July 2005
Volume 46, Issue 7
Free
Physiology and Pharmacology  |   July 2005
Chemical Toxicity of Indocyanine Green Damages Retinal Pigment Epithelium
Author Affiliations
  • Hiroshi Ikagawa
    From the Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan; the
  • Masahiko Yoneda
    Aichi Prefectural College of Nursing and Health, Nagoya, Aichi, Japan; the
  • Masayoshi Iwaki
    From the Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan; the
  • Zenzo Isogai
    Department of Dermatology, National Center for Geriatrics and Gerontology, Aichi, Japan; the
  • Kaoru Tsujii
    Nanotechnology Research Center, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan; and the
  • Ritsuko Yamazaki
    Kao Corporation, Sumida-ku, Tokyo, Japan.
  • Tetsurou Kamiya
    Kao Corporation, Sumida-ku, Tokyo, Japan.
  • Masahiro Zako
    From the Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan; the
Investigative Ophthalmology & Visual Science July 2005, Vol.46, 2531-2539. doi:10.1167/iovs.04-1521
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Hiroshi Ikagawa, Masahiko Yoneda, Masayoshi Iwaki, Zenzo Isogai, Kaoru Tsujii, Ritsuko Yamazaki, Tetsurou Kamiya, Masahiro Zako; Chemical Toxicity of Indocyanine Green Damages Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2005;46(7):2531-2539. doi: 10.1167/iovs.04-1521.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. To investigate the chemical toxicity of indocyanine green (ICG).

methods. Surface active and precipitating effects of ICG were quantitatively analyzed by determining bovine serum albumin dissolved or precipitated in the presence or absence of salt solutions. The effects of precipitation on serum and cytotoxicity were evaluated by measuring the viability of retinal pigment epithelium (RPE) in vitro.

results. ICG functioned as a surfactant without salts, but with nearly physiological concentrations of balanced salts, it functioned as a unique precipitating factor. This rendered the soluble molecules in serum that are indispensable in the culture of RPE cells insoluble during a 12-hour exposure, resulting in poor cell survival in vitro. Cytotoxicity in serum-free medium was also shown during brief exposures.

conclusions. Commonly used dosages of ICG directly applied into the vitreous cavity, which not only contact the retina but also invade the space between the retina and RPE through a macular hole, may be sufficient to induce retinal disorders after the damaging chemical property of ICG has disturbed the microenvironment.

Before 1990, there was no treatment available for macular holes. Kelly and Wendel 1 first reported that vitreous surgery improved visual acuity in some eyes with macular holes, and since then an effective internal limiting membrane (ILM) peeling technique has been used to improve outcomes. 2 However, it is generally very difficult to perform efficient and complete removal of the ILM, because the ILM is thin and transparent. After indocyanine green (ICG) was introduced for selective staining of the ILM, 3 ICG-facilitated ILM peeling appeared to be beneficial, because it facilitated functional and anatomic success. 4 5 6 7 8 9 10  
Although some reports have shown no negative effects and/or excellent anatomic results and visual retinal function after ICG-assisted ILM peeling for macular hole surgery, 9 11 12 13 14 15 16 recent clinical outcomes have clearly shown adverse effects of ICG on retinal function despite improved anatomic macular hole closure. Inadvertent removal of some small amounts of retinal elements peeled away with the ILM have suggested actual physical retinal damage. 17 18 19 Patients who had undergone ICG-assisted vitrectomy for an idiopathic macular hole were analyzed and compared with patients operated on without the use of ICG. 20 It was shown that functional outcomes in patients with ICG-assisted vitrectomy were significantly lower, and that the incidence of visual field defects was 50%. No statistically significant improvement of postoperative visual acuity, but rather unexpected visual field defects, were revealed after ICG-assisted ILM peeling. 18 21 Unusual atrophic changes in the retinal pigment epithelium (RPE) at the site of the macular hole and undesirable postoperative visual acuity were reported, despite successful anatomic closure of the macular hole. 22 After macular hole surgery with ICG-guided ILM peeling, hypertrophic and atrophic RPE changes were apparent. 23 24  
Laboratory data also show that ICG has some undesirable effects. Brief exposure of cultured RPE cells to ICG resulted in decreased mitochondrial enzyme activity. 25 Morphologic and functional damage of the retina was evaluated after intravitreous ICG injection, and functional damage evaluated by electroretinography was shown, even at low dosages of ICG (0.025 mg/mL) without any apparent morphologic damage. 26 ICG injected into the vitreous cavity showed b- and a-wave amplitude, and latency abnormalities, suggesting impairment of retinal function. 27 ICG caused cytotoxicity in cultured RPE cells in a dose- and time-dependent manner, and necrotic cell death occurred. 28 The incubation of RPE cells with ICG increased the number of apoptotic RPE cells. 29 ICG injected into the subretinal space caused destructive degeneration of photoreceptors and RPE, 30 and apoptosis was confirmed in photoreceptor layers. 31 Subretinal ICG injection resulted in damage to the outer nuclear layer, photoreceptor inner and outer segments, and RPE. 32  
Although all these data obviously indicate toxic effects of ICG on retinal function, we hardly consider these results to be caused by ICG alone. ICG-induced cytotoxicity in cultured RPE was reduced with the removal of sodium from the solvent. 33 Toxic effects of ICG on cultured RPE cells have been related to osmolarity of the solvent. 34 Factors such as sodium or osmolarity modulate the degree of toxicity, implying that some chemical mechanisms may be involved in the effects. The dye ICG is an amphipathic molecule and has both hydrophilic (two anions and one ammonium cation) and hydrophobic (mainly aromatic series) properties, suggesting that ICG is a surface active compound and may have unknown chemical and toxic properties that affect the retina. 
In the present study, we demonstrated unique chemical properties of ICG—acting as a precipitating factor in the presence of salts, but as a surface active compound in the absence of salts. Our data support the proposition that the clinically used dosage of ICG in buffered salt conditions directly applied into the vitreous cavity and subretinal space through a macular hole may be sufficient to induce retinal disorder. 
Materials and Methods
Solubilization of the Denatured-BSA
BSA (Sigma-Aldrich, Tokyo, Japan) was dissolved in H2O at a concentration of 5 mg/mL. BSA solution (100 μL) was put into 14 new microtubes and heated at 95°C for 30 minutes. After centrifugation at 10,000g for 30 minutes, precipitates were collected. Less than 0.1 μg/mL BSA was detected in the supernatant. An aqueous solution of 30 μL sodium dodecyl sulfate (SDS) or ICG in various concentrations was added to the precipitates. Vortex mixing was undertaken for 20 seconds every 10 minutes for 1 hour. Next, samples were left at room temperature overnight. After further centrifugation of the samples, 10 μL supernatant was used for SDS polyacrylamide gel electrophoresis (10%). The gel was stained with Bio-Safe (Bio-Rad, Tokyo, Japan) and scanned after drying. 
Precipitation of Dissolved Native BSA by SDS or ICG in Dulbecco’s PBS
BSA solution (100 μL 1 mg/mL in PBS) was prepared in each of 16 microtubes. ICG and SDS were obtained from Daiichi Pharmaceutical (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. SDS or ICG was dissolved at a concentration of 12.5 mg/mL in H2O. With 2.5× dilutions, the highly concentrated solution was adjusted to appropriate concentrations (12.5, 5.0, 2.0, 0.8, 0.3, 0.15, and 0.05 mg/mL). PBS (20 μL 10×), 40 μL H2O, and 40 μL SDS or ICG, adjusted to various concentrations, were added to the tubes. The final concentrations of SDS or ICG were 2.5, 1.0, 0.4, 0.16, 0.06, 0.03, and 0.01 mg/mL. Samples were then incubated at room temperature overnight, after which the solutions were centrifuged at 10,000g for 30 minutes and 10 μL sample supernatants were used for SDS polyacrylamide gel electrophoresis (10%). 
Precipitation of Dissolved Native BSA by ICG in H2O or PBS
BSA (1 mg/mL) was prepared with H2O or PBS solutions. One hundred microliters of each solution was added to new microtubes. ICG was dissolved to a concentration of 5 mg/mL in H2O. The highly concentrated solution was adjusted to appropriate concentrations (5.0, 2.0, 0.8, 0.32, 0.13, and 0.05 mg/mL) at a 2.5× dilution. PBS (20 μL, 10×), and 80 μL ICG at various concentrations was added to the tubes for the PBS experimental series. In the H2O system, H2O was used instead of 10× PBS. The final concentrations of ICG were 2.0, 0.8, 0.32, 0.13, 0.05, and 0.02 mg/mL. Samples were incubated at room temperature overnight, after which the solutions were centrifuged at 10,000g for 30 minutes, and 10 μL of the sample supernatants was used for 10% SDS polyacrylamide gel electrophoresis. 
Measurement of Surface Tension
Distilled water was filtered and deionized before use in the following experiments. Specific resistance of water was always kept at 18.3 MΩ/cm. ICG dissolved in H2O, with or without PBS was diluted to appropriate concentrations. The surface tension and CMC were measured and determined with a single-fiber tensiometer (model K14; Kruss, Hamburg, Germany). 35 36 A series of measurements was performed, mainly with a range of ICG concentrations of 0.01 to 1.0 (wt). Surface tension was measured after the plate had stood at room temperature for 2 hours. As a reference, we performed similar measurements using appropriate concentrations of SDS solutions, with or without PBS. 
Precipitation of Dissolved BSA by ICG in Various Concentrations of Different Balanced Salt Solutions
ICG was dissolved in H2O at a concentration of 25 mg/mL. BSA was dissolved in Dulbecco’s phosphate-buffered saline (PBS), BSS (Alcon, Fort Worth, TX), Hanks’ BSS (Ca2+, Mg2+ free, including NaHCO3), or Dulbecco’s modified Eagle’s medium (DMEM) at a concentration of 0.5 mg/mL, and 10-μL solutions of each composition were put into each of five tubes. Ninety microliters of different concentrations of these buffers diluted with H2O were added to each BSA-containing tube and the buffers with 11.1%, 33.3%, 55.5%, or 77.7% (vol/vol) and 100% (no dilution with H2O), were prepared. ICG solution (10 μL) was added to each 90-μL of buffer solution. Finally, buffer content in the solutions was adjusted to 10%, 30%, 50%, 70%, and 90% (vol/vol) of the original buffer. As a control, H2O was used instead of buffers. Samples were left at room temperature overnight. After incubation, samples were centrifuged at 10,000g for 30 minutes. After the supernatant was removed, 20 μL SDS sample buffer was added and then applied to 10% SDS polyacrylamide gel electrophoresis. 
Precipitation of Serum Proteins by ICG in DMEM
DMEM including 10% (vol/vol) fetal calf serum was prepared and 300 μL were transferred into each of eight microtubes. ICG was dissolved at a concentration of 12.5 mg/mL in H2O. The highly concentrated solution was adjusted to appropriate concentrations (12.5, 5.0, 2.0, 0.8, 0.3, 0.15, and 0.05 mg/mL) with a 2.5× dilution. Seventy-five microliters ICG of various concentrations was added to each tube. Final concentrations of ICG in the samples were 2.5, 1.0, 0.4, 0.16, 0.06, 0.03, and 0.01 mg/mL. Samples were incubated at 37°C overnight; after which the medium was spun and filtered through 5-μm pore size filters (Ultrafree-MC; Millipore Co., Bedford, MA). Precipitation on the filters was resolved with 50 μL SDS sample buffer, and 10 μL of this was then applied to 10% SDS polyacrylamide gel electrophoresis. 
Human RPE Culture
Human ARPE19 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in 1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s and Ham’s F12 medium (DF; Invitrogen-Gibco, Rockville, MD) containing 3 mM l-glutamine, 10% fetal bovine serum (Invitrogen-Gibco, Grand Island, NY), and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin sulfate; Invitrogen-Gibco). 37 In three passages, RPE cells grew to 70% confluence at 37°C with 5% CO2 balanced with air for ICG treatments. 
Cell Viability Evaluation
Cell viability was assessed by MTS colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) (Promega Corp, Madison, WI). This quantitative assay detects only living cells. 38 In our experiments, 3 × 103 cells in 100 μL culture medium at appropriate ICG concentrations (0, 0.01, 0.03, 0.06, 0.16, 0.4, 1.0, and 2.5 mg/mL) were seeded into each well of a 96-well plate. After 12 hours of culture, the culture medium was removed, and the cells were rinsed with PBS. For brief (0–60 minutes) exposure to ICG, cells were incubated in culture medium without fetal bovine serum and then were washed twice and seeded onto a 96-well plate for another 12 hours of culture in regular culture medium without ICG. The MTS assay was performed by adding 100 μL culture medium and 20 μL MTS to each well. After 3 hours of incubation at 37°C, the absorbance at 490 nm was recorded. Five wells were evaluated for each ICG concentration. 
Quantitative Analysis
After appropriate BSA amounts (1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 μg) were applied to 10% SDS-polyacrylamide gel electrophoresis, gels were stained with Bio-Safe (Bio-Rad, Tokyo, Japan) for the same period as the gels used for the experimental samples. An image-analysis program (NIH image; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) was used to measure each band after drying. We evaluated each targeted BSA band according to these standardized densities. 
Statistical Analysis
All results in experiments repeated at least four times are given as the mean ± SE. 
Results
Surface-Active Effects of ICG in H2O
Surface-active effects of ICG were evaluated in salt-free conditions by comparing with SDS as a control. Solubilities of denatured-BSA at each concentration of SDS or ICG in H2O after standing overnight are shown in Figure 1after standing overnight. Each band, stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis, represented the amount of dissolved denatured-BSA in H2O (Fig. 1A) . Quantitative analysis was performed for the density of each band measured by NIH image software, according to the standardized denatured-BSA bands (Fig. 1B) . ICG at 0.32 mg/mL in H2O showed the maximum surfactant effect, whereas SDS showed a simple increasing one that depended on concentration. Dissolved denatured-BSA for the SDS lane reached a plateau of more than 0.8 mg/mL. 
Precipitating Effects of ICG in PBS
Precipitating effects of ICG in saline conditions were confirmed by measuring the reduced solubility of dissolved BSA in PBS after the solution was allowed to stand overnight (Fig. 2) . Initially, 5 μg BSA was dissolved in each lane before the application of SDS or ICG. Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis showed the remaining amount of dissolved BSA with each concentration of SDS or ICG (Fig. 2A) . Quantitative analysis was performed from the density of each band measured by NIH Image software according to the standardized dissolved-BSA bands (Fig. 2B) . ICG of more than 0.16 mg/mL in PBS showed striking precipitating effects, depending on concentration, while SDS showed no precipitating effects. 
The reduced solubility of dissolved BSA due to the precipitating effects of ICG after the solution was allowed to stand overnight is shown in Figure 3 . Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represented the remaining amount of dissolved BSA in H2O or PBS with each concentration of ICG (Fig. 3A) . Initially, 5 μg BSA was dissolved in each lane before the application of ICG, and then further quantitative analysis was performed from the density of each band measured by the NIH Image software according to the standardized denatured BSA bands (Fig. 3B) . ICG of more than 0.13 mg/mL in PBS showed remarkable precipitating effects depending on concentration, whereas ICG in H2O showed no precipitating effects. 
Surface tension of SDS or ICG dissolved in H2O was measured with a single fiber tensiometer (model K14; Kruss). The measured surface-tension values of SDS or ICG in H2O clearly decreased depending on their concentrations and showed an obvious CMC (Fig. 4) . These results clearly indicate that ICG is a surface-active compound with medium activity. Microscopic examinations showed that these solutions were always transparent without any precipitation. Surface tension of SDS or ICG dissolved in PBS was also measured. Surface tension of SDS in PBS decreased depending on concentration and showed CMC; however, that of ICG showed ambiguous values without indicating any clear CMC. Microscopic examinations showed that SDS solutions were always transparent without any precipitation, but insoluble ICG precipitations were identified in all ICG solutions. All CMC values determined are summarized in Table 1
Effect of Various Balanced Salt Solutions in Almost Physiologic Concentrations on Precipitation of ICG
Precipitating effects of ICG in various concentrations of balanced saline solutions were examined by measuring precipitated BSA after standing overnight. Dulbecco’s PBS, BSS (Alcon), Hanks’ BSS (Ca2+ and Mg2+ free, including NaHCO3), and DMEM were the balanced salt solutions. Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis showed BSA precipitated with 2.5 mg/mL of ICG (Fig. 5A) . Quantitative analysis was also performed from the density of each band measured by NIH image software, according to the standardized dissolved BSA bands. Similar acute ascending precipitations of BSA were seen at 70% and above with all balanced salt solutions, whereas moderate increased precipitation at 50% had already been observed in DMEM (Fig. 5B)
Effect of Serum Proteins Coprecipitated by ICG on the Viability of RPE Cells In Vitro
Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis showed coprecipitated molecules derived from 10 μL fetal calf serum in DMEM under various ICG concentrations after standing overnight (Fig. 6A) . Some bands increased linearly, depending on ICG concentration (arrowheads), but some bands that were coprecipitated at low concentrations showed no changes at higher concentrations (arrows). Quantitative analysis was performed from the density of each band measured by NIH Image software, according to the standardized dissolved BSA bands. Total coprecipitated molecules increased, depending on ICG concentration, especially if it was more than 0.4 mg/mL. Next, we evaluated the viability of RPE cells with each ICG concentration. Viability greatly decreased, depending on ICG concentration, especially if it was more than 0.4 mg/mL, and correlated closely with precipitation data (Fig. 6B) . Thus, depending on the ICG concentration, the coprecipitation of desirable molecules of fetal calf serum in DMEM may result in the reduced viability of RPE. 
Cytotoxicity of ICG for a Brief Exposure Evaluated by Viability of RPE In Vitro
Viabilities of RPE cells after contact with ICG at several concentrations for several brief periods are shown in Figure 7 . Viabilities were reduced, depending on the increase in ICG concentration, at all exposure times except 0 minutes. Cells were incubated in serum-free culture medium for brief exposure to ICG, to evaluate the cytotoxicity of ICG without serum interference. Viabilities with 0 mg/mL ICG decreased, depending on the increase in exposure time, suggesting detrimental effects caused by serum-free conditions, and these were regarded as a control for each exposure time. The culture medium was immediately removed by centrifugation after contact with ICG medium for 0 minutes of incubation. 
Discussion
The present study showed that the concentrations of ICG used clinically are hazardous for retina and RPE in association with salts. Damage increased, depending on the ICG concentrations applied, 26 27 28 29 30 31 39 40 whereas removal of sodium from an ICG solution reduced toxicity. 33 Generally, ICG is dissolved in sterile water and then diluted with balanced saline solution before use in surgery. The final concentrations of ICG reported to have been used are various, such as 0.25 to 1.25, 7 41 0.5, 18 20 23 42 0.625, 3 1.0, 22 25 1.25, 43 2.5, 10 44 45 or 4.16 mg/mL, 8 but in most cases, the concentration was 5.0 mg/mL. 4 5 6 11 17 19 21 24 32 46 47 48 49 The accompanying balanced saline solution dilution ratios for each preparation was 50% to 90%, 9 50%, 45 60%, 21 75%, 8 43 or 80% 19 22 23 25 48 49 ; but in most cases it was 90%. 4 5 7 11 20 24 32 42 Our recent data suggest that an ICG concentration of more than 0.4 mg/mL and a balanced saline concentration of 70% or more create precipitable conditions; implying that most of the reported cases involved risky conditions. When ICG solution was injected into the vitreous cavity, it became diluted to a considerable degree. If the 0.1 mL of 5.0 mg/mL ICG was diluted to 2.0 mL of hypothesized posterior vitreous hemisphere during the operation, the ratio of dilution would be 1:20, resulting in a final concentration of 0.25 mg/mL, which still showed precipitating effects, as demonstrated in the present study. Safety levels suggested by recent data are an ICG concentration of less than 0.05 mg/mL and a balanced saline concentration of less than 50%. Furthermore, viability of RPE was reduced and the death of RPE cells increased, depending on the exposure time to ICG. 28 In the present study, cytotoxicity by ICG with physiological salts in serum-free conditions was shown for short exposure periods except immediately after removal of ICG. In this study, we used serum-free conditions, because ICG in the balanced saline solution used for staining in ILM peeling contained no serum, reflecting reality more accurately. Exposure of RPE cells to ICG for more than 5 minutes at concentrations of more than 0.4 mg/mL reduced cell viability, implying that a quick elimination of ICG from the vitreous cavity including the macular hole is clinically advisable. As persistent ICG fluorescence has been found localized in the central macula up to 8 to 9 months after ICG-assisted ILM peeling for macular hole surgery, 44 49 this severe condition is a lasting one and may be intolerable to the retina. 
Less damage was observed after epiretinal ICG injection, but subretinal ICG injection resulted in more severe damage to the outer nuclear layer, the inner and outer photoreceptor segments, and RPE in rabbit eyes. 32 Subretinal ICG injection induced apparent morphologic damage of retina, especially to the outer retinal layer in a dose-dependent manner, and apoptosis was confirmed in the photoreceptor layers. 31 ICG injected into the subretinal space in rabbits caused destructive degeneration of photoreceptors and RPE cells. 30 No histologic damage was detected in retina that had no macular hole, even at higher doses of ICG followed by illumination. 50 ICG-assisted peeling of the epiretinal membrane in eyes without a macular hole led to an expedited resolution of macular pucker and an improvement in visual acuity without clinical evidence of ICG toxicity. 47 Morris et al. 51 commented that ICG’s effects on functional outcomes of macular pucker surgery were satisfactory. Retina with a single retinal hole exposed to ICG concentrations used in human vitreoretinal surgery had greater RPE atrophy and outer retinal degeneration than eyes the underwent the same surgery but without ICG. 40 These laboratory data support the observation that ICG causes cytotoxicity in cultured human RPE. 25 28 29 33 34 39 ICG injection into the subretinal space through a macular hole during this type of operation may be the primary trigger that is followed by retinal disorder. This clearly indicates that a surgical technique that can avoid the damaging effects of ICG is a necessity. However, as adverse effects have also been reported after ICG-assisted ILM peeling in patients with macular pucker or epiretinal membrane, 42 48 further investigations are necessary to elucidate this inconsistency. 
CMC is the most important characteristic in evaluating a surfactant. We determined CMC as 0.42 wt % for ICG and 0.18 wt % for SDS, respectively, in H2O. ICG decreased the surface tension depending on its concentration and reached 37 dyn/cm at its CMC, demonstrating that ICG had surface-active effects in H2O. The CMC in SDS shown herein is in agreement with that already reported, 35 indicating the reliability of the method used in our study. The CMC of 0.42 wt % and surface tension of 37 dyn/cm corresponded to a medium surface activity, although it was less than that of SDS. In contrast, surface tensions of ICG with PBS tended to vary with every concentration and showed no obvious CMC. With the addition of PBS into ICG dissolved in H2O, ICG became insoluble and precipitation was found at all concentrations examined. Once precipitation formed in PBS, surface tension data for ICG became meaningless. 
A surfactant effect of ICG was observed only in H2O, a special situation for actual clinical use. Therefore, this effect must be minor or nonexistent in practice, whereas the precipitating effects result in major toxicity, as the dissolving solution usually contains salts. We hypothesize that the surfactant and precipitating effects may be closely related to each other by unknown mechanisms, and these reactions may be in equilibrium, although the major reaction is toward precipitation in salt solution. 
The surface-active effect of ICG in H2O was supported by surface tension and by the dissolving denatured BSA. Although an increase in dissolved denatured BSA depending on the ICG concentration is understandable, the cause of the decrease in concentrations of more than 0.32 mg/mL is not clear. ICG in H2O may form a complex with the denatured BSA to be dissolved but may precipitate over the threshold of ICG concentration by an unknown mechanism. Molecules, including BSA, that are dissolved may form a complex with ICG with salts and reach some limits for coprecipitation of more than 0.16 mg/mL of ICG concentration. As shown in Figure 8 , PBS, BSS (Alcon), and Hanks’, with and without NaHCO3 showed a significant increase in ICG precipitation at concentrations of ≥0.4 mg/mL, though the degree of increase varied slightly, depending on the solution. Measurement of the osmolarity of the solutions was performed with an osmometer (OM-6030; Daiichi Kagaku, Kyoto, Japan). All solutions were iso-osmotic, and there was no significant difference. This suggests, on the one hand, that some chemical properties of ICG vary at the borderline concentration in the presence or absence of salts. On the other hand, coprecipitating effects of ICG differ depending on the particular molecule, as the amount of precipitation increased with the increase in ICG concentration with some molecules but not with others (Fig. 6A) . Further investigations are necessary to understand this behavior. 
The chemical toxicity of ICG causes initial damage to RPE cells and retinal cells. As the concentration of ICG and the exposure time in the globe become lower and briefer, respectively, the chemical toxicity may pose less risk. Actually, no or fewer negative anatomic or functional effects were described in some recent reports, despite the persisting presence of ICG after surgery. We hypothesize that one of the ultimate conditions that may finally provoke death of the damaged cells is additional physical damage such as phototoxicity derived from certain wavelengths, osmotic effects, and air. 34 39 40 50 52 53 Clinical disorders such as loss of visual acuity or visual field defect may vary, depending on the type of damaged cells and the degree of damage. As physical factors are largely influenced by procedures performed by each surgeon, outcomes of macular hole surgery may be variable, as practically reported. However, to prevent these supplementary fatal conditions for RPE and retinal cells, lower concentrations of ICG combined with flushing with balanced saline solution after ILM peeling, no application of ICG in an air-filled eye, and a briefer exposure time to ICG and light are desirable. 
Components of Dulbecco’s PBS and balanced saline solution are similar except for the NaHCO3 and glucose contents. In the case of balanced saline solution, it is important that it include NaHCO3, but this is not the case with PBS. The maintenance of a stable CO2 concentration is critical to keep the pH accurate, and this need is fulfilled by NaHCO3. We used PBS instead of balanced saline for the experiments referred to in Figure 4because we found it hard to keep a stable CO2 environment for evaluating the surface tension by using the single-fiber tensiometer (Kruss). A small pH error may induce an inappropriate surface tension in each sample solution by using this method. Samples of Hanks’ BSS, with and without NaHCO3, showed slight differences in pH, as shown by the color of the solutions at 0 mg/mL in Figure 8 , and they also showed different precipitation profiles. However, PBS and Hanks’ BSS solution without NaHCO3, showed quite similar ICG precipitation profiles. Furthermore, it was practically impossible to make BSA samples with different concentrations of ICG in 100% balanced saline solution for the experiments referred to in Figures 2 and 3by using commercially available balanced saline solution (due to unavailability of concentrated balanced saline solutions). Thus, we used PBS instead of balanced saline for the experiments referred to in Figures 2 3 and 4 . Although there were slight differences in ICG precipitation results, depending on the use of PBS or balanced saline (Fig. 8) , no differences were detected for precipitation with BSA (Fig. 5)
DMEM and Hanks’ BSS with Ca2+ and Mg2+ ions were also used to evaluate the precipitating effects, as two values of ions may influence precipitation. However, we failed to detect any differences between the presence and absence of these ions, suggesting that these ions are not critical for precipitation (data not shown). 
ICG contains iodine to enhance its solubility and it must be dissolved in pure H2O. Infracyanine green does not contain iodine and precipitates in H2O, and 5% glucose is often used as a solvent to avoid a hypo-osmotic preparation. Infracyanine green dissolved in glucose has been used in macular hole surgery, and resulted in a high incidence of anatomic closure, with good visual outcomes, 54 55 whereas the use of infracyanine green followed by illumination induced adverse effects similar to those induced by ICG, but to a varying degree. 56 57 The chemical toxicity of infracyanine green is unclear, but in comparison with ICG, its toxicity may be similar or worse, because infracyanine green contains no iodine, which may result in less solubility in replacing physiological intraocular aqueous humor after surgery. 
Almost all the data in this study indicate the general chemical properties of this reagent. We believe that all medical doctors, not just ophthalmologists, who use this widely accepted reagent should be aware of its inconvenient properties and seek to avoid the side effects of ICG—especially as large dosages of this reagent are generally infused into the systemic circulation to evaluate cardiac and hepatic circulation. ICG damaged RPE cells and may also disable vascular endothelial cells. Induction of coprecipitation of serum proteins was shown in the present study. Although very rare, it has been reported that several patients every year have significant side effects from the use of ICG. The coprecipitating phenomenon of human serum, as shown in this study, may be involved in these incidents, as human serum includes a wide variety of proteins and salts in physiological conditions. Precipitated ICG complexes formed in blood vessels may induce lethal embolization and are especially risky in capillaries, as the blood flow is very slow and the caliber quite thin, and more so in cases of dehydration, as electrolytes and proteins are concentrated. 
In the present study, we showed that the chemical properties of ICG in the presence and absence of balanced salts can show toxicity in both cases. First, BSA was used to show clearly the surface-active and precipitating effects of ICG. Then we indicated that ICG has a coprecipitating effect on a wide variety of serum molecules, an effect that makes it difficult for cells to survive. Practitioners need to understand this chemical behavior when applying ICG, and immediate steps should be taken to prevent further retinal disorder. 
 
Figure 1.
 
Solubility of denatured-BSA in H2O indicates that the surfactant effect depends on the concentration of SDS or ICG. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved, denatured BSA in H2O in various concentrations of SDS or ICG after standing overnight (arrows). (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized denatured BSA bands. ICG at 0.32 mg/mL in H2O shows a maximum surfactant effect, whereas SDS shows an increasing effect, depending on the concentration. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 1.
 
Solubility of denatured-BSA in H2O indicates that the surfactant effect depends on the concentration of SDS or ICG. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved, denatured BSA in H2O in various concentrations of SDS or ICG after standing overnight (arrows). (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized denatured BSA bands. ICG at 0.32 mg/mL in H2O shows a maximum surfactant effect, whereas SDS shows an increasing effect, depending on the concentration. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 2.
 
Reduced solubility of dissolved BSA in PBS indicates that the precipitating effects depend on ICG concentration. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in PBS in various concentrations of SDS or ICG, after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to the standardized dissolved BSA bands. ICG of >0.16 mg/mL in PBS showed a striking precipitating effect, depending on concentration, whereas SDS showed none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 2.
 
Reduced solubility of dissolved BSA in PBS indicates that the precipitating effects depend on ICG concentration. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in PBS in various concentrations of SDS or ICG, after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to the standardized dissolved BSA bands. ICG of >0.16 mg/mL in PBS showed a striking precipitating effect, depending on concentration, whereas SDS showed none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 3.
 
Reduced solubility of dissolved BSA indicates that the precipitating effect depends on the concentration of ICG in PBS. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in H2O or PBS in various concentrations of ICG after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. ICG of more than 0.13 mg/mL in PBS shows a remarkable precipitating effect depending on concentration, whereas ICG in H2O shows none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 3.
 
Reduced solubility of dissolved BSA indicates that the precipitating effect depends on the concentration of ICG in PBS. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in H2O or PBS in various concentrations of ICG after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. ICG of more than 0.13 mg/mL in PBS shows a remarkable precipitating effect depending on concentration, whereas ICG in H2O shows none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 4.
 
Surface tensions of ICG and SDS dissolved in H2O or PBS were measured and showed a clear decrease, depending on the concentration, and obvious CMC. The surface tensions of SDS in PBS decreased, depending on the concentration, and also showed CMC, but those of ICG showed ambiguous values without a clear CMC. The CMCs shown are summarized in Table 1 .
Figure 4.
 
Surface tensions of ICG and SDS dissolved in H2O or PBS were measured and showed a clear decrease, depending on the concentration, and obvious CMC. The surface tensions of SDS in PBS decreased, depending on the concentration, and also showed CMC, but those of ICG showed ambiguous values without a clear CMC. The CMCs shown are summarized in Table 1 .
Table 1.
 
Critical Micelle Concentrations
Table 1.
 
Critical Micelle Concentrations
H2O (wt %) PBS (wt %)
SDS 0.18 0.02
ICG 0.42
Figure 5.
 
Precipitation of BSA dissolved in different concentrations of various balanced salt solutions. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis shows precipitated BSA at each concentration of balanced salt solution with 2.5 mg/mL of ICG after standing overnight. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Each group of histograms represents (left to right) 0%, 10%, 30%, 50%, 70%, and 90% (vol/vol) dilutions of each solution. Quite similar acute ascending precipitations of BSA were seen at ≥70%, with all balanced salt solutions. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 5.
 
Precipitation of BSA dissolved in different concentrations of various balanced salt solutions. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis shows precipitated BSA at each concentration of balanced salt solution with 2.5 mg/mL of ICG after standing overnight. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Each group of histograms represents (left to right) 0%, 10%, 30%, 50%, 70%, and 90% (vol/vol) dilutions of each solution. Quite similar acute ascending precipitations of BSA were seen at ≥70%, with all balanced salt solutions. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 6.
 
Precipitation of indispensable molecules of fetal calf serum in DMEM resulted in decreased viability of RPE depending on ICG concentration. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents precipitated molecules derived from fetal calf serum in DMEM in various concentrations of ICG after standing overnight. Quantitative analysis was performed from the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Some bands increased linearly, depending on ICG concentration (arrowheads), but others that precipitated in low concentrations showed no increase at higher concentrations (arrows). Total precipitated molecules increased depending on ICG concentration, especially if it was more than 0.4 mg/mL. For each concentration, n = 4. All data are represented as the mean ± SE. (B) Viability of RPE sharply decreased depending on ICG concentration. Viability decreases especially if more than 0.4 mg/mL. For each concentration, n = 5. All data are represented as the mean ± SE.
Figure 6.
 
Precipitation of indispensable molecules of fetal calf serum in DMEM resulted in decreased viability of RPE depending on ICG concentration. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents precipitated molecules derived from fetal calf serum in DMEM in various concentrations of ICG after standing overnight. Quantitative analysis was performed from the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Some bands increased linearly, depending on ICG concentration (arrowheads), but others that precipitated in low concentrations showed no increase at higher concentrations (arrows). Total precipitated molecules increased depending on ICG concentration, especially if it was more than 0.4 mg/mL. For each concentration, n = 4. All data are represented as the mean ± SE. (B) Viability of RPE sharply decreased depending on ICG concentration. Viability decreases especially if more than 0.4 mg/mL. For each concentration, n = 5. All data are represented as the mean ± SE.
Figure 7.
 
Viability of RPE cells after exposure to several concentrations of ICG for several brief periods. Viability decreased, depending on increasing ICG concentrations for all exposure periods except 0 minutes. Cells were incubated in medium in serum-free conditions for brief contact with ICG, to evaluate the cytotoxicity of ICG without interference of serum. The culture medium was immediately removed by centrifugation after contact with ICG for 0 minutes of incubation. All data shown are the mean at each concentration (n = 5).
Figure 7.
 
Viability of RPE cells after exposure to several concentrations of ICG for several brief periods. Viability decreased, depending on increasing ICG concentrations for all exposure periods except 0 minutes. Cells were incubated in medium in serum-free conditions for brief contact with ICG, to evaluate the cytotoxicity of ICG without interference of serum. The culture medium was immediately removed by centrifugation after contact with ICG for 0 minutes of incubation. All data shown are the mean at each concentration (n = 5).
Figure 8.
 
ICG in PBS, BSS (Alcon), and Hanks’ with and without NaHCO3 after overnight incubation at room temperature. Precipitation significantly increased in all solutions at an ICG concentration of ≥0.4 mg/mL, though the degree of precipitation varied slightly, depending on each solution. Hanks’ solutions include phenolsulfonphthalein to indicate pH. Slight differences in pH between Hanks’ solutions are shown by a change in color of the solution at 0 mg/mL and are caused by NaHCO3. The precipitation profiles were all different. Arrows: The minimum concentration at which ICG precipitation was detected.
Figure 8.
 
ICG in PBS, BSS (Alcon), and Hanks’ with and without NaHCO3 after overnight incubation at room temperature. Precipitation significantly increased in all solutions at an ICG concentration of ≥0.4 mg/mL, though the degree of precipitation varied slightly, depending on each solution. Hanks’ solutions include phenolsulfonphthalein to indicate pH. Slight differences in pH between Hanks’ solutions are shown by a change in color of the solution at 0 mg/mL and are caused by NaHCO3. The precipitation profiles were all different. Arrows: The minimum concentration at which ICG precipitation was detected.
KellyNE, WendelRT. Vitreous surgery for idiopathic macular holes: results of a pilot study. Arch Ophthalmol. 1991;109:654–659. [CrossRef] [PubMed]
ParkDW, LeeJH, MinWK. The use of internal limiting membrane maculorrhexis in treatment of idiopathic macular holes. Korean J Ophthalmol. 1998;12:92–97. [CrossRef] [PubMed]
KadonosonoK, ItohN, UchioE, NakamuraS, OhnoS. Staining of internal limiting membrane in macular hole surgery. Arch Ophthalmol. 2000;118:1116–1118. [CrossRef] [PubMed]
BurkSE, Da MataAP, SnyderME, RosaRH, Jr, FosterRE. Indocyanine green-assisted peeling of the retinal internal limiting membrane. Ophthalmology. 2000;107:2010–2014. [CrossRef] [PubMed]
Da MataAP, BurkSE, RiemannCD, et al. Indocyanine green-assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for macular hole repair. Ophthalmology. 2001;108:1187–1192. [CrossRef] [PubMed]
GandorferA, MessmerEM, UlbigMW, KampikA. Indocyanine green selectively stains the internal limiting membrane. Am J Ophthalmol. 2001;131:387–388. [CrossRef] [PubMed]
KwokAK, LiWW, PangCP, et al. Indocyanine green staining and removal of internal limiting membrane in macular hole surgery: histology and outcome. Am J Ophthalmol. 2001;132:178–183. [CrossRef] [PubMed]
SheidowTG, BlinderKJ, HolekampN, et al. Outcome results in macular hole surgery: an evaluation of internal limiting membrane peeling with and without indocyanine green. Ophthalmology. 2003;110:1697–1701. [CrossRef] [PubMed]
KwokAK, LaiTY, YuenKS, TamBS, WongVW. Macular hole surgery with or without indocyanine green stained internal limiting membrane peeling. Clin Experiment Ophthalmol. 2003;31:470–475. [CrossRef] [PubMed]
WolfS, ReichelMB, WiedemannP, SchnurrbuschUE. Clinical findings in macular hole surgery with indocyanine green-assisted peeling of the internal limiting membrane. Graefes Arch Clin Exp Ophthalmol. 2003;241:589–592. [CrossRef] [PubMed]
WeinbergerAW, KirchhofB, MazinaniBE, SchrageNF. Persistent indocyanine green (ICG) fluorescence 6 weeks after intraocular ICG administration for macular hole surgery. Graefes Arch Clin Exp Ophthalmol. 2001;239:388–390. [CrossRef] [PubMed]
WeinbergerAW, SchlossmacherB, DahlkeC, HermelM, KirchhofB, SchrageNF. Indocyanine-green-assisted internal limiting membrane peeling in macular hole surgery—a follow-up study. Graefes Arch Clin Exp Ophthalmol. 2002;240:913–917. [CrossRef] [PubMed]
SlaughterK, LeeIL. Macular hole surgery with and without indocyanine green assistance. Eye. 2004;18:376–378. [CrossRef] [PubMed]
HoriguchiM, NagataS, YamamotoN, KojimaY, ShimadaY. Kinetics of indocyanine green dye after intraocular surgeries using indocyanine green staining. Arch Ophthalmol. 2003;121:327–331. [CrossRef] [PubMed]
Da MataAP, BurkSE, FosterRE, 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;111:2246–2253. [CrossRef] [PubMed]
Ben SimonGJ, DesatnikH, AlhalelA, TreisterG, MoisseievJ. Retrospective analysis of vitrectomy with and without internal limiting membrane peeling for stage 3 and 4 macular hole. Ophthalmic Surg Lasers Imaging. 2004;35:109–115. [PubMed]
GandorferA, HaritoglouC, GassCA, UlbigMW, KampikA. Indocyanine green-assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol. 2001;132:431–433. [CrossRef] [PubMed]
HaritoglouC, GandorferA, GassCA, SchaumbergerM, UlbigMW, KampikA. 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. [CrossRef] [PubMed]
NakamuraT, MurataT, HisatomiT, et al. Ultrastructure of the vitreoretinal interface following the removal of the internal limiting membrane using indocyanine green. Curr Eye Res. 2003;27:395–399. [CrossRef] [PubMed]
GassCA, HaritoglouC, SchaumbergerM, KampikA. Functional outcome of macular hole surgery with and without indocyanine green-assisted peeling of the internal limiting membrane. Graefes Arch Clin Exp Ophthalmol. 2003;241:716–720. [CrossRef] [PubMed]
AndoF, SasanoK, OhbaN, HiroseH, YasuiO. 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. [PubMed]
EngelbrechtNE, FreemanJ, SternbergP, Jr, et al. Retinal pigment epithelial changes after macular hole surgery with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol. 2002;133:89–94. [CrossRef] [PubMed]
MaiaM, HallerJA, PieramiciDJ, et al. Retinal pigment epithelial abnormalities after internal limiting membrane peeling guided by indocyanine green staining. Retina. 2004;24:157–160. [CrossRef] [PubMed]
HirataA, InomataY, KawajiT, TaniharaH. Persistent subretinal indocyanine green induces retinal pigment epithelium atrophy. Am J Ophthalmol. 2003;136:353–355. [CrossRef] [PubMed]
SippyBD, EngelbrechtNE, HubbardGB, et al. Indocyanine green effect on cultured human retinal pigment epithelial cells: implication for macular hole surgery. Am J Ophthalmol. 2001;132:433–435. [CrossRef] [PubMed]
EnaidaH, SakamotoT, HisatomiT, GotoY, IshibashiT. Morphological and functional damage of the retina caused by intravitreous indocyanine green in rat eyes. Graefes Arch Clin Exp Ophthalmol. 2002;240:209–213. [CrossRef] [PubMed]
MaiaM, MargalitE, LakhanpalR, et al. Effects of intravitreal indocyanine green injection in rabbits. Retina. 2004;24:69–79. [CrossRef] [PubMed]
HoJD, TsaiRJ, ChenSN, ChenHC. Cytotoxicity of indocyanine green on retinal pigment epithelium: implications for macular hole surgery. Arch Ophthalmol. 2003;121:1423–1429. [CrossRef] [PubMed]
RezaiKA, Farrokh-SiarL, ErnestJT, Van SeventerGA. Indocyanine green induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol. 2004;137:931–933. [CrossRef] [PubMed]
LeeJE, YoonTJ, OumBS, LeeJS, ChoiHY. Toxicity of indocyanine green injected into the subretinal space: subretinal toxicity of indocyanine green. Retina. 2003;23:675–681. [CrossRef] [PubMed]
KawajiT, HirataA, InomataY, KogaT, TaniharaH. Morphological damage in rabbit retina caused by subretinal injection of indocyanine green. Graefes Arch Clin Exp Ophthalmol. 2004;242:158–164. [CrossRef] [PubMed]
MaiaM, KellnerL, de JuanE, Jr, et al. Effects of indocyanine green injection on the retinal surface and into the subretinal space in rabbits. Retina. 2004;24:80–91. [CrossRef] [PubMed]
HoJD, TsaiRJ, ChenSN, ChenHC. Removal of sodium from the solvent reduces retinal pigment epithelium toxicity caused by indocyanine green: implications for macular hole surgery. Br J Ophthalmol. 2004;88:556–559. [CrossRef] [PubMed]
StalmansP, Van AkenEH, VeckeneerM, FeronEJ, StalmansI. Toxic effect of indocyanine green on retinal pigment epithelium related to osmotic effects of the solvent. Am J Ophthalmol. 2002;134:282–285. [CrossRef] [PubMed]
HolmbergK, JonssonB, KronbergB, LindmanB. Surfactants and Polymers in Aqueous Solution. 2002; 2nd ed.John Wiley & Sons West Sussex, UK.
IsraelachviliJN. Intermolecular and Surface Forces: with Applications to Colloidal and Biological Systems. 1992; 2nd ed.Academic Press London, UK.
DunnKC, Aotaki-KeenAE, PutkeyFR, HjelmelandLM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res. 1996;62:155–169. [CrossRef] [PubMed]
MosmannT. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. [CrossRef] [PubMed]
YamHF, KwokAK, ChanKP, et al. Effect of indocyanine green and illumination on gene expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2003;44:370–377. [CrossRef] [PubMed]
CzajkaMP, McCuenBW, II, CummingsTJ, NguyenH, StinnettS, WongF. Effects of indocyanine green on the retina and retinal pigment epithelium in a porcine model of retinal hole. Retina. 2004;24:275–282. [CrossRef] [PubMed]
KwokAK, LaiTY, YewDT, LiWW. Internal limiting membrane staining with various concentrations of indocyanine green dye under air in macular surgeries. Am J Ophthalmol. 2003;136:223–230. [CrossRef] [PubMed]
HaritoglouC, GandorferA, GassCA, SchaumbergerM, UlbigMW, KampikA. The effect of indocyanine-green on functional outcome of macular pucker surgery. Am J Ophthalmol. 2003;135:328–337. [CrossRef] [PubMed]
HorioN, HoriguchiM. Effect on visual outcome after macular hole surgery when staining the internal limiting membrane with indocyanine green dye. Arch Ophthalmol. 2004;122:992–996. [CrossRef] [PubMed]
CiardellaAP, SchiffW, BarileG, et al. Persistent indocyanine green fluorescence after vitrectomy for macular hole. Am J Ophthalmol. 2003;136:174–177. [CrossRef] [PubMed]
TadayoniR, PaquesM, GirmensJF, MassinP, GaudricA. Persistence of fundus fluorescence after use of indocyanine green for macular surgery. Ophthalmology. 2003;110:604–608. [CrossRef] [PubMed]
KusakaS, HayashiN, OhjiM, HayashiA, KameiM, TanoY. Indocyanine green facilitates removal of epiretinal and internal limiting membranes in myopic eyes with retinal detachment. Am J Ophthalmol. 2001;131:388–390. [CrossRef] [PubMed]
SorcinelliR. Surgical management of epiretinal membrane with indocyanine-green-assisted peeling. Ophthalmologica. 2003;217:107–110. [CrossRef] [PubMed]
UemuraA, KandaS, SakamotoY, KitaH. Visual field defects after uneventful vitrectomy for epiretinal membrane with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol. 2003;136:252–257. [CrossRef] [PubMed]
AshikariM, OzekiH, TomidaK, SakuraiE, TamaiK, OguraY. Retention of dye after indocyanine Green-assisted internal limiting membrane peeling. Am J Ophthalmol. 2003;136:172–174. [CrossRef] [PubMed]
GrisantiS, SzurmanP, GeliskenF, AisenbreyS, Oficjalska-MlynczakJ, Bartz-SchmidtKU. Histological findings in experimental macular surgery with indocyanine green. Invest Ophthalmol Vis Sci. 2004;45:282–286. [CrossRef] [PubMed]
MorrisR, WitherspoonCD, KuhnF, TaylorSW, BreaudS. The effect of indocyanine-green on functional outcome of macular pucker surgery. Am J Ophthalmol. 2003;136:778–779.
HaritoglouC, GandorferA, SchaumbergerM, TadayoniR, GandorferA, KampikA. Light-absorbing properties and osmolarity of indocyanine-green depending on concentration and solvent medium. Invest Ophthalmol Vis Sci. 2003;44:2722–2729. [CrossRef] [PubMed]
GandorferA, HaritoglouC, GandorferA, KampikA. Retinal damage from indocyanine green in experimental macular surgery. Invest Ophthalmol Vis Sci. 2003;44:316–323. [CrossRef] [PubMed]
Van De MoereA, StalmansP. Anatomical and visual outcome of macular hole surgery with infracyanine green-assisted peeling of the internal limiting membrane, endodrainage, and silicone oil tamponade. Am J Ophthalmol. 2003;136:879–887. [CrossRef] [PubMed]
RivettK, KrugerL, RadloffS. Infracyanine-assisted internal limiting membrane peeling in macular hole repair: does it make a difference?. Graefes Arch Clin Exp Ophthalmol. 2004;242:393–396. [CrossRef] [PubMed]
JacksonTL, VoteB, KnightBC, El-AmirA, StanfordMR, MarshallJ. Safety testing of infracyanine green using retinal pigment epithelium and glial cell cultures. Invest Ophthalmol Vis Sci. 2004;45:3697–3703. [CrossRef] [PubMed]
HaritoglouC, GandorferA, GassCA, KampikA. Histology of the vitreoretinal interface after staining of the internal limiting membrane using glucose 5% diluted indocyanine and infracyanine green. Am J Ophthalmol. 2004;137:345–348. [CrossRef] [PubMed]
Figure 1.
 
Solubility of denatured-BSA in H2O indicates that the surfactant effect depends on the concentration of SDS or ICG. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved, denatured BSA in H2O in various concentrations of SDS or ICG after standing overnight (arrows). (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized denatured BSA bands. ICG at 0.32 mg/mL in H2O shows a maximum surfactant effect, whereas SDS shows an increasing effect, depending on the concentration. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 1.
 
Solubility of denatured-BSA in H2O indicates that the surfactant effect depends on the concentration of SDS or ICG. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved, denatured BSA in H2O in various concentrations of SDS or ICG after standing overnight (arrows). (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized denatured BSA bands. ICG at 0.32 mg/mL in H2O shows a maximum surfactant effect, whereas SDS shows an increasing effect, depending on the concentration. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 2.
 
Reduced solubility of dissolved BSA in PBS indicates that the precipitating effects depend on ICG concentration. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in PBS in various concentrations of SDS or ICG, after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to the standardized dissolved BSA bands. ICG of >0.16 mg/mL in PBS showed a striking precipitating effect, depending on concentration, whereas SDS showed none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 2.
 
Reduced solubility of dissolved BSA in PBS indicates that the precipitating effects depend on ICG concentration. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in PBS in various concentrations of SDS or ICG, after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to the standardized dissolved BSA bands. ICG of >0.16 mg/mL in PBS showed a striking precipitating effect, depending on concentration, whereas SDS showed none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 3.
 
Reduced solubility of dissolved BSA indicates that the precipitating effect depends on the concentration of ICG in PBS. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in H2O or PBS in various concentrations of ICG after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. ICG of more than 0.13 mg/mL in PBS shows a remarkable precipitating effect depending on concentration, whereas ICG in H2O shows none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 3.
 
Reduced solubility of dissolved BSA indicates that the precipitating effect depends on the concentration of ICG in PBS. (A) Each band stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents the amount of dissolved BSA in H2O or PBS in various concentrations of ICG after standing overnight (arrows). Initially, 5 μg of dissolved BSA was applied in each lane. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. ICG of more than 0.13 mg/mL in PBS shows a remarkable precipitating effect depending on concentration, whereas ICG in H2O shows none. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 4.
 
Surface tensions of ICG and SDS dissolved in H2O or PBS were measured and showed a clear decrease, depending on the concentration, and obvious CMC. The surface tensions of SDS in PBS decreased, depending on the concentration, and also showed CMC, but those of ICG showed ambiguous values without a clear CMC. The CMCs shown are summarized in Table 1 .
Figure 4.
 
Surface tensions of ICG and SDS dissolved in H2O or PBS were measured and showed a clear decrease, depending on the concentration, and obvious CMC. The surface tensions of SDS in PBS decreased, depending on the concentration, and also showed CMC, but those of ICG showed ambiguous values without a clear CMC. The CMCs shown are summarized in Table 1 .
Figure 5.
 
Precipitation of BSA dissolved in different concentrations of various balanced salt solutions. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis shows precipitated BSA at each concentration of balanced salt solution with 2.5 mg/mL of ICG after standing overnight. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Each group of histograms represents (left to right) 0%, 10%, 30%, 50%, 70%, and 90% (vol/vol) dilutions of each solution. Quite similar acute ascending precipitations of BSA were seen at ≥70%, with all balanced salt solutions. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 5.
 
Precipitation of BSA dissolved in different concentrations of various balanced salt solutions. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis shows precipitated BSA at each concentration of balanced salt solution with 2.5 mg/mL of ICG after standing overnight. (B) Quantitative analysis was performed, with the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Each group of histograms represents (left to right) 0%, 10%, 30%, 50%, 70%, and 90% (vol/vol) dilutions of each solution. Quite similar acute ascending precipitations of BSA were seen at ≥70%, with all balanced salt solutions. For each concentration, n = 4. All data are represented as the mean ± SE.
Figure 6.
 
Precipitation of indispensable molecules of fetal calf serum in DMEM resulted in decreased viability of RPE depending on ICG concentration. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents precipitated molecules derived from fetal calf serum in DMEM in various concentrations of ICG after standing overnight. Quantitative analysis was performed from the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Some bands increased linearly, depending on ICG concentration (arrowheads), but others that precipitated in low concentrations showed no increase at higher concentrations (arrows). Total precipitated molecules increased depending on ICG concentration, especially if it was more than 0.4 mg/mL. For each concentration, n = 4. All data are represented as the mean ± SE. (B) Viability of RPE sharply decreased depending on ICG concentration. Viability decreases especially if more than 0.4 mg/mL. For each concentration, n = 5. All data are represented as the mean ± SE.
Figure 6.
 
Precipitation of indispensable molecules of fetal calf serum in DMEM resulted in decreased viability of RPE depending on ICG concentration. (A) Each lane stained with Coomassie brilliant blue after SDS polyacrylamide gel electrophoresis represents precipitated molecules derived from fetal calf serum in DMEM in various concentrations of ICG after standing overnight. Quantitative analysis was performed from the density of each band measured by NIH Image, according to standardized dissolved BSA bands. Some bands increased linearly, depending on ICG concentration (arrowheads), but others that precipitated in low concentrations showed no increase at higher concentrations (arrows). Total precipitated molecules increased depending on ICG concentration, especially if it was more than 0.4 mg/mL. For each concentration, n = 4. All data are represented as the mean ± SE. (B) Viability of RPE sharply decreased depending on ICG concentration. Viability decreases especially if more than 0.4 mg/mL. For each concentration, n = 5. All data are represented as the mean ± SE.
Figure 7.
 
Viability of RPE cells after exposure to several concentrations of ICG for several brief periods. Viability decreased, depending on increasing ICG concentrations for all exposure periods except 0 minutes. Cells were incubated in medium in serum-free conditions for brief contact with ICG, to evaluate the cytotoxicity of ICG without interference of serum. The culture medium was immediately removed by centrifugation after contact with ICG for 0 minutes of incubation. All data shown are the mean at each concentration (n = 5).
Figure 7.
 
Viability of RPE cells after exposure to several concentrations of ICG for several brief periods. Viability decreased, depending on increasing ICG concentrations for all exposure periods except 0 minutes. Cells were incubated in medium in serum-free conditions for brief contact with ICG, to evaluate the cytotoxicity of ICG without interference of serum. The culture medium was immediately removed by centrifugation after contact with ICG for 0 minutes of incubation. All data shown are the mean at each concentration (n = 5).
Figure 8.
 
ICG in PBS, BSS (Alcon), and Hanks’ with and without NaHCO3 after overnight incubation at room temperature. Precipitation significantly increased in all solutions at an ICG concentration of ≥0.4 mg/mL, though the degree of precipitation varied slightly, depending on each solution. Hanks’ solutions include phenolsulfonphthalein to indicate pH. Slight differences in pH between Hanks’ solutions are shown by a change in color of the solution at 0 mg/mL and are caused by NaHCO3. The precipitation profiles were all different. Arrows: The minimum concentration at which ICG precipitation was detected.
Figure 8.
 
ICG in PBS, BSS (Alcon), and Hanks’ with and without NaHCO3 after overnight incubation at room temperature. Precipitation significantly increased in all solutions at an ICG concentration of ≥0.4 mg/mL, though the degree of precipitation varied slightly, depending on each solution. Hanks’ solutions include phenolsulfonphthalein to indicate pH. Slight differences in pH between Hanks’ solutions are shown by a change in color of the solution at 0 mg/mL and are caused by NaHCO3. The precipitation profiles were all different. Arrows: The minimum concentration at which ICG precipitation was detected.
Table 1.
 
Critical Micelle Concentrations
Table 1.
 
Critical Micelle Concentrations
H2O (wt %) PBS (wt %)
SDS 0.18 0.02
ICG 0.42
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×