Apical periodontitis (AP) originates from the host's immune defense as a response to microorganisms and their products. Several studies have been carried out in order to clarify the etiology of the periodontal disease as well as the several factors that can interfere in the immunoinflammatory responses. The chronic stress (CS) have been related as an additional risk factor to periodontal disease. Once CS leads to alveolar bone loss, it is possible that the AP can also be affected due to the similarities in the biological processes. The aim of this study is to evaluate the AP development in mice with and without a CS protocol. Mice will be maintained in plastic cages for 1-2 weeks before pulp exposure containing animal paper bedding for acclimation. In vivo: wild-type (WT) C57BL/6J mice at 8 weeks of age will be subjected to dental pulp exposure. The access cavity will be left open allowing contamination of root. On the first day of exposure to stress, we will exchange the paper bedding with water to a depth of 1.5 cm and the lighting conditions will be changed from light/dark to constant darkness. Animals will be anesthetized and killed after 28 days of AP induction. Before euthanasia, blood samples will be collected in order to determine the levels of corticosterone to validate de model. The mandibles will be isolated and hemisected. One hemimandible will be fixed in 4% paraformaldehyde and subjected to micro-computed tomography, histology, and will be immunohistochemically stained for macrophages, inducible NO synthase (iNOS), and arginase 1. The other hemimandible will be frozen and stored in -80 °C until total RNA extraction for real-time RT-PCR. In vitro: resident peritoneal macrophages will be isolated from WT mice. The levels of IL-1±, TNF± and IL-6 will be determined by ELISA. For Western Blot, monoclonal Anti-rat/mouse IL-1±, TNf-±, IL-6, and anti-²-actin antibody will be used. After the Shapiro-Wilk normality test, Student's t-test or Mann-Whitney test will be used with p<0.05.
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