Background Myocardial infarction (MI) is the leading cause of death worldwide. Upon reperfusion further damage occurs in a phenomenon called ischemia/reperfusion (I/R) injury. Additional loss of cells, inflammation and remodelling happens during the subsequent tissue healing. This multi-facetted process is hard to fully characterize using traditional methodology. Light sheet-based MI analysis has been established recently, based on vascular staining, which is insufficient to reflect the full extent of tissue injury. We have here established a 3D analysis pipeline for cardiac tissue damage.
Methods & Results We subjected mice to an in vivo ischemia/reperfusion (I/R) protocol with 45 min of ischemia and 24 h to 28 d of reperfusion. To determine tissue damage, explanted hearts were fixated in formaldehyde, dehydrated with ethanol, bleached with peroxide and stained with eosin. Samples were again dehydrated with ethanol and cleared with ethyl cinnamate. The hearts were imaged 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.We determined TTCneg marked areas of damaged tissue in heart slices, which were similarly processed as whole hearts. These areas were comparable in size and localisation as Eosinhigh volumes, indicating eosin staining as a marker for acute tissue damage. We also investigated eosin staining at 5d and 28d of reperfusion. At 5d, we could show a co-localization of Eosinneg staining with CD31high signals, which also correlated with granulation tissue in traditional histology. At 28d, we found Eosinhigh staining at sites of fibrosis, which was confirmed by H&E and Picrosirius red staining. These areas were also less vascularized than surrounding tissue as shown by CD31 staining.
Conclusion We have shown here for the first time a 3D analysis pipeline for the whole heart to investigate cardiac tissue damage. This approach salvages low wavelength channels by using eosin, leaving several options for multiplex analysis e.g. by combination with CD31 for vascular staining. We could further show that this methodology is applicable at all time points of healing in experimental myocardial infarction providing a robust framework for investigation.
