The purpose of the statistical analyses was to determine if there were significant variations on the histological structures observed and on the levels of expression of target genes according to presence of experimental periodontal disease in each period. Comparison of the results in each experimental period according to the control group was performed using unpaired

Student’s t-test. Moreover, we also wanted to determine if the area of bone resorption in the lingual surface varied within the experimental periods. One-way Analysis of Variance test (ANOVA) followed by the CHIR-99021 cost Tukey post hoc test was used to evaluate significant differences among experimental periods. Significance level was set to 5%. All calculations were performed using

GraphPad Prism 5 software (GraphPad, Inc., San Diego, CA, USA). There was a significant increase on the number of inflammatory cells and vascular structures already at 7 days post-ligature placement. The overall changes on the composition of the connective tissue, including a decrease ABT-263 supplier on the number of fibroblasts and on the density of collagen is a common finding in periodontal disease. The severity of inflammation was significantly higher in comparison to the control group throughout the 30-day experimental period; but a decrease in inflammatory cell density is observed after 15 days, as well as a trend of increasing number of fibroblasts and extracellular matrix at 15 and 30 days (Fig. 1 and Fig. 2). Cytokine gene expression in the gingival tissues however corroborate these findings, with a maximum increase of mRNA expression for bone-related cytokines RANKL and OPG and pro-inflammatory

cytokines TNF-α and IL-6 at 7 days, followed by a decrease at 15 and 30 days. These results also agree with the finding that anti-inflammatory cytokine IL-10 tended to increase over the 30 day-experimental period (Fig. 4). Expression of SOCS1 and 3 proteins were significantly increased already at 7 days in the disease-induced group, followed by a significant decrease on remaining experimental periods, although their expression remained higher than in the control group (Fig. 5). These results mirror those of the macroscopic analysis of bone resorption and of the stereometry indicating a strong correlation of the inflammatory status and the expression of SOCS (Fig. 1, Fig. 2 and Fig. 3). It is well documented that SOCS is expressed at low levels in healthy periodontal tissues.11 Our results are in accordance with these findings. Interestingly, activation of STAT1 and STAT3 in both total and phosphorylated forms followed the expression of SOCS1 and SOCS3 proteins, respectively. A significant activation of STAT1 and STAT3 was observed already at 7 days in animals with ligature-induced periodontal disease.