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| Author(s): |
Ana Paula Gomes e Moura
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2024-08-21 |
| Examining board members: |
Raquel Assed Bezerra Segato;
Paulo Sérgio Cerri;
Paulo Nelson Filho;
Thais Marchini de Oliveira Valarelli
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| Advisor: | Léa Assed Bezerra da Silva |
| Abstract | |
Neutrophil Extracellular Traps (NETs) are one of the mechanisms through which neutrophils participate in the host\'s inflammatory immune response, but excessive NET formation can lead to tissue damage. Concurrently, previous studies have demonstrated the positive effects of probiotics in pulp infections. However, the impact of probiotics on NETs remains unknown. Thus, the primary objective of this study was to evaluate the effect of local administration of the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) HN019, used as an irrigant solution, on the expression of NETs and the antimicrobial peptide CRAMP in experimentally induced periapical lesions in rats. After experimental induction of periapical lesions, the animals were divided into three groups and four subgroups according to the irrigant solution and the number of irrigation sessions, as follows: Group I (9 animals): no treatment; Group II: 2.5% sodium hypochlorite (18 animals) - IIa: one irrigation (9 animals) and IIb: two irrigations (9 animals); and Group III: Probiotic (18 animals) - IIIa: one irrigation (9 animals) and IIIb: two irrigations (9 animals). The probiotic B. lactis HN019 was used as an irrigant solution (2.7×109 CFU in an aqueous medium with 2% carboxymethylcellulose) with a volume of 600µL, with concurrent aspiration, along with the addition of 5µL maintained in the root canal. After 21 days of periapical lesion induction, the animals were euthanized, and the right hemimaxillae and hemimandibles were processed for microscopic, genetic, and molecular analyses. Slides were stained with hematoxylin and eosin (HE) and analyzed using light microscopy for descriptive and semi-quantitative evaluation of periodontal ligament parameters, inflammatory infiltrate, cementum resorption, and bone resorption, as well as neutrophil counts and histomorphometry of the periapical lesion area. Additionally, immunohistochemistry for CRAMP (human LL-37 ortholog) and indirect immunofluorescence for NETs detection using neutrophil elastase (NE), citrullinated histone H3 (H3Cit), and myeloperoxidase (MPO) markers were performed. The right hemimaxillae were processed for real-time polymerase chain reaction (qRT-PCR) to assess the mRNA expression of the genes ELANE, H3F3B, MPO, and CAMP. The results were statistically analyzed using the Shapiro-Wilk or KruskalWallis (H) tests, with Dwass-Steel-Critchlow-Fligner post hoc analysis, and a significance level of 5%. The comparison of semi-quantitative scores indicated significant differences between the groups concerning the periodontal ligament and inflammatory infiltrate (p=0.03 and p=0.004, respectively). Regarding bone tissue, 100% resorption was observed in the non-irrigated group (Group I) and the groups irrigated with 2.5% sodium hypochlorite (Group IIa and IIb), while the probioticirrigated groups showed 28.57% and 75% resorption in Groups GIIIa and GIIIb, respectively. Cementum resorption was noted in the non-irrigated group (Group I) and in 100% of cases in Group IIa (one irrigation with 2.5% sodium hypochlorite), while Group IIb (two irrigations with 2.5% sodium hypochlorite) had 71.43% resorption, and the probiotic groups showed 14.29% and 50% resorption in Groups GIIIa and GIIIb, respectively. Neutrophil counts did not differ significantly between groups (p=0.064). In terms of periapical lesion area (mm²), a significant difference was found between groups (p=0.002), indicating that the group receiving one irrigation with probiotic solution had significantly smaller periapical lesions compared to the non-irrigated group. Regarding the percentage of CRAMP-labeled area, a significant difference was observed between groups (p=0.001), demonstrating that one irrigation with probiotic solution and two irrigations with 2.5% sodium hypochlorite were associated with a larger immunolabeled area compared to the nonirrigated group. Additionally, a significant statistical difference was found in NETs area percentage for H3Cit (p=0.002), MPO (p<0.001), and NE (p=0.001). Gene expression of CAMP also showed significant differences between groups (p<0.001), as did MPO and ELANE (p=0.002 and p=0.001, respectively), while no significant difference was observed for H3F3B expression (p=0.307). Based on the results of this study, it can be concluded that the probiotic-based irrigant solution reduced NET formation. Additionally, the probiotic solution stimulated CRAMP expression and had a positive effect on reducing cementum and bone resorption. (AU) | |
| FAPESP's process: | 22/16612-4 - PROBIOTICS-BASED IRRIGATION SOLUTION: MICROSCOPIC AND MOLECULAR EVALUATION OF EXTRACELLULAR NEUTROPHIL TRAPS EXPRESSION IN TEETH WITH PERIAPICAL LESION INDUCED IN RATS |
| Grantee: | Ana Paula Gomes e Moura |
| Support Opportunities: | Scholarships in Brazil - Master |