Original Paper Ophthalmic Res 2014;52:147–150 DOI: 10.1159/000364881
Received: February 24, 2014 Accepted after revision: May 16, 2014 Published online: October 4, 2014
The Effects of Vital Dyes on Retinal Pigment Epithelium Cells in Oxidative Stress Shani Golan a Ran Levi b Michal Entin-Meer b Adiel Barak a Departments of a Ophthalmology and b Cardiology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Key Words Indocyanine green · Trypan blue · Oxidative stress · Retinal pigment epithelium
Abstract Purpose: To determine the effect of the most commonly used vital dyes in vitrectomy [trypan blue at 0.15% concentration and indocyanine green (ICG) at 0.5% concentration] on the viability of human retinal pigment epithelium (RPE) cell lines (ARPE-19) exposed to oxidative stress. Methods: ARPE-19 cells unexposed or exposed to oxidative stress (hypoxic chamber) were treated for 1 min with one of the dyes. RPE proliferation was measured by 3H-thymidine incorporation, adhesion by ability to adhere to fibronectin, and safety by annexin V staining. Results: Proliferation: The dyes affected the proliferation of RPE cells differently under non-hypoxic and hypoxic conditions (p = 0.001). In non-hypoxic conditions, there was no statistically significant difference between the proliferation of the treated (both dyes) and untreated RPE cells (p = 0.279). Under hypoxia, both dyes significantly suppressed proliferation, more so with ICG (p = 0.001). Adhesion: The dyes affected adhesion differently under non-hypoxic and hypoxic conditions (p = 0.04). In nonhypoxic conditions, both increased the adhesive properties of RPE cells to fibronectin, ICG more than trypan blue (p = 0.001). Under hypoxia, both dyes suppressed adhesion, with
© 2014 S. Karger AG, Basel 0030–3747/14/0523–0147$39.50/0 E-Mail [email protected] www.karger.com/ore
no statistically significant difference between treated and non-treated RPE cells. Apoptosis: Both dyes increased early apoptosis of RPE cells compared with no treatment (p = 0.001), ICG more than trypan blue. Hypoxia increased the apoptosis of both dyes compared to non-hypoxic conditions (p = 0.02). Conclusions: In hypoxic conditions, both dyes showed an inhibition of RPE adhesion to fibronectin and proliferation capacity and an increase in early apoptosis compared with non-hypoxic conditions. Apoptosis was greater in ICG-treated RPE cells than in trypan blue-treated cells. © 2014 S. Karger AG, Basel
Introduction
Removal of the internal limiting membrane is widely used in the surgical treatment of macular pathology, including macular hole formation and macular oedema due to inflammatory or vascular diseases [1]. Staining is considered essential to the complete removal of the internal limiting membrane, as it avoids complications of missed tissues and ensures the detection of the membrane around the rim of macular holes, thus increasing the closure rate [2–5]. The most common surgical dye in ophthalmology is indocyanine green (ICG), introduced in 2000. However, complications associated with its use over the past decade have called its safety into question. ICG was found Shani Golan, MD Department of Ophthalmology, Tel Aviv Sourasky Medical Center 6 Weizmann Street Tel Aviv 64239 (Israel) E-Mail shanigol2 @ walla.com
to be associated with the risk of retinal damage [6–9], atrophy of the retinal pigment epithelium (RPE) [10], damage to the photoreceptors and RPE cells [11, 12], lower visual function outcome [13–16], loss of epiretinal cellular integrity [17], cellular toxicity [18–20], and other harmful effects. Recently, research groups also reported that ICG may persist in the ocular cavity up to 6 weeks after its application in surgery [21, 22]. The search for new dyes for vitrectomy has led to the introduction of a number of alternative stains to vitreoretinal surgery, and their number is constantly growing [5, 23, 24]. The question of which dye is most effective in reducing toxicity, raising affinity for targeted tissues, and minimizing residual permanence remains to be clarified. Trypan blue is used in microscopy for staining dead tissues and was shown to have preferential affinity for the epiretinal membrane [25–27]. While it may not enable visualization of the internal limiting membrane as well as ICG, it does exhibit better biocompatibility [27–29]. The present study compares the in vitro effect of these two most commonly used stains in vitrectomy on human RPE cells exposed to oxidative stress. The effects of therapeutic concentrations of the two dyes were examined under the same experimental conditions, providing a more precise comparison of the specific effects of each dye. Methods ARPE-19 cells were seeded at 1 × 106/10 cm on plates with DMEM containing 10% fetal bovine serum, 1% L-glutamine solution, and 10% penicillin-streptomycin, and incubated at 37 ° C for 2 days. Serum was withdrawn into DMEM + 1% bovine serum albumin for 3 days to make the cells quiescent. The cells were then put in a hypoxic chamber (2% oxygen) for 12 h. The control group of cells was treated in the same way but was not exposed to oxidative stress. The cells were then treated with therapeutic concentrations of ICG (Pulsion Medical Systems GmbH, Feldkirchen, Germany) or trypan blue (Membrane Blue; DORC France, Paris, France) at 0.05 and 0.15%, respectively. The concentrations of trypan blue were prepared by using serial dilutions of the dye with PBS and of ICG with distilled water. The solution of dye was then added directly to the cells in order to obtain a uniform concentration of drug in each well of the tissue culture plate for exactly 1 min. Treated cells were compared with control cells that had not been exposed to either dye. All experiments were compared to two controls: ARPE-19 cells that were exposed to oxidative stress and ARPE-19 cells treated with one of the two dyes. The results are normalized to both controls.
Measuring the Effects of the Dyes Proliferation Assay Given that proliferation is a functional property that characterizes activated RPE cells, we examined the effect of treatment with oxidative stress on the proliferation capacity of the cells. The pro-
148
Ophthalmic Res 2014;52:147–150 DOI: 10.1159/000364881
liferation index was determined by first synchronizing the cells at the G1-S boundary by culturing cells for 24 h in a serum-free medium, after which 4 × 103 ARPE-19 cells/well were seeded in 96-well plates and 3H-thymidine (1 μCi/well) was added for 16 h before proliferation was assayed by scintillation counting (beta counter). Adhesion Assay Fibronectin is a component of the extracellular matrix and the ligand of integrins expressed in RPE cells [30, 31]. Because high RPE adhesion capacity plays a crucial role in RPE growth and activation [32], we assessed the ability of the ARPE-19 cell line to adhere to fibronectin following treatment with the dyes under non-hypoxic and hypoxic conditions, using a previously described attachment assay [33]. Adhesion to fibronectin was evaluated by seeding 104 H5V cells/well in 96-well plates coated with fibronectin for 30 min. Non-adherent cells were washed away and the adherent cells were stained with XTT-based colorimetry (Biological Industries, Beit Haemek, Israel). Optical density at 450 nm, proportional to viable cell numbers, was measured using an ELISA reader. Fibronectin-coated wells served as a background. Toxicity Measures Toxicity measures included any amount of cell toxicity, annexin V detection of dead cells, necrotizing cells, and cells in early stages of apoptosis. The cells were washed twice with cold PBS and re-suspended in 85 μl 1× binding buffer (MEBCYTO Apoptosis Kit®, Cat. No. 4700) at a concentration of 2 × 105 cells/ml. Ten microlitres of annexin V-FITC and 5 μl of propidium iodide were added to an 85-μl cell suspension and incubated for 15 min at room temperature. The stained cells were analysed by flow cytometry using a single laser-emitting excitation light at 488 nm. Statistical Analysis Each experiment was performed in triplicate. Significance was calculated with t tests and graphing software (Sigma Plot; Systat, Chicago, Ill., USA); p < 0.05 was considered significant. The results are expressed as means ± SD.
Results
The results are summarized in table 1. Proliferation Capacity Under non-hypoxic conditions, there was no statistically significant difference in the proliferation of treated and untreated ARPE-19 cells (p = 0.279). Under hypoxic conditions, both treated and untreated RPE cells exhibited inhibited proliferation (p = 0.01), with ICG inhibiting proliferation capacity significantly more than trypan blue (p = 0.01). Adhesive Properties In non-hypoxic conditions, both dyes increased ARPE-19 cell adhesive properties compared with untreated cells (p = 0.001). Trypan blue increased the adheGolan /Levi /Entin-Meer /Barak
Table 1. ARPE-19 cell properties after exposure to either ICG or trypan blue under hypoxic and non-hypoxic conditions
ICG
Trypan blue
hypoxic nonhypoxic hypoxic Proliferation ↓↓a Adhesion to fibronectin ↓ Early apoptosis ↑↑a
↓ ↑↑a ↑
↓a ↓ ↑↑a
nonhypoxic ↓ ↑a ↑
The position of the arrowhead indicates whether the dye increased (↑) or decreased (↓) the viability property measured. a The difference between the effects was statistically significant.
sive properties of ARPE-19 cells more than ICG (p = 0.001). Under hypoxic conditions, there was a decrease in the adhesive properties of cells treated with dyes compared with untreated cells (p = 0.004). A statistically nonsignificant difference was found between the two dyes (p = 0.887). Apoptotic Features of RPE Cells Early apoptosis was greater in cells exposed to oxidative stress than in those not exposed, regardless of whether they were stained (p = 0.02). Safety of ICG and Trypan Blue Both dyes showed an increase in RPE apoptotic cells compared with controls (untreated cells) in both hypoxic and non-hypoxic conditions, reaching statistical significance only with ICG.
ture and could be explained by the early increase in adhesion capacity followed by apoptosis of cells. Our experimental design allowed us to focus on the specific biological effects associated with each of the dyes and to determine which dye is more appropriate in a given situation, mainly vitreoretinal surgery for ischaemic retinal areas, where there is a relatively hypoxic environment in the eye. Indeed, our experimental conditions mimicked surgical conditions as closely as possible: exposure of the cells to oxidative stress, like in the operating field, short exposure times (1 min), and dye concentrations that are typically administered to patients during surgery. ICG was the first dye to be used for intraocular surgery and is currently the dye of choice; it is more extensively characterized than any of the other dyes. However, it is also one of the most controversial dyes, having yielded contradictory results even under similar conditions. The present study corroborates reports that ICG is associated with partial toxic effects. The effects of trypan blue are also well characterized, probably because it is the second most commonly used dye. Because it does not stain as well as ICG, its use is usually restricted to the staining of the epiretinal membrane [25–27, 34]. Our finding that trypan blue inhibits proliferation and enhances apoptosis to a significantly lesser degree than ICG argues for its greater safety. Nevertheless, as with ICG, there is little consensus about its effects [10–17]. In summary, the present study demonstrated that ICG inhibited several aspects of cell viability at therapeutic doses, more so than trypan blue, leading to the conclusion that, in vitro, trypan blue has a significant advantage over ICG.
Discussion
In our study designed to determine whether ICG or trypan blue at therapeutic concentrations has toxic effects on proliferating RPE cells under hypoxic conditions, we found that ICG inhibited their proliferation capacity more than trypan blue, and that it had a significant toxic effect on proliferation in hypoxic conditions. We did not try to establish the LD50 concentration for either of the two compounds in the current work. In addition, ICGtreated RPE cells exhibited increased adhesive capacity, greater than trypan blue-treated RPE cells and untreated cells, reaching statistical significance only in non-hypoxic conditions. This increased adhesiveness of treated cells in non-hypoxic conditions is not described in the literaEffects of Vital Dyes on RPE Cells in Oxidative Stress
References
1 Abdelkader E, Lois N: Internal limiting membrane peeling in vitreoretinal surgery. Surv Ophthalmol 2008;53:368–396. 2 Rodrigues EB, Costa EF, Penha FM, Melo GB, Bottós J, Dib E, Furlani B, Lima VC, Maia M, Meyer CH, Höfling-Lima AL, Farah ME: The use of vital dyes in ocular surgery. Surv Ophthalmol 2009;54:576–617. 3 Farah ME, Maia M, Rodrigues EB: Dyes in ocular surgery: principles for use in chromovitrectomy. Am J Ophthalmol 2009;148:332– 340.
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4 Rodrigues EB, Penha FM, de Paula Fiod Costa E, Maia M, Dib E, Moraes M Jr, Meyer CH, Magalhaes O Jr, Melo GB, Stefano V, Dias AB, Farah ME: Ability of new vital dyes to stain intraocular membranes and tissues in ocular surgery. Am J Ophthalmol 2010; 149: 265– 277. 5 Morales MC, Freire V, Asumendi A, Araiz J, Herrera I, Castiella G, Corcóstegui I, Corcóstegui G: Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2010; 51: 6018–6029. 6 Gandorfer A, Haritoglou C, Gass CA, Ulbig MW, Kampik A: Indocyanine green-assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol 2001;132:431–433. 7 Sippy BD, Engelbrecht NE, Hubbard GB, Moriarty SE, Jiang S, Aaberg TM Jr, Aaberg TM Sr, Grossniklaus HE, Sternberg P Jr: Indocyanine green effect on cultured human retinal pigment epithelial cells: implication for macular hole surgery. Am J Ophthalmol 2001;132:433–435. 8 Enaida H, Sakamoto T, Hisatomi T, Goto Y, Ishibashi T: Morphological and functional damage of the retina caused by intravitreous indocyanine green in rat eyes. Graefes Arch Clin Exp Ophthalmol 2002;240:209–213. 9 Gandorfer A, Haritoglou C, Gandorfer A, Kampik A: Retinal damage from indocyanine green in experimental macular surgery. Invest Ophthalmol Vis Sci 2003;44:316–323. 10 Hirata A, Inomata Y, Kawaji T, Tanihara H: Persistent subretinal indocyanine green induces retinal pigment epithelium atrophy. Am J Ophthalmol 2003;136:353–355. 11 Engelbrecht NE, Freeman J, Sternberg P Jr, Aaberg TM Sr, Aaberg TM Jr, Martin DF, Sippy BD: Retinal pigment epithelial changes after macular hole surgery with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol 2002;133:89–94. 12 Maia M, Kellner L, de Juan E Jr, Smith R, Farah ME, Margalit E, Lakhanpal RR, Grebe L, Au Eong KG, Humayun MS: Effects of indocyanine green injection on the retinal surface and into the subretinal space in rabbits. Retina 2004;24:80–91.
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13 Haritoglou C, Gandorfer A, Gass CA, Schaumberger M, Ulbig MW, Kampik A: Indocyanine green-assisted peeling of the internal limiting membrane in macular hole surgery affects visual outcome: a clinicopathologic correlation. Am J Ophthalmol 2002;134: 836–841. 14 Uemura A, Kanda S, Sakamoto Y, Kita H: Visual field defects after uneventful vitrectomy for epiretinal membrane with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol 2003;136:252–257. 15 Ando F, Sasano K, Ohba N, Hirose H, Yasui O: Anatomic and visual outcomes after indocyanine green-assisted peeling of the retinal internal limiting membrane in idiopathic macular hole surgery. Am J Ophthalmol 2004; 137:609–614. 16 Tsuiki E, Fujikawa A, Miyamura N, Yamada K, Mishima K, Kitaoka T: Visual field defects after macular hole surgery with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol 2007;143:704–705. 17 Rezai KA, Farrokh-Siar L, Ernest JT, van Seventer GA: Indocyanine green induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 2004;137:931–933. 18 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. 19 Ikagawa H, Yoneda M, Iwaki M, Isogai Z, Tsujii K, Yamazaki R, Kamiya T, Zako M: Chemical toxicity of indocyanine green damages retinal pigment epithelium. Invest Ophthalmol Vis Sci 2005;46:2531–2539. 20 Sato Y, Tomita H, Sugano E, Isago H, Yoshida M, Tamai M: Evaluation of indocyanine green toxicity to rat retinas. Ophthalmologica 2006; 220:153–158. 21 Weinberger AW, Kirchhof B, Mazinani BE, Schrage NF: Persistent indocyanine green (ICG) fluorescence 6 weeks after intraocular ICG administration for macular hole surgery. Graefes Arch Clin Exp Ophthalmol 2001;239: 388–390. 22 Sayanagi K, Ikuno Y, Soga K, Sawa M, Oshima Y, Kamei M, Kusaka S, Tano Y: Residual indocyanine green fluorescence pattern after vitrectomy for idiopathic macular hole with internal limiting membrane peeling. Br J Ophthalmol 2007;91:939–944. 23 Lüke C, Lüke M, Dietlein TS, Hueber A, Jordan J, Sickel W, Kirchhof B: Retinal tolerance to dyes. Br J Ophthalmol 2005;89:1188–1191.
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24 Rodrigues EB, Maia M, Meyer CH, Penha FM, Dib E, Farah ME: Vital dyes for chromovitrectomy. Curr Opin Ophthalmol 2007; 18: 179–187. 25 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;87:216–219. 26 Rodrigues EB, Meyer CH, Schmidt JC, Kroll P: Trypan blue stains the epiretinal membrane but not the internal limiting membrane. Br J Ophthalmol 2003;87:1431–1432. 27 Lee KL, Dean S, Guest S: A comparison of outcomes after indocyanine green and trypan blue assisted internal limiting membrane peeling during macular hole surgery. Br J Ophthalmol 2005;89:420–424. 28 Haritoglou C, Eibl K, Schaumberger M, Mueller AJ, Priglinger S, Alge C, Kampik A: Functional outcome after trypan blue-assisted vitrectomy for macular pucker: a prospective, randomized, comparative trial. Am J Ophthalmol 2004;138:1–5. 29 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;138:64–69. 30 Kim BS, Mooney DJ: Engineering smooth muscle tissue with a predefined structure. J Biomed Mater Res 1998;41:322–332. 31 Bouhadir KH, Mooney DJ: In vitro and in vivo models for the reconstruction of intercellular signaling. Ann NY Acad Sci 1998; 842: 188–194. 32 Inumaru J, Nagano O, Takahashi E, Ishimoto T, Nakamura S, Suzuki Y, Niwa S, Umezawa K, Tanihara H, Saya H: Molecular mechanisms regulating dissociation of cell-cell junction of epithelial cells by oxidative stress. Genes Cells 2009;14:703–716. 33 Vacca A, Iurlaro M, Ribatti D, Minischetti M, Nico B, Ria R, Pellegrino A, Dammacco F: Antiangiogenesis is produced by nontoxic doses of vinblastine. Blood 1999; 94: 4143– 4155. 34 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;147:251–259.
Golan /Levi /Entin-Meer /Barak
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: February 24, 2014 Accepted after revision: May 16, 2014 Published online: October 4, 2014
The Effects of Vital Dyes on Retinal Pigment Epithelium Cells in Oxidative Stress Shani Golan a Ran Levi b Michal Entin-Meer b Adiel Barak a Departments of a Ophthalmology and b Cardiology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Key Words Indocyanine green · Trypan blue · Oxidative stress · Retinal pigment epithelium
Abstract Purpose: To determine the effect of the most commonly used vital dyes in vitrectomy [trypan blue at 0.15% concentration and indocyanine green (ICG) at 0.5% concentration] on the viability of human retinal pigment epithelium (RPE) cell lines (ARPE-19) exposed to oxidative stress. Methods: ARPE-19 cells unexposed or exposed to oxidative stress (hypoxic chamber) were treated for 1 min with one of the dyes. RPE proliferation was measured by 3H-thymidine incorporation, adhesion by ability to adhere to fibronectin, and safety by annexin V staining. Results: Proliferation: The dyes affected the proliferation of RPE cells differently under non-hypoxic and hypoxic conditions (p = 0.001). In non-hypoxic conditions, there was no statistically significant difference between the proliferation of the treated (both dyes) and untreated RPE cells (p = 0.279). Under hypoxia, both dyes significantly suppressed proliferation, more so with ICG (p = 0.001). Adhesion: The dyes affected adhesion differently under non-hypoxic and hypoxic conditions (p = 0.04). In nonhypoxic conditions, both increased the adhesive properties of RPE cells to fibronectin, ICG more than trypan blue (p = 0.001). Under hypoxia, both dyes suppressed adhesion, with
© 2014 S. Karger AG, Basel 0030–3747/14/0523–0147$39.50/0 E-Mail [email protected] www.karger.com/ore
no statistically significant difference between treated and non-treated RPE cells. Apoptosis: Both dyes increased early apoptosis of RPE cells compared with no treatment (p = 0.001), ICG more than trypan blue. Hypoxia increased the apoptosis of both dyes compared to non-hypoxic conditions (p = 0.02). Conclusions: In hypoxic conditions, both dyes showed an inhibition of RPE adhesion to fibronectin and proliferation capacity and an increase in early apoptosis compared with non-hypoxic conditions. Apoptosis was greater in ICG-treated RPE cells than in trypan blue-treated cells. © 2014 S. Karger AG, Basel
Introduction
Removal of the internal limiting membrane is widely used in the surgical treatment of macular pathology, including macular hole formation and macular oedema due to inflammatory or vascular diseases [1]. Staining is considered essential to the complete removal of the internal limiting membrane, as it avoids complications of missed tissues and ensures the detection of the membrane around the rim of macular holes, thus increasing the closure rate [2–5]. The most common surgical dye in ophthalmology is indocyanine green (ICG), introduced in 2000. However, complications associated with its use over the past decade have called its safety into question. ICG was found Shani Golan, MD Department of Ophthalmology, Tel Aviv Sourasky Medical Center 6 Weizmann Street Tel Aviv 64239 (Israel) E-Mail shanigol2 @ walla.com
to be associated with the risk of retinal damage [6–9], atrophy of the retinal pigment epithelium (RPE) [10], damage to the photoreceptors and RPE cells [11, 12], lower visual function outcome [13–16], loss of epiretinal cellular integrity [17], cellular toxicity [18–20], and other harmful effects. Recently, research groups also reported that ICG may persist in the ocular cavity up to 6 weeks after its application in surgery [21, 22]. The search for new dyes for vitrectomy has led to the introduction of a number of alternative stains to vitreoretinal surgery, and their number is constantly growing [5, 23, 24]. The question of which dye is most effective in reducing toxicity, raising affinity for targeted tissues, and minimizing residual permanence remains to be clarified. Trypan blue is used in microscopy for staining dead tissues and was shown to have preferential affinity for the epiretinal membrane [25–27]. While it may not enable visualization of the internal limiting membrane as well as ICG, it does exhibit better biocompatibility [27–29]. The present study compares the in vitro effect of these two most commonly used stains in vitrectomy on human RPE cells exposed to oxidative stress. The effects of therapeutic concentrations of the two dyes were examined under the same experimental conditions, providing a more precise comparison of the specific effects of each dye. Methods ARPE-19 cells were seeded at 1 × 106/10 cm on plates with DMEM containing 10% fetal bovine serum, 1% L-glutamine solution, and 10% penicillin-streptomycin, and incubated at 37 ° C for 2 days. Serum was withdrawn into DMEM + 1% bovine serum albumin for 3 days to make the cells quiescent. The cells were then put in a hypoxic chamber (2% oxygen) for 12 h. The control group of cells was treated in the same way but was not exposed to oxidative stress. The cells were then treated with therapeutic concentrations of ICG (Pulsion Medical Systems GmbH, Feldkirchen, Germany) or trypan blue (Membrane Blue; DORC France, Paris, France) at 0.05 and 0.15%, respectively. The concentrations of trypan blue were prepared by using serial dilutions of the dye with PBS and of ICG with distilled water. The solution of dye was then added directly to the cells in order to obtain a uniform concentration of drug in each well of the tissue culture plate for exactly 1 min. Treated cells were compared with control cells that had not been exposed to either dye. All experiments were compared to two controls: ARPE-19 cells that were exposed to oxidative stress and ARPE-19 cells treated with one of the two dyes. The results are normalized to both controls.
Measuring the Effects of the Dyes Proliferation Assay Given that proliferation is a functional property that characterizes activated RPE cells, we examined the effect of treatment with oxidative stress on the proliferation capacity of the cells. The pro-
148
Ophthalmic Res 2014;52:147–150 DOI: 10.1159/000364881
liferation index was determined by first synchronizing the cells at the G1-S boundary by culturing cells for 24 h in a serum-free medium, after which 4 × 103 ARPE-19 cells/well were seeded in 96-well plates and 3H-thymidine (1 μCi/well) was added for 16 h before proliferation was assayed by scintillation counting (beta counter). Adhesion Assay Fibronectin is a component of the extracellular matrix and the ligand of integrins expressed in RPE cells [30, 31]. Because high RPE adhesion capacity plays a crucial role in RPE growth and activation [32], we assessed the ability of the ARPE-19 cell line to adhere to fibronectin following treatment with the dyes under non-hypoxic and hypoxic conditions, using a previously described attachment assay [33]. Adhesion to fibronectin was evaluated by seeding 104 H5V cells/well in 96-well plates coated with fibronectin for 30 min. Non-adherent cells were washed away and the adherent cells were stained with XTT-based colorimetry (Biological Industries, Beit Haemek, Israel). Optical density at 450 nm, proportional to viable cell numbers, was measured using an ELISA reader. Fibronectin-coated wells served as a background. Toxicity Measures Toxicity measures included any amount of cell toxicity, annexin V detection of dead cells, necrotizing cells, and cells in early stages of apoptosis. The cells were washed twice with cold PBS and re-suspended in 85 μl 1× binding buffer (MEBCYTO Apoptosis Kit®, Cat. No. 4700) at a concentration of 2 × 105 cells/ml. Ten microlitres of annexin V-FITC and 5 μl of propidium iodide were added to an 85-μl cell suspension and incubated for 15 min at room temperature. The stained cells were analysed by flow cytometry using a single laser-emitting excitation light at 488 nm. Statistical Analysis Each experiment was performed in triplicate. Significance was calculated with t tests and graphing software (Sigma Plot; Systat, Chicago, Ill., USA); p < 0.05 was considered significant. The results are expressed as means ± SD.
Results
The results are summarized in table 1. Proliferation Capacity Under non-hypoxic conditions, there was no statistically significant difference in the proliferation of treated and untreated ARPE-19 cells (p = 0.279). Under hypoxic conditions, both treated and untreated RPE cells exhibited inhibited proliferation (p = 0.01), with ICG inhibiting proliferation capacity significantly more than trypan blue (p = 0.01). Adhesive Properties In non-hypoxic conditions, both dyes increased ARPE-19 cell adhesive properties compared with untreated cells (p = 0.001). Trypan blue increased the adheGolan /Levi /Entin-Meer /Barak
Table 1. ARPE-19 cell properties after exposure to either ICG or trypan blue under hypoxic and non-hypoxic conditions
ICG
Trypan blue
hypoxic nonhypoxic hypoxic Proliferation ↓↓a Adhesion to fibronectin ↓ Early apoptosis ↑↑a
↓ ↑↑a ↑
↓a ↓ ↑↑a
nonhypoxic ↓ ↑a ↑
The position of the arrowhead indicates whether the dye increased (↑) or decreased (↓) the viability property measured. a The difference between the effects was statistically significant.
sive properties of ARPE-19 cells more than ICG (p = 0.001). Under hypoxic conditions, there was a decrease in the adhesive properties of cells treated with dyes compared with untreated cells (p = 0.004). A statistically nonsignificant difference was found between the two dyes (p = 0.887). Apoptotic Features of RPE Cells Early apoptosis was greater in cells exposed to oxidative stress than in those not exposed, regardless of whether they were stained (p = 0.02). Safety of ICG and Trypan Blue Both dyes showed an increase in RPE apoptotic cells compared with controls (untreated cells) in both hypoxic and non-hypoxic conditions, reaching statistical significance only with ICG.
ture and could be explained by the early increase in adhesion capacity followed by apoptosis of cells. Our experimental design allowed us to focus on the specific biological effects associated with each of the dyes and to determine which dye is more appropriate in a given situation, mainly vitreoretinal surgery for ischaemic retinal areas, where there is a relatively hypoxic environment in the eye. Indeed, our experimental conditions mimicked surgical conditions as closely as possible: exposure of the cells to oxidative stress, like in the operating field, short exposure times (1 min), and dye concentrations that are typically administered to patients during surgery. ICG was the first dye to be used for intraocular surgery and is currently the dye of choice; it is more extensively characterized than any of the other dyes. However, it is also one of the most controversial dyes, having yielded contradictory results even under similar conditions. The present study corroborates reports that ICG is associated with partial toxic effects. The effects of trypan blue are also well characterized, probably because it is the second most commonly used dye. Because it does not stain as well as ICG, its use is usually restricted to the staining of the epiretinal membrane [25–27, 34]. Our finding that trypan blue inhibits proliferation and enhances apoptosis to a significantly lesser degree than ICG argues for its greater safety. Nevertheless, as with ICG, there is little consensus about its effects [10–17]. In summary, the present study demonstrated that ICG inhibited several aspects of cell viability at therapeutic doses, more so than trypan blue, leading to the conclusion that, in vitro, trypan blue has a significant advantage over ICG.
Discussion
In our study designed to determine whether ICG or trypan blue at therapeutic concentrations has toxic effects on proliferating RPE cells under hypoxic conditions, we found that ICG inhibited their proliferation capacity more than trypan blue, and that it had a significant toxic effect on proliferation in hypoxic conditions. We did not try to establish the LD50 concentration for either of the two compounds in the current work. In addition, ICGtreated RPE cells exhibited increased adhesive capacity, greater than trypan blue-treated RPE cells and untreated cells, reaching statistical significance only in non-hypoxic conditions. This increased adhesiveness of treated cells in non-hypoxic conditions is not described in the literaEffects of Vital Dyes on RPE Cells in Oxidative Stress
References
1 Abdelkader E, Lois N: Internal limiting membrane peeling in vitreoretinal surgery. Surv Ophthalmol 2008;53:368–396. 2 Rodrigues EB, Costa EF, Penha FM, Melo GB, Bottós J, Dib E, Furlani B, Lima VC, Maia M, Meyer CH, Höfling-Lima AL, Farah ME: The use of vital dyes in ocular surgery. Surv Ophthalmol 2009;54:576–617. 3 Farah ME, Maia M, Rodrigues EB: Dyes in ocular surgery: principles for use in chromovitrectomy. Am J Ophthalmol 2009;148:332– 340.
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