB either alone or in combination with BBG. These results were seen only after an incubation for 30 minutes; for a 5-minute incubation, no toxicity was seen for any of the preparations. CONCLUSIONS: For short incubation times, all dyes appear equally safe. For longer incubation times, TB preparations were more toxic than BBG preparations. The toxicity of TB was reduced by the addition of PEG. Further studies are required to determine the clinical impact of this finding.

Two cases of toxic anterior segment syndrome from generic trypan blue.

We report 2 cases of toxic anterior segment syndrome (TASS) resulting from impurities in generic trypan blue that was administered intracamerally to improve visualization of the capsule. Histology of the corneal buttons revealed foci of inflammatory response and complete loss of endothelial cells. Cell culture analysis showed that the generic trypan blue was approximately twice as toxic to the endothelium as a proprietary trypan blue. Ophthalmologists should be aware that any substance administered intraocularly can be a source of complications, and they should know the source of all material used in surgery.

Functional and structural effect of intravitreal indocyanine green, triamcinolone acetonide, trypan blue, and brilliant blue g on rat retina.

PURPOSE: The purpose of this study was to evaluate the functional and structural damage of the retina after intravitreal injections of four different dyes in the rat. METHODS: Rats were injected intravitreally with indocyanine green (ICG), trypan blue, triamcinolone acetonide, or brilliant blue G in the right eye. The other eye was injected with saline and served as a control. Simultaneous bilateral electroretinograms were recorded before injection and 7 and 28 days after injection. Histology and immunohistochemistry analyses with antibodies recognizing glial fibrillary acidic protein and protein kinase C were performed 28 days after the initial injection on both eyes. RESULTS: Seven days after dye injection, the electroretinogram response of the treated eyes was altered in each group. At 1 month, eyes injected with triamcinolone acetonide, trypan blue, or brilliant blue G fully recovered, whereas eyes treated with ICG had A-wave and B-wave reduction of 65% and 63%, respectively. The inner nuclear layer thickness was statistically decreased in the ICG group (P = 0.003) but not with other dyes. Protein kinase C staining was decreased in the ICG group only, but no abnormal qualitative staining was found with either glial fibrillary acidic protein or protein kinase C antibodies with any dye. CONCLUSION: Among the four tested dyes, only ICG led to functional and structural retinal damage.

Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells.

PURPOSE: To evaluate and compare the effects of the following dyes on human pigmented epithelial cells: indocyanine green (ICG), infracyanine green (IfCG), trypan blue (TB), bromophenol blue (BrB), patent blue (PB), and Brilliant Blue G (BBG). METHODS: ARPE-19 cells cultured in vitro were exposed to these dyes, and acute and chronic toxicity were evaluated. Cell viability was measured by colorimetry (MTT assay), morphology was observed by phase-contrast microscopy, membrane permeability (CMP) was evaluated by flow cytometry with propidium iodide (PI), and mitochondrial membrane potential (ΔΨm) was measured with 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)). RESULTS: Each of the studied dyes exhibited toxicity after acute exposure at surgical doses. The presence of light often reduced cell viability, especially when measured 3 hours after incubation in the case of ICG, TB, BrB, and BBG. Morphologic changes were induced by ICG, IfCG, and BBG. Both CMP and ΔΨm were altered after exposure to surgical doses of ICG, TB, PB, and a fourfold surgical dose of BrB. Chronic exposure to residual amounts of some dyes was associated with reduced proliferation and even cell death. CONCLUSIONS: It appears to be prudent to use the lowest possible dose of each dye, to minimize the risk of toxic effects. This precaution may be particularly important in the case of BrB, which should not be used in excess of 0.5%. In addition, abundant irrigation of the vitreous cavity after surgery to completely remove traces of dye may be of crucial importance, particularly in the case of ICG, in minimizing chronic toxicity.

Normal physiological and pathophysiological effects of trypan blue on the retinas of albino rabbits.

PURPOSE: To determine whether intravitreal injection of trypan blue is toxic to the retina of the albino rabbit. METHODS: Sixteen albino rabbits were studied for the effects of intravitreal trypan blue (eight with 0.06% solution and eight with 0.15% solution). Saline was injected into the fellow control eye of all rabbits. The electroretinogram and visual evoked potentials were recorded from each rabbit at different time intervals after injection. The rabbits were killed at the termination of the follow-up period, and their retinas were prepared for histologic examination under light microscopy. RESULTS: In all rabbits, short-term follow-up showed significant reduction of ERG responses in the experimental eye, with the b-wave more affected than the a-wave. Partial to complete recovery was observed during follow-up. After 4 weeks, negligible ERG deficit was observed in the rabbits treated with 0.06% trypan blue, whereas significant ERG deficit was measured in rabbits tested by the 0.15% trypan blue. No differences in flash VEP responses between experimental and control rabbit eyes were found. Light microscopy showed no significant histologic effects in the retinas exposed to the 0.06% solution. Marked disorganization of all retinal layers was observed in areas close to the site of injection in the rabbits injected with the 0.15% solution. CONCLUSIONS: Trypan blue exerts transient physiological effects on the distal retina of the rabbit, but in concentrations of 0.15% it can induce permanent toxic effects. Therefore, caution should be used when using this dye in vitreoretinal surgery.