Background Myocardial infarction (MI) is the leading cause of death worldwide. Upon reperfusion of, some capillary vessels remain dysfunctional and leading to the phenomenon of microvascular obstruction (MVO). MVO size may serve as a desirable therapeutic target to salvage damaged myocardium. Light sheet-based MI analysis has been established recently, based on staining of vascular damage, which was speculated to also show MVO. We set out to quantify MVO in the whole murine heart and incorporate this analysis into an established pipeline.

Methods & Results We subjected mice to an in vivo ischemia/reperfusion (I/R) protocol with 30 min or 45 min of ischemia and 24 h of reperfusion. To determine endothelial integrity and immune cell population sizes and localisation, mice were intravenously injected with fluorophore labelled CD31 and Ly6G antibodies, as well as 4% thioflavin S solution. Mice were sacrificed 10 min after injection and hearts were extracted. Hearts were then cut into horizontal slices and stained in 1% triphenyl tetrazoliumchloride (TTC) solution. Images of the slices were taken under UV and white light to visualize thioflavin S and TTC staining. Following, slices were fixated in formaldehyde, dehydrated with ethanol, bleached with peroxide and stained with eosin. Slices were again dehydrated with ethanol and cleared with ethyl cinnamate. Heart slices 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. In the hearts subjected to 30 min of ischemia, TTC^neg clearly marked damaged tissue, while no thioflavin S^neg areas could be detected, indicating no MVO. In the hearts subjected to 45 min of ischemia, TTC^neg also marked areas of damaged tissue, but there were still no detectable thioflavin S^neg areas. In contrast to that, CD31^neg volume resembled TTC^neg areas, as well as eosin^high volumes, indicating a match between tissue and vascular damage. In both models, Ly6G^pos staining surrounded damaged tissue areas and cardiac damage was apparent from elevated plasma troponin levels.

Conclusion CD31^neg volumes following myocardial I/R seem to not indicate sites of MVO, but rather show distinct vascular damage apart from the no reflow phenomenon. Whether this results from loss of CD31 signal or other obstruction of the endothelial cells from the staining antibody needs to be determined. In addition, changes in endothelial damage between different ischemic times and the impact of the resulting possible dysfunctional vascular network on tissue recovery warrants further research.