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'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="Female pelvic floor diagram; prolapse and uterosacral ligament suspension process illustrated." class="xfigimg" src="/files/ftp_upload/64311/64311fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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>

<table><tbody><tr><td>Alcohol prep pad</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Artificial Tear Ointment</td><td>American Health Service Sales Corp</td><td>PH-PARALUBE-O</td><td></td></tr><tr><td>Bluehill software</td><td>Instron</td><td>Bluehill 3</td><td></td></tr><tr><td>Cavicide 1 disinfectant</td><td>Fisher Scientific</td><td>22 998 800</td><td></td></tr><tr><td>Compression platean</td><td>Instron</td><td>2501-163</td><td></td></tr><tr><td>Cotton swabs</td><td>Puritan Medical</td><td>806-WC</td><td></td></tr><tr><td>Gauze Sponge, 8-Ply</td><td>VWR</td><td>95038-728</td><td></td></tr><tr><td>Mosquito Forceps</td><td>Medline Industries</td><td>MMDS1222115</td><td></td></tr><tr><td>Needle Holder</td><td>Medline Industries</td><td>DYND04045</td><td></td></tr><tr><td>Operating Scissors, 5&amp;frac12;&quot;, Sharp</td><td>American Health Service Sales Corp</td><td>4-222</td><td></td></tr><tr><td>Opioid Analgesic (Buprenorphine XR)</td><td>Fidelis Animal Health</td><td>Ethiqa XR</td><td>0.65&amp;nbsp;mg/kg&amp;nbsp;SC&amp;nbsp;Q72</td></tr><tr><td>NSAID Analgesic (Meloxicam SR)</td><td>Wildlife Pharmaceuticals, LLC</td><td>Meloxicam SR</td><td>1&amp;nbsp;mg/kg&amp;nbsp;SC&amp;nbsp;q72</td></tr><tr><td>PDS II, 3-0 Polydioxanone Suture, SH-1</td><td>Ethicon</td><td>Z316H</td><td></td></tr><tr><td>PDS II, 5-0 P olydioxanone Suture, RB-1</td><td>Ethicon</td><td>Z303H</td><td></td></tr><tr><td>Retractor</td><td>Medline Industries</td><td>MDS1862107</td><td></td></tr><tr><td>Scalpel Blade Stainless Surgical #10</td><td>Miltex</td><td>4-310</td><td></td></tr><tr><td>Scalpel Handle</td><td>Medline Industries</td><td>MDS15210</td><td></td></tr><tr><td>Scissor, Micro, Curved, 4.5&quot;</td><td>Westcott</td><td>MDS0910311</td><td></td></tr><tr><td>Single Column Universal Testing System</td><td>Instron</td><td>5943 S3873</td><td>1 kN force capacity, 10 N load cell</td></tr><tr><td>Sterile Natural Rubber Latex Gloves</td><td>Accutech</td><td>91225075</td><td></td></tr><tr><td>Suture,Vicryl,6-0,P-3</td><td>Ethicon</td><td>J492G</td><td></td></tr><tr><td>Tape,Umbilical,Cotton,1/8X18&quot;</td><td>Ethicon</td><td>U10T</td><td></td></tr><tr><td>Tension and Compression Load Cell</td><td>Instron</td><td>2530-10N</td><td>10N load cell (1 kgf, 2 lbf)</td></tr><tr><td>Veterinary surgical adhesive (skin glue)</td><td>Covetrus</td><td>31477</td><td></td></tr></tbody></table>

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 ...

development-of-a-uterosacral-ligament-suspension-rat-model

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JoVE Journal

Medicine

3.7K Views.  University of Virginia. 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.

Video: Development of a Uterosacral Ligament Suspension Rat Model

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Bioengineering

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Transvaginal Mesh Insertion in the Ovine Model

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms. Parous sheep underwent the dissection of the rectovaginal septum, followed by the insertion of an implant with or without four anchoring arms, both designed to fit the ovine anatomy. The anchoring arms were put in place using a trocar and an "outside-in" technique. The cranial arms were passed through the obturator, gracilis, and adductor magnus muscles. The caudal arms were fixed near the sacrotuberous ligament, through the coccygeus muscles. This technique allows for the mimicking of surgical procedures performed in women suffering from pelvic organ prolapse. The anatomical spaces and elements are easily identified. The most critical part of the procedure is the insertion of the cranial trocar, which can easily penetrate the peritoneal cavity or the surrounding pelvic organs. This can be avoided by a more extensive retroperitoneal dissection and by guiding the trocar more laterally. This approach is designed only for experimental testing of novel implants in large animal models, as trocar-guided insertion is currently not used clinically.

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided ...

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer from AUFI. These women were deprived of the option to have children until the advent of UTx. The clinical application of UTx was driven by experimental studies in animals, and the first successful UTx was achieved in rats. Given their physiological, immunological, genetic, and reproductive characteristics, rats are a suitable model system for such transplants. In particular, their short gestation period is a clear advantage, as the usual endpoint of experimental UTx is successful pregnancy with live birth. The biggest challenge for rat models remains the small anatomy, which requires advanced microsurgical skills and experience. Although UTx has led to pregnancy in the clinic, the procedure is not established and requires continuous experimental optimization. Here, a detailed protocol is presented, including essential troubleshooting for rat UTx, which is expected to make the entire procedure easier to grasp for those without experience in this type of microsurgery.

The present protocol describes all the essential steps for successful uterine transplantation (UTx) in rats. The rat model has proven suitable to promote the clinical implementation of UTx; however, rat UTx is a highly complex procedure requiring careful instructions.

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer ...

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

Effects of Manual Therapy on a Chronic Non-Specific Low Back Pain Murine Model

Manual Therapy for a Chronic Non-Specific Low Back Pain Rat Model

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed with chronic non-specific low back pain (CNLBP) due to an unknown pathological cause. Manual therapy (MT) is an integral aspect of traditional Chinese medicine and is recognized as Tuina in China. It involves techniques like bone-setting and muscle relaxation manipulation. Despite its clinical efficacy in treating CNLBP, the underlying mechanisms of MT remain unclear. In animal experiments aimed at investigating these mechanisms, one of the main challenges is achieving normative MT on CNLBP model rats. Improving the stability of finger strength is a key issue in MT. To address this technical limitation, a standardized procedure for MT on CNLBP model rats is presented in this study. This procedure significantly enhances the stability of MT with the hands and alleviates common problems associated with immobilizing rats during MT. The findings of this study are of reference value for future experimental investigations of MT.

This study proposes a standardized procedure for manual therapy on chronic low back pain model rats, which will be of reference value for future experimental research on manual therapy.

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed ...

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Biology

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

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic floor are supported by different anatomical structures, including muscles, ligaments, and pelvic fascia. The uterosacral ligament (USL) is a critical load-bearing structure, and injury to the USL results in a higher risk of developing POP. The present protocol describes the dissection of murine USLs and the pelvic floor organs alongside the acquisition of unique data on the USL biochemical composition and function using Raman spectroscopy and the evaluation of mechanical behavior. Mice are an invaluable model for preclinical research, but dissecting the murine USL is a difficult and intricate process. This procedure presents an approach to guide the dissection of murine pelvic floor tissues, including the USL, to enable multiple assessments and characterization. This work aims to aid the dissection of pelvic floor tissues by basic scientists and engineers, thus expanding the accessibility of research on the USL and pelvic floor conditions and the preclinical study of women's health using mouse models.

This article presents a detailed protocol for dissecting uterosacral ligaments and other pelvic floor tissues, including the cervix, rectum, and bladder in mice, to expand the study of female reproductive tissues.

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic ...