• epiretinal membrane
• indocyanine green
• infracyanine green
• internal limiting membrane
• trypan blue

In an article in this issue of the BJO by Stalmans and colleagues (p 713) and another recently by Li and colleagues,¹ the novel vital stains trypan blue (TB) and infracyanine green are investigated clinically. These vital stains are promising in vitreoretinal surgery as alternatives to the by now familiar agent indocyanine green (ICG). Firstly, a brief history of membrane staining. ICG, a fluorescent dye used in choroidal angiography, was recognised for its potential in the operating room by cataract surgeons, who first used it to stain the anterior capsule to facilitate capsulorrhexis in difficult cases. Vitreoretinal surgeons, taking notice of their anterior segment colleagues’ success, soon found that ICG similarly stained the internal limiting membrane (ILM) of the retina. This was important because peeling of the nearly invisible ILM from the retinal surface had recently been advocated in macular hole surgery^2,3; this was easier said than done, however, until the arrival of ICG made ILM peeling easier, faster, and less traumatic. Thus, a trend in posterior segment surgery was born, and ICG soon enjoyed widespread attention. A flurry of articles appeared, with several early clinical series reporting impressive success rates in macular hole surgery with ICG assisted ILM peeling.^4,5 ICG staining has by now entered the mainstream of retinal surgery as a useful tool in macular hole repair and epiretinal membrane removal.

But, more recently, some doubts have arisen around ICG, as a critical reassessment has followed the initial enthusiasm. As discussed by the authors of both of these papers, greater clinical experience has begun to reveal possible toxic effects of ICG on the RPE and neural retina. Engelbrecht et al published a clinical series that found central RPE atrophy with poor visual outcomes after some cases of ICG assisted macular hole repair, possibly caused by direct contact of ICG with RPE cells in the area of the hole.⁶ Laboratory studies, in a perhaps inverted sequence, have followed the initial clinical reports and have raised further concerns of overlooked ICG toxicity. Histological and electron microscopic examinations of surgically excised ILM specimens have demonstrated disruption of retinal elements after ICG application. Muller cell fragments, as well as astrocytes and cellular debris, were observed in ILM specimens peeled with ICG; this was in contrast with those peeled using no ICG,^5,7 suggesting an alteration of the “cleavage plane” between the ILM and underlying neural retina. In vitro studies have indicated toxicity of ICG and its aqueous solvent to cultured RPE cells,^8,9 a possible correlate to the clinical study of central RPE atrophy after macular hole surgery using ICG in some patients.⁶ Such concerns have led some surgeons to attempt to minimise ICG exposure during surgery, with reduced concentrations and application times of the dye, as well as circumscribed indications for adjunctive ICG use.

Here’s where the work presented by Stalmans et al comes in. They performed double staining with trypan blue and infracyanine green to enhance visualisation during macular pucker surgery. One of two important aspects of this paper is the introduction of infracyanine green as an alternative to indocyanine green for vital staining in posterior segment surgery. Under the theory that retinal toxicity may be a consequence of the hypotonicity of standard indocyanine green in its aqueous solvent, the authors have turned to infracyanine green as an iso-osmotic alternative.

Indocyanine green is prepared for surgical use by reconstituting it with pure water as a solvent, before bringing it to its final concentration in balanced saline solution; it is the hypo-osmolarity of the solvent and the final solution that is suspected by the authors of disrupting cellular elements of the neural retinal. In contrast, infracyanine green, which uses 5% glucose solution as its solvent for an iso-osmotic final solution, offers comparable staining of the ILM while reducing the untoward osmotic effects. In previous in vitro studies by this group, infracyanine green in 5% glucose did not demonstrate cytotoxicity to cultured RPE cells, while ICG exposure led to significantly increased cell death.⁹ Similarly, in the present study ILM specimens excised with infracyanine green were not noted to include Muller cell footplates or other evidence of neural retinal disruption when examined by histopathology and electron microscopy. Comparing ICG and infracyanine green, maybe the grass is actually greener on the other side of the fence.

Another central aspect of the paper by Stalmans et al and also that of Li et al is the use of trypan blue for epiretinal membrane surgery. The introduction of trypan blue is more than just a change in colour schemes; its properties as a vital stain are quite distinct from those we’re accustomed to with indocyanine green (or even infracyanine green). Unlike the green dyes which selectively stain the acellular ILM but not overlying membranes and vitreous, trypan blue directly stains epiretinal membranes (ERMs), making it valuable in cases such as macular pucker removal. Whereas ICG can only indicate the presence of the ERM by its lack of green staining within an area of stained ILM (referred to as “negative staining”), trypan blue has an affinity for the cellular material composing epiretinal membranes (ERMs), providing visualisation and localisation. One advantage of this is noted by the authors, who found in some cases that trypan blue reveals the extent of the ERM to be much larger than suspected clinically. It is possible that macular pucker surgery unaided by trypan blue can underestimate the size of the membrane, resulting in incomplete ERM removal and residual macular distortion. Trypan blue has other potential surgical benefits; Li et al found that it not only stains ERMs but also provides adequate staining of the ILM, and they forwarded the use of trypan blue as yet another alternative to ICG in macular hole repair with ILM peeling. Finally, in another twist, Stalmans et al exploit the “complementarity” of these dyes in a double staining technique for macular pucker removal, first with trypan blue to peel the ERM, then with infracyanine green to peel the underlying ILM. It would appear that no membrane is safe from vitreoretinal surgeons these days.

The introduction of trypan blue and infracyanine green by no means lays to rest the issue of retinal toxicity. Trypan blue stained ILM fragments were found by Li et al to contain glial elements from the neurosensory retina, raising again the spectre of retinal disruption which can affect visual outcomes. As with ICG, with more study and clinical experience the limitations of trypan blue and infracyanine green will be defined while their dosages and application times are optimised. Other barriers also remain to be overcome, not the least of which is that trypan blue and infracyanine green are unavailable for surgical use in many countries including the United States. But the work presented in these two papers marks an important development in this relatively new technology, moving beyond ICG as the sole dye to a second generation of vital stains for intraocular surgery. As more types of vital stains with distinct properties are added to our arsenal, further innovations may enable novel surgical strategies. One can envision in the future an array of vital stains, a sort of surgical palette, with different intraoperative dyes or even non-invasive dyes having distinct affinities for specific membranes or cell types. Diabetic fibrovascular membranes, vitreous cortical hyaloid, proliferative vitreoretinopathy membranes, neurosensory retina, choroidal neovascular membranes, active tumour cells—all might be promising targets for vital stains. Such advances will probably require that we move beyond “off the shelf” agents and instead turn our attention to the research and development of new vital stains specifically for ophthalmic surgery.