Apoptosis and participation of macrophages in the establishment of the eruptive pa...
| Grant number: | 16/09264-9 |
| Support type: | Regular Research Grants |
| Duration: | December 01, 2016 - February 28, 2019 |
| Field of knowledge: | Biological Sciences - Morphology - Histology |
| Principal Investigator: | Paulo Sergio Cerri |
| Grantee: | Paulo Sergio Cerri |
| Home Institution: | Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil |
| Assoc. researchers: | Estela Sasso Cerri |
Abstract
The degradation of tissues of the eruptive pathway occurs during tooth eruption, including the lamina propria and bone located between tooth germ and oral mucosa. The eruptive pathway degradation depends on a complex cascade of cellular and molecular events that occur at specific sites and time. It is known that TNF-± and IL-1 are involved in osteoclastogenesis and activation of metalloproteinases (MMPs). Thus, peaks in the expression of these cytokines may occur during tooth eruption. The purpose of this study is to evaluate the effects of Infliximab (TNF-± blocker) and Anakinra (competitive recombinant receptor for IL-1) in the TNF- ± and IL-1 immunoexpression in the lamina propria during tooth eruption in rat molars. Whereas TNF-± and IL-1 are important signaling molecules to tissue remodeling and resorption processes, we intend to evaluate whether Infliximab and Anakinra cause delay in the eruption process. Furthermore, we will evaluate whether this delay may be associated with interference of these cytokines in the osteoclastogenesis and/or in the degradation process of the eruptive pathway.Ninety-six male 6-day-old and 12-day-old rats will be distributed into 4 groups (n=32 rats per group): CG (control group) - the rats will receive vehicle solution (physiological solution); INFG - rats will receive daily injections of 5mg/kg of Infliximab (anti-TNF-± - Remicade - Janssen Biotech.); ANKG - rats will receive daily injections of 5 mg/kg Anakinra (anti-IL-1 - Kineret - SOBI); INF+ANKG - rats will receive daily injections of 5mg/kg infliximab + 5mg/kg Anakinra. The 6-day-old rats will receive the treatment during 3 and 5 days. The rats aged 12 days will be treated during 4 days. After 24 hours of the last injection, the rats will be euthanized. With aid of a stereomicroscope, the upper maxillae will be analyzed and the images will be captured. Fragments of oral mucosa will be frozen at -80 ºC to detection of TNF-± and IL-1 by Western blot. The hemimaxillae containing the first molars will be immersed in fixative solution. For light microscopy (n=60; 5 rats/group/period), the fragments will be fixed in 4% formaldehyde (pH 7.2) for 48 hours. After decalcification, the fragments will be dehydrated and embedded in paraffin. The sections containing the eruptive pathway of the first molar will be stained with hematoxylin and eosin (HE) for morphological analysis. Some sections will be submitted to immunohistochemistry reactions for TNF-a, IL-1, IL-6, RANKL, OPG, MMP-1 and MMP-9 detection. The number of immunolabeled cells (for each immunolabeling) in the lamina propria will be computed. Sections will also be submitted to histochemical reaction for tartrate resistant acid phosphatase (TRAP), used as osteoclast marker, and the number of TRAP-positive osteoclasts in the linear bone surface will be estimated. Picro-sirius red-stained sections will be used to estimate the birefringent collagen content in the lamina propria. Data will be analyzed using a one-way ANOVA with the post-hoc Kruskal-Wallis or Turkey test (significance level of pd0.05). In order to verify whether Infliximab and Anakinra interfere in the bone resorption and in the degradation of the extracellular matrix components of lamina propria, the ultrastructural analysis of osteoclasts and lamina propria cells will be performed; For this purpose, some samples (n=3 rats/group/period) will be fixed in glutaraldehyde/formaldehyde and embedded in Araldite. Semithin sections will be stained with 1% toluidine blue, examined under light microscope and suitable regions (eruptive pathway of the first molar) will be carefully selected for trimming the blocks. Ultrathin sections will be examined using a TECNAI transmission electron microscope. (AU)