Abstract
AIM: Imaging mass cytometry (IMC) affords simultaneous immune-labelling/imaging of multiple antigens in the same tissue. Methods utilizing multiplex data beyond co-registration are lacking. This study developed and applied an innovative spatial analysis workflow for multiplex imaging data to IMC data determined from cardiac tissues and revealed the mechanism(s) of neutrophil-mediated post-myocardial-infarction damage. METHODS: IMC produced multiplex images with various redox/inflammatory markers. The cardiac peri-infarct zone (PIZ) was determined to be up to 240 µm from the infarct border based on the presence of neutrophils. The tissue region beyond the infarct was defined as the remote area (RA). ImageJ was used to quantify the immunoreactivity. Functional assessments included infarct size, cell necro/apoptosis, total thiol assay and echocardiogram. RESULTS: Expression of damage markers decreased in order from the infarct area to PIZ and then RA, reflecting the neutrophil density in the regions. Concentrically spaced "shoreline contour analysis" around the cardiac infarct extending into the PIZ showed that immunoreactivity for damage markers decreased linearly with increasing distance from the infarct, concomitant with a decreasing neutrophil-myeloperoxidase (MPO) gradient from the infarct to the PIZ. Stratifying by concentric bands around individual MPO(+) -signal identified that the immunoreactivity of haem-oxygenase-1 (HO-1) and phosphorylated-p38 mitogen-activated protein kinase (pP38) peaked near neutrophils. Furthermore, spatial dependence between neutrophils and markers of cardiac cellular damage was confirmed by nearest-neighbour distance analysis. Post-infarction tissue exhibited declined functional parameters that were associated with neutrophil migration from the infarct to PIZ. CONCLUSION: This image-based quantitative protocol revealed the spatial association and provided potential molecular pathways responsible for neutrophil-mediated damage post-infarction.