Specular microscopy of the different regions of the cornea in enucleated swine eyes: ex vivo evaluation / Microscopia especular das diferentes regiões da córnea de suínos: estudo ex vivo

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.

O objetivo do presente estudo foi avaliar a densidade endotelial e a hexagonalidade das células endoteliais nas diferentes regiões da córnea 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 células/mm2, na região inferior foi de 1854 células/mm2, na região lateral foi de 1847 células/mm2 e na região medial foi de 1831 células/mm2. Na região central, a hexagonalidade foi de 53%, na região superior foi de 54%, na região inferior foi de 54%, na região lateral foi de 54%, na região medial foi de 54%. Não foram observadas diferenças significativas na densidade celular e na hexagonalidade nas diferentes regiões da córnea analisadas. Este estudo demonstrou que a densidade endotelial e a hexagonalidade da área central da córnea representam todo o mosaico endotelial.

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. 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.

Acute effects of ropivacaine hydrochloride on corneal endothelial cell ultrastructure of horses: ex vivo study / Efeitos agudos do cloridrato de ropivacaína na ultraestrutura das células endoteliais da córnea de equinos: estudo ex vivo

ABSTRACT: 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.

RESUMO: 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.

Acute effects of ropivacaine hydrochloride on corneal endothelial cell ultrastructure of horses: ex vivo study / 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.

Acute effects of ropivacaine hydrochloride on corneal endothelial cell ultrastructure of horses: ex vivo study / 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.(AU)

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.(AU)

Evaluation of equine (Equus cabbalus) corneal endothelium stored in EUSOL-C® preservation medium / Avaliação do endotélio da córnea de equinos (Equus cabbalus) acondicionado em meio de preservação de córnea Eusol-C®

The objective of this study was evaluate the maintenance of the corneal endothelium of horses in cold EUSOL-C® preservation medium over different periods (seven and 14 days) using scanning electron microscopy. A total of 20 pairs of eyes from horses were analysed. The corneas were divided into four groups of 10 corneas each (G1, G2, G3 and G4): G1 - the samples were kept in the preservation medium for seven days; G3 - the samples were kept in the preservation medium for for 14 days; G2 and G4 were formed by the control corneal buttons of G1 and G3, respectively. The average cell loss observed in G1 was 7.62%, in G2 it was 7.04%, in G3 9.12% and in G4 7.16%. No statistically significant differences were observed between the four groups. It was concluded that the Eusol-C® hypothermic preservation medium provided satisfactory preservation of the corneal endothelium in equine species for up to 14 days.

Objetivou-se avaliar a manutenção do endotélio da córnea de equinos em meio de preservação a frio EUSOL-C® em diferentes períodos de acondicionamento (sete e 14 dias) utilizando microscopia eletrônica de varredura. Foram avaliados 40 bulbos oculares de 20 equinos. Os bulbos oculares foram divididos em quatro grupos (G1, G2, G3 e G4), nos quais: G1 foi composto por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante sete dias; o G3 foi formado por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante 14 dias. O G2 e G4 foram formados pelas córneas controle armazenadas imediatamente em glutaraldeído. A média da perda celular observada no G1 foi de 7,62%, no G2 foi de 7,04 %, no G3 foi de 9,12% e o no G4 foi de 7,16%. Não foram observadas diferenças estatisticamente significativas entre os quatro grupos. Foi possível concluir que o meio de preservação hipotérmico Eusol-C® proporcionou de forma satisfatória a preservação do endotélio da córnea na espécie equina durante o período de até 14 dias.

Evaluation of equine (Equus cabbalus) corneal endothelium stored in EUSOL-C® preservation medium / Avaliação do endotélio da córnea de equinos (Equus cabbalus) acondicionado em meio de preservação de córnea Eusol-C®

The objective of this study was evaluate the maintenance of the corneal endothelium of horses in cold EUSOL-C® preservation medium over different periods (seven and 14 days) using scanning electron microscopy. A total of 20 pairs of eyes from horses were analysed. The corneas were divided into four groups of 10 corneas each (G1, G2, G3 and G4): G1 - the samples were kept in the preservation medium for seven days; G3 - the samples were kept in the preservation medium for for 14 days; G2 and G4 were formed by the control corneal buttons of G1 and G3, respectively. The average cell loss observed in G1 was 7.62%, in G2 it was 7.04%, in G3 9.12% and in G4 7.16%. No statistically significant differences were observed between the four groups. It was concluded that the Eusol-C® hypothermic preservation medium provided satisfactory preservation of the corneal endothelium in equine species for up to 14 days.(AU)

Objetivou-se avaliar a manutenção do endotélio da córnea de equinos em meio de preservação a frio EUSOL-C® em diferentes períodos de acondicionamento (sete e 14 dias) utilizando microscopia eletrônica de varredura. Foram avaliados 40 bulbos oculares de 20 equinos. Os bulbos oculares foram divididos em quatro grupos (G1, G2, G3 e G4), nos quais: G1 foi composto por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante sete dias; o G3 foi formado por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante 14 dias. O G2 e G4 foram formados pelas córneas controle armazenadas imediatamente em glutaraldeído. A média da perda celular observada no G1 foi de 7,62%, no G2 foi de 7,04 %, no G3 foi de 9,12% e o no G4 foi de 7,16%. Não foram observadas diferenças estatisticamente significativas entre os quatro grupos. Foi possível concluir que o meio de preservação hipotérmico Eusol-C® proporcionou de forma satisfatória a preservação do endotélio da córnea na espécie equina durante o período de até 14 dias.(AU)

Deep anterior lamellar keratoplasty (DALK) as a treatment of a deep stromal ulcer in a dog: case report / Ceratoplastia lamelar anterior profunda(DALK) como tratamento de úlcera estromal profunda em cão: relato de caso

Characterized by the rupture of the corneal epithelium, corneal ulcers threaten ocular integrity, with multifactorial development, such as decreased protection and excessive abrasion applied to the corneal surface. Keratoplasty restores the corneal anatomy, removing the inflamed or infected tissues, in order to promote visual acuity. The technique of deep anterior lamellar keratoplasty (DALK) selectively removes the diseased layers of the cornea, keeping the Descemet membrane and endothelium healthy, reducing the risk of rejection, aimed at tissue recovery. In the present report, a canine, from the Shih Tzu breed, a female, that has not been castrated, 2 years and 6 months old, was treated, she had a history of ocular discomfort and mucous secretion in both eyes. In the ophthalmic examination, keratoconjunctivitis sicca was found in the left eye and ectopic cilia was found in both eyes. With the aid of a fluorescein test, the presence of a deep stromal ulcer affecting the right eye was confirmed. The patient underwent ectopic cilia removal using the wedge resection technique and an allogeneic corneal transplantation using DALK, with only the diseased epithelium and stroma being replaced, reducing the risk of rejection and failures related to surgery. 90 days after the procedure, there was no reaction to the graft, there was complete epithelial repair and maintenance of

Caracterizada pela ruptura do epitélio corneano, as úlceras de córnea ameaçam a integridade ocular, tendo desenvolvimento multifatorial, como a diminuição da proteção e o excesso de abrasão aplicado na superfície da córnea. A ceratoplastia restaura a anatomia corneana, removendo tecidos inflamados ou infectados, com objetivo de promover acuidade visual. A técnica de ceratoplastia lamelar anterior profunda (DALK -deep anterior lamelar keratoplasty) remove seletivamente as camadas doentes da córnea, mantendoa membrana de Descemet e endotélio saudáveis, reduzindo o risco de rejeição, visando recuperação tecidual. No presente relato, um canino, raça Shih Tzu, fêmea não castrada, 2 anos e 6 meses de idade, foi atendido com histórico de desconforto ocular e secreção mucosa em ambos os olhos. No exame oftálmico constatou-se ceratoconjuntivite seca no olho esquerdo e cílios ectópicos em ambos os olhos. Com auxílio do teste de fluoresceína, confirmou-se a presença de úlcera estromal profunda acometendo o olho direito. O paciente foi submetido a remoção dos cílios ectópicos pela técnica de secção em cunha e ao transplante de córnea alógena utilizando a DALK, sendo substituído apenas o epitélio e estroma doentes, reduzindo o risco de rejeição e falhas relacionadas a cirurgia. Após 90 dias do procedimento, constatou-se ausência de reação ao enxerto, completa reparação epitelial e manutenção da função visual com discreta opacidade corneana.

Deep anterior lamellar keratoplasty (DALK) as a treatment of a deep stromal ulcer in a dog: case report / Ceratoplastia lamelar anterior profunda(DALK) como tratamento de úlcera estromal profunda em cão: relato de caso

Characterized by the rupture of the corneal epithelium, corneal ulcers threaten ocular integrity, with multifactorial development, such as decreased protection and excessive abrasion applied to the corneal surface. Keratoplasty restores the corneal anatomy, removing the inflamed or infected tissues, in order to promote visual acuity. The technique of deep anterior lamellar keratoplasty (DALK) selectively removes the diseased layers of the cornea, keeping the Descemet membrane and endothelium healthy, reducing the risk of rejection, aimed at tissue recovery. In the present report, a canine, from the Shih Tzu breed, a female, that has not been castrated, 2 years and 6 months old, was treated, she had a history of ocular discomfort and mucous secretion in both eyes. In the ophthalmic examination, keratoconjunctivitis sicca was found in the left eye and ectopic cilia was found in both eyes. With the aid of a fluorescein test, the presence of a deep stromal ulcer affecting the right eye was confirmed. The patient underwent ectopic cilia removal using the wedge resection technique and an allogeneic corneal transplantation using DALK, with only the diseased epithelium and stroma being replaced, reducing the risk of rejection and failures related to surgery. 90 days after the procedure, there was no reaction to the graft, there was complete epithelial repair and maintenance of(AU)

Caracterizada pela ruptura do epitélio corneano, as úlceras de córnea ameaçam a integridade ocular, tendo desenvolvimento multifatorial, como a diminuição da proteção e o excesso de abrasão aplicado na superfície da córnea. A ceratoplastia restaura a anatomia corneana, removendo tecidos inflamados ou infectados, com objetivo de promover acuidade visual. A técnica de ceratoplastia lamelar anterior profunda (DALK -deep anterior lamelar keratoplasty) remove seletivamente as camadas doentes da córnea, mantendoa membrana de Descemet e endotélio saudáveis, reduzindo o risco de rejeição, visando recuperação tecidual. No presente relato, um canino, raça Shih Tzu, fêmea não castrada, 2 anos e 6 meses de idade, foi atendido com histórico de desconforto ocular e secreção mucosa em ambos os olhos. No exame oftálmico constatou-se ceratoconjuntivite seca no olho esquerdo e cílios ectópicos em ambos os olhos. Com auxílio do teste de fluoresceína, confirmou-se a presença de úlcera estromal profunda acometendo o olho direito. O paciente foi submetido a remoção dos cílios ectópicos pela técnica de secção em cunha e ao transplante de córnea alógena utilizando a DALK, sendo substituído apenas o epitélio e estroma doentes, reduzindo o risco de rejeição e falhas relacionadas a cirurgia. Após 90 dias do procedimento, constatou-se ausência de reação ao enxerto, completa reparação epitelial e manutenção da função visual com discreta opacidade corneana.(AU)

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.[...]

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.[...](AU)

Effects of atracurium besylate on corneal endothelium of chickens: in vitro study / Avaliação dos efeitos do besilato de atracúrio no endotélio corneano de galinhas: estudo in vitro

The aim of this study was to investigate the acute effects of atracurium besylate on cellular damage in corneal endothelium of chickens. Twenty healthy chicken eyes were assigned to the following groups: Group 1 (G1), experimental group (n=10); and Group 2 (G2), control (n=10). Excised corneoscleral buttons were immediately placed on glass microscopy slides with endothelial region faced up. Corneal endothelium of eyes in G1 were covered with AB (0.2mL, 10mg/mL) for 3 min and then rinsed with balanced salt solution (BSS), while the corneal endothelium of eyes in G2 were covered with BBS for 3 min. Corneas from both groups were stained with alizarin red/trypan blue and visualized by light microscopy. Ten random photographs were taken from each cornea. The area of cellular damage was measured by software in all samples and cell loss of each group was averaged and compared. Endothelial area of denudation and Descemet's membrane exposure were higher in G1 than G2. In conclusion, atracurium besylate induced an acute damage on corneal endothelium of chickens.(AU)'

Objetivou-se avaliar os efeitos agudos do besilato de atracúrio sobre o endotélio corneano de galinhas. Vinte olhos saudáveis de galinhas foram aleatoriamente separados em dois grupos com 10 olhos cada, sendo G1 o grupo controle e G2 o grupo tratamento. Imediatamente após a excisão dos botões corneoesclerais estes foram colocados em lâminas de microscopia de vidro com o lado endotelial voltado para cima. No Grupo 1, o endotélio corneano foi recoberto com 0,2ml de besilato de atracúrio (10mg/ml) durante 3 minutos e depois lavado com solução salina balanceada. No Grupo 2, o endotélio corneano foi recoberto apenas com solução salina balanceada durante 3 min. As córneas de ambos os grupos foram coradas com vermelho de alizarina e azul de tripano e visualizadas com microscópio óptico. Foram obtidas dez fotografias aleatórias de cada amostra. As imagens foram analisadas e com auxílio de um software as áreas com ausência de células endoteliais calculadas. A perda celular endotelial foi significativamente maior no grupo tratamento comparativamente ao grupo controle. Com base nos resultados apresentados foi possível concluir que o besilato de atracúrio induziu dano agudo nas células do endotélio da córnea de galinhas.(AU)

Effects of atracurium besylate on corneal endothelium of chickens: in vitro study / Avaliação dos efeitos do besilato de atracúrio no endotélio corneano de galinhas: estudo in vitro

The aim of this study was to investigate the acute effects of atracurium besylate on cellular damage in corneal endothelium of chickens. Twenty healthy chicken eyes were assigned to the following groups: Group 1 (G1), experimental group (n=10); and Group 2 (G2), control (n=10). Excised corneoscleral buttons were immediately placed on glass microscopy slides with endothelial region faced up. Corneal endothelium of eyes in G1 were covered with AB (0.2mL, 10mg/mL) for 3 min and then rinsed with balanced salt solution (BSS), while the corneal endothelium of eyes in G2 were covered with BBS for 3 min. Corneas from both groups were stained with alizarin red/trypan blue and visualized by light microscopy. Ten random photographs were taken from each cornea. The area of cellular damage was measured by software in all samples and cell loss of each group was averaged and compared. Endothelial area of denudation and Descemet's membrane exposure were higher in G1 than G2. In conclusion, atracurium besylate induced an acute damage on corneal endothelium of chickens.(AU)'

Objetivou-se avaliar os efeitos agudos do besilato de atracúrio sobre o endotélio corneano de galinhas. Vinte olhos saudáveis de galinhas foram aleatoriamente separados em dois grupos com 10 olhos cada, sendo G1 o grupo controle e G2 o grupo tratamento. Imediatamente após a excisão dos botões corneoesclerais estes foram colocados em lâminas de microscopia de vidro com o lado endotelial voltado para cima. No Grupo 1, o endotélio corneano foi recoberto com 0,2ml de besilato de atracúrio (10mg/ml) durante 3 minutos e depois lavado com solução salina balanceada. No Grupo 2, o endotélio corneano foi recoberto apenas com solução salina balanceada durante 3 min. As córneas de ambos os grupos foram coradas com vermelho de alizarina e azul de tripano e visualizadas com microscópio óptico. Foram obtidas dez fotografias aleatórias de cada amostra. As imagens foram analisadas e com auxílio de um software as áreas com ausência de células endoteliais calculadas. A perda celular endotelial foi significativamente maior no grupo tratamento comparativamente ao grupo controle. Com base nos resultados apresentados foi possível concluir que o besilato de atracúrio induziu dano agudo nas células do endotélio da córnea de galinhas.(AU)

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)

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)

Morphology of endothelial cells from different regions of the cornea of dogs / Morfologia das células endoteliais de diferentes regiões da córnea de cães

The aim of this study was to evaluate the morphology of endothelial cells from different areas of the cornea of dogs. Twenty healthy eyes from 10 dogs, females or males, of different ages were studied. Corneal endothelium morphology of superior, inferior, central, nasal and temporal areas was assessed by 0.2% alizarin red staining using an optic microscope. One hundred endothelial cells from each corneal area were analyzed. In all areas of the cornea studied were found endothelial cells with four sides, five sides, six sides and seven sides. There was no significant difference regarding endothelial cell morphology in all corneal regions evaluated. Thus, the morphology of the central cornea area represents the entire endothelial mosaic and may be applied to peripheral areas. Therefore, analysis of the central area is sufficient to estimate the shape of endothelial cells of peripheral areas of healthy dog corneas.(AU)

Objetivou-se avaliar a morfologia das células endoteliais de diferentes regiões da córnea de cães. Vinte olhos saudáveis de 10 cães, fêmeas ou machos, de diferentes idades foram estudados. A morfologia do endotélio corneano das regiões superior, inferior, central, nasal e temporal foi avaliada pela coloração vermelho de alizarina 0,2% com microscópio óptico. Foram analisadas 100 células endoteliais de cada região da córnea. Em todas as regiões da córnea estudadas foram encontradas células endoteliais com quatro lados, cinco lados, seis lados e sete lados. Não houve diferença significativa em relação à morfologia de células endoteliais da córnea em todos as regiões estudadas. Assim, a morfologia da região central da córnea representa todo o mosaico endotelial e pode ser aplicada em áreas periféricas. Portanto, a análise da área central é suficiente para estimar a forma das células endoteliais das áreas periféricas de córneas de cães saudáveis.(AU)

Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study / 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 cَrnea de equinos. Vinte e quatro cَrneas de 12 equinos saudلveis, machos ou fêmeas, de diferentes idades foram estudadas. Somente olhos hيgidos foram utilizados. Aleatoriamente, um olho foi incluيdo 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 cَrnea foi corado com azul de tripano e vermelho de alizarina, analisado e fotografado usando microscَpio َptico. As لreas com células endoteliais danificadas foram aferidas e quantificadas utilizando um software para anلlise morfométrica. Os valores encontrados foram expressos como percentual de perda celular. Em todos os olhos examinados foram observadas لreas 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 nمo diferiram. Foi possيvel concluir que a ICG 0,5% nمo causou dano agudo nas células do endotélio da cَrnea de equinos.

Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study / 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.(AU)

O objetivo do estudo foi investigar se indocianina verde (ICG) induz dano nas células do endotélio da cَrnea de equinos. Vinte e quatro cَrneas de 12 equinos saudلveis, machos ou fêmeas, de diferentes idades foram estudadas. Somente olhos hيgidos foram utilizados. Aleatoriamente, um olho foi incluيdo 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 cَrnea foi corado com azul de tripano e vermelho de alizarina, analisado e fotografado usando microscَpio َptico. As لreas com células endoteliais danificadas foram aferidas e quantificadas utilizando um software para anلlise morfométrica. Os valores encontrados foram expressos como percentual de perda celular. Em todos os olhos examinados foram observadas لreas 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 nمo diferiram. Foi possيvel concluir que a ICG 0,5% nمo causou dano agudo nas células do endotélio da cَrnea de equinos.(AU)

Corneal endothelial cell density and morphology in rabbits eyes using contact specular microscopy / Densidade celular e morfologia das células do endotélio da córnea de coelhos obtidas com microscopia especular

The goal of this study was to describe the normal values of corneal endothelial cell density and hexagonality in healthy rabbits eyes using contact specular microscopy. Both eyes of 18 rabbits, males and females, of different ages were evaluated. The eyes were divided into three groups of 12 each according to the age range of the animals: G1 (6 months old), G2 (12 months old), and G3 (48 months old). Parameters studied included mean endothelial cell density and hexagonality. For Group I, the mean cell density was 2307 cells per mm2. For GII, the mean cell density was 1895 cells per mm2. For G III, the mean cell density was 1818 cells per mm2. Cell density decreased significantly with aging (P 0.001) among the three groups. Pleomorphism in the rabbits of GI was 74.33±10.08. In the rabbits of GII it was 71.83±11.38. In the rabbits of GIII it was 64.02±28.80. Significant differences (P 0.001) were evident between GI and GIII and GII and GIII but not between Groups I and II. The results showed a decrease in number of cells and hexagonality with aging. Corneal endothelium of rabbits suffers changes due to advancing age both in terms of cell density and morphology.

Objetivou-se avaliar a densidade celular e a hexagonalidade do endotélio da cَrnea de coelhos saudلveis usando um microscَpio especular de contato. Os dois olhos de 18 coelhos, machos ou fêmeas, de diferentes idades foram avaliados. Os olhos foram separados em três grupos de 12 olhos cada em funçمo da idade: G1 (6 meses de idade), G2 (12 meses de idade) e G3 (48 meses de idade). Os parâmetros estudados incluيram a densidade celular e a hexagonalidade. Para o Grupo I, a densidade celular média foi de 2307 células por mm2. Para GII, a densidade celular média foi de 1895 células por mm2. Para G III, a densidade celular média foi de 1818 células por mm2. A densidade celular diminuiu significativamente com o envelhecimento (P 0,001) entre os três grupos. O pleomorfismo nos coelhos de GI foi de 74,33±10,08. Nos coelhos do GII foi 71,83±11,38. Nos coelhos de GIII foi de 64,02±28,80. Diferenças significativas (P 0,001) foram evidentes entre GI e GIII e GII e GIII, mas nمo entre os Grupos I e II. Os resultados revelaram diminuiçمo da densidade celular e da hexagonalidade com o avanço da idade. O endotélio da cَrnea de coelhos sofre alteraçُes devido ao avanço da idade tanto na densidade celular e quanto na morfologia.

Corneal endothelial cell density and morphology in rabbits eyes using contact specular microscopy / Densidade celular e morfologia das células do endotélio da córnea de coelhos obtidas com microscopia especular

The goal of this study was to describe the normal values of corneal endothelial cell density and hexagonality in healthy rabbits eyes using contact specular microscopy. Both eyes of 18 rabbits, males and females, of different ages were evaluated. The eyes were divided into three groups of 12 each according to the age range of the animals: G1 (6 months old), G2 (12 months old), and G3 (48 months old). Parameters studied included mean endothelial cell density and hexagonality. For Group I, the mean cell density was 2307 cells per mm2. For GII, the mean cell density was 1895 cells per mm2. For G III, the mean cell density was 1818 cells per mm2. Cell density decreased significantly with aging (P 0.001) among the three groups. Pleomorphism in the rabbits of GI was 74.33±10.08. In the rabbits of GII it was 71.83±11.38. In the rabbits of GIII it was 64.02±28.80. Significant differences (P 0.001) were evident between GI and GIII and GII and GIII but not between Groups I and II. The results showed a decrease in number of cells and hexagonality with aging. Corneal endothelium of rabbits suffers changes due to advancing age both in terms of cell density and morphology.(AU)

Objetivou-se avaliar a densidade celular e a hexagonalidade do endotélio da cَrnea de coelhos saudلveis usando um microscَpio especular de contato. Os dois olhos de 18 coelhos, machos ou fêmeas, de diferentes idades foram avaliados. Os olhos foram separados em três grupos de 12 olhos cada em funçمo da idade: G1 (6 meses de idade), G2 (12 meses de idade) e G3 (48 meses de idade). Os parâmetros estudados incluيram a densidade celular e a hexagonalidade. Para o Grupo I, a densidade celular média foi de 2307 células por mm2. Para GII, a densidade celular média foi de 1895 células por mm2. Para G III, a densidade celular média foi de 1818 células por mm2. A densidade celular diminuiu significativamente com o envelhecimento (P 0,001) entre os três grupos. O pleomorfismo nos coelhos de GI foi de 74,33±10,08. Nos coelhos do GII foi 71,83±11,38. Nos coelhos de GIII foi de 64,02±28,80. Diferenças significativas (P 0,001) foram evidentes entre GI e GIII e GII e GIII, mas nمo entre os Grupos I e II. Os resultados revelaram diminuiçمo da densidade celular e da hexagonalidade com o avanço da idade. O endotélio da cَrnea de coelhos sofre alteraçُes devido ao avanço da idade tanto na densidade celular e quanto na morfologia.(AU)