Our protocol is significant because it provides the first quantitative ex vivo method for assessing the spontaneous motility of an intact uterus. This novel approach provides an easy and less expensive alternative for testing the relaxant potential of naturally occurring remedies in synthetic compounds. A major advantage of this technique is that all the intra-uterine cellular interactions remain intact throughout the experiment.
Over contractility of the uterus is associated with dysmenorrhea, for which uterine relaxants may be useful for treatment. This method provides an easy way to test new relaxants. After confirming the estrous cycle stage, place the mouse in the supine position, and spread the extremities to expose the abdominal pelvic region.
Wet the abdomen with 70%ethanol, and use forceps to carefully lift the skin superior to the clitoris. Make small transverse incisions on the lateral aspects of the lower abdominal area up to the upper extremities to expose the peritoneum. Carefully cut through the peritoneum to expose the gastrointestinal tract without damaging the uterine horns, and use forceps to remove the fascia and adipose tissue covering the gastrointestinal tract.
Remove the duodenum, jejunum, ileum, cecum, and the ascending and transverse colon. Locate the urinary bladder to locate the vagina which is found directly under the urinary bladder. Locate the pubic symphysis at the confluence of the pubic bones, caudal to the bladder, and use scissors to carefully make an incision on the lateral sides of the pubic symphysis through the interpubic fibrocartilaginous tissue to gain access and to provide a route for vaginal extraction.
Cut through the perineum located between the anus and lower part of the vulva, and use forceps to lift the vagina to allow a slow excision of the rectum. Identify the two uterine horns that bifurcate into a fork rostrally to the vagina and locate a convoluted oviduct and ovary at the end of each horn. Correctly identifying the uterine horns is critical to the procedure to ensure the tissues are not damaged.
Use small dissecting scissors to remove any ligaments that connect to and support the horns, oviducts and ovaries within the abdominal cavity, and remove the vagina, uterus, oviducts and ovaries. Transfer the entire isolated reproductive tract into a 100 millimeter Petri dish containing 10 milliliters of Dulbecco's PBS. Use forceps and surgical scissors to remove any connective and adipose tissue surrounding the uterine horns and vagina as well as any fur in the pubic region that could hinder the image quality.
Remove the broad ligament to allow motility of the uterine horns and wash the tissue two times with fresh Dulbecco's PBS per wash before transferring the isolated reproductive tract into a 35 millimeter Petri dish containing three milliliters of oxygenated Krebs solution. For spontaneous motility imaging, place the Petri dish on a black surface at room temperature, and allow 15 to 30 minutes for spontaneous contractions to begin. When movement can be observed, record the spontaneous uterine motility for 10 minutes from an axial plane using any type of digital video equipment.
Next, transfer the preparation into a Petri dish containing oxygenated Krebs buffer supplemented with the test compound and record the spontaneous uterine motility for an additional 10 minutes in the same manner. At the end of the recording period, wash the entire reproductive tract in 10 milliliters of Dulbecco's PBS in a 100 millimeter Petri dish, and transfer the sample into a new Petri dish of freshly oxygenated Krebs buffer to assess the reversibility of the treatment. Record the spontaneous uterine motility for about 10 minutes in the same manner, as for the last two recordings.
Then transfer the preparation to another 35 millimeter Petri dish filled with oxygenated Krebs buffer supplemented with the vehicle for the test compound to ensure that there are no mechanically induced changes in the spontaneous uterine motility. The reproductive tract preparation exhibited high motility in panels A and C in the absence of epinephrine. But this phenotype is dormant in panel B with the presence of one micro muller epinephrine.
Here, a sample data analysis of epinephrine administration and spontaneous motility recovery is shown, illustrating how the effects of the hormone on reproductive tract spontaneous motility can be quantified from the recordings of the tissue taken before, during, and after test compound treatment. It is important to avoid compressing or overstretching the uterine horns otherwise no spontaneous contractility will be observed. Our method may also be used for testing compounds that may increase the contractility of the uterus.
This procedure may be applied to pregnant mouse uteri at the earlier stages of pregnancy. This experiment has the potential to be upgraded to a six well plate format for throughput drug screening.
