Our protocol is one of the first that uses a rat model to describe the uterosacral ligament suspension surgery, and therefore can really serve as a blueprint for future investigators to study preclinical outcomes and modifications of the surgery. The rad uterosacral ligament is similar in anatomy, cellularity, histological architecture, and composition compared to the human uterosacral ligament. These components combined with the cost benefits and feasibility make it a really great model to study augmentation and outcomes of the uterosacral ligament suspension surgery.
The tensile testing method could be adapted for use in other pelvic floor animal models. Additionally, future investigators could use a general surgery setup to inform other prolapse surgeries and rodents like sacrocolpopexy. To begin, wear a surgical gown, head cover, mask, and sterile gloves.
Drape the animal with a sterile field leaving only the abdomen exposed. There may be some bleeding due to trauma from clipping around the caudal pair of nipples. If necessary, this can be made hemostatic with pressure or silver nitrate.
Make a seven centimeter incision down the linea alba from just below the xiphoid process to the lower nipple line using a scalpel blade. The incision should end 0.5 to one centimeter rostro from the urethral orifice. Then make an incision through the muscle layer underneath.
Assemble the abdominal retractor and inspect the abdominal cavity. Using iris forceps, gently locate the left uterine horn. Gently elevate the left uterine horn with a grasper or mosquito clamp.
Begin hysterectomy by ligating the horn below the ovary and oviduct using a mosquito clamp. To continue the hysterectomy, clamp and trim the adjacent vasculature, connective tissue, and fat from the uterine horn using micro scissors. Remember to clamp the connective tissue prior to removal to reduce bleeding.
Place the clamps as close to the uterine interface as possible, and up to the uterocervical junction. Clamp across the uterine horn near the point of bifurcation using mosquito forceps. Excise the ipsilateral horn just cephalad to the clamp to avoid bleeding.
Due to the small caliber of the rat vessels, ligation of the uterine stumps with a temporary clamp was sufficient for this surgery. However, this technique can be modified as needed by either sealing the pedicles with electrocautery or suture ligation. Adjust the abdominal retractor to expose the lower pelvis.
Inspect the exposed vaginal vault and the pelvic floor support ligamental and connective tissues attached to the vagina and cervix. Identify the ureter bilaterally, which is just medial to the ovaries. Identify the uterosacral ligaments which can be found attached to the cervix just below the remaining stumps of the uterine horns.
The ligament is traced in cephalad medial orientation toward the sacrum. Put a three zero polydioxanone suture on a small tapered needle and place a stitch through the left uterosacral ligament located high on the ligament and close to the sacrum. Tug on the stitch to ensure it has captured the uterosacral ligament ensuring that the ligament structure inserts into the cervix with the origin dividing behind the rectum where it attaches to the sacrum.
Again, identify the ureter to ensure it has not been incorporated into or kinked with the uterosacral stitch. Then pass the left polydioxanone stitch through the left aspect of the vaginal vault while incorporating both the anterior and posterior aspects of the vaginal cuff. Repeat these steps to complete the uterosacral ligament suspension procedure on the right side.
After the uterosacral stitches are placed bilaterally, securely tie the suture using a square knot such that the vaginal vault is elevated cephalad toward the sacrum. First, dissect the adipose tissue until the vaginal vault is visible. Continue to remove the abdominal fat pads until either the intact uterosacral ligament is clearly visible or at the junction between the uterosacral ligament and the vaginal vault is visible.
Using a flexible ruler, measure the distance between the uterosacral insertion and the vaginal vault to estimate the original length of the tissue. Thread umbilical tape behind the intact uterosacral ligament or the uterosacral ligament suspension junction such that the tissue is centered on the umbilical tape. Measure the height and width of the tissue where it intersects with the umbilical tape using digital caliper.
To estimate the values, used to calculate cross-sectional area. For tensile testing, position the animal such that the specimen is centered beneath the grip. Immobilize the pelvic region surrounding the specimen by securing the animal to the platen.
Lowered the load cells such that the tails of the umbilical tape easily reach the grip. Secure the umbilical tape in the grip leaving the tape slack to avoid specimen manipulation. Then open the pre-conditioning test in the software interface and label the test with the sample name.
Ensure that the pre-conditioning method includes the preload step. Click to start the pre-conditioning test which will preload the sample at 0.015 Newton. Once the preload force is stable, the test will precondition the sample at an elongation rate of 0.1 millimeters per second for 30 seconds.
After 30 seconds, allow the tissue to rest for one minute. In the meantime, load the pull to failure testing regime. Label the test with the sample name and click on Next to get to the next window.
Input the gauge length of the sample and then click on next to transition to the test page. Balance all and click on Start. Allow the test to run at an elongation rate of 0.1 millimeters per second until the tissue has been pulled to failure.
The test will produce load displacement data. Following placement of the sutures using this protocol, the newly formed junction between the uterosacral ligament and vaginal vault was elevated cephalad toward the sacrum and prevented movement of the cervical stump. For intact uterosacral ligament and uterosacral ligament suspension sample, tensile testing was evaluated by plots such as load displacement curve, stress strain analysis, and slope of the line curve fit equation.
The data showed that all structural material properties of the repair sample were lower than that of the native uterosacral ligament. The structures are small, and therefore identifying them requires practice of meticulous dissection. Theater specifically can be challenging to identify, so a dissecting scope may be useful.
We'd also suggest practicing with cadaver surgeries before survival surgeries. We describe a pathway for augmentation of the uterosacral ligament suspension as well as a novel methodology for mechanical testing of the ligament. The goal is that the augmentation describe in our protocol will make prolapse surgery more successful in the future, and therefore better for patients.
