Efeitos do Fucoidam de Fucus vesiculosus sobre a morfologia, a ploidia e o metabolismo do endotélio corneal de coelhos
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Data
2019-01-10
Autores
Silva, Germana Alegro da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A córnea atua como principal elemento refrativo do olho, para tal tem que manter-se transparente. O endotélio corneal é um peça chave para essa manutenção, possuindo mecanismos como a bomba de Na+ e K+ que ajudam a garantir a desturgescência corneal. Descompensações endoteliais constituem uma importante indicação de transplante da córnea, uma vez que as células endoteliais corneais apresentam limitada capacidade proliferativa. Entretanto, transplantes são procedimentos cirúrgicos complicados e de elevado custo. Alternativas e coadjuvantes medicamentosos vêm sendo estudadas. O fucoidam é uma substância que, dentre outras propriedades, promove migração e proliferação celular, portanto objetivou-se, com a presente pesquisa, avaliar seus efeitos sobre a morfologia, a ploidia e o metabolismo de células endoteliais da córnea de coelhos, após criolesão experimental. Para tal, foram realizadas avaliações quanto à morfologia e viabilidade celular empregando-se dupla marcação com corantes vitais (alizarina vermelha e azul de tripan) e ultraestrutural pela microscopia eletrônica de transmissão e de varredura. Outrossim, avaliou-se a morfometria in vivo e in vitro de seguintes parâmetros: número de células, densidade, hexagonalidade e coeficiente de variação celulares. Estudou-se o metabolismo dessas células empregando-se a imunofluorescência com observação em confocal dos anticorpos Ki-67, Na+K+/ATPase, n-Caderina e Zo-1, conjugados com Alexafluor-488. Visando-se a conseguirem conhecimentos adicionais relativos à atividade nuclear durante o processo de cicatrização do endotélio corneal na presença de fucoidam foram analisados, a partir da reação de feulgen, perímetro e área nucleares, além da supraorganização da cromatina. A criolesão ensejou piora de todos parâmetros morfométricos, com diminuição das células, densidade celular, do percentagem de células padrão com 6 lados e aumento no coeficiente de variação, a retomada aos valores normais com o decorrer do tempo foi melhor em olhos tratados com fucoidam. Nas córneas que não receberam fucoidam observou-se células desvitalizadas em todo decorer do tempo de avaliação. Houve diminuição da expressão das proteínas de junção celular (n-Caderina e Zo-1) logo após a lesão e aumento progressivo no decorrer do tempo, em contrapartida o contrário ocorreu com Ki-67, revelando aumento na proliferação celular após lesão e baixa das expressão das demais proteínas, com notável estimulação na presença do fucoidam. As características do comportamento nuclear, como descompactação da cromatina na presença do fucoidam puderam ratificar essa proliferação. O fucoidam atuou na modulação à reparação cicatricial, revelando cicatrizes menos opacas, estimulando a proliferação celular e mediando a expressão das proteínas de ligação e atuando na proteção contra morte celular.
The cornea as the main refractive element of the eye, which has to remain transparent. The corneal endothelium is a key piece for this maintenance, having mechanisms such as the Na + and K + pump that help to ensure corneal dysurgescence. Endothelial decompensations are an important indication for corneal transplantation, since the corneal endothelial cells have limited proliferative capacity. However, transplants are complicated and expensive surgical procedures. Alternatives and drug coadjuvants have been studied. Fucoidam is a substance that, among other properties, promotes cell migration and proliferation, therefore, the objective of this research was to evaluate its effects on the morphology, ploidy and metabolism of rabbit cornea endothelial cells after experimental cryoinjury. For this, evaluations were made for morphology and cell viability using double labeling with vital dye (alizarin red and tripan blue) and ultrastructural analysis by transmission and scanning electron microscopy. Morphometry (in vivo and in vitro) of the following parameters were also evaluated: cell number, cell density, hexagonality and coefficient of variation. The metabolism of these cells was studied using immunofluorescence with confocal observation of the Ki-67, Na + K + / ATPase, n-Caderin and Zo-1 antibodies, conjugated to Alexafluor-488. In order to obtain additional knowledge regarding the nuclear activity during the healing process of the corneal endothelium in the presence of fucoidam were analyzed, from the feulgen reaction, nuclear perimeter and area, in addition to the chromatin supraorganization. Cryoinjury resulted in worsening of all morphometric parameters, with cell decrease, cell density, the percentage of standard cells with 6 sides and increase in coefficient of variation, the recovery to normal values with time was better in eyes treated with fucoidam. In the corneas that did not receive fucoidam devitalised cells were observed throughout the evaluation time. There was a decrease in the expression of the cellular junction proteins (n-Caderin and Zo-1) shortly after injury and progressive increase over time, whereas the opposite occurred with Ki-67, revealing increased cell proliferation after injury and low expression of the other proteins, with remarkable stimulation in the presence of fucoidam. The characteristics of nuclear behavior, such as the decomposition of chromatin in the presence of fucoidam, could confirm this proliferation. Fucoidam has been involved in modulation of scar repair, revealing less opaque scars, stimulating cell proliferation and mediating the expression of binding proteins, and acting to protect against cell death.
The cornea as the main refractive element of the eye, which has to remain transparent. The corneal endothelium is a key piece for this maintenance, having mechanisms such as the Na + and K + pump that help to ensure corneal dysurgescence. Endothelial decompensations are an important indication for corneal transplantation, since the corneal endothelial cells have limited proliferative capacity. However, transplants are complicated and expensive surgical procedures. Alternatives and drug coadjuvants have been studied. Fucoidam is a substance that, among other properties, promotes cell migration and proliferation, therefore, the objective of this research was to evaluate its effects on the morphology, ploidy and metabolism of rabbit cornea endothelial cells after experimental cryoinjury. For this, evaluations were made for morphology and cell viability using double labeling with vital dye (alizarin red and tripan blue) and ultrastructural analysis by transmission and scanning electron microscopy. Morphometry (in vivo and in vitro) of the following parameters were also evaluated: cell number, cell density, hexagonality and coefficient of variation. The metabolism of these cells was studied using immunofluorescence with confocal observation of the Ki-67, Na + K + / ATPase, n-Caderin and Zo-1 antibodies, conjugated to Alexafluor-488. In order to obtain additional knowledge regarding the nuclear activity during the healing process of the corneal endothelium in the presence of fucoidam were analyzed, from the feulgen reaction, nuclear perimeter and area, in addition to the chromatin supraorganization. Cryoinjury resulted in worsening of all morphometric parameters, with cell decrease, cell density, the percentage of standard cells with 6 sides and increase in coefficient of variation, the recovery to normal values with time was better in eyes treated with fucoidam. In the corneas that did not receive fucoidam devitalised cells were observed throughout the evaluation time. There was a decrease in the expression of the cellular junction proteins (n-Caderin and Zo-1) shortly after injury and progressive increase over time, whereas the opposite occurred with Ki-67, revealing increased cell proliferation after injury and low expression of the other proteins, with remarkable stimulation in the presence of fucoidam. The characteristics of nuclear behavior, such as the decomposition of chromatin in the presence of fucoidam, could confirm this proliferation. Fucoidam has been involved in modulation of scar repair, revealing less opaque scars, stimulating cell proliferation and mediating the expression of binding proteins, and acting to protect against cell death.
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Palavras-chave
criolesão endotelial, polissacarídeo sulfatado, reparação cicatricial, cromatina nuclear, proliferação celular, cryoinjury, sulphate polysaccharide, wound healing, nuclear chromatin, cell proliferation