Experimental Study Of Trypan Blue On The Retinal Toxicity

[Objective] To investigate the effect of histopathology and function of retina during exposed to trypan blue (TB) solutions in different concentrations and durations. Acute histopathological analysis was performed in experimental macular surgery with TB staining on porcine eyes. To study the histopathological and functional changes in rabbit's retina after the vitreous cavity injected with TB for a period of time.[Methods] For the first part, nine adult porcine eyes were used within 4 hours after enucleation and randomized into three TB staining groups: 0.06%, 0.1% and 0.2%, 3 eyes in each group. The eyes were hemi-sected and the vitreous removed. The anterior segments were discarded. 0.5 ml of Different concentration of TB was applied over the trephined macula, and the remainder of the eyecup was filled with balanced salt solution (BSS). Both the TB and the BSS were removed after 1 minute and the complete eye cup irrigated and filled with fresh BSS. Both the treated and untreated retinas were illuminated with a standard surgical light pipe and light source at maximum power for 10 minutes. Both the TB exposed retina and non-treated surrounding retina were processed for light microscopy (LM) observation. For the second part, eighteen New Zealand white rabbits were randomized into 3 TB injection groups: 0.06%, 0.1% and 0.2%, 6 rabbits in each group. The right eyes were used for experiment and the left served as controls. All eyes received injections of 0.4ml C-3F₈ into mid-vitreous cavity for gas compression vitrectomy. At the time of maximum gas expansion (72 hours of gas compression), the mid-vitreous cavity of right eyes were injected with 0.1ml 0.06% TB, 0.1ml 0.1% TB or 0.1ml 0.2% TB. The left eyes were injected with 0.1ml BSS in the same way. Slit lamp biomicroscopy, indirect ophthalmoscopy and intraocular pressure (IOP) measurement were performed at regular intervals in all eyes pre- andpostoperatively. Electroretinogram (ERG) was performed pre- and 4 weeks postoperatively in all eyes and recorded the amplitude of a wave and b wave value. The eyes were enucleated 30 days postoperatively and processed for light microscopy (LM) and transmission electron microscope (TEM) to examination. [Results] For the first part, exposure of the retina of the porcine eyes to TB for 1 minute followed by illumination caused no histological detectable damage compared to the controls. The vitreoretinal interface was unaffected and the architectures of all layers of retina were well preserved, no cellular disruption was detected. For the second part, Clinical examination showed posterior capsule transient opacification in all animals, which cleared as the gas bubble resolved. The IOP was no waved pre and postoperatively. The ratios of experimental and control eyes of the amplitude of a wave and b wave value were no different Pre- and 4 weeks postoperatively in all eyes (P>0.05). The superior and inferior architectures of all layers of retina were well preserved compared to the controls in all animals. No histological damage was detected. The ultrastructure of all superior retina and inferior retina in 0.06%TB group was normal under the TEM. The ultrastructure of inferior retina in 0.1 %TB group was slightly and reversible. But in 0.2% group, the obvious changes of the ultrastructure in inferior retina were detected. The neural fibers partly degenerated. The mainly nuclear membrane of neural ganglion cells and bipolar cells dissolved, the nuclear was obscure. Lots of neural fibers degenerated in inner and outer plexiform layers. The cell architecture of outer nuclear layer was blurred. The outer segment membrane disc of rod and cone cells was irregular. The mainly part of damages were irreversible.[Conclusions] 1. The ex porcine retina exposed to 0.06%, 0.1 % and 0.2%TB for 1 minute followed by illumination with a standard surgical light pipe and light source at maximum power for 10 minutes caused no acute histological changes. 2. Nohistopathological and functional changes in rabbit's retina after the vitreous cavity injected with 0.06%TB for 4 weeks. 3. The ultrastructure changes were slightly and reversible in rabbit's retina after the vitreous cavity injected with 0.1 %TB for 4 weeks. The retinal function was normal. 4. The ultrastructure changes were obvious and irreversible in rabbit's retina after the vitreous cavity injected with 0.2%TB for 4 weeks. But ERG a wave and b wave were unaffected. 5. The higher concentration of TB prolonged presence in the vitreous cavity may toxicity to the retina and it is dose relevant. The minimal effective concentration of TB should be used in clinical vitrectomy and shorten the touching time of the TB with retina and it should be washed out as complete as possible.