Effect of vital dyes on human corneal endothelium and elasticity of Descemet’s membrane

Abstract

The purpose of this study was to evaluate the effects of vital dyes on human Descemet's membranes (DMs) and endothelia. DMs of 25 human cadaveric corneas with research consent were treated with dyes routinely used in Descemet membrane endothelial keratoplasty (DMEK), 0.05% Trypan blue (TB) or a combination of 0.15% Trypan blue, 0.025% Brilliant blue and 4% Polyethylene glycol (commercial name Membrane Blue Dual; MB). The effects of these two dyes on (i) endothelial cell viability, (ii) DM mechanical properties as assessed by atomic force microscopy, and iii) qualitative DM dye retention were tested for two varying exposure times (one or four minutes). No significant differences in cell toxicity were observed between treatments with TB and MB at the two different exposure times (P = 0.21). Further, both dyes led to a significant increase in DM stiffness: exposure to TB and MB for one minute increased the apparent elastic modulus of the DM by 11.2% (P = 8*10−3) and 17.7%, respectively (P = 4*10−6). A four-minute exposure led to an increase of 8.6% for TB (P = 0.004) and 13.6% for MB (P = 0.03). Finally, at 25 minutes, the dye retention of the DM was considerably better for MB compared to TB. Taken together, a one-minute exposure to MB was found to improve DM visibility compared to TB, with a significant increase in DM stiffness and without detrimental effects on endothelial cell viability. The use of MB could therefore improve (i) visibility of the DM scroll, and (ii) intraoperative unfolding, enhancing the probability of successful DMEK surgery.

Evolution of therapies for the corneal endothelium: past, present and future approaches

“…82 Nevertheless, the exposure of the DMEK grafts to these vital dyes should be restricted as studies have reported timedependent toxic effects of these dyes to the CE. 83 To prevent iatrogenic graft failure caused by an inadvertent 'up-side-down' graft, intraoperative orientation of the DMEK graft is essential. 84 However, graft orientation can often be difficult following the intracameral transfer of the graft.…”

“… 82 Nevertheless, the exposure of the DMEK grafts to these vital dyes should be restricted as studies have reported time-dependent toxic effects of these dyes to the CE. 83 …”

Outcomes of Descemet Membrane Endothelial Keratoplasty in Bangkok, Thailand

“… 29 At the same time, treating of DM tissue with the higher concentration of 0.15% trypan blue (MembraneBlue ® , DORC International, Zuidland, The Netherlands), typically used for vitreoretinal surgery, up to 3–4 minutes led to a deeper blue stain for a prolonged period without detrimental effects on endothelial cell viability. 29 , 30 However, a longer staining time of 5 minutes with 0.15% concentration resulted in a decrease in ECD, approaching statistical significance. 29 In general, a DMEK graft is clinically stained with 0.06% trypan blue for about 30 seconds to 5 minutes before transplantation.…”

“…Regarding the concentration and exposure time of trypan blue and ECL, there have been a few in vitro studies attempting to determine the proper concentration and the duration of application of trypan blue dye to DMEK donor corneal tissue to enhance visibility while minimizing endothelial toxicity. 29 , 30 Staining DM grafts with 0.06% trypan blue (VisionBlue ® , DORC International, Zuidland, The Netherlands), which is often used in DMEK, up to 5 minutes was found to be effective and safe. 29 At the same time, treating of DM tissue with the higher concentration of 0.15% trypan blue (MembraneBlue ® , DORC International, Zuidland, The Netherlands), typically used for vitreoretinal surgery, up to 3–4 minutes led to a deeper blue stain for a prolonged period without detrimental effects on endothelial cell viability.…”

“…Staining of DM graft with 0.1% trypan blue up to 4 minutes theoretically allowed more intense stain without additional adverse effects as the use of the higher concentration of trypan blue (0.15% MembraneBlue ® ) was proven to be effective and safe for staining DMEK donor tissue for 3–4 minutes. 29 , 30 Stained DM roll was aspirated into the curved glass injector system (DMEK surgical disposable set, DORC International ® Zuidland, The Netherlands) ready for injection into the recipient eye.…”