1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2Universitätsklinikum Bonn Klinik für Anästhesiologie Bonn, Deutschland; 3Universität Bonn Anatomisches Institut Bonn, Deutschland
Background:
With mortality rates ranging from 15 to 40% under optimal medical treatment, infectious endocarditis (IE) remains one of the deadliest infectious diseases worldwide, with Staphylococcus aureus being the most common pathogen and the aortic valve being the most affected by IE. In the early stages of IE, diagnosis and treatment initiation are often difficult and research is limited due to unreliable models. Most murine IE models rely on permanent prosthetic wire placement and i.v. bacterial injection without considering valvular pathologies as the key predisposing factor, thus making it inadequate to study IE pathophysiology.
Aim:
We established a novel reproducible murine IE model, based on aortic valve stenosis preceding i.v. S. aureus bacterial challenge.
Methods:
Based on a wire-injury model we induced aortic valve stenosis (AVS). 24h to 72h after wire injury, we performed i.v. bacterial challenge using S. aureus. Echocardiography was performed before wire injury and after bacterial challenge to confirm bacterial vegetations on the valve leaflets. Cross sections of valvular leaflets were prepared for transmission electron microscopy (TEM). Macrophage, neutrophil and S. aureus-specific immunofluorescence staining was performed and visualized by immunofluorescence microscopy 1d, 3d and 7d after bacterial challenge. Bacterial cultivation was performed from peripheral blood and valve tissue. Systemic immune response was analyzed using flow cytometry.
Results:
Wire injury induced AVS was observed in all mice including endothelial dysfunction and superficial fibrin deposition in TEM imaging. We reliably found IE using 105 and 106 CFU S. aureus as bacterial challenge via in vivo echocardiography as well as ex vivo S.aureus immunofluorescence staining, TEM imaging and bacterial cultivation of aortic valve tissue (s. Figure 1). Mice undergoing bacterial challenge responded with significant neutrophilia in the blood. Using 106 CFU S. aureus was associated with an increased mortality, liver and kidney abscess formation due to systemic bacterial spreading compared to 105 CFU. Our results indicate that bacterial challenge 72h following wire injury, was superior in IE induction compared to 24h, analyzed by immunofluorescence microscopy. Aortic regurgitation was more prevalent after bacterial challenge mediating increased left ventricular volumes, meanwhile ejection fraction was not altered. Immunofluorescence staining revealed early (1d) valvular macrophage and neutrophil infiltration.
Conclusion:
In vivo echocardiography and ex vivo histological staining revealed reliable IE induction using our new model with S. aureus concentrations of 105 CFU. IE causes neutrophilia in peripheral blood counts as well as early macrophage and neutrophile infiltration of valvular tissue. A further analysis of early immune cell response and biomarker expression may yield potential makers for early IE diagnosis.
Figure 1 Intravalvular S. aureus infiltration after bacterial challenge
Exemplary image: 8μm thick horizontal cross section of aortic valve tissue after wire-injury and bacterial challenge; staining for S. aureus and Dapi nuclear counterstaining.