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. Author manuscript; available in PMC: 2014 Dec 9.
Published in final edited form as: Ophthalmologica. 2013 Sep 6;230(0 2):41–50. doi: 10.1159/000354251

Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy

Fernando M Penha 1, Marianne Pons 2, Elaine Fiod Costa 1, Eduardo B Rodrigues 1, Mauricio Maia 1, Maria E Marin-Castaño 2, Michel Eid Farah 1
PMCID: PMC4260821  NIHMSID: NIHMS520962  PMID: 24022718

Abstract

Purpose

To investigate in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line.

Methods

ARPE-19 cells were exposed to brilliant blue-BriB, evans blue-EB, bromophenol blue-BroB, indocyanine green-ICG, infracyanine green-IfCG, light green-LG, fast green-FG, indigo carmine-IC and congo red-CR. BSS was used as the control. Five different concentrations and two times were tested. Cell viability was determined by MTS assay and apoptosis by Bax expression on western blot.

Results

All dyes significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01), except for BriB that was safe at concentrations up to 0.25mg/mL and CR up to 0.05mg/mL, while LG was safe in all concentrations. Toxicity was higher after 30 minutes of exposure. Expression of Bax was upregulated after all dyes exposure, except BriB; ICG had the highest Bax expression (p<0.01).

Conclusions

Overall the safest dye was BriB followed by LG, IfCG, FG, CR, IC, BroB, RB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BriB was the only dye that did not induce apoptosis in ARPE-19 cells.

Introduction

Removal of the internal limiting membrane (ILM) has been an important maneuver for anatomical and functional success in macular hole and other macular surgeries [1-3]. However due to its anatomical characteristics, the identification of the ILM during surgery is a difficult step in the surgical procedure. Therefore, the use of dyes to identify structures during vitreoretinal surgery, “chromovitrectomy,” has become a popular technique in recent years [4]. Indocyanine green (ICG) was the first dye to be used in macular surgery promoting a good contrast with retinal tissue and making ILM removal technically easier [5]. Although, in the past few years, several studies have demonstrated toxicity to the retinal pigment epithelium (RPE) and neurosensory retina, as well as cases of optic nerve atrophy, after the use of ICG [1, 6-9]. Other alternative dyes have emerged for staining the ILM with less toxicity profile.

Trypan blue (TB) demonstrated a lower toxicity profile to RPE cells and retinal tissue when compared to ICG, with an excellent affinity for epiretinal membranes, but it is not a good dye for acellular membranes, such as the ILM [7, 10, 11]. Brilliant blue (BriB) have been emerged as second generation dye with an outstanding staining to ILM [12-16]. Moreover, BriB was recently released on the European market in a concentration of 0.25 mg/ml – Brilliant Peel™ (DORC, The Netherlands). This presentation of the dye was shown to provide a good staining capacity to the ILM and was not toxic in experimental studies and case series in humans [17]. However, BriB could induce RPE changes after accidental subretinal dye injection in humans [18-20].

Currently, ICG, TB and BriB have been used in chromovitrectomy [4]. However, a dye with little toxicity, and with a good affinity for the ILM is yet to be found. The aim of this study was to provide a detailed in vitro toxicity investigation of seven dyes: evans blue (EB), bromophenol blue (BroB), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and congo red (CR); and compare it to indocyanine green (ICG) and brilliant blue (BriB), which are in clinical use. Five dye concentrations (1, 0.5, 0.25, 0.05 and 0.005 mg/ml) and two exposure times (3 and 30 min) were used.

The evaluation of apoptosis in retinal toxicity studies of dyes has become an important issue, since it was shown that residual ICG can be found months after surgery [21]. For this reason, in the present study we also evaluated the link between cell toxicity and apoptosis in ARPE-19 cells exposed to these vital dyes.

Methods

Compounds

The dyes ICG, LG, CR, FG, EB, BroB, IC and BriB and cell culture reagents were obtained from Sigma-Aldrich (Munich, Germany). IfCG was obtained from Serb (Paris, France). Balanced salt solution - BSS (BSS®) was obtained from Alcon Laboratories (Fort Worth, TX). MTS CellTiter 96 Aqueous One Solution Cell Proliferation Assay was purchased from Promega (Madison, WI). LDH-Cytotoxicity assay kit was purchased form Abcam Inc (Cambridge, MA). Primary antibody Bax was purchased from EMD Millipore Corporation (Billerica, MA). The secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Dye preparation

Initially, 5 mg of each dye were measured using an analytical balance (Mettler-Toledo Inc., Columbus, OH) and dissolved in sterile BSS to obtain stock solutions. ICG and IfCG required different methods of preparation. ICG was first diluted in distilled water and IfCG with 5% glucose solution as recommended by the manufacturer. Subsequent dilutions were performed with sterile BSS to obtain final concentrations of 1, 0.5, 0.25, 0.05 and 0.005 mg/ml. This serial dilution was made to evaluate a wide range of concentrations that could be present in the surgical field due to variations of dilution technique and dye injection (air or fluid filled vitreous cavity).

Afterwards, the pH and osmolarity of each dye solution were determined using a previously calibrated pH meter and osmometer (Advanced Instruments Inc., Norwood, MA). These measurements were made to minimize deleterious conditions related to unexpected variations of pH and osmolarity, which could have an influence on the toxicity found in the experiments.

Cell-viability assay

All experiments were performed using the immortalized human retinal pigment cell line ARPE-19 (American Type Culture Collection, Manassas, VA), a well-established model to test the safety of vital dyes in RPE cells. The rationale for using this cell line in dye toxicological studies is that during macular hole surgery the dye could come in direct contact with the RPE through the macular hole. Other reason is that the dye can penetrate trough retinal layers and cause RPE toxicity as well. Therefore, a meticulous study of this cell layer is very important in these pre-clinical toxicity evaluations.

ARPE-19 cells were grown in Dulbecco's Modified Eagles Medium/ Ham's F-12 (DMEM/F12; Gibco, Carlsbad, CA) (1:1 vol/vol) medium supplemented with 10% fetal bovine serum (FBS), 1mM L-glutamine, 100 ∓g/ml penicillin/streptomycin, and 0.348% Na2CO3 in a 5% CO2 humidified air incubator at 37°C. Cells were used between passage 5 and 8. For experiments, cells were seeded at a concentration of 5 × 103 cells/well in 96-well, flat-bottom tissue culture plates in 200 μl of culture medium and grown for 24 hours before the experiments in a 5% CO2 humidified incubator at 37°C. Subsequently, the cell culture medium was removed and the cells washed three times with BSS. Subsequently, cells were incubated with ICG, IfCG, FG, LG, BriB, BroB, EB, IC or CR (1, 0.5, 0.25, 0.05 and 0.005 mg/ml) for 3 or 30 minutes. After incubation, cells were washed three times with 200 μl of phosphate buffered saline (PBS). The number of surviving cells was measure by cell count (Coulter ZI cell counter; Beckman Coulter, Hialeah, FL) and by MTS (a tetrazolium salt) assay (Cell Titer 96 AQueous One Solution kit; Promega, Madison, WI). The results for MTS were obtained by measuring absorbance at 490 nm with an ELISA plate reader (Bio-Rad, Hercules, CA). All experiments were performed in quadruplicate and repeated three times.

The rationale of these two time exposures, 3 and 30 minutes, is to simulate an acute exposure to these dyes that occurs during vitreoretinal surgery (3 min) and also a prolonged exposure that might happen if the dye is not entire washed out from the vitreous cavity (30 min).

Apoptosis Assay

Background

Recent studies have delineated one key mechanism responsible for initiating the executioner phase of apoptosis. It is widely recognized that apoptosis is mediated by Bax cascade through mitochondrial stress [22]. Bax is a pro-apoptotic Bcl-2-family protein that resides in the cytosol and translocates to mitochondria upon induction of apoptosis [23]. Recently, Bax has been shown to induce cytochrome c release and caspase activation in vivo and in vitro [24, 25].

Some of the tested dyes were really toxic to RPE cells and were excluded from the apoptosis assay. We therefore examined the expression of pro apoptotic protein, Bax by ICG, IfCG, FG, LG and BriB in ARPE-19 cells exposed to 0.05 mg/ml dyes for 3 minutes, which are the concentration and time mostly used in vitreoretinal surgery.

Western blot analysis

After treatment, lysates of ARPE-19 cells were obtained, and total protein was extracted in protein lysis buffer M-PER (Pierce, Rockford, IL) and quantified by a Detergent Compatible protein assay (Bio-Rad, Hercules, CA). Protein extracts (20 to 40 μg) were denatured in Laemmli's sample buffer, followed by boiling for 5 minutes, and then resolved on a 4 to 20% Tris-glycine gel. After electrophoresis (120 V for 2 hours), proteins were transferred in 1X transfer buffer [25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS, and 20% methanol (pH 8.4)] to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) using constant current (350 mA for 45 minutes). Membranes were blocked in 5% nonfat dry milk–TBS solution for 1 hour at room temperature. Blots were incubated overnight at 4°C with primary antibodies against Bax, cytochrome c, caspase-9, Bcl-2, and GAPDH. Membranes were washed three times with TBS-Tween 20, then incubated with horseradish peroxidase–linked donkey anti-rabbit antibody or donkey anti-mouse for 2 hours at room temperature, and finally washed three times in TBS-Tween 20. Immunoreactive bands were determined by exposing the nitrocellulose blots to a chemiluminescent solution and exposing to X-Omat AR film (Eastman Kodak Co., Rochester, NY). Three independent experiments were performed in triplicate.

Statistical analyses

All experiments were performed three times, showing reproducible results. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad, La Jolla, CA). Data are expressed as mean ± SEM of percentage of cell viability/toxicity with respect to control. Statistical comparisons were performed using one-way analysis of variance followed by Tukey post hoc test for multiple comparisons. Values of P < 0.05 were considered statistically significant.

Results

Effect of tested dyes on cell viability

In order to determine the range of dyes toxicity, the cells were treated with nine different dyes, in five concentrations (1, 0.5, 0.25, 0.05, and 0.005 mg/ml) for 3 or 30 minutes and the cell toxicity determined by MTS reduction assay and cell count.

As shown in Figure 1, all concentrations tested for ICG, IfCG and FG during 3 minutes of exposure significantly decreased cell viability when compared to control (BSS). This effect was dose-dependent (Table I). However for LG no toxicity was observed at 3 minutes. Moreover, treatment with these dyes for 30 minutes dramatically reduced the viability when compared to control (Figure 3 Table I). ICG (1 mg/ml) for 3 minutes decreased cell viability by approximately 57% (43.1±3.3%;P<0.01) whereas 0.5 mg/ml and 0.25 mg/ml ICG decreased cell viability by approximately 49% (51.6%±3.5%; P<0.01, 55.2%±2.7%; P<0.01) and concentrations of 0.050 mg/ml and 0.005 mg/ml by approximately 38% (61.9%±4.2%; P<0.01, 66.3%±3.9%; P<0.01) (Figure. 1). However, after 30 minutes of treatment the decrease in cell viability was ~70% for all the assayed doses (Figure 3 and Table I).

Figure 1.

Figure 1

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Effect of different doses of indocyanine green (ICG), infracyanine green (IfCG), fast green (FG) and light green (LG) on ARPE-19 cell viability assessed by MTS cell assay. Cells were exposed to dyes for 3 minutes. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by **P < 0.01 and for comparison with the control (BSS solution).

Table 1.

Effect of vital dyes on retinal pigmented epithelial cell viability, at different dye concentration and exposure times

Exposure time Concentrations (mg/ml)
Dyes 1 (Mean ± SD) 0,5 (Mean ± SD) 0,25 (Mean ± SD) 0,05 (Mean ± SD) 0,005 (Mean ± SD)
BSS 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0
BriB 64.8 ± 3.6** 77.4 ± 4.5** 94.4 ± 4.9 94.9 ± 6.2 98.2 ± 6.5
ICG 43.1 ± 3.3** 51.6 ± 3.5** 55.2 ± 2.7** 61.9 ± 4.2** 66.3 ± 3.9**
IfCG 56.5 ± 3.5** 62.0 ± 2.9** 68.1 ± 2.9** 74.3 ± 3** 79.2 ± 3.5**
LG 92.7 ± 6.8 93.6 ± 4.3 96.9 ± 4.2 97.7 ± 4.8 101.2 ± 5.1
FG 69.2 ± 5.1** 69.8 ± 8.3** 77.4 ± 4.8** 79.7 ± 4.6** 80.1 ± 3.4**
3 minutes IC 47.3 ± 4.9** 53.0 ± 3.5** 56.4 ± 3.5** 59.7 ± 4.5** 66.6 ± 3.5**
BroB 49.6 ± 3.1** 57.2 ± 3.7** 61.4 ± 2.4** 62.1 ± 4.9** 66.6 ± 5.3**
EB 50.4 ± 3.6** 58.7 ± 6.3** 62.6 ± 7.2** 67.3 ± 8.1** 61.9 ± 5.3**
CR 55.8 ± 3.1** 64.3 ± 4.1** 73.7 ± 3.4** 89.1 ± 5 93.9 ± 6.7
BSS 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0
BriB 62.3 ± 3.1** 72.8 ± 5.1** 81.6 ± 6.1** 87.4 ± 8.3* 94.2 ± 9.3
ICG 33 ± 4.6** 31.4 ± 3.3** 33.5 ± 3.3** 37.2 ± 3.6** 37.2 ± 5**
IfCG 52.4 ± 6.7** 52.3 ± 7.8** 54.6 ± 7.7** 53.4 ± 8** 48.5 ± 9.1**
LG 39.4 ± 2.6** 44.9 ± 4.5** 47.1 ± 2.8** 45.5 ± 3.3** 46.7 ± 4**
FG 46.3 ± 3.7** 51.1 ± 2.4** 44 ± 4.3** 41.3 ± 1.5** 56.1 ± 2**
30 minutes IC 42.7 ± 2.4** 44.3 ± 3.3** 49.2 ± 2** 50.4 ± 3.1** 53.7 ± 3.1**
BroB 37.2 ± 5.1** 51.6 ± 2.5** 56.9 ± 4.2** 57.3 ± 6.2** 60.4 ± 2**
EB 39.9 ± 6.2** 52.2 ± 7** 55.4 ± 3.2** 60 ± 2.5** 58.6 ± 7.5**
CR 39.9 ± 4.1** 42.5 ± 2** 49.9 ± 4.2** 52.3 ± 4** 59.9 ± 3.3**
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Analysis of variance (ANOVA) –Tukey test

Observations:

I– Lower the values higher the dye toxicity.

II– Values are means of at least 3 independent experiments.

III – In regard to the P values presented in this table, there was a significant difference between the dyes with all concentrations and exposure times (* P < 0.05, ** P < 0.01) when compared with non-treated control cells

Figure 3.

Figure 3

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Effect of different doses of indocyanine green (ICG), infracyanine green (IfCG), fast green (FG) and light green (LG) on ARPE-19 cell viability assessed by MTS cell assay. Cells were exposed to dyes for 30 minutes. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by **P < 0.01 and for comparison with the control (BSS solution).

Exposure for 3 minutes to 1 and 0.5 mg/ml BriB showed a statistically significant reduction in cell viability of ~ 35% and 23% respectively (64.8%±3.6%; P<0.01, 77.4%±4.5%; P<0.01). However, doses of 0.25, 0.05 and 0.005 mg/ml, did not modify cell viability (Figure 2, Table I). When cells were exposed to this dye for 30 minutes, cell viability decreased by ~37% and 27% at concentrations of 1 to 0.05 mg/ml respectively (62.3%±3.1%; P<0.01, 72.8%±5.1%; P<0.01) and by ~18% and 13% (81.6%±6.1%; P<0.01, 87.4%±8.3%; P<0.05) at concentrations of 0.25 and 0.05 respectively (Figure 4, Table I). BriB (0.005 mg/ml) did not modify cell viability.

Figure 2.

Figure 2

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Effect of different doses of indigo carmine (IC), bromophenol blue (BroB), evans blue (EB), congo red (CR) and brilliant blue (BriB) on ARPE-19 cell viability assessed by MTS cell assay. Cells were exposed to dyes for 3 minutes. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by **P < 0.01 and for comparison with the control (BSS solution).

Figure 4.

Figure 4

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Effect of different doses of indigo carmine (IC), bromophenol blue (BroB), evans blue (EB), congo red (CR) and brilliant blue (BriB) on ARPE-19 cell viability assessed by MTS cell assay. Cells were exposed to dyes for 30 minutes. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by **P < 0.01 and for comparison with the control (BSS solution).

Our data also show that IC, BroB and EB for 3 minutes induced decrease in cell viability at all tested doses. On the other hand CR induced decrease in RPE cell viability just in doses higher than 0.05 mg/ml (55.7%±5.81%; P<0.01, 64.2%±8.1%; P<0.01, 73.7%±6.7%; P<0.01) whereas it had no effect at concentrations of 0.05 mg/ml and 0.005 mg/ml (Figure 3, Table I). However, 30 minutes of exposure IC, BroB, EB and CR reduced cell viability significantly in all tested concentrations (Figure 4, Table I).

When exposure time was evaluated as an isolated factor, it was shown that for the all concentrations at 30 minutes induced higher toxicity when compared with 3 minutes of exposure (Figure 5).

Figure 5.

Figure 5

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Combined results of effect of vital dyes on ARPE-19 cells. The effects of dyes on cell viability were combined for each concentration and compared between 3 and 30 minutes of exposure. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by *P < 0.01 for comparison with the control.

In regard to the dye itself, when all values were analyzed independently of concentration or exposure time, all dyes induced a reduction of RPE cell viability except BriB. The order of cell viability reduction (lower to higher) is: BriB, LG, IfCG, FG, CR, IC, BroB, EB and ICG (Figure 6). Comparable results were observed for cell viability measured by cell count (data not shown).

Figure 6.

Figure 6

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Combined results of effects of vital dyes on ARPE-19 cell viability. The effects of dyes on cell viability were combined for all concentrations and both exposure times and the dyes compared. Bars correspond to means of three independent experiments. Data are expressed as percentage of control, and the results shown are means ± SEM of three independent experiments run in triplicate. Statistical significance is indicated by *P < 0.01 for comparison with the control (BSS solution).

Expression of Bax by vital dyes in ARPE-19 cells

Dysregulated expression of Bax by RPE may be involved in the cell susceptibility to apoptosis [26]. Therefore, based on the observations obtained for cell viability, it was explored whether the tested dyes induces apoptosis through Bax overexpression in ARPE-19 cells. With the use of Western blot analysis, it was investigated the expression of Bax in ARPE-19 cells treated with or without 0.05 mg/ml ICG, IfCG, LG, FG, BriB and CR for 3 minutes. Our results revealed that ICG robustly increases Bax expression at protein levels (383.1±2.38, p<0.01; Figure 7). The other dyes, LG, FG, IfCG and CR also induced an overexpression of Bax by 2, 2.18, 2.07 and 2.17-times respectively (p<0.05; Figure 7). BriB revealed a slightly increase in Bax expression, but not significantly when compared to untreated cells (Figure 7).

Figure 7.

Figure 7

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Bax protein expression in ARPE-19 cells following treatment with vital dyes. Confluent ARPE-19 cells were treated with 0.05 mg/ml BriB, LG, ICG, FG, IfCG and CR for 3 minutes. Total proteins were extracted to assess Bax protein expression by Western blot. GAPDH was used as the internal control. Representative Western blot gel (Top) of proapoptotic protein Bax. The numbers to the left are molecular weights in kilodaltons (kDa). Bottom: average densitometry results from three independent experiments. Data are means± SE and represent the average results of 3 independent experiments run in duplicate. * is p<0.05 and ** is p<0.01 versus control.

Discussion

In recent years the use of vital dyes in vitrectomy, chromovitrectomy, has become the standard method to facilitate intraoperative surgical procedures such as ILM or ERM peeling. The two dyes available for chromovitrectomy, ICG and TB, may lead to complications in macular surgery [27-31]. BriB has been used as an alternative and safer dye, but even though they may migrate to the subretinal space and produce alterations in the RPE and campimetric and papillary defects. The ideal, non-toxic dye is yet to be determined. Therefore, in this study, a detailed in vitro toxicity investigation of nine dyes: ICG, IfCG, LG, FG, BriB, BroB, EB, IC and CR were done.

Five different dye concentrations (1, 0.5, 0.25, 0.05 and 0.005 mg/ml) and two exposure times (3 and 30 min) were selected to simulate the possible dye dilutions and injection techniques that can be performed by surgeons during chromovitrectomy [27].

ICG is a tricarbocyanine anionic vital dye with a molecular formula of C43H47N2NaO6S2 and mass of 775 daltons [32]. ICG adheres well to the extracellular matrix components of the ILM, such as collagen type 4, laminin, and fibronectin [4]. Wollensak et al showed, in a porcine model, that ICG with light exposure produces a significant increase in biomechanical stiffness, thereby facilitating ILM peeling [33]. Following the Kadonosono et al publication of ICG use macular hole surgery, many authors have reported easier and less traumatic ICG-guided peeling with good clinical results [34]. Controversial publications have been shown ICG related toxicity such as perimetric defects, vision loss, optic nerve atrophy and RPE lesions. The present study reinforces the toxic profile of ICG showing a reduction of RPE cell viability in all tested concentrations and time exposure. More recently some papers have been dedicated to reduce the ICG-related toxicity with free radical scarvengers (tampol), less concentration, short time exposure, or iodine-free solution such as IfCG [35] [36, 37].

IfCG also binds with a high affinity acellular membranes, such as ILM [38]. Differently from ICG, IfCG is a iodine-free solution and should be dissolved with 5% glucose solution generating an iso-osmotic solution (294-314 mOsm); both characteristics that makes IfCG a safer dye. Several experimental and clinical studies reported positive results with little retinal toxicity in concentrations lower than 0.05% [6, 37, 39, 40]. Indeed, the present study shows that IfCG had a safer profile when compared to ICG, but still reduced RPE cell viability in all tested concentrations and time exposure.

Bromophenol blue (BroB), also named tetrabro-mophenolsulfonephthalein, is a hydroxytriarylmethane color marker dye with a molecular weight of 670 daltons, and has been proposed as an alternative biostain for chromovitrectomy. An experimental in vitro study showed that BroB can stain ILM and did not induce RPE cell toxicity at concentrations of 0.2 and 0.02% [41, 42]. Moreover in vivo studies in rodent and porcine eyes demonstrated that BroB at concentrations of 0.5% and 0.02% promoted less toxicity when compared to LG, Chicago blue and E68 [42, 43]. Clinically BroB 0.2% was used in patients with ERM with good staining affinity and no side effects [44]. The present paper shows that in a wide range of concentrations and time exposure BroB may produce RPE toxicity. Similar effect was also observed in our previous in vivo study where BroB induced significant retina toxicity after intravitreal injections in rabbits [18]. Moreover, its clinical use in macular hole surgery should be look with caution.

Light green and FG are anionic amino triarylmethane with a molecular weight of 792 and 809 daltons respectively. Sorsby in 1939, first applied FG in vitreoretinal surgery for retinal breaks identification after intravenous injection [45]. In vitro and in vivo studies showed that FG did not induce remarkable retinal toxicity [18, 46]. In regard to LG, Haritoglou et al. found in their in vitro study, no toxic effect on ARPE-19 and primary RPE cell line [42]. Our group also showed that no remarkable histologic and functional retinal abnormalities was observed in a rabbit model with LG [18]. The present study showed that FG was toxic to ARPE-19 cell line, but in contrast LG did not induce reduction in cell viability in all concentrations after 3 minutes exposure. However after 30 minutes, both FG and LG were toxic in all tested concentrations.

Azo dyes constitute a group of dyes containing an azo chemical group linked to benzene, naphthalene, or aromatic heterocylic rings. EB and CR are part of this group with a molecular weight of 960 and 696 daltons. In ophthalmology, EB has been used as a dye for endothelial function evaluation, and animal studies have demonstrated that the dye is not toxic to corneal endothelial cells [47]. The toxicity and staining affinity of EB for retinal structures has been evaluated in cell culture, where EB induced slight retinal damage at 0.02%, and it showed favorable staining affinity for ILM [46]. In contrast, an in vivo investigation showed that EB caused severe functional and morphologic retinal toxicity at the higher dose of 0.5% [18].The current study showed that EB could reduce RPE cell viability in all experiments conducted. Based on these observations, EB at least at high doses should not be considered for human application in chromovitrectomy. CR on the other hand did not induce RPE cell toxicity in concentrations up to 0.05 mg/ml. Besides a safer profile, CR is not a good candidate for ILM staining due to it poor contrast with the red fundus background color.

Indigo carmine, with 466 daltons, is part of the thiazine dyes, which are small and cationic molecules containing a chromophore called thiazinium. Our group was the first to evaluate IC for vitreoretinal surgery, where it showed overall good safety about the retina [18]. However the present in vitro analysis showed that IC might be toxic to RPE cells in all tested concentrations and time exposure. Such findings may indicate that IC and possibly other thiazine dyes may be useful in chromovitrectomy, but staining affinity issues and avoid the contact to RPE cells should be considered.

Brilliant Blue G (BriB) is a synthetic triarylmethane, the same group of BroB, and has a molecular weight of 854 daltons. The safety profile of BriB was investigated by Enaida et al., and later evaluated by other groups in pre-clinical and clinical experiments [48]. BriB emerged as the first real safe alternative for ICG and IfCG in chromovitrectomy due to its remarkable affinity for the ILM, and no significant toxicity findings have been reported in uneventful retinal surgeries. The current study showed that BriB is safe to RPE cells up to 0.25 mg/ml after 3 minutes and up to 0.005 mg/ml after 30 minutes of exposure.

Based in our viability data and since it was shown that residual ICG can be found months after surgery, the evaluation of apoptosis in retinal toxicity studies of this dye has become an important issue [49]. Therefore, the present study also evaluated the link between dye exposure and apoptosis in RPE cells by studying Bax protein, which is involved in controlling apoptosis events [50]. The present study shows that ICG remarkably induced Bax expression after 3 minutes of exposure at 0.05 mg/ml, a common concentration still in use by some surgeons. This upregulation of Bax protein was also noticed after exposure to LG, FG, IfCG and CR (Figure 7). In contrast, BriB had Bax expression similar to control, BSS. Balaiya et al. also showed similar results, where ICG but not BriB induces apoptosis in retinal cells [37].

One limitation of this study is that the results of this paper should not be extrapolated to entire retina toxicity induced by the dye exposure. The present paper evaluated dye toxicity and induction apoptosis only in an immortalized RPE human cell line (ARPE-19). Besides this drawback, this design of study is necessary since direct dye-RPE contact may occur during macular hole surgery and it may induce RPE defects and vision loss.

In summary, the safety profiles of nine different dyes were compared. The safest dye with regard to RPE cells was BriB followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. Moreover ICG had the highest Bax expression, an indicative of apoptosis cascade activation. On the other hand BriB was the safest dye and the only that did not induce apoptosis in our experiments. Based in the literature and supported by our results, BriB seems to be the best alternative for ICG in vitreoretinal surgery. Indeed, it has been postulated that BriB could have a protective effect on retinal tissue, but it should be further investigated [51].

Acknowledgments

From the Sorocaba Eye Bank; Sorocaba, SP, Brazil

Financial support: Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), NIH Center Core Grant P30EY014801, Research to Prevent Blindness Unrestricted Grant, and Department of Defense (DOD- Grant#W81XWH-09-1-0675).

Footnotes

Presented as poster at 2011 American Academy of Ophthalmology Annual Meeting, Orlando, FL, USA

No conflicting relationship exists for any author

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