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G. P. Holley, D. Patel, S. Pieramici, H. F. Edelhauser; Methylene Blue Toxicity - A Comparative in vitro Study versus Trypan Blue Evaluating Corneal Endothelial Structure and Function Following Exposure. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2689.
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To compare and evaluate the direct effect of 1% Methylene Blue versus 0.1% Trypan Blue exposure on corneal endothelial cell function and ultrastructure.
Paired fresh rabbit corneas (n=5) were obtained from New Zealand white rabbits weighing 2.0 to 2.5 kg. Paired human corneas (n=5 Trypan Blue, n=3 Methylene Blue) stored in Optisol-GS were obtained from the Georgia Eye Bank. The human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion and were perfused with a control solution for 1 hour, BSS Plus for human corneas, and glutathione bicarbonate Ringer’s solution (GBR) for rabbit corneas. Corneal thickness was measured every 15 minutes. After the 1 hour stabilization period, one cornea of each matched pair was exposed to either 1% Methylene Blue or 0.1 % Trypan Blue for 3 minutes followed immediately by a washout with the control solution. The endothelial perfusion for each cornea was then continued for an additional 3 hours with the respective control solutions. At the end of the experiment, the corneas were fixed for scanning (SEM) and transmission (TEM) electron microscopy.
In rabbit corneas, the mean ( ± std. error) corneal swelling rate was 63.3 ± 6.2 µm/hr for Methylene Blue exposed corneas vs. 9.4 ± 2.3 µm/hr for control GBR (p<0.0001). Comparatively, the mean corneal swelling rate for Trypan Blue exposed corneas was 0.4 ± 2.0 µm/hr vs. 5.0 ± 2.4 µm/hr for GBR. The human corneal swelling data shows the same trend with significant swelling for the Methylene Blue exposed corneas versus control. This is contrasted with Trypan Blue which swelled 2.8 ± 2.5µm/hr versus 7.2 ± 2.8 µm/hr for control BSS Plus. Electron microscopy shows normal appearing corneal endothelial ultrastructure in both rabbit and human corneas exposed to Trypan Blue. Contrastingly, the TEM and SEM reveal cell damage (vacuolization and significant cell death) to the endothelium of the Methylene Blue exposed corneas in both rabbit and human.
Methylene Blue exposure to the corneal endothelium for 3 minutes causes endothelial cell death with the resultant corneal swelling. Trypan Blue does not cause corneal swelling nor changes in endothelial cell ultrastructure.
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