A Microscopic 2,3,5-Triphenyltetrazolium Chloride Assay for Accurate and Reliable Analysis of Myocardial Injury

My research focuses on improving TTC staining methods to enhance image quality for accurate detection of myocardial ischemia reperfusion injury. Recent advances include improved TTC assay using cryosectioning and microscopic imaging to enhance myocardial injury visualization. To begin, pre-fill the system with ice cold PBS using a 10 milliliter syringe through the PBS port.

Use a three milliliter syringe to pre-fill the TTC port with freshly prepared 1%staining solution. Secure a Langendorff retro-perfusion apparatus horizontally on a 150 millimeter Petri dish with adhesive tape to prevent movement. Position the cannula tip one millimeter above the dish bottom, aligning it with the aortic root when the heart is placed flat.

Check the perfusion system for any unintentional air bubbles. If bubbles are visible, withdraw and refill PBS while gently tapping the three-way stopcock in the upright cannula position. Next, form a loose half-square knot using a 6-0 silk suture and place it on the distal end of the cannula to secure the aortic root later.

With micro-dissecting forceps and scissors, remove the pelt from the mid-abdomen to the mid-forepaw region of a euthanized and secured mouse. Then make a two centimeter transverse incision into the abdominal muscle wall just below the diaphragm to allow for subsequent thoracotomy. Now, use fine scissors to spread the diaphragm and expose the heart.

Then make bilateral incisions through the skin, muscle, and ribs to open the thoracic cavity. Cut the diaphragm back bilaterally and lift the sternum to fully expose the heart. Immediately pour 20 milliliters of ice cold PBS onto the heart surface to arrest the heartbeat.

Then excise the heart along with the surrounding tissues. Transfer the tissue mass into a 50 milliliter beaker containing ice cold PBS for about one minute to allow the heartbeat to subside. Next, transfer the heart onto a 150 millimeter Petri dish.

Trim the aortic root free of connective tissue under a binocular microscope using fine forceps and scissors. Then gently remove the thymus and expose the ascending aorta. Trim the aortic root by dissecting the connective tissue.

Pick up the aortic stump with fine tweezers and mount it over the cannula that is already connected to a pre-filled perfusion apparatus. Now, secure the aorta using the preformed loose knot on the cannula. Inspect the cannula and confirm heart filling by observing ventricle bulging during PBS perfusion.

Leave the extra cardiac tissues, including lungs and esophagus, in place to support the heart in a natural and appropriate position. Cover the heart with moist tissue paper once the cannulation is secured. Begin rinsing the heart with ice cold PBS using a pre-connected 10 milliliter syringe until the cardiac eluate runs clear.

Inspect the heart under a binocular microscope to confirm there is no leakage at the aortic root, and that it inflates properly under perfusion pressure. Now, turn the three-way stopcock toward the TTC solution and slowly infuse one milliliter of staining solution through the TTC port over 30 seconds. Inspect the heart surface microscopically.

At this stage, viable myocardium should appear deep red while necrotic tissue appears light gray. Then remove all non-cardiac tissues using fine scissors and transfer the stained heart into a 15 milliliter conical tube containing three milliliters of TTC solution. Place the conical tube at four degrees Celsius overnight to allow a second round of staining by immersion.

To create a tissue block, first fill a blunted curved 20 gauge cannula with optimal cutting temperature compound using a one milliliter syringe. Excise the tricuspid valve using fine scissors. Then insert the cannula into the right ventricle.

Infuse 50 to 80 microliters of OCT compound into the cavity and stop the infusion when backflow is observed. Carefully place the heart into a cylindrical aluminum foil capsule filled with 300 microliters of OCT compound in a vertical apex down position. Then immerse the capsule in a 2-methylbutane solution pre-chilled to between minus 30 and minus 40 degrees Celsius.

Mount the tissue block onto a cryostat chuck using OCT. Place the chuck into the cryostat and set the temperature to approximately minus 24 degrees Celsius. Slice the tissue block until the apex of the heart is visible.

Then set the section thickness to 50 micrometers and collect every second slice to maintain 100 micrometer intervals between sections. Align the collected slices sequentially on glass microscope slides placing eight sections per slide for a total of 10 slides per heart. Dry the slides with a hairdryer on the lowest setting using room temperature airflow for approximately 10 minutes.

Submerge the slides in a staining chamber filled with Zamboni's fixative containing 2%paraformaldehyde and 0.4%picric acid by weight. Fix the heart tissue at four degrees Celsius overnight to ensure the tissue surface remains free of air bubbles formed during fixation. Mount the slides using mounting medium and cover the sections with cover slips to finalize preparation for image acquisition.

Store the completed slides in a slide box at room temperature. To image the sections, set up a light microscope using a 1.25X objective lens and run the CellSens imaging software. Position the heart section in the center of the field of view.

Adjust the camera's exposure time, focus, and white balance as needed. Then capture images of each section and save all images from the same heart in a dedicated folder. Import all images from each heart into the ImageJ platform for 3D reconstruction.

Stack all images in sequential order using the pop-up menu of ImageJ. Then create a 3D construction using the surface plot function. Lastly, open the Volume Viewer window and adjust parameters for 3D projection.

Next, copy the R script to the RStudio platform and run the automated algorithm to assess myocardial infarction. The algorithm calculates infarct sizes for each slice and computes total infarct volume. Export the dataset to an Excel file and analyze the data as needed.

In each section, deep red staining of viable cardiomyocytes was clearly distinguishable from yellow necrotic tissue, yielding a sharply defined border. Compared to conventional staining, the microscopic protocol provided clearer and sharper boundaries and an improved viable to infarct signal ratio. Filling the ventricular cavities with OCT maintained the heart's physiological shape and preserved the endocardial layer without altering infarct size.

Deep red formazan precipitates were found in the cytoplasm of viable cardiomyocytes and overlapped with cardiac troponin-I staining. In the mouse myocardial infarction model with 50 minutes of ischemia, the average global infarct size was 32.14%matching the planometric size above the mid-ventricular level. Infarct size varied across heart levels, peaking at 71.21%in the apical region, and gradually declining to zero near the ligation site.

The microscopic TTC assay revealed infarct areas even three days post myocardial infarction with yellow staining in the necrotic core and pinkish gray in the periphery. At seven days post-myocardial infarction, fibrotic scar tissue showed distinguishable TTC staining between myocytes and non-myocytes. 30 minutes of cardiac ischemia induced a wavefront pattern of injury starting in the endocardium and progressing toward the epicardium.

A transmural infarct was consistently induced when ischemia duration exceeded 40 minutes. We demonstrated that our microscopic TTC assay significantly improved image quality. Our method enhances image clarity and boundary definitions, allowing reliable infarct zone definition, and thereby advancing myocardial infarct assessment accuracy.

Our standardized method opens new options in staining dynamics, correlating image precision with molecular changes, and improving cardiac repair assessment.