RESUMO
PURPOSE: To quantify endothelial cell loss (ECL) caused by orientation stamps on prestripped and preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts, and to examine a method for reducing ECL using a smaller stamp. METHODS: Ten prestripped and 10 preloaded DMEK grafts were prepared with S-stamps. Ten additional preloaded DMEK grafts were prepared with both an S-stamp and a smaller F-stamp in different paracentral areas of the graft. The footprint of each stamp was measured using ink on cardstock. DMEK grafts were stored in viewing chambers filled with 20 mL of Optisol-GS for 3 days at 4°C. ECL was quantified using Calcein-AM staining and FIJI Weka Segmentation. RESULTS: S-stamps on prestripped DMEK grafts contributed an average ECL of 1.1% ± 0.5% (range: 0.6%-2.2%) toward total graft damage, whereas S-stamps on preloaded DMEK grafts contributed approximately twice that amount (average ECL: 2.0% ± 0.7%, range: 1.3%-3.1%, P = 0.004). Overall ECL for prestripped grafts (average: 7.1% ± 3.3%, range: 3.3%-13.7%) and preloaded grafts (average: 11.3% ± 4.2%, range: 6.9%-19.4%) was similar to previous reports. The footprint of the S-stamp was approximately 45% larger than that of the F-stamp. In 10 preloaded grafts marked with both stamps, the S-stamp caused an average ECL of 1.9% ± 0.6% (range: 1.2%-3.2%), whereas the smaller F-stamp caused an average ECL of 1.0% ± 0.2% (range: 0.8%-1.4%, P = 0.0002). CONCLUSIONS: Loss of endothelial cells associated with graft-stamping was greater in preloaded tissue than in prestripped tissue and was less with a smaller F-stamp than with a larger S-stamp. Using a smaller stamp could help minimize ECL in prestripped and preloaded DMEK grafts.
Assuntos
Perda de Células Endoteliais da Córnea/prevenção & controle , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Bancos de Olhos/métodos , Coleta de Tecidos e Órgãos/métodos , Idoso , Sobrevivência Celular , Perda de Células Endoteliais da Córnea/patologia , Lâmina Limitante Posterior/citologia , Lâmina Limitante Posterior/cirurgia , Endotélio Corneano/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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
PURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemet's membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS: The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06%, the Group two showed an index of 3.58% and the control group an index of 0.18% of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 microm thickness of stroma remained attached to the Descemet's membrane. CONCLUSION: This technique should be better investigated because it is a viable and efficient alternative of Descemet's membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06%. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58%.
Assuntos
Transplante de Células/métodos , Lâmina Limitante Posterior/cirurgia , Endotélio Corneano/citologia , Endotélio Corneano/transplante , Animais , Transplante de Córnea , Lâmina Limitante Posterior/citologia , Lâmina Limitante Posterior/lesões , Masculino , Estudos Prospectivos , Coelhos , Reprodutibilidade dos TestesRESUMO
OBJETIVO: Avaliar a porcentagem de dano endotelial induzido por uma técnica cirúrgica para a separação da membrana de Descemet contendo endotélio sadio, analisar a viabilidade e eficácia desta técnica, e avaliar a porcentagem de dano endotelial causado pela inversão da córnea em câmara anterior artificial. MÉTODOS: As córneas de três grupos de 12 coelhos da linhagem Nova Zelândia foram avaliadas. O grupo 1 foi usado como controle; portanto, as córneas foram analisadas após coletadas e trepanadas. O grupo 2 foi analisado após a inversão da córnea (endotélio para cima na posição convexa), montada em câmara anterior artificial, para o cálculo da porcentagem do dano endotelial induzido por esta inversão. O grupo 3 foi avaliado após a separação entre a membrana de Descemet e o estroma com o uso de substância viscoelástica em córneas invertidas e montadas em câmara anterior artificial. O dano endotelial foi avaliado por meio de fotografias digitais tiradas no microscópio após impregnar o endotélio com vermelho de alizarina. Amostras do grupo 3 foram processadas para avaliação histopatológica. RESULTADOS: O grupo 3 (separação viscoelástica) apresentou um índice de lesão celular endotelial de 10,06 por cento, o grupo 2 apresentou um índice de 3,58 por cento e o grupo controle um índice de 0,18 por cento de lesão celular endotelial (p<0,05). A avaliação histológica das córneas do grupo 3 revelou que aproximadamente 120 µm de espessura estromal manteve-se aderido à membrana de Descemet. CONCLUSÃO: Esta técnica deve ser melhor investigada, pois é uma alternativa viável e eficaz de separação da membrana de Descemet para transplante celular endotelial, já que o porcentual de dano celular induzido é de 10,06 por cento. A porcentagem de dano celular causado pela inversão da córnea em câmara anterior artificial foi de 3,58 por cento.
PURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemet's membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS: The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06 percent, the Group two showed an index of 3.58 percent and the control group an index of 0.18 percent of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 µm thickness of stroma remained attached to the Descemet's membrane. CONCLUSION: This technique should be better investigated because it is a viable and efficient alternative of Descemet's membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06 percent. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58 percent.