The exposure of pulp to the oral environment causes endodontic periapical lesions, allowing bacteria to penetrate the pulp tissue and reach the tooth apex. The interaction between microbial infection and the host pulpal cells often causes an inflammatory reaction, culminating in the pulp tissue necrosis. The bacteria infection and its products spread towards the tooth apex, inducing the recruitment and activation of leukocytes in the periapical region (Graves et al., 2011, Cavalla et al., 2021). Pulp necrosis during tooth root formation can compromise tooth development and function since the root canal walls remain thin and, therefore, fragile and the root length is often reduced. Thus, therapies have been evaluated to allow the thickening of dentine walls, root growth, and apexification.

Root canal infection causes an immunoinflammatory response that leads to the release of several inflammatory chemokines. Among them, interleukin-6 (IL-6) has an important participation in the initiation and progression of inflammatory reaction in the periapical region (Graves et al., 2011, Lin et al., 2015). IL-6 transcription is induced by other cytokines (IL-1, IL-17) and tumour necrosis factor-α (TNF-α), lipopolysaccharide (LPS), and pathogen-associated molecular patterns (PAMPs) exerting multiple functions in various cells (Wolf et al., 2014). IL-6 is secreted by macrophages, B cells, T cells, dendritic cells and non-immune cells including fibroblasts, keratinocytes, and endothelial cells (de Oliveira et al., 2017, da Fonseca et al., 2019). High levels of IL-6 stimulate the immunoglobulin production by plasma cells (Lin et al., 2015), induce Th 17 differentiation (Heink et al., 2017) and inhibit the differentiation of Treg cells (Bettelli et al., 2006). IL-6 also stimulates the release and activation of matrix metalloproteinases (MMPs), which degrade the components of the extracellular matrix, including collagen and amorphous substances (Okada and Murakami, 1998, Cerri et al., 2010, de Oliveira et al., 2017; de Pizzol Júnior et al., 2018; Silva et al., 2022).

In addition to its role as a pro-inflammatory cytokine, IL-6 modulates bone resorption (da Fonseca et al., 2019, Barbosa et al., 2021, de Oliveira et al., 2024) through the synthesis of receptor activator of nuclear factor-B ligand (RANKL), which interacts with its receptor (RANK) on the surface of osteoclast precursors. The RANKL/RANK interaction leads to the fusion of mononuclear precursors and differentiation of osteoclasts promoting bone resorption (Longhini et al., 2014, Florencio-Silva et al., 2015, Yu et al., 2024). In contrast, osteoprotegerin (OPG) is a membrane-bound TNF-related factor that binds to RANKL preventing the RANKL/RANK interaction (Graves et al., 2011, Yu et al., 2024) and, consequently, reducing the number of osteoclasts (de Oliveira et al., 2024, Graves et al., 2011, Šromová et al., 2023). Thus, IL-6 stimulates bone resorption by increasing the RANKL/OPG ratio and, consequently, inducing osteoclast formation (de Oliveira et al., 2024, Šromová et al., 2023).

The orchestrated cascade of cytokines following periapical lesions culminates in structural changes of periodontal tissues in the periapical region. Although apical periodontitis has been widely investigated in adult rodents (rats and mice), there is still no consensus on the induction protocol. The periapical lesion is often induced by dental pulp exposure to the oral environment for 7 (de Oliveira et al., 2017), 15 (Duarte Faria et al., 2024), 21 (Lima et al., 2021) and 30 (Ferraz et al., 2024, França et al., 2017, Shrestha et al., 2024) days. After periapical lesion formation, these root canals are treated for the evaluation of the effectiveness of intracanal medication in the disinfection and periodontal tissue repair (Lima et al., 2021). This induction model has also been used to assess the effect of drugs such as zoledronic acid (França et al., 2017) and amoxicillin-clavulanate (Duarte Faria et al., 2024) on the impact of a high-fat diet in the periapical lesion development (Shrestha et al., 2024) as well as possible correlation between apical periodontitis and rheumatoid arthritis (Damiani et al., 2024), and whether apical periodontitis induces hepatic changes (Ferraz et al., 2024). To promote periapical lesion without excessive tissue damage, it was proposed an injection of LPS from Porphyromonas gingivalis into the root canals after dental pulp extirpation. After infection with LPS, the molars were kept sealed with glass ionomer for 14, 28 and 42 days. A significant increase in TNF-α immunoexpression was detected at 28 and 42 days while IL-6 immunoexpression was increased at 42 days after LPS-induced periapical periodontitis, suggesting, therefore, that high TNF-α levels are associated with the type of inflammatory reaction. Moreover, elevated IL-6 level may exert a control on the size of periapical lesions (Prasetyo et al., 2023).

Few studies have induced periapical lesions in developing teeth (de Oliveira et al., 2017, Duarte et al., 2014, Scarparo et al., 2011) to evaluate the histopathological features of periapical lesions in developing molars, which exhibit roots with thin dentine walls and large apical foramen filled with pulp tissue in continuity with the periodontal ligament in differentiation. Periapical lesions in developing molars of 4-week-old rats were induced by exposing the root canal to the oral environment for 3 weeks to evaluate different disinfection protocols using antibiotic pastes for 3, 6 or 9 weeks. The treatment caused a reduction in the size of periapical lesions and allowed the cementum formation on the outer root surface of 50 % of the samples (Scarparo et al., 2011). The progression of apical periodontitis induced for 7, 14 and 21 days in developing molars was accentuated in mice treated with rosiglitazone, causing large lesions and an increase in the number of osteoclasts (de Oliveira et al., 2017). A marked periapical lesion was also found after root canals exposure to the oral environment for 30 and 90 days in 5-week-old rats (Duarte et al., 2014). In 3-week-old female rats, the apical lesion was induced by insertion of Fusobacterium nucleatum ATCC 25586 in developing roots. The infection of root canals with F. nucleatum caused changes in the bacterial flora in several organs including liver, kidney, gut, and heart (Haraga et al., 2022). Therefore, describing cellular events and structural changes in periodontal tissues is essential for a better understanding of the different stages of periapical lesion formation. Furthermore, the correlation between the pulp exposure time to the oral environment and the size of the periapical lesion is essential to evaluate the effectiveness of different endodontic therapies.

This study aimed to evaluate the histopathological features of periapical lesions induced by pulp exposure to oral environment at different time intervals in molars from young rats. The spatiotemporal pattern of IL-6 immunoexpression according to the exposure time for induction of periapical lesion and with the characterization of the inflammatory reaction was also evaluated. Furthermore, a possible correlation between IL-6 immunoexpression and bone resorption during the establishment of periapical lesions was also investigated.