Elaine de Paula Fiod Costa

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.

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.

Comparative analysis of the nuclear lens opalescence by the Lens Opacities Classification System III with nuclear density values provided by Oculus Pentacam: a cross-section study using Pentacam Nucleus Staging software

PURPOSE To compare the clinical classification of cataract using the Lens Opacities Classification System (LOCS) III with the mean values of lens density provided by the Pentacam Scheimpflug System in nuclear cataracts. METHODS One hundred and one eyes from 101 patients with age-related nuclear cataract were submitted to clinical examination for lens grading score using LOCS III. According to LOCS III, nuclear opalescence was divided in six groups. Patients were evaluated by the Pentacam Scheimpflug System for the mean lens density using the Pentacam lens densitometry program (PLDP), the Pentacam Nucleus Staging (PNS) mean value and the PNS cataract grading score. RESULTS A positive correlation between the mean values of lens density and LOCS III classification, considering groups 1 to 5, could be noticed with PLDP and PNS mean value. The mean values between the groups were similar using the PLDP and the PNS mean value. However, when the PNS cataract grading score was evaluated, there was low correspondence with LOCS III classification. CONCLUSION Pentacam Scheimpflug device offers an objective measure of the lens nuclear density on nuclear cataracts. PLDP and the PNS mean value were both useful to evaluate age-related nuclear cataract up to LOCS III group 5.

Current Concepts of Trypan Blue in Chromovitrectomy

Trypan blue (TB) is a blue vital dye with fine color properties to stain the anterior lens capsule and thereby may facilitate capsulorrhexis during cataract surgery. In addition, the blue stain may assist in the visualization of various preretinal membranes and tissues during vitreoretinal surgery in a procedure also called chromovitrectomy. TB has demonstrated great binding affinity for the glial epiretinal membranes, although it remains yet to be determined in which circumstances the dye may color the vitreous and internal limiting membrane. Most studies suggest that 0.06% TB does not pose harm to the retina, but at higher concentrations further investigation is necessary. In this paper, various aspects of the application of TB for chromovitrectomy are discussed including laboratory investigations, surgical technique and clinical outcomes.

Structure-function correlations in glaucoma using matrix and standard automated perimetry versus time-domain and spectral-domain OCT devices.

PURPOSE We examined the structure-function relationship between two perimetric tests, the frequency doubling technology (FDT) matrix and standard automated perimetry (SAP), and two optical coherence tomography (OCT) devices (time-domain and spectral-domain). METHODS This cross-sectional study included 97 eyes from 29 healthy individuals, and 68 individuals with early, moderate, or advanced primary open-angle glaucoma. The correlations between overall and sectorial parameters of retinal nerve fiber layer thickness (RNFL) measured with Stratus and Spectralis OCT, and the visual field sensitivity obtained with FDT matrix and SAP were assessed. The relationship also was evaluated using a previously described linear model. RESULTS The correlation coefficients for the threshold sensitivity measured with SAP and Stratus OCT ranged from 0.44 to 0.79, and those for Spectralis OCT ranged from 0.30 to 0.75. Regarding FDT matrix, the correlation ranged from 0.40 to 0.79 with Stratus OCT and from 0.39 to 0.79 with Spectralis OCT. Stronger correlations were found in the overall measurements and the arcuate sectors for both visual fields and OCT devices. A linear relationship was observed between FDT matrix sensitivity and the OCT devices. The previously described linear model fit the data from SAP and the OCT devices well, particularly in the inferotemporal sector. CONCLUSIONS The FDT matrix and SAP visual sensitivities were related strongly to the RNFL thickness measured with the Stratus and Spectralis OCT devices, particularly in the overall and arcuate sectors.

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.

Novel Vitreous Modulators for Pharmacologic Vitreolysis in the Treatment of Diabetic Retinopathy

Vitreous constitutes about 80% of the volume of the human eye. It is an extended extracellular matrix that is composed of collagen, hyaluronan, and other extracellular matrix molecules, but mostly water. In both health as well as disease, especially diabetic retinopathy (DR), special attention should be drawn to the posterior vitreous cortex and its relation to the retinal surface. The important role of vitreous in the pathogenesis of proliferative DR has already been demonstrated by several experimental and clinical studies. Thus, vitreo-retinal separation by pharmacologic vitreolysis and/or removal by surgical means are appropriate approaches to interrupt the pathogenic contribution of vitreous and prevent progression of diabetic retinopathy to more advanced stages. This review describes various aspects of the molecular morphology and structural anatomy of vitreous and the vitreo-retinal interface, as well as the role of vitreous in the pathophysiology of DR. Lastly, this treatise provides a critical analysis of novel vitreous modulators for pharmacologic vitreolysis in the treatment of DR. Microplasmin is currently the most promising approach to treat vitreoretinal traction by pharmacologic vitreolysis.

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.

Effect of Vital Dyes on Retinal Pigmented Epithelial Cell Viability and Apoptosis: Implications for Chromovitrectomy

Purpose: To investigate the in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial cell line. Methods: ARPE-19 cells were exposed to brilliant blue (BBG), Evans Blue (EB), bromophenol blue (BroB), indocyanine green (ICG), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and Congo red (CR). Balanced salt solution was used as the control. Five different concentrations and 2 exposure times were tested. Cell viability was determined by the MTS (1-solution methyl thiazolyl tetrazolium) assay and apoptosis by Bax expression on Western blot. Results: All dyes significantly reduced cell viability after 3 min of exposure at all concentrations (p < 0.01), except for BBG that was safe at concentrations up to 0.25 mg/ml and CR up to 0.05 mg/ml, while LG was safe at all concentrations. Toxicity was higher after 30 min of exposure. Expression of Bax was upregulated after all dye exposures, except BBG; ICG had the highest Bax expression (p < 0.01). Conclusions: Overall the safest dye was BBG followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BBG was the only dye that did not induce apoptosis in ARPE-19 cells.