Temporal immunohistochemical analysis of macrophage, neutrophil, and osteoclast dynamics during metformin-mediated suppression of experimentally induced apical periodontitis in mice

Apical periodontitis (AP) is a chronic inflammatory disease characterized by immune cell infiltration and progressive periapical bone resorption. Metformin (MET), an oral biguanide antidiabetic drug, possesses anti-inflammatory and anti-bone-resorptive properties. However, the stage at which MET exerts effects during AP progression and the associated inflammatory cell populations remain unclear. We analyzed the temporal changes in inflammatory cell infiltration, osteoclast distribution, and bone resorption following systemic MET administration in an experimentally induced AP mouse model to determine when MET exerts its protective effects and which inflammatory cell responses are associated. Experimental AP was induced by pulp exposure of mandibular first molars in male mice. MET (50 mg/kg) or phosphate-buffered saline (PBS) was administered daily via intraperitoneal injection beginning 1 day before AP induction. Mandibles were harvested on postoperative days (POD) 7, 14, and 21 and analyzed using micro-computed tomography, hematoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry with antibodies against F4/80 (pan-macrophage marker), arginase-1 (M2 macrophage marker), and Ly6G (neutrophil marker). Data were analyzed using ordinary two-way ANOVA followed by Šídák’s post hoc test. Periapical lesion volume increased progressively in both groups. No significant differences were observed between the groups on POD 7 or 14. However, MET-treated mice exhibited significantly smaller lesion volumes on POD 21. Histological analysis demonstrated progressive inflammatory cell infiltration over time in both groups. On POD 21, inflammatory cell infiltration was markedly reduced in the MET-treated group compared with PBS-treated controls. TRAP staining revealed significantly fewer osteoclasts along the alveolar bone surface on POD 14 and 21 in MET-treated mice, whereas osteoclast numbers within periapical lesions were significantly reduced only on POD 21. Immunohistochemical analysis showed significant reductions in F4/80-positive macrophages, arginase-1–positive cells, and Ly6G-positive neutrophils in MET-treated lesions on POD 21. Furthermore, significant time × treatment interactions were observed for lesion size, osteoclast numbers, and F4/80-positive macrophages. Systemic MET administration attenuates AP progression and is associated with reduced macrophage/neutrophil infiltration and osteoclast numbers during the actively expanding phase of lesion development. These findings provide histological evidence supporting the potential of MET as an anti-inflammatory agent in AP.