Aa EV-soaked gel induced proinflammatory cytokine release in a mouse model of ligature-induced periodontal disease (PD).

(A) Schematic diagram of the in vivo experimental design. Gelatin gels (50 μl) including DiD-labeled Aa EVs (approximately 2.25 × 109 particles in 50 μl of PBS) were administered to mice. Created with BioRender.com. (B-G) Aa EV signals were captured and quantified using IVIS in both dorsal and ventral regions of the brain at 12 h (n = 8 per each group; B-D) and 24 h (n = 7 to 9 per each group; F-G) of EV-soaked gel treatments. (H) 3D rendering of confocal fluorescence image analysis showed increased Aa EV particles (red) in the brains (cerebral cortex) of mice with ligature-induced PD. DAPI signals are shown in blue. Scale bar = 80 μm. (I) Immunostaining of IL-6 (green), TNF-α (red), and DAPI (blue) in the brains (cerebral cortex) is shown. Scale bar = 80 μm. Also see the S3 Fig. IL-6 (J) and TNF-α (K) quantification using ELISA showed their increased level in response to Aa EV intragingival injection. (L) Aa-20050 in the brain was measured using qRT-PCR following intragingival injection of Aa EVs. For qRT-PCR, Cel-miR-39-3p was added (spike-in) for normalization. (J-L) Data are presented as mean ± SD from six independent experiments. One-way ANOVA with Tukey’s post-hoc test was used to compare each test group.