Avaliação do endotélio da córnea de suínos (Sus scrofa domesticus) utilizando a microscopia especular

O endotélio corneano é uma monocamada de células poligonais justapostas que recobrem a superfície posterior da córnea. É uma estrutura fundamental para a manutenção da transparência corneana. A espécie suína é bastante empregada como modelo experimental na oftalmologia. No entanto, os estudos avaliando o endotélio da córnea com microscópio especular nessa espécie são escassos. Objetivou-se avaliar a densidade endotelial e o percentual de células hexagonais nas diferentes regiões do endotélio corneano de suínos utilizando a microscopia especular de contato. Foram estudados 24 bulbos oculares de 12 suínos (Sus scrofa domesticus), machos, com seis meses de idade e da raça Large White. A microscopia especular de contato foi realizada nas regiões central, superior, inferior, lateral e medial da córnea. A densidade endotelial média na região central foi de 1865 células/mm², na região superior foi de 1877,3 células/mm², na região inferior foi de 1854,9 células/mm², na região lateral foi de 1847 células/mm² e na região medial foi de 1831,7 células/mm². Na região central, a porcentagem média de células hexagonais foi de 53,3%, na região superior foi de 53,8%, na região inferior foi de 54,4%, na região lateral foi de 53,8%, na região medial foi de 53,7%. Não foram observadas diferenças significativas na densidade celular e na hexagonalidade nas diferentes regiões analisadas da córnea. Os valores da densidade endotelial e da hexagonalidade da região central do endotélio podem ser extrapolados para as demais regiões da córnea.The corneal endothelium is a monolayer of juxtaposed polygonal cells that cover the posterior surface of the cornea. It is a fundamental structure for the maintenance of corneal transparency. The swine species is widely used as an experimental model in ophthalmology. However, studies evaluating the corneal endothelium with a specular microscope in this species are scarce. The objective was to evaluate the endothelial density and cell morphology of different regions of the porcine corneal endothelium using specular contact microscopy. Twenty-four eyeballs from 12 male, six-month-old, Large White pigs (Sus scrofa domesticus) were studied. Contact specular microscopy was performed in the central, superior, inferior, lateral and medial regions of the cornea. The mean endothelial density in the central region was 1865 cells/mm², in the upper region it was 1877.3 cells/mm², in the lower region it was 1854.9 cells/mm², in the lateral region it was 1847 cells/mm² and in the medial was 1831.7 cells/mm². In the central region, the average percentage of hexagonal cells was 53.3%, in the upper region it was 53.8%, in the lower region it was 54.4%, in the lateral region it was 53.8%, in the medial region was 53.7%. No significant differences were observed in cell density and hexagonality in the different analyzed regions of the cornea. The endothelial density and hexagonal values of the central region of the endothelium can be extrapolated to the other regions of the cornea

Adesivos sintéticos e biológicos na superfície ocular : revisão bibliográfica

A córnea é a porção mais externa do bulbo ocular e está constantemente sujeita a traumas. Quando as afecções corneanas não são tratadas adequadamente, pode ocorrer perda da visão e até mesmo do globo ocular. Dependendo da gravidade, somente o tratamento medicamentoso não é suficiente, e diversas técnicas cirúrgicas são recomendadas, incluindo os flaps conjuntivais, os enxertos, a ceratoplastia penetrante, o uso de lentes de contato, adesivos, entre outras, cada uma apresentando vantagens e desvantagens. A busca por métodos seguros, de fácil e rápida aplicação, com mínima interferência na cicatrização da córnea, deu início ao estudo das substâncias adesivas. Os adesivos cirúrgicos para o tratamento de úlceras e perfurações corneanas devem ser de fácil aplicação, ter rápida polimerização, possuírem mínima toxicidade e a permanecerem no local da lesão durante o tempo necessário para a cicatrização. Entre as vantagens da utilização dos adesivos na superfície ocular, encontram-se a rapidez da aplicação, o menor desconforto ocular e a inflamação menos exacerbada. Os adesivos sintéticos derivados do cianocrilato são monômeros líquidos que em contato com a água polimerizam-se e solidificam-se rapidamente, e a placa formada serve de suporte para a cicatrização corneana. O adesivo biológico a base de fibrina mimetiza a fase final da cascata de coagulação através da polimerização do fibrinogênio pela trombina, levando a formação de um coágulo firme de fibrina. Ao contrário do adesivo de cianocrilato, esse bioadesivo forma uma vedação suave ao longo da borda da lesão proporcionando maior conforto pós-operatório e com menos complicações. Objetiva-se com esta revisão bibliográfica salientar, principalmente indicações, técnicas de aplicação e limitações dos adesivos sintéticos e biológicos utilizados na superfície ocular.The cornea is the outer portion of the ocular globe and is constantly subject to trauma. When corneal conditions are not treated properly, loss of vision and even of the eyeball may occur. Depending on the severity, only drug treatment is not sufficient, and several surgical techniques are recommended, including conjunctival flaps, grafts, penetrating keratoplasty, contact lens wear, adhesives, among others, each presenting advantages and disadvantages. The search for safe methods, with easy and quick application, with minimal interference in the healing of the cornea, began the study of the adhesive substances. Surgical adhesives for the treatment of corneal ulcers and perforations should be easy to apply, have rapid polymerization, possess minimal toxicity and remain at the site of injury for as long as necessary for healing. Among the advantages of the use of adhesives on the ocular surface are speed of application, less ocular discomfort and less exacerbated inflammation. Synthetic adhesives derived from cyanoacrylate are liquid monomers which in contact with water polymerize and solidify rapidly, and the formed plaque serves as a support for corneal healing. The fibrin-based biological adhesive mimics the final phase of the coagulation cascade through the polymerization of fibrinogen by thrombin, leading to the formation of a firm clot of fibrin. Unlike the cyanoacrylate adhesive, this bioadhesive forms a soft seal along the edge of the lesion, providing greater postoperative comfort and less complications. This literature review aims to highlight, mainly indications, application techniques and limitations of synthetic and biological adhesives used on the ocular surface

Specular microscopy of the different regions of the cornea in enucleated swine eyes - ex vivo evaluation

The objective of this study was to determine the endothelial cell density (ECD) and hexagonality of the cornea in the different regions of healthy swine corneal endothelium using specular microscopy. Twenty-four eyeballs from 12 male, 6-month-old Large White pigs (Sus scrofa domesticus) were studied. Contact specular microscopy was performed in the central, superior, inferior, lateral and medial regions. The corneal parameters analysed in this study were ECD and hexagonality. The ECD in the central region was 1865 cells/mm²; in the upper region, it was 1877 cells/mm², in the lower region, it was 1854 cells/mm², in the lateral region, it was 1847 cells/mm², in the medial region, it was 1831 cells/mm². Hexagonality in the central region, was 53%; in the upper region, it was 54%, in the lower region, it was 54%, in the lateral region, it was 54%, in the medial region, it was 54%. There was no significant difference regarding to the evaluated parameters in all corneal regions evaluated. No statistically significantly differences were observed in ECD and hexagonality between the left and the right eyes. This study demonstrates that ECD and hexagonality of the central cornea area represent the entire endothelial mosaic. Keywords: cornea; endothelium; morphology; cell count, swine

Treatment of Limbal Melanocytoma with Full Excision and a Fresh Homologous Corneoscleral Graft in a Dog

Background: Limbal melanoma has been diagnosed in dogs and due to progression may cause vision loss and eyeball removal. Definitive diagnosis is made through histopathological examination. Therapeutic options include full thickness resection and repair by homologous corneal tissue, synthetic graft material, and enucleation. In this report, we describe a case of limbal melanocitoma in a dog that has been treated successfully with fresh homologous corneoscleral graft.Case: A 5-year-old female Labrador was referred to the Ophthalmology Veterinary Section of the Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil, with a history of a pigmented mass located on the left eye. Ophthalmic examination revealed a pigmented mass located at the left temporal limbus with corneal involvement. Surgical excision followed by reconstruction using fresh homologous corneoscleral was recommended. The patient was premedicated with acepromazine (0.05 mg/kg, IM) and meperidine (20 mg/kg, IM). Anaesthesia was induced with propofol (10 mg/kg, IV) and maintained with isoflurane. Atracurium (0.2 mg/kg, IV) was administered to maintain a central eye position. The mass and a free margin were removed by full-thickness corneoscleral resection. A corneoscleral graft was harvested from a dogthat had been euthanised for reasons unrelated to this study and sutured with 9-0 polyglactin 910 using a simple interrupted pattern. The mass was immediately fixed in 10% neutral buffered formalin and submitted for histological sectioning and routine staining. Based on the histopathological analysis it was confirmed limbal melanocytoma. Postoperative treatment consisted of topical administration of 0.3% flurbiprofen every 6 h for 15 days, and a combination of topical ciprofloxacin/dexamethasone eye drops every 6 h for 30 days. Systemic carprofen (4 mg/kg per day, VO) was prescribed for 10 days. Topical tropicamide was used twice daily for 1 week. Cyclosporin 0.2% eye drops were applied twice daily for 2 months. Examination of the left eye two months after surgery revealed decreased corneal vascularization, and the results of pupillary light response and vision testing were normal. The values of intraocular pressure remained normal in all postoperativeevaluations. The patient was followed for 36 months postoperatively, during which time there was no recurrence.Discussion: Limbal melanomas are the most common ocular melanomas in dogs. Most of them develop slowly and are located closely to the superior limbus. The tumors tend to grow more rapidly in younger dogs and more slowly in older dogs. Labrador Retrievers and German shepherds appear to be affected more frequently than other breeds. The present case involved a 5-year-old Labrador breed. The clinical presentation included a pigmented limbal mass extending intothe adjacent cornea, sclera and conjunctival tissue. A tissue biopsy is necessary to confirm the diagnosis. In the present case, the diagnosis of limbal melanocytoma was based on clinical signs and confirmed by histopathological examination. The choice of treatment is influenced by the tumour size and location, availability of equipment and materials, clinician expertise and the cost of treatment. Some techniques for removing the mass and repairing the resultant corneoscleral defect has been described. In the present case, due to the location and size of the mass and the absence of intraocular invasion, surgical removal including a margin of normal tissue was performed. With this surgical procedure, the intention was to preserve the eyeball and maintain vision. In this case, surgical excision of a limbal melanocytoma combined with homologous corneoscleral graft was effective for repairing a full-thickness corneoscleral defect and preserving ocular function

Conjunctival Melanoma in a Horse Treated by Tumor Resection and Cryotherapy

Background: Ocular melanoma is very rare compared to cutaneous melanoma in horses. Definitive diagnosis is made through histopathological examination and treatment options include surgical excision associated with cryotherapy, radiation therapy, and chemotherapy. In this report, we describe a case of conjunctival melanoma in a horse that has been treated successfully with surgical excision associated with cryotherapy.Case: A 15-year-old male Percheron male was referred to the Ophthalmology Veterinary Section of the Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil, with a history of a pigmented mass located on the lower eyelid of the left eye. Ophthalmologic examination revealed ocular discomfort, secretion and a pigmented mass in the left inferior bulbar conjunctiva. The dermatological examination revealed other melanomas in the perineal region. Complete blood count and serum chemistry profile were within normal ranges and prior to surgery the horse was treated with flunixin meglumine (1.1 mg/kg, IV, q 12 h). Sedation was performed with xylazine (0.4 mg/kg, IV) and detomidine hydrochloride(0.01 mg/kg, IV) and then the animal was placed in a retention trunk. The conjunctival mass was resected with a margin of safety. Liquid nitrogen was applied to the tumor site and the adjacent conjunctiva with a copper cryoprobe with one unit of liquid nitrogen. Histopathological examination revealed neoplastic cells containing pigmented melanocytes in the conjunctival submucosa, confirming the diagnosis of conjunctival melanoma. Postoperative treatment was performed with flunixin meglumine (1.1 mg/kg, IV, q 12 h) for 3 days and topical ophthalmic ointment containing neomycin, polymyxin B sulfate and dexamethasone twice daily for one week. Seven days after surgery, the lesion was healed. The patient was followed for 24 months after excision and there was no evidence of recurrence.Discussion: Older horses are considered more predisposed to melanoma development, possibly because of the proliferation of melanocytes as a manifestation of aging, and in addition, cutaneous melanomas are common in gray horses and rare in other horse colors. In this case, the horse was a 15-year-old Percheron horse with gray hair. In horses, there is only one case of conjunctival melanoma documented in the literature. In both cases, the ophthalmic examination revealed a large, raised, heavily pigmented mass protruding from the bulbar conjunctiva. The only difference is that in the present case the location of the mass was in the inferior bulbar conjunctiva and in the case cited in the literature. The mass was located in the bulbar conjunctiva under the lateral comer. In this case, the diagnosis of conjunctive melanoma was based on clinical signs and confirmed by histopathological examination. It was decided to perform an excisional biopsy for treatment and to confirm the diagnosis of conjunctival melanoma. The choice of treatment depends very much on the clinical presentation, that in this animal, despite the neoplasia being extended, it was located only in the conjunctiva without involvement of the sclera and the eyelid. Therefore the decision was made to perform an excisional biopsy associated with cryotherapy. The purpose of such adjuvant therapy is to kill all residual tumor cells and prevent the recurrence of malignant tumors. In the present case, the surgical wound was cured one week after surgery. The surgical procedure in the case reported was performed under local anesthesia and sedation with the horse standing. To make this decision, consideration should be given to patient health, anesthetic risk, and additional risks during recovery from general anesthesia. In this case, surgical excision of the mass associated with cryotherapy was effective in the treatment of conjunctival melanoma in a horse.Keywords: ocular, equine, melanocytic neoplasia, cryosurgery

Modified Eye Evisceration in a Tropical Screech Owl (Megascops choliba)

Background: An adult owl was presented with an injury to the right eye that rendered it blind in that eye. The left eye was normal. Removal of the right eye was recommended and a modified eye evisceration was performed. No complications were observed during or after surgery. The objective of this paper is to describe the modified eye evisceration technique that was successfully used in a tropical screech owl (Megascops choliba). Case: An adult owl was presented with an injury to the right eye that rendered it blind in that eye. Two previous surgical treatments have been carried out but have not been successful. Using a portable slit-lamp (Kowa SL-15®), both eyes were examined. The left eye was normal. Upon ophthalmic examination of the right eye, the owl demonstrated blepharospasm and large central corneal ulcer. Removal of the right eye was recommended. The bird received midazolam hydrochloride [Dormire® - 1 mg/kg, IM] and ketamine hydrochloride [Ketamina® - 5 mg/kg IM] as pre-anesthetic medications. Subsequently, the bird was anesthetized with isoflurane (Isoforine®) by facemask for induction, and then maintained with isoflurane vaporized in 100% oxygen through an endotracheal tube. With the aid of a surgical microscope and microsurgery materials, a modified eye evisceration was performed. Post-operatively, the owl received meloxicam [Maxicam® - 0.5 mg/kg, IM] and tramadol hydrochloride [Cronidor® - 15 mg/kg, orally for 4 days]. The day after surgery, the owl was comfortable and its usual appetite was regained. The patient remained hospitalized for 3 weeks and was evaluated daily. The skin sutures were removed 10 days after the surgical procedure and the surgical wound had healed normally. The patient was reintroduced into the wild after 2 months. During the 6 months post-release, the bird was evaluated once a month, and no complications were observed.Discussion: Severe eye trauma and complicated corneal ulcers are common causes of eyeball removal in birds. In birds, there is a high risk of complications during enucleation. The fragility of the orbital bones makes them susceptible to trauma during the surgery. Evisceration involves the removal of the inner contents of the eye while leaving the cornea and the sclera intact. In the current case, evisceration was chosen because the eye was blind, and maintaining a blind eye would be a source of pain and infection. In the modified evisceration technique, the risk of complications is minimal compared to enucleation, mainly because surgical manipulation is minimal. In our case, the total surgery time was 20 min. Another complication reported after enucleation in birds is the possibility of disfiguring the bird because the removal of the globe disturbs the natural head balance. To avoid these complications, the use of an intraocular prosthesis after evisceration in birds has been performed. However, owls have a tubular-shaped globe with scleral ossicles. These factors could hinder or even prevent the accommodation of a cylindrical silicone prosthesis. In the present case, an intraocular prosthesis implant was never considered due to the unavailability of the prosthesis and to avoid the risk of postoperative complications that have been reported from the literature in dogs. In this case, the owl recovered well from anesthesia without complications, and no postoperative hemorrhage was observed. No signs of pain were observed during the postoperative period and the owl had already shown an appetite and fed on the first postoperative day. The previously published reports using the modified evisceration technique also demonstrated an absence of pain signs during the postoperative period

Sheep corneal endothelium morphology : evaluation with trypan blue and alizarin red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quan¬titative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research

Sheep Corneal Endothelium Morphology - Evaluation with Trypan Blue and Alizarin Red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods &amp; Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P &lt; 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research. Keywords: ovine, endothelial cells, ex vivo model, vital staining, hexagonality