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Safety Testing of Indocyanine Green and Trypan Blue using Retinal Pigment Epithelium and Glial Cell
Author Response: Safety Testing of Indocyanine Green and Trypan Blue...
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Timothy Lai
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tyylai{at}netvigator.com Timothy Lai
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Safety Testing of Indocyanine Green and Trypan
Blue using Retinal Pigment Epithelium and Glial Cell Cultures
We read with great interest the article by Jackson et al.,1 which investigated the safety of indocyanine green (ICG) and trypan blue dyes in retinal pigment epithelium (RPE) and glial cell cultures. The authors concluded that although no evidence of cell damage to RPE and Muller cell cultures was found with ICG at dosages used clinically, reduced viability of RPE and glial cells did occur at higher ICG concentrations or when applied under osmolarity lower than those used clinically. The authors also concluded that trypan blue is safe in the cell culture model. We have also conducted a similar experiment to investigate the effects of trypan blue on cultured RPE cells and would like to share some of our findings with the authors concerning the safety of trypan blue.2 We evaluated the effects of trypan blue on ARPE19 cell viability using MTT assay for 6 consecutive days following application without illumination for 1 minute. In contrast with the results by Jackson et al., we found that there was a concentration-dependent effect of trypan blue on the viability of RPE cells. At a low concentration of 0.006% trypan blue, RPE cell viability was similar to the control group. However, at higher concentrations of 0.06% and 0.4% trypan blue, statistically significant reductions in RPE cell viability were observed. This reduction in RPE cell viability was transient and was more marked within the first 2 days of trypan blue exposure. At 6 days after the application of trypan blue, significant reduction in cell viability was only limited to the 0.4% trypan blue group. We also studied the expression of apoptosis-related gene in ARPE19 cells in response to trypan blue. There was a significant increase in the number of cells with p21 expression at all three concentrations of trypan blue compared with the unexposed control group. Increase in p21 expression in ARPE19 cells implies a transient arrest in the G1 phase for DNA repair and therefore cell growth is retarded. This was reflected by the MTT assay as reduction of cell viability was most substantial in the first 2 days after the trypan blue application and the reduction of cell viability was less significant after 6 days. We think that one of the reasons for the differences in the results of our study and the study by Jackson et al. may be due to the cell sampling time intervals. The more frequent serial measurements in our study allowed the monitoring of cell viability at various time points. This is important since the acute effect of trypan blue on cell culture is time-dependent and the reduction in cell viability might be difficult to detect if the samples are measured either too early or too late. Another difference between our experiments was the level of confluence of RPE cells in the two studies. We used RPE cells of five passages that were grown to 70% confluence, compared with the RPE cells of 23 passages in the study by Jackson et al., which has higher confluence. Since RPE cells at different levels of confluence may have different physiological properties,3 effects of trypan blue in the two studies may vary. Differences in the experiments might also be due to the slightly lower osmolarity of trypan blue solution we used than the one employed by Jackson et al. Jackson et al. suggested that trypan blue might be a safer intraoperative dye to use clinically compared with ICG. However, trypan blue may not provide adequate staining for the internal limiting membrane (ILM) as it mainly stains epiretinal membrane and higher concentration of trypan blue may therefore be required.4 Since our results show that trypan blue may cause a concentration-dependent reduction in RPE cell viability, surgeons should utilise the lowest possible concentration of intraocular dye whenever possible. Further in vivo and in vitro studies on the effect of the use of intraocular dyes in vitreoretinal surgery are warranted. Timothy Y.Y. Lai1 1Department of Ophthalmology & Visual Sciences, The Chinese University
of Hong Kong, Hong Kong, People's Republic of China References 1. Jackson TL, Hillenkamp J, Knight BC, et al. Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures. Invest Ophthalmol Vis Sci. 2004;45:2778-2785. |
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Timothy L. Jackson
Send letter to journal:
timljackson{at}hotmail.com Timothy L. Jackson
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Author Response: Safety Testing of Indocyanine Green and Trypan Blue Using Retinal Pigment Epithelium and Glial Cell Cultures
We are grateful for the comments of Lai et al. Their finding of damage in ARPE19 exposed to 0.06% to 0.4% trypan blue differs from our result using the same cells.1 It also differs from the results of Gale2 and Stalmans,3 who, like us, failed to show ARPE19 toxicity. Lai's suggestion that this discrepancy might reflect differences in sampling times may be partly valid, but it does not explain why they found toxicity at day 1, whereas we did not. Their other explanations for the difference in terms of passage and level of confluency are valid hypotheses. For example, it is possible that non-confluent cells expose relatively more surface area to dyes than confluent monolayers. One strength of their study is the use of exposure times and concentrations that simulate the clinical use of trypan blue. Another is their use of daily sampling routines and two outcome measures. It might be argued that extending cell culture beyond 6 days would be worthwhile given our findings with indocyanine green,1 but 6 days is longer than most published experimental studies. It would of course be important to study the details of their methodology. For example, were appropriate rinsing routines or assay corrections4 applied to prevent trypan blue interfering with the mitochondrial (MTT) enzyme assay, as there is a spectral overlap of trypan blue and the blue formazan reaction product measured in this assay. Assuming that their experimental routines were similar to previous, well-designed studies from their laboratory, then their findings imply that 0.15% trypan blue may be unsafe as a macular vital stain. There is indirect support for this conclusion in the literature. Rabbit experiments by Funahashi5 showed that subtenons injection of trypan blue produced scleral and optic nerve damage. Veckeneer6 injected trypan blue into the vitreous cavity of rabbits and also showed toxicity with prolonged contact to a 0.2% solution, but 0.06% trypan blue did not produce retinal damage. It would not yet be appropriate to abandon the use of trypan blue on the basis on one cell culture study, particularly when results differ from relatively similar experiments by Gale, Stalmans and ourselves. This difference does not, however, invalidate their data, which could have considerable clinical impact. We therefore hope that a full account of their methods and results will be expedited. Timothy L. Jackson Department of Ophthalmology, The Rayne Institute, St Thomas' Hospital, London References 1. Jackson TL, Hillenkamp J, Knight BC, et al. Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures. Invest Ophthalmol Vis Sci. 2004;45:2778-2785. |
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