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Primary culture and characterization of the viability and steroidogenic function of adrenal cortex cells of dogs

Grant number: 18/24250-0
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2019
Effective date (End): December 31, 2019
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Paula de Carvalho Papa Keohane
Grantee:Cíntia Pinto da Silva
Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Disturbances in the secretion of hormones produced by the adrenal cortex lead to the most varied systemic disorders. The most well known of these, hyperadrenocorticism (HAC), affects both humans and a significant portion of dogs. In this context, a deep knowledge of the adrenal physiology and possible interferences in the production of its hormones is necessary. The present project aims to establish a primary cell culture of glomerulosa, fasciculate and reticular cells derived from adrenal dogs. During one week of cultivation, characterize the viability of the cells and evaluate their ability to preserve steroidogenic production. In addition, we aimed to evaluate and compare the effect of the hormones insulin, adrenocorticotrophic hormone (ACTH) and angiotensin II under hormonal production in the cultures. Soon after the collection of the adrenal glands, we proceed with the separation of the layers and respective zones of the adrenal gland. After establishing cell viability, 30x103 cells / well will be grown in a 96-well plate and 70x103 cells / well in a 24-well plate, with half of each plate being cultured in two different media: DMEM and DMEM / F12 medium. The Trypan blue staining technique will be used to evaluate cell viability before plating and every 48 hours for a total period of 124 hours. To evaluate the hormonal effect on steroidogenic adrenal production, the cells will be maintained in culture medium without overnight fetal bovine serum and subjected to the stimulation of 100 pM at 1 ¼M angiotensin II (Sigma-Aldrich, USA), 10 ng / ml at 1 mg / ml insulin (Sigma-Aldrich, USA) and 1nM at 100nM ACTH (Sigma-Aldrich, USA), and evaluated after 1 hour and 6 hours of treatment. The data obtained will be evaluated by the ANOVA parametric test, followed by the Newman-Keuls multiple comparison test, and the non-parametric Kruskal Wallis test will be applied, followed by the Dunn multiple comparisons test. These data will be analyzed through the statistical program GraphPad Prism (Software Inc., San Diego, CA, USA), being considered statistical differences when p <0.05.

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