Multimodal Study of Murine Cardiovascular Remodeling: Four-Dimensional Ultrasound and Mass Spectrometry Imaging

Our lab uses preclinical ultrasound imaging to study cardiovascular biomechanics in various diseases and physiological states. We also use mass spectrometry imaging to study the spatial distribution of lipids in cardiovascular and brain tissue. We are trying to see where in tissue do we have functional and molecular changes.

Although common molecular imaging techniques, like histology and immunohistochemistry, can yield molecular data, they are limited by currently available stains and antibodies. On the other hand, mass spectrometry imaging is an untargeted approach for multiomic studies that still maintains spatial integrity of the tissue. With our technique, we can expand multiomics to include lipids, glycans, and peptides, and couple these with functional imaging techniques, such as ultrasound.

With these techniques, researchers can now couple functional imaging with molecular imaging techniques. In our lab, we have two main focus areas for cardiovascular disease, and one of those is looking at cardiac remodeling with heart attacks or cancer treatments, so cardiotoxicity. And our second big cardiovascular aim is looking at aging and how aging impacts the vasculature.

To perform cardiac four-dimensional ultrasound, place the mouse in the supine position on the imaging plate. Then, place the transducer in the holder in a semi-locked position. Align the raised dot on the transducer with the blue dot on the screen, positioning it toward the mouse's right side.

Turn the transducer to align along the sagittal plane of the mouse with the raised notch pointing caudally. Apply a generous amount of ultrasound gel to the ventral surface of the thoracic cavity for acoustic coupling with a transducer. Lower the transducer until it contacts the ultrasound gel.

Make precise adjustments with the XY knobs at the base of the plate for fine-tuning. Next, ensure the peristernal long-axis view on the screen includes the apex, left ventricular outflow tract, and aorta, aligned horizontally for accurate imaging. Select Name Image in the bottom corner of the screen to save the image to the current series.

Rotate the transducer 90 degrees clockwise to obtain a peristernal short-axis view. Ensure the left ventricle is clearly defined in the image and the papillary muscles are visible. Select the cube icon in the top left corner of the screen to set up a four-dimensional image.

After resetting the transducer, adjust the start position to just below the apex and the stop position to the aortic arch. Set the step size to 0.08 to 0.13 millimeters and the frame rate to 200 to 300 hertz. Ensure vital signs and the EKG signal remain stable before initiating the scan.

After completing the scan and processing, turn Save EKV/4D data and Respiration Gating on for post-processing. Select Name Image in the bottom right corner and include the mouse ID in the name. To visualize each plane of view through the cardiac cycle, select More Controls, and choose Load Into Four-Dimensional.

Review each plane view of the heart, confirming that the center of the heart remains stable throughout the cardiac cycle. To begin, prepare aluminum foil boats for flash freezing of mouse tissues. Using forceps, tent the skin of the euthanized mouse and cut through the tented skin with scissors over the neck for vascular access or just below the sternum for cardiac access.

Continue cutting through the skin and muscle layers to expose the vasculature. For heart removal, cut through the bone to expose the heart. Using blunt dissection with cotton swabs, isolate the heart or vasculature from surrounding tissues, including fat.

Carefully separate the carotid vessel from the nerve. Then, remove the heart or vasculature using surgical tools. Place the carotid vessel, heart, and vasculature on a pre-labeled aluminum foil boat.

Then, place the boats in liquid nitrogen for flash freezing. To begin, fill a beaker with HPLC water and set it aside along with five-and one-milliliter syringes. Then, set the cryostat temperature to minus 25 degrees Celsius and insert the blade.

Place a pair of forceps inside the cryostat chamber to cool prior to mounting the tissue. Draw five milliliters of HPLC water into a syringe and place it in the cryostat to partially freeze the water. Before the water in the syringe fully freezes, dispense it onto the metal chuck and allow it to fully freeze.

Now, fill a one-milliliter syringe with HPLC water and place it in the cryostat. After 30 to 60 seconds, place a small daub of partially solidified water onto the center of the chuck. Using forceps, quickly place the extracted mouse heart into the water droplet and hold it there until the surrounding water is completely frozen.

To begin, remove the slide with the mounted mouse heart from the freezer and place it in a desiccator to dry. Turn on the HTX M3+Sprayer. Open the HTX app on the laptop, and in the Method, set the nozzle temperature to 75 degrees Celsius, the flow rate to 100 microliters per minute, and the pressure to 10 psi.

Record the sample name, polarity, matrix, solvent, and concentration in the lab notebook. Then, calculate the matrix amount required for the desired concentration. Next, weigh the desired amount of 2, 5-dihydroxybenzoic acid for the positive mode matrix.

Dissolve the matrix in 70%methanol in a 15-milliliter tube. Sonicate the matrix solution for 10 minutes. During sonication, remove the slide from the desiccator.

Open the Sprayer tray. Then, place the slide in the bottom left corner and tape down the edges. Select the sample spray region and close the tray.

Using a syringe and filter, draw the matrix solution into the syringe. Filter the matrix solution through the syringe into the vial with the black lid located on the left side of the Sprayer. Place the vial back into its designated spot on the Sprayer and insert the D line tubing securely into the vial.

Next, turn on the inert gas and confirm the gauge on the Sprayer reads 10 psi. Press Start, and once the Sprayer reaches the set temperature, select the flashing Start button to begin spraying. After the spray is complete, remove the sample from the Sprayer and place it in the MALDI slide holder.

Scan the MALDI slide holder and slide using a scanner. Save the image on a flash drive for use with mass spectrometry imaging. Select the Wash option on the Sprayer and move the D line from the matrix vial to the waste beaker.

Finally, spray methanol onto the Sprayer tray and wipe it clean. Turn off the nitrogen. MALDI mass spectrometry imaging of infarcted myocardial tissue identified molecular ion mass to charge 577.52, likely corresponding to COHb in C or D, suggesting involvement in myocardial remodeling.

The four-dimensional ultrasound image showed myocardial regions with less than 20%surface area strained magnitude, visualized as green-yellow tissue, indicating infarcted zones. In the long-axis view of the myocardial infarction tissue, lipid delocalization was visible, complicating the correlation between tissue biomechanics and molecular composition.