Department of Ophthalmology and Visual Science, Eye & ENT Hospital of Fudan University, Shanghai, China.
Eur Rev Med Pharmacol Sci. 2018 May;22(10):2923-2933. doi: 10.26355/eurrev_201805_15046.
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.
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 nick-end labeling (TUNEL) was applied to determine retinal apoptosis.
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.
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.
确定在大鼠模型中用于视网膜下注射的合适锥虫蓝（TB）浓度，并提供一种安全性概况，在保持染料可见度的同时限制视网膜毒性。
成年大鼠接受不同浓度的 TB 或磷酸盐缓冲盐水（PBS）的视网膜下注射；接受假注射的大鼠作为额外的对照。在手术显微镜下观察注射区域。进行视网膜电图（ERG）以测量视网膜功能。然后处死动物，对眼睛进行切片并用光学显微镜检查。末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）用于确定视网膜细胞凋亡。
视网膜下注射后 1 天，在 0.2%、0.08%和 0.04%TB 注射组中可在手术显微镜下观察到 TB 染色，但在 0.02%TB 注射组中不可见。在注射后超过 2 周，在 0.2%、0.08%和 0.04%TB 注射组中，TB 染色可在视网膜和巩膜中检测到。然而，在 0.2%TB 注射组中，a 和 b 波的振幅受到影响，明显低于 PBS 或假注射组的振幅。此外，在 0.2%和 0.08%TB 注射组中，在视网膜下注射后 1 天和 7 天，在外核层（ONL）、神经节细胞层（GCL）和视网膜色素上皮（RPE）层中出现 TUNEL+细胞。相比之下，在 0.04%TB 或 PBS 注射组中发现的 TUNEL+细胞很少。注射后 2 周，在 0.2%TB 注射组中，ONL 明显比 0.04%TB、PBS 或假注射组薄。
TB 注射对视网膜细胞产生剂量依赖性神经毒性作用。视网膜下注射 0.04%TB 相对安全且对视网膜下染色有效。
