Bacterial Culture and Infection Model

Pg ATCC 33277 was cultured in trypticase soy broth (BD Biosciences) containing 1% yeast extract, 5 µg/mL hemin, and 1 µg/mL menadione and grown at 37°C in an anaerobic atmosphere of 10% H2, 5% CO2, and 85% N2 (22). Pg infection was carried out as previously described (23) with some modifications ( Figure 1A ). Briefly, age- and sex-matched WT and App KI mice (6 weeks of age) were randomly assigned to control or Pg-infected groups. Mice were provided drinking water containing kanamycin (1 mg/ml) for 7 days to reduce the indigenous oral microflora. After removing antibiotics for 3 days, experimental mice were given 100 µl of freshly harvested Pg (10¹⁰ CFU/ml) in 2% carboxymethylcellulose (CMC) and control groups received 100 µl of 2% CMC by oral gavage 3 times per week for 6 weeks. Mice were refrained from food and water intake for 1 hour (h) after infection.

Pg-induced alveolar bone loss and periodontal inflammation in App KI and WT mice following oral infection. (A) Schematic of the experimental design used in this study. m, month. (B) Representative methylene blue-stained maxillae from non-infected (NC) and Pg-infected WT and App KI mice (n=9 mice/group). Bone loss was assessed in a total of 7 buccal sites (red arrows) per mouse. (C) Alveolar bone loss in Pg-infected and non-infected WT and App KI mice; mm, millimeter; CEJ-ABC, cemento-enamel junction-alveolar bone crest. (D) Inflammatory cytokine and complement gene expression in gingival tissues from non-infected and Pg-infected WT and App KI mice. Gene expression was normalized to GAPDH and expressed as fold changes. Samples were done in duplicate (n=7 mice/group). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way ANOVA followed with Tukey correction.