A novel approach to studying infective endocarditis: Ultrasound-guided wire injury and bacterial challenge in mice

Figures

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  Fig 1. Experimental protocol and bacterial growth in mice after bacterial challenge. (a) Bacterial challenge was performed using 104, 105, and 106 CFU for i.v. injection. Blood and valve samples were collected 24h after bacterial challenge and microbiologically examined. (b-d) Successful IE induction was determined 24h after bacterial challenge in %. CFU concentration in blood and valvular samples was measured 18h after incubation using agar. (e) Mice underwent wire injury (WI) to induce endothelial damage, bacterial challenge was performed either 24h or 72h after WI using 104, 105, and 106 CFU, blood and valve samples were collected 24h and 72h after bacterial challenge. (f-h) Successful IE induction was determined 24h after bacterial challenge in %. CFU concentration in blood and valvular samples was measured 18h after incubation using agar. (i) Immunofluorescence microscopy data for S. aureus. Data in the quantitative plots are presented as mean ± SEM, and statistical significance was determined using unpaired one-way ANOVA. ***P < 0.001; **P < 0.01; * P < 0.05. BC = bacterial challenge.

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  Fig 2. Scanning electron microscopy after bacterial challenge. (a-c) Scanning electron microscopy images (2.00 KV LEI, 14mm WD) of aortic valve leaflet cross-sections of BC animals three days after bacterial challenge. (d-f) Wire injury causes endothelial damage with only few endothelial cells remaining (EC) and enables S. aureus (S.a., violet) infiltration via fibrin layers and activated platelets (AP). BC = bacterial challenge, WI = wire injury.

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  Fig 3. Echocardiographic analysis. (a) Aortic regurgitation was more prevalent in mice after wire injury and bacterial challenge. (b-c) Exemplary images of a healty aortic valve (b) and aortic valve endocarditis (c) vegetations in parasternal long-axis view after wire injury and bacterial challenge. (d) Left ventricular ejection fraction (LVEF), left ventricular volumes and fractional shortening (%) were measured in parasternal long-axis view after wire injury and bacterial challenge. Data is presented as mean ± SEM, and statistical significance was determined using unpaired one-way ANOVA. ***P < 0.001; **P < 0.01; * P < 0.05. BC = bacterial challenge, WI = wire injury.

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  Fig 4. Immunofluorescence staining and cytokine expression levels. (a-c) Immunofluorescence microscopy was performed after mice were sacrificed and the hearts were collected. Representative images and quantitative analysis of CD45 (a), CD68 (b), Ly6G (c) in WI + BC mice. (d-g) Pro-inflammatory cytokines IL-1α, IL-1β, IL-10 and macrophage-colony stimulating-factor (M-CSF) were measured from sera taken immediately before sacrifice 3d after bacterial. Data in the quantitative plots are presented as mean ± SEM, and statistical significance was determined using unpaired one-way ANOVA. ***P < 0.001; **P < 0.01; * P < 0.05. BC = bacterial challenge, WI = wire injury.

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  Fig 5. Enterotoxin and Alpha-Toxin levels 1d and 3d after bacterial challenge. (a) There was no difference in Enterotoxin expression between BC and WI+BC mice and no difference between different time points. (b) Alpha-Toxin was not differently expressed between BC and WI+BC, however Alpha-Toxin was overexpressed in BC animals after 3d compared to 1d. Data in the quantitative plots are presented as mean ± SEM, and statistical significance was determined using unpaired one-way ANOVA. ***P < 0.001; **P < 0.01; * P < 0.05. BC = bacterial challenge, WI = wire injury.