Name: development of a uterosacral ligament suspension rat model
Uploaded: 2022-08-17T14:30:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about Development of a Uterosacral Ligament Suspension Rat Model at JoVE.com

We thank Prof. Silvia Blemker for use of her Instron and Prof. George Christ for use of his surgical space as well as the 3D printed holder and grip. This work was supported by the UVA-Coulter Translational Research Partnership and the DoD (W81XWH-19-1-0157).

The authors have nothing to disclose.

The protocol is notable for several advantages. To our knowledge, it is the first published description of USLS in the rat model and will provide future investigators with reproducible steps for performing this procedure in the research setting. Second, we include a novel protocol for tensile testing of the native and surgical interface of the USL. The tensile testing protocol could be utilized in similar studies that investigate new tissue engineering approaches to augment native tissue repairs such as USLS. Moreover, the rat model itself is useful for the study of pelvic floor disorders due to ease of handling/boarding, short lifespan, and cost efficiency compared to larger animal models. Limitations of the protocol include an inability to assess one of the main complications of USLS, ureteral kinking. Despite this, we had no cases of presumed ureteral injury in this study. Another consideration is that the horizontal orientation of the pelvis, small fetal head-to-birth canal ratio, and lack of spontaneous prolapse in the rat model does limit some applicability of results to humans. However, the use of multiparous rats is a strength of this study since this accounts for the leading risk factor in the development of POP3.
The establishment of a successful protocol for hysterectomy and USLS in the Lewis rat will be a useful tool for future researchers investigating surgical components of POP, while minimizing variability in testing the mechanical behavior of the USL. Surgical animal models are beneficial in that they allow researchers to design clinically relevant experiments that control for parity, body mass, disease, and nutrition34 while mitigating the ethical risk of initial study in humans. Further, standardized models for POP allow researchers to bypass the limitations of human tissue collection. In particular, the tensile testing methods described in this protocol will enable consistency between studies. Previous rodent models tested the mechanical properties of the entire pelvic region, which includes the cervix, vagina, and the multiple pelvic support ligaments29,42. The methods described here allow for measurement of the USL in a way that maintains the native spinal and cervical attachments. It should be noted that the tensile testing methods do not assess the USL alone, but rather the USL in combination with its insertion at the sacrum and cervix. This is a strength of the study as it reflects the usual in situ forces to which the ligament is subjected. We acknowledge that the mechanical behavior of the isolated ligament would be different if it were tested ex vivo without its native attachments. This is especially true since the rat structures are small and limit the feasibility of collecting a sample suitable for ex vivo testing. The USLs do experience loading in multiple directions in situ, so the uniaxial nature of the test is a limitation, but using this method allows for meaningful comparisons between previous&#160;studies of rat USL mechanics29,42. While there is currently no widely accepted standard mechanical testing protocol, this model will be a useful tool for future tissue engineering studies in the field.
Several steps described in this protocol are critical to the health and well-being of the animals as well as the reproducibility of the USLS surgery and subsequent tensile testing. First, it is essential to obtain both the analgesic and the anti-inflammatory drugs described as the analgesic alone was found to be inadequate for pain management. The prophylactic antibiotic decreases the risk of surgical site infection and is the standard of care in human surgery. Regarding the USLS surgical procedure, avoiding damage to the ovaries and minimizing blood loss are essential for a successful surgery. Steps 1.3.3 and 1.3.4 describe separating the top of the uterine horn from the adjacent ovary; care should be taken to maintain this dissection on the side of the uterine horn to prevent disruption of delicate vessels around the ovary, which can result in excessive bleeding. Of note, other investigators have shown that ovarian function is preserved after removal of the uterine horns43. Moreover, if the ovaries are disrupted or removed, the overall collagen fibril architecture will be disturbed, altering the mechanical properties of its tissues44,45. Once the uterine horn is safely separated from the ovary, there is a clear plane of dissection allowing isolation of the uterine horn from the surrounding fat pads and vasculature. Despite the clear plane of dissection, the pedicles along the uterine horn should be secured with a clamp prior to transection with micro scissors. Contrary to surgical practice in humans, we have found that suture ligation of the hysterectomy pedicles is unnecessary, as clamping the pedicle prior to transection ensures adequate hemostasis. Step 1.3.6 of the protocol describes this careful process to minimize blood loss. As the hysterectomy is being performed, great care should be taken to identify the ureters as mentioned in steps 1.3.6 and 1.3.8. Understanding the anatomical proximity of the ureter is critical, as one of the most common complications associated with the USLs in humans is ureteral injury46.
In conclusion, we present a novel protocol for performing hysterectomy, uterosacral ligament suspension, and tensile testing of the USL in a rat model. We anticipate that our findings will assist future basic science investigators by providing a clear, reproducible description of these procedures and thereby allow for advancement of pelvic organ prolapse research.

Pelvic organ prolapse (POP) is a common pelvic floor disorder affecting millions of women worldwide with the potential to significantly impact many aspects of a woman&#39;s life, particularly with age1. Notably, approximately 13% of women in the United States will undergo surgery for prolapse or urinary incontinence2. A condition most common after pregnancy and childbirth, prolapse is characterized by the descent of pelvic organs, predominantly the various compartments of the vagina and/or uterus, beyond their normal position in the peritoneal cavity. This leads to bothersome symptoms of vaginal bulge or pressure, bowel, bladder, and sexual dysfunction, and overall reduced quality of life. Other risk factors for POP include obesity, tobacco use, chronic cough, and constipation3.
In healthy women, the pelvic floor organs are supported by the levator ani muscles, uterosacral ligaments (USLs), cardinal ligaments, connective tissue attachments to the pelvic sidewall and the distal structures of the perineal body4,5. The USLs are among the most important apical supportive structures for both the uterus and apical vagina, and thus, are often used in surgical correction of POP (Figure 1). Structural support from the USL stems from the dense collagenous connective tissue in the sacral region that transitions into closely packed smooth muscle. Due to this compositional gradient, the USL becomes interwoven with the uterine and vaginal musculature to provide sturdy support for the pelvic organs6,7. In the uterosacral ligament suspension (USLS), the USLs are secured to the vaginal vault following a hysterectomy, restoring the vagina and the surrounding structures to their anatomical position in the abdominal compartment. However, regardless of a transvaginal or laparoscopic route, the USLS procedure is plagued by a relatively high failure rate of up to 40% in some studies8,9. The recurrence rate of bothersome vaginal bulge symptoms at 5 years post-repair for apical compartment prolapse, such as USLs, was approximately 40% in a large multicenter randomized controlled trial9. In the same trial, retreatment for recurrent prolapse at 5 years was approximately 10%. The mechanism of this high failure rate has not been studied, but restoring the vagina and the surrounding structures to their anatomical position requires suture placement in the dense collagenous region of the USL10,11 rather than the smooth muscle region. Therefore, the high failure rate could be due to the mechanical and compositional mismatch of the surgically formed vagina-USL interface compared to the complete integration seen in the native cervical-USL attachment.
The economic impact of treating these disorders is also notable, with approximately $300 million spent annually in the US on ambulatory care12, and more than $1 billion spent annually in direct costs for surgical procedures13. Despite the vast economic resources dedicated to treating these conditions, the complications arising from many prolapse surgeries remain discouraging. For example, polypropylene mesh-based apical prolapse repairs, such as sacrocolpopexy, offer higher success rates compared to native tissue repairs14, but at the cost of potential complications such as mesh exposure or erosion. The FDA received nearly 3,000 complaints related to mesh complications between 2008 and 2010 alone. This culminated in an order by the FDA to halt the manufacture and sale of all transvaginally-placed mesh products for POP in April 201915. Therefore, there is a strong clinical need for materials other than polypropylene, and models with which to test them, that may augment native tissue prolapse repairs and increase success rates compared to traditional techniques with suture alone.
Since the FDA announcement in 2019, most pelvic surgeons have stopped using transvaginally-placed mesh for prolapse repairs, prompting investigators to seek new tissue engineering approaches to augment native tissue repairs16,17,18 such as with mesenchymal stromal cells (MSCs)9,20. With this shift in focus, there is an urgent need for the refinement of animal models that can assist with development of new materials; the challenge in this process is balancing clinical relevance with cost. To this end, basic science and clinical investigators studying pelvic organ prolapse have taken advantage of several animal models thus far, including rats, mice, rabbits, sheep, swine, and non-human primates19. The process of identifying an optimal animal model is challenging, as humans are bipedal, have no tail, and have a traumatic birth process compared to other mammalian species20. Swine21 have been utilized to simulate robotic sacrocolpopexy, while sheep have been used to simulate vaginal prolapse repairs22. These animal models, while clinically relevant, are limited in feasibility by cost and maintenance. Non-human primates have been used to study the pathogenesis of prolapse; squirrel monkeys in particular are one of the only species other than humans that can develop spontaneous prolapse, making them one of the most relevant animal models20. Non-human primates have also been used to study gynecologic surgical procedures such as sacrocolpopexy23 and uterine transplantation24. Similar to their sheep and swine counterparts, the primary limitation of non-human primates as an animal model of prolapse is the cost of maintenance, care, and boarding19.
Although the rodent pelvis is oriented horizontally with a much smaller head-to-birth canal size ratio compared to humans19, rats are suitable for small animal studies of USLS surgery since they have similar USL anatomy, cellularity, histological architecture, and matrix composition compared to the human USL25. Moreover, they are beneficial in terms of maintenance and boarding. Despite these beneficial attributes, there are no published reports of a rat model of USLS repair. Therefore, the aim is to describe a protocol for hysterectomy and USLS in the multiparous Lewis rat. This protocol will be beneficial for investigators who aim to study the pathophysiology and surgical components of POP using this accessible animal model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64311/64311fig01.jpg" /> 
Figure 1: Pelvic organ prolapse. (A) The normal orientation of organs in the peritoneal cavity and (B) the dramatic organ descension when prolapse occurs. Following hysterectomy, (C) uterosacral ligament suspension restores the vagina and surrounding structures to their proper anatomical position. <a href="https://www.jove.com/files/ftp_upload/64311/64311fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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development of a uterosacral ligament suspension rat model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Surgical feasibility and uterosacral suture placement 
There were no intraoperative complications related to hysterectomy or uterosacral ligament suspension in any of the animals. There was minimal bleeding during removal of the uterine horns, provided the adjacent vasculature was clamped prior to removal. Limited bleeding allowed for good visualization of the uterosacral ligaments for suture placement and prevented intra-operative bowel, rectum, ureteral, or bladder injury. Following placement of the sutures, the newly formed USL-vaginal vault junction prevented movement of the cervical/uterine stump as shown in Figure 5C. During the first three postoperative days, the animals were checked on daily, and then bi-weekly basis until the end of the experiment. With the extended-release opioid and NSAID analgesics administered at the time of surgery, additional analgesics were found to be unnecessary. Based on our experience with 16 animal surgeries (n = 8 for both control and USLS groups), a drop in weight should be expected in the first week following surgery with an average loss of 5.7 &#177; 1.4% from surgery day weight. As expected, the rats slowly gained weight over the subsequent 23 weeks, with an average weight gain of 15.1 &#177; 4.5% over the course of the experiment.
Mechanical testing of the USLS repair 
To demonstrate the functionality of the USLS repair, uniaxial tensile testing was performed. After euthanasia of the animal at the chosen post-operative timepoint, 24 weeks in this study, the surgical area should be carefully dissected to visualize the USL-vaginal vault junction as shown in Figure 6A. Compared to other methodologies for testing the rat USLs together with other supportive structures and pelvic organs29,42, the method described here is the first to test the rat USL in an isolated manner. The umbilical tape used in this study was strategically chosen for its flexibility as the tape compliance allowed for minimal disruption of the tissue during tensile testing preparation. Load displacement data, therefore, must be adjusted to account for the small amount of stretch contributed by the umbilical tape. Figure 9 provides an example of data obtained via tensile testing with Figure 9A providing an example of a typical stress-strain plot. Reporting of stress-strain data is recommended as this information is normalized and independent of the size of the specimens34 and can be better compared across studies. For the intact uterosacral ligament, we report structural properties such as ultimate load (2.9 &#177; 0.5 N) and stiffness (0.4 &#177; 0.1 N/mm) as well as normalized material properties such as ultimate stress (2.1 &#177; 0.4 MPa), ultimate strain (1.6 &#177; 0.5) and tangent modulus (4.0 &#177; 1.1 MPa). In the uniaxial tests performed on the rat reproductive organs and all their supportive tissue connections by Moalli et al., they reported an ultimate load at failure (13.2 &#177; 1.1 N) and stiffness (2.9 &#177; 0.9 N/mm) higher than the isolated USL29. The work done by Moalli et al. and other literature34,35 mention the high variability between tested specimens as shown in the data presented here. For the uterosacral ligament suspension repair, we found all structural material properties (stiffness, 0.33 &#177; 0.13 N/mm; ultimate load, 2.6 &#177; 1.3 N) and normalized material properties (ultimate stress, 1.8 &#177; 0.7 MPa; ultimate strain 1.3 &#177; 0.3; tangent modulus, 3.0 &#177; 0.9 MPa) to be lower than that of the native USL.

Watch this Scientific Journal Video about Development of a Uterosacral Ligament Suspension Rat Model at JoVE.com

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

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