Modulation of blood redox status by the progression of induced apical periodontitis in rats

**Background:** Apical periodontitis (AP) is an inflammatory disease caused by microbial infection of the root canal system, leading to periapical bone resorption. Chronic inflammation can generate reactive oxygen species (ROS), causing oxidative stress when antioxidant defenses are overwhelmed. This study hypothesized that AP progression is associated with increasing oxidative damage and decreasing antioxidant response, and aimed to investigate how AP at different phases modulates blood oxidative biochemistry in Wistar rats.

**Methods:** Twenty-four male Wistar rats (90 days old) were randomly divided into three groups: control (sham, n=8), AP induced for 14 days (AP14, n=8), and AP induced for 28 days (AP28, n=8). AP was induced by exposing the pulp of mandibular first molars with a carbide bur and confirming access with a #10 file, leaving the teeth open to the oral environment. Body weight was monitored weekly. After 14 or 28 days, blood samples were collected via cardiac puncture for biochemical analysis: reduced glutathione (GSH) levels (Ellman method), Trolox equivalent antioxidant capacity (TEAC), and thiobarbituric acid reactive substances (TBARS) as a marker of lipid peroxidation. Hemimandibles were analyzed by micro-CT for alveolar bone resorption volume and bone quality parameters (bone volume BV, percent bone volume BV/TV, trabecular thickness Tb.Th, trabecular number Tb.N, trabecular spacing Tb.Sp). Histopathological evaluation was performed on the other hemimandible after demineralization and H&E staining. Statistical analyses used one-way ANOVA with Tukey post hoc test, MANOVA, and repeated measures two-way ANOVA (p<0.05).

**Key Results:** Body weight did not differ significantly among groups (p=0.42). Micro-CT showed that bone destruction volume was significantly higher in AP14 vs control (9.54±0.03 vs 4.93±0.93 mm³, p=0.0089) and in AP28 vs AP14 (13.17±1.10 vs 9.54±0.03, p=0.03) and vs control (13.17±1.10 vs 4.93±0.93, p=0.0002). Bone quality parameters: BV was lower in AP14 and AP28 vs control (10.10±1.17 and 9.95±0.59 vs 14.06±0.93, p<0.05) but not different between AP14 and AP28 (p=0.99). BV/TV was significantly reduced in AP28 vs control (65.10%±3.12% vs 79.3%±4.55%, p=0.03) but not in AP14 vs control (70.7±2.2 vs 79.3±4.55, p=0.24). Tb.Th was lower in AP14 and AP28 vs control (p<0.05), and AP28 had lower Tb.Th than AP14 (0.19±0.01 vs 0.25±0.009, p=0.03). Tb.N was higher in both AP groups vs control (p<0.05) but not different between AP14 and AP28 (3.03±0.05 vs 2.95±0.16, p=0.91). Tb.Sp was significantly increased in AP28 vs control (0.19±0.11 vs 0.11±0.01, p=0.004) and vs AP14 (0.19±0.11 vs 0.13±0.01, p=0.03). Histopathology: control showed no inflammation; AP14 had moderate mononuclear infiltration; AP28 exhibited more severe inflammatory infiltrate with necrosis. Biochemical results: GSH levels were significantly higher in AP14 vs control (6.05±0.35 vs 3.69±0.57 μmol/mL, p=0.0016) and vs AP28 (6.05±0.35 vs 4.50±0.15, p=0.0058), but control vs AP28 not significant (3.69±0.57 vs 4.50±0.15, p=0.31). TEAC was higher in AP14 and AP28 vs control (1.61±0.07 and 1.68±0.03 vs 1.12±0.12 mmol/L, p<0.05) but not different between AP14 and AP28 (p=0.73). TBARS levels were significantly higher in AP28 vs AP14 (76.1±2.44 vs 61.1±2.97 nmol/mL, p=0.0033) and vs control (76.1±2.44 vs 29.0±4.05, p<0.0001), and AP14 vs control (61.1±2.97 vs 29.0±4.05, p<0.0001). MANOVA confirmed significant group effects for bone quality (Pillai's Trace=1.37, p=0.001) and oxidative biochemistry (Pillai's Trace=1.17, p<0.001).

**Clinical Implications:** This study demonstrates that apical periodontitis progression is associated with a systemic redox imbalance, characterized by an initial increase in GSH at 14 days that is not sustained at 28 days, along with a progressive rise in lipid peroxidation (TBARS). The findings suggest that oxidative stress markers may reflect the severity and chronicity of periapical inflammation. Clinically, this raises the possibility that antioxidant therapies could be explored as adjuncts to endodontic treatment to mitigate systemic oxidative damage. However, as an animal study, results cannot be directly extrapolated to humans, and further clinical research is needed to verify these biochemical changes in patients with apical periodontitis before and after treatment.