Histopathological analysis of submandibular gland of SARS-CoV-2-infected k18-hACE2 mice

Abstract

In December 2019, the first case of the new coronavirus SARS-CoV-2 was detected in Wuhan, and the virus has spread through the world, causing a fatal disease named COVID-19. Viruses can spread through small droplets of saliva, a common and transient medium for viral transmission. Thus, studies have demonstrated that the salivary glands are important organs involved in infection, replication, and transmission of SARS-CoV-2 due to the presence of angiotensin-converting enzyme 2 (ACE2), as well as the expression of transmembrane serine protease enzyme II (TMPRSS2) in submandibular gland cells of infected patients. A new transgenic mouse k18-hACE2 has been recently performed for the study of COVID-19. These animals express the human ACE2 (hACE2), and the viral spike (S) protein binds to hACE2 receptors in the cells of these transgenic animals, causing the disease. During COVID-19, cytokines such as IL-1, IL-2, IL-6, IL-8, and TNF-± mediate the inflammatory response induced by COVID-19. The increase of these cytokines leads to hypercytokinemia named "cytokines storm", which stimulates the inflammatory reaction and activates matrix metalloproteinases (MMPs), such as MMP-1, which is able to degrade types I and III collagen, normally present in the extracellular matrix of salivary glands. In this study, the immunoexpression of IL-6, TNF-±, and MMP-1 will be evaluated in submandibular glands of SARS-CoV-2-infected k18-hACE2 mice. The impact of the infection on the structural integrity of the glandular tissue as well as the immunoexpression of epidermal growth factor (EGF) will also be evaluated. Ten k18-hACE2 male mice will be divided into two groups: 5 days (5DG) and control (CG). The animals from 5DG will be intranasally inoculated with 5x104 PFU (in 40 µL). The animals from CG will be inoculated with the same volume of DMEM. After 5 days of infection, the salivary glands will be fixed and embedded in paraffin or historesin. In H.E-stained-historesin sections, the volume density of acini, ducts, connective tissue, and blood vessels will be analyzed. Paraffin sections will be submitted to immunofluorescence reactions for detection of SARS-CoV-2 spike protein and hACE2. IL-6, TNF-alpha, and EGF immunoexpression will also be evaluated by immunofluorescence, and immunohistochemistry for detection of MMP-1 will also be performed. The quantitative analyzes of the immunoexpression of each marker (cytokines and proteins) will be performed, and the results will be statistically analyzed by Student t-test (pd0.05). (AU)