Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy.

Complex cellular interactions determine functional and structural tissue remodeling during reperfused acute myocardial infarction (repAMI). These processes show distinct spatial distribution as the injured heart muscle is segmented into different areas (damage area, area at risk (AAR), and remote area). Three-dimensional (3D) visualization of these areas is essential for the analysis of various interactions between resident cardiac cells and infiltrating immune cells, enabling the identification of possible treatment targets. Here, a protocol is described for simultaneous and automatable 3D visualization and quantification of the cardiac damage area, AAR, and infiltrating immune cells (e.g., neutrophils) after repAMI. This includes intravital antibody-mediated staining of cardiac damage area (CD31^neg) and neutrophil infiltration (Ly6G⁺) following ex vivo visualization of AAR by retrograde antibody perfusion and further non-toxic tissue clearing for light sheet fluorescence microscopy (LSFM) imaging. This technique allows the spatial analysis of target cells, e.g., infiltrating immune cells and damaged areas in an intact mouse heart after repAMI. Traditional histology and immunohistochemistry can be performed after non-toxic tissue clearing and image acquisition with computer-assisted post-processing. This allows multiplexing information gain within the same mouse heart, strengthening data robustness and being especially significant in a highly complex injury like repAMI.