Clin Res Cardiol (2022).

3D cardiac damage analysis using a combined immunofluorescent and whole mount histological staining approach
S. Korste1, E. Haj-Yehia1, P. Stock1, U. Hendgen-Cotta1, T. Rassaf1, M. Totzeck1
1Klinik für Kardiologie und Angiologie, Universitätsklinikum Essen, Essen;

Background Myocardial infarction (MI) is the leading cause of death worldwide. In the past years, multi-target therapies to simultaneously increase cardiomyocyte survival, preserve endothelial integrity and modulate immune response have been proposed as the most promising approach to maximise cardioprotection. Specific analysis tools to characterize all these components are a prerequisite for the assessment of these strategies. To solve this task, we developed a light sheet fluorescence microscopy (LSFM) based workflow, incorporating immunofluorescent and whole mount histological staining of an intact murine heart.

Methods & Results We subjected mice to an in vivo ischemia/reperfusion (I/R) protocol with 45 min of ischemia and 24 h or 5 d reperfusion. To determine endothelial integrity and immune cell population sizes and localisation, mice were intravenously injected with fluorophore labelled CD31, Ly6G and F4/80 antibodies. Mice were sacrificed 10 min after injection and hearts were extracted. After fixation, hearts were dehydrated with ethanol, bleached with peroxide and stained with eosin and TO-PRO-3 for nuclear visualization. Hearts were again dehydrated with ethanol and cleared with ethyl cinnamate. Hearts were imaged in total with a z step size of 10µm using a LaVision Biotec Blaze light sheet microscope. 3D modelling was done using Imaris software and custom python scripts were used for HE-pseudocolor conversion. At 24 h of reperfusion, the infarct area presented as CD31neg and eosinhigh, while being surrounded by Ly6Gpos cells. Furthermore, CD31neg and eosinhigh volumes were comparable in size, with eosinhigh volumes being slightly larger. At 5 d of reperfusion, infarct area was characterized by eosinlow staining and a mixture of CD31neg and unorganized CD31pos tissue (CD31curly). Ly6Gpos cells were still found at the edge of eosinlow and CD31curly volumes, while F4/80pos cells were located throughout the infarct zone. Additional analyses using HE-pseudocolor conversion with eosin and TO-PRO-3 allowed for similar assessments regarding tissue damage and immune cell infiltration into the myocardium. Furthermore, infarct size determined by CD31neg and eosinhigh staining at 24 h of reperfusion correlated with ejection fraction (EF) reduction and plasma troponin levels.

Conclusion We here provide a readily available protocol for analysis of all three parameters of importance in myocardial infarction: tissue survival, endothelial integrity and immune response. Furthermore, infarct size determined by our approach is comparable to other parameters such as EF reduction and troponin levels. In addition, HE-pseudocolor conversion allows for traditional histological analysis in 3D, drastically reducing time and effort and allowing for fine structure volumetric analysis.