RESUMO
Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians.
Assuntos
Córnea/ultraestrutura , Ovinos/anatomia & histologia , Fatores Etários , Animais , Argentina , Lâmina Limitante Anterior/ultraestrutura , Córnea/inervação , Substância Própria/citologia , Substância Própria/inervação , Substância Própria/ultraestrutura , Lâmina Limitante Posterior/citologia , Lâmina Limitante Posterior/ultraestrutura , Células Endoteliais/ultraestrutura , Endotélio Corneano/citologia , Endotélio Corneano/ultraestrutura , Epitélio Corneano/ultraestrutura , Feminino , Processamento de Imagem Assistida por Computador , Microscopia Confocal/veterinária , Microscopia Eletrônica de Transmissão/veterináriaRESUMO
The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.
Assuntos
Acanthamoeba , Amebíase , Epitélio Corneano , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/enzimologia , Amebíase/patologia , Animais , Cricetinae , Cães , Epitélio Corneano/metabolismo , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Junções Intercelulares/metabolismo , Junções Intercelulares/parasitologia , Junções Intercelulares/ultraestrutura , Células Madin Darby de Rim Canino , Masculino , MesocricetusRESUMO
The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman s membrane in 3h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested.
Assuntos
Acanthamoeba castellanii/fisiologia , Córnea/parasitologia , Acanthamoeba castellanii/patogenicidade , Acanthamoeba castellanii/ultraestrutura , Técnicas de Cocultura , Lentes de Contato/parasitologia , Córnea/ultraestrutura , Meios de Cultivo Condicionados , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de VarreduraRESUMO
The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [(3)H]thymidine ([(3)H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium.
Assuntos
Proliferação de Células , Túnica Conjuntiva/ultraestrutura , Epitélio Corneano/ultraestrutura , Limbo da Córnea/ultraestrutura , Animais , Autorradiografia , Túnica Conjuntiva/fisiologia , Epitélio Corneano/fisiologia , Imuno-Histoquímica , Limbo da Córnea/fisiologia , Masculino , Microscopia Eletrônica de Varredura , CoelhosRESUMO
Endothelial cell function is essential to maintain corneal transparency, but unfortunately the regenerative capacity of the endothelium is limited. There are only a few reports describing the effect of age on morphologic appearance of corneal endothelial cells of dogs. Studies of normal corneal endothelial cells in humans and dogs have shown a decrease in endothelial cell density (ECD) and an increase in pleomorphism and polymegethism with advancing age. The purpose of this study was to investigate the effect of age on ECD and endothelial cell morphology in dogs. A total of 30 dogs were divided into three groups (10 dogs/group) based on age: group 1 (2-12 months old), group 2 (24-72 months old), and group 3 (84 months or older). Corneas were processed for light and scanning electron microscopy. Results showed only difference in cell density between group 1 and groups 2 and 3, showing an initial decrease in cell density as the animal matured. Whereas there was significantly greater variation in cell size within the dogs in group 3 than there was within the other two groups, suggesting that there was increased polymegethism and pleomorphism with advancing age.
Assuntos
Envelhecimento/fisiologia , Cães/fisiologia , Epitélio Corneano/ultraestrutura , Microscopia Eletrônica de Varredura/veterinária , Fatores Etários , Animais , Contagem de Células/veterinária , Tamanho Celular , Epitélio Corneano/citologia , Epitélio Corneano/patologia , Feminino , Masculino , Microscopia Eletrônica de Varredura/métodosRESUMO
The corneal endothelium is essential for the maintenance of the corneal transparency. The aim of this study was to examine the morphology of the endothelial surface and perform morphometric analysis of the normal corneal endothelial cells of the Magellanic penguin (Spheniscus magellanicus) using scanning electron microscopy. The present work demonstrates that the corneal endothelium of the Magellanic penguin is similar to those described in other vertebrates.
Assuntos
Epitélio Corneano/ultraestrutura , Microscopia Eletrônica de Varredura/veterinária , Spheniscidae , Animais , Epitélio Corneano/citologia , Microscopia Eletrônica de Varredura/métodos , Spheniscidae/anatomia & histologiaRESUMO
OBJETIVO: Observar os efeitos da aplicaçäo tópica de 5-fluorouracil (5-FU) sobre o epitélio corneano íntegro. MÉTODOS: Foram utilizados 10 coelhos albinos (14 olhos), divididos em: grupo controle (GC), 4 olhos nos quais näo se administraram drogas, grupo 1 (G1), 5 olhos que receberam 5-fluorouracil na concentraçäo 1,25 por cento e grupo 2 (G2), 5 olhos que receberam 5-fluorouracil na concentraçäo de 2,5 por cento. A droga foi instilada 4 vezes por dia, durante 7 dias, quando os animais foram sacrificados, os olhos removidos, separando-se a córnea que foi preparada de modo convencional para estudo em microscópico eletrônico de varredura. RESULTADOS: GC: observaram-se células de formato hexagonal, claras, escuras e intermediárias, compondo o epitélio corneano de coelhos. Presença de numerosas microplicas, principalmente nas células claras. Cada célula possui cerca de 1 a 3 criptas. Nos animais do G1, observou-se maior número de células escuras, regiöes com diminuiçäo no número de criptas; alteraçöes da superfície celular, protusäo na regiäo do núcleo e descamaçäo de células epiteliais. Os do G2 tiveram aumento de microprojeçöes na superfície celular, modificaçöes nas junçöes intercelulares até separaçäo de células adjacentes; diminuiçäo do número e variabilidade no tamanho das criptas. As alteraçöes mais freqüentes ocorreram nas células da periferia da córnea. CONCLUSÄO: O 5-fluorouracil teve efeitos deletérios no epitélio íntegro corneano de coelhos. As alteraçöes observadas foram mais importantes nos animais que receberam a droga mais concentrada (G2) e mais freqüentes na periferia da córnea
Assuntos
Animais , Coelhos , Córnea , Epitélio Corneano/ultraestrutura , Fluoruracila , Imunossupressores , Fluoruracila , Imunossupressores , Microscopia Eletrônica de Varredura/métodosRESUMO
OBJETIVO: Padronizar cultivo primário de células epiteliais germinativas do limbo de olhos doadores do Banco de Olhos da Santa Casa de São Paulo. MÉTODOS: Por meio de biópsias de aproximadamente 4 mm² realizadas na região do limbo cirúrgico do anel córneo-escleral remanescente de olhos doadores, foram obtidos explantes, que passaram por métodos de dissociação para obtenção de células epiteliais germinativas do limbo, as quais foram semeadas em placas de cultivo celulare encubadasna estufa a 37°C em atmosfera a 5 por cento de CO2. As placas foram observadas em 21 dias, sendo avaliadas a aderência, a morfologia e a multiplicação celular. RESULTADOS: Observamos que as células no decorrer dos 21 dias multiplicaram-se, passando da forma arredondada inicial ao processo de dissociação, para a forma poligonal como in vivo, além de demonstrarem maior aderência à placa de cultivo. CONCLUSÃO: Neste estudo foi possível demonstrar que o cultivo de células epiteliais límbicas in vitro pode ser realizado.
Assuntos
Células Cultivadas , Córnea , Epitélio Corneano/ultraestrutura , Células Epiteliais , Técnicas In Vitro , Limbo da Córnea/citologia , Adesão Celular , Biópsia , Transplante de Células , MicroscopiaRESUMO
Lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were studied during corneal epithelial growth and differentiation in cell culture. LDH and G-6-PDH activities increased up to 60 and 150-fold, respectively, when corneal epithelial cells constituted a differentiated four to five layered epithelium; these increases showed a similar time-course to the expression of K3 keratin. Immunostaining experiments showed that in growing colonies, LDH staining is stronger in those cells that are K3 positive; in contrast, in confluent four to five layered epithelia LDH and K3 were located in all cell layers, similar to the pattern found in frozen sections from rabbit central cornea. During growth and differentiation, the LDH isoenzyme set from corneal epithelial cells did not change; and it was different from those observed in cultured conjunctival, esophageal and epidermal cells. The augment in LDH activity was due to a 25-fold increase in the LDH-H mRNA and a 12-fold augment in LDH-M mRNA. A computer-assisted search led to identify AP2 and Sp1 binding sites in the LDH and G-6-PDH promoters, suggesting that their expression might share common regulatory mechanisms with the regulation of the differentiation-linked keratins. It is proposed that LDH may be an early marker of corneal epithelial differentiation, and its isozyme pattern could be distinctive from other epithelial cell lineages.
Assuntos
Células Epiteliais/enzimologia , Epitélio Corneano/enzimologia , L-Lactato Desidrogenase/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/ultraestrutura , Epitélio Corneano/ultraestrutura , Imunofluorescência , Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Isoenzimas/metabolismo , Queratinas/metabolismo , L-Lactato Desidrogenase/genética , Masculino , RNA Mensageiro/genética , CoelhosRESUMO
A autora realizou o estudo clínico e ultra-estrutural da Ceratoplastia Lamelar Inversa em coelhos objetivando verificar a validade desta técnica cirúrgica que permite à córnea doadora fornecer dois enxertos lamelares. Dezessete coelhos foram operados e posteriormente quinze foram submetidos ao exame biomicroscópio periódico. Os exames ultra-estruturais foram realizados imediatamente após a cirurgia e na primeira, segunda, quarta, oitava e décima-segunda semanas de pós-operatório. A epitelizaçäo completa do enxerto ocorreu entre o oitavo e o décimo-quarto dia. A partir do quinto dia de pós-operatório todos os enxertos estavam transparentes. O endotélio do doador degenerou no per-operatório. A membrana de Descemet do doador permaneceu íntegra até a décima-segunda semana de pós-operatório e foi recoberta pelo epitélio corneano do receptor. O epitélio era morfologicamente normal. Hemidesmosomas associados a fragmentos de membrana basal foram evidenciados a partir da quarta semana. Em síntese, os resultados desta investigaçäo experimental indicam, juntamente com os dados da literatura, que uma única córnea doadora propicia duas Ceratoplastias Lamelares em coelhos: a Ceratoplastia Lamelar Inversa e a Ceratoplastia Lamelar Convencional.