Apical periodontitis (AP) is an inflammatory disease that arises due to an infectious process within the root canal system of a tooth. This condition results in persistent inflammation and bone resorption in the apical region (Sehirli et al., 2019). The initial inflammatory changes in the pulp are primarily a consequence of microbial infection (Segura-Egea et al., 2015; Tibúrcio-Machado et al., 2021) As the infection progresses, there is an influx of polymorphonuclear leukocytes (PMNs) and monocytes into the pulp, leading to an intensified cellular infiltrate (Stashenko et al., 1998; Cosme-Silva et al., 2020). The interconnection between the root canal system and periradicular tissues through the apical foramen and foramina enables the spread of intracanal infections to these tissues (Sehirli et al., 2019). Consequently, pulp infection leads to chronic inflammation and bone resorption in the apical region, resulting in a periapical lesion (Sehirli et al., 2019; Tibúrcio-Machado et al., 2021). Persistent aggressive stimuli can induce tissue damage in the host (Aksoy et al., 2019; Lopes and Siqueira, 2020), due to the release of inflammatory cytokines that stimulate bone-resorbing cells, such as osteoclasts (Siqueira and Rôças, 2007; Cosme-Silva et al., 2020).

In addition to its local effects, apical periodontitis may contribute to the systemic immune response through a variety of pathways, including changes in interleukin expression (Baeza et al., 2016; Garrido et al., 2019; Haug and Marthinussen, 2019; Sirin et al., 2021), immunoglobulins (Garrido et al., 2019; Sirin et al., 2021), C-reactive protein (Garrido et al., 2019; Sirin et al., 2019), and biochemical factors (Chen et al., 2021; Bakhsh et al., 2022). Consequently, there is evidence suggesting that AP contributes to increased systemic inflammation (Inchingolo et al., 2013; Zhang et al., 2016; Hernández-Ríos et al., 2017; Georgiou et al., 2021; Poornima et al., 2021; Bakhsh et al., 2022; Niazi and Bakhsh, 2022) as chronic infections activate inflammatory mediators that exert systemic effects (Gomes et al., 2013; Niazi and Bakhsh, 2022).

The generation of excessive free radicals (FR) and depletion of antioxidants by pro-inflammatory mediators can lead to several cellular adverse effects (Khansari et al., 2009), triggering or exacerbating the pathogenesis of various diseases FRs are characterized by unpaired electrons centered on oxygen or nitrogen atoms, resulting in the generation of reactive oxygen species (ROS) or nitrogen species (RNS) (Birben et al., 2012; Sies, 2015; Sies et al., 2017). While free radicals play crucial biological roles, an imbalance between their generation and the action of antioxidants, which are the primary defense against pro-oxidant damage, can result in oxidative stress (OS) (Palacios et al., 2015; Rodrigues de Araujo et al., 2018). This oxidative stress can cause lipid peroxidation of membranes and damage enzymes, proteins, carbohydrates, RNA, and DNA (Barreiros et al., 2006; Miricescu et al., 2011).

The association between blood redox and inflammation is complex. Chronic inflammation is characterized by persistent immunological activation and the production of inflammatory cytokines. Inflammatory cells such as macrophages, neutrophils, and eosinophils contribute to inflammation by generating inflammatory cytokines and chemokines (Khansari et al., 2009). Importantly, as part of the immunological response, these immune cells can produce ROS. The increased generation of reactive oxygen species (ROS) at the site of inflammation results in endothelial dysfunction and tissue injury. The vascular endothelium plays a crucial role in the transmigration of macromolecules and inflammatory cells from the bloodstream to the surrounding tissue (Mittal et al., 2014). When the equilibrium between ROS production and antioxidant defense systems is disrupted, excessive ROS can further exacerbate inflammation by activating pro-inflammatory signaling pathways and damaging tissues (Khansari et al., 2009; Mittal et al., 2014).

Animal models have been extensively used in dental research to investigate the apical and periapical repair processes, partly due to their anatomical and physiological similarities with humans. The utilization of these models facilitates the establishment of predictable periradicular lesions after experimental pulp exposure (Stashenko et al., 1992), with rapid outcomes due to their high growth rate (Wang et al., 2010). While ferrets (Verma et al., 2017), dogs (Torabinejad et al., 1995), and monkeys (FABRICIUS et al., 1982) have been reported as suitable animal models (Richert et al., 2022), rats remain the most used (Kakehashi et al., 1965; Kalatzis-Sousa et al., 2017). Although animal models have their limitations in studying systemic parameters and oral infection, they do provide a means of investigating the underlying mechanisms of such diseases and utilizing methodologies that would otherwise be unfeasible in human studies (Cintra et al., 2021). Thus, rats were selected for the present investigation.

Rat experiments have a rich history in the study of apical periodontitis and have played a crucial role in elucidating the complex cellular and molecular events involved in its development and progression. For instance, a study examined the kinetics of macrophages and lymphoid cells during the induction of experimental periapical lesions in rat molars (Kawashima et al., 1996). This quantitative immunohistochemical investigation shed light on the dynamic cellular processes that contribute to the formation and progression of AP. Moreover, animal experiments allow researchers to investigate the interactions between apical lesions and systemic conditions, such as cardiovascular diseases (Brilhante Wolle et al., 2012) and diabetes (Prieto et al., 2017). In addition, rat models have facilitated the exploration of potential therapeutic approaches, including the use of adjuvant substances that can mitigate bone resorption and promote tissue repair (Aksoy et al., 2019; Sehirli et al., 2019).

This study hypothesized that the progression of apical periodontitis is associated with increasing oxidative damage and subsequently decreasing the antioxidant response. Therefore, this study aimed to investigate if apical periodontitis at different phases modulates the oxidative biochemistry response in Wistar rats.

Our findings were the first to demonstrate that blood oxidative biochemistry is modulated by the duration and severity of the periapical injury since the oxidative biochemistry reaction varied over time. This occurred by reducing the antioxidant response mediated by GSH and increasing lipid peroxidation proportionally to bone destruction. Secondly, we demonstrated that, after 14 days of apical periodontitis, rats exhibited significant inflammatory reactions and bone destruction, prompting a systemic biochemical response.

The literature points out different protocols of induction of apical periodontitis in experimental models, such as access and inoculation of bacteria with and without sealing (Yamauchi et al., 2011; Palma et al., 2017) and pulp exposure with or without cavity sealing (Sehirli et al., 2019; Tibúrcio-Machado et al., 2021; Aksoy et al., 2019; Verma et al., 2017; da Silva et al., 2010). These experimental models were performed on different animals such as dogs (Yamauchi et al., 2011; da Silva et al., 2010; Wang et al., 2010), monkeys (MÖLLER et al., 1981; FABRICIOUS et al., 1982; FABRICIUS et al., 1982), ferrets (Verma et al., 2017) and rats (Wang and Stashenko, 1991; Brilhante Wolle et al., 2012; Prieto et al., 2017; Aksoy et al., 2019; Dal-Fabbro et al., 2019; Sehirli et al., 2019; Cosme-Silva et al., 2020; Chen et al., 2021; Tibúrcio-Machado et al., 2021). In this work, we chose to induce apical periodontitis in rats by accessing the pulp and leaving it open to the oral environment. Previous studies have widely described this well-established experimental model (Aksoy et al., 2019; Dal-Fabbro et al., 2019; Sehirli et al., 2019; Cosme-Silva et al., 2020; Tibúrcio-Machado et al., 2021).

The choice of different time points in this study was based on literature reports indicating significant increases in periapical lesion volume between 14 and 21 days, followed by stability until 28 days of pulp exposure (Minhoto et al., 2021). The progression of the lesion can be divided into an active or developing phase from day 0 to day 15, and a chronic phase from days 20–30 (Wang and Stashenko, 1991). The progression of the periapical lesion at two distinct periods results in different reactions. Micro-CT scans showed that the lesions were visible after 14 days and reached their maximum size after 28 days (Wang and Stashenko, 1991), demonstrating a correlation between resorbing activity and lesion expansion (Wang and Stashenko, 1991; Tani‐Ishii et al., 1995). Thus, our study found that by 14 days, there was already a well-established apical lesion with an inflammatory profile and bone damage capable of generating a systemic oxidative response. Micro-CT evaluations revealed that the lesion volume was smaller at 14 days compared to 28 days, with changes in trabecular thickness and number. Histological examination indicated ongoing bone resorption, likely due to the presence of osteoclasts and mononuclear infiltrates. These findings suggest a relationship between the number of osteoclasts and the higher posterior lesion volume (Sun et al., 2021).

Hence, after 28 days, a higher degree of periapical bone resorption and changes in certain indicators of bone quality was observed, consistent with previous studies (Balto et al., 2000; Chen et al., 2019). The remaining bone volume and the bone volume to tissue volume ratio were similar to those at 14 days, although the BV/TV showed a significant decrease compared to the control. Additionally, there was a notable decrease in trabecular thickness after 28 days, potentially leading to increased trabecular spacing. The histopathological examination further confirmed these findings, demonstrating areas of more pronounced inflammatory infiltrate and necrosis, as well as significant loss of interradicular and apical alveolar bone.

Many research studies have focused on investigating biomarkers associated with periapical injury and their relationship with systemic diseases (Inchingolo et al., 2013; Kimak et al., 2015; Gomes et al., 2018; Sirin et al., 2021). Interleukins and tumor necrosis factors have been found to play a role in the pathogenesis of chronic inflammatory diseases like pulpitis and apical periodontitis (Tani‐Ishii et al., 1995). Various biomarkers, such as IL-10, TNF2, TNFB, and bacterial components (LPS), have been identified in infected root canals and periapical lesions, triggering periapical bone resorption (Wang and Stashenko, 1991; Kawashima and Stashenko, 1999). Reactive oxygen species (ROS) have also been linked to bone health, inhibiting osteoblast differentiation and increasing osteoclastogenesis, leading to bone resorption (Wauquier et al., 2009; Hernández-Ríos et al., 2017; Sies et al., 2017; Vengerfeldt et al., 2017). RANKL, expressed by osteoblasts, plays a critical role in osteoclast differentiation and subsequent bone resorption. Oxidative stress disrupts this bone interaction, decreasing osteoblast-mediated bone formation and increasing osteoclast activities and bone resorption (Wauquier et al., 2009). As observed in our study, at 28 days, an increase in bone loss, necrosis, and inflammation, along with a decline in GSH and an increase in lipid peroxidation, indicated a redox imbalance.

In this study, we investigated three important parameters that have considerable applicability in clinical research: GSH, TEAC, and TBARS (Spanidis et al., 2016; Dumitrescu et al., 2017). Glutathione is the most abundant antioxidant in this system and serves as the primary regulator, controlling the inflammatory process (Bains and Bains, 2015). As a result of the periodontal damage associated with the loss of this antioxidant, the inflammatory reaction becomes more severe. We found a rise in reduced glutathione levels in the AP14 group in response to apical periodontitis, which might indicate a significant procedure to the inflammatory process and/or response. However, the AP28 group did not maintain this increase since it did not exhibit a statistically significant difference from the healthy controls. These findings suggest a limited response to the long-term maintenance of high glutathione levels.

The potential of antioxidant capacity as a predictor of oral cavity diseases has been investigated (Öngöz Dede et al., 2016). Plasma antioxidant capacity, measured using the TEAC assay, showed elevated levels after inducing apical periodontitis at 14 and 28 days. Although the measurement of plasma antioxidant capacity lacks specificity, a reduction in plasma antioxidant capacity suggests oxidative stress, while an increase may indicate an early response to inflammation (Brock et al., 2004). The reduction of enzymatic antioxidants such as glutathione reductase occurs concomitantly with the progression of chronic inflammation, thereby restricting the capacity to mend the damage caused by free radicals. Our study revealed a substantial and time-dependent elevation in lipid peroxidation, suggesting a decline in the capacity of the body to counteract oxidative stress and an amplified inflammatory reaction that produces reactive oxygen species. However, TEAC levels were higher and comparable to the AP14 group, implying that antioxidant defense can be modulated through alternative pathways apart from glutathione. Moreover, with the progression of the inflammatory process towards a chronic state, there could be a decrease in the levels of enzymatic antioxidants, resulting in an elevation of lipid peroxidation levels and a decline in the capacity to mend the damage caused by free radicals.

Nevertheless, this investigation presents some limitations. First, because this is an animal study, the results cannot be extrapolated to humans. Thus, there is a need for clinical studies verifying oxidative biochemistry before and after endodontic treatment, and also comparing it to healthy patients. In addition, we checked the systemic biochemistry using only the blood of the rats. Finally, we explored only three of the various parameters to investigate the oxidative response.

Therefore, our findings stimulate new investigations regarding the response of other antioxidant parameters, such as the antioxidant enzymes superoxide dismutase (SOD), responsible for converting the superoxide anion (O₂ ⁻) into hydrogen peroxide (H₂O₂), and catalase (CAT), responsible for removing H₂O₂. Also, it is essential to investigate the modulation of other oxidative stress parameters, such as damage to nucleic acids, proteins, and carbohydrates. Besides, clinically our findings encourage further research into the remission of this condition by endodontic treatment and the duration of this remission. At the same time, our study suggests that using antioxidants as an adjuvant therapy strategy, as is done in chronic periodontitis (Castro et al., 2019), may reduce the biochemical changes caused or possibly interfere with the progression of apical periodontitis.

Our findings indicate that oxidative biochemistry parameters are associated with different stages of periapical lesions. After 14 days, there was a decrease in reduced glutathione levels, which was associated with a mononuclear inflammatory response and an increase in lipid peroxidation, indicating that cellular damage had already occurred. As a result of this mechanism, an osteoclast activation response occurred later in the process, resulting in more significant periapical bone loss, which may be related to a decrease in reduced glutathione and an increase in lipid peroxidation.