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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2019.tde-22032019-113127
Document
Author
Full name
Matheus Garcia de Fragas
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2018
Supervisor
Committee
Michelini, Lisete Compagno (President)
Campos Junior, Ruy Ribeiro de
Canteras, Newton Sabino
Silva, Bruno Moreira
Title in Portuguese
Efeitos da hipertensão e do treinamento aeróbio sobre a expressão/atividade de diferentes componentes da barreira hematoencefálica.
Keywords in Portuguese
ratos espontaneamente hipertensos; barreira hematoencefálica; treinamento aeróbio; hipotálamo;
Abstract in Portuguese
A hipertensão arterial cursa com disfunção autonômica e lesão da barreira hematoencefálica (BHE) em áreas de controle autonômico. Demonstramos recentemente que o treinamento aeróbio corrige a lesão da BHE, e a disfunção autonômica, a qual se encontra correlacionada com a integridade da BHE observada nos hipertensos treinados. O objetivo deste trabalho é avaliar a expressão gênica e proteica de componentes da BHE envolvidos na mediação das respostas cardiovasculares à hipertensão e ao treinamento aeróbio (T). Ratos espontaneamente hipertensos (SHR) e seus controles normotensos (WKY) (250-300g) foram submetidos ao protocolo de T em esteira ou mantido sedentários (S) por 4 semanas. Ao final do T os animais dos grupos experimentais foram canulados para aquisição das variáveis hemodinâmicas. A seguir procedeu-se à infusão intra-arterial de 2 corantes (Rodamina-d, 70 KD, e FITC-d,10 KD) e 20 min após os encéfalos foram coletados para realização de ensaios de fluorescência no Núcleo Paraventricular do Hipotálamo (PVN). Outros ratos dos grupos experimentais foram perfundidos com salina via transcardíaca e realizada a microdissecção do PVN. O mRNA foi extraído e sua concentração de foi analisada pela técnica de RT-PCR. Para investigar os efeitos da hipertensão e do T nos componentes da BHE, foram utilizados os seguintes primers: Occludina, Claudina-5, Zônula Ocludens 1 (proteínas da junção oclusiva), Caveolina-1 (indicador de transporte transcelular), Laminina alfa 1 e Colágeno 4 (componentes da membrana basal), PDGFRβ (marcador de pericitos)e Aquaporina-4 (indicador de podócitos de astrócitos), todos eles normalizados para o HPRT endógeno. Os dados de PCR em tempo real foram quantificados pelo método 2Δ ΔCT. Além disso, outros ratos dos mesmos grupos experimentais foram perfundidos com paraformaldeído 4% para a fixação do encéfalo. O tecido foi crioprotegido e seccionado em criostato, 30 um, os cortes foram incubados em anticorpos primários (Reca-1(marcador endotelial), Claudina-5, Caveolina-1, PDGFRβ e Aquaporina-4) e secundários (Alexa Flúor 488 e 594), e sua quantificação no PVN foi realizada através da densidade integrada. Não foi observada diferença na pressão arterial entre os grupos T e S, porém, houve bradicardia de repouso nos animais T (SHR-T:317±3 e WKY-T:308±2) comparados com os animais S (SHR-S: 344±4 e WKY-S: 323±3). A permeabilidade da BHE foi reduzida e normalizada pelo T nos animais hipertensos (SHR-S: 13,6±1,2% e SHR-T: 3,8±0,4%; WKY-S: 3,9±0,2% e WKY-T: 4,1±0,16%), e análise do RT-PCR não mostrou nenhuma diferença para Claudina, PDGFRβ e Aquaporina-4 entre os T e S. A expressão gênica de Caveolina-1 estava aumentada nos SHR comparado aos WKY, e o T foi capaz de reduzir sua expressão (SHR-T: 1,05±0,1). O que foi confirmado pela expressão proteica no PVN: a Caveolina-1 encontrava-se aumentada significativamente nos SHR-S em relação aos WKY, e o T reduziu sua expressão no PVN dos SHR. Conclusão: Nossos dados sugerem que o aumento da permeabilidade da BHE no PVN de hipertensos é devida ao aumento de transcitose, identificada pela expressão de Caveolina-1 e que o treinamento aeróbio reverte esta permeabilidade ao reduzir o transporte transcelular sem alterar o transporte paracelular.
Title in English
Effects of hypertension and aerobic training on the expression / activity of different components of the blood-brain barrier.
Keywords in English
spontaneously hypertensive rats; blood-brain barrier aerobic training; hypothalamus;
Abstract in English
The arterial hypertension courses with autonomic dysfunction and Blood Brain Barrier (BBB) damage in areas of autonomic control. We recently demonstrated that aerobic training corrects the damage to the BBB, and autonomic dysfunction, which is correlated with the integrity of the BBB observed in trained hypertense subjects. The objective of this work is to evaluate the gene and protein expression of BBB components involved in mediating cardiovascular responses to hypertension and aerobic training (T). Spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) (250-300g) were submitted to treadmill protocol or maintained sedentary (S) for 4 weeks. At the end of the T, the animals of the experimental groups were cannulated to acquire the hemodynamic variables. Intra-arterial infusion of two dyes (Rhodamine-d, 70 KD, and FITC-d, 10 KD) and 20 min after brains were collected for fluorescence assays in the Paraventricular Nucleus of hypothalamus (PVN). Other rats from the experimental groups were perfused with transcardiacally with saline and the PVN was microdissected. The mRNA was extracted and its concentration was analyzed by the RT-PCR technique. To investigate the effects of hypertension and T in the BBB components, the following primers were used: Occludin, Claudin-5, Zonula Ocludens 1 (tight junction proteins), Caveolina-1 (indicator of transcellular transport), Laminin α 1 and Collagen-4 (basement membrane components), PDGFRβ (pericyte marker) and Aquaporin-4 (astrocyte podocyte indicator), all standardized for endogenous HPRT. Real-time PCR data were quantified by the method 2ΔΔCT. In addition, other rats from the same experimental groups were perfused with 4% paraformaldehyde for fixation of the brain. The tissue was cryoprotected and cross-sectioned, 30 m, sections were incubated on primary (Reca-1 (endothelial marker), Claudin-5, Caveolin-1, PDGFRβ and Aquaporin-4) and secondary antibodies (Alexa Fluor 488 and 594), and its quantification in thePVN was performed through the integrated density. There was no difference in blood pressure between the T and S groups, but there was resting bradycardia in the T animals (SHR-T: 317 ± 3 and WKY-T: 308 ± 2) compared to S controls (SHR-S: 344 ± 4 and WKY-S: 323 ± 3). The permeability of BBB was reduced and normalized by T in hypertensive animals (SHR-S: 13.6 ± 1.2% and SHR-T: 3.8 ± 0.4%; WKY-S: 3.9 ± 0, 2% and WKY-T: 4.1 ± 0.16%), and RT-PCR analysis showed no difference for Claudin-5, PDGFRβ and Aquaporin-4 between T and S. Caveolin-1 gene expression was increased in SHR compared to WKY, and T was able to reduce its expression (SHR-T: 1.05 ± 0.1). This was confirmed by protein expression in PVN: Caveolin-1 was significantly increased in SHR-S relative to WKY, and T reduced its expression in the PVN of SHR. Conclusion: Our data suggest that increased permeability of BBB in the PVN of hypertense individuals is due to the increase transcytosis as identified by Caveolin-1 expression and that aerobic training reverses this permeability by reducing transcellular transport without altering the paracellular transport.
 
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Release Date
2021-03-21
Publishing Date
2019-05-08
 
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