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. D., Maher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcome+assessment+of+pelvic+organ+prolapse.">Epidemiology and outcome assessment of pelvic organ prolapse.</a> International Urogynecology Journal and Pelvic Floor Dysfunction. 24 (11), 1783-1790 (2013).</li><li>Becker, W. R., 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=Biaxial+mechanical+properties+of+swine+uterosacral+and+cardinal+ligaments.">Biaxial mechanical properties of swine uterosacral and cardinal ligaments.</a> Biomechanics and Modeling in Mechanobiology. 14 (3), 549-560 (2015).</li><li>Donaldson, K., Huntington, A., 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=Mechanics+of+uterosacral+ligaments:+Current+knowledge,+existing+gaps,+and+future+directions.">Mechanics of uterosacral ligaments: Current knowledge, existing gaps, and future directions.</a> Annals of Biomedical Engineering. 49 (8), 1788-1804 (2021).</li><li>Amundsen, C. L., Flynn, B. J., Webster, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+correction+of+vaginal+vault+prolapse+by+uterosacral+ligament+fixation+in+women+who+also+require+a+pubovaginal+sling.">Anatomical correction of vaginal vault prolapse by uterosacral ligament fixation in women who also require a pubovaginal sling.</a> Journal of Urology. 169 (5), 1770-1774 (2003).</li><li>Jelovsek, J. E., Maher, C., Barber, M. D. <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> The Lancet. 396 (9566), 1027-1038 (2007).</li><li>Blomquist, J. L., Mu&#241;oz, A., Carroll, M., Handa, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+delivery+mode+with+pelvic+floor+disorders+after+childbirth.">Association of delivery mode with pelvic floor disorders after childbirth.</a> Journal of the American Medical Association. 320 (23), 2438-2447 (2018).</li><li>Iwanaga, R., 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=Comparative+histology+of+mouse,+rat,+and+human+pelvic+ligaments.">Comparative histology of mouse, rat, and human pelvic ligaments.</a> International Urogynecology Journal. 27 (11), 1697-1704 (2016).</li><li>Zhu, Y. P., 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=Evaluation+of+extracellular+matrix+protein+expression+and+apoptosis+in+the+uterosacral+ligaments+of+patients+with+or+without+pelvic+organ+prolapse.">Evaluation of extracellular matrix protein expression and apoptosis in the uterosacral ligaments of patients with or without pelvic organ prolapse.</a> International Urogynecology Journal. 32 (8), 2273-2281 (2021).</li><li>Jimenez, J. M., 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=Multiscale+mechanical+characterization+and+computational+modeling+of+fibrin+gels.">Multiscale mechanical characterization and computational modeling of fibrin gels.</a> bioRxiv. , (2022).</li><li>Fischenich, K. M., 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=Human+articular+cartilage+is+orthotropic+where+microstructure,+micromechanics,+and+chemistry+vary+with+depth+and+split-line+orientation.">Human articular cartilage is orthotropic where microstructure, micromechanics, and chemistry vary with depth and split-line orientation.</a> Osteoarthritis and Cartilage. 28 (10), 1362-1372 (2020).</li><li>Luetkemeyer, C. M., Neu, C. P., Calve, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+defining+tissue+injury+criteria+reveals+ligament+deformation+thresholds+are+multimodal.">A method for defining tissue injury criteria reveals ligament deformation thresholds are multimodal.</a> bioRxiv. , (2023).</li><li>O'Brien, C. M., 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=In+vivo+Raman+spectroscopy+for+biochemical+monitoring+of+the+human+cervix+throughout+pregnancy.">In vivo Raman spectroscopy for biochemical monitoring of the human cervix throughout pregnancy.</a> American Journal of Obstetrics and Gynecology. 218 (5), 1-18 (2018).</li><li>Louwagie, E. M., 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=et+al.+ultrasonic+dimensions+and+parametric+solid+models+of+the+gravid+uterus+and+cervix.">et al. ultrasonic dimensions and parametric solid models of the gravid uterus and cervix.</a> PLoS One. 16 (1), 0242118 (2021).</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.">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. D., 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=The+effects+of+hormone+replacement+on+the+biomechanical+properties+of+the+uterosacral+and+round+ligaments+in+the+monkey+model.">The effects of hormone replacement on the biomechanical properties of the uterosacral and round ligaments in the monkey model.</a> American Journal of Obstetrics and Gynecology. 192 (5), 1741-1751 (2005).</li><li>Shahryarinejad, A., Vardy, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+to+macaque+uterosacral-cardinal+ligament+complex+and+its+relationship+to+pelvic+organ+prolapse.">Comparison of human to macaque uterosacral-cardinal ligament complex and its relationship to pelvic organ prolapse.</a> Toxicological Pathology. 36 (7), 101 (2008).</li><li>Smith, T. M., Luo, J., Hsu, Y., Ashton-Miller, J., DeLancey, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+to+measure+in+vivo+uterine+suspensory+ligament+stiffness.">A novel technique to measure in vivo uterine suspensory ligament stiffness.</a> American Journal of Obstetrics and Gynecology. 209 (5), 1-7 (2013).</li><li>Vandamme, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+rodents+as+models+for+human+diseases.">Use of rodents as models for human diseases.</a> Journal of Pharmacy and Bioallied Sciences. 6 (1), 2-9 (2014).</li></ol>

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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Research

JoVE Journal

Biology

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...