April 11, 2012
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Dyes play vital role in vitreoretinal surgery

Authors discuss different aspects of available dyes and which ones are appropriate for which cases.

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Daniele Veritti, MD
Daniele Veritti

Vitreoretinal surgeons often have to remove very thin membranes, such as the epiretinal membrane and the internal limiting membrane.

Several reports show that the removal of the internal limiting membrane (ILM) during macular pucker or macular hole surgery improves anatomical and functional outcomes, but its transparency can make peeling a challenge even for the experienced surgeon. For this reason, the use of vital dyes to stain the epiretinal membrane (ERM) and ILM has become popular among vitreoretinal surgeons and has made the peeling procedure safer and easier, reducing the operating time and the mechanical trauma to the retina. Several dyes are used today to selectively visualize target structures.

Trypan blue

The anionic disazo dye trypan blue was initially used to stain the lens capsule in 0.06% concentration and then to stain preretinal structures in 0.15% concentration. Feron and colleagues initially focused their attention on the behavior of trypan blue when used for removing membranes in proliferative vitreoretinopathy, after which its usefulness for ERM and ILM removal in concentrations ranging from 0.06% to 0.2% was reported.

The commercial concentration of 0.15% is helpful in staining epimacular membranes; however, its level of staining ILM is quite poor. Another drawback of the commercially available product is the dilution in phosphate-buffered saline, necessitating an air-fluid exchange before injection.

And what about safety? Studies on rabbit retina found that the 0.15% concentration was not toxic if removed quickly, and tests conducted on cultured retinal pigment epithelial cells showed that it is safe in concentrations of 0.15% to 0.25% for 5 minutes of exposure. Haritoglou and colleagues described a similar improvement in vision among eyes operated with and without trypan blue. This may suggest that trypan blue does not have a negative influence on visual outcome. The safety profile of trypan blue led a number of European surgeons to use it in vitreoretinal surgery even with its less optimal staining characteristics when compared with indocyanine green.

Indocyanine green

Cyanine dyes such as indocyanine green are most selective for ILM staining and assure a better visualization of the membrane. However, potential damage to the retina and retinal pigment epithelium caused by indocyanine green has been reported. The circumstances under which indocyanine green may be harmful to retinal tissues are not completely understood.

Several hypotheses have been postulated, including harmful hypo-osmolarity of the solution and direct toxicity caused by 5% iodine contained in the indocyanine green molecules. In addition, the absorption spectrum of indocyanine green overlaps with different types of illumination, posing the risk of phototoxicity to the retina.

Moreover, indocyanine green does not appear to be an ideal candidate for ILM staining because its maximum absorption is in the near infrared and not within the spectral sensitivity of the human eye. As a consequence, relatively high dye concentrations are required to achieve a sufficient contrast on the vitreoretinal interface.

Infracyanine green

To avoid the toxicity of indocyanine green while preserving the good clarity of ILM staining ensured by cyanine dyes, another molecule with similar pharmacologic properties has been studied: infracyanine green.

When compared with indocyanine green, infracyanine green possesses some notable differences. Infracyanine green is believed to have less potential for retinal pigment epithelium toxicity because it contains no sodium iodine. Moreover, infracyanine green is used in an isosmotic solution of 294 mOsm/kg to 314 mOsm/kg due to the dilution in 5% glucose.

Finally, some theoretical reasons explain why infracyanine green is less likely to produce retinal phototoxicity than indocyanine green. The shift of the absorption spectrum to longer wavelengths noticed for infracyanine green (690 nm vs. 630 nm) reduces the amount of absorption when using a typical endoillumination Xenon light (400 nm to 500 nm). Balaiya and colleagues showed that infracyanine green appears to be significantly less toxic on retinal ganglion cells and retinal pigment epithelial cell lines when compared with indocyanine green, and it was also the least toxic among newer dyes such as bromophenol blue and brilliant blue green. Both indocyanine green and brilliant blue green selectively stain the ILM, but the staining effect is more pronounced with indocyanine green. In contrast to the narrow safety margin of indocyanine green, brilliant blue green has demonstrated good biocompatibility.

Combinations

At the Innovations in Ophthalmology symposium in Kyoto, Japan, in 2007, Andreas Mohr, MD, suggested that “a combination of trypan blue and [brilliant blue green] administered simultaneously might be even more advantageous for visualizing the ILM as well as ERMs.” To be clinically usable, however, this combination of dyes must not present major toxicity compared with the individual components. Awad and colleagues report that the addition of brilliant blue green at 0.025% completely neutralizes the toxicity of a 0.25% trypan blue solution.

To obtain a better settling of the dye on the ERM or ILM, it would be desirable to have a solution with a higher density and viscosity. The addition of glucose has previously been suggested. Because glucose strongly affects the osmolarity of the solution, other additives with a high molecular weight, such as polyethylene glycol, are preferable. Awad and colleagues tested the toxicity of a polyethylene glycol solution alone and in combination with the trypan blue and brilliant blue green dyes and found that the addition of polyethylene glycol to the dyes did not increase the toxicity of the solution.

Time on retinal surface

Another important point is the time in which the dye solution remains on the retinal surface. This is important in testing the toxicity of the compound and a key factor especially for beginners in vitreoretinal surgery, who usually require longer handling times and may apply the dye several times. Also, for longer incubation times, trypan blue, brilliant blue green and polyethylene glycol preparations have been shown to be less toxic. Therefore, we found new formulations composed of brilliant blue G 0.025% and trypan blue 0.15%, diluted in a 4% polyethylene glycol solution of 338 mOsm/kg, to be promising.

At present, several other dyes have been subjects of in vivo and ex vivo experiments including, among others, methyl violet, crystal violet, eosin Y, Sudan black B, methylene blue, toluidine blue, light green, indigo carmine, fast green, Congo red, Evans blue and bromophenol blue.

To conclude, we think that an ideal candidate dye would incorporate the excellent contrast provided by indocyanine green and the high biocompatibility of brilliant blue green or infracyanine green or include characteristics of strong absorption at visible wavelengths, convenient tissue binding, nontoxicity and physiological degradation at a practical time scale.

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For more information:

Daniele Veritti, MD, can be reached at the Department of Ophthalmology, University of Udine, p.le S. Maria della Misericordia, 33100 Udine, Italy 33100; +39-0432-559907; email: verittidaniele@gmail.com.

Disclosures: Dr. Veritti and Dr. Toneatto have no relevant financial disclosures.