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

Abstract

Background: Infective endocarditis (IE) is frequently caused by Staphylococcus aureus (S. aureus) and most commonly affects the aortic valve. Early diagnosis and treatment initiation are challenging because the involved immunological processes are poorly understood due to a lack of suitable in vivo models.

Objectives: To establish a novel reproducible murine IE model, based on ultrasound-guided wire injury (WI) induced endothelial damage.

Methods: IE was established by inducing endothelial damage via ultrasound-guided wire injury followed by bacterial challenge with S. aureus using 104-6 colony-forming units (CFU) 24h to 72h after wire injury. Cross-sections of valvular leaflets were prepared for scanning electron microscopy (SEM) and immunofluorescence microscopy to visualize valvular invasion of macrophages, neutrophils, and S. aureus. Bacterial cultivation was carried out from blood and valve samples. Systemic immune response was assessed using flow cytometry.

Results: Wire injury induced endothelial damage was observed in all mice after wire-injury in SEM imaging. We reliably induced IE using 105 (85%) and 106 (91%) CFU S. aureus after wire injury. Aortic regurgitation was more prevalent in wire injury mice after bacterial challenge. Mice undergoing bacterial challenge responded with significant neutrophilia and elevated pro-inflammatory cytokines in the blood. Immunofluorescence staining revealed significantly increased immune cell accumulations using our proposed model compared to controls.

Conclusion: Echocardiography and ex vivo histological staining demonstrated consistent infective endocarditis induction in our new model, combining a wire injury-induced endothelial damage and S. aureus administration. Further exploration of the initial immune cell response and biomarker expression could potentially identify indicators for early IE diagnosis and novel treatment targets.

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.

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.

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.

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.

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.