Vital dyes increase the rigidity of the internal limiting membrane

Abstract

To assess the stiffness of the natural human internal limiting membrane (ILM) and evaluate potential changes of the mechanical properties following staining with brilliant blue (BB) and indocyanine green (ICG). Unstained ILM specimens were obtained during ophthalmic surgical procedures. After removal, the specimens were dissected into five parts. Two fragments were stained with BB and ICG, respectively, for 1 min, another two specimens were stained similarly followed by additional subsequent illumination using a standard light source (PENTA LUX x 50, Ophthalmologische Systeme GmbH Fritz Ruck). The fifth part served as an untreated control. All specimens were then analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 × 10 μm were chosen and stiffness was determined by using AFM in a force spectroscopy mode. The force curves were plotted with a data rate of 5000 Hz. In all specimens both the retinal side and vitreal side were analyzed. Staining resulted in a significant increase in tissue stiffness. An increase was seen both for the vitreal (BB: P<0.001; ICG: P<0.01) and retinal side (BB: P<0.01; ICG: P<0.01), with the retinal side being significantly stiffer in all control and stained samples. Additional illumination after staining did further increase tissue rigidity in most samples but not significantly. Staining significantly increases the stiffness of the human ILM. This might explain the fact that the stained ILM can be removed more easily and in larger fragments during vitreoretinal surgical procedures compared with unstained ILM.