Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs

This protocol will expand the accessibility of research on the uterosacral ligament and pelvic floor conditions, like pelvic organ prolapse, or POP, and facilitate the dissection of mirroring pelvic floor tissue. This technique allows the isolation of both uterosacral ligaments from a mouse while maintaining anatomical attachments and retrieving the surrounding pelvic floor organs. Our method establishes a preclinical model that allows for this study of how pregnancy or disease influences tissue mechanical behavior.

This method establishes a new tool for women's health-related research. The main struggle is identifying and handling the uterosacral ligament. My advice is to take adequate time and care because these tissues are small and fragile.

Begin by placing a euthanized mouse on a pad and pinning the four limbs down. Make a one to 1.5-centimeter long incision on the abdomen with scissors. Gently separate the skin at the cranial, caudal, and lateral sides of the incision.

Flip the mouse to face the dorsal side up and gently peel back the skin towards the hind limbs. Pin the limbs and make a one-centimeter long incision into the abdomen from the thorax to the pelvis. Gently push the organs towards the thorax for a clear view.

After irrigation with PBS using a pair of forceps, pull and clear the pelvic fat tissue. Identify the uterine horns and then cut them from the ovaries. Now, pull them out of the field of view and sever the cervical connection.

Sever the ureters from the bladder connection. Cut the colon as close to the cervix as possible. Place the mouse with the pad under a dissecting scope to visualize the uterosacral ligaments.

Use forceps to gently clear any surrounding fat from the uterosacral ligaments. Tie a 5-0 suture around the cervical end of both ligaments. Cut the cervical end of the uterosacral ligament, leaving a piece of the cervix attached.

Now, cut a piece of muscle from the bottom of the ligament. Place the dissected tissue in a bath containing PBS to maintain tissue hydration. Cut the cervical end of the remaining tissue, leaving a piece of cervix attached for mechanical testing and imaging.

Once the fat has been cleared, hold the bladder with forceps and gently lift it at an angle of approximately 40 degrees. Use scissors to cut the bladder at the distal side right above the cervix. Place the bladder in a bath with PBS to maintain hydration.

Now, use the forceps to lift the cervix at an approximate angle of 40 degrees. Identify the rectovaginal fascia connecting the rectum and the cervix and sever the connection with the scalpel. Use scissors to cut the pubic bone at the pubic synthesis.

Gently widen the pelvic floor to increase the visibility of the tissue insertions. Hold the cervix with forceps and cut it as close to the vulva as possible. Place the tissue in PBS to maintain hydration.

Use the forceps to gently pull the rectum towards the thorax. Identify the rectum and cut it at the anus. Place the rectum in PBS to maintain hydration.

Once all tissues of interest are harvested dislocate the femurs from the pelvis. Cut the pelvic bone from the distal and proximal ends. Place the dissected tissue in PBS.

The enzyme treatment appeared to reduce the average axial peak and relaxed stressors that the uterosacral ligament experiences at prescribed global displacements, indicating that collagen and glycosaminoglycans contributed to axial stiffness of the ligaments. The average axial strains for each mechanically-tested specimen indicate that the global axial strain in the enzyme-treated samples was similar to or larger than the controlled sample, suggesting that the reduction in macroscopic stress was due to enzyme treatment, rather than smaller strains. Raman spectroscopy of the uterosacral ligament's intermediate section suggests that enzyme treatments altered the biochemical composition of the murine uterosacral ligaments.

The collagenase-treated uterosacral ligaments had decreased collagen content, as well as decreased water and hyaluronic acid. Chondroitinase-treated uterosacral ligaments had less hyaluronic acid, as well as lower water and collagen content. Attention to detail is very important, especially when clearing the fact that deposits around the USL and pelvic organs here is needed to avoid damaging the USL and its surrounding tissues.

Further investigation can be conducted on the USL and other pelvic floor tissues using mice that have genetic variations that hypothesized to affect POP. Our protocol is already enabling a range of new studies, including investigation into how the mechanical behavior of USLs changes a function of pregnancy, which is a key risk factor for POP.