Abstract
Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes.
Highlights
The established tools for the characterization of I/R injury and response assessment have several limitations
We hypothesized that light sheet fluorescence microscopy (LSFM) was capable of characterizing myocardial ischemia/ reperfusion (I/R) injury in conjunction with significant I/R injury response mechanisms, immune cell infiltration
We could demonstrate the applicability of our clearing protocol to other murine tissues[9,21,22] and the human heart
Summary
The established tools for the characterization of I/R injury and response assessment have several limitations. The mainstay for cardiac injury analysis in animal models is measurement of metabolic activity of cardiomyocytes in serial thick sections using triphenyl tetrazolium chloride (TTC)[8] This does typically not include advanced histological and immunohistochemical co-assessments and fails to provide an accurate 3D reconstruction of the affected tissue. While ECi performed well for murine organs, such as kidney and bone, it suffered, like other approaches, from extremely high tissue autofluorescence of the heart muscle. We wished to (i) develop a readily applicable, non-toxic workflow with chemicals and tools that are commercially available, (ii) benchmark this work flow against standard I/R histology techniques in compliance with recent guideline recommendations[14], (iii) quantify I/R injury parameters in 3D, (iv) assess the long-term impact of I/R injury following days after reperfusion and (v) relate the I/R injury zones to immune response mechanisms. We introduce an ECi-based 3D myocardial I/R injury assessment workflow, termed BALANCE (Bleaching-Augmented soLvent-bAsed Non-toxic ClEaring), to overcome the limitations of the current analysis tools
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