Safety evaluation of subretinal injection of trypan blue in rats.

OBJECTIVE: To determine the appropriate concentration of trypan blue (TB) for subretinal injection in a rat model and to provide a safety profile that limits retinal toxicity while maintaining dye visibility. MATERIALS AND METHODS: Adult rats were subretinally injected with various concentrations of either TB or phosphate-buffered saline (PBS); rats which received sham injections served as an additional control. The injected areas were visualized under a surgical microscope. Electroretinography (ERG) was performed to measure retinal function. Animals were then sacrificed, and the eyes were sectioned and examined by light microscopy. Terminal deoxynucleotidy1 transferase dUTP nickend labeling (TUNEL) was applied to determine retinal apoptosis. RESULTS: One day after the subretinal injection, TB stains were visible under the surgical microscope in the 0.2%, 0.08%, and 0.04% TB-injected groups, but not in the 0.02% TB-injected group. TB stain was detectable in the retina and sclera of the 0.2%, 0.08%, and 0.04% TB-injected groups for over 2 weeks after injection. However, the amplitudes of ERGa- and b-waves were affected and became significantly lower in the 0.2% TB-injected group than the amplitudes in the PBS-, or sham-injected group. Moreover, TUNEL+ cells appeared in the outer nuclear layer (ONL), ganglion cell layer (GCL), and retinal pigment epithelium (RPE) layer of the 0.2% and 0.08% TB-injected groups at 1 and 7 days after subretinal injection. In contrast, very few TUNEL+ cells were found in the 0.04% TB- or PBS-injected group. Two weeks after injection, the ONL was significantly thinner in the 0.2% TB-injected group than in the 0.04% TB-, PBS- or sham-injected group. CONCLUSIONS: TB injection induces a dose-dependent neurotoxic effect on retinal cells. Subretinal injection of 0.04% TB is relatively safe and effective for subretinal staining. [ABSTRACT FROM AUTHOR]