Tracking the evolution of CNS remyelinating lesion in mice with neutral red dye.

Animal models of central nervous system (CNS) demyelination, including toxin-induced focal demyelination and immune-mediated demyelination through experimental autoimmune encephalomyelitis (EAE), have provided valuable insights into the mechanisms of neuroinflammation and CNS remyelination. However, the ability to track changes in transcripts, proteins, and metabolites, as well as cellular populations during the evolution of a focal lesion, has remained challenging. Here, we developed a method to label CNS demyelinating lesions by the intraperitoneal injection of a vital dye, neutral red (NR), into mice before killing. We demonstrate that NR-labeled lesions can be easily identified on the intact spinal cord in both lysolecithin- and EAE-mediated demyelination models. Using fluorescence microscopy, we detected NR in activated macrophages/microglia and astrocytes, but not in oligodendrocytes present in lesions. Importantly, we successfully performed RT-qPCR, Western blot, flow cytometry, and mass spectrometry analysis of precisely dissected NR-labeled lesions at 5, 10, and 20 d postlesion (dpl) and found differential changes in transcripts, proteins, cell populations, and metabolites in lesions over the course of remyelination. Therefore, NR administration is a simple and powerful method to track and analyze the detailed molecular, cellular, and metabolic changes that occur within the lesion microenvironment over time following CNS injury. Furthermore, this method can be used to identify molecular and metabolic pathways that regulate neuroinflammation and remyelination and facilitate the development of therapies to promote repair in demyelinating disorders such as multiple sclerosis.

Investigating retinal toxicity of a lutein-based dye in a model of isolated and perfused bovine retina.

PURPOSE: Retidyne™ is a new lutein-based dye for internal limiting membrane staining. It uses the intrinsic staining characteristics of lutein which is already known to act as an antioxidant and blue-light filter in the human retina. We investigated retinal tolerance to different staining times measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. METHODS: For functionality, testing bovine retinas were prepared and perfused with an oxygen saturated standard solution and the ERG was recorded until stable b-wave amplitudes were reached. Then the perfusion was stopped and Retidyne™ was applied directly onto the retinal surface for exposure times of 60 or 120 s. After restarting the perfusion with standard solution, the ERG amplitudes were monitored for 75 min. To investigate the effects on photoreceptor function alone, 1 mM asparate was added to block b-waves. RESULTS: For an exposure time of 60 s amplitudes of a- and b-waves remained stable throughout the experiment. Exposure times of 120 s caused an initial drop of amplitudes that reached statistical significance only for a-waves (a, - 21%, p = 0.047; b, - 14%, p = 0.052). This effect was only seen during the first minutes of the washout and the ERG recovered completely. CONCLUSIONS: In the model of isolated and perfused bovine retina, Retidyne™ showed a good safety profile for common intraoperatively used staining times. An initial toxic effect regarding the transient drop of amplitudes cannot be ruled out but the effect might also be explained by the partial blockage of the flashlight due to a more intense staining effect at the beginning of the washout.

Determination of trypan blue efficacy in the mitigation of ex vivo canine PCO formation.

PURPOSE: To determine whether trypan blue (TB) reduces canine lens epithelial cell (LEC) or corneal endothelial cell (CEC) viability in vitro; if cell death is noted, to subsequently evaluate the molecular mechanism. METHODS: Cellular viability was determined using a lactate dehydrogenase (LDH) assay. In TB-treated LECs, caspase 3/7 activity was assessed to evaluate apoptosis; autophagy was evaluated using immunoblotting against LC3 and p62. To evaluate the effects of TB on ex vivo posterior capsule opacification (PCO), following mock cataract surgery, lens capsules were treated with TB and subsequently maintained in culture to determine LEC migration and proliferation. RESULTS: Following acute exposure, TB did not significantly reduce LEC or CEC viability at any of the concentrations tested. Increased caspase 3/7 activity was found in LEC cultures treated with TB for an extended period of time; no change in LC3 or p62 expression was noted. Ex vivo PCO formation was not significantly altered by TB treatment. CONCLUSIONS: Acute exposure to TB did not reduce LEC or CEC viability, and only longer exposure to TB was able to initiate apoptosis. Treatment with intraocular TB at the time of cataract surgery is likely safe to the CECs but will not prevent PCO formation.

Retinal Toxicity of Medical Devices Used during Vitreoretinal Surgery: A Critical Overview.

Retinal toxicity/biocompatibility of medical devices in direct contact with the retina is an important subject for clinicians and scientists. As these effects are not very frequent, there is also a relative lack of information for many clinicians. The past has taught us multiple times that there is a significant safety problem associated with severe loss of vision in affected patients. In this review, we want to classify medical products that are used in the back of the eye, describe recent examples of toxicity, critically reflect on the regulations that exist and suggest improvements that can be done to ensure patient safety without hindering innovation. METHODS: Critical review of the recent papers and personal experience of the authors in this issue. Medical devices used in the back of the eye and recent examples of toxicity are described, regulations that exist are critically reflected and improvements suggested that can ensure patient safety without hindering innovation. RESULTS: There is clear evidence of toxicity after intraocular surgery in any category. Some cytotoxic indirect methods have failed in detecting this toxicity. Some ISO rules do not seem appropriate. Postmarketing safety is missing. There is little data on this issue. CONCLUSIONS: The absence of a clear regulation of the production, purification and evaluation of the toxic effects of the medical devices supposes the possibility that products are not sufficiently safe to obtain the CE mark.

Sentinel lymph node biopsy in cutaneous melanoma of the head and neck using the indocyanine green SPY Elite system.

PURPOSE: Lymph node status is the single most important prognostic factor for patients with early-stage cutaneous melanoma. Sentinel lymph node biopsy (SLNB) has become the standard of care for intermediate depth melanomas. Modern SLNB implementation includes technetium-99 lymphoscintigraphy combined with local administration of a vital blue dye. However, sentinel lymph nodes may fail to localize in some cases and false-negative rates range from 0 to 34%. Here we demonstrate the feasibility of a new sentinel lymph node biopsy technique using indocyanine green (ICG) and the SPY Elite near-infrared imaging system. MATERIALS AND METHODS: Cases of primary cutaneous melanoma of the head and neck without locoregional metastasis, underwent SLNB at a single quaternary care institution between May 2016 and June 2017. Intraoperatively, 0.25 mL of ICG was injected intradermal in 4 quadrants around the primary lesion. 10-15 minute circulation time was permitted. SPY Elite identified the sentinel lymph node within the nodal basin marked by lymphoscintigraphy. Target first echelon lymph nodes were confirmed with a gamma probe and ICG fluorescence. RESULTS: 14 patients were included with T1a to T4b cutaneous melanomas. Success rates for sentinel lymph node identification using lymphoscintigraphy and the SPY Elite system were both 86%. Zero false negatives occurred. Median length of follow-up was 323 days. CONCLUSIONS: In this pilot study, Indocyanine green near-infrared fluorescence demonstrates a safe, and facile method of sentinel lymph node biopsy for cutaneous melanoma of the head and neck compared with lymphoscintigraphy and vital blue dyes.

The Vital Dye CDr10b Labels the Zebrafish Mid-Intestine and Lumen.

We describe the use of the fluorescent reporter compound CDr10b to label mid-intestinal structures in zebrafish larvae after simple immersion. CDr10b is deposited into the gut where it initially fills the lumen and is excreted. Using laser-mediated injury of the intestine, we show that CDr10b provides a useful readout of the integrity and repair of the epithelial cell barrier. In addition, CDr10b specifically labels the absorptive mid-intestine segment that is analogous to the mammalian small intestine. By perturbing retinoic acid signaling, which regulates the size of the mid-intestine segment, we show that CDr10b is a valuable tool to rapidly assess developmental malformations of the intestine in live animals.

Application of a non-hazardous vital dye for cell counting with automated cell counters.

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

Use of the vital stain FUN-1 indicates viability of Phytophthora capsici propagules and can be used to predict maximum zoospore production.

The fluorescent vital dye FUN®-1 (2-chloro-4-[2,3-dihydro-3-methyl-{benzo-1,3-thiazol-2-yl}-methylidene]-1-phenylquinolinium iodide) was evaluated as a tool to assess Phytophthora capsici sporangia and zoospore metabolic activity and viability. Under aerobic conditions, mycelia, sporangia and zoospores cultured on agar medium and stained with FUN-1 exhibited red fluorescent cylindrical intravacuolar structures (CIVS) that were clearly visible at 100× magnification. Encysted zoospores did not exhibit CIVS after exposure to FUN-1 dye. Over 7 d there was a significant reduction in the percent of sporangia containing CIVS, which corresponded with a significant increase in zoospore formation and release. The decline in the percentage of metabolically active sporangia and increase in the number of zoospores fit both a linear and log regression model. The FUN-1 dye was suitable for distinguishing between live and dead sporangia and effective in monitoring the change in metabolic activity of sporangia over time. It will be useful in determining parameters, including P. capsici culture age, that maximize production of zoospores in vitro.

Assessment of the cytolytic potential of a multivirus-targeted T cell therapy using a vital dye-based, flow cytometric assay.

Reliable and sensitive characterization assays are important determinants of the successful clinical translation of immunotherapies. For the assessment of cytolytic potential, the chromium 51 (51Cr) release assay has long been considered the gold standard for testing effector cells. However, attaining the approvals to access and use radioactive isotopes is becoming increasingly complex, while technical aspects [i.e. sensitivity, short (4-6 hours) assay duration] may lead to suboptimal performance. This has been the case with our ex vivo expanded, polyclonal (CD4+ and CD8+) multivirus-specific T cell (multiVST) lines, which recognize 5 difficult-to-treat viruses [Adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus 6 (HHV6)] and when administered to allogeneic hematopoietic stem cell (HCT) or solid organ transplant (SOT) recipients have been associated with clinical benefit. However, despite mediating potent antiviral effects in vivo, capturing in vitro cytotoxic potential has proven difficult in a traditional 51Cr release assay. Now, in addition to cytotoxicity surrogates, including CD107a and Granzyme B, we report on an alternative, vital dye -based, flow cytometric platform in which superior sensitivity and prolonged effector:target co-culture duration enabled the reliable detection of both CD4- and CD8-mediated in vitro cytolytic activity against viral targets without non-specific effects.

Internal limiting membrane peeling in macular hole surgery.

Since the era when macular hole was considered untreatable, macular hole surgery has come a long way to being one of the most successful surgeries. Internal limiting membrane (ILM) peeling has been an essential step of macular hole surgery since the establishment of the role of ILM in the aetiopathogenesis and progression of macular hole. However, the novel technique was not all virtuous. It had some vices which were not evident immediately. With the advent of spectral domain optical coherence tomography, short- and long-term effects of ILM peeling on macular structures were known; and with microperimetry, its effect on the function of macula could be evaluated. The technique has evolved with time from total peeling to inverted flap to just temporal peeling and temporal flap in an attempt to mitigate its adverse effects and to improve its surgical outcome. ILM abrasion technique and Ocriplasmin may eliminate the need of ILM peeling in selected cases, but they have their own limitations. We here discuss the role of ILM in the pathogenesis of macular hole, the benefits and adverse effects of ILM peeling, and the various modifications of the procedure, to then explore the alternatives.

The Influence of Crescent-Shaped Selective Internal Limiting Membrane Staining on Vital Dye Toxicity in Temporal Inverted Flap Technique.

Purpose: To investigate the macular function and morphology after temporal inverted internal limiting membrane (ILM) flap technique with and without staining of the ILM flap in contact with the retinal pigment epithelium (RPE).Materials and Methods: This retrospective study included 30 patients with idiopathic macular hole (MH), who underwent 27 G vitrectomy and temporal inverted ILM flap technique with brillant blue G (BBG) assisted ILM staining. In Group 1 (n = 16), a large bubble of perfluorocarbon liquid (PFCL) measuring approximately 6-disc diameters was used to cover the hole and central part of the ILM flap whereas in Group 2 (n = 14), only a small drop of PFCL to merely cover the MH was used. Complete ophthalmic examination including microperimetry (MP), optical coherence tomography (OCT) was performed preoperatively, 6 months after surgery.Results: MH closure was achieved in all the eyes in both groups. The sizes of ellipsoid zone (EZ) and external limiting membrane (ELM) defect significantly decreased after surgery relative to the baseline width in both groups (p < .05 for all). The mean improvement in visual acuity (p = .896) and retinal sensitivity was similiar between groups (p = .409). Accordingly, the postoperative mean lengths of the EZ (p = .254) and ELM disruption (p = .406) on OCT scans were similiar between groups. However, 3 of the eyes in Group 2 developed cystoid macular edema between postoperative month-1 and month-6.Conclusion: The crescent-shaped selective staining of the ILM flap could prevent prolonged retinal toxicity of vital dyes in inverted ILM flap technique. Further studies involving larger number of patients with longer follow up are needed to determine the impact of this technique in the management of vital dye toxicity.

Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria.

Rapidly assaying cell viability for diverse bacteria species is not always straightforward. In eukaryotes, cell viability is often determined using colorimetric dyes; however, such dyes have not been identified for bacteria. We screened different dyes and found that erythrosin B (EB), a visibly red dye with fluorescent properties, functions as a vital dye for many Gram-positive and -negative bacteria. EB worked at a similar concentration for all bacteria studied and incubations were as short as 5 min. Given EB's spectral properties, diverse experimental approaches are possible to rapidly visualize and/or quantitate dead bacterial cells in a population. As the first broadly applicable colorimetric viability dye for bacteria, EB provides a cost-effective alternative for researchers in academia and industry.

Outer Retinal Healing After Internal Limiting Membrane Peeling in Diabetic Macular Oedema with Vitreomacular Interface Abnormality Using Three Different Dyes.

Purpose: To evaluate the difference in ellipsoid zone and external limiting membrane (EZ/ELM) restoration after vitrectomy with internal limiting membrane (ILM) peeling in diabetic macular oedema with vitreomacular interface abnormalities (VMA DME) patients using 3 different dyes.Methods: Patients with type 2 diabetes mellitus and VMA DME indicated for ILM peelings were included. Disruption of the EZ/ELM was graded and compared among groups (preoperative and 6 months postoperative). Patients were divided into 3 groups according to the dye used: group A: indocyanine green (ICG); group B: trypan blue (TB); and group C: brilliant blue G(BBG).Results: Twenty-six eyes were included in group A, 29 eyes were included in group B, and 28 eyes were included in group C. Improvement in EZ/ELM integrity was observed in 34.6% of group A, 27.6% of group B, and 32.1 % of group C (p = .84). Deterioration of EZ/ELM integrity was observed in 19.2% of group A, 6.9% of group B and 0% of group C (p = .03).Conclusion: ICG resulted in a greater percentage of deteriorated EZ/ELM integrity at 6 months after surgery in cases with pre-treatment ELM interruption. Therefore, ICG should be used with caution in cases with ELM disruption, yet, further studies are recommended.

Computed tomography-guided dye localization for deeply situated pulmonary nodules in thoracoscopic surgery.

BACKGROUND: Increased lung cancer screening of asymptomatic adults using low-dose computed tomography (CT) with high-resolution imaging modalities has increased the identification of small and deeply situated pulmonary nodules. This study aimed to evaluate the role of preoperative patient blue vital (PBV) dye localization for an undiagnosed nodule deeply situated in the lung parenchyma followed by minimally invasive lung resection. METHODS: From July 2013 to December 2016, 27 consecutive patients (16 women, median age: 62 years) with small undiagnosed pulmonary nodules at a depth of more than 30 mm underwent preoperative CT-guided PBV dye localization followed by thoracoscopic diagnostic resection of the nodule at National Taiwan University Hospital. The clinical characteristics were collected retrospectively to evaluate the efficacy and safety of the procedure. RESULTS: The median size of pulmonary nodule in preoperative CT images was 11 mm with a median depth of 31.6 mm (range, 30.0-48.6 mm). Of the 27 nodules, 8 were pure ground-glass nodules, 3 were pure solid nodules, and 16 were partially solid nodules. The diagnostic yield of CT-guided dye localization following diagnostic wedge resection was 100%. The final pathological diagnoses were: primary adenocarcinoma of the lung (n=20), adenocarcinoma in situ (n=1), and benign nodules (n=6). Only asymptomatic complications were noted after localization, and the median hospital stay was 3 days [interquartile range (IQR), 3-4 days]. All of 21 patients were cancer-free after a median follow-up of 39.0 months (IQR, 29.5-50.0 months). CONCLUSIONS: This study indicated that preoperative, percutaneous CT-guided PBV dye localization for undiagnosed nodules at a depth of more than 30 mm could be a safe and feasible procedure. Furthermore, it was considerably advantageous for preserving the lung parenchyma, especially for benign nodules.

Protocol for Murine/Mouse Platelets Isolation and Their Reintroduction in vivo.

Platelets and coagulation have long been known to be essential for metastasis in experimental models. In order to study the interactions between tumor cells, platelets and endothelium, we have adapted methods used in coagulation research for the isolation of platelets and their reintroduction into mice. Anti-coagulated murine blood served as the source for platelets. Platelets were separated from other elements of the whole blood by centrifugation. Here the critical elements are first inhibition of coagulation and second isolation and maintenance of the platelets in the presence of inhibitors of platelet activation. We then used the vital dye PKH26 to fluorescently label the platelets. Infusion of these labelled platelets allows microscopic observation of the introduced platelets. After reintroduction, these platelets appear to function normally and comprise approximately 50% of the total platelets. Because they are fluorescently labelled, they can easily be identified. Finally it would be possible to use these methods for the determination of specific effects of altered gene expression in platelets by using platelets from genetically engineered mice. These methods have facilitated study of the interactions between platelets and tumor cells in tissue culture and in murine models. They would also be applicable to video microscopy. Here we provide details of the methods we have used for platelet isolation from mice and their staining for further microscopy and re-introduction into mice.

Controversies over the role of internal limiting membrane peeling during vitrectomy in macular hole surgery.

Surgical management of an idiopathic macular hole consists of vitrectomy to release vitreofoveal traction and intraocular tamponade to flatten and reappose the hole's edges. The intentional atraumatic removal of the internal limiting membrane has been proposed as cost-effective option in macular hole surgery. The internal limiting membrane contributes to tangential traction at the edges of the hole and acts as a platform on which glial cells proliferate. Removal of the internal limiting membrane increases the elasticity of the denuded macula and improves the anatomical success rate; however, the visual consequences of this surgical maneuver are still not fully known. We discuss the beneficial and adverse effects associated with internal limiting membrane peeling in macular hole surgery, highlighting the internal limiting membrane's role in macular hole etiology and pathogenesis and the anatomical and functional findings after its removal.

Computed tomography-guided patent blue vital dye localization of pulmonary nodules in uniportal thoracoscopy.

OBJECTIVE: Due to the limitations of the small single incision, an ideal preoperative localization technique is essential for surgical resection of small pulmonary nodules by uniportal video-assisted thoracoscopic surgery (VATS). The aim of this study is to evaluate the usefulness and safety of preoperative computed tomography (CT)-guided patent blue vital (PBV) dye localization in patients with small indeterminate pulmonary nodules who have undergone uniportal VATS for lung resection. METHODS: In this retrospective study, 177 consecutive patients (196 pulmonary nodules) who underwent preoperative CT-guided PBV dye localization and uniportal VATS from January 2013 to September 2015 were enrolled. RESULTS: The CT-dye localization procedure was performed successfully and correctly for 99.5% (195/196) of the nodules within a mean procedure time of 30 minutes. The mean size of the nodules was 7.8 mm, and their mean depth from the pleural surface was 18.3 mm. Most of the nodules (78.6%, 154/196) were pure ground-glass nodules (GGNs) and part-solid GGN with ground-glass opacity (GGO) of 50% or more. Asymptomatic pneumothorax occurred in 29.4% (52/177) of patients after the localization procedure, but none required invasive treatment. All nodules were successfully resected using uniportal VATS without any conversion to thoracotomy. The postoperative course was smooth, with a short mean hospital stay (3.3 ± 1.2 days) and a low morbidity rate (0.6%, 1/177). CONCLUSIONS: Preoperative CT-guided PBV dye localization is a feasible, safe, and accurate procedure. It makes uniportal VATS easy for small, poorly located pulmonary nodules with GGO predominance and synchronous multiple nodules.

Vital dyes in ophthalmology: a chemical perspective.

Vital dyes have advanced diagnosis and surgical technique in various specialties, including oncology, gastroenterology and ophthalmology. Intra-operative and diagnostic dyes are finding uses in all areas of ophthalmology, including cornea, cataract, retina, glaucoma, orbit and conjunctiva. We provide a summary of current knowledge of the chemical concepts of vital dyes in ophthalmology. 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.

Evaluation of equine corneal endothelium after exposure to 0. 05% brilliant blue - an in vitro study / Avaliação do endotélio corneano de equinos após contato com o corante azul brilhante 0, 05% - estudo in vitro

The aim of this study was to evaluate the immediate effects of 0.05% brilliant blue on corneal endothelium of horses. Thirty-eight corneas of 19 horses, male or female, of different ages were studied. Corneas were randomly divided into two groups. Group 1: Corneal endothelium was covered with 0.3mL of brilliant blue 0.05% for 60 seconds followed by rinsing with a balanced salt solution. Group 2: Corneal endothelium was covered with BSS for 60 seconds. The corneas were excised with an 8mm trephine and prepared to analyze posterior endothelial surface using a light microscope (24 corneas) and a scanning electron microscope (14 corneas). The equine posterior corneal endothelium surface observed by optical microscopy and scanning electron microscopy revealed a continuous layer of polygonal cells of uniform size and shape in both the control and treatment groups. Due to non-normal residuals at ANOVA mean comparison, a generalized linear model was utilized at 5% level of significance. The chi-square test stated that treatment and control group were not different statistically. The 0.05% brilliant blue did not cause damage to equine corneal endothelium.(AU)

Objetivou-se avaliar os efeitos imediatos de uma solução de 0,05% de azul brilhante sobre o endotélio da córnea de equinos. Trinta e oito córneas de 19 cavalos, machos ou fêmeas, de diferentes idades foram estudadas. As córneas foram divididas aleatoriamente em dois grupos. Grupo 1: O endotélio corneano foi perfundido com 0,3mL de azul brilhante 0,05% durante 60 segundos seguido por irrigação com uma solução salina balanceada. Grupo 2: O endotélio corneano foi perfundido com BSS durante 60 segundos. As córneas foram posteriormente excisadas com trépano de 8mm e preparadas para análise endotelial utilizando um microscópio óptico (24 córneas) e um microscópio eletrônico de varredura (14 córneas). A análise da superfície posterior do endotélio da córnea equina observada por microscopia óptica e microscopia eletrônica de varredura revelou uma camada contínua de células poligonais de tamanho e forma uniformes tanto no grupo controle quanto no grupo tratamento. Devido aos resíduos não normais na comparação da média de ANOVA, utilizou-se um modelo linear generalizado com nível de significância de 5%. Evidenciou-se com o teste qui-quadrado que não houve diferença estatística entre o grupo controle e o grupo tratamento. O azul brilhante de 0,05% não causou dano ao endotélio corneano de equinos.(AU)