Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The overall goal of this procedure is to demonstrate how optical projection tomography can be used for visualization and three dimensional quantitation of atherosclerotic and neointimal lesions in the mouse. This is accomplished by first generating arterial lesions in mice, either surgically or by feeding a high cholesterol diet to mice with a predisposition to develop atherosclerosis. The next step is to isolate the target artery and scan it using optical projection tomography.

The final steps are to perform image reconstruction, identify the lesion, and measure the lesion volume. Ultimately, results can show anatomical reconstructions of the lesion and quantification of lesion volume and arterial narrowing through image analysis. Our group is interested in understanding the mechanisms underlying the formation of lesions that cause arterial narrowing leading to life-threatening complications such as heart attack and stroke.

This application of optical projection tomography was developed in our group by Dr.Nicholas Kirkby, working with Dr.Lucy Lowe, and it will be demonstrated by Dr.Jung Shi Wu, who's a postdoctoral scientist working in our laboratories. The main advantage of this technique over existing methods such as histology and immunohistochemistry, is that it is less labor intensive and it allow three dimensional rather than two dimensional analysis. This technique to induce femoral artery lesions is modified from those by R and company and Sada and company.

The text protocol explains how to induce atherosclerotic lesions by dietary manipulation. Begin with a 10 to 12 week old C 57 black six mouse weighing between 25 and 30 grams. Once anesthetized, place the mouse on a warm pad to maintain its body temperature and provide isof fluorine at two to 3%via a nose cone.

When the mouse is not responsive to a toe pinch, administer buprenorphine at 0.1 milligrams per kilogram. Next, put the mouse on its back and shave the ventral surface of the left hind limb. Then make an incision to expose the muscles of the upper hind limb between the bifurcation of the popliteal artery and the abdominal wall.

Make a blunt dissection to isolate the femoral nerve from the femoral artery and vein throughout the procedure. Keep the tissues moist with 1%weight by volume li canine. Next place temporary 6.0 Mers silk ligatures around the femoral artery and vein close to the abdominal wall and immediately below the branch with the popliteal artery.

These are to control the blood flow. Then isolate the popliteal artery, two to five millimeters distal to the branch with the femoral artery, and ligate the popliteal artery under the popliteal artery. Place a second ligature.

Now, make a small incision for the wire insertion in the popliteal artery immediately distal to the branch with the femoral artery. Apply pressure to the proximal temporary ligature to prevent bleeding through the incision and into the femoral artery towards the abdominal wall. Insert a 0.014 inch G wire about one to 1.5 centimeters.

Leave it in place for 30 seconds and then remove it. Then using the ligatures ligate, the popliteal artery above the incision. Be careful to avoid occluding the femoral artery.

Now remove the temporary ligatures. Close the wound with discontinuous running sutures using 5.0 mercal. Then apply EMLA cream.

Allow the animal to regain consciousness and movement in the recovery cage before returning it to the home cage. The leg is expected to show lameness for two or three days, but this does not mean the mouse must be isolated. For OPT, the mouse must be euthanized, induce terminal anesthesia from pentobarbital.

Then make a trans cardiac perfusion fixation with exsanguination. Now isolate the femoral arteries or the aortic arch and its branches. Remove any extraneous perial material in the process.

Then postfix the tissue in 10%neutral buffered formalin overnight. The next day either store the tissue in 70%ethanol or proceed by embedding the arteries in filtered 1.5%LMP aeros. Gently lower the tissue into the molten aeros at about 40 degrees Celsius.

Trim the aeros to make a conical shape. Now dehydrate this preparation in 100%methanol for at least 12 hours. Once dehydrated, clear the vessels in a one to two mixture of benzo alcohol and benzo benzoate.

Let the vessels incubate in this solution for 12 to 24 hours. The next day, load the cleared sample into the tomograph, which should be pre calibrated. The resolution is set at 1024.

By 1024 pixels. Set the magnification for the area of interest. The Z axis will automatically be set to the same resolution.

The voxel size will be about 200 microns. Next, using the Brightfield channel position the sample so it rotates around its own axis at the center of the field of view. Using the GFP one filter emission channel, set the focus and adjust the exposure to maximize the dynamic range of the image.

Avoid oversaturation. Now scan the vessel in the GFP one emission channel only with 0.9 degrees steps. The samples should then be returned to 100%methanol for at least 24 hours prior to embedding in wax.

For histological analysis, open the acquired images in n recon software or similar software using data viewer software. Check the quality of the image reconstruction, improve the image quality, adjust the image intensity, and have the software compensate for any misalignment of the sample from its ideal rotational axis. For each scan, define the vertical region of interest containing the lesion.

In every 50th scan line, trace the border between the media and the position of the internal elastic lamina. The software can then interpolate the border in all the other scans, manually check these interpolations and correct them as needed. Now produce a binary image set with the white pixels representing neointima and black pixels representing patent lumen.

This is accomplished by defining the intensity thresholds. Once this is accomplished, the object volume is the total lesion volume, and the luminal volume is the total volume, less the object volume. Thus, the distribution of the lesion and lumen along the axial length is assessed.

Murine femoral arteries were harvested 28 days after wire induced or ligation induced injury. Neointimal thickening was evident in the emission projections in reconstructed two dimensional slides. Concentric neointimal lesions could be distinguished.

Optical projection tomography emission images of whole aortic arch from an APOE deficient mouse identified the expected lesions in the lesser curvature of the aortic arch, the brachiocephalic artery, and in the origins of the left carotid and subclavian arteries. The same lesion was segmented and rendered in red. To emphasize the distribution of plaques when superimposed on the original image, the media and lumen could be distinguished in cross-sectional images showing that these were eccentric lesions.

Here, as the scan moves distally along these branches, the lesions gradually reduce and disappear first in the subclavian artery, then in the carotid, and finally in the brachiocephalic artery. Interestingly, this lesion in the brachiocephalic artery moves on to the flow divider as this vessel divides into the right carotid and right subclavian arteries. Following OPT procedure, other methods like histology and immunohistochemistry can be performed in order to answer additional questions like what is the composition of the lesion?

So after its development, this technique paved the way for scientists working in the field of vascular research to investigate the role of endothelial receptors, lectins, and steroid metabolizing enzymes in the development of neointimal and atherosclerotic lesions. After watching this video, you should have a good understanding of how to obtain and analyze lesions from rine arteries using optical projection tomography.