Name: isolation and characterization of the murine uterosacral ligaments and pelvic floor organs
Uploaded: 2023-03-03T13:25:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs at JoVE.com

This work was supported by the CU Boulder Summer Underground Research Opportunities Program (UROP) grant (C.B.), the NSF Graduate Research Fellowship (L.S.), the Schmidt Science Fellowship (C.L.), the University of Colorado Research &#38; Innovation Seed Grant Program (2020 award to V.F., S.C., and K.C.), and the Anschutz Boulder Nexus Seed Grant at the University of Colorado (to V.F. and K.C.). Special acknowledgment goes to Dr. Tyler Tuttle for help with the loading chamber design as well as the Calve lab members for helpful discussions.

All animal experiments and procedures were performed according to protocol #2705, approved by the Animal Care and Use Committee of the University of Colorado Boulder. Six week old female C57BL/6J mice were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Animal preparation
<ol>
	<li>Euthanize the animal following the institutionally approved method. 
	NOTE: The present study used CO2 ...

<ol>
	<li>Maldonado, P. A., Wai, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelvic+organ+prolapse.">Pelvic organ prolapse.</a> Obstetrics and Gynecology Clinics of North America. 43 (1), 15-26 (2016).</li><li>Drewes, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelvic+organ+prolapse+in+fibulin-5+knockout+mice:+Pregnancy-induced+changes+in+elastic+fiber+homeostasis+in+mouse+vagina.">Pelvic organ prolapse in fibulin-5 knockout mice: Pregnancy-induced changes in elastic fiber homeostasis in mouse vagina.</a> American Journal of Pathology. 170 (2), 578-589 (2007).</li><li>Barber, M. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelvic+organ+prolapse+in+fibulin-5+knockout+mice.">Pelvic organ prolapse in fibulin-5 knockout mice.</a> The American Journal of Pathology. 170 (2), 578-589 (2007).</li><li>Rahn, D. D., Ruff, M. D., Brown, S. A., Tibbals, H. F., Word, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+properties+of+the+vaginal+wall:+Effect+of+pregnancy,+elastic+fiber+deficiency,+and+pelvic+organ+prolapse.">Biomechanical properties of the vaginal wall: Effect of pregnancy, elastic fiber deficiency, and pelvic organ prolapse.</a> American Journal of Obstetrics and Gynecology. 198 (5), 1-6 (2008).</li><li>Roman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+alternative+materials+for+the+treatment+of+stress+urinary+incontinence+and+pelvic+organ+prolapse:+A+comparison+of+the+in+vivo+response+to+meshes+implanted+in+rabbits.">Evaluating alternative materials for the treatment of stress urinary incontinence and pelvic organ prolapse: A comparison of the in vivo response to meshes implanted in rabbits.</a> Journal of Urology. 196 (1), 261-269 (2016).</li><li>Couri, B. M., Lenis, A. T., Borazjani, A., Paraiso, M. F., Damaser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+female+pelvic+organ+prolapse:+Lessons+learned.">Animal models of female pelvic organ prolapse: Lessons learned.</a> Expert Review of Obstetrics &amp; Gynecology. 7 (3), 49 (2012).</li><li>Abramowitch, S. D., Feola, A., Jallah, Z., Moalli, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+mechanics,+animal+models,+and+pelvic+organ+prolapse:+A+review.">Tissue mechanics, animal models, and pelvic organ prolapse: A review.</a> European Journal of Obstetrics &amp; Gynecologyand Reproductive Biology. 144, S146-S158 (2009).</li><li>Tan, T., Cholewa, N. M., Case, S. W., De Vita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-structural+and+biaxial+creep+properties+of+the+swine+uterosacral-cardinal+ligament+complex.">Micro-structural and biaxial creep properties of the swine uterosacral-cardinal ligament complex.</a> Annals of Biomedical Engineering. 44 (11), 3225-3237 (2016).</li><li>Tan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histo-mechanical+properties+of+the+swine+cardinal+and+uterosacral+ligaments.">Histo-mechanical properties of the swine cardinal and uterosacral ligaments.</a> Journal of the Mechanical Behavior of Biomedical Materials. 42, 129-137 (2015).</li><li>Baah-Dwomoh, A., Alperin, M., Cook, M., De Vita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+the+uterosacral+ligament:+Swine+vs.+human.">Mechanical analysis of the uterosacral ligament: Swine vs. human.</a> Annals of Biomedical Engineering. 46 (12), 2036-2047 (2018).</li><li>Vardy, M. 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The effect of structural damage on female reproductive tissues is understudied, and an easily accessible animal model for POP research is needed. The mouse is a cost-effective model that can mimic human reproductive studies16. Due to the growing interest in the study of the female reproductive system, there is a need for methods that aid the study of these tissues. To address this need, in this work, a method is established to dissect and prepare murine pelvic floor tissues for structural and func...

Approximately 50% of women are affected by pelvic organ prolapse (POP)1,2. About 11% of these women fit the criteria for undergoing surgical repair, which has a poor success rate (~30%)3,4. POP is characterized by the descent of any or all of the pelvic organs (i.e., bladder, uterus, cervix, and rectum) from their natural position due to the failure of the USL and the pelvic floor muscles to provide adequate support5. This condition involves anatomical dysfunction and disruption of the connective tissue, as well as neuromuscular injury, in addition to predisposing factors3,6. POP is associated with multiple factors such as age, weight, parity, and delivery type (i.e., vaginal or caesarian births). These factors are thought to affect the mechanical integrity of all the pelvic floor tissues, with pregnancy and parity thought to be the main drivers of POP5,7,8. 
The uterosacral ligaments (USLs) are important supportive structures for the uterus, cervix, and vagina and tether the cervix to the sacrum4. Damage to the USLs puts women at increased risk of developing POP. It is believed that pregnancy and childbirth impose additional strain on the USL, which potentially induces injury and increases the chances of POP. The USL is a complex tissue composed of smooth muscle cells, blood vessels, and lymphatics distributed heterogeneously along the ligament, which can be divided into three distinct sections: cervical, intermediate, and sacral region9. The mechanical integrity of the USL is derived from extracellular matrix (ECM) components like collagens, elastin, and proteoglycans5,9,10. Type I collagen fibers are known to be a major load-bearing tensile component of ligamentous tissues and are, therefore, likely involved in USL failure and POP11. 
There is a lack of knowledge regarding the causes, prevalence, and effects of POP in women. The development of an appropriate animal model of POP is necessary to advance our understanding of the female pelvic floor. Mice and humans have similar anatomical landmarks within the pelvis, such as the ureters, rectum, bladder, ovaries, and round ligaments9, as well as similar intersection points of the USL with the uterus, cervix, and sacrum. Further, mice offer ease of genetic manipulation and have the potential to be an easily accessible, cost-effective model for the study of POP9. 
This study developed a method to access and isolate the USL and the different pelvic floor tissues from nulliparous (i.e., never pregnant) mice. The extracted USLs were subjected to enzymatic digestion (i.e., to remove collagens and glycosaminoglycans), tested to determine the mechanical response under tensile loading, and evaluated for biochemical composition in a proof-of-concept study. The ability to isolate intact tissues will facilitate further mechanical and biochemical characterizations of the pelvic floor components, which is a crucial first step toward improving our understanding of the injury risks related to childbirth, pregnancy, and POP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>11 Blade</td>
			<td>Fisher</td>
			<td>3120030</td>
			<td>Removable blade</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Fisher</td>
			<td>BP399-1</td>
			<td>Diluted from 10x concentration</td>
		</tr>
		<tr>
			<td>Chondroitinase ABC</td>
			<td>Sigma</td>
			<td>C3667-10UN</td>
			<td>Enzyme&#160;</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004194</td>
			<td>Enzyme&#160;</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica</td>
			<td>STELLARIS 5</td>
			<td>Upright configuration</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Leica</td>
			<td>S9E</td>
			<td>With camera</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fisher</td>
			<td>NC9626652</td>
			<td>Thin tip</td>
		</tr>
		<tr>
			<td>Female C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>strain #: 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoTools Micromanipulator</td>
			<td>FemtoTools</td>
			<td>FT-RS1002</td>
			<td>100 mN load cell</td>
		</tr>
		<tr>
			<td>FST Curved Forceps</td>
			<td>Fisher</td>
			<td>NC9639443</td>
			<td>Curved tip</td>
		</tr>
		<tr>
			<td>FST Sharp 9 mm Scissors&#160;</td>
			<td>Fisher</td>
			<td>NC9639443</td>
			<td>Dissection scissors</td>
		</tr>
		<tr>
			<td>Ghost Dye 780&#160;</td>
			<td>Tonbo</td>
			<td>13-0865-T500</td>
			<td>Free amine stain</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher</td>
			<td>06-666</td>
			<td>Box of 50 wipes</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue Tek</td>
			<td>4583</td>
			<td>Used for tissue preservation</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Thermo Fisher</td>
			<td>044764.AK</td>
			<td>Follow manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Petri Dishes 35 mm</td>
			<td>Fisher</td>
			<td>FB0875711A</td>
			<td>Used for dissected tissue</td>
		</tr>
		<tr>
			<td>Polyglactin 5-0 Suture</td>
			<td>Veter.Sut</td>
			<td>VS385VL</td>
			<td>With needle</td>
		</tr>
		<tr>
			<td>Renishaw InVia Raman Microscope</td>
			<td>Renishaw</td>
			<td>PN192(EN)-02-A</td>
			<td>With confocal objectives</td>
		</tr>
		<tr>
			<td>Rocking Platform</td>
			<td>VWR</td>
			<td>10127-876</td>
			<td>2 tier platform</td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Fisher</td>
			<td>52818</td>
			<td>For dissection&#160;</td>
		</tr>
		<tr>
			<td>Sytox</td>
			<td>Thermo Fisher</td>
			<td>S11381</td>
			<td>Nuclear stain&#160;</td>
		</tr>
		<tr>
			<td>T-pins</td>
			<td>Fisher</td>
			<td>S99385</td>
			<td>For dissection&#160;</td>
		</tr>
		<tr>
			<td>Transfer Pipets</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td>For dissection&#160;</td>
		</tr>
		<tr>
			<td>Underpads</td>
			<td>Fisher</td>
			<td>22037950</td>
			<td>To cover dissection pad</td>
		</tr>
	</tbody>
</table>

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.

Each step of the dissection of a wild-type mouse is detailed in the associated video and figures related to the protocol. For this study, 6 week old female C57BL/6J mice were used (Supplementary Table 1). Three sample groups with USLs treated with different enzymes were analyzed: control (no treatment), collagenase-treated, and chondroitinase-treated groups. The smooth muscle, nerves, and lymphatics in the USL are surrounded by an ECM rich in fibrillar collagens and glycosaminoglycans (GAGs)<sup class="x...

Watch this Scientific Journal Video about Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs at JoVE.com

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

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 ...

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.

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JoVE Journal

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