Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Our research focuses on understanding the role of the nervous system in atherosclerosis progression. Specifically each interaction with the ridge, we aim to answer how the nerve changes during atherosclerosis and how its interaction influences the disease progression. Several cutting-edge technologies are currently used to advance atherosclerosis research to improve understanding of disease mechanisms.

These includes tissue clearing and 3D imaging, advanced microscopic techniques, artificial intelligence, single cell sequencing, and machine learning. During atherosclerosis progression, arterial wall components undergo robust restructuring. Delineating the changes in 3D and high resolution imaging of the intact tissues are the key experimental challenges.

The imaging tools described here will help researchers and cardiologists to better understand the disease and eventually help treat patients. The protocol described here offers several advantages over traditional techniques such as deep tissue imaging of the intact tissues, as well as visualization of their structure that are otherwise inaccessible. These protocols will help to better understand cell-cell and cell-tissue interaction to improve the understanding of the disease progression.

Visualizing the cellular and structural changes in 3D in the intact tissue can provide new insight to better understand the unanswered biological questions in cardiovascular research. To begin, place the anesthetized mouse onto a foam plate covered with surgical paper towels. Fix the mouse's arms and legs in a supine position using adhesive tapes.

Disinfect the chest with 75%ethanol. Collect the blood from the left ventricle using a one milliliter disposable syringe. Then make a midline incision on the chest and a small incision in the right atrium.

Perfuse the mouse's left ventricle with 10 milliliters of five millimolar EDTA in PBS for five minutes until the blood is flushed out, followed by 20 milliliters of PBS for 5 to 10 minutes. Finally, perfuse with 10 milliliters of 4%paraformaldehyde for 20 minutes. Under a dissecting stereo microscope, remove the internal organs, including gastrointestinal and reproductive organs.

Leave the heart aorta and kidneys intact. Then carefully remove the thymus and surrounding adipose tissue. Expose the whole aorta from the ascending aorta to the iliac bifurcation.

Harvest the entire aorta, and place it into a Petri dish filled with PBS. Separate the aorta into different segments and split it longitudinally for two 2, 2'Thiodiethanol, or TDE clearing. Pin the end face aorta onto a flat black wax plate in a Y shape, and fix it in 4%paraformaldehyde overnight at four degrees Celsius.

The next day, unpin the aorta and transfer it into PBS for a five-minute wash. Then transfer the aorta into a blocking solution for two hours for blocking and permeabilization. Next, incubate the aorta with primary antibodies in the blocking solution for 24 hours.

Wash the aorta in PBS for five minutes, repeating five times before incubating with secondary antibodies in 10%normal donkey serum and DAPI for nuclear staining overnight, Transfer the stained aorta into increasing concentration of TDE solution. Next, stick the double-sided sticky rectangular imaging spacer well to a clean glass slide and transfer the cleared aorta, ensuring the end face aortic adventitia faces the cover slip. Mount the aorta with drops of 60%TDE solution and carefully attach a cover slip to the well avoiding air bubbles.

Turn on the inverted confocal laser scanning microscope equipped with a 20x oil immersion objective. Tune the hybrid diode detectors based on the stain dyes. Adjust the display settings and select the 1024x1024 pixel XY format for imaging.

Drop immersion oil onto the cover slip and move the 63x objective coarsely towards the sample until it touches the immersion oil and cover slip. Then identify the region of interest and acquire Z-stacks from the adventitia side of the end face aorta at two to four micrometer step size up to 60 micrometer depth for three-dimensional imaging. Name the file with sample details and scan details and save the data.

Using an upright multi-photon microscope equipped with a 20x objective, acquire Z-stacks from the abluminal side at a 10 to 15 micrometer step size up to 700 micrometer depth. After anesthetizing and perfusing the mouse with PBS and paraformaldehyde, dissect the body part above the diaphragm level. Fix the lower body part with 4%paraformaldehyde for one to two days at four degrees Celsius.

Next, thoroughly wash the sample in PBS for 10 minutes, repeating three times. Incubate the sample in 20%cubic solution for 48 hours. After washing with PBS, incubate the sample in PGST solution overnight for permeabilization and blocking.

The next day, incubate the sample with primary antibodies in PGST solution at four degrees Celsius with gentle shaking for 10 to 12 days. After washing the sample in PGST, add secondary antibodies in DAPI in PGST at four degrees Celsius for seven days. Thoroughly wash the sample in PGST for one hour repeating five times.

Transfer the stained sample to a series of increased concentrations of tetrahydrofuran working solutions for dehydration, incubating 12 hours per concentration. After dehydration, place the sample in an absolute dichloromethane solution for three hours for lipid removal. Next, incubate the sample in a refractive index matching benzyl alcohol benzyl benzoate solution for three to six hours.

To begin load the Z-stack-tiled images of mouse aorta and lower body part images to the image processing workstation. To color code the volume depth of a cell or structure, use image restoration software to deconvolute the raw image. In the Fiji software, generate a maximum intensity projection of the deconvoluted data using temporal color coding.

Load the Z-stack-tiled TIFF image series into the software. Using Fiji stitching plugin, stitch the images and save them in TIFF format. Next, load the stitched images into three-dimensional visualization software for image segmentation.

Manually trace the neuronal structures in the X-Y-Z axis along the entire path between the aorta and ganglia. Load pre-processed images into image analysis software. Use autofluorescence to segment the aorta and connective tissues.

Apply separate pseudocolors to visualize the distinct aortic wall and plaque. Finally, use the contrast-limited adaptive histogram equalization function to enhance the local contrast over the background of the processed images. The TDE cleared atherosclerotic aorta revealed CD3e stained T cells in plaques, an extensive neogenesis of NF200-stained nerve fibers in arterial tertiary lymphoid organs.

Multi-photon imaging of the diseased abdominal aorta in APOE knockout mice showed NF200-expressing axon sprouting in regions lacking B220 positive B cells within artery tertiary lymphoid organs. Light sheet imaging of iDISCO-cleared mouse abdomen visualized the spatial relationship between the aorta and sympathetic ganglia with newly formed NF200-stained axons on the adventitia adjacent to atherosclerotic plaque.