Evaluation of periapical lesions in the absence of adaptor protein for toll-like receptors activation: study in Knockout mice

Abstract

The adaptor protein Myeloid Differentiation primary response gene 88 (MyD88) is involved in the Toll-like receptors (TLRs) activation and they are considered molecules responsible for early recognition of invading pathogens and triggering of immune response. The aim of the present study will be to characterize the development and progression of periapical lesions experimentally induced in the teeth of MyD88 knockout (MyD88 KO) mice compared to wild-type (WT) mice. Periapical lesions will be induced in the lower first molars of 30 WT and 30 MyD88 KO mice. After 7, 21 and 42 of periapical lesion induction, the animals will be euthanized in a CO2 chamber, and the mandibles will be removed and subjected to histotechnical processing. The femurs will also be removed for measurement of bone mineral density by dual-energy x-ray absorptiometry (DXA). Representative histological sections will be stained with hematoxylin and eosin (HE) for description of the features of the pulp tissue and the apical and periapical regions and to count the inflammatory cells number (neutrophils) under conventional optical microscopy, and for determination of the size of the periapical lesions under fluorescence microscopy. Sequential specimens will be evaluated by: TRAP histo-enzymology for identification e counting of osteoclasts; Brown & Brenn staining for localization of bacteria; and immunohistochemistry for identification of osteoclastogenesis markers (RANK, RANKL, OPG). Data from the morphometric evaluation of the size of periapical lesions, the number of osteoclasts, the number of inflammatory cells and bone mineral density will be subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests, using the SAS (Statistical Analysis System) software for Windows version 9.1.3. A significance level of 5% was set for all analyses. (AU)