Eduardo Dib

The Use of Vital Dyes in Ocular Surgery

Vital dyes have advanced diagnosis and surgical technique in various specialties, including oncology, gastroenterology, and ophthalmology. In ocular surgery vital dyes are widely used in cataract and vitreoretinal surgery. Worldwide, intra-operative use of trypan blue during cataract surgery has enhanced visualization of the anterior capsule during capsulorrhexis, and patent blue has been recently licensed in Europe for cataract surgery. For chromovitrectomy, the vital dyes indocyanine green, infracyanine green, and brilliant blue stain the internal limiting membrane, and trypan blue and triamcinolone acetonide help visualize epiretinal membranes and vitreous, respectively. Intra-operative vital dyes are finding uses in corneal, glaucoma, orbit, strabismus, and conjunctival surgery. We provide a summary of current knowledge of the use of vital dyes in ocular surgery. We review the properties of dyes, techniques of application, indications, and complications in ocular surgery. Vital dyes represent an expanding area of research, and novel dyes deserve further investigation.

Vital dyes for chromovitrectomy.

Purpose of review The aim of this article is to present the current state-of-the-art in regard to the application of vital dyes during vitreoretinal surgery, ‘chromovitrectomy’, as well as to overview the current literature regarding the properties of dyes, techniques of application, indications, and complications in chromovitrectomy. Recent findings A large body of published research has recently addressed the toxicity profile of indocyanine green for chromovitrectomy. Experimental data demonstrate dose-dependent toxicity of indocyanine green to various retinal cells. Newer generation vital dyes for chromovitrectomy include trypan blue, patent blue, triamcinolone acetonide, infracyanine green, sodium fluorescein, bromophenol blue, fluorometholone acetate and brilliant blue. Novel instruments may enable a selective painting of preretinal tissues during chromovitrectomy. Summary This review suggests that the field of chromovitrectomy represents an expanding area of research. The first line agents for internal limiting membrane staining in chromovitrectomy are indocyanine green, infracyanine green, and brilliant blue. Patent blue, bromophenol blue and trypan blue arose as outstanding biostains for visualization of epiretinal membranes. Novel dyes available for chromovitrectomy deserve further investigation.

Ability of New Vital Dyes to Stain Intraocular Membranes and Tissues in Ocular Surgery

PURPOSE To evaluate the ability of novel dyes to stain lens capsule (LC), internal limiting membrane (ILM), epiretinal membrane (ERM), and vitreous. DESIGN Experimental study in animal and human donor eyes. METHODS Thirteen dyes, methyl violet, crystal violet, eosin Y, sudan black B, methylene blue, toluidine blue, light green, indigo carmine, fast green, congo red, evans blue, brilliant blue, and bromophenol blue, were injected onto the LC and ILM of enucleated porcine eyes. The vitreous was stained with 2 mL of dyes for 1 minute. Six dyes (indigo carmine, evans blue, fast green, light green, bromophenol blue, and brilliant blue) were selected for experiments in human donor eyes and freshly removed ERM. RESULTS In the porcine eyes, ILM staining with methylene blue, toluidine blue, indigo carmine, evans blue, bromophenol blue, and fast green was moderate, and methyl violet, crystal violet, brilliant blue, or sudan black resulted in strong staining. Methyl violet, crystal violet, sudan black, toluidine blue, and methylene blue caused histologic damage in porcine retinas. Vitreous examination revealed moderate staining with congo red, crystal violet, fast green, eosin Y, methylene blue, toluidine blue, brilliant blue, bromophenol blue, and methyl violet and strong staining with light green and evans blue. ERMs showed strong staining with 0.5% evans blue and moderate staining with 0.5% light green, fast green, brilliant blue, and bromophenol blue. Evaluation of donor eyes disclosed moderate staining with evans blue, light green, and bromophenol blue and strong staining with 0.5% brilliant blue. Moderate or strong staining of the vitreous occurred with most dyes. LC evaluation showed moderate staining with 0.5% evans blue, fast green, and brilliant blue, whereas 0.5% light green produced strong LC staining. CONCLUSIONS Brilliant blue shows the best ILM staining, whereas bromophenol blue, evans blue, and light green also stain ILM. Most dyes bind well to LC, vitreous, and ERM.

Subretinal bevacizumab detection after intravitreous injection in rabbits.

PURPOSE To evaluate subretinal detection of bevacizumab 2 hours after intravitreous injection of 1.25 mg in rabbit eyes. METHODS Anterior chamber paracentesis using a 30-gauge needle was performed in nine female Dutch-belted rabbits by removal of 0.05 mL of aqueous humor. Transscleral retinal detachment was performed with a modified 25-gauge infusion cannula connected to a bottle of physiologic saline solution (PSS). The animals were divided into experimental group 1, with intravitreous injection of 0.05 mL of (1.25 mg) with a 30-gauge needle (n = 6) and the control group 2, with intravitreous injection of 0.05 mL of PSS with a 30-gauge needle (n = 3). Two hours after the intravitreous bevacizumab or PSS injection, subretinal fluid was aspirated and immunoassayed to measure the level of bevacizumab. The rabbits were killed by intravenous pentobarbital injection. The eyes were enucleated and fixed in 10% formaldehyde. The pars plana site at which the transscleral cannula was introduced was analyzed by light microscopy, to exclude iatrogenic retinal tears. Eyes with accidental retinal tears were excluded. RESULTS Subretinal bevacizumab molecules were detected in the six eyes that received an intravitreous bevacizumab injection. No subretinal bevacizumab was detected in the control eyes. Light microscopy showed no evidence of retinal tears or holes in any rabbits used for the bevacizumab detection and control group. CONCLUSIONS Bevacizumab molecules were detected in the subretinal space after intravitreous injection of 1.25 mg of bevacizumab, possibly as the result of diffusion through the retina in a rabbit model.

Fluid dynamics in three 25-gauge vitrectomy systems: principles for use in vitreoretinal surgery.

Purpose:  To compare infusion and extrusion fluid volumes with three 25‐gauge vitrectomy systems.

Morphologic and Clinical Effects of Subretinal Injection of Indocyanine Green and Infracyanine Green in Rabbits

PURPOSE The aim of this study was to determine the effects of subretinal injection of indocyanine green (ICG), infracyanine (IfCG), and balanced salt solution (BSS) in rabbits. METHODS Ten (10) animals were subjected to a subretinal injection of 0.05% ICG (279 mOsm), 0.5% IfCG (276 mOsm), and BSS (300 mOsm) used as a control. Animals were examined at 6, 12, and 24 h and 14 days following the surgical procedure by indirect binocular ophthalmoscopy, fluorescein angiography (FA), and light and transmission electron microscopy. RESULTS The subretinal injection of ICG caused damage to all retinal layers and retinal pigment epithelium (RPE) during the entire follow-up. Subretinal injection of IfCG resulted in abnormalities of the photoreceptor outer segments (POSs) during the entire follow-up; however, abnormalities of the photoreceptor inner segments (PISs) and outer nuclear layer (ONL) were observed only 24 h and 14 days after surgery; no RPE damage was observed. FA showed that window defects were more prominent in the subretinal ICG bleb position than the IfCG-related area. BSS caused only abnormalities of the POS layer and no RPE alterations. CONCLUSIONS Subretinal injection of 0.05% ICG results in more significant retinal damage than 0.5% IfCG. In this model, iodine-free IfCG demonstrates a safer profile than a tenfold lower concentration of ICG, which contains iodine in its composition.

Perspective on Fluid and Solid Dynamics in Different Pars Plana Vitrectomy Systems

PURPOSE To summarize current concepts and recent data from the literature about different vitrectomy machines and small-gauge systems based on physical laws. DESIGN Interpretive essay. METHODS Review and synthesis of selected recent literature with interpretation and perspective. RESULTS Pars plana vitrectomy can be performed with a wide variety of treatment strategies, for multiple diseases, and with different materials (solids and fluids). We discuss a variety of machines and system settings (peristaltic pump, duty cycle, aspiration, and infusion controls) targeting safer and the most effective surgery with detailed explanations of the physical properties. CONCLUSIONS Effective management of new surgical strategies is based on recognizing and addressing various physical characteristics in disease and ocular settings. Although the properties of machine are expanding, the long-term efficacy and safety of most new approaches have yet to be established in controlled clinical trials.

Retinal Pigmented Epithelial Cells Cytotoxicity and Apoptosis through Activation of the Mitochondrial Intrinsic Pathway: Role Of Indocyanine Green, Brilliant Blue and Implications for Chromovitrectomy

Purpose To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. Methods ARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. Results ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. Conclusions The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

Toxicological considerations for intravitreal drugs

Introduction: Intravitreal injections are a very common procedure and are the most effective route of drug delivery to the retina. There are currently several drugs available and even more are in development; therefore, safety is a very important concern. Areas covered: The toxicological considerations of the most common drugs used for intravitreal pharmacotherapy such as anti-VEGFs, corticosteroids and antibiotics. Emerging agents such as anti-TNFs, VEGF-trap and kinase inhibitors are also discussed. An assessment of the efficacy and safety issues of the most relevant drugs including bevacizumab, ranibizumab and triamcinolone is presented. Expert opinion: The toxicology and safety profiles are available for several drugs that are either in use or will be available for intravitreal injections. Retinal pharmacotherapy is very effective for different retinal diseases; however safety is a very important issue when intravitreal injections are applied and the possibility of retinal toxicity should always be kept in mind. Bevacizumab and ranibizumab are effective for the therapy of wet-age-related macular degeneration and macular edema, while triamcinolone remains an alternative agent to treat secondary macular edema. It is important, as some of these drugs will be used for extended periods of time, that their long-term toxicological effects are better understood.

In vivo, in vitro toxicity and in vitro angiogenic inhibition of sunitinib malate.

Purpose: To evaluate the in vivo and in vitro toxicity of sunitinib malate, a multikinase inhibitor molecule. Design: Experimental, Prospective, Controlled. Methods: Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelialcells (HUVECS) were used in a culture toxicity test and exposed to different concentrations of sunitinib malate for 18 hours. The HUVECs also were cultured to evaluate the angiogenesis inhibitory effect of sunitinib malate. Fundus photography and angiographic, electrophysiologic, and histopathologic evaluations with light and electron microscopy were performed in two groups of five rabbits each that received different intravitreal concentrations of the drug. Each rabbit received 0.1 ml of sunitinib malate in the right eye (one group with 12.5 mg/ml, the other group with 25 mg/ml); all animals received 0.1 ml of physiologic saline solution in the left eye. After sacrifice, the eyes were enucleated and fixed with modified Karnovsky solution. Results: No toxicity related to sunitinib malate was observed using an in vitro model with the 12.5 and 25 mg/ml solutions in HUVEC and ARPE cell cultures. No toxicity was observed in the in vivo model with 12.5 mg/ml, but light microscopy showed that the 25 mg/ml solution damaged the photoreceptors layer. No functional changes in the electroretinogram were observed in any group. Conclusions: Sunitinib malate 12.5 mg/ml caused no toxicity in in vivo and in vitro models, but the 25 mg/ml concentration caused retinal changes suggesting toxicity in the in vivo model. Further research with the drug is needed in models of ocular neovascularization.