Participation of macrophages M1 and M2 in the genesis, development and treatment of periodical lesions

Abstract

Macrophages are phagocytic cells known for presenting considerable heterogeneity in morphological terms, functional and markers expression. Depending on the microenvironment in which monocytes, their progenitor cells, are exposed, resulting in the definition of their phenotype in "classically activated" (macrophage M1) or "alternatively activated" (macrophage M2a, M2b and M2c). Macrophages M1 promote an highly microbicide environment and have a role in mediating the pathogens destruction and tumor cells. On the other hand the macrophages M2 plays a central role in the parasitic infestations answers, tissue remodeling, angiogenesis and allergic diseases. Thus, the aim of this study is to evaluate the participation of macrophages (M1 and M2) in the genesis and development of periapical lesions induced in mice's teeth, through histopathological, immunohistochemical and gene expression analysis. In addition, in cell culture, will be assessed the macrophages polarisation, when in contact with different materials used for the periapical lesions treatment. A total of 195 male mice C57BL/6 will be divided into two groups (sound teeth; n=75 animals) and experimental (teeth with periapical lesions induced experimentally; n=120 animals). After periods of 2, 7, 14, 21 and 42 days, control and experimental groups will be euthanized and the specimens submitted to histotechnical processing, for a description of the apical and periapical tissues characteristics and morphological analysis of macrophages in HE stained sections, under conventional microscopy. Will be performed immunostaining for identification of macrophages. Also it will be done the qRT-PCR assay for gene expression analysis (genes Cxcl10, CxCL9, iNos2, Arg1, Ym1, Fizz1 e MRC1) and Luminex® assay for macrophages identification (M1 and M2) for the and identification of different cytokines (GM-CSF, IFN-³, IL-4, IL-13, IL- 10, IL-6, IL-1² e TNF-±). In cell cultures macrophages derived from bone marrow from those animals, will be performed the cell viability assessment, dosage, gene expression analysis through the qRT-PCR (genes Cxcl10, CxCL9, iNos2, Arg1, Ym1, Fizz1 e MRC1) and cytokines quantification by Luminex® (GM-CSF, IFN-³, IL-4, IL-13, IL- 10, IL-6, IL-1² e TNF-±), after exposure to five materials used in the periapical lesions treatment (AH PlusTM, Sealapex XpressTM, EndosequenceTM, BioRootTM, EndomethasoneTM and calcium hydroxide based paste). The data will be analyzed with Graph Pad Prism 5, using the appropriate tests for each data. The significance level will be 5%. (AU)