The overall goal of these procedures is to study the formation of the embryonic and perinatal aortic walls during normal development as well as, perturbations in this process during disease. This method can help answer key questions in the vascular development field such as, what is the source of the cells that contribute to the embryonic and perinatal vascular medium. The main advantage of these techniques is that they allow the evaluation of the smooth muscle pathogenesis and morphogenesis that occur during the embryonic and perinatal stages of development.
The implication of these techniques stem towards the therapy of vascular diseases such as, supravalvular aortic stenosis of periodic disease that causes excessive sumo cell production. The mini pump placement procedure will be performed by Dr.Jiasheng Zhang, a research scientist directing a microsurgery core and Yale University. Begin by setting up mating pairs between two to six month old mice with Cre ER and mice with a cre reporter.
Using a metal probe, check for vaginal plugs each morning, separating the female from the male when a plug is detected. For early embryonic induction, inject tamoxifen intraperitoneally into the pregnant damp. To mark cells in the mid to late gestational period and to trace their fate or clonality in the post-natal mouse, inject progesterone into the intraperitoneal cavity of the pregnant damp incompetently with tamoxifen in a one to two dose ratio.
To harvest the embryos on the appropriate embryonic date, cleanse the abdomen of the pregnant damp with 70%ethanol and use scissors to open the abdomen to remove the uterus. Collect the embryos from the uterus using forceps and a stereo microscope to carefully separate them from the placenta and yolk sac. Fix the embryos in 4%paraformaldehyde at four degrees celsius for two hours.
Followed by three washes in PBS to remove the excess fixative. After the third wash, incubate the embryos in 30%sucrose in PBS, in individual 15 milliliter tubes until the embryos sink. Next, fill plastic freezing molds with optimum cutting temperature compound up to two thirds of the maximum volume and place one embryo vertically in each mold.
After 10 minutes at room temperature, freeze the tissues upright in a dry ice 70%ethanol bath and set the cryostat temperature to 22 degrees celsius. Section the tissue blocks to generate 10 to 20 micrometer thick transverse tissue slices through the entirety of each aorta, capturing each section on a glass microscope slide as they are obtained. When all of the samples have been acquired, dry the slides at room temperature for 30 minutes prior to 80 degree celsius storage.
To stain the tissue samples, thaw the slides at room temperature, protect it from light to avoid fluorophore bleaching and wash the slides in 0.1%PBS twin 20. For clonal analysis, stain the tissues with an appropriate nuclear dye and directly image the other flurophores of the rainbow cre reporter on a fluorescent microscope. For fate mapping, with the mTmG cre reporter, visualize the GFP expression by direct imaging with or without immunostaining.
To culture and explant an embryonic aorta, place the embryo in the supine position and secure the extended limbs to a dissection board under a stereo microscope. Using scissors, make a vertical incision from the abdomen through the sternum to the upper thorax. Dissect away the finest tracheal lungs, esophagus, liver and intestine.
Use forceps to gently pull the heart ventrally, using scissors to dissect the aorta away from the dorsal aspect of the thoracic and abdominal cavities. Cut the aorta at the proximal root and the distal abdominal positions to release it and place the harvested aorta in sterile ice cold PBS in a cell culture hood. After a brief wash, transfer the aorta to one well of a 24 well plate, containing DMEM medium supplemented with 0.5%PBS and place the plate in a cell culture incubator for up to 24 hours.
At the end of the incubation, wash the aorta two times with PBS and fix the tissue in 4%paraformaldehyde for 20 minutes. After three more PBS washes, transfer the aorta to a 1.5 milliliter tube containing 30%sucrosine PBS until the tissue sinks. For perinatal aorta smooth muscle cell isolation, use a 23 gauge needle to puncture the left ventricle of a postnatal day 0.5 mouse and use plastic tubing to connect the needle to a sterile PBS containing syringe, held vertically in an elevated position, so the PBS flows into the left ventricle by gravity.
When the liver blanches, use forceps to remove the adventitial tissue on the outside of the aorta and harvest the aorta as just demonstrated. Dissociate the aortic tissue in 500 microliters of digestion solution with manual shaking every five to 10 minutes. After 45 minutes, transfer the tube to a sterile cell culture hood and use a 200 microliter pipette to triturate the tissue slurry until a single cell suspension has been obtained.
Transfer the cells into a 15 milliliter conical tube and then, add two milliliters of DMEM and collect the cells by centrifugation. Resuspend the pellet in three milliliters of smooth muscle cell culture medium and seed the cells onto a 35 millimeter culture plate. Then, place the plate into a 37 degree celsius incubator, refreshing the cell culture medium every three days.
To place a subcutaneous osmotic mini pump, begin by shaving the lower back of an anesthetized mouse with clippers and place the mouse in the prone position on a heated surgical plate. Scrub the back with betadine and isopropyl alcohol and place the mouse under a stereo microscope. Then, use a scalpel to make a 0.5 to one centimeter horizontal skin incision approximately three to five centimeters rostral to the base of the tail, without injuring the underlying muscle.
Insert a hemostat into the incision and open and close the hemostat jaws to create a subcutaneous pocket. Insert an osmotic mini pump filled with the agent of interest into the pocket and use 6 o non-absorbable sutures to close the incision. Then, allow the mouse to recover with monitoring until the achievement of sternal recumbency.
Embryos mutant for the gene encoding the extracellular matrix protein, elastin accumulate excess smooth muscle cells in the inner layers of the aorta, beginning after embryonic day 15.5. Clonal analysis of the smooth muscle cells with the multicolor cre reporter during this process, indicates that the cells are of multiple colors and thus, are polyclonal. Immunostaining explanted aorta sections reveals that elastin mutant embryonic day 15.5 aortas become hyper muscular and stenodic within 18 hours of culture, a process that is attenuated by the addition of an anti antegrin beta three blocking antibody.
Smooth muscle cells isolated from the aorta of wild type mice that post natal day 0.5 express smooth muscle cell markers but not CD31. Continuous infusion of an integrin beta three inhibitor into pregnant damps beginning on embryonic day 13.5, significantly attenuates aortic stenosis and hyper muscularization in mice mutant for the extracellular matrix protein, elastin. But do not alter the aortas of wild type mice.
Once mastered, the osmotic mini pump placement technique can be completed in less than 10 minutes if it is performed properly. Following these procedures, these approaches can also be extrapolated to study smooth muscle coded, non-vascular such as, tissue of gastrointestinal tract or the lung areas. After watching this video, you should have a good understanding of how to conduct in-vivo embryonic fit mapping and clonal analysis, culture explanted embryonic aortas, perinatal aortic smooth muscle cells and explant subcutaneous osmotic mini pumps.
Don't forget that working with mice, tamoxifen and a fixed reducer can be hazardous and that precautions such as, wearing gloves and a lab coat and taking extra care with should always be taking while performing these procedures.
