Propriedades birrefringentes e organização macromolecular dos colágenos fibrilares do estroma corneal de cães
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Data
2019-01-31
Autores
Silveira, Camila Pinho Balthazar da
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Universidade Estadual Paulista (Unesp)
Resumo
Visando compreender como se comporta a organização das fibras colágenas do estroma corneal de cães por meio de propriedades birrefringentes, objetivou–se com a pesquisa mapear a supraorganização das fibras do estroma corneal de cães, de maneira setorizada e comparativa, nos diferentes sítios e fragmentos. Córneas (n=22) foram removidas de 11 cães submetidos a eutanásia por razões não relacionadas à pesquisa. Quatro córneas foram estudadas in totum, após confecção de montagens totais, e 18 foram incluídas em parafina e transversalmente seccionadas para 7 µm. Todo o material foi estudado sob microscópio de luz polarizada (Olympus BX-53 Pol, Tóquio, Japão) munido de luz policromática e monocromática de 546 nm, compensadores de Sénarmont e Brace-Köhler, e sistema para vídeo-análise de imagens. As fibras colágenas foram posicionadas a 45 e 90 graus do plano da luz polarizada (PLP) e avaliadas setorialmente, ou seja, nas regiões anterior, média e profunda dos estromas superior, inferior, central, nasal e temporal das córneas. A supraorganização das fibras colágenas foi estabelecida a partir dos valores da diferença do caminho óptico (OPD) quantificados pelo método de Sénarmont e Brace-Köhler. O tensor de estrutura, foi processado com auxílio do plugin Java “OrientationJ”. Diferenças com P<0,05 foram consideradas significativas. Não foram observadas diferenças em alinhamento das fibras colágenas entre as regiões superior, inferior e central da córnea (P>0,05). As fibras colágenas nos sítios anterior e médio dos fragmentos nasal e temporal das córneas foram menos alinhadas e mais onduladas do que as fibras colágenas dos demais fragmentos corneais (P<0,05). As fibras colágenas dos fragmentos centrais das córneas apresentaram valores altos de OPD, ou seja, foram mais compactadas, se comparadas com as fibras das outras regiões da córnea (P<0,01). Em contraste, as fibras do fragmento inferior foram menos compactadas (valores<OPD) do que nos outros fragmentos (P<0,01). As fibras colágenas estromais da córnea canina apresentam birrefringência de forma (BF) positiva. A contribuição da BF para a Birrefringência total (BT) foi superior a 80%. Relativamente a birrefringência intrínseca (BI), fibras colágenas nos fragmentos corneais central e inferior apresentaram, respectivamente, o menor e o maior valor. Em conclusão, a birrefringência total (forma e intrínseca) em cães é positiva em todos os fragmentos e profundidades da córnea, e a córnea canina apresenta dispersão anômala de birrefringência em sua periferia. Além disso, o padrão de ordenação das fibras colágenas não é constante em toda a córnea, sendo diferente entre os fragmentos estudados.
In order to comprehend the behavior of the organization of the canine cornea stromal collagen fibers by birefringent properties, this research aimed to map the supraorganization of the canine corneal stroma fibers, sectorial and comparatively, in different sites and fragments. Corneas (n =22) were removed from 11 dogs submitted to euthanasia for reasons not related to this research. Four corneas were fully studied, after the total montage confection, and eighteen of those were embedded in Histosec® and transversely sectioned to 7 µm. All material were studied under a polarized-light advanced microscope (Olympus BX-53 Pol, Tokyo, Japan) equipped with polychromatic and 546 nm monochromatic light, Sénarmont and Brace-Köhler compensators, and an image analysis system. The collagen fibers were positioned at 45 e 90 degrees to the polarized light plane (PPL) and evaluated by sector, that is, at the anterior, medium and deep stroma of the superior, inferior, central, nasal and temporal corneal fragments. The supraorganization of the collagen fibers was defined based on values of optical path difference (OPD) quantified by Sénarmont and Brace-Köhler method. The structure tensor, a 2x2 matrix that calculates the coherency coefficient of local pixels to inform about the orientation of the fibers in the X and Y microscopy images planes, was processed with a Java plugin named “OrientationJ”. Differences were considered significant when P<0.05. No differences regarding the alignment of the collagen fibers among superior, inferior and central regions of the cornea (P>0.05) were observed. Collagen fibers in the anterior and middle sites of the nasal and temporal corneal fragments were less aligned and more wavy than the collagen fibers from other corneal fragments (P<0.05). The collagen fibers in the central fragments of the corneas presented high OPD values, that is, they were more compact, when compared to the fibers from other regions of the cornea (P<0.01). In contrast, the fibers in the inferior fragments were less compact (values<OPD) than in the other fragments (P<0,01). The stromal collagen fibers of canine cornea present positive BF. The contribution of BF to BT was greater than 80%. Regarding BI, collagen fibers in the central and inferior corneal fragments presented, respectively, the lowest and the greatest value. In conclusion, total (form and intrinsic) birefringence in dogs is positive in all corneal fragments and depths, and canine cornea presents anomalous dispersion of birefringence in its periphery. Also, the ordering pattern of the collagen fibers is not constant through all the cornea, being different among the fragments studied.
In order to comprehend the behavior of the organization of the canine cornea stromal collagen fibers by birefringent properties, this research aimed to map the supraorganization of the canine corneal stroma fibers, sectorial and comparatively, in different sites and fragments. Corneas (n =22) were removed from 11 dogs submitted to euthanasia for reasons not related to this research. Four corneas were fully studied, after the total montage confection, and eighteen of those were embedded in Histosec® and transversely sectioned to 7 µm. All material were studied under a polarized-light advanced microscope (Olympus BX-53 Pol, Tokyo, Japan) equipped with polychromatic and 546 nm monochromatic light, Sénarmont and Brace-Köhler compensators, and an image analysis system. The collagen fibers were positioned at 45 e 90 degrees to the polarized light plane (PPL) and evaluated by sector, that is, at the anterior, medium and deep stroma of the superior, inferior, central, nasal and temporal corneal fragments. The supraorganization of the collagen fibers was defined based on values of optical path difference (OPD) quantified by Sénarmont and Brace-Köhler method. The structure tensor, a 2x2 matrix that calculates the coherency coefficient of local pixels to inform about the orientation of the fibers in the X and Y microscopy images planes, was processed with a Java plugin named “OrientationJ”. Differences were considered significant when P<0.05. No differences regarding the alignment of the collagen fibers among superior, inferior and central regions of the cornea (P>0.05) were observed. Collagen fibers in the anterior and middle sites of the nasal and temporal corneal fragments were less aligned and more wavy than the collagen fibers from other corneal fragments (P<0.05). The collagen fibers in the central fragments of the corneas presented high OPD values, that is, they were more compact, when compared to the fibers from other regions of the cornea (P<0.01). In contrast, the fibers in the inferior fragments were less compact (values<OPD) than in the other fragments (P<0,01). The stromal collagen fibers of canine cornea present positive BF. The contribution of BF to BT was greater than 80%. Regarding BI, collagen fibers in the central and inferior corneal fragments presented, respectively, the lowest and the greatest value. In conclusion, total (form and intrinsic) birefringence in dogs is positive in all corneal fragments and depths, and canine cornea presents anomalous dispersion of birefringence in its periphery. Also, the ordering pattern of the collagen fibers is not constant through all the cornea, being different among the fragments studied.
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Palavras-chave
Cão, Córnea, Colágeno, Tensor de estrutura, Microscopia de luz polarizada, Dog, Cornea, Collagen, Structure tensor, Polarized light microscopy