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Clinical and Experimental Ophthalmology 2015; ••: ••–•• doi: 10.1111/ceo.12568
Original Article Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G Saffar Mansoor PhD,1 Ashish Sharma MD,1,2 Javier Cáceres-del-Carpio MD,1 Leandro C Zacharias MD,1,3 A Jayaprakash Patil MS, FRCS,4 Navin Gupta MD,1 G Astrid Limb PhD,5 M Cristina Kenney MD, PhD1,5 and Baruch D Kuppermann MD, PhD1 1
Gavin Herbert Eye Institute, School of Medicine, University of California, Irvine, California, USA; 2Department of Ophthalmology, Lotus Eye Care Hospital, Coimbatore, TN, India; 3Serviço de Oftalmologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; 4Department of Ophthalmology, University Hospitals of Morecambe Bay NHS Foundation Trust, Kendal, LA9 5JE, and 5 Department of Paediatric Ophthalmology, University Hospitals of Morecambe Bay NHS Foundation Trust, Kendal LA9 5JE, UK
ABSTRACT Background: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. Method: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. Result: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their
respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. Conclusion: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells. Key words:
retina, retinal light toxicity, surgery.
INTRODUCTION The internal limiting membrane (ILM) is the innermost layer of the retina which forms a boundary between the vitreous and retina. The ILM is a basement membrane for the Müller glial cells, which are essential for maintenance of the retina’s normal structure and function. ILM removal may be necessary during surgeries for epiretinal membranes and macular holes1,2 but removal of ILM requires a delicate procedure due to its fragility and translucency. Therefore, selective staining of ILM may enhance the visualization and facilitate its removal. Trypan blue (TB) and indocyanine green (ICG) have been used for several years to stain the ILM.3–9
■ Correspondence: Dr Ashish Sharma, Lotus Eye Care Hospital, Coimbatore, TN 641014, India. Email: [email protected] Received 26 December 2014; accepted 22 April 2015. Conflict of interest: None. Funding sources: Supported by the Discovery Eye Foundation, Henry L. Guenther Foundation, The Iris and B. Gerald Cantor Foundation, The Skirball Molecular Ophthalmology Program, Poly and Michael Smith Foundation and Research to Prevent Blindness Foundation. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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TB appears to be safe for intraocular application but provides a far less pronounced staining and lacks the selective staining of ILM.10 In addition, TB toxicity has been reported in cultured retinal pigment epithelial (RPE) cells.11 ICG is a dye that stains ILM selectively,12 but several in vitro, ex vivo and in vivo animal investigations as well as clinical reports demonstrated intravitreal ICG-related toxicity. The reported ICG toxicities include direct biochemical injury to the ganglion cells/neuroretinal cells,13 RPE cells14 and superficial retinal vessels,15 apoptosis and gene expression alterations to either RPE cells or neuroretinal cells,16–18 osmolarity effects of ICG solution on the vitreoretinal interface,19 mechanical cleavage effects to the ILM/inner retina20 and lightinduced injury to the retinal cells.21 In 2006, in the search for a safer dye, Brilliant Blue G 250 (BBG, also known as acid blue 90 or Coomassie blue) was shown to stain the ILM well with no significant in vivo toxicity in animals.22,23 BBG is commonly used for protein staining and gel electrophoresis because it binds non-specifically to virtually all proteins,24–31 which makes it an excellent candidate dye for staining ILMs. Prior to the reports in 2006,22,23,32 ophthalmic uses of BBG had never been reported. However, since then BBG has been studied with respect to its selective staining of ILM as well as possible toxic effects on rats, primates and humans22,23 after intravitreal injection. BBG has demonstrated an efficient ILM staining which allows for its easy peeling. Moreover, there have been no reports of apoptosis in rat eyes22 or glial cells in vitro.33 All of the above safety data of BBG has encouraged vitreoretinal surgeons to use this dye for staining and peeling of the ILM in human patients.12,23,34–37 However, what still remains to be investigated is the effects of combined phototoxicity along with BBG-related toxicity. Although neither BBG nor ICG absorbs visible light, ICG is reported to cause light-induced toxicity to retinal cells.8,21,38–40 In the present study, we intend to analyse the effects of light on BBG-treated human retinal pigment epithelial cells (ARPE-19), rat neurosensory retinal cells (R28) and human Müller cells (MIO-M1). Our in vitro approach allows us to identify subtle, molecular changes caused by the BBG plus light combination that might be very easily missed in in vivo studies.
METHODS Cell culture Human retinal pigment epithelial cell line (ARPE19) was obtained from American type culture collection (ATCC; Manassas, VA). Cells were grown in 1:1 mixture (vol./vol.) of Dulbecco’s modified Eagle’s
and Ham’s nutrient mixture F-12 medium (DMEM F-12, Gibco, Carlsbad, CA), 10 mM non-essential amino acids, 0.37% sodium bicarbonate, 0.058% L-glutamine, 10% fetal bovine serum and antibiotics (100 U/mL penicillin G, 0.1 mg/mL streptomycin sulfate, 10 μg/mL gentamicin, and 2.5 μg/mL fungizone-Amphotericin B). Rat embryonal neurosensory precursor retinal (R28) cells were derived from day 6 post-natal rat retina from the laboratory of Dr Gail M. Seigel, Buffalo, NY.41 R28 cells express genes characteristic of neurons as well as functional neuronal properties.42 The cell line was cultured in Dulbecco’s modified Eagle’s medium, high glucose (DMEM high glucose, Gibco, Carlsbad, CA) with 10% fetal bovine serum, 1x minimum essential medium, 10 mM nonessential amino acids, 0.37% sodium bicarbonate and 10 μg/mL gentamicin.43,44 The human Müller cells line (MIO-M1) (courtesy of Dr G. Astrid Limb, Institute of Ophthalmology, University College London, UK) was grown in Dulbecco’s modified Eagle medium (DMEM) (1x) high glucose SKU#10569-044 (GlutaMAX-1 medium substituted on a molar equivalent basis for L-glutamine, 4500 mg/L D-glucose, 110 mg/L sodium pyruvate, 1x penicillin/streptomycin and 10% fetal bovine serum). ARPE-19, R28 and MIO-M1 cells were plated in 6-well and 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) for cell viability (5 × 105 cells/well), caspase-3/7 (1.2 × 105 cells/well) and mitochondrial potential (ΔΨm) (1.2 × 105 cells/ well) assays and were incubated at 37°C in 5% CO2 until monolayer confluence was achieved. Cells were incubated further in serum-free DMEM (SFDMEM) or 1.5% serum DMEM for 24 h to make them relatively non-proliferating. This was followed by exposure to BBG with or without light.
Exposure to BBG In this study, BBG powder (Brilliant Blue G; Sigma– Aldrich, St Louis, MO) was dissolved in balanced salt solution (BSS-Plus, Alcon Laboratories, Fort Worth, TX) to achieve 0.125 mg/mL, 0.25 mg/mL and 0.50 mg/mL solutions. The mean osmolarity (measured by Fiske 2400 Osmometer, Fiske Associates, Two Tech Way, Norwood, MA) and pH of all BBG and BSS-Plus solutions were determined over a six-month period as shown in Table 1. The proposed clinical concentration of BBG is 0.25 mg/mL, and this was assigned as 1x clinical dose. The concentrations used in our experiments were equivalent to 0.5x, 1x and 2x of the clinical concentration. ARPE19, R28 and MIO-M1 cells were treated with one of the three different concentrations of BBG for 10, 15 or 30 min.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells Table 1.
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Characterization of BBG solution
Solution BBG, 0.125 mg/mL BBG, 0.25 mg/mL BBG, 0.5 mg/mL BSS-Plus
Osmolarity (/KgH2O)
pH
304–305 303 301 303–305
7.41 7.42 7.41 7.20
BBG, Brilliant Blue G; BSS, balanced salt solution.
Exposure to halogen light BBG solutions were rinsed off the cells three times with culture medium and replaced with fresh culture medium. Thereafter, cells were exposed to a halogen light source (L) (Pilling Surgical, Model 529317, Dual Active 150 Halogen; Teleflex Medical, 2917 Weck Drive, Research Triangle Park, NC 27709). The intensity of the illumination was 3000 lux (378.7 foot candle), which was maintained by using a light meter (Light Meter Foot Candles, Sper Scientific, Model 840021, Taiwan) throughout exposure phase of the experiment. The durations of illumination were as follows: (i) the 10-min BBG cells were exposed for 10 min; (ii) the 15-min BBG cells were exposed for 15 min; and (iii) the 30-min BBG cells were exposed for 30 min. Cells illuminated with L alone (for 30 min) but without exposure to BBG solution served as a positive control. Cells receiving neither L nor BBG served as a negative control. All cells were then incubated an additional 24 h at 37°C with 5% CO2 balanced with air and analysed using different assays.
Cell viability assay Viable cells were determined using the trypan blue dye exclusion test. It is based on the principle that live cells possess intact cell membranes that exclude dyes, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye where a viable cell will have a clear cytoplasm and a non-viable cell will have a blue cytoplasm. Briefly, cells were harvested from the six-well plates after treatment with 0.2% trypsin-EDTA followed by their incubation at 37°C for 5 min. The cells were then centrifuged at 1000 rpm for 5 min and re-suspended in 1 mL of culture medium. Cell viability analyses were performed using a ViCell counter (Beckman-Coulter, Fullerton, CA). The machine automatically performs trypan blue dye exclusion assay and gives the percentage of viable cells.
Caspase-3/7 assay In order to identify apoptosis within the cultures, caspase-3/7 activity was detected using the carboxyfluorescein FLICA apoptosis detection kit
(Immunochemistry Technologies LLC, Bloomington, MN). The FLICA reagent has an optimal excitation range from 488 nm to 492 nm, and emission range from 515 nm to 535 nm. Caspase-3/7 activities were measured using the FMBIO III (549-6 Shinano-cho, Totsuka-ku, Yokohama 244-0801, Japan) instrument that measures apoptosis as the amount of green fluorescence emitted from FLICA probes bound to caspase-3/7. Non-apoptotic cells do not stained while cells undergoing apoptosis stain brightly. Caspase-3/7 assays were performed according to the supplier’s instructions. Briefly, after 24 h incubation, cells were rinsed with fresh culture medium (500 μl/well) and replaced with 300 μl FLICA solution (prepared by adding 1 μl of FLICA in each 300 μl culture media) and incubated for 30 min at 37°C. Thereafter, the FLICA solution in each well was washed three times with wash buffer (500 μl/ well) that was supplied with the kit. Finally, 300 μl of washing buffer was placed in each well. In addition to the experimental groups, the following control groups were included: (i) untreated cells without FLICA for background native cell autofluorescence; (ii) untreated cells with FLICA to determine native caspase activity in the cell groups; and (iii) culture media only wells. The scans were performed using a scanning unit instrument (FMBIO III, Hitachi, Yokohama, Japan).
Mitochondrial membrane potential (ΔΨm) assay Loss of the mitochondrial membrane potential (ΔΨm) is a hallmark for early apoptosis and can be measured using the JC-1 mitochondrial membrane potential detection kit (Biotium, Hayward, CA). JC-1 contains a cationic dye (5,5',6,6'-tetrachloro-1,1',3,3'tetraethyl-benzimidazolyl-carbocyanine-iodide) that fluoresces red in the mitochondria of living cells. In cells undergoing early apoptosis, the mitochondrial membrane potential collapses, but the cationic dye remains in the cytoplasm and fluoresces green. The ratio of red to green fluorescence is higher in healthy cells and comparatively lower in apoptotic cells. JC-1 assays were conducted as per the supplier’s instructions. Briefly, 24 h after incubation, the cells were rinsed with fresh media and incubated for 15 min with 500 μl per well of JC-1 reagent in culture media. Then JC-1 reagent in each well was washed three times with the buffer (500 μl/well) included in the kit. Finally, 300 μl of washing buffer was placed in each well. In addition to the experimental groups, three control groups were included as mention earlier in caspase-3/7 assay. Images were captured using a fluorescence image scanning unit instrument (FMBIO III) and red/green ratios were calculated.
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Statistical analysis Data were subjected to statistical analysis by analysis of variance (Prism version 3.0 statistics program; GraphPad Software, San Diego, CA). The Newman– Keuls multiple-comparison test was used to compare the data within each experiment. P < 0.05 was considered statistically significant. Error bars in the graphs represent the standard error of the mean.
RESULTS Cell viability assay None of the tested concentrations of BBG with or without exposure to light (2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, L) for 10 min, 15 min or 30 min caused a significant reduction in cell viabilities in the ARPE-19, R28 or MIO-M1 cultures (Fig. 1). The mean % cell viability of ARPE-19 cells after BBG treatment with or without exposure to light for 30 min were 91.5 ± 0.5, 92.1 ± 1.3, 92.5 ± 1.5, 92.7 ± 0.5, 93.5 ± 0.6, 94.0 ± 1, and 95.5 ± 1 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, and L, respectively, compared to 95.6 ± 1.1 in untreated ARPE-19 control cells (P > 0.05 for all treated concentrations, Fig. 1a). The mean % cell viability of R28 cells after BBG treatment with or without exposure to light for 30 min were 89 ± 0.8, 90.5 ± 0.5, 91 ± 1, 91.5 ± 0.5, 92.3 ± 0.7, 93.0 ± 1 and 93.2 ± 0.4 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 93.5 ± 0.5 in untreated R28 control cells (P > 0.05 for all treated concentrations, Fig. 1b). The mean % cell viability of MIO-M1 cells after BBG treatment with or without exposure to light for 30 min were 91.5 ± 0.5, 92.5 ± 1.3, 93.3 ± 1.3, 93.5 ± 0.5, 93.5 ± 0.5, 94.3 ± 0.6, 94.7 ± 1.1, and 95 ± 0.5 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 95.1 ± 0.5 in untreated MIO-M1 control cells (P > 0.05 for all treated concentrations, Fig. 1c).
Apoptosis assay Caspase-3/7 activities were not significantly increased after BBG treatment with or without exposure to light (2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, L) for 10 min, 15 min or 30 min in ARPE-19, R28 or MIO-M1 cells (Fig. 2). The fluorescent values for ARPE-19 cells after 24 h were 3377.5 ± 100 msi, 3350 ± 100 msi, 3307 ± 92.5 msi, 3225 ± 100 msi, 3215 ± 135 msi, 3185 ± 115 msi and 3160 ± 90 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 3165 ± 95 msi in untreated control ARPE-19 cells (P > 0.05 for all treated concentrations, Fig. 2a).
Similarly, the fluorescent values for R28 cells after 24 h were 1747 ± 17.5 msi, 1708.5 ± 41.5 msi, 1666.5 ± 38.5 msi, 1656 ± 56 msi, 1649 ± 41 msi, 1587 ± 32.5 msi and 1592.5 ± 37.5 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 1593.5 ± 31.5 msi for untreated R28 control cells (P > 0.05 for all treated concentrations, Fig. 2b). The fluorescent values for MIO-M1 cells after 24 h were 559 ± 16 msi, 557.5 ± 17.5 msi, 554 ± 14 msi, 535 ± 10 msi, 530 ± 15 msi, 520 ± 15 msi and 509 ± 21 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, and L, respectively, compared to 510 ± 10 msi for untreated MIO-M1 control cells (P > 0.05 for all treated concentrations, Fig. 2c).
ΔΨm assay ΔΨm in ARPE-19, R28 and MIO-M1 cells (following exposure to BBG with or without light for 30 min) was significantly reduced at 2x + L treatment and 2x treatment compared to untreated control cells (Fig. 3). At the lower BBG concentrations tested, with and without exposure to light (1x + L, 1x, 0.5x + L, 0.5x and L), the ΔΨm values were not significantly reduced. Importantly, the ΔΨm values were not reduced significantly at any concentrations of BBG ± 10 min or 15 min light exposure. This result shows that mitochondrial function may be impaired at 2x clinical BBG concentrations in the presence or absence of 30 min light exposure. The ΔΨm value in ARPE-19 cells for 2x BBG plus 30 min light treatment (2x + L) was 14.75 ± 0.25 (P < 0.05) and for BBG alone (2x) was 15.5 ± 0.5 (P < 0.05) as compared to 19.25 ± 0.75 for untreated ARPE-19 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 17.5 ± 0.5 (P > 0.05), 18.11 ± 0.7 (P > 0.05), 18.2 ± 0.7 (P > 0.05), 17.95 ± 0.9 (P > 0.05) and 18.25 ± 0.25 (P > 0.05), respectively, compared to 19.25 ± 0.8 for untreated ARPE-19 (Fig. 3a). In R28 cells, the ΔΨm values after 2x BBG plus 30 min light treatment (2x + L) was 3.75 ± 0.25 (P < 0.05) and 4.25 ± 0.75 (P < 0.05) for 2x BBG alone compared to 7.7 ± 0.7 for untreated R28 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 6.07 ± 0.12 (P > 0.05), 6.1 ± 0.4 (P > 0.05), 6.2 ± 0.6 (P > 0.05), 6.25 ± 0.25 (P > 0.05) and 6.35 ± 0.7 (P > 0.05), respectively, compared to 7.7 ± 0.7 for untreated R28 cells (Fig. 3b). The cultures exposed to light for 10 min and 15 min showed ΔΨm values similar to controls. In the MIO-M1 cells, the ΔΨm values for 2x BBG plus 30 min light treatments (2x + L) were 9.9 ± 0.5 (P < 0.01) and 11.2 ± 0.2 (P < 0.05) for 2x BBG alone as compared to 14.9 ± 0.6 for untreated MIO-M1 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 12.4 ± 0.5 (P > 0.05), 13.2 ± 0.2 (P > 0.05), 13.7 ± 0.2 (P > 0.05), 14.3 ± 0.5 (P > 0.05) and
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells ARPE-19 Cells Treated with BBG and BBG + Light
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Figure 1. Bar graph showing cell viability (CV) assay in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after 10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. The concentrations of BBG were 2x, 1x or 0.5x. There were no significant decreases in % CV in any of BBG-treated cells with or without exposure to light compared to untreated control (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate and the experiments repeated three times. Values are mean ± standard error mean (SEM).
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14.3 ± 0.5 (P > 0.05), respectively, compared to 14.9 ± 0.6 for untreated MIO-M1 cells (Fig. 3c). The ΔΨm values for the cultures exposed to light for 10 min and 15 min showed were similar to controls.
DISCUSSION All vitreoretinal surgeries require illumination, and some retinal surgeries require dyes as well. The dye
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is injected into the posterior chamber to stain selectively the ILM or the epiretinal membrane, and the dye is usually washed away within a few seconds. The stained membrane is then well visualized and can be removed with the aid of endoillumination. Even with immediate washing, some dye may remain in contact with the retinal cells and be retained for long durations. The potential toxicity may be compounded by phototoxicity from light
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Figure 2. Bar graph showing caspase-3/7 activities in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after 10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. The concentrations of BBG were 2x, 1x or 0.5x. There were no significant differences in caspase-3/7 activities in any of BBG-treated cultures with or without exposure to light compared to the untreated control (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate, and the experiments repeated three times. Values are mean ± standard error mean.
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exposure. The choice of different BBG concentrations used in our experiments is based on several recent clinical trials12,23,34–37 as well as BBG-related reports.33 Before treating cells, the osmolarity and pH of different concentrations of BBG solutions maintained up to six months were measured (Table 1). The BBG solution values for osmolarity and pH were similar to those of BSS plus, the intraocular irrigating solution used during vitrectomies. This is important because from the safety standpoint, the osmolarity of intravitreal dyes plays a key role in cell survival.45
Previous studies screening the osmolarity effects of different concentrations of BBG, ICG, TB and other vital dyes in animal and human eyes demonstrated that the highest safety profile was found for BBG.22,32 In our experiment, cells were exposed to varying concentrations of BBG followed by up to 30 min of light exposure. Even though this extended duration does not represent the light exposure time during a typical vitreoretinal surgery, it was chosen to approximate the extreme upper limit of surgical exposure in a complicated case.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells ARPE-19 Cells Treated with BBG and BBG + Light ∗
ΔΨm (Fluorescence Red/Green)
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Figure 3. Bar graph showing ratio of red:green fluorescence (mitochondrial membrane potential) in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. There were significant decreases in mitochondrial membrane potential in the cultures treated for 30 min with 2x BBG with or without light compared to control (**P < 0.01 and *P < 0.05, respectively for 2x + L, and 2x). However, there were no statistically significant changes in mitochondrial membrane potential in the cultures treated 30 min with 1x BBG or 0.5x BBG with or without light compared to untreated control. Also, there were no statistically significant changes in mitochondrial membrane potential for the cultures treated with 2x BBG, 1x BBG or 0.5x BBG with or without exposure to light for 10 or 15 min compared to untreated control cultures (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate and the experiments repeated three times. Values are mean ± standard error mean.
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Our study is the first report using three different retinal cell lines exposed to BBG for up to 30 min in presence or absence of light. Our findings showed that ARPE-19, R28 and MIO-M1 cells incubated with 0.25 mg/mL of BBG (clinical dose) and exposed to light for up to 30 min did not decrease cell viability, or cause apoptosis or mitochondrial damage. However, when cells were treated with 0.5 mg/mL of BBG (twice the clinical dose) with or without exposure
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to light for 30 min, mitochondrial damage was observed, though no decrease in cell viability or apoptosis were noted. In contrast to our results, Yuen et al.46 demonstrated significant reduction in ARPE-19 cell viability after exposure to BBG (0.25 mg/mL) for 30 min. However, we found no significant reduction in cell viability in RPE cells, MIO-M1 or R28 cells using the same concentration of BBG dye and same duration of treatment. This discrepancy
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may be due to different assays being used to measure cell viability. Yuen and co-workers used the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to examine the cell viability while in our work the trypan blue dye exclusion assay was used. The MMT method is a colorimetric assay that measures enzyme activities needed to reduce MTT to a formazan dye which can then be measured. Changes in metabolic activity can change the MMT levels without necessarily decreasing the numbers of viable cells. The trypan blue assay uses a vital dye that is normally excluded from the cells but when the cell membrane is damaged, then the dye enters into the cells and can be measured. Therefore while both assays can provide information about the health of the cells, the trypan blue assay represents cells that are more damaged, and this may account for the disparity of our findings. In another study, Morales et al.47 reported significant loss of cell viability, altered cell membrane permeability (CMP) and mitochondrial membrane potential in ARPE-19 cells after treatment with two different concentrations of BBG (0.5 mg/mL and 0.05 mg/mL) plus 3 min light exposure. The discrepancy with our results may be based upon differences in dye + light treatment protocols. They exposed cells to the surgical intensity light in the presence of the BBG dye (0.5 mg/mL and 0.05 mg/ mL) and then used the MTT assay for the assessment of cell viability. In our work, we exposed cells to the dye at different times, removed the dye, washed the cells three times with culture media and then exposed the cells for up to 30 min with light of surgical intensity. We then used the trypan blue dye exclusion assay to show that the BBG did not decrease the cell viability. However, comparing our results to the Morales findings suggests that light exposure in the presence of BBG dye greatly increases the toxicity to cells which emphasizes the importance of removing the dye prior to using the light during the retinal surgeries. Our safety findings for BBG are supported by some cell culture studies. Kawahara et al.33 demonstrated that Müller cells in vitro did not show apoptosis after being treated with BBG (0.25 mg/mL) for 15 min, while treatment with ICG dye for the same duration did induce apoptosis in cells. Awad et al.48 reported the lower toxicity in ARPE-19 cells following exposure of polyethylene glycol with BBG (0.025%), TB (0.15%), a mixture of BBG (0.025%) with TB (0.15% and 0.25%) and BBG (0.025% in phosphate-buffered saline). Two ex vivo studies have also shown more favourable effects of BBG on isolated perfused bovine retina as compared to ICG or TB.42,49 Several clinical studies have reported BBG as a safe alternative and potential dye for vitreoretinal surgery. In 2006, Enaida et al.22,23,32 introduced BBG as an
intraocular vital stain after a comparative study of various dyes for their safety and ability to stain membranes in pigs, rats, monkey and human eyes. It was observed that BBG effectively stained the anterior capsule in pig eyes without showing any toxic effect to the cornea.32 Also BBG was found to be safe to the posterior segment of rats and primates eyes in preclinical studies.22 Additional safety studies of BBG were conducted on 78 Brown Norway rats. The animals were injected with BBG following vitrectomy, and the eyes were enucleated after two weeks to two months. There were no abnormalities in pathology, apoptosis or reduction in the electroretinograms waveform amplitudes in the eyes.22 Using the same protocol, two cynomolgus monkeys were intravitreally injected with BBG and underwent vitrectomy.22 The ILMs were adequately stained by BBG, visualized and taken out easily. There were no clinical issues or angiographic toxicities related to BBG after two weeks to six months postoperatively. Following the various preclinical investigations, interventional non-comparative, prospective clinical series were conducted to investigate the use of BBG for ILM staining during macular hole and epiretinal membrane surgeries in human subjects.23 Twenty eyes from 20 consecutive patients with macular holes or epiretinal membrane (five men, five women for each pathology) initially underwent vitrectomy, and then a 0.5-mL solution containing 0.25 mg/mL of BBG was injected into the vitreous cavity, and immediately washed out. The BBG sufficiently stained the ILM similar to ICG making easy and complete removal of ILM. All specimens in this investigation showed the presence of the ILM under transmission electron microscope. BBG did not induce any clinical or toxic signs to the eyes after two weeks of postoperative period.23 Subsequently, by establishing the safety profile of BBG in humans, it has opened the door for ophthalmologists to use this dye in surgeries for ILM,34–37 macular holes12 and capsulorhexis,50 without generating toxic effects. BBG is now available in the European Union market as ILM Blue (manufactured by DORC Zuidland, the Netherlands) in prefilled syringes, ready-to-use for intraocular applications. ILM Blue is expected to be approved soon in the United States.51 In the present study, exposure of ARPE-19, R28 or MIO-M1 cells in culture to clinically utilized concentrations of BBG dye (0.25 mg/mL) plus light treatments for up to 30 min did not decrease cell viability, cause apoptosis or generate mitochondrial damage. However, when twice the clinical dose (0.5 mg/mL) with or without light was utilized for 30 min, mitochondrial damage was observed, though no decrease in cell viability or increased in apoptosis were noted. In our experience, we have often found
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells that in vitro assays are far more sensitive in detecting cellular toxicities, which can often be missed in in vivo studies. Hence, our study is a much stronger evidence of safety of BBG at a more fundamental cellular and molecular level.
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REFERENCES 1. Brooks HL Jr. Macular hole surgery with and without internal limiting membrane peeling. Ophthalmology 2000; 107: 1939–48. 2. Kuhn F. Point: to peel or not to peel, that is the question. Ophthalmology 2002; 109: 9–11. 3. Burk SE, Da Mata AP, Snyder ME, Rosa RH Jr, Foster RE. Indocyanine green-assisted peeling of the retinal internal limiting membrane. Ophthalmology 2000; 107: 2010–4. 4. Da Mata AP, Burk SE, Riemann CD et al. Indocyanine green-assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for macular hole repair. Ophthalmology 2001; 108: 1187–92. 5. Kadonosono K, Itoh N, Uchio E, Nakamura S, Ohno S. Staining of internal limiting membrane in macular hole surgery. Arch Ophthalmol 2000; 118: 1116–8. 6. Da Mata AP, Burk SE, Foster RE et al. Long-term follow-up of indocyanine green-assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for idiopathic macular hole repair. Ophthalmology 2004; 111: 2246–53. 7. Li K, Wong D, Hiscott P, Stanga P, Groenewald C, McGalliard J. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003; 87: 216–9. 8. Jackson TL, Hillenkamp J, Knight BC et al. Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures. Invest Ophthalmol Vis Sci 2004; 45: 2778–85. 9. Gale JS, Proulx AA, Gonder JR, Mao AJ, Hutnik CM. Comparison of the in vitro toxicity of indocyanine green to that of trypan blue in human retinal pigment epithelium cell cultures. Am J Ophthalmol 2004; 138: 64–9. 10. Lesnik Oberstein SY, Mura M, Tan SH, de Smet MD. Heavy trypan blue staining of epiretinal membranes: an alternative to infracyanine green. Br J Ophthalmol 2007; 91: 955–7. 11. Rezai KA, Farrokh-Siar L, Gasyna EM, Ernest JT. Trypan blue induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 2004; 138: 492–5. 12. Remy M, Thaler S, Schumann RG et al. An in vivo evaluation of Brilliant Blue G in animals and humans. Br J Ophthalmol 2008; 92: 1142–7. 13. Nakamura H, Hayakawa K, Imaizumi A, Sakai M, Sawaguchi S. Persistence of retinal indocyanine green dye following vitreous surgery. Ophthalmic Surg Lasers Imaging 2005; 36: 37–45. 14. Sippy BD, Engelbrecht NE, Hubbard GB et al. Indocyanine green effect on cultured human retinal
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
pigment epithelial cells: implication for macular hole surgery. Am J Ophthalmol 2001; 132: 433–5. Kwok AK, Leung DY, Hon C, Lam DS. Vision threatening vitreous haemorrhage after internal limiting membrane peeling in macular surgeries. Br J Ophthalmol 2002; 86: 1449–50. Friedlander RM. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med 2003; 348: 1365–75. Rezai KA, Farrokh-Siar L, Ernest JT, van Seventer GA. Indocyanine green induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 2004; 137: 931–3. Doonan F, Cotter TG. Apoptosis: a potential therapeutic target for retinal degenerations. Curr Neurovasc Res 2004; 1: 41–53. Ho JD, Tsai RJ, Chen SN, Chen HC. Toxic effect of indocyanine green on retinal pigment epithelium related to osmotic effects of the solvent. Am J Ophthalmol 2003; 135: 258. Wollensak G, Spoerl E, Wirbelauer C, Pham DT. Influence of indocyanine green staining on the biomechanical strength of porcine internal limiting membrane. Ophthalmologica 2004; 218: 278–82. Engel E, Schraml R, Maisch T et al. Light-induced decomposition of indocyanine green. Invest Ophthalmol Vis Sci 2008; 49: 1777–83. Enaida H, Hisatomi T, Goto Y et al. Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G. Retina 2006; 26: 623–30. Enaida H, Hisatomi T, Hata Y et al. Brilliant blue G selectively stains the internal limiting membrane/ brilliant blue G-assisted membrane peeling. Retina 2006; 26: 631–6. Congdon RW, Muth GW, Splittgerber AG. The binding interaction of Coomassie blue with proteins. Anal Biochem 1993; 213: 407–13. Laughton C. Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein. Anal Biochem 1984; 140: 417–23. Westermeier R. Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels. Proteomics 2006; 6 (Suppl. 2): 61–4. Said-Fernandez S, Gonzalez-Garza MT, Mata-Cardenas BD, Navarro-Marmolejo L. A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250. Anal Biochem 1990; 191: 119–26. Burgess-Cassler A, Johansen JJ, Santek DA, Ide JR, Kendrick NC. Computerized quantitative analysis of Coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis. Clin Chem 1989; 35: 2297–304. Saoji AM, Kulkarni M, Naghi T. Coomassie brilliant blue R-250 as a highly sensitive pre-stain for immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis and raising
10
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Mansoor et al. monospecific antibodies. Indian J Pathol Microbiol 1989; 32: 286–90. Oshima I, Kohama T, Kodaira T. A sensitive assay for serum acid soluble proteins with Coomassie brilliant blue G 250 and its application for autoimmune diseases. Tokushima J Exp Med 1987; 34: 23–7. Bergqvist Y, Karlsson L, Fohlin L. Total protein determined in human breast milk by use of Coomassie brilliant blue and centrifugal analysis. Clin Chem 1989; 35: 2127–9. Hisatomi T, Enaida H, Matsumoto H et al. Staining ability and biocompatibility of brilliant blue G: preclinical study of brilliant blue G as an adjunct for capsular staining. Arch Ophthalmol 2006; 124: 514–9. Kawahara S, Hata Y, Miura M et al. Intracellular events in retinal glial cells exposed to ICG and BBG. Invest Ophthalmol Vis Sci 2007; 48: 4426–32. Schumann RG, Remy M, Grueterich M, Gandorfer A, Haritoglou C. How it appears: electron microscopic evaluation of internal limiting membrane specimens obtained during brilliant blue G assisted macular hole surgery. Br J Ophthalmol 2008; 92: 330–1. Cervera E, Diaz-Llopis M, Salom D, Udaondo P. High dose intravitreal brilliant blue G. Arch Soc Esp Oftalmol 2007; 82: 473. Cervera E, Diaz-Llopis M, Salom D, Udaondo P, Amselem L. Internal limiting membrane staining using intravitreal brilliant blue G: good help for vitreo-retinal surgeon in training]. Arch Soc Esp Oftalmol 2007; 82: 71–2. Mochizuki Y, Enaida H, Hisatomi T et al. The internal limiting membrane peeling with brilliant blue G staining for retinal detachment due to macular hole in high myopia. Br J Ophthalmol 2008; 92: 1009. Yam HF, Kwok AK, Chan KP et al. Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2003; 44: 370–7. Wu WCHD, Roberts JE. Phototoxicity of indocyanine green on human retinal pigment epithelium in vitro and its reduction by lutein. Photochem Photobiol 2004; 81: 537–40. Narayanan R, Kenney MC, Kamjoo S et al. Toxicity of indocyanine green (ICG) in combination with light
41.
42.
43.
44.
45.
46.
47.
48.
49. 50.
51.
on retinal pigment epithelial cells and neurosensory retinal cells. Curr Eye Res 2005; 30: 471–8. Seigel GM. Establishment of an E1A-immortalized retinal cell culture. In Vitro Cell Dev Biol Anim 1996; 322: 66–8. Wei S, Seigel GM, Salvi RJ. Retinal precursor cells express functional ionotropic glutamate and GABA receptors. Neuro Report 2002; 13: 2421–4. Patil AJ, Gramajo AL, Sharma A, Seigel GM, Kuppermann BD, Kenney CM. Differential effect of nicotine on retinal and vascular cells in vitro. Toxicol 2009; 259: 69–76. Sharma A, Patil JA, Gramajo AL, Seigel GM, Kuppermann BD, Kenney CM. Effect of hydroquinone on retinal and vascular cells in vitro. Indian J Ophthalmol 2012; 60: 189–93. Narayanan R, Kenney MC, Kamjoo S et al. Trypan blue: effect on retinal pigment epithelial and neurosensory retinal cells. Invest Ophthalmol Vis Sci 2005; 46: 304–9. Yuen D, Gonder J, Proulx A, Liu H, Hutnik C. Comparison of the in vitro safety of intraocular dyes using two retinal cell lines: a focus on brilliant blue G and indocyanine green. Am J Ophthalmol 2009; 147: 251–9. Morales MC, Freire V, Asumendi A et al. Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2010; 51: 6018– 29. Awad D, Schrader I, Bartok M, Mohr A, Gabel D. Comparative toxicology of trypan blue, brilliant blue G, and their combination together with polyethylene glycol on human pigment epithelial cells. Invest Ophthalmol Vis Sci 2011; 52: 4085–90. Luke C, Luke M, Dietlein TS et al. Retinal tolerance to dyes. Br J Ophthalmol 2005; 89: 1188–91. Udaondo P, Diaz-Llopis M, Salom D, Garcia-Delpech S, Cervera E. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch Soc Esp Oftalmol 2007; 82: 471–2. Kagimoto HTS, Hisatomi T, Enaida H, Ishibashi T. Brilliant blue G for ILM staining and peeling. Retina Today 2011; 45–8. Accessed July 2013. Available from: http://retinatoday.com/2011/04/global-perspectives -brilliant-blue-g-for-ilm-staining-and-peeling/
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Clinical and Experimental Ophthalmology 2015; ••: ••–•• doi: 10.1111/ceo.12568
Original Article Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G Saffar Mansoor PhD,1 Ashish Sharma MD,1,2 Javier Cáceres-del-Carpio MD,1 Leandro C Zacharias MD,1,3 A Jayaprakash Patil MS, FRCS,4 Navin Gupta MD,1 G Astrid Limb PhD,5 M Cristina Kenney MD, PhD1,5 and Baruch D Kuppermann MD, PhD1 1
Gavin Herbert Eye Institute, School of Medicine, University of California, Irvine, California, USA; 2Department of Ophthalmology, Lotus Eye Care Hospital, Coimbatore, TN, India; 3Serviço de Oftalmologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; 4Department of Ophthalmology, University Hospitals of Morecambe Bay NHS Foundation Trust, Kendal, LA9 5JE, and 5 Department of Paediatric Ophthalmology, University Hospitals of Morecambe Bay NHS Foundation Trust, Kendal LA9 5JE, UK
ABSTRACT Background: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. Method: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. Result: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their
respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. Conclusion: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells. Key words:
retina, retinal light toxicity, surgery.
INTRODUCTION The internal limiting membrane (ILM) is the innermost layer of the retina which forms a boundary between the vitreous and retina. The ILM is a basement membrane for the Müller glial cells, which are essential for maintenance of the retina’s normal structure and function. ILM removal may be necessary during surgeries for epiretinal membranes and macular holes1,2 but removal of ILM requires a delicate procedure due to its fragility and translucency. Therefore, selective staining of ILM may enhance the visualization and facilitate its removal. Trypan blue (TB) and indocyanine green (ICG) have been used for several years to stain the ILM.3–9
■ Correspondence: Dr Ashish Sharma, Lotus Eye Care Hospital, Coimbatore, TN 641014, India. Email: [email protected] Received 26 December 2014; accepted 22 April 2015. Conflict of interest: None. Funding sources: Supported by the Discovery Eye Foundation, Henry L. Guenther Foundation, The Iris and B. Gerald Cantor Foundation, The Skirball Molecular Ophthalmology Program, Poly and Michael Smith Foundation and Research to Prevent Blindness Foundation. © 2015 Royal Australian and New Zealand College of Ophthalmologists
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TB appears to be safe for intraocular application but provides a far less pronounced staining and lacks the selective staining of ILM.10 In addition, TB toxicity has been reported in cultured retinal pigment epithelial (RPE) cells.11 ICG is a dye that stains ILM selectively,12 but several in vitro, ex vivo and in vivo animal investigations as well as clinical reports demonstrated intravitreal ICG-related toxicity. The reported ICG toxicities include direct biochemical injury to the ganglion cells/neuroretinal cells,13 RPE cells14 and superficial retinal vessels,15 apoptosis and gene expression alterations to either RPE cells or neuroretinal cells,16–18 osmolarity effects of ICG solution on the vitreoretinal interface,19 mechanical cleavage effects to the ILM/inner retina20 and lightinduced injury to the retinal cells.21 In 2006, in the search for a safer dye, Brilliant Blue G 250 (BBG, also known as acid blue 90 or Coomassie blue) was shown to stain the ILM well with no significant in vivo toxicity in animals.22,23 BBG is commonly used for protein staining and gel electrophoresis because it binds non-specifically to virtually all proteins,24–31 which makes it an excellent candidate dye for staining ILMs. Prior to the reports in 2006,22,23,32 ophthalmic uses of BBG had never been reported. However, since then BBG has been studied with respect to its selective staining of ILM as well as possible toxic effects on rats, primates and humans22,23 after intravitreal injection. BBG has demonstrated an efficient ILM staining which allows for its easy peeling. Moreover, there have been no reports of apoptosis in rat eyes22 or glial cells in vitro.33 All of the above safety data of BBG has encouraged vitreoretinal surgeons to use this dye for staining and peeling of the ILM in human patients.12,23,34–37 However, what still remains to be investigated is the effects of combined phototoxicity along with BBG-related toxicity. Although neither BBG nor ICG absorbs visible light, ICG is reported to cause light-induced toxicity to retinal cells.8,21,38–40 In the present study, we intend to analyse the effects of light on BBG-treated human retinal pigment epithelial cells (ARPE-19), rat neurosensory retinal cells (R28) and human Müller cells (MIO-M1). Our in vitro approach allows us to identify subtle, molecular changes caused by the BBG plus light combination that might be very easily missed in in vivo studies.
METHODS Cell culture Human retinal pigment epithelial cell line (ARPE19) was obtained from American type culture collection (ATCC; Manassas, VA). Cells were grown in 1:1 mixture (vol./vol.) of Dulbecco’s modified Eagle’s
and Ham’s nutrient mixture F-12 medium (DMEM F-12, Gibco, Carlsbad, CA), 10 mM non-essential amino acids, 0.37% sodium bicarbonate, 0.058% L-glutamine, 10% fetal bovine serum and antibiotics (100 U/mL penicillin G, 0.1 mg/mL streptomycin sulfate, 10 μg/mL gentamicin, and 2.5 μg/mL fungizone-Amphotericin B). Rat embryonal neurosensory precursor retinal (R28) cells were derived from day 6 post-natal rat retina from the laboratory of Dr Gail M. Seigel, Buffalo, NY.41 R28 cells express genes characteristic of neurons as well as functional neuronal properties.42 The cell line was cultured in Dulbecco’s modified Eagle’s medium, high glucose (DMEM high glucose, Gibco, Carlsbad, CA) with 10% fetal bovine serum, 1x minimum essential medium, 10 mM nonessential amino acids, 0.37% sodium bicarbonate and 10 μg/mL gentamicin.43,44 The human Müller cells line (MIO-M1) (courtesy of Dr G. Astrid Limb, Institute of Ophthalmology, University College London, UK) was grown in Dulbecco’s modified Eagle medium (DMEM) (1x) high glucose SKU#10569-044 (GlutaMAX-1 medium substituted on a molar equivalent basis for L-glutamine, 4500 mg/L D-glucose, 110 mg/L sodium pyruvate, 1x penicillin/streptomycin and 10% fetal bovine serum). ARPE-19, R28 and MIO-M1 cells were plated in 6-well and 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) for cell viability (5 × 105 cells/well), caspase-3/7 (1.2 × 105 cells/well) and mitochondrial potential (ΔΨm) (1.2 × 105 cells/ well) assays and were incubated at 37°C in 5% CO2 until monolayer confluence was achieved. Cells were incubated further in serum-free DMEM (SFDMEM) or 1.5% serum DMEM for 24 h to make them relatively non-proliferating. This was followed by exposure to BBG with or without light.
Exposure to BBG In this study, BBG powder (Brilliant Blue G; Sigma– Aldrich, St Louis, MO) was dissolved in balanced salt solution (BSS-Plus, Alcon Laboratories, Fort Worth, TX) to achieve 0.125 mg/mL, 0.25 mg/mL and 0.50 mg/mL solutions. The mean osmolarity (measured by Fiske 2400 Osmometer, Fiske Associates, Two Tech Way, Norwood, MA) and pH of all BBG and BSS-Plus solutions were determined over a six-month period as shown in Table 1. The proposed clinical concentration of BBG is 0.25 mg/mL, and this was assigned as 1x clinical dose. The concentrations used in our experiments were equivalent to 0.5x, 1x and 2x of the clinical concentration. ARPE19, R28 and MIO-M1 cells were treated with one of the three different concentrations of BBG for 10, 15 or 30 min.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells Table 1.
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Characterization of BBG solution
Solution BBG, 0.125 mg/mL BBG, 0.25 mg/mL BBG, 0.5 mg/mL BSS-Plus
Osmolarity (/KgH2O)
pH
304–305 303 301 303–305
7.41 7.42 7.41 7.20
BBG, Brilliant Blue G; BSS, balanced salt solution.
Exposure to halogen light BBG solutions were rinsed off the cells three times with culture medium and replaced with fresh culture medium. Thereafter, cells were exposed to a halogen light source (L) (Pilling Surgical, Model 529317, Dual Active 150 Halogen; Teleflex Medical, 2917 Weck Drive, Research Triangle Park, NC 27709). The intensity of the illumination was 3000 lux (378.7 foot candle), which was maintained by using a light meter (Light Meter Foot Candles, Sper Scientific, Model 840021, Taiwan) throughout exposure phase of the experiment. The durations of illumination were as follows: (i) the 10-min BBG cells were exposed for 10 min; (ii) the 15-min BBG cells were exposed for 15 min; and (iii) the 30-min BBG cells were exposed for 30 min. Cells illuminated with L alone (for 30 min) but without exposure to BBG solution served as a positive control. Cells receiving neither L nor BBG served as a negative control. All cells were then incubated an additional 24 h at 37°C with 5% CO2 balanced with air and analysed using different assays.
Cell viability assay Viable cells were determined using the trypan blue dye exclusion test. It is based on the principle that live cells possess intact cell membranes that exclude dyes, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye where a viable cell will have a clear cytoplasm and a non-viable cell will have a blue cytoplasm. Briefly, cells were harvested from the six-well plates after treatment with 0.2% trypsin-EDTA followed by their incubation at 37°C for 5 min. The cells were then centrifuged at 1000 rpm for 5 min and re-suspended in 1 mL of culture medium. Cell viability analyses were performed using a ViCell counter (Beckman-Coulter, Fullerton, CA). The machine automatically performs trypan blue dye exclusion assay and gives the percentage of viable cells.
Caspase-3/7 assay In order to identify apoptosis within the cultures, caspase-3/7 activity was detected using the carboxyfluorescein FLICA apoptosis detection kit
(Immunochemistry Technologies LLC, Bloomington, MN). The FLICA reagent has an optimal excitation range from 488 nm to 492 nm, and emission range from 515 nm to 535 nm. Caspase-3/7 activities were measured using the FMBIO III (549-6 Shinano-cho, Totsuka-ku, Yokohama 244-0801, Japan) instrument that measures apoptosis as the amount of green fluorescence emitted from FLICA probes bound to caspase-3/7. Non-apoptotic cells do not stained while cells undergoing apoptosis stain brightly. Caspase-3/7 assays were performed according to the supplier’s instructions. Briefly, after 24 h incubation, cells were rinsed with fresh culture medium (500 μl/well) and replaced with 300 μl FLICA solution (prepared by adding 1 μl of FLICA in each 300 μl culture media) and incubated for 30 min at 37°C. Thereafter, the FLICA solution in each well was washed three times with wash buffer (500 μl/ well) that was supplied with the kit. Finally, 300 μl of washing buffer was placed in each well. In addition to the experimental groups, the following control groups were included: (i) untreated cells without FLICA for background native cell autofluorescence; (ii) untreated cells with FLICA to determine native caspase activity in the cell groups; and (iii) culture media only wells. The scans were performed using a scanning unit instrument (FMBIO III, Hitachi, Yokohama, Japan).
Mitochondrial membrane potential (ΔΨm) assay Loss of the mitochondrial membrane potential (ΔΨm) is a hallmark for early apoptosis and can be measured using the JC-1 mitochondrial membrane potential detection kit (Biotium, Hayward, CA). JC-1 contains a cationic dye (5,5',6,6'-tetrachloro-1,1',3,3'tetraethyl-benzimidazolyl-carbocyanine-iodide) that fluoresces red in the mitochondria of living cells. In cells undergoing early apoptosis, the mitochondrial membrane potential collapses, but the cationic dye remains in the cytoplasm and fluoresces green. The ratio of red to green fluorescence is higher in healthy cells and comparatively lower in apoptotic cells. JC-1 assays were conducted as per the supplier’s instructions. Briefly, 24 h after incubation, the cells were rinsed with fresh media and incubated for 15 min with 500 μl per well of JC-1 reagent in culture media. Then JC-1 reagent in each well was washed three times with the buffer (500 μl/well) included in the kit. Finally, 300 μl of washing buffer was placed in each well. In addition to the experimental groups, three control groups were included as mention earlier in caspase-3/7 assay. Images were captured using a fluorescence image scanning unit instrument (FMBIO III) and red/green ratios were calculated.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
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Statistical analysis Data were subjected to statistical analysis by analysis of variance (Prism version 3.0 statistics program; GraphPad Software, San Diego, CA). The Newman– Keuls multiple-comparison test was used to compare the data within each experiment. P < 0.05 was considered statistically significant. Error bars in the graphs represent the standard error of the mean.
RESULTS Cell viability assay None of the tested concentrations of BBG with or without exposure to light (2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, L) for 10 min, 15 min or 30 min caused a significant reduction in cell viabilities in the ARPE-19, R28 or MIO-M1 cultures (Fig. 1). The mean % cell viability of ARPE-19 cells after BBG treatment with or without exposure to light for 30 min were 91.5 ± 0.5, 92.1 ± 1.3, 92.5 ± 1.5, 92.7 ± 0.5, 93.5 ± 0.6, 94.0 ± 1, and 95.5 ± 1 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, and L, respectively, compared to 95.6 ± 1.1 in untreated ARPE-19 control cells (P > 0.05 for all treated concentrations, Fig. 1a). The mean % cell viability of R28 cells after BBG treatment with or without exposure to light for 30 min were 89 ± 0.8, 90.5 ± 0.5, 91 ± 1, 91.5 ± 0.5, 92.3 ± 0.7, 93.0 ± 1 and 93.2 ± 0.4 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 93.5 ± 0.5 in untreated R28 control cells (P > 0.05 for all treated concentrations, Fig. 1b). The mean % cell viability of MIO-M1 cells after BBG treatment with or without exposure to light for 30 min were 91.5 ± 0.5, 92.5 ± 1.3, 93.3 ± 1.3, 93.5 ± 0.5, 93.5 ± 0.5, 94.3 ± 0.6, 94.7 ± 1.1, and 95 ± 0.5 for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 95.1 ± 0.5 in untreated MIO-M1 control cells (P > 0.05 for all treated concentrations, Fig. 1c).
Apoptosis assay Caspase-3/7 activities were not significantly increased after BBG treatment with or without exposure to light (2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, L) for 10 min, 15 min or 30 min in ARPE-19, R28 or MIO-M1 cells (Fig. 2). The fluorescent values for ARPE-19 cells after 24 h were 3377.5 ± 100 msi, 3350 ± 100 msi, 3307 ± 92.5 msi, 3225 ± 100 msi, 3215 ± 135 msi, 3185 ± 115 msi and 3160 ± 90 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 3165 ± 95 msi in untreated control ARPE-19 cells (P > 0.05 for all treated concentrations, Fig. 2a).
Similarly, the fluorescent values for R28 cells after 24 h were 1747 ± 17.5 msi, 1708.5 ± 41.5 msi, 1666.5 ± 38.5 msi, 1656 ± 56 msi, 1649 ± 41 msi, 1587 ± 32.5 msi and 1592.5 ± 37.5 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x and L, respectively, compared to 1593.5 ± 31.5 msi for untreated R28 control cells (P > 0.05 for all treated concentrations, Fig. 2b). The fluorescent values for MIO-M1 cells after 24 h were 559 ± 16 msi, 557.5 ± 17.5 msi, 554 ± 14 msi, 535 ± 10 msi, 530 ± 15 msi, 520 ± 15 msi and 509 ± 21 msi for 2x + L, 2x, 1x + L, 1x, 0.5x + L, 0.5x, and L, respectively, compared to 510 ± 10 msi for untreated MIO-M1 control cells (P > 0.05 for all treated concentrations, Fig. 2c).
ΔΨm assay ΔΨm in ARPE-19, R28 and MIO-M1 cells (following exposure to BBG with or without light for 30 min) was significantly reduced at 2x + L treatment and 2x treatment compared to untreated control cells (Fig. 3). At the lower BBG concentrations tested, with and without exposure to light (1x + L, 1x, 0.5x + L, 0.5x and L), the ΔΨm values were not significantly reduced. Importantly, the ΔΨm values were not reduced significantly at any concentrations of BBG ± 10 min or 15 min light exposure. This result shows that mitochondrial function may be impaired at 2x clinical BBG concentrations in the presence or absence of 30 min light exposure. The ΔΨm value in ARPE-19 cells for 2x BBG plus 30 min light treatment (2x + L) was 14.75 ± 0.25 (P < 0.05) and for BBG alone (2x) was 15.5 ± 0.5 (P < 0.05) as compared to 19.25 ± 0.75 for untreated ARPE-19 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 17.5 ± 0.5 (P > 0.05), 18.11 ± 0.7 (P > 0.05), 18.2 ± 0.7 (P > 0.05), 17.95 ± 0.9 (P > 0.05) and 18.25 ± 0.25 (P > 0.05), respectively, compared to 19.25 ± 0.8 for untreated ARPE-19 (Fig. 3a). In R28 cells, the ΔΨm values after 2x BBG plus 30 min light treatment (2x + L) was 3.75 ± 0.25 (P < 0.05) and 4.25 ± 0.75 (P < 0.05) for 2x BBG alone compared to 7.7 ± 0.7 for untreated R28 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 6.07 ± 0.12 (P > 0.05), 6.1 ± 0.4 (P > 0.05), 6.2 ± 0.6 (P > 0.05), 6.25 ± 0.25 (P > 0.05) and 6.35 ± 0.7 (P > 0.05), respectively, compared to 7.7 ± 0.7 for untreated R28 cells (Fig. 3b). The cultures exposed to light for 10 min and 15 min showed ΔΨm values similar to controls. In the MIO-M1 cells, the ΔΨm values for 2x BBG plus 30 min light treatments (2x + L) were 9.9 ± 0.5 (P < 0.01) and 11.2 ± 0.2 (P < 0.05) for 2x BBG alone as compared to 14.9 ± 0.6 for untreated MIO-M1 cells. The ΔΨm values for 1x + L, 1x, 0.5x + L, 0.5x and L were 12.4 ± 0.5 (P > 0.05), 13.2 ± 0.2 (P > 0.05), 13.7 ± 0.2 (P > 0.05), 14.3 ± 0.5 (P > 0.05) and
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells ARPE-19 Cells Treated with BBG and BBG + Light
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Figure 1. Bar graph showing cell viability (CV) assay in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after 10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. The concentrations of BBG were 2x, 1x or 0.5x. There were no significant decreases in % CV in any of BBG-treated cells with or without exposure to light compared to untreated control (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate and the experiments repeated three times. Values are mean ± standard error mean (SEM).
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14.3 ± 0.5 (P > 0.05), respectively, compared to 14.9 ± 0.6 for untreated MIO-M1 cells (Fig. 3c). The ΔΨm values for the cultures exposed to light for 10 min and 15 min showed were similar to controls.
DISCUSSION All vitreoretinal surgeries require illumination, and some retinal surgeries require dyes as well. The dye
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is injected into the posterior chamber to stain selectively the ILM or the epiretinal membrane, and the dye is usually washed away within a few seconds. The stained membrane is then well visualized and can be removed with the aid of endoillumination. Even with immediate washing, some dye may remain in contact with the retinal cells and be retained for long durations. The potential toxicity may be compounded by phototoxicity from light
© 2015 Royal Australian and New Zealand College of Ophthalmologists
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Figure 2. Bar graph showing caspase-3/7 activities in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after 10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. The concentrations of BBG were 2x, 1x or 0.5x. There were no significant differences in caspase-3/7 activities in any of BBG-treated cultures with or without exposure to light compared to the untreated control (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate, and the experiments repeated three times. Values are mean ± standard error mean.
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exposure. The choice of different BBG concentrations used in our experiments is based on several recent clinical trials12,23,34–37 as well as BBG-related reports.33 Before treating cells, the osmolarity and pH of different concentrations of BBG solutions maintained up to six months were measured (Table 1). The BBG solution values for osmolarity and pH were similar to those of BSS plus, the intraocular irrigating solution used during vitrectomies. This is important because from the safety standpoint, the osmolarity of intravitreal dyes plays a key role in cell survival.45
Previous studies screening the osmolarity effects of different concentrations of BBG, ICG, TB and other vital dyes in animal and human eyes demonstrated that the highest safety profile was found for BBG.22,32 In our experiment, cells were exposed to varying concentrations of BBG followed by up to 30 min of light exposure. Even though this extended duration does not represent the light exposure time during a typical vitreoretinal surgery, it was chosen to approximate the extreme upper limit of surgical exposure in a complicated case.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells ARPE-19 Cells Treated with BBG and BBG + Light ∗
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Figure 3. Bar graph showing ratio of red:green fluorescence (mitochondrial membrane potential) in ARPE-19 cells (a), R28 cells (b) and MIO-M1 cells (c) after10 min Brilliant Blue G (BBG) + 10 min light exposure, 15 min BBG + 15 min light exposure or 30 min treatment + 30 min light exposure. There were significant decreases in mitochondrial membrane potential in the cultures treated for 30 min with 2x BBG with or without light compared to control (**P < 0.01 and *P < 0.05, respectively for 2x + L, and 2x). However, there were no statistically significant changes in mitochondrial membrane potential in the cultures treated 30 min with 1x BBG or 0.5x BBG with or without light compared to untreated control. Also, there were no statistically significant changes in mitochondrial membrane potential for the cultures treated with 2x BBG, 1x BBG or 0.5x BBG with or without exposure to light for 10 or 15 min compared to untreated control cultures (P > 0.05). 1x = 0.25 mg/mL BBG. Assays were performed in triplicate and the experiments repeated three times. Values are mean ± standard error mean.
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Our study is the first report using three different retinal cell lines exposed to BBG for up to 30 min in presence or absence of light. Our findings showed that ARPE-19, R28 and MIO-M1 cells incubated with 0.25 mg/mL of BBG (clinical dose) and exposed to light for up to 30 min did not decrease cell viability, or cause apoptosis or mitochondrial damage. However, when cells were treated with 0.5 mg/mL of BBG (twice the clinical dose) with or without exposure
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to light for 30 min, mitochondrial damage was observed, though no decrease in cell viability or apoptosis were noted. In contrast to our results, Yuen et al.46 demonstrated significant reduction in ARPE-19 cell viability after exposure to BBG (0.25 mg/mL) for 30 min. However, we found no significant reduction in cell viability in RPE cells, MIO-M1 or R28 cells using the same concentration of BBG dye and same duration of treatment. This discrepancy
© 2015 Royal Australian and New Zealand College of Ophthalmologists
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may be due to different assays being used to measure cell viability. Yuen and co-workers used the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to examine the cell viability while in our work the trypan blue dye exclusion assay was used. The MMT method is a colorimetric assay that measures enzyme activities needed to reduce MTT to a formazan dye which can then be measured. Changes in metabolic activity can change the MMT levels without necessarily decreasing the numbers of viable cells. The trypan blue assay uses a vital dye that is normally excluded from the cells but when the cell membrane is damaged, then the dye enters into the cells and can be measured. Therefore while both assays can provide information about the health of the cells, the trypan blue assay represents cells that are more damaged, and this may account for the disparity of our findings. In another study, Morales et al.47 reported significant loss of cell viability, altered cell membrane permeability (CMP) and mitochondrial membrane potential in ARPE-19 cells after treatment with two different concentrations of BBG (0.5 mg/mL and 0.05 mg/mL) plus 3 min light exposure. The discrepancy with our results may be based upon differences in dye + light treatment protocols. They exposed cells to the surgical intensity light in the presence of the BBG dye (0.5 mg/mL and 0.05 mg/ mL) and then used the MTT assay for the assessment of cell viability. In our work, we exposed cells to the dye at different times, removed the dye, washed the cells three times with culture media and then exposed the cells for up to 30 min with light of surgical intensity. We then used the trypan blue dye exclusion assay to show that the BBG did not decrease the cell viability. However, comparing our results to the Morales findings suggests that light exposure in the presence of BBG dye greatly increases the toxicity to cells which emphasizes the importance of removing the dye prior to using the light during the retinal surgeries. Our safety findings for BBG are supported by some cell culture studies. Kawahara et al.33 demonstrated that Müller cells in vitro did not show apoptosis after being treated with BBG (0.25 mg/mL) for 15 min, while treatment with ICG dye for the same duration did induce apoptosis in cells. Awad et al.48 reported the lower toxicity in ARPE-19 cells following exposure of polyethylene glycol with BBG (0.025%), TB (0.15%), a mixture of BBG (0.025%) with TB (0.15% and 0.25%) and BBG (0.025% in phosphate-buffered saline). Two ex vivo studies have also shown more favourable effects of BBG on isolated perfused bovine retina as compared to ICG or TB.42,49 Several clinical studies have reported BBG as a safe alternative and potential dye for vitreoretinal surgery. In 2006, Enaida et al.22,23,32 introduced BBG as an
intraocular vital stain after a comparative study of various dyes for their safety and ability to stain membranes in pigs, rats, monkey and human eyes. It was observed that BBG effectively stained the anterior capsule in pig eyes without showing any toxic effect to the cornea.32 Also BBG was found to be safe to the posterior segment of rats and primates eyes in preclinical studies.22 Additional safety studies of BBG were conducted on 78 Brown Norway rats. The animals were injected with BBG following vitrectomy, and the eyes were enucleated after two weeks to two months. There were no abnormalities in pathology, apoptosis or reduction in the electroretinograms waveform amplitudes in the eyes.22 Using the same protocol, two cynomolgus monkeys were intravitreally injected with BBG and underwent vitrectomy.22 The ILMs were adequately stained by BBG, visualized and taken out easily. There were no clinical issues or angiographic toxicities related to BBG after two weeks to six months postoperatively. Following the various preclinical investigations, interventional non-comparative, prospective clinical series were conducted to investigate the use of BBG for ILM staining during macular hole and epiretinal membrane surgeries in human subjects.23 Twenty eyes from 20 consecutive patients with macular holes or epiretinal membrane (five men, five women for each pathology) initially underwent vitrectomy, and then a 0.5-mL solution containing 0.25 mg/mL of BBG was injected into the vitreous cavity, and immediately washed out. The BBG sufficiently stained the ILM similar to ICG making easy and complete removal of ILM. All specimens in this investigation showed the presence of the ILM under transmission electron microscope. BBG did not induce any clinical or toxic signs to the eyes after two weeks of postoperative period.23 Subsequently, by establishing the safety profile of BBG in humans, it has opened the door for ophthalmologists to use this dye in surgeries for ILM,34–37 macular holes12 and capsulorhexis,50 without generating toxic effects. BBG is now available in the European Union market as ILM Blue (manufactured by DORC Zuidland, the Netherlands) in prefilled syringes, ready-to-use for intraocular applications. ILM Blue is expected to be approved soon in the United States.51 In the present study, exposure of ARPE-19, R28 or MIO-M1 cells in culture to clinically utilized concentrations of BBG dye (0.25 mg/mL) plus light treatments for up to 30 min did not decrease cell viability, cause apoptosis or generate mitochondrial damage. However, when twice the clinical dose (0.5 mg/mL) with or without light was utilized for 30 min, mitochondrial damage was observed, though no decrease in cell viability or increased in apoptosis were noted. In our experience, we have often found
© 2015 Royal Australian and New Zealand College of Ophthalmologists
Brilliant Blue G and light on retinal cells that in vitro assays are far more sensitive in detecting cellular toxicities, which can often be missed in in vivo studies. Hence, our study is a much stronger evidence of safety of BBG at a more fundamental cellular and molecular level.
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REFERENCES 1. Brooks HL Jr. Macular hole surgery with and without internal limiting membrane peeling. Ophthalmology 2000; 107: 1939–48. 2. Kuhn F. Point: to peel or not to peel, that is the question. Ophthalmology 2002; 109: 9–11. 3. Burk SE, Da Mata AP, Snyder ME, Rosa RH Jr, Foster RE. Indocyanine green-assisted peeling of the retinal internal limiting membrane. Ophthalmology 2000; 107: 2010–4. 4. Da Mata AP, Burk SE, Riemann CD et al. Indocyanine green-assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for macular hole repair. Ophthalmology 2001; 108: 1187–92. 5. Kadonosono K, Itoh N, Uchio E, Nakamura S, Ohno S. Staining of internal limiting membrane in macular hole surgery. Arch Ophthalmol 2000; 118: 1116–8. 6. Da Mata AP, Burk SE, Foster RE et al. Long-term follow-up of indocyanine green-assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for idiopathic macular hole repair. Ophthalmology 2004; 111: 2246–53. 7. Li K, Wong D, Hiscott P, Stanga P, Groenewald C, McGalliard J. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003; 87: 216–9. 8. Jackson TL, Hillenkamp J, Knight BC et al. Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures. Invest Ophthalmol Vis Sci 2004; 45: 2778–85. 9. Gale JS, Proulx AA, Gonder JR, Mao AJ, Hutnik CM. Comparison of the in vitro toxicity of indocyanine green to that of trypan blue in human retinal pigment epithelium cell cultures. Am J Ophthalmol 2004; 138: 64–9. 10. Lesnik Oberstein SY, Mura M, Tan SH, de Smet MD. Heavy trypan blue staining of epiretinal membranes: an alternative to infracyanine green. Br J Ophthalmol 2007; 91: 955–7. 11. Rezai KA, Farrokh-Siar L, Gasyna EM, Ernest JT. Trypan blue induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 2004; 138: 492–5. 12. Remy M, Thaler S, Schumann RG et al. An in vivo evaluation of Brilliant Blue G in animals and humans. Br J Ophthalmol 2008; 92: 1142–7. 13. Nakamura H, Hayakawa K, Imaizumi A, Sakai M, Sawaguchi S. Persistence of retinal indocyanine green dye following vitreous surgery. Ophthalmic Surg Lasers Imaging 2005; 36: 37–45. 14. Sippy BD, Engelbrecht NE, Hubbard GB et al. Indocyanine green effect on cultured human retinal
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
© 2015 Royal Australian and New Zealand College of Ophthalmologists
pigment epithelial cells: implication for macular hole surgery. Am J Ophthalmol 2001; 132: 433–5. Kwok AK, Leung DY, Hon C, Lam DS. Vision threatening vitreous haemorrhage after internal limiting membrane peeling in macular surgeries. Br J Ophthalmol 2002; 86: 1449–50. Friedlander RM. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med 2003; 348: 1365–75. Rezai KA, Farrokh-Siar L, Ernest JT, van Seventer GA. Indocyanine green induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 2004; 137: 931–3. Doonan F, Cotter TG. Apoptosis: a potential therapeutic target for retinal degenerations. Curr Neurovasc Res 2004; 1: 41–53. Ho JD, Tsai RJ, Chen SN, Chen HC. Toxic effect of indocyanine green on retinal pigment epithelium related to osmotic effects of the solvent. Am J Ophthalmol 2003; 135: 258. Wollensak G, Spoerl E, Wirbelauer C, Pham DT. Influence of indocyanine green staining on the biomechanical strength of porcine internal limiting membrane. Ophthalmologica 2004; 218: 278–82. Engel E, Schraml R, Maisch T et al. Light-induced decomposition of indocyanine green. Invest Ophthalmol Vis Sci 2008; 49: 1777–83. Enaida H, Hisatomi T, Goto Y et al. Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G. Retina 2006; 26: 623–30. Enaida H, Hisatomi T, Hata Y et al. Brilliant blue G selectively stains the internal limiting membrane/ brilliant blue G-assisted membrane peeling. Retina 2006; 26: 631–6. Congdon RW, Muth GW, Splittgerber AG. The binding interaction of Coomassie blue with proteins. Anal Biochem 1993; 213: 407–13. Laughton C. Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein. Anal Biochem 1984; 140: 417–23. Westermeier R. Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels. Proteomics 2006; 6 (Suppl. 2): 61–4. Said-Fernandez S, Gonzalez-Garza MT, Mata-Cardenas BD, Navarro-Marmolejo L. A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250. Anal Biochem 1990; 191: 119–26. Burgess-Cassler A, Johansen JJ, Santek DA, Ide JR, Kendrick NC. Computerized quantitative analysis of Coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis. Clin Chem 1989; 35: 2297–304. Saoji AM, Kulkarni M, Naghi T. Coomassie brilliant blue R-250 as a highly sensitive pre-stain for immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis and raising
10
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Mansoor et al. monospecific antibodies. Indian J Pathol Microbiol 1989; 32: 286–90. Oshima I, Kohama T, Kodaira T. A sensitive assay for serum acid soluble proteins with Coomassie brilliant blue G 250 and its application for autoimmune diseases. Tokushima J Exp Med 1987; 34: 23–7. Bergqvist Y, Karlsson L, Fohlin L. Total protein determined in human breast milk by use of Coomassie brilliant blue and centrifugal analysis. Clin Chem 1989; 35: 2127–9. Hisatomi T, Enaida H, Matsumoto H et al. Staining ability and biocompatibility of brilliant blue G: preclinical study of brilliant blue G as an adjunct for capsular staining. Arch Ophthalmol 2006; 124: 514–9. Kawahara S, Hata Y, Miura M et al. Intracellular events in retinal glial cells exposed to ICG and BBG. Invest Ophthalmol Vis Sci 2007; 48: 4426–32. Schumann RG, Remy M, Grueterich M, Gandorfer A, Haritoglou C. How it appears: electron microscopic evaluation of internal limiting membrane specimens obtained during brilliant blue G assisted macular hole surgery. Br J Ophthalmol 2008; 92: 330–1. Cervera E, Diaz-Llopis M, Salom D, Udaondo P. High dose intravitreal brilliant blue G. Arch Soc Esp Oftalmol 2007; 82: 473. Cervera E, Diaz-Llopis M, Salom D, Udaondo P, Amselem L. Internal limiting membrane staining using intravitreal brilliant blue G: good help for vitreo-retinal surgeon in training]. Arch Soc Esp Oftalmol 2007; 82: 71–2. Mochizuki Y, Enaida H, Hisatomi T et al. The internal limiting membrane peeling with brilliant blue G staining for retinal detachment due to macular hole in high myopia. Br J Ophthalmol 2008; 92: 1009. Yam HF, Kwok AK, Chan KP et al. Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2003; 44: 370–7. Wu WCHD, Roberts JE. Phototoxicity of indocyanine green on human retinal pigment epithelium in vitro and its reduction by lutein. Photochem Photobiol 2004; 81: 537–40. Narayanan R, Kenney MC, Kamjoo S et al. Toxicity of indocyanine green (ICG) in combination with light
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43.
44.
45.
46.
47.
48.
49. 50.
51.
on retinal pigment epithelial cells and neurosensory retinal cells. Curr Eye Res 2005; 30: 471–8. Seigel GM. Establishment of an E1A-immortalized retinal cell culture. In Vitro Cell Dev Biol Anim 1996; 322: 66–8. Wei S, Seigel GM, Salvi RJ. Retinal precursor cells express functional ionotropic glutamate and GABA receptors. Neuro Report 2002; 13: 2421–4. Patil AJ, Gramajo AL, Sharma A, Seigel GM, Kuppermann BD, Kenney CM. Differential effect of nicotine on retinal and vascular cells in vitro. Toxicol 2009; 259: 69–76. Sharma A, Patil JA, Gramajo AL, Seigel GM, Kuppermann BD, Kenney CM. Effect of hydroquinone on retinal and vascular cells in vitro. Indian J Ophthalmol 2012; 60: 189–93. Narayanan R, Kenney MC, Kamjoo S et al. Trypan blue: effect on retinal pigment epithelial and neurosensory retinal cells. Invest Ophthalmol Vis Sci 2005; 46: 304–9. Yuen D, Gonder J, Proulx A, Liu H, Hutnik C. Comparison of the in vitro safety of intraocular dyes using two retinal cell lines: a focus on brilliant blue G and indocyanine green. Am J Ophthalmol 2009; 147: 251–9. Morales MC, Freire V, Asumendi A et al. Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2010; 51: 6018– 29. Awad D, Schrader I, Bartok M, Mohr A, Gabel D. Comparative toxicology of trypan blue, brilliant blue G, and their combination together with polyethylene glycol on human pigment epithelial cells. Invest Ophthalmol Vis Sci 2011; 52: 4085–90. Luke C, Luke M, Dietlein TS et al. Retinal tolerance to dyes. Br J Ophthalmol 2005; 89: 1188–91. Udaondo P, Diaz-Llopis M, Salom D, Garcia-Delpech S, Cervera E. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch Soc Esp Oftalmol 2007; 82: 471–2. Kagimoto HTS, Hisatomi T, Enaida H, Ishibashi T. Brilliant blue G for ILM staining and peeling. Retina Today 2011; 45–8. Accessed July 2013. Available from: http://retinatoday.com/2011/04/global-perspectives -brilliant-blue-g-for-ilm-staining-and-peeling/
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