Cardiovascular disease is the main cause of death globally. Mouse models are useful tools for studying this disease and our protocol can be used to quantify atherosclerosis in mice. Using this protocol, lesion size can be measured in three vascular locations.
The aortic root, the aortic arch and the brachiocephalic artery. The quantification of atherosclerosis in mice can be a tedious task. We have provided detailed steps that we hope can speed up the process.
Some of these steps are difficult to describe in writing. We hope that this video will help you in obtaining robust atherosclerotic lesion size analysis. After harvesting a mouse heart according to standard protocols, place the heart on a cork bed, ventral side up under a dissecting microscope and use a needle to fix the heart to the cork through the apex.
Holding the base of the heart with anatomical forceps, use a scalpel held at a 20 degree angle caudally in the sagittal plane, and 20 degrees cranially in the transversal plane, to cut away the apical two thirds of the heart. Embed the aortic root and the base of the heart in optimum cutting temperature compound, or OCT, and gently squeeze the heart with the forceps to fill the aortic root with compound and to remove any air bubbles. Transfer the specimen to the bottom of a cryomold filled with OCT with the aortic root perpendicular to the bottom surface and freeze the compound on dry ice.
Then store the tissue sample in a freezer bag at minus 80 degrees Celsius until cryosectioning. To prepare a pinning bed for en face analysis, fold a segment of paraffin wax film eight times to make a flat 25 by 25 mm surface and wrap black electric insulation tape around the film to make a dark background for the aorta. Place a label on the back side of the pinning bed and use a lead pencil to record the mouse identification number.
Transfer the aortic arch to the pinning bed and add a drop of PBS to the tissue. Using a stereo microscope, clean the aorta from the remaining periadventitial adipose tissue and use Vannas scissors and Dumont forceps to gently peel away all of the surrounding adipose tissue without manipulating or damaging the aorta. Next, introduce the Vannas scissors into the aortic lumen to expose the intimal surface.
Begin cutting the outer curvature of the ascending arch in the distal direction, continuing to cut open the branches, including the brachiocephalic artery and sparing the dorsal part of the descending thoracic region. To display the intimal surface, cut open the lesser curvature and fold open the aorta. Using a micro Castroviejo needle holder, secure the open arch to the pinning bed with the blunt end of minuten insect pins without stretching the specimen, gently bending the pins away from the specimen as the tissue is held in place.
Then store the pinned arch facedown in a Petri dish of PBS at four degrees Celsius. For Sudan IV staining, rinse the specimen for five minutes in a Petri dish of 70%ethanol with the arch facing down, before transferring the specimen to a dish of Sudan IV working solution for seven minutes. At the end of the incubation, rinse the sample with two three-minute washes in 80%ethanol to de-stain the normal intimal surface, followed by a final rinse in PBS before returning the tissue to its original Petri dish.
Then acquire micrographs under a stereo microscope connected to a digital camera at a 10X magnification, obtaining images of the pinned arch submerged in PBS, using small metal weights to hold the pinning bed to the bottom of the Petri dish. To obtain cryosections of the embedded aortic root, set the cryostat temperature to minus 20 degrees Celsius and the section thickness to 10 micrometers. Mount the OCT block containing the aortic root on the specimen holder with the ventricular tissue facing outward.
Secure the positioning with some additional OCT if needed. The aortic root should now be positioned perpendicular to the knife blade. Collect the initial control sections containing heart muscle tissue on ordinary microscope slides, checking the progression through the tissue every 200 micrometers under a light microscope.
When the first aortic cusp appears, tilt the specimen toward the point zero cusp to align the section plane with the two other cusps and count every 10 micrometer section that is cut from point zero onwards. Begin collecting sections for analysis from 90 micrometers and onwards according to the slide planned, until 800 micrometers from point zero has been reached. Calculating the lesion fraction of the total vessel area of the aortic root makes the data less sensitive to possible angle differences during sectioning.
In addition, it could be illustrative to calculate the area under the curve or the atheroto lesion size per mouse and present the data in a dot plot to further visualize individual variations within groups. Lesional lipid accumulation can be quantified by color thresholding oil red O positive areas of the total lesion area. The right and left coronary arteries usually diverge from the aorta around 250 micrometers from the aortic sinus which often coincides with the most prominent lesion sizes.
Removing the dark background in representative images of the en face aortic arches can enhance the visual display. The lesion size is typically normally distributed within groups, allowing statistical analysis using Student's t-test between groups. We'd recommend practicing with some test material until you have obtained sufficient skills for processing your experimental samples.
If the lesion size is different between groups, you should determine the mechanism, as a lesion composition analysis is usually the next step to pursue.
