Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy

We're looking at the interplay of immune cells and damaged tissue after myocardial infarction. We aim to understand the immune response and improve cardiac function. It was proposed that after myocardial infarction, all three major cellular entities, cardiomyocytes and cells and immune cells have to be modulated by multi-target strategies at the same time.

In addition to simultaneous analysis of cardiac damage and response, we could show 3D localization to these damaged areas, which was previously unknown. 3D analysis, especially when multiplexing various targets, provides a more robust way to assess the cellular composition after myocardial infarction. To begin, place the euthanized mouse under a stereo microscope.

Using fine scissors, open the chest cavity and incise the exposed superior vena cava close to the exit of the vessel from the rib cage to the neck. Using a 27-gauge needle attached to a roller pump, perfuse the heart in situ via the right and left atrial oracles with PBS containing heparin. Confirm sufficient cardiac blood removal by observing the fading of the superficial vascular drawing of the heart and the whitening of the lung and liver.

Excise the heart along with at least five millimeters of the remaining aorta and surrounding lung tissue using forceps and scissors, and transfer it to a six-centimeter Petri dish prefilled with ice-cold PBS and gauze. Now, cut away the remaining lung, fat and vessel tissue until only the heart with approximately five millimeters of aorta remains. Transfer the heart to a dish prepared for aortic cannulation and pull the remaining aorta over a 20-gauge blunt steel cannula.

Secure it to the cannula notch with a prettied 5-0 suture. Legate the left anterior descending artery permanently with a 6-0 suture at the location used for induction of reperfused acute myocardial infarction. Inject 400 microliters of PBS containing 20 micrograms of mouse NT CD31 antibody, labeled with a LexaFluor 546 retrogradely via the inserted cannula.

Carefully pull out the cannula from the aorta and transfer the heart to a 15-milliliter polypropylene reaction tube containing 10 milliliters of 4%paraformaldehyde. Incubate the tube overnight at four degrees Celsius in the dark without agitation. Wash the fixated heart in three changes of five milliliters of PBS for 10 minutes each at room temperature in the dark, using near-overhead rotation for agitation inside five-milliliter polypropylene reaction tubes.

Dehydrate the fixated heart in five milliliters of 50%ethanol in deionized water for at least two hours at room temperature in the dark, agitating with near-overhead rotation in five-milliliter reaction tubes. To bleach the dehydrated heart, transfer it to a 15-milliliter reaction tube containing 10 milliliters of 100%ethanol with 5%dimethyl sulphoxide and 5%hydrogen peroxide, and incubate it at four degrees Celsius in the dark for four hours without agitation. Wash the bleached heart in five milliliters of 100%ethanol three times for 12 hours each at room temperature in the dark, using near-overhead rotation for agitation in five-milliliter reaction tubes.

Transfer the heart to a five milliliter reaction tube containing four milliliters of ethyl cinnamate and incubate it without agitation for three days at room temperature in the dark. Cut cleared gel and gum blocks to a size that stabilizes the cleared heart in the microscope sample holder. Use a 0.1 numerical aperture objective lens with a working distance of 17.6 millimeters for image acquisition.

Then detect the fluorescence signals using appropriate excitation and emission filter settings. For light sheet microscopy imaging, choose a sheet numerical aperture of 0.079, resulting in a light sheet thickness of five micrometers. Set the distance between two individual Z planes to 10 micrometers.

Use a sheet width of 100%to achieve homogeneous illumination of the sample. The representative heart slices localizing the area at risk, endothelial injury and infiltrated neutrophils using multichannel antibody staining and autofluorescence are illustrated in this figure. The area at risk was identified by absent CD31 staining after retrograde injection, endothelial injury by intravenous CD31 negativity and infiltrating neutrophils by Ly6G-positive spots, mostly near injury zones.

3D renderings showed overlap between AAR, endothelial injury and neutrophil clusters, suggesting region-specific immune localization. Quantification showed 90.2%of neutrophils in AAR versus 9.8%in remote regions, indicating localized inflammation. Within AAR, 79.3%of neutrophils were outside endothelial injury volumes and 20.7%inside, suggesting peripheral clustering around damage.