Avaliação do endotélio corneano de equinos após exposição à indocianina verde 0,5% : estudo in vitro

The purpose of the study was to investigate whether indocyanine green (ICG) dye damages the corneal endothelium of horses. Twenty-four corneas of 12 healthy equines, males or females, of different ages were used in this study. Only eyes with no ocular findings were used. Randomly, one eye was included in the treatment group and one in the control group. The eyes of the treatment group were exposed for 1 minute to dye ICG 0.5%. After that the endothelium of all eyes was stained with trypan blue and alizarin red S and analyzed and photographed under an optical microscope. Areas with damaged endothelial cells were manually measured and quantified using software for morphometric analysis and expressed as a percentage of cell damage. In all eyes examined areas of cell damage were observed in both corneas of the control group and the treatment group. The mean endothelial damage was 0.8 ± 0.37% in the treatment group and 0.97 ± 0.39% in the control. The Qui-square test stated that treatment and control group were not different. The ICG 0.5% did not cause acute damage to equine corneal endothelium.O objetivo do estudo foi investigar se indocianina verde (ICG) induz dano nas células do endotélio da córnea de equinos. Vinte e quatro córneas de 12 equinos saudáveis, machos ou fêmeas, de diferentes idades foram estudadas. Somente olhos hígidos foram utilizados. Aleatoriamente, um olho foi incluído no grupo controle e outro no grupo tratamento. Os olhos do grupo tratamento foram expostos durante um minuto à indocianina verde a 0,5%. Posteriormente, o endotélio da córnea foi corado com azul de tripano e vermelho de alizarina, analisado e fotografado usando microscópio óptico. As áreas com células endoteliais danificadas foram aferidas e quantificadas utilizando um software para análise morfométrica. Os valores encontrados foram expressos como percentual de perda celular. Em todos os olhos examinados foram observadas áreas de dano celular, tanto no grupo controle quanto no grupo tratamento. A perda celular endotelial média foi de 0,8±0,37% no grupo tratamento e 0,97 ± 0,39% no grupo controle. O teste Qui-quadrado confirmou que os grupos tratamento e controle não diferiram. Foi possível concluir que a ICG 0,5% não causou dano agudo nas células do endotélio da córnea de equinos

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

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

Morphology of Endothelial Cells from Different Regions of the Swine Cornea

Background: The corneal endothelium is a monolayer of polygonal cells which constitute the last layer of the cornea. The integrity of this layer is critical to cornea transparency. The characterization of normal corneal endothelial morphology is important not only to clinical evaluation but also to selection of areas of the cornea with better quality to be employed as donor tissue. The aim of the present study was to evaluate the morphology of endothelial cells from different regions of the swine cornea after alizarin red staining using optical microscopy.Materials, Methods & Results: Twenty-four healthy eyes from 12 swine Large White breed, with 14-month-old, males or females obtained from a licensed Brazilian commercial slaughterhouse were studied. Immediately after humane slaughter, the eyes were enucleated and submitted to ophthalmic examination. Eyes with signs of diseases of the anterior segment were excluded. The cornea, with 3 mm of the sclera, was removed and placed on a glass microscope slide with the endothelial side up. Four radial incisions were made in the periphery of the cornea to better accommodate the cornea on the microscope slide. Alizarin red was diluted in isotonic solution (0.2 g/100 mL) and the pH was adjusted to 4.2 with hydrochloric acid. Three drops of alizarin red were placed on the corneal endothelium. After 90 s, the dye was removed from the cornea with balanced saline solution. The corneal endothelium was examined and photographed using an optical microscope. All evaluations were performed by the same investigator. Photomicrographs were taken of central, superior, inferior, nasal and temporal corneal areas. Parameter studied included endothelial cell morphology. For the statistical analysis, was employed the ANOVA variance test (repeated measures). Differences were considered statistically significant at P < 0.05. Normal endothelium cells were mainly hexagonal (83.7%), pentagonal (7.45%) and heptagonal (8.8%), with a minimal number of cells of other shapes present. There were no significant statistical differences in the proportion of the morphology and the different regions of the cornea (P = 0.31).Discussion: Different techniques are available for the analysis of corneal endothelium, including mainly scanning electron microscopy, specular microscopy and optical microscopy. 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 the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and to obtain images of the swine endothelium of all regions of the cornea.  The analysis of the cellular morphology or the percentage of hexagonal cells are among the main parameters used to evaluate the health of the corneal endothelium. In this study, the endothelium had the predominance of the hexagonal shape in all regions studied. In swine, there are no studies evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea

Efeitos agudos do cloridrato de ropivacaína na ultraestrutura das células endoteliais da córnea de equinos : estudo ex vivo

The objective of this study was to evaluate the acute effects of ropivacaine hydrochloride on the corneal endothelium of horses. Forty-eight eyes were obtained from a commercial slaughterhouse and were randomly divided into three groups. In group A, the corneal endothelium was exposed to 0.75% ropivacaine hydrochloride for 60 seconds. In group B, the corneal endothelium was exposed to 0.75% ropivacaine hydrochloride for 15 minutes. In group C, the corneal endothelium was exposed to a balanced saline solution for 60 seconds. Afterwards, all samples were prepared for evaluation with scanning electron microscopy. Random electromicrographs were obtained from each sample. The images were analysed and, with the aid of software, areas with no endothelial cells were measured. The average endothelial loss, expressed as a percentage in relation to the total area, of the samples in group A was 5.28%. The average endothelial loss of samples from group B, expressed as a percentage in relation to the total area, was 20.39%. The damage to the corneal endothelium was significantly greater in group B compared to groups A and C. It was possible to conclude that 0.75% ropivacaine hydrochloride induced acute damage to corneal endothelium cells.Objetivou-se avaliar os efeitos agudos do cloridrato de ropivacaína no endotélio da córnea de equinos. Quarenta e oito olhos de equinos foram divididos aleatoriamente em três grupos. No grupo A o endotélio da córnea foi exposto a cloridrato de ropivacaína a 0,75% por 60 segundos. No grupo B o endotélio da córnea foi exposto a cloridrato de ropivacaína a 0,75% por 15 minutos. No grupo C o endotélio da córnea foi exposto à solução salina balanceada por 60 segundos. As amostras foram preparadas para avaliação com microscopia eletrônica de varredura. Eletromicrografias eletrônicas de varredura foram obtidas aleatoriamente de cada amostra. As imagens foram analisadas e, com o auxílio de um programa para morfometria foram medidas as áreas sem células endoteliais. A perda endotelial média foi expressa em porcentagem em relação à área total das amostras do grupo A foi de 5,28%. A perda endotelial média de amostras do grupo B foi expressa em porcentagem em relação à área total, foi de 20,39%. O dano ao endotélio da córnea foi significativamente maior no grupo B, comparado aos grupos A e C. O cloridrato de ropivacaína a 0,75% induziu dano agudo nas células do endotélio da córnea de equinos

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

Morphology of endothelial cells from different regions of the swine cornea

Background: The corneal endothelium is a monolayer of polygonal cells which constitute the last layer of the cornea. The integrity of this layer is critical to cornea transparency. The characterization of normal corneal endothelial morphology is important not only to clinical evaluation but also to selection of areas of the cornea with better quality to be employed as donor tissue. The aim of the present study was to evaluate the morphology of endothelial cells from different regions of the swine cornea after alizarin red staining using optical microscopy. Materials, Methods & Results: Twenty-four healthy eyes from 12 swine Large White breed, with 14-monthold, males or females obtained from a licensed Brazilian commercial slaughterhouse were studied. Immediately after humane slaughter, the eyes were enucleated and submitted to ophthalmic examination. Eyes with signs of diseases of the anterior segment were excluded. The cornea, with 3 mm of the sclera, was removed and placed on a glass microscope slide with the endothelial side up. Four radial incisions were made in the periphery of the cornea to better accommodate the cornea on the microscope slide. Alizarin red was diluted in isotonic solution (0.2 g/100 mL) and the pH was adjusted to 4.2 with hydrochloric acid. Three drops of alizarin red were placed on the corneal endothelium. After 90 s, the dye was removed from the cornea with balanced saline solution. The corneal endothelium was examined and photographed using an optical microscope. All evaluations were performed by the same investigator. Photomicrographs were taken of central, superior, inferior, nasal and temporal corneal areas. Parameter studied included endothelial cell morphology. For the statistical analysis, was employed the ANOVA variance test (repeated measures). Differences were considered statistically significant at P < 0.05. Normal endothelium cells were mainly hexagonal (83.7%), pentagonal (7.45%) and heptagonal (8.8%), with a minimal number of cells of other shapes present. There were no significant statistical differences in the proportion of the morphology and the different regions of the cornea (P = 0.31). Discussion: Different techniques are available for the analysis of corneal endothelium, including mainly scanning electron microscopy, specular microscopy and optical microscopy. 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 the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and to obtain images of the swine endothelium of all regions of the cornea. The analysis of the cellular morphology or the percentage of hexagonal cells are among the main parameters used to evaluate the health of the corneal endothelium. In this study, the endothelium had the predominance of the hexagonal shape in all regions studied. In swine, there are no studies evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea

Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study

The purpose of the study was to investigate whether indocyanine green (ICG) dye damages the corneal endothelium of horses. Twenty-four corneas of 12 healthy equines, males or females, of different ages were used in this study. Only eyes with no ocular findings were used. Randomly, one eye was included in the treatment group and one in the control group. The eyes of the treatment group were exposed for 1 minute to dye ICG 0.5%. After that the endothelium of all eyes was stained with trypan blue and alizarin red S and analyzed and photographed under an optical microscope. Areas with damaged endothelial cells were manually measured and quantified using software for morphometric analysis and expressed as a percentage of cell damage. In all eyes examined areas of cell damage were observed in both corneas of the control group and the treatment group. The mean endothelial damage was 0.8 ± 0.37% in the treatment group and 0.97 ± 0.39% in the control. The Qui-square test stated that treatment and control group were not different. The ICG 0.5% did not cause acute damage to equine corneal endothelium