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From The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom.
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Abstract |
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METHODS. Experiments were undertaken in a cell culture model used previously to perform safety testing of indocyanine green (ICG). Human retinal pigment epithelium (RPE) and Müller cells were exposed to IFCG for 5 minutes, over a range of concentrations up to 0.5%. Experiments were repeated, using double-staining with trypan blue. Cell viability was measured at days 1, 5, and 15 using a mitochondrial dehydrogenase assay and a fluorescent live–dead probe containing calcein and ethidium homodimer-1. Viability was measured after exposure to 0.5% IFCG and 5 minutes of illumination with a vitrectomy endolight powered by a xenon light source.
RESULTS. RPE viability was not reduced over the range of concentrations and follow-up intervals. RPE cells exposed to IFCG and illumination had reduced viability relative to the negative control (cells exposed to saline), but not relative to those exposed to saline and illumination. Glial cells showed reduced viability at days 1 and 5, but not day 15. Illumination did not further reduce viability.
CONCLUSIONS. IFCG has been advocated as a safer macular vital stain than ICG. These results suggest that it is less likely to produce phototoxicity, but despite being nearly iso-osmolar, IFCG also produces damage in cultured glial cells.
ICG is usually dissolved in distilled water that is then mixed with a balanced saline solution (BSS). This results in an approximately 275-mOsM solution that is hypo-osmotic relative to ocular tissue. Some investigators believe that it is the combination of low osmolarity and ICG exposure that damages cells.6 Others have suggested that it is the interaction of surgical endoillumination and ICG that produces a phototoxic effect on the RPE and neuroretina.7 8 9
More recently, researchers have advocated infracyanine green (IFCG) as an alternative vital stain.6 10 11 IFCG differs from ICG, in that it contains no iodine and can be dissolved directly into a 5% glucose solution, avoiding a hypo-osmotic preparation. It is therefore postulated that IFCG is less likely to produce cell damage than ICG, and a brief experimental report suggests that this is the case.6
There are theoretical reasons why IFCG is less likely to produce retinal phototoxicity than ICG, as absorption has been found to be lower between 600 and 700 nm.12 It is therefore postulated that IFCG is less likely to produce phototoxic damage when used in conjunction with surgical endoillumination. Despite a potentially safer absorption spectrum, a brief report by Haritoglou et al.13 showed retinal damage in cadaveric eyes exposed to IFCG and light.
The experiments presented in this article were designed to test the safety of IFCG and to compare findings with those in experiments using ICG. To do this, we adapted a cell culture model used to test the safety of ICG.14 If IFCG is to be used clinically, then further safety testing is certainly worthwhile.
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Methods |
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Cell Culture
Experiments were undertaken on RPE and glial cell lines, by using culture techniques described previously.14 Briefly, human RPE cells (ARPE-19, passage 23; American Type Culture Collection, Manassas, VA) were cultured in Ham’s F-10 medium (pH 7.4; Sigma-Aldrich, Poole, UK), supplemented with 2 mM glutamine, 25 mM/L HEPES, 10 IU/mL penicillin, 10 µg/mL streptomycin, and 15% heat-inactivated fetal calf serum (Sigma-Aldrich). Glial cell experiments were undertaken in a spontaneously immortalized Müller cell line (passage 57) obtained from a 68-year-old female donor. Cells were grown in Dulbecco’s modified Eagle’s medium containing a commercial cell culture medium (glutaMAX; Invitrogen-Gibco, Paisley, Scotland, UK), supplemented with 2 mM glutamine, 10 IU/mL penicillin, 10 µg/mL streptomycin, and 10% heat-inactivated fetal calf serum (Sigma-Aldrich). The isolation and characterization of these cells is described elsewhere.15 Cells were grown to confluence in an incubator with a humidified atmosphere of 5% CO2, 95% air at 37°C and were trypsinized and seeded into 96-well flat-bottomed plates (TPP, Switzerland) and 8-well chamber slides (Nunc, Inc., Naperville, IL).
Dye Exposure
Once cells reached confluence, the culture medium was removed and 50 µL IFCG was placed in each well. After 5 minutes, IFCG was removed with a suction pipette, and the wells were rinsed three times with phosphate-buffered saline (PBS; sodium chloride 120 mM, potassium chloride 2.7 mM, phosphate buffer salts 10 mM [pH 7.4] at 25°C; 294 mOsM; Sigma-Aldrich). The culture medium was then replaced, and cells were returned to the incubator. Cells were also exposed to 0.15% trypan blue, as this has been used in conjunction with IFCG—so-called double staining.10 Monolayers undergoing double staining were stained first with trypan blue, then IFCG, to simulate the reported clinical use of these agents.10 Cells were rinsed three times after exposure to each agent. Other cells were also exposed to trypan blue alone, to determine whether the cellular response to double-staining more closely matched that of IFCG, trypan blue, or otherwise. After dye exposure the culture medium was replaced, and cell viability was measured at days 1, 5, and 15.
Negative control cultures were cells exposed to PBS. These had undergone identical rinsing routines. At least 16 wells were used as the negative control for each 96-well plate. The positive control was cells killed by exposure to 30% methanol for 1 minute. Previous experiments have shown that the cell culture grade trypan blue is comparable to medical grade trypan blue (DORC, Zuidland, The Netherlands) used clinically.14
Light Exposure
Experiments using IFCG were repeated in the presence of illumination from a surgical endolight powered by a medical, 300-W, xenon light source with a total lamp output of 5000 lumens. A xenon light source was selected over the more commonly used a quartz halogen light source, because it provided more stimulus for IFCG excitation and more chance of detecting IFCG-mediated phototoxicity.16 Some of the major manufacturers of vitrectors are now offering the option of xenon light sources for endoillumination, because these units provide brighter illumination than halogen. This light source was coupled to a standard fiber-optic endoilluminating "light pipe" with a 45% illumination transmittance and an angular spread of 8 NA (manufacturer’s information). The cone angle of the light was measured in PBS containing 3% fat-free milk and found to be 36°. The output from the endolight was measured with a light meter (Graseby Optronics, Orlando, FL) and found to be 4.86 x 10–2 mW/mm2 at 5 mm. Cells were first exposed to IFCG for 5 minutes and then were rinsed only once with PBS, leaving a residual stain on the monolayer. The wells were then filled with PBS, and the endolight was held in a retention stand, 5 mm above the monolayer for 5 minutes. Unilluminated cells exposed to PBS acted as the main negative control, but controls were also provided in the form of cells incubated with PBS and exposed to light in the same manner. All cells were exposed to ambient illumination for the duration of the experiment. After illumination, wells were rinsed twice with PBS, and the growth medium was replaced. Cells were then returned to the incubator, and viability was measured at day 1.
Cell-Viability Assays
A quantitative measure of cell viability was made with an MTT assay, based on our study of ICG.14 There is a theoretical risk that residual IFCG and trypan blue will interfere with the assay of the blue formazan reaction product that is measured with the MTT assay. Although previous control experiments14 with ICG and trypan blue suggested that this might not occur, preliminary analysis of results with IFCG suggest that it produces more residual staining than ICG and that this interferes with the MTT assay. To establish whether this was the case, cells were exposed to IFCG and rinsed in the usual manner; then, MTT was added. Immediately after this the optical density was measured with a microplate reader, so that results reflected the effect of residual IFCG, as the cells had not had time to produce the formazan reaction product. The results of this preliminary analysis are shown in Figure 1 . Monolayers had decreased optical density, relative to the negative control, that was statistically significant for up to 24 hours in RPE cells and 15 days in glia. This interaction could produce a falsely low reading that might simulate cell damage.
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A qualitative assessment of cell viability was made with a commercially available fluorescent live–dead probe (Molecular Probes, Eugene, OR). Live cells were stained green with calcein-AM (CAM), dead cells were stained red with ethidium homodimer-1 (EH-1). Cells were grown in eight-well chamber slides and underwent the same exposure routines as those in the 96-well plates. Negative control cultures were also included on each chamber slide, but not all chambers included a positive (dead-cell) control, given the uniformity of dead-cell staining. For light exposure experiments, only the cells immediately underneath the endolight were examined, as the area of the well extended beyond the area of illumination. Staining routines, microscopy, and photography were as described previously.14
Statistics
Cells were defined as having reduced viability if their optical density on the MTT assay was below two standard deviations of the negative control. Group comparisons were also made using the unpaired t-test, with the Welch correction, if assumption tests showed a significant difference in the standard deviation of the groups. Nonparametric (Mann-Whitney) tests were used if normality tests (Kolmogorov-Smirnov) indicated that data had a non-Gaussian distribution. P 
0.05 was considered significant. The data presented as confidence intervals (CIs) are expressed in relation to the mean of the negative control for each experiment, which was set at 1.0. The 95% CI shows the difference between the mean result of the negative control (cells exposed to PBS) and the comparison group, unless otherwise stated.
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Results |
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Staining with CAM and EH-1 did not reveal any consistent difference from the negative control.
Glial Cells
MTT Assay.
MTT assays of Müller cells exposed to IFCG over a range of concentrations from 0.03125% to 0.5% did not show toxicity, in that all data points were within the predetermined limit. However, the 0.25% and 0.5% group were lower than the negative control (Fig. 5) . This difference was significant in the 0.5% group (P = 0.0003; CI 0.10–0.31), but not the 0.25% group (P = 0.218; CI –0.14729 to 0.57). There was a tendency toward a linear decrease in viability with increasing concentration, but this association was not close (R2 = 0.5226).
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Discussion |
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Another response has been to try other agents that may prove safer.21 Hence, Stalmans et al.10 recently reported the clinical use of IFCG. As already noted, this agent is similar to ICG but can be dissolved directly into glucose, avoiding hypo-osmotic preparations. It is therefore possible that it may be less toxic. Preliminary ex vivo work by the same researchers did not show significant damage in RPE cells exposed to IFCG, although average results were lower than the control.6 Alternatively, IFCG’s chemical similarity to ICG may mean that it has the same effect on retinal tissue, either in isolation or in combination with endoillumination. As is the case of ICG, the clinical use of IFCG has predated extensive ex vivo experiments. We sought to investigate this issue by performing safety testing of IFCG, using RPE and glial cell cultures.
The main findings were as follows. There was no evidence of acute or delayed damage when RPE cells were exposed to IFCG up to a concentration of 0.5%. This was also true when IFCG was used in conjunction with trypan blue, referred to as double staining. The combination of IFCG and endoillumination significantly reduced RPE viability relative to the negative control, but not relative to cells that had undergone illumination without IFCG exposure. Glial cells were more vulnerable, with 0.5% IFCG producing acute damage that persisted at day 5, but had recovered by day 15. There was no apparent phototoxicity when glial cells were exposed to IFCG and endoillumination.
Cell damage was more apparent with the MTT assay than the live–dead fluorescent probe. This was similar to our observations using ICG,14 and other reports that found more damage with the MTT assay than was seen with light and electron microscopy.7 This may suggest that ICG and IFCG have more effect on mitochondrial function than on cytoplasmic esterase activity and cell wall integrity, as the former is measured using the MTT assay and the latter two are assessed using the live–dead probe.
Another possibility is that IFCG interferes with the MTT assay. Our earlier studies14 of ICG suggested that this was not the case, but there is a theoretical risk given the spectral overlap of ICG and the blue formazan reaction product measured by the MTT assay. Unlike the situation with ICG, preliminary studies using IFCG suggested that higher concentrations interfere with the MTT assay. For this reason, the assay was adapted by subtracting a baseline reading from the final reading, so that the effect of any residual dye was negated. MTT assays of cells exposed to IFCG without this adjustment may produce falsely low estimates of cell viability.
It is not known why IFCG produced more surface staining than ICG. It is possible that the saline-based fluid used to rinse the wells (PBS) produced flocculation of residual IFCG on the monolayers. A glucose solution may have been more effective in rinsing the monolayers, but would differ from the saline-based solutions used clinically for vitreous infusion. Internationally, surgeons select from several proprietary brands of intraocular solutions, and those originally designed for intravenous use, such as Hatmann’s solution. In the absence of a universal intraocular infusion, PBS was selected because it is similar to most solutions used clinically and is well established for use with cell cultures.
Some other findings of this study are more similar to our observations with ICG.14 Both experiments showed that glial cells were more vulnerable to damage than RPE cells, and that the maximum damage appears at day 5.The viability of glial cells exposed to IFCG and endoillumination was not reduced relative to those exposed to IFCG alone, unlike the situation with ICG. One hypothesis is that the shift in the absorbance spectrum of IFCG relative to ICG makes it less likely to mediate phototoxic damage.12
Initial inspection of the results in RPE suggests a phototoxic effect, with the viability of cells exposed to IFCG with illumination significantly reduced relative to the negative control (PBS without illumination). Closer inspections of these results suggests otherwise, as viability was not reduced relative to the PBS with illumination group. Although the difference between the PBS with and without illumination groups did not reach significance, the data in Figure 4 suggest that illumination was primarily responsible for cell damage, and this effect was not significantly altered by the presence of IFCG.
Although the statistical tests do not support the conclusion that the xenon light source was damaging to RPE, this trend merits further investigation, particularly as these light sources have recently been introduced for endoillumination during vitreoretinal surgery. Compared with halogen light sources, xenon produces much more intense output over a broad spectral range. Despite this increased potential for phototoxic damage, it is noted that our previous study14 did not show reduced viability in the control groups that underwent illumination without ICG, despite using the same xenon light source and illumination distance. In the present study, a longer exposure time was used; 5 minutes instead of 1. This suggests a dose–response effect.
Yam et al.8 also investigated ARPE-19 cells exposed to light and ICG. Although light alone produced less damage than it did in combination with ICG, the expression of the c-Fos gene was increased in BSS control groups exposed to a standard surgical endolight, suggesting cell damage. It was not our intent to investigate the effect of xenon light sources on cells in culture, but it would be interesting to repeat these observations with the interposition of optical filters in the light path and to compare halogen and xenon light sources directly. Direct comparison of both light sources might be problematic, however, as the power sources, filters, and endoscopic probes differ between manufacturers, making standardization difficult. Given what is known about the spectral absorption of IFCG12 and emission of these light sources,16 22 23 24 halogen is probably less likely to cause phototoxic damage than xenon.
The purpose of this study was instead to perform safety testing of IFCG. A cell culture model was selected for several reasons. Cell culture affords relatively precise control of experimental parameters, including those that might not be possible to control clinically or in animals. It has the obvious advantage that it does not subject humans or animals to potentially damaging conditions and is certainly appropriate for preliminary testing. This method also allows individual cell lines to be studied. The two chosen are probably the most relevant, as clinical and experimental reports suggest that RPE may be damaged by ICG,5 as may Müller cells.9 Müller cells are also in intimate contact with dyes that stain the ILM. An important disadvantage of a cell culture model is that it does not fully replicate the situation that occurs in vivo, and findings cannot be directly extrapolated to the clinical environment. It is possible also that the immortalized cell lines used in this study behave differently from those in vivo and may be more robust than primary cell cultures. This latter fact may mean that our findings tend to underestimate the potential damage caused by IFCG.
In this study, we repeated several experiments that were undertaken on cells exposed to ICG,14 but used IFCG instead. A comparison of both these studies is summarized in Table 1 . One notable difference was that there was no evidence of IFCG-mediated phototoxicity, despite the longer illumination times. Although IFCG avoids the hypo-osmolarity of ICG preparations, it remains damaging to glial cells when used at higher concentrations.
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Acknowledgements |
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Footnotes |
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Disclosure: T.L. Jackson, Keeler Instruments UK (F); B. Vote, None; B.C. Knight, None; A. El-Amir, None; M.R. Stanford, None; J. Marshall, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Timothy L. Jackson, Academic Department of Ophthalmology, The Rayne Institute, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK; tim.jackson{at}nhs.net.
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