Análise imunoistoquímica e histomorfométrica do endométrio de ratas em estro permanente tratadas com melatonina, metformina e citrato de clomifeno
Arquivos
Data
2017
Autores
Mattos, Leandro Sabará de 
Orientadores
Simões, Manuel de Jesus
Tipo
Dissertação de mestrado
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Resumo
Objetivo: Avaliar os efeitos da melatonina, metformina e citrato de clomifeno no endométrio de ratas modelo de síndrome dos ovários policísticos (SOP) em estro permanente induzido por iluminação contínua. As variações na histomorfometria do endométrio, sua espessura e porcentagem de fibras colágenas birrefringentes foram analisadas, bem como o número de glândulas e eosinófilos. Determinou-se também a imunoexpressão do Ki67 (marcador de proliferação celular) e VEGFA (fator de crescimento endotelial vascular). Métodos: Setenta e oito ratas foram divididas em 13 grupos. Os grupos foram divididos em 2 experimentos. O Experimento I (prevenção) incluiu ratas na fase de estro fisiológico (GSham), ratas em estro permanente induzido pela iluminação contínua (GCtrl), além de animais que receberam melatonina, metformina ou citrato de clomifeno, de forma isolada e associada, durante um período de 60 dias de exposição a iluminação contínua. Já o experimento II (tratamento) incluiu animais em estro permanente previamente induzidos pela exposição de 60 dias a iluminação contínua que, após a exposição a luz, permaneceram expostos e receberam melatonina, metformina e citrato de clomifeno como forma de tratamento. Os animais do GSham permaneceram com período de luz das 7:00 as 19:00 horas, enquanto que os animais do grupo controle e demais grupos tratados permaneceram sob iluminação contínua (400 Lux), durante um período de 60 dias para o experimento I e um período total de 120 dias para os animais do experimento II. Ao final desse período todos os animais foram anestesiados e tiveram retirados os cornos uterinos que foram fixados em formol a 10% e processados para inclusão em parafina. Três cortes histológicos com 4 μm com distância de 50 μm entre os cortes foram coletados em lâminas e submetidos a coloração pelo H.E e imunoistoquímica (Ki67 e Vegf). Os dados obtidos foram submetidos ao teste t de Student (p<0,05). Resultados: Após os 180 dias entre prevenção e tratamento, o presente estudo mostrou os seguintes resultados histomorfométricos referentes a espessura do endométrio: não houve efeitos tróficos para os grupos experimentais em comparação ao grupo Ctrl. A quantidade de glândulas mostrou resultados tróficos para os grupos MM1 e Metf+CC1 no exp. I e para os grupos Sham e Metf+CC2 no exp. II em relação ao grupo Ctrl. Já para a quantidade de eosinófilos os efeitos tróficos foram observados para os grupos Mel1, MM1 e Mel+CC1 no exp. I e para todos os grupos, exceto para o grupo Sham no exp. II. Quanto ao percentual de colágeno, o grupo Ctrl mostrou maior expressão em relação aos outros grupos no exp. I e mostrou resultados tróficos para os grupos Mel2 e Metf+CC2 para no exp. II. Para resultados imunoistoquímicos a detecção de Ki67 foi significantemente maior nos grupos Ctrl seguido dos grupos Sham, tanto no exp. I como no exp. II. Na detecção do (VEGF), notamos no exp. I que o grupo com maior marcação também foi o controle, seguido dos grupos Sham, e para o exp. II, o grupo de maior marcação foi o Ctrl seguido do grupo Mel2 Conclusão: A melatonina, a metformina e o citrato de clomifeno não promovem efeitos proliferativos e angiogênicos no endométrio de ratas em estro permanente, porém, a melatonina tem efeito trófico mais significativo no estroma endometrial, não interferindo na sua espessura, enquanto que a metformina apresentou efeito trófico mais significante no epitélio glandular do endométrio, portanto, estes efeitos são semelhantes tanto antes quanto após a indução de SOP por exposição à luz contínua.
Objective: To evaluate the effects that polycystic ovarian syndrome (PCOS) can cause in the histomorphometry of the uterus of rats in permanent estrus. The thickness and number of glands, as well as quantify the number of eosinophilic cells, the percentage of birefringent collagen fibers and to evaluate Ki67 (cell proliferation) and VegfA (angiogenic factor) immunoexpression of rat uterus in permanent estrus Treated with melatonin, metformin, and clomiphene citrate. Methods: Seventyeight rats were divided into thirteen groups. The groups were divided into 2 experiments. Experiment I (prevention) was composed of rats in the physiological estrus phase (GSham), continuous light induced permanent estrus rats (GCtrl), as well as animals that received melatonin, metformin or clomiphene citrate in isolation and associated for a Period of 60 days of exposure to continuous illumination, being considered a prevention group. Experiment II (treatment) consisted of permanent estrus animals previously induced by 60 day exposure to continuous lighting, which after exposure to light remained exposed and received melatonin, metformin and clomiphene citrate as a treatment. GSham animals remained in the light period from 7:00 am to 7:00 p.m., while animals from the control group and other treated groups remained under continuous illumination (400 Lux) for a period of 60 days for experiment I and A total of 120 days the animals of experiment II. At the end of this period all animals were anesthetized and had removed the uterine horns that were fixed in 10% formalin and processed for inclusion in paraffin. Three histological sections with 4 μm with a distance of 50 μm between the cuts were collected on slides and submitted to staining by H.E and immunohistochemistry for each animal. Data were submitted to Student's ttest (p <0.05). Results: After 180 days between prevention and treatment, the present study showed histomorphometric results regarding endometrial thickness: there were no trophic results for the groups compared to the Ctrl group. The quantity of glands showed trophic results for groups MM1 and Metf + CC1 in exp. I and for the Sham and Metf + CC2 groups in exp. II in relation to the Ctrl group. For the eosinophils, the results were trophic for the groups Mel1, MM1 and Mel + CC1 in the exp. I and for all groups except for the Sham group in exp. II. As for the percentage of collagen, the Ctrl group showed greater expression in relation to the other groups in exp. I and showed trophic results for the groups Mel2 and Metf + CC2 for exp. II. For immunohistochemical results the detection of Ki67 was significantly higher in the Ctrl groups followed by the Sham groups, both in exp. I as not exp. II. Regarding the marking for the detection of vascular endothelial growth factor (Vegf), we note in exp. I that the group with the highest marking was also the control, followed by the Sham groups, and for the exp. II, the group with the highest marking was Ctrl followed by the Mel2 group Conclusion: Melatonin, metformin, and clomiphene citrate do not promote proliferative and angiogenic effects on the endometrium of rats in permanent estrus. However, melatonin has a more significant trophic effect on the endometrial stroma and does not interfere with its thickness, whereas metformin has a trophic effect more significant in the glandular epithelium of the endometrium, therefore, these effects are similar both before and after the induction of PCOS by exposure to continuous light.
Objective: To evaluate the effects that polycystic ovarian syndrome (PCOS) can cause in the histomorphometry of the uterus of rats in permanent estrus. The thickness and number of glands, as well as quantify the number of eosinophilic cells, the percentage of birefringent collagen fibers and to evaluate Ki67 (cell proliferation) and VegfA (angiogenic factor) immunoexpression of rat uterus in permanent estrus Treated with melatonin, metformin, and clomiphene citrate. Methods: Seventyeight rats were divided into thirteen groups. The groups were divided into 2 experiments. Experiment I (prevention) was composed of rats in the physiological estrus phase (GSham), continuous light induced permanent estrus rats (GCtrl), as well as animals that received melatonin, metformin or clomiphene citrate in isolation and associated for a Period of 60 days of exposure to continuous illumination, being considered a prevention group. Experiment II (treatment) consisted of permanent estrus animals previously induced by 60 day exposure to continuous lighting, which after exposure to light remained exposed and received melatonin, metformin and clomiphene citrate as a treatment. GSham animals remained in the light period from 7:00 am to 7:00 p.m., while animals from the control group and other treated groups remained under continuous illumination (400 Lux) for a period of 60 days for experiment I and A total of 120 days the animals of experiment II. At the end of this period all animals were anesthetized and had removed the uterine horns that were fixed in 10% formalin and processed for inclusion in paraffin. Three histological sections with 4 μm with a distance of 50 μm between the cuts were collected on slides and submitted to staining by H.E and immunohistochemistry for each animal. Data were submitted to Student's ttest (p <0.05). Results: After 180 days between prevention and treatment, the present study showed histomorphometric results regarding endometrial thickness: there were no trophic results for the groups compared to the Ctrl group. The quantity of glands showed trophic results for groups MM1 and Metf + CC1 in exp. I and for the Sham and Metf + CC2 groups in exp. II in relation to the Ctrl group. For the eosinophils, the results were trophic for the groups Mel1, MM1 and Mel + CC1 in the exp. I and for all groups except for the Sham group in exp. II. As for the percentage of collagen, the Ctrl group showed greater expression in relation to the other groups in exp. I and showed trophic results for the groups Mel2 and Metf + CC2 for exp. II. For immunohistochemical results the detection of Ki67 was significantly higher in the Ctrl groups followed by the Sham groups, both in exp. I as not exp. II. Regarding the marking for the detection of vascular endothelial growth factor (Vegf), we note in exp. I that the group with the highest marking was also the control, followed by the Sham groups, and for the exp. II, the group with the highest marking was Ctrl followed by the Mel2 group Conclusion: Melatonin, metformin, and clomiphene citrate do not promote proliferative and angiogenic effects on the endometrium of rats in permanent estrus. However, melatonin has a more significant trophic effect on the endometrial stroma and does not interfere with its thickness, whereas metformin has a trophic effect more significant in the glandular epithelium of the endometrium, therefore, these effects are similar both before and after the induction of PCOS by exposure to continuous light.
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Citação
MATTOS, Leandro Sabará de. Análise imunoistoquímica e histomorfométrica do endométrio de ratas em estro permanente tratadas com melatonina, metformina e citrato de clomifeno. São Paulo, 2017. [119] p. Dissertação (Mestrado em Medicina: ginecologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2017.