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Structural and functional study of epididymis of K18-hACE2 transgenic mice infected with SARS-CoV-2

Grant number: 21/05802-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): September 01, 2021
Effective date (End): August 31, 2022
Field of knowledge:Biological Sciences - Morphology - Histology
Principal researcher:Estela Sasso Cerri
Grantee:André Acácio Souza da Silva
Home Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

COVID-19, a disease induced by SARS-CoV-2 virus, is a serious respiratory disease responsible for the pandemic declared by the World Health Organization in 2020. A transgenic mice (K18-hACE2) expressing the human ACE2, an essential protein that mediates viral entry into host cells, was created as a model of study to investigate the impact of COVID-19 in the organism. The epididymal cells produce a specialized luminal fluid responsible for the maintenance of spermatozoa and protection against pathogens. The maintenance of luminal fluid pH is, in part, controlled by clear cells via crosstalk between these cells and basal cells, which in association with angiotensin 2 mediates the translocation of V-ATPase (proton pump) to the apical surface. Epididymis also contains inflammatory cells that trigger immune responses, producing cytokines such as TNF-±, IL-1 e IL-6. Therefore, considering the intense levels of cytokines released during Covid-19, and that men tested for Covid have demonstrated either sex hormonal or testicular changes, we will try to localize the presence of SARS-CoV-2 virus in the epididymis of SARS-CoV-2-infected K18-hACE2 transgenic mice as well as to evaluate the integrity of epididymal tissues, quantity of macrophages and TNF-±, IL-6 and V-ATPase immonexpression in the epididymis of these mice. Twelve K18-hACE2 mice will be divided into two groups (n=6): control group (CG) and 5-day infected group (5DG), whose animals will be inoculated with DMEM and SARS-CoV-2, respectively, by intranasal route. After 5 days, the epididymis will be immersed in fixative solution and processed for parafin or Araldite resin (for transmission electron microscopy - TEM). The paraffin sections will be subjected to immunohistochemistry and immunofluorescence for the quantitative analyses of TNF-±, IL-6, V-ATPase immunoexpression as well as the number of CD163-immunopositive macrophages. The results will be statistically analyzed by Student`s-t test (pd0.05). In the ultra-thin sections, the possible presence of virus in the epididymal tissues will be checked and the ultrastructural integrity of epithelial, muscle and connective cells will be evaluated by TEM. (AU)

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