trypan blue not toxic for retinal pigment epithelium in vitro

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  • plastin time, antithrombin III activity, factor VII activity andantigen, factor XII activity, protein C and protein S activity,heparin cofactor II, plasma homocysteine level, lupus antico-agulant (Exner, DRVVT), anticardiolipin antibodies (IgG,IgM), activated protein C-response, plasminogen activity,fibrinogen, tissue plasminogen activator antigen, and plas-minogen activator inhibitor activity and antigen. Plasmahistidine-rich glycoprotein levels were recurrently found to beelevated at 220%, however (reference range, 60%148%,electroimmunodiffusion according to Laurell).

    Since its first description in 1972, a few authors havereported an association between abnormal plasma levels ofhistidine-rich glycoprotein and systemic thromboembolicevents.4,5 We are unaware, however, of previous reports ofan association between increased plasma levels of histi-dine-rich glycoprotein and retinal vessel occlusion andcould find no reference to it in a computerized search usingMEDLINE.

    Histidine-rich glycoprotein is a nonenzymatic proteinpresent in human plasma and platelets. It has beendemonstrated that histidine-rich glycoprotein interactswith the high-affinity lysine-binding site of plasminogen,which appears to play an important role in the regulationof fibrinolysis. Moreover, it has been found to interactstrongly with heparin, resulting in neutralization of theanticoagulant activity of heparin.6 Interestingly, both,increased4 and decreased levels5 of histidine-rich glyco-protein have been found in patients with deep veinthrombosis, lung embolism, or myocardial infarction.

    In view of the absence of cardiovascular risk factors,we believe this case represents a coagulopathy secondaryto increased plasma levels of histidine-rich glycoprotein.It is plausible that increased plasma histidine-rich gly-coprotein levels are a risk factor for development ofocular vascular complications as they are for othervascular occlusions.


    1. Greiner K, Hafner G, Dick B, Dirk PE, Prellwitz W, Pfeiffer N.Retinal vascular occlusion and deficiency in the protein cpathway. Am J Ophthalmol 1999;128:6974.

    2. Incorvaia C, Lamberti G, Parmeggiani F, et al. Idiopathiccentral retinal vein occlusion in a thrombophilic patient withthe heterozygous 20210 G/A prothrombin genotype. Am JOphthalmol 1999;128:247248.

    3. Hattenbach LO, Klais C, Scharrer I. Heparin cofactor IIdeficiency in central retinal vein occlusion. Acta OphthalmolScand 1998;76:758759.

    4. Hennis BC, van Boheemen PA, Koeleman BPC, et al. Aspecific allele of the histidine-rich glycoprtein (HRG) locus islinked with elevated plasma levels of HRG in a Dutch familywith thrombosis. Br J Haematol 1995;89:845852.

    5. Souto JC, Gari M, Falkon L, Fontcuberta J. A new case ofhereditary histidine-rich glycoprotein defieciency with famil-ial thrombophilia. Thromb Haemost 1996;75:374375.

    6. Lijnen HR, Hoylaerts M, Collen D. Isolation and character-ization of human plasma protein with high affinity for lysinebinding sites in plasminogen. J Biol Chem 1980;225:1021410222.

    Trypan Blue Not Toxic for RetinalPigment Epithelium In VitroPeter Stalmans, MD, PhD,Elisabeth H. Van Aken, MD, PhD,Gerrit Melles, MD, PhD, Marc Veckeneer, MD,Eric J. Feron, MD, andIngeborg Stalmans, MD, PhD

    PURPOSE: To investigate whether trypan blue has a toxiceffect on cultured retinal pigment epithelial (retinalpigment epithelium) cells.DESIGN: Experimental study with a direct live/dead cellstaining technique using fluorescent dyes.METHODS: Cultured human retinal pigment epitheliumcells were exposed for 5 minutes to various concentra-tions of trypan blue (0.06%, 0.15%, 0.30%), and cellviability was confocally measured.RESULTS: No increased cell death was found in culturesincubated in any of the trypan blue concentrations used.CONCLUSION: These findings indicate that a short expo-sure of trypan blue does not have a toxic effect oncultured retinal pigment epithelium cells. (Am J Oph-thalmol 2003;135:234236. 2003 by Elsevier Sci-ence Inc. All rights reserved.)

    OVER THE PAST FEW YEARS, TRYPAN BLUE 0.06% HASbeen widely used for staining the anterior lenscapsule to facilitate the capsulorhexis during cataractsurgery in mature white cataract.1 Trypan blue is notapproved or available in the United States at this time,however.

    More recently, clinical trials have been published stud-ies on trypan blue as an aid in vitreoretinal surgery forstaining epiretinal membranes in proliferative vitreoreti-nopathy2 or for macular pucker removal.3 After a contacttime of 1 to 3 minutes, trypan blue stains epiretinalmembranes at a concentration of 0.15% without stainingthe underlying neural retina.2,3

    Accepted for publication Sept 4, 2002.From the Department of Ophthalmology (P.S., I.S.), UZLeuven,

    Leuven, Belgium; Department of Ophthalmology (E.H.v.A.), GhentUniversity Hospital, Ghent, Belgium; Netherlands Institute for Innova-tive Ocular Surgery (G.M.) and Rotterdam Eye Hospital (M.V., E.J.F.),Rotterdam, The Netherlands; the Center for Transgene Technology andGene Therapy (I.S.), Flanders Interuniversity Institute for Biotechnol-ogy, KU Leuven, Belgium.

    This study was supported by a grant from the Foundation for Researchin Ophthalmology, Belgium. Doctors Elisabeth H. Van Aken andIngeborg Stalmans are research assistants of the Fonds voor Wetenschap-pelijk OnderzoekFlanders, Brussels, Belgium.

    Dr. Melles has proprietary and financial interest in commerciallyavailable solutions of trypan blue for intraocular use (VisionBlueTM andMembraneBlueTM, D.O.R.C., Zuidland, The Netherlands).

    Inquiries to Peter Stalmans, MD, PhD, Department of Ophthalmology,UZLeuven, Capucijnenvoer 33, B3000 Leuven, Belgium; e-mail:


  • Toxicity of trypan blue on the neural retina has beenevaluated in rabbit experiments,4 but toxicity on theretinal pigment epithelium remains to be determined.Because trypan blue may potentially contact the retinalpigment epithelium during vitreoretinal surgery, we inves-tigated the cytotoxicity on trypan blue on retinal pigmentepithelium cells in vitro.

    In an experimental study, retinal pigment epitheliumcells were obtained from human cadaver eyes as describedearlier.5 Primary culture was obtained by explanting retinalpigment epithelium fragments in RPMI 1640 medium,supplemented with 20% fetal calf serum, 2 mmol/l L-glu-tamine, 100 IU/ml penicillin, 100 g/ml streptomycin, and2.5g/ml fungizone. The retinal pigment epithelium cell linewas isolated by trypsin treatment from the primary culture.The epithelial origin of the cultured retinal pigmentepithelium cells was demonstrated by cytokeratin.

    Cell viability was measured using a Zeiss confocalmicroscope with a 10 objective as described previously.5Briefly, the cells were loaded by a 30-minute incubationwith a live/dead fluorescent kit. They were excited at 488nm to stain viable cells with calcein-AM and at 540 nm tostain dead cells with ethidium homodimer-1 dye. The488-nm images obtained were processed using imagingsoftware. After thresholding the images, the fluorescentcells were automatically counted using the particle analyzefunction. The 540-nm images were manually analyzed to

    avoid false measurements related to the autofluorescense ofthe trypan blue dye at its higher concentration.

    For control experiments, a phosphate-buffered solutionwas used. A concentration of typan blue 0.3% was ob-tained by dissolving 60 mg in 2 ml phosphate bufferedsolution. The 0.06% concentration was obtained by dilut-ing the 0.3% concentration five times in buffered saltsolution. The concentration 0.15% (Membrane Blue,D.O.R.C., Zuidland, The Netherlands) was provided bythe manufacturer.

    Osmolarity measurements were performed with a cryo-scopic osmometer. Statistical analysis was performed usinga Student t test with unpaired and two-tailed data and withunequal variance. A significance of P .01 was predeter-mined.

    A measurement of the osmolarity of the solutions wasperformed to exclude any cellular toxicity related to theosmolarity of the dyes.5 All solutions (phosphate bufferedsolution and trypan blue concentrations) were found to bein the iso-osmotic range (phosphate buffered solution: 289mOsm/kg, trypan blue 0.06%: 296 mOsm/kg, trypan blue0.15%: 298 mOsm/kg, trypan blue 0.30: 309 mOsm/kg).

    To determine whether any of the solutions showedautofluorescence that could induce false-positive measure-ment, all conditions were measured without any live/deadsolution. No autofluorescense was found in either the488-nm or 540-nm spectrum after incubation in the

    FIGURE 1. Ratio of living vs dead cells (Y axis) measured in several conditions. Measurements statistically different from controlconditions are indicated with an asterisk (*). Error bars indicate standard error measurements. Aqueous solution 20 minutes: P


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