Ability of New Vital Dyes to Stain Intraocular Membranes and Tissues in Ocular Surgery

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Ability of New Vital Dyes to Stain IntraocularMembranes and Tissues in Ocular Surgery

EDUARDO B. RODRIGUES, FERNANDO M. PENHA, ELAINE DE PAULA FIOD COSTA, MAURICIO MAIA,EDUARDO DIB, MILTON MORAES, JR, CARSTEN H. MEYER, OCTAVIANO MAGALHAES, JR,

GUSTAVO BARRETO MELO, VINICIUS STEFANO, ANA BEATRIZ DIAS, AND MICHEL EID FARAH

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PURPOSE: To evaluate the ability of novel dyes to stainens capsule (LC), internal limiting membrane (ILM),piretinal membrane (ERM), and vitreous.DESIGN: Experimental study in animal and human

onor eyes.METHODS: Thirteen dyes, methyl violet, crystal violet,

osin Y, sudan black B, methylene blue, toluidine blue,ight green, indigo carmine, fast green, congo red, evanslue, brilliant blue, and bromophenol blue, were injectednto the LC and ILM of enucleated porcine eyes. Theitreous was stained with 2 mL of dyes for 1 minute. Sixyes (indigo carmine, evans blue, fast green, light green,romophenol blue, and brilliant blue) were selected forxperiments in human donor eyes and freshly removedRM.RESULTS: In the porcine eyes, ILM staining withethylene blue, toluidine blue, indigo carmine, evanslue, bromophenol blue, and fast green was moderate,nd methyl violet, crystal violet, brilliant blue, or sudanlack resulted in strong staining. Methyl violet, crystaliolet, sudan black, toluidine blue, and methylene blueaused histologic damage in porcine retinas. Vitreousxamination revealed moderate staining with congo red,rystal violet, fast green, eosin Y, methylene blue, tolu-dine blue, brilliant blue, bromophenol blue, and methyliolet and strong staining with light green and evans blue.RMs showed strong staining with 0.5% evans blue andoderate staining with 0.5% light green, fast green,rilliant blue, and bromophenol blue. Evaluation ofonor eyes disclosed moderate staining with evans blue,ight green, and bromophenol blue and strong stainingith 0.5% brilliant blue. Moderate or strong staining of

he vitreous occurred with most dyes. LC evaluationhowed moderate staining with 0.5% evans blue, fastreen, and brilliant blue, whereas 0.5% light greenroduced strong LC staining.CONCLUSIONS: Brilliant blue shows the best ILM

taining, whereas bromophenol blue, evans blue, and

ccepted for publication Aug 18, 2009.From the Vision Institute (IPEPO), Department of Ophthalmology,

ederal University of São Paulo, São Paulo, Brazil (E.B.R., F.M.P.,.d.P.F.C., M.M., E.D., M.M.Jr., O.M., G.B.M., V.S., A.B.D., M.E.F.);nd the Department of Ophthalmology, University of Bonn, Bonn,ermany (C.H.M.).

BInquiries to Michel Eid Farah, R. Botucatu 820, 04023-062 São Paulo

P, Brazil; e-mail: [email protected]

© 2010 BY ELSEVIER INC. A002-9394/10/$36.00oi:10.1016/j.ajo.2009.08.020

ight green also stain ILM. Most dyes bind well to LC,itreous, and ERM. (Am J Ophthalmol 2010;149:65–277. © 2010 by Elsevier Inc. All rights reserved.)

NTRAOPERATIVE APPLICATION OF VITAL DYES FOR THE

visualization of intraocular membranes and tissues hasfacilitated surgical techniques and outcomes in recent

ears.1 For vitreoretinal surgery, indocyanine green (ICG)ye initially was introduced for staining the internalimiting membrane (ILM).2 This was followed by manyublications reporting signs of retinal toxicity after intra-itreous ICG injection.3–6 In cataract surgery, trypan blueas provided since the 1990s a better visualization of thenterior lens capsule (LC) when the red reflex is notossible.1 Following ICG and trypan blue, other dyes suchs brilliant blue and patent blue were presented as newerlternative vital dyes for ocular surgery.7,8 However, con-erns also were raised about the safety of brilliant blue,rypan blue, and patent blue, and their selective affinity forome intraocular membranes such as epiretinal membranesERMs), LC, or ILM, but not for all of them.9–11

For this study, a total of 13 vital dyes were selected: lightreen, fast green, methyl violet, crystal violet, congo red,osin Y, sudan black B, evans blue, brilliant blue, bromo-henol blue, methylene blue, toluidine blue, and indigoarmine. In addition, ICG, trypan blue, and patent blueere included in our in vivo examinations for comparison.he goal of this study was to investigate the staining abilityf these dyes regarding the retinal ILM, vitreous, ERM,nd LC in a series of systematic experiments in freshlynucleated porcine eyes, freshly removed ERM, and enu-leated human donor eyes.

METHODS

SELECTION OF DYES AND PREPARATION: A total of0 mg of dye powder of light green, fast green, bromophe-ol blue, brilliant blue, patent blue, methyl violet, crystaliolet, congo red, eosin Y, sudan black, evans blue,ethylene blue, toluidine blue, indigo carmine, ICG, or

rypan blue (Merck, Darmstadt, Germany, for all dyes,xcept for Sigma-Aldrich, Munich, Germany, in the casef bromophenol blue and Ophthalmos Ind, São Paulo,
razil, for ICG and trypan blue) was weighed with an
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nalytic balance (Mettler-Toledo, Inc, Columbus, Ohio,SA) and dissolved in 10 mL balanced salt solution (BSS

lus; Alcon Laboratories, Inc, Fort Worth, Texas, USA) tobtain a concentration of 0.5% for the initial experiments.he mixtures were shaken for 5 minutes and sonicated

Unique Ind, Idaiatuba, Brazil) to obtain a homogeneousolution. For donor eye experiments, the selected dyes lightreen, ICG, trypan blue, patent blue, fast green, indigoarmine, evans blue, brilliant blue, and bromophenol blueere prepared as 0.5% and 0.05% solutions in balanced

alt solution. The choice of agents was based on a rationaleo validate the experimental method; thiazine, carbonyl,iazo, aminoarylmethane, and hydroxyxanthene dyes werenalyzed to search for an optimal staining agent.12

STAINING OF PORCINE INTERNAL LIMITING MEM-

RANE, VITREOUS, AND LENS CAPSULE: All animal ex-eriments were conducted according to the ARVOtatement for the Use of Animals in Ophthalmic andision Research. The experiments for ILM, LC, anditreous staining were conducted on an ex vivo setup.inety-six pig eyes were obtained from a slaughterhouse

nd were prepared within 6 hours after death. The dyesnitially were used to stain the LC of pigs. Immediatelyfter eye removal, the eyes were placed on a glass support,nd a 360-degree limbal incision was performed for cornealemoval, followed by total iris removal. Next, 0.05 mL ofconcentrations of 0.05% and 0.5% of each of the 16 dyesere placed onto the LC for 1 minute. Afterward, thenterior surface of the lens was irrigated carefully withaline solution. An anterior curvilinear continuous capsu-orrhexis was performed within the stained capsule usingorceps.

For evaluation of ILM and vitreous staining, the remain-ng eye cups had the anterior segment removed with a-mm incision behind the limbus, and eyes with fundus-opic red reflex were included in the experiments. Theitreous was gently extracted completely with cotton budsnd forceps and was placed in a glass vial for its evaluation.ext, 0.05 mL of 0.5% of the 16 dyes was injected slowly

ver the retinal surface to avoid dispersion. The dye waseft on the retina for 2 minutes and the retinal surfaceas rinsed twice with 2 mL saline. Each experiment was

epeated with 4 samples with each dye. In all eyes, peelingf the ILM was attempted with a bent 27-gauge needle.To evaluate vitreous staining, the vitreous removed

rom fresh enucleated pig eyes was placed in a 5-mL vial forminute with 2 mL of each of the 13 dyes at a concen-

ration of 0.5%. The vitreous then was removed, rinsedhoroughly, and placed on a white background for stainingvaluation under the microscope. Photographs of all ex-eriments were obtained with a Canon camera (CanonowerShot A2000 IS; Canon Electronics, Saitama, Japan)tted to the operating microscope. The intensity of ILM,
C, and vitreous staining was graded in a masked fashion d
AMERICAN JOURNAL OF66

s no staining (0), faint staining (�), moderate staining��), or strong staining (��).13

HISTOLOGIC EVALUATION OF PORCINE EYES BY

IGHT MICROSCOPY: LCs were placed in 10% bufferedormaldehyde and 4% buffered glutaraldehyde (Karnowskyxative) for a minimum of 48 hours. Specimens of LC thenere evaluated macroscopically by light microscopy afterreparation with routine hematoxylin and eosin staining.Immediately after ILM removal, some specimens of both

LM and peeled retinas of porcine eyes were placed in aixture of 4% buffered formaldehyde and 4% buffered

lutaraldehyde (Karnowsky fixative) for 15 to 20 minuteso achieve rapid and proper fixation of the retina followedy storage in 10% buffered formaldehyde for 48 hours.ross examinations of the tissues were performed. Semi-

hin sections were cut along a superior–inferior plane, andhey then were embedded in paraffin. Next, the retinasere sectioned at a thickness of 6 �m, stained using

outine hematoxylin and eosin and examined by lighticroscopy. In screening for the most suitable dye for

xperiments in donor eyes, each dye was considered to beoxic to the retina if any of the following findings werebserved: major signs of disorganization, cell loss, vacuol-zation, condensation, edema or swelling; necrosis of ateast one retinal layer; or fragments, debris, as well asieces of retinal cell elements within the removed ILMs.

STAINING OF FRESHLY EXCISED HUMAN EPIRETINAL

EMBRANE: This study was approved by local ethicalommittee and informed consent was obtained from eachatient before surgery. Staining pattern of human ERMsas examined after exposure to 0.5% of the 16 dyes

ncluded in this study. The vital dyes (0.3 mL) werepplied onto freshly surgically removed ERMs obtainedrom patients with proliferative diabetic retinopathy androliferative vitreoretinopathy of rhegmatogenous retinaletachment. The staining intensity of ERMs was graded asor other membranes in as masked fashion. The stainedRM tissues also were fixed with 10% buffered formalde-yde followed by hematoxylin and eosin preparation for

ight microscopy to detect vitreous or ILM remnants andhe composition of the tissue.

STAINING OF LENS CAPSULE, VITREOUS, AND INTER-

AL LIMITING MEMBRANE IN HUMAN DONOR EYES:

uman donor eyes were obtained from the Eye Bank of theederal University of São Paulo after institutional reviewoard approval. Six dyes were chosen for this setup basedn their safety and staining profile on the porcine eyes:ight green, fast green, brilliant blue, indigo carmine,romophenol blue, and evans blue. In addition, analysis oftaining in these donor eyes was conducted with ICG,rypan blue, and patent blue to compare the currently used
yes with the novel future dyes. Initially, human cadaveric
OPHTHALMOLOGY FEBRUARY 2010

TABLE 1. Staining Properties of Vital Dyes in Porcine Eyes

Dye

Biochemical Properties and Principles of Biological Staining

Ex Vivo Staining in Porcince Eyes at 0.5%

Ex Vivo Histologic

Results at 0.5%

Chemical Group Chemical Formula Weight Staining Principle

Lens Capsule

Staining

ILM

Staining Color on the Retina

Vitreous

Staining

Retina Histologic

Damage

Methylene blue Thiazine C16H18N3ClS 373 Absorptive � �� Bright blue �� Toxic

Toluidine blue Thiazine C15H16N3SCl 305 Absorptive �� Dark blue �� Toxic

Indigo carminea Carbonyl C16H8N2Na2O8S2 466 Contrast � �� Dark blue � Safe

Evans bluea Anionic dis-azo C34H24N6Na4O14S4 960 Unknown �� Blue �� Safe

Congo red Anionic dis-azo C32H22N6Na2O6S2 696 Contrast/reactive � � Red �� Safe

Light green Sfa Anionic amino triarylmethane C37H34N2Na2O9S3 792 Contrast �� Light greenish �� Safe

Eosin Yb Acidic hydroxyxanthene C20H8O5Br4 648 Unknown � 0 Light reddish �� Safe

Sudan black B Anionic dis-azo C29H24N4 457 Unknown �� Black � Toxic

Crystal violet Cationic amino arylmethane C25H30N3 372 Contrast/reactive �� Red violet �� Toxic

Fast greena Anionic amino arylmethane C37H34N2Na2O10S3 809 Unknown �� Dark green �� Safe

Methyl violet Cationic amino arylmethane C24H28ClN3 393 Contrast �� Red violet �� Toxic

Patent blue Anionic amino arylmethane C27H31N2NaO6S2 582 Absorptive � � Blue �� Safe

Indocyanine green Tricarbocyanine C43H47N2NaO6S2 774 Unknown �� Green �� Safe

Trypan blue Anionic dis-azo C34H24N6Na4O14S4 961 Contrast �� Dark-blue �� Safe

Brilliant bluea Triarylmethane C47H48N3S2O7Na 854 Unknown �� Violet blueish �� Safe

Bromophenol bluea Triarylmethane C19H10Br4O5S 670 Reactive/contrast �� Dark blue violet �� Safe

ILM � internal limiting membrane.

Grade of staining intensity: 0 � no staining; � � faint staining; �� moderate staining; �� strong staining. See ref. 13.aSelected for further in vivo experiments in rabbits.bEosin is a vital stain derivate of fluorescein.

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IGURE 1. After a 360-degree limbal incision, corneal removal was performed, followed by total iris removal. Then, 0.05 mL ofconcentrations of 0.05% and 0.5% of each dye were placed onto the lens capsule (LC) for 1 minute followed by irrigation with

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yes up to 48 hours after death were examined in theaboratory to determine their integrity. To stain the LC, a60–degree-limbal incision was performed for cornealemoval, followed by segmented iris removal. Next, 0.05L of 0.05% and 0.5% concentrations of each dye were

pplied to the LC for 1 minute, followed by rinsing withaline solution. An anterior curvilinear capsulorrhexis wasarried out with forceps, and selectively removed mem-ranes were sent for histologic analysis after bufferedormaldehyde fixation and periodic acid–Schiff staining.

The entire anterior segment of the eye was then excisedo gain access to the vitreous cavity. After open skyitrectomy (Accurus; Alcon Laboratories), 0.2 mL of dyeolution was injected into the posterior vitreous cavityver the macula for 2 minutes and then was removed byechanical aspiration, trying to leave the stained ILM

learly visible. A bent 27-gauge needle was used to createhe initial ILM tear, followed by grasping with a 23-gaugeorceps applied to peel the ILM.

RESULTS

SELECTION OF DYES AND PREPARATION: All dyes butCG could be diluted successfully in balanced salt solution;CG was diluted further in glucose 5% solution. Table 1ummarizes the chemical and staining properties of theital dyes selected for investigation in this study. Fourtaining agents, evans blue, congo red, sudan black, andrypan blue, belong to the azo dyes, and 7 dyes, light green,rystal violet, fast green, methyl violet, patent blue, bril-iant blue, and bromophenol blue, are in the group of therylmethane agents. The mechanism of staining of theyes varies and can involve binding, contrast, or reactivity,lthough some staining agents such as fast green and evanslue bind to living tissues through unknown mecha-isms.

STAINING OF PORCINE INTERNAL LIMITING MEM-

RANE, VITREOUS, AND LENS CAPSULE: The grading ofhe staining effect of each dye in porcine eyes is shown inable 1. The effect in surgically stained LC varied for theifferent dyes and dye concentrations (Figure 1). With theid of most dyes, continuous capsulorrhexis was completeduccessfully, whereas adequate contrast and visibility waseported by the surgeon. Indigo carmine, congo red,rilliant blue, and methylene blue did not stain the LCufficiently at a concentration of 0.05%; however, patent

aline solution. An anterior curvilinear continuous capsulorrhexnd dye concentrations from weak (�) to strong (��) colorRight) 0.5% FG induced moderate (��) staining. (Second rowtaining, whereas (Right) 0.5% LG generated moderate (��) stid not color the LC sufficiently, but (Right) the 0.5% concentrith (Left) 0.05% bromophenol blue (BroB) caused faint (�

taining.

NEW VITAL DYES IN OOL. 149, NO. 2

lue, trypan blue, evans blue, light green, sudan black,rystal violet, methyl violet, ICG, and bromophenol blueroduced only light (�) staining of the LC, whereas fastreen and trypan blue promoted moderate (��) staining.t the higher concentration of 0.5%, toluidine blue, evans

lue, light green, sudan black, crystal violet, fast green,ethyl violet, ICG, brilliant blue, and bromophenol blue

rovided moderate (��) and trypan blue provided strong��) staining of the LC (Table 1).

Experiments with freshly enucleated porcine eyes withyes at 0.5% demonstrated no (0) retinal ILM stainingfter eosin Y exposure, whereas congo red, light green,atent blue, and trypan blue generated only faint (�)etinal ILM staining. Exposure of the retinal ILM toethylene blue, toluidine blue, indigo carmine, evans

lue, bromophenol blue, and fast green resulted in moder-te (��) staining, and methyl violet, crystal violet, sudanlack, brilliant blue, and ICG yielded strong (��)etinal ILM staining in porcine eyes (Figure 2). With mostyes, a continuous 1-piece ILM peeling was difficult tochieve, an expected finding in porcine eyes.

Most of the vital dyes used in this investigation at 0.5%tained the vitreous very well. Vitreous examination dem-nstrated moderate (��) staining with congo red, crystaliolet, fast green, patent blue, eosin Y, methylene blue,oluidine blue, ICG, and methyl violet; demonstratedtrong staining (��) with trypan blue, light green, andvans blue; and demonstrated faint (�) staining withndigo carmine and sudan black (Figure 3).

HISTOLOGIC EVALUATION BY LIGHT MICROSCOPY:

ight microscopy examination revealed the typical fibro-lastic characteristics of the anterior LC. No anteriorapsule or lens epithelium abnormality in the removedrea was observed in any eyes except for surface-parallelntracapsular splits and delamination of the capsulorrhexisesulting from fixation artifact (Figure 4).

Gross examination of porcine retinal specimens showedo hemorrhage, inflammation, or detachment. In the casesf methyl violet, toluidine blue, and methylene bluetaining, additional deeper layers of the retina also wereemoved during the ILM peeling maneuver, as cellularlements were present in the histopathologic examinationFigure 4). In addition, the vital dyes crystal violet andudan black at 0.5% were found to be toxic because theynduced severe morphologic damage to the retina, such asroducing regions of deformation and destruction of inner

s performed. Surgically stained LC varied for the different dyesTop row) LC staining with (Left) 0.05% fast green (FG) andstaining with (Left) 0.05% light green (LG) caused faint (�)

g. (Third row) Staining with (Left) 0.05% brilliant blue (BriB)promoted moderate (��) staining. (Bottom row) LC staining

aining, whereas (Right) 0.5% LG generated moderate (��)

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IGURE 2. Histologic specimens of retinas, lens capsules, internal limiting membranes (ILMs), and epiretinal membranes (ERMs). (Topeft) Gross examination of specimen of peeled retina of porcine eyes after application of 0.5% bromophenol blue dye. Histologicxamination revealed no major toxicity changes in the inner and outer nuclear layer cells or in the nerve fiber layer, whereas successfuleparation of the ILM was achieved. Arrow separates the ILM peeled area (right) from the unpeeled area (left; hematoxylin andosin, �40 magnification). (Top middle) Gross examination of specimen of peeled retina of porcine eyes after application of 0.5% crystaliolet dye. The dye was considered to be toxic to the retina because it promoted disorganization and diffuse vacuolization within the nerveber layer and ganglion cell layer (hematoxylin and eosin, �40 magnification). (Top right) In the case of 0.5% methyl violet staining forhe ILM peeling maneuver, examination of the ILM disclosed cellular fragments of the retina besides the collagen structure (hematoxylinnd eosin, �25 magnification). (Middle center) Freshly removed ERMs stained with 0.5% evans blue showing strong staining (�10agnification). (Middle right) Freshly removed ERMs stained with 0.5% light green (LG) showing moderate staining (hematoxylin and

osin, �10 magnification). (Bottom left) Gross examination of specimen of peeled retina of porcine eyes after application of 0.5% lightreen (LG) dye. Histologic examination revealed no signs of retinal cellular damage, and successful separation of the ILM was completed.rrow separates the ILM peeled area (left) from the unpeeled area (right; hematoxylin and eosin, �40 magnification). (Bottom middle)ight microscopy examination with periodic acid–Schiff (PAS) staining after application of 0.5% fast green onto the lens capsule (LC)evealing the typical fibroblastic characteristics. No anterior capsule or lens epithelium abnormality in the removed area was observedhematoxylin and eosin, �25 magnification). (Bottom right) Light microscopy with PAS staining after application of 0.5% LG onto theC showing the fibroblastic characteristics of the capsule without any other abnormality (hematoxylin and eosin, �25 magnification).

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uclear layers and outer nuclear layers, as well as focalreas of damage to photoreceptors. In contrast, eosin Y,vans blue, congo red, brilliant blue, bromophenol blue,ndigo carmine, light green, patent blue, ICG, trypan blue,nd fast green yielded no major morphologic alterations inorcine retinas, whereas the ILM could be peeled success-ully. Histologic examination revealed that these dyesromoted no major toxicity changes in the inner and outeruclear layer cells, nerve fiber layer, retinal pigmentpithelium, and choriocapillaris. Histologic evaluationlso demonstrated the successful separation of the ILM inases of peeling with the aid of evans blue, light green,rilliant blue, bromophenol blue, patent blue, ICG, trypanlue, and fast green, without remnants of retinal cellsFigure 4).

STAINING OF FRESHLY EXCISED HUMAN EPIRETINAL

EMBRANE: The results showed that most dyes possess at

IGURE 3. For evaluation of internal limiting membrane (ILMith a 3-mm incision behind the limbus, and eyes with fundusco

taining is shown, which was obtained after placement of 0.05 mtaining with (Left) fast green FG, and weak staining after (Rndocyanine green (ICG), and faint staining with (Right) congEB) and (Right) indigo carmine (IC). (Fourth row) Moderate stTB) caused faint coloring.

east some binding affinity to freshly enucleated human 0


RM. ERM examination revealed strong (��) stainingith evans blue and trypan blue; moderate (��) stainingith 0.5% light green, fast green, brilliant blue, bromo-henol blue, and patent blue; and faint (�) staining afterxposure to indigo carmine and ICG (Table 1). At theower dose of 0.05%, evans blue, fast green, patent blue,CG, brilliant blue, and bromophenol blue provided onlyaint (�) staining, and light green and trypan blue pro-uced moderate ERM staining (Figure 4). Histopathologicxamination disclosed the highly cellular composition ofhe ERM with retinal pigment epithelium elements withome connective tissue components.

STAINING OF LENS CAPSULE, VITREOUS, AND INTER-

AL LIMITING MEMBRANE IN HUMAN DONOR EYES:

ost of the dyes stained the LC homogenously, where itsdge could be observed, and the circular capsulorrhexisould be performed in all cases. The concentration of

ining, the porcine eye cups had the anterior segment removeded reflex were included in the experiments. In this figure, ILM% of several dyes onto the retinal surface. (Top row) Moderate

) patent blue (PB). (Second row) Strong staining with (Left)(CR). (Third row) Moderate staining with (Left) evans blueg with (Left) toluidine blue (ToB), whereas (right) trypan blue

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CULAR SURGERY 271

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ith clear visualization with most dyes. Exceptionally,ndigo carmine produced no staining, and no merit coulde found in using this dye at this concentration. However,rypan blue enabled strong staining of the anterior LC.he staining was enhanced as the concentration of theyes was increased to 0.5%, where mild (�) stainingccurred with indigo carmine; moderate (��) stainingccurred with evans blue, fast green, patent blue, ICG, andrilliant blue; and strong (��) staining occurred withromophenol blue, light green, and trypan blue (Figure 5).To achieve ILM staining, the dye solution was injected

irectly over the macula, where it tended to settle withinimal dispersion (Figure 6). After its removal from the

itreous cavity, the underlying ILM that had been inontact with the dye was stained brightly (��) with.5% ICG and brilliant blue. However, 0.5% evans blue,ight green, and bromophenol blue yielded moderate (��)

IGURE 4. Human donor eyes obtained from the eye bank unemoval. An anterior curvilinear capsulorrhexis was carried oromoted strong lens capsule (LC) coloring. (Middle row) Ataining. (Bottom row) Staining with 0.5% indigo carmine (IC

taining of the human ILM, whereas 0.5% indigo carmine, i


ast green, patent blue, and trypan blue had only a mildffect. The flap of ILM was seen easily, and it could berasped using intraocular forceps, aided by the distinctontrast between the stained ILM and the unstainedetina. The posterior hyaloid of 3 eyes remained attachedfter limited open sky vitrectomy. The grading of thetaining effect of each dye and dye concentration for everyntraocular membrane, vitreous, anterior LC, and ILM ishown in Table 2.

DISCUSSION

N RECENT YEARS, DYE-ENHANCED CATARACT AND VIT-

eoretinal surgery with trypan blue and ICG has becomehe standard method for cases of poor visualization of theC, ERMs, or ILM. The dyes currently available for

ent application of vital dyes for staining after corneal and irisith forceps. (Top row) Staining with 0.5% light green (LG)ation of 0.5% bromophenol blue (BroB) induced strong LClded mild LC coloring.

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re: ICG (ICG-Pulsion; Munich, Germany; Indocianinaerde; Ophthalmos, São Paulo, Brazil; and IC-Green; Akorn

nc, Buffalo Grove, Illinois, USA); trypan blue (VisionBluend MembraneBlue; DORC International, Zuidland,etherlands), patent blue (Blueron; Fluoron/Geuder, Ulm,ermany), IfCG (Infracyanine green; Serb, Paris, France),

nd brilliant blue (Brilliant peel; Fluoron/Geuder, Ulm,

IGURE 5. Vitreous was removed from fresh enucleated pig ey3 dyes at a concentration of 0.5%. The vitreous then was rtaining evaluation. Most vital dyes at 0.5% stained the vitreoCV), fast green (FG), patent blue (PB), eosin Y (EY), methyleethyl violet (MV); strong staining with trypan blue (TB), lig

armine (IC), and sudan black (SB).

ermany).1 However, recent research has shown that b


ome of the available dyes may be toxic to ocular tissues.1,3

herefore, we selected 13 alternative vital dyes to conductn investigation of their staining potential with regard tohe LC, vitreous, ERMs, and ILM.12,14

In this study, we performed a systematic series ofxperiments to determine the better new vital dyes for eyeurgery. The dye concentrations of 0.5% and 0.05% were

d placed in a 5-mL vial for 1 minute with 2 mL of each of theed, rinsed thoroughly, and placed on a white background forreatly: moderate coloring with congo red (CR), crystal violetlue (MB), toluidine blue (ToB), indocyanine green (ICG), andeen (LG), and evans blue (EB); and faint staining with indigo

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ased on their previous application in other fields of

CULAR SURGERY 273

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edicine and enabled comparison between dyes.12,15 Ini-ially, all new dyes at 0.5% tested in enucleated porcineyes enabled exclusion of some vital dyes for 3 reasons:eak or no staining of intraocular tissues, any major signsf toxicity in porcine retinal tissue, or the presence ofetinal cell fragments in the ILM. When methyl violet,oluidine blue, and methylene blue were used for the ILMeeling maneuver, cellular elements were observed,hereas the dyes crystal violet and sudan black at 0.5%

nduced retinal damage. Eosin Y and congo red did notroduce significant staining to make further testing worthursuing. These results left the 6 dyes evans blue, brilliantlue, bromophenol blue, indigo carmine, light green, andast green for further investigation in human donor eyes.hese 6 dyes did not cause damage, whereas a considerable

ntense staining was achieved.During cataract surgery, trypan blue and ICG dyes at

oncentrations from 0.1% to 0.5% may stain the LC.1

owever, trypan blue should be avoided in fertile orregnant women and in children because of possible

IGURE 6. Human donor eyes underwent vitreous and removaTop left) Bromophenol blue (BroB) at 0.5% promoted modenerated mild staining of the ILM. (Bottom left) Evans blue (ight green (LG) at 0.5% induced moderate staining of the IL

eratogenic and mutagenic effects.16 Besides, the dyes have s


een recently described as possible toxic agents for endo-helial cells in certain circumstances.17 Moreover, LCtaining in vitrectomized patients may lead to inadvertenttaining of the posterior LC because of dye diffusion intohe vitreous, thereby obscuring the red reflex. Based onhese drawbacks, our group decided to pursue the testing ofovel dyes also for LC. We found that most dyes providedome staining of the LC, where the best staining agents forhat task may be bromophenol blue, light green, fast green,vans blue, and brilliant blue. In regard to fast green andvans blue, these dyes promoted significant staining of theC; interestingly, there has been no previous report ofheir use for LC visualization. In regard to brilliant blue,ur study found good staining of the LC at 0.5%, but onlymild effect was achieved with a lower concentration.ther studies have described opposite results concerning

rilliant blue; Hisatomi and associates analyzed the effec-iveness of brilliant blue for LC visualization at concen-rations from 0.001% to 1% in porcine eyes.18 Theinimal concentration needed to produce high-quality

examination of the internal limiting membrane (ILM) staining.staining of the ILM. (Top right) Fast green (FG) at 0.5%

at 0.5% caused moderate staining of the ILM. (Bottom right)

l forerateEB)M.

taining with clear visualization was found to be 0.025%,


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alf of the lower dose we chose to study. With regard toromophenol blue, Schuettauf and associates and Harito-lou and associates reported in surgically extracted LC thatmong several novel dyes, bromophenol blue demon-trated satisfactory staining characteristics at concentra-ions of 0.02% to 0.5%.10,19 These results correlateartially with our findings in this study, because bromo-henol blue was able to stain the LC at the 2 concentra-ions investigated, whereas at the lower concentration,nly mild staining was found. Haritoglou and associatesnvestigated the staining characteristics of 6 new dyes,ncluding light green, at concentrations from 0.05% to.0% for staining of the LC. They reported that light greenid not stain the LC sufficiently at concentrations of 0.5%r less.19 In contrast to those results, we found excellenttaining with light green at the concentration of 0.5%.ome explanations for different results for this dye includehe time of experiments after enucleation, solvents for dyereparation, the presence of salts or chemical substances inhe dye, or surgical technique for dye application. There-ore, more clinical studies are necessary before final con-lusions can be drawn with regard to those new vital dyes.

ICG has become a great tool for ILM identification, andore recently, brilliant blue has been approved in Europe

t a concentration of 0.025% for visualization of theLM.14 Indeed, we observed a strong staining of the ILMith brilliant blue, confirming previous reports.1,3,20 How-ver, a number of studies that suggest remarkable retinaloxicity of ICG and brilliant blue; such observationsrompted our group to investigate the staining potential ofther dyes. The vital dyes selected as better for the ILMere bromophenol blue, light green, and fast green (Table). Fast green produced moderate staining of the porcineLM and faint staining of the human ILM, whereas lightreen provided faint staining at the low dose and moderate

TABLE 2. Staining Properties in Hum

Dye

Ex Vivo Staining

Excised ERM

Concentration 0.05% 0.5% 0.05%

Indigo carmine 0 � 0

Evans blue � ��

Light green SF ��

Fast green � ��

Patent bluea � �� 0

Indocyanine greena � � ��

Trypan bluea ��

Brilliant blue � ��

Bromophenol blue � ��

ERM � epiretinal membrane; ILM � internal limiting membrane.

Grade of staining intensity: 0 � no staining; � � faint staining; �aNot considered novel, but they were included for comparison o

taining with higher concentration. Nevertheless, mild i


taining by light green and fast green was enough to allowLM peeling, and no major damage to the retina was noted.nterestingly, fast green was applied first in vitreoretinalurgery by Sorsby in 1939 as an endovenous agent fordentification of breaks in the treatment of retinal detach-ents.21 In 2005, Jackson and associates examined the

taining potential of different types of dyes including fastreen in bovine retina.11 Their results showed that fastreen produced homogenous but weak staining in someetinal specimens at concentrations as low as 0.05%. Withegard to bromophenol blue, in vivo animal studies showedhat bromophenol blue at concentrations of 0.5% and.02% did not result in any significant retinal toxicity,19

hereas providing enhanced ILM staining, which confirmshe good staining of the ILM achieved in our study withromophenol blue. Therefore, light green, brilliant blue,romophenol blue, and fast green should be considered inuture trials for macular hole surgery.

Staining the ERMs may be particularly advantageousecause they rarely contain pigment and usually areifficult to recognize during surgery. In this investigation,taining of the ERM obtained from retinal detachment oriabetic retinopathy surgery could be achieved successfullyith most dyes, whereas the strongest staining agents were

ight green, followed by fast green, evans blue, brilliantlue, and bromophenol blue. Human ERM exposed to fastreen, evans blue, brilliant blue, and bromophenol blueemonstrated moderate staining with the higher dose of.5% and faint staining at 0.05%. Haritoglou and associ-tes investigated staining characteristics of ERMs with dyeoncentrations of 1.0%, 0.5%, 0.2%, and 0.05%, andound that among 6 stains, including light green and E68,romophenol blue stained the ERM best.19 There is notuch information to date concerning the staining capacity

f other dyes such as fast green and evans blue. However,

onor Eyes of Novel Selected Dyes

Ex Vivo Staining in Donor Eyes

Vitreous Lens Capsule

0.5% 0.05% 0.5% 0.05% 0.5%

� � � 0 �

��

��

� � ��

� � � � ��

��

� ��

��

��

moderate staining; �� strong staining. See ref. 13.

ing affinity.

an D

ILM

� �

t can be assumed that evans blue may possess chemical

CULAR SURGERY 275

ml

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fI

rasrtasrtamtsaa

bIIgnctac

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2

oieties that bind to ERMs, because they also are azo dyesike trypan blue, which is an excellent dye for the ERM.

Vitreous staining was achieved with all dyes tested inhis study in porcine eyes. Trypan blue, light green, andvans blue led to strong vitreous staining (��), whereasoluidine blue, congo red, crystal violet, bromophenollue, brilliant blue, fast green, methyl violet, ICG, patentlue, eosin Y, and methylene blue caused moderate stain-ng. Our results revealed that all 6 dyes stained to someegree the vitreous tissue in human cadaveric eyes, al-hough indigo carmine to a lesser extent. We postulatehat the affinity of the dyes for the vitreous is the result ofhe hydrophilic characteristic of the dyes in relation to theigh water content of the vitreous. Considering futurepplications, there are 2 methods to choose a dye. First,ne may consider applying dyes that also stain both theRM and ILM, because costs and product registering cane reduced. However, when one dye stains more than onentraocular target, it can lead to intraoperative confusions to which membrane has been stained. In this regard, itan be proposed to restrict intraoperative use of dyes tohose that stain only the vitreous and not other targetedissues.

In our current research, we elected to use porcine eyesor several reasons. First, the morphologic features of the
LM in pigs are similar to those of humans, and research t
Ophthalmologica 2006;220:190–193.

1

1

1

1

1


elated to ILM removal has been performed already.22,23 Inddition, porcine eyes are more readily available, and thehort interval between animal death and experiments mayeduce the incidence of autolysis in the retina.24 Never-heless, the results of this study may not be totallypplicable to humans, because pigs may have differentubtypes of ILM collagen. In another branch of ouresearch, we recently determined the biocompatibility ofhe 6 dyes investigated thoroughly in this study. Evans bluend light green induced some signs of functional andorphologic retinal damage in rabbits.25 Some strengths of

he present study include a systematic investigation ofeveral dyes, concern for obtaining preliminary data innimals before human testing, and better quality of data ofnimal studies in comparison with cell culture models.

In summary, this study showed that brilliant blue,romophenol blue, fast green, and light green stain theLM well, and they therefore are good alternative dyes toCG, and that ERMs can be stained successfully by lightreen, fast green, evans blue, brilliant blue, and bromophe-ol blue. All dyes stained the vitreous and may beonsidered for human use in the future. Future studies ofhese vital dyes may improve the indications for retinalnd vitreous staining, may increase the precision of surgi-al techniques, and may enhance the discrimination be-
ween the several intravitreous targets.26
HE AUTHORS INDICATE NO FINANCIAL SUPPORT OR FINANCIAL CONFLICT OF INTEREST. INVOLVED IN DESIGN ANDonduct of the study (E.B.R., F.M.P., E.d.P.F.C., E.D., M.M.Jr., O.M., G.B.M., V.S., M.E.F.); Collection, management, analysis, and interpretation ofhe data (E.B.R., F.M.P., E.d.P.F.C., M.M., M.M.Jr., C.H.M., A.B.D., V.S., M.E.F.); and preparation, review, or approval of the manuscript (E.B.R.,.M., E.D., G.M.L., E.d.P.F.C., M.M., C.H.M., A.B.D., M.E.F.). The study was approved by the ethics committee of the Federal University of São Paulo,ão Paulo, Brazil. Care of the animals was conducted according to Association for Research in Vision and Ophthalmology guidelines. The authors thankhe German Academic Exchange Service (DAAD), Fehr Foundation (Germany), Pan-American Ophthalmological Foundation (PAOF), and Fundaçãde Amparo à Pesquisa do Estado de São Paulo (FAPESP).

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Ability of New Vital Dyes to Stain Intraocular Membranes and Tissues in Ocular Surgery

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