Effects of intracameral brilliant blue on the corneal endothelium of swine: in vitro study

Avaliação do endotélio corneano suíno por microscopia eletrônica de varredura após aplicação de azul brilhante a 0,05% na câmara anterior: estudo in vitro

Mariana Terzariol Paula S. Hünning Gustavo Brambatti Luciane de Albuquerque Carolina Neumann João A.T. Pigatto About the authors

Abstract:

The aim was to investigate the ultrastructural changes in the corneal endothelium of pigs induced by intracameral 0.05% brilliant blue. Twenty swine corneas were separated into two groups, the right eye bulbs (control group) and the left eye bulbs (experimental group) of the same animal. All the eye bulbs were evaluated with specular microscopy. The cornea of the right eye bulbs was excised and in the left eye bulbs 0.2ml of 0.05% brilliant blue vital dye (OPTH-blue±) was injected into the anterior chamber, where it remained for one minute. Then the anterior chamber was cleaned with a balanced salt solution injection and the cornea was excised too. All the corneas were evaluated by scanning electron microscopy to evaluate the changes on the endothelium caused by the brilliant blue dye. There were no significant differences between the right corneal endothelium cells and the left corneal endothelium cells with scanning electron microscopy after intracameral use of 0.05% brilliant blue dye. The 0.05% brilliant blue dye concentration did not cause deleterious effects for the swine corneal endothelium after intracameral use and can be a choice for safe staining of the anterior capsule of the lens in cataract surgery.

Index Terms:
Eye; brilliant blue 0.05%; toxicity; corneal endothelium; swine.

Resumo:

Com o objetivo de avaliar as alterações ultraestruturais no endotélio corneano suíno induzidas pela aplicação intracameral do corante azul brilhante 0,05%, vinte córneas suínas foram separadas em dois grupos: olhos direitos (grupo controle) e olhos esquerdos (grupo experimental). Inicialmente todos os bulbos oculares foram avaliados por microscopia especular. As córneas dos bulbos oculares do grupo controle foram excisadas, enquanto nos bulbos oculares do grupo experimental foram injetados 0,2ml do corante vital azul brilhante 0,05% na câmara anterior e mantido por um minuto. Após esse período a câmara anterior foi lavada com solução salina balanceada e as córneas foram excisadas. Todas as córneas excisadas foram avaliadas por microscopia eletrônica de varredura para verificar a ocorrência possíveis alterações no endotélio corneano causadas pelo corante. Não foram observadas diferenças ultraestruturais durante a avaliação endotelial por microscopia eletrônica de varredura das córneas do grupo controle e experimental após a aplicação do azul brilhante 0,05% na câmara anterior. O corante azul brilhante na concentração 0,05% não causou danos endoteliais após seu uso intracameral em suínos e pode ser uma alternativa segura para a coloração da cápsula anterior da lente na cirurgia de catarata.

Termos de Indexação:
Olho; azul brilhante 0,05%; toxicidade; endotélio corneano; suíno.

Introduction

Continuous curvilinear capsulorhexis (CCC) is a critical step in cataract surgery, specifically in white cataracts when the red reflex is poor or absent (Chang et al. 2005Chang Y.S., Tseng S.Y. & Tseng S.H. 2005. Comparison of dyes for cataract surgery. 2. Efficacy of capsule staining in a rabbit model. J. Cataract and Refract. Surg. 31:799-804., Jacobs et al. 2006Jacobs D.S., Cox T.A., Wagoner M.D., Ariyasu R.G. & Karp C.L. 2006. Capsule staining as an adjunct to cataract surgery: a report from the American Academy of Ophthalmology. Ophthalmol. 113:707-713.). In these cases, one of the options to facilitate the visualisation of the anterior capsule is the use of vital dyes to stain the anterior capsule of the lens before proceeding with capsulorhexis (Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Dada et al. 2004Dada V.K., Sharma N., Sudan R., Sethi H., Dada T. & Pangtey M.S. 2004. Anterior capsule staining for capsulorhexis in cases of white cataract: Comparative clinical study. J. Cataract and Refract. Surg. 30: 326-333.). The importance of having a better visualisation of the flap during capsulorhexis is that it increases surgery success, decreases the chance of anterior capsule tears during phacoemulsification, and ensures the safe in-the-bag implantation of the intraocular lens (Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Marback et al. 2001Marback E.F., Freitas L.L., Fernandes F.P., Branco B.C. & Belfort Jr R. 2001. Anterior capsule staining using 0.025% trypan blue in cataracts without red reflex. Arq. Bras. Oftalmol. 64:333-335.).

Another suggested use for vital dyes in cataract surgery is to help trainee cataract surgeons during CCC confection in order to reduce the learning curve for those students (Waziri-Erameh et al. 2006Waziri-Erameh J.M., Omoti A.E. & Edema M.T. 2006. The value of Trypan blue dye I the training of ophthalmology resident doctors hands on capsulotomy. Annals Biomed. Sci. 5(1/2):73-76., Udaondo et al. 2007Udaondo P., Díaz-Llopis M., Salom D., García-Delpech S. & Cervera E. 2007. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch. Soc. Esp. Oftalmol. 82:471-472.). However, the toxic effects of each vital dye have to be evaluated before the start of its use in the hospital routine (Remy et al. 2008Remy M., Thaler S., Schumann R.G., May C.A., Fiedorowicz M., Schuettauf F., GrüTerich M., Priglinger S.G., Nentwich, M.M., Kampik A. & Haritoglou C. 2008. An in vivo evaluation of brilliant blue G in animals and humans. Braz. J. Ophthalmol. 92:1142-1147.). An ideal vital dye for facilitating CCC during cataract surgery needs to satisfactorily stain the anterior capsule of lens and cause minimal toxic effects to the corneal endothelium (Hisatomi et al. 2006Hisatomi T., Enaida H., Matsumoto H., Kagimoto T., Ueno A., Hata Y., Kubota T., Goto Y. & Ishibashi T. 2006. Staining ability and biocompatibility of Brilliant Blue G. Arch. Ophthalmol.124:514-519.).

The fluorescein sodium solution was the first vital dye used to facilitate capsulorhexis in 1993. From then on, several other vital dyes have been studied and used to stain the anterior capsule of the lens during cataract surgeries, e.g. gentian violet, autologous blood, trypan blue, green indocyanine, and brilliant blue (Melles et al. 1999Melles G.R.J., Waard P.W.T., Pameyer J.H. & Beekhuis W.H. 1999. Trypan blue staining to visualize the capsulorhexis in catarata surgery. J. Cataract and Refract. Surg. 25:7-9., Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Ünlü et al. 2000Ünlü K., Askünger A., Söker S., Kilinç N., Karaca C. & Erdinc M. 2000. Gentian violet solution for staining the anterior capsule. J. Cataract and Refrac. Surg. 26:1228-1232., Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991., Chang et al. 2005Chang Y.S., Tseng S.Y. & Tseng S.H. 2005. Comparison of dyes for cataract surgery. 2. Efficacy of capsule staining in a rabbit model. J. Cataract and Refract. Surg. 31:799-804.).

The endothelial cell loss due to cataract surgery is inevitable. For this reason, corneal endothelium of the patient should be evaluated before surgery, safer techniques should be chosen and intraocular solutions should not damage the endothelium to avoid large cell loss that can induce corneal decompensation after cataract surgery (Ishikawa 2002Ishikawa A. 2002. Risk factors for reduced corneal endothelial cell density before cataract surgery. J. Cataract and Refract. Surg. 28:1982-1992., Parikh & Edelhauser 2003Parikh C.H. & Edelhauser H.F. 2003. Ocular surgical pharmacology: corneal endothelial safety and toxicity. Curr. Opin. Ophthalmol. 14(14):178-184.).

Some research has studied the use of the brilliant blue vital dye to facilitate CCC and have suggested that this is a safe dye for the corneal endothelium (Hisatomi et al. 2006Hisatomi T., Enaida H., Matsumoto H., Kagimoto T., Ueno A., Hata Y., Kubota T., Goto Y. & Ishibashi T. 2006. Staining ability and biocompatibility of Brilliant Blue G. Arch. Ophthalmol.124:514-519., Udaondo et al. 2007Udaondo P., Díaz-Llopis M., Salom D., García-Delpech S. & Cervera E. 2007. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch. Soc. Esp. Oftalmol. 82:471-472.). However, this is the first research to evaluate the endothelium effects of this dye through scanning electron microscopy in swine eyes.

Materials and Methods

Twenty normal eye bulbs from ten male and female one-year-old Landrace pigs were studied. These eye bulbs were obtained from a licensed Brazilian commercial company (Avisui food company) that breeds these animals for meat production. The eye bulbs were divided into two groups: the control group and the experimental group, consisting respectively of the right eye bulbs and the left eye bulbs. All procedures were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research. All animals were sacrificed for reasons unrelated to this study.

The eye bulbs were enucleated and transported to the laboratory in a moist chamber containing physiologic saline immediately after the swine were sacrificed. The eye bulbs were mounted on an eyeball holder and examined using a fluorescein test, biomicroscopy (Kowa, SL 15, Japan), and a contact specular microscope (Celmax, Medical Service, Brazil) with software for corneal endothelium analysis. Specular microscopy was performed on all eye bulbs in order to determine the conditions of the corneal endothelium and those with evidence of alteration were excluded.

Immediately after the specular microscopy, a clear corneal incision with a 15° knife was made in the left eye bulbs using surgical microscopy. One eye bulb received a 0.2ml intracameral injection of 0.05% brilliant blue vital dye (OPHT-BLUE±, Ophthalmos, São Paulo/SP, Brazil) and the fellow eye bulb served as a control. The vital dye remained in the left eye bulb in the anterior chamber for one minute and was then removed with the application of a balanced saline salt solution (Ophthalmos, São Paulo/SP, Brazil).

The corneas and a 2.0mm rim of sclera were excised, transferred to 2.5% glutaraldehyde in 0.1 sodium cacodylate buffer at pH 7.4 and stored for 24 hours at 4oC for fixation. The corneas were washed in a cacodylate buffer and dehydrated through an increasing series of ethanol solutions. Thereafter, the specimens were submitted to critical point drying using liquid carbon dioxide. The corneas were placed on 10 mm aluminium stubs with adhesive tape and sputter coated with gold-palladium. The posterior endothelial surfaces were examined and photographed using a scanning electron microscope (JEOL JSM 6060, Japan) operating at 15 kV.

The photomicrographs were scanned into the computer, and the percentage of cell loss was calculated by observing the surrounding areas which had an absence of endothelial cells. Image analysis was performed with image analyser software (Leica, Quantimed 500 IW, Leica Cambridge Ltd, Cambridge, UK) by a skilled observer who did not know the source of the photographs. Ultrastructural characteristics were described using photomicrographs obtained at 1.000-3.500x. Statistical data analysis was conducted using the F test if intercellular empty spaces were found in the endothelium samples. Values of P<0.05 were considered significant.

Results

Specular microscopy showed that a normal swine corneal endothelium was characterised by a continuous monolayer of polygonal cells of uniform size and shape (Fig.1). Reproducible images were obtained from all eye bulbs. The main cell density obtained with the contact specular microscope in control groups and experimental groups was 3,136 cells/mm2 and 3,150 cells/mm2, respectively (Table 1). Regarding the morphology of the endothelium, were found cells with six-sided (68%), five-sided (18%), and seven-sided (14%). The parameters evaluated did not differ significantly between the right eye bulbs and the left eye bulbs from the same pig.

Fig.1:
Specular micrograph of a normal corneal endothelium of a pig from the experimental group.

The swine posterior corneal endothelium surface observed on the scanning electron microscope revealed a continuous layer of polygonal cells of uniform size and shape. The scanning electron microscopy images showed that corneas in both the control and experimental sample group characterised similar morphological endothelia. All the samples had high hexagonal cell counts, an evident nucleus, the presence of interdigitation, microvilli, and the absence of intercellular empty spaces. The endothelial cells were interdigitated and adhered in both the experimental group and the control group (Fig.2). In the experimental group and the control group there were no areas with cell loss.

Fig.2:
Image of experimental swine ocular bulb corneal endothelium (pig #9) in scanning electron microscope showing the interdigitation (red arrow), microvillus (green arrow), and evident nucleus (yellow arrow). It is possible to note tight intercellular adhesion. 2000x.

The similarity of the control and experimental scanning electron microscope images obtained during the search are shown in Figure 3, which represents part of the sample images. No intercellular empty areas were evident and the cellular morphology is similar to all the sample images.

Fig.3:
Comparative swine corneal endothelium scanning electron microscope images from each control and experimental group, respectively. 1000x.

Discussion

The evaluation of the appearance of the corneal endothelium is almost universal nowadays in testing potential toxicity of ophthalmic solutions (Doughty 1989Doughty M.J. 1989. Toward a quantitative analysis of corneal endothelial cell morphology: a review of techniques and their application. Optometry Vision Sci. 66(9):626-642.). The brilliant blue vital dye has been considered safe and effective for facilitating CCC in human white cataract surgery and in the training of cataract surgeons (Hisatomi et al. 2006Hisatomi T., Enaida H., Matsumoto H., Kagimoto T., Ueno A., Hata Y., Kubota T., Goto Y. & Ishibashi T. 2006. Staining ability and biocompatibility of Brilliant Blue G. Arch. Ophthalmol.124:514-519., Udaondo et al. 2007Udaondo P., Díaz-Llopis M., Salom D., García-Delpech S. & Cervera E. 2007. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch. Soc. Esp. Oftalmol. 82:471-472.). To describe the corneal endothelial effects of its vital dye, this study evaluated the in vitro effects to the swine corneal endothelial cells with scanning electron microscopy.

Swine eyes are considered the standard experimental model for cataract surgeries (Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122.). The ocular bulbs and cornea sizes of pigs, their morphological endothelium characteristics, their wide availability and their low sale prices are some of the biggest advantages of this choice in medicine and veterinary medicine (Proulx & Brunette 2012Proulx S. & Brunette I. 2012. Methods being developed for preparation, delivery and transplantation of a tissue-engineered corneal endothelium. Exp. Eye Res. 95:68-75.). Due to all of these reasons, the ocular models for this research were swine ocular bulbs. Furthermore, pigs were chosen as experimental animals because of the morphological similarities between swine and human corneas (Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122., Proulx & Brunette 2012Proulx S. & Brunette I. 2012. Methods being developed for preparation, delivery and transplantation of a tissue-engineered corneal endothelium. Exp. Eye Res. 95:68-75.).

Both ocular bulbs from the same animal were used to get homogeneous samples with similar corneal endothelia. The parameters evaluated by specular microscopy did not differ significantly between the right eye bulb and the left eye bulb from the same pig. It is well established that the density of endothelial cells from both healthy eyes of the same patient is similar (Pigatto et al. 2005Pigatto J.A.T., Abib F.C., Pizzeti J.C., Laus J.L., Santos J.M. & Barros P.S.M. 2005. Análise morfométrica do endotélio de coelhos à microscopia eletrônica de varredura. Acta Scient. Vet.33(1):41-45., Gum et al. 2007Gum G.G., Gelatt K.N. & Esson D.W. 2007. Physiology of the eye, p.149-182. In: Gelatt K.N. (Ed.), Veterinary Ophthalmology. Vol.1. 4th ed. Blackwell Publishing, Iowa., Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122., Albuquerque et al. 2015Albuquerque L., Freitas L.V.R. & Pigatto J.A.T. 2015. Analysis of the corneal endothelium in eyes of chickens using contact specular microscopy. Semin, Ciênc. Agrárias 36(6):4199-4206., Bercht et al. 2015Bercht B.S., Albuquerque L., Araujo A.C.P. & Pigatto J.A.T. 2015. Specular microscopy to determine corneal endothelial cell morphology and morphometry in chinchilas (Chinchilla lanigera) in vivo. Vet. Ophthalmol. 18(1):137-142., Coyo, et al. 2015Coyo N., Peña M.T., Costa D., Rıos J., Lacerda R. & Leiva M. 2015. Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Vet. Ophthalmol. 19(1):1-6.). The examination of specular microscopy performed before starting of the experiment allowed to only be selected healthy eyes. The results observed in this study show that normal swine corneal endothelium consisted of a continuous monolayer with mainly hexagonal cells, but were also found cells with five sides and with seven sides. Previous studies conducted involving vertebrate animals have shown that there is predominance of cells with hexagonal shape and, in lower proportion, there is presence of five- and seven-sided cells (Collin & Collin, 1998Collin S.P. & Collin H.B. 1998. A comparative study of the corneal endothelium in vertebrates. Clin. Exp. Optometry 8(6):245-254., Pigatto et al. 2005Pigatto J.A.T., Abib F.C., Pizzeti J.C., Laus J.L., Santos J.M. & Barros P.S.M. 2005. Análise morfométrica do endotélio de coelhos à microscopia eletrônica de varredura. Acta Scient. Vet.33(1):41-45., Gum et al. 2007Gum G.G., Gelatt K.N. & Esson D.W. 2007. Physiology of the eye, p.149-182. In: Gelatt K.N. (Ed.), Veterinary Ophthalmology. Vol.1. 4th ed. Blackwell Publishing, Iowa., Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122., Albuquerque et al. 2015Albuquerque L., Freitas L.V.R. & Pigatto J.A.T. 2015. Analysis of the corneal endothelium in eyes of chickens using contact specular microscopy. Semin, Ciênc. Agrárias 36(6):4199-4206., Bercht et al. 2015Bercht B.S., Albuquerque L., Araujo A.C.P. & Pigatto J.A.T. 2015. Specular microscopy to determine corneal endothelial cell morphology and morphometry in chinchilas (Chinchilla lanigera) in vivo. Vet. Ophthalmol. 18(1):137-142., Coyo, et al. 2015Coyo N., Peña M.T., Costa D., Rıos J., Lacerda R. & Leiva M. 2015. Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Vet. Ophthalmol. 19(1):1-6.).

The endothelial cellular membranes' integrity for scanning electron microscopy evaluation depends on adequate tissue preservation and fixation from the time after the death of the animal until the use of the tissue samples (Collin & Collin 1998Collin S.P. & Collin H.B. 1998. A comparative study of the corneal endothelium in vertebrates. Clin. Exp. Optometry 8(6):245-254.). In humans, corneal tissues for transplants must be used within six hours of the donor's death and need to be safely maintained to avoid endothelial damage (Franzen et al. 2010Franzen A.A., Pigatto J.A.T., Abib F.C., Albuquerque L. & Laus J.L. 2010. Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes. Vet. Ophthalmol. 13(4):222-226.). The swine corneas in this research were maintained in moist chamber for less than six hours until glutaraldehyde fixation to avoid endothelial damage. This methology has also been used with excellent results in previous studies (Pigatto et al. 2005Pigatto J.A.T., Abib F.C., Pizzeti J.C., Laus J.L., Santos J.M. & Barros P.S.M. 2005. Análise morfométrica do endotélio de coelhos à microscopia eletrônica de varredura. Acta Scient. Vet.33(1):41-45., Franzen et al. 2010Franzen A.A., Pigatto J.A.T., Abib F.C., Albuquerque L. & Laus J.L. 2010. Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes. Vet. Ophthalmol. 13(4):222-226., Albuquerque et al. 2015Albuquerque L., Freitas L.V.R. & Pigatto J.A.T. 2015. Analysis of the corneal endothelium in eyes of chickens using contact specular microscopy. Semin, Ciênc. Agrárias 36(6):4199-4206., Coyo et al. 2015Coyo N., Peña M.T., Costa D., Rıos J., Lacerda R. & Leiva M. 2015. Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Vet. Ophthalmol. 19(1):1-6.). SEM has been widely used to compare the endothelial ultrastructure of vertebrates, and to evaluate the effects of medication, chemicals or surgical procedures on the endothelium (Pigatto et al. 2005Pigatto J.A.T., Abib F.C., Pizzeti J.C., Laus J.L., Santos J.M. & Barros P.S.M. 2005. Análise morfométrica do endotélio de coelhos à microscopia eletrônica de varredura. Acta Scient. Vet.33(1):41-45., Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122.).

The previous studies conducted using SEM demonstrated that the corneal endothelial cells have cilia and microvillus (Tuft & Coster 1990Tuft S.J. & Coster D.J. 1990. The corneal endothelium. Eye4:389-424., Pigatto et al. 2005Pigatto J.A.T., Abib F.C., Pizzeti J.C., Laus J.L., Santos J.M. & Barros P.S.M. 2005. Análise morfométrica do endotélio de coelhos à microscopia eletrônica de varredura. Acta Scient. Vet.33(1):41-45., Pigatto et al. 2008Pigatto J.A.T., Cerva C., Freire C.D., Abib F.C., Bellini L.P., Barros P.S.M. & Laus J.L. 2008. Morphological analysis of the corneal endothelium in eyes of dogs using specular microscopy. Pesq. Vet. Bras. 8(9):427-430.). Humans and rabbits have twenty to thirty microvilli in the cell superficies, and these structures react to toxic substances changing their sizes (Collin & Collin 1998Collin S.P. & Collin H.B. 1998. A comparative study of the corneal endothelium in vertebrates. Clin. Exp. Optometry 8(6):245-254.). Both the endothelium control and experimental groups presented almost the same morphological characters in scanning electron microscopy analyses, and the microvillus and interdigitation were evident in all sample images. The cilia in swine corneal endothelial cells are described by Tamayo-Arango et al. (2009)Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122., although in this research neither cell presented these structures. (Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122.). The cilia absence or the presence of a few numbers of them in swine endothelium research is due to their fragility during sample production (Collin & Collin 1998Collin S.P. & Collin H.B. 1998. A comparative study of the corneal endothelium in vertebrates. Clin. Exp. Optometry 8(6):245-254., Tamayo-Arango et al. 2009Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Boteon F.C. 2009. Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig. Ciência Rural 39(1):117-122.).

To obtain a better visibility of the lens capsule, capsular staining techniques that had to be done using a biocompatible dye were developed. The use of vital dyes in ophthalmology to facilitate CCC is actually indicated in white cataracts without fundus reflex (Marback et al. 2001Marback E.F., Freitas L.L., Fernandes F.P., Branco B.C. & Belfort Jr R. 2001. Anterior capsule staining using 0.025% trypan blue in cataracts without red reflex. Arq. Bras. Oftalmol. 64:333-335.). The CCC technique can be used in planned extracapsular cataract extraction and in phacoemulsification to reduce the risks of tearing during nucleus and cortex removal and intraocular lens implantation. Unsuccessful CCC increases the risk of posterior capsule rupture, vitreous loss, nucleus drop and intraocular lens displacement. Rodrigues and collaborators tested the anterior lens stain capacity of the brilliant blue dye in some concentrations and concluded that the concentration of 0.05% was enough to stain the anterior swine lens capsule homogenously and to promote the observation of the flap portion (tissue being incised) during CCC, although its effects on corneal endothelium are not completely known (Rodrigues 2009Rodrigues E.B., Costa E.F., Penha F.M., Melo G.B., Botto's J., Dib E., Furlani B., Lima V.C., Maia M., Meyer C.H., Höfling-Lima A.L. & Farah M.E. 2009. The use of vital dyes in ocular surgery. Survey Ophthalmol. 54(5):576-617.).

Several techniques for staining the anterior lens capsules have been used, such as direct vital dye application in the anterior chamber under an air bubble or not, dye application under a viscoelastic substance in the anterior chamber, and subcapsular application (Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991., Xiao et al. 2004Xiao Y., Wang Y., Fu Z. & Hong H. 2004. Staining the anterior lens capsule with indocyanine green or trypan blue for capsulorhexis in eyes with white cataract. Int. Ophthalmol. 25:273-276., Allen et al. 2006Allen R.C., Russell S.R., Schluter M.L. & Oetting T.A. 2006. Retained posterior segment indocyanine green dye after phacoemulsification. J. Cataract and Refract. Surg. 32:357-360.). The last three techniques are indicated for reducing the possible toxic effects of the dye to the corneal endothelium (Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991., Allen et al. 2006Allen R.C., Russell S.R., Schluter M.L. & Oetting T.A. 2006. Retained posterior segment indocyanine green dye after phacoemulsification. J. Cataract and Refract. Surg. 32:357-360.). In this research, the use of the brilliant blue intracameral dye without another substance or air bubble was done to produce direct contacts between the vital dye and the corneal endothelium.

The vital dyes that have been used to stain the anterior lens capsule are trypan blue, fluorescein, green indocyanine, violet gentian, autologous blood and brilliant blue (Melles et al. 1999Melles G.R.J., Waard P.W.T., Pameyer J.H. & Beekhuis W.H. 1999. Trypan blue staining to visualize the capsulorhexis in catarata surgery. J. Cataract and Refract. Surg. 25:7-9., Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Ünlü et al. 2000Ünlü K., Askünger A., Söker S., Kilinç N., Karaca C. & Erdinc M. 2000. Gentian violet solution for staining the anterior capsule. J. Cataract and Refrac. Surg. 26:1228-1232., Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991., Chang et al. 2005Chang Y.S., Tseng S.Y. & Tseng S.H. 2005. Comparison of dyes for cataract surgery. 2. Efficacy of capsule staining in a rabbit model. J. Cataract and Refract. Surg. 31:799-804.) although some of them are not considered safe or effective (Pandey et al. 2000Pandey S.K.., Werner L., Escobar-Gomez M., Roig-Melo E.A. & Apple D.J. 2000. Dye-enhanced cataract surgery. I. Anterior capsule staining for capsulorrhexis in advanced/white cataract. J. Cataract and Refract. Surg. 26:1052-1059., Marback et al. 2001Marback E.F., Freitas L.L., Fernandes F.P., Branco B.C. & Belfort Jr R. 2001. Anterior capsule staining using 0.025% trypan blue in cataracts without red reflex. Arq. Bras. Oftalmol. 64:333-335.). Trypan blue and green indocyanine selectively stain dead endothelial cells and because the endothelial cells are alive in cataract surgery they do not obstruct the surgeon's view (Horiguchi et al. 1998Horiguchi M., Miyake K., Ohta I. & Ito Y. 1998. Staining of the lens capsule for circular continuous capsulorhexis in eyes with white cataract. Arch. Ophthalmol. 116:535-537.). Nowadays, trypan blue is the vital dye of choice used to stain the anterior lens capsule. Several studies have demonstrated no toxic effects from its use for corneal endothelium and the effective in the staining of anterior lens capsule (Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991.). Both trypan blue and green indocyanine stain the anterior lens capsule uniformly, although considered the staining provided by trypan blue slightly superior. In this study, brilliant blue was effectively for staining the anterior lens capsule and did not provide endothelial cell pigmentation during the experiment, as revealed by Horiguchi et al. (1998)Horiguchi M., Miyake K., Ohta I. & Ito Y. 1998. Staining of the lens capsule for circular continuous capsulorhexis in eyes with white cataract. Arch. Ophthalmol. 116:535-537. and Xiao et al. (2004)Xiao Y., Wang Y., Fu Z. & Hong H. 2004. Staining the anterior lens capsule with indocyanine green or trypan blue for capsulorhexis in eyes with white cataract. Int. Ophthalmol. 25:273-276. who used trypan blue and green indocyanine to facilitate CCC.

Despite positive points related to its use, the trypan blue is among the carcinogen substances Merck Indices and due to its long-lasting effects, which are not still known, its use in pregnant women and children must be avoided (Yetik et al. 2002Yetik H., Devranoglu K. & Ozkan S. 2002. Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule. J. Cataract and Refract. Surg. 28:988-991.). The green indocyanine stains the anterior lens capsule satisfactorily and does not have toxic effects on the corneal endothelium, but its use has been associated with retinal toxicity in lens subluxation after cataract surgeries (Allen et al. 2006Allen R.C., Russell S.R., Schluter M.L. & Oetting T.A. 2006. Retained posterior segment indocyanine green dye after phacoemulsification. J. Cataract and Refract. Surg. 32:357-360.). Because of this, when choosing the vital dye to facilitate CCC during cataract surgeries their posterior chamber toxic effect must be considered (Jacobs et al. 2006Jacobs D.S., Cox T.A., Wagoner M.D., Ariyasu R.G. & Karp C.L. 2006. Capsule staining as an adjunct to cataract surgery: a report from the American Academy of Ophthalmology. Ophthalmol. 113:707-713.).

An advantage of the brilliant blue over the trypan blue and green indocyanine for staining the anterior lens capsule is that this vital dye has been shown to have fewer in vitro toxic effects on corneal endothelial cells (Udaondo et al. 2007Udaondo P., Díaz-Llopis M., Salom D., García-Delpech S. & Cervera E. 2007. Brilliant blue G-assisted capsulorhexis: a good help for phacoemulsification surgeons in training. Arch. Soc. Esp. Oftalmol. 82:471-472.). Eggeling et al. 2000Eggeling P., Pleyer U., Hartmann C. & Rieck P.W. 2000. Corneal endothelial toxicity of different lidocaine concentrations. J. Cataract and Refract. Surg. 6:1403-1408. examined the potential in vitro damaging effects on swine corneal endothelium of lidocaine in concentrations of 1%, 5%, and 10% with scanning electron microscopy. The 1% concentration did not cause any changes in the corneal endothelium. In the 5% and 10% concentrations, cell borders were hard to distinguish, single groups of two to three necrotic cells were scattered over the entire endothelium and some of them had developed a generally swollen aspect with no intact intercellular borders. Furthermore, extended areas of denuded Descemet's membrane impressed with remainders of detached cells were also seen in the last two groups. In this research, the corneas treated with 0.05% brilliant blue dye showed a normal endothelium in scanning electron microscopy, similar to that seen in the 1% lidocaine group studied by Eggeling et al. (2000)Eggeling P., Pleyer U., Hartmann C. & Rieck P.W. 2000. Corneal endothelial toxicity of different lidocaine concentrations. J. Cataract and Refract. Surg. 6:1403-1408.. Endothelial cells in our experimental groups were hexagonal in the majority and presented well-defined cell borders with the typical appearance of the normal cells structures, such as the presence of the nucleus, microvillus, intercellular adhesion and the presence of interdigitation, showing no toxic effects of the 0.05% brilliant blue in the corneal endothelium. Previous studies with a similar methodology using scanning electron microscopy revealed morphological abnormalities in eyes injected with 2% lidocaine, which caused morphological abnormalities, such as decreased corneal endothelial cells with irregularities, damage to the cellular organelles with larger spaces between the endothelial cells, and fewer microvilli (Eggeling et al. 2000Eggeling P., Pleyer U., Hartmann C. & Rieck P.W. 2000. Corneal endothelial toxicity of different lidocaine concentrations. J. Cataract and Refract. Surg. 6:1403-1408.).

Conclusion

The results of this study showed that the 0.05% brilliant blue vital dye does not damage swine endothelial cells of the eye after one minute of intracameral injection, and suggested that this vital dye seems to be a safe choice for facilitating CCC in cataract surgery.

Acknowledgements

We thank Avisui food company for supplying the eyes used in this research and Eletron Mocroscopy Center (CME/UFRGS) for support for realization of this search.

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Publication Dates

  • Publication in this collection
    Aug 2016

History

  • Received
    22 Mar 2016
  • Accepted
    13 May 2016
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