The overall goal of this procedure is to assess the maternal vascular changes necessary for normal fetal and placental growth during mouse pregnancy. So this method is useful to characterize carefully the maternal vascular adaptation that occur during pregnancy, and this adaptation may have significant implications for fetal growth. The main advantage of this technique is that it provides a robust and reproducible stereo logical readout, which can be validated by visualizing residual vascular smooth muscle actin by immunohistochemistry.
Visual demonstration of this method is critical as the correct preparation orientation of the uterine tissue, which changes markedly throughout gestation are key to the success of this procedure. On gestational day 9.5, carefully thread a piece of dental floss under one uterine horn of a pregnant time mated C 57 black six mouse proximal to the ovary and gently but tightly tie the floss to ligate the uterine artery. Repeat the ligation under the other horn and around the cervix to prevent the arteries from collapsing.
Then quickly excise the entire uterus distally to the three ligation points at the tip of the horns and the cervix. Position the uterus on a piece of polystyrene with both uterine horns aligned along the same long axis in opposite directions from the cervix. Then gently stretch the tissue using two to 3 25 gauge needles to pin it into place.
Immerse the polystyrene in a 50 milliliter conical tube and fix the uterine tissue with up to 50 milliliters of 10%formalin at room temperature. Uterine tissue is very delicate and needs to be handled carefully prior to fixation to avoid damage or tearing. After five to six hours, replace the formin with 50 milliliters of PBS for a five minute wash.
Then dissect the uterine horns proximately to the ligation points at both ends and proximately to the cervical ligation point in the middle. Trim any excess fat from the implantation sites. Then wash the uterus two more times in PBS for five minutes each to remove any final traces of formalin and store the tissue in 70%ethanol at four degrees Celsius using an automated processing system with the parameters set as indicated in the table.
Embed the horns individually in paraffin so that they can be cut along the plane perpendicular to the long axis of the uterus. It is essential to embed all of the samples in the same orientation as this will ensure the same cross-sectional profile across the implantation sites upon sectioning. After obtaining seven micron thick serial sections on a microtome stain, one section every 49 microns with hematin and eoin, and store the sections at room temperature for further downstream analysis.
To assess the vessel size and tissue remodeling status for each implantation site, select three sections at 49 micron intervals that are close to the midsagittal point. After scanning the selected slides, restrict the analyses to the central two quarters of each implantation site to avoid including veins. To assess the lumen size, select a region of interest around the inside of the vessels and record the area of the region to obtain the total vessel size.
Draw a region of interest around the outer wall of the vessel, including the endothelial cells, parasites, and intramural leukocytes. Finally, taking care to avoid measuring the same artery multiple times, record measurements in triplicate from the five vessels in each implantation site with the largest and roundest lumens as a result of the reduced remodeling exhibited by Immunodeficient Rag two IL two receptor gamma chain knockout mice. These animals also demonstrate significantly smaller luminal surface areas indicative of overall smaller vessels that can be visualized by h and e staining as shown here for wild type mice and quantified stereo logically.
Furthermore, the relative vessel to lumen ratio is higher in the spiral arteries of these mice suggesting a reduced blood supply and a higher blood velocity in immunodeficient mice. The quantitative assessment of this representative experiment was confirmed by immunohistochemical detection of smooth muscle actin, most of which is lost from the vascular media of wild type spiral arteries by mid gestation. If this NK cell driven process does not take place, however, smooth muscle actin remains in the media of the vessels, which can be detected as rings of brown staining around the blood vessels, Once the sections have been fully prepared, the measurements and validation by immunohistochemistry can reasonably be completed within the course of a few days To reduce the number of required animals.
This protocol can be combined with other techniques such as flow cytometry to investigate maternal leukocyte populations or immune function. After watching this video, you should have a good understanding of how to assess the maternal vascular adaptations that occur during most pregnancy.
