The work was funded by NSF CAREER award grant #1751050.

Nulliparous 4-6 months female C57BL6J mice (29.4 &#177; 6.8 grams) at estrus were used for this study. All procedures were approved by the Institute Animal Care and Use Committee at Tulane University. After delivery, the mice acclimated for one week before euthanasia and were housed under standard conditions (12-hour light/dark cycles).
1. Mouse sacrifice at estrus
<ol>
	<li>Determine the estrous cycle: The estrous cycle was monitored by visual assessment in accordance to previous studies<su...

The protocol provided in this article presents a method for determining the mechanical properties of the murine vagina and cervix. The mechanical properties analyzed in this protocol include both the passive and basal tone conditions of the organs. Passive and basal tone conditions are induced by altering the biochemical environment in which the organ is submerged. For this protocol, the media involved in basal testing contains calcium. Testing the basal tone condition permits isolation of the smooth muscle cell mechanic...

The vaginal wall is composed of four layers, the epithelium, lamina propria, muscularis, and adventitia. The epithelium is primarily composed of epithelial cells. The lamina propria has a large amount of elastic and fibrillar collagen fibers. The muscularis is also composed of elastin and collagen fibers but has an increased amount of smooth muscle cells. The adventitia is comprised of elastin, collagen, and fibroblasts, albeit in reduced concentrations compared to the previous layers. The smooth muscle cells are of interest to biomechanically motivated research groups as they play a role in the contractile nature of the organs. As such, quantifying the smooth muscle cell area fraction and organization is key to understanding the mechanical function. Previous investigations suggest that the smooth muscle content within the vaginal wall is primarily organized in the circumferential and longitudinal axis. Histological analysis suggests that the smooth muscle area fraction is approximately 35% for both the proximal and distal sections of the wall1.
The cervix is a highly collagenous structure, that until recently, was thought to have minimal smooth muscle cell content2,3. Recent studies, however, have suggested that smooth muscle cells may have a greater abundance and role in the cervix4,5. The cervix exhibits a gradient of smooth muscle cells. The internal os contains 50-60% smooth muscle cells where the external os only contains 10%. Mouse studies, however, report the cervix to be composed of 10-15% smooth muscle cells and 85-90% fibrous connective tissue with no mention of regional differences6,7,8. Given that the mouse model differs from the frequently reported human model, further investigations concerning the mouse cervix are needed.
The purpose of this protocol was to elucidate the mechanical properties of the murine vagina and cervix. This was accomplished by using a pressure myograph device that enables assessment of mechanical properties in the circumferential and axial directions simultaneously while maintaining native cell-matrix interactions and organ geometry. The organs were mounted on two custom cannulas and secured with silk 6-0 sutures. Pressure-diameter tests were performed around the estimated physiological axial stretch to determine the compliance and tangent moduli9. Force-length tests were conducted to confirm the estimated axial stretch and to ensure that mechanical properties were quantified in the physiological range. The experimental protocol was performed on the nonpregnant murine vagina and cervix at 4-6 months of age in estrus.
The protocol is divided into two main mechanical testing sections: basal tone and passive testing. A basal tone is defined as the baseline partial constriction of smooth muscle cells, even in the absences of external local, hormonal, and neural stimulation10. This baseline contractile nature of the vagina and cervix yields characteristic mechanical behaviors which are then measured by the pressure myograph system. The passive properties are assessed by removing the intercellular calcium that maintains the baseline state of contraction, resulting in relaxation of the smooth muscle cells. In the passive state, collagen and elastin fibers provide the dominant contributions for the mechanical characteristics of the organs.
The murine model is used extensively to study pathologies in women&#8217;s reproductive health. The mouse offers several advantages for quantifying the evolving relationships between ECM and mechanical properties within the reproductive system11,12,13,14. These advantages include short and well-characterized estrous cycles, relatively low cost, ease of handling, and a relatively short gestational time15. Additionally, the genome of laboratory mice is well-mapped and genetically-modified mice are valuable tools to test mechanistic hypotheses16,17,18.
Commercially available pressure myograph systems are used extensively to quantify the mechanical responses of various tissues and organs. Some notable structures analyzed on the pressure myograph system include elastic arteries19,20,21,22, veins and tissue engineered vascular grafts23,24, the esophagus25, and the large intestines26. The pressure myograph technology permits simultaneous assessment of properties in the axial and circumferential directions while maintaining the native cell-ECM interactions and in vivo geometry. Despite the extensive use of myograph systems in soft tissue and organ mechanics, a protocol utilizing the pressure myograph technology had not previously been developed for the vagina and cervix. Prior investigations into the mechanical properties of the vagina and cervix were assessed uniaxially27,28. These organs, however, experience multiaxial loading within the body29,30, thus quantifying their biaxial mechanical response is important.
Moreover, recent work suggests smooth muscle cells may play a potential role in soft tissue pathologies5,28,31,32. This provides another attraction of utilizing the pressure myograph technology, as it preserves the native cell-matrix interactions, thus permitting delineation of the contribution that smooth muscle cells play in physiological and pathophysiological conditions. Herein, we propose a protocol to quantify the multiaxial mechanical properties of the vagina and cervix under both basal tone and passive conditions.

<table>
	<tbody>
		<tr>
			<td>2F catheter</td>
			<td>Millar</td>
			<td>SPR-320</td>
			<td>catheter to measure cervical pressure</td>
		</tr>
		<tr>
			<td>6-0 Suture</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
			<td>larger suture ties</td>
		</tr>
		<tr>
			<td>CaCl2 (anhydrous)</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td>HBSS concentration: 140 mg/ mL</td>
		</tr>
		<tr>
			<td>CaCl2-2H20</td>
			<td>Fischer chemical</td>
			<td>BDH9224-1KG</td>
			<td> 
			KRB concentration: 3.68 g/L</td>
		</tr>
		<tr>
			<td>Dextrose (D-glucose)</td>
			<td>VWR</td>
			<td>101172-434</td>
			<td>HBSS concentration: 1000 mg/mL 
			KRB concentration: 19.8 g/L</td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>curved forceps</td>
		</tr>
		<tr>
			<td>Dumont SS Forceps</td>
			<td>Fine Science Tools</td>
			<td>11203-25</td>
			<td>straight forceps</td>
		</tr>
		<tr>
			<td>Eclipse</td>
			<td>Nikon</td>
			<td>E200</td>
			<td>microscope used for imaging</td>
		</tr>
		<tr>
			<td>Flow meter</td>
			<td>Danish MyoTechnologies</td>
			<td>161FM</td>
			<td>flow meter within the testing apparatus</td>
		</tr>
		<tr>
			<td>Force Transducer - 110P</td>
			<td>Danish MyoTechnologies</td>
			<td>100079</td>
			<td>force transducer</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>SciJava</td>
			<td>ImageJ1</td>
			<td>used to measure volume</td>
		</tr>
		<tr>
			<td>Instrument Cases</td>
			<td>Fine Science Tools</td>
			<td>20830-00</td>
			<td>casing to hold dissection tools</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Chemical</td>
			<td>97061-566</td>
			<td>HBSS concentration: 400 mg/ mL 
			KRB concentration: 3.5 g/L</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>G-Biosciences</td>
			<td>71003-454</td>
			<td>HBSS concentration: 60 mg/ mL</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>VWR</td>
			<td>97064-150</td>
			<td> 
			KRB concentration: 1.14 g/L</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>VWR</td>
			<td>BDH9244-500G</td>
			<td>HBSS concentration: 100 mg/ mL</td>
		</tr>
		<tr>
			<td>MgSO4-7H20</td>
			<td>VWR</td>
			<td>97062-134</td>
			<td>HBSS concentration: 48 mg/ mL</td>
		</tr>
		<tr>
			<td>Mircosoft excel</td>
			<td>Microsoft</td>
			<td>6278402</td>
			<td>program used for spreadsheet</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic anhydrous)</td>
			<td>VWR</td>
			<td>97061-588</td>
			<td>HBSS concentration: 48 mg/mL 
			KRB concentration: 1.44 g/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>97061-274</td>
			<td>HBSS concentration: 8000 mg/mL 
			KRB concentration: 70.1 g/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>VWR</td>
			<td>97062-460</td>
			<td>HBSS concentration: 350 mg/ mL 
			KRB concentration: 21.0 g/L</td>
		</tr>
		<tr>
			<td>Pressure myograph systems</td>
			<td>Danish MyoTechnologies</td>
			<td>110P and 120CP</td>
			<td>Pressure myograph system: 
			prorgram, cannulation device, 
			and controller unit</td>
		</tr>
		<tr>
			<td>Pressure Transducer</td>
			<td>Danish MyoTechnologies</td>
			<td>100106</td>
			<td>pressure transducer</td>
		</tr>
		<tr>
			<td>Student Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>straight forceps</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td>micro-scissors</td>
		</tr>
		<tr>
			<td>Tissue dye</td>
			<td>Bradley Products</td>
			<td>1101-3</td>
			<td>ink to measure in vivo stretch</td>
		</tr>
		<tr>
			<td>Ultrasound transducer</td>
			<td>FujiFilm Visual Sonics</td>
			<td>LZ-550</td>
			<td>ultrasound transducer used; 256 elements, 40 MHz center frequency</td>
		</tr>
		<tr>
			<td>VEVO2100</td>
			<td>FujiFilm Visual Sonics</td>
			<td>VS-20035</td>
			<td>ultrasound used for imaging</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>Fine Science Tools</td>
			<td>14069-12</td>
			<td>larger scissors</td>
		</tr>
	</tbody>
</table>

biaxial basal tone and passive testing of the murine reproductive system using a pressure myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

Successful analysis of the mechanical properties of the female reproductive organs is contingent on appropriate organ dissection, cannulation, and testing. It is imperative to explant the uterine horns to the vagina without any defects (Figure 1). Depending on the organ type, the cannula size will vary (Figure 2). Cannulation must be done so that the organ cannot move during the experiment but also not damage the wall of the organ during the procedure (Figure 3</...

Watch this Scientific Journal Video about Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph at JoVE.com

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

This protocol investigates the biaxial mechanical properties of the reproductive organs elucidating the contribution from the smooth muscle cells and the passive matrix. This technique maintains organ geometry and native smooth muscle cell to matrix interactions permitting subjection of the organ to a physiologic range of pressures and axial extensions. Investigating the organ biaxial function under physiologically relevant loads may aid in a better understanding of the etiology of reproductive pathologies.Biaxially testing the cervix and vagina provides insight into the fields of women's health and biomechanics. Similar methods are utilized to study blood vessels, the esophagus and large intestines. When working with a new organ, it may take time to finalize the technical details such as how to best suture the tissue or how to determine the unloaded length.To collect the reproductive system from the experimental animal, place the mouse on an absorbent pad under a dissecting microscope and use angled tweezers and scissors to lift the skin around the abdomen. Make an initial incision at the base of the abdomen above the pubic bone shallow enough to not puncture the abdominal muscle wall and continue the incision superiorly toward the rib cage and deep through the abdominal muscles. After removing the superficial fat and carefully cutting the pubic symphysis, use straight tweezers to grasp the bladder to create tension and use blunt dissection to separate the surrounding tissue from the vagina.Then cut the base of the reproductive system to remove the tissues from the body cavity. After determining the appropriate size cannula for the harvested system, mount the cervical side on the force transducer portion of the cannulation device and mount the opposite end of the organ on the micrometer portion of the device. Then tighten both ends with sutures.To find the unloaded length of the mounted tissue, first stretch the organ so that the wall is not in tension. For the vagina, observe the grooves on the vaginal wall. For the cervix, cut immediately below the ink dots located above and below the central cervix mark.Use calipers to measure the length of the system from suture to suture and increase the pressure of the myograph system from zero to 10 millimeters of mercury in one millimeter of mercury increments. The pressure in which the organ is no longer collapsed can be determined as the largest jump in the outer diameter at a given pressure. After recording the pressure and the outer diameter, note these data as the first point wherein the organ is not collapsed and zero the force.To find the experimental in vivo stretch, adjust the organ to the estimated in vivo length while at the unloaded pressure and click start to assess the pressure versus force values for the pressure values ranging from the unloaded pressure to the maximum pressure. Then click stop and save the file. For pressure diameter pre-conditioning, set the pressure to zero millimeters of mercury, the length to the experimental in vivo length, and the gradient to 1.5 millimeters of mercury per second.Run a sequence that takes the pressure from zero millimeters of mercury to the maximum pressure plus the unloaded pressure, hold for 30 seconds and returns to zero millimeters of mercury with an additional 30-second hold period. After repeating this sequence for a total of five cycles, click stop and save the file. For force length pre-conditioning, enter 1/3 of the maximum pressure plus the unloaded pressure for both the inlet and outlet pressures and adjust the organ to minus 2%of the in vivo length.Click start and adjust the length to plus 2%in vivo length and back down to minus 2%at a 10 micrometer per second rate. Repeat the axial extension for a total of five cycles before clicking stop and saving the file. For pressure diameter testing of the in vivo length, click start and adjust the organ to the in vivo length, set the pressure to zero millimeters of mercury, and set the gradient to 1.5 millimeters of mercury per second.Increase the pressure from zero millimeters of mercury to the maximum pressure before bringing the pressure back down to zero millimeters of mercury with a 20-second hold period. Then repeat the test for five cycles before stopping the system and saving the data. For force length testing of 1/3 of the maximum pressure plus the unloaded pressure, set the pressure to 1/3 of the maximum pressure plus the unloaded pressure and adjust the organ to minus 2%of the in vivo length.After clicking start, stretch the organ to plus 2%of the in vivo length and back to minus 2%of the in vivo length at a rate of 10 micrometers per second. Then repeat the test for a total of three cycles before stopping the system and saving the data. After the last test, remove the KRB testing medium and wash the organ with calcium-free KRB medium.After the wash, incubate the organ with fresh calcium-free KRB solution supplemented with two millimolar EGTA for 30 minutes before replacing the treatment solution with fresh calcium-free KRB. At the completion of the basal tone testing, for pressure diameter pre-conditioning, click start and set the pressure to zero millimeters of mercury, the length as the estimated in vivo length, and the gradient to 1.5 millimeters of mercury per second. Begin running a sequence that takes the pressure from zero millimeters of mercury to the maximum pressure and back to zero millimeters of mercury.Repeat this process through five cycles with a 30-second hold time. For force length pre-conditioning, adjust the organ to the in vivo length and manually enter the unloaded pressure for both pressures. Click start and set the gradient to 1.5 millimeters of mercury and the pressure to 1/3 of the maximum.Stretch the organ up to plus 2%and back down to minus 2%stretch at 10 micrometers per second and repeat the cycle for a total of five times. For pressure diameter testing, with the organ at minus 2%of the experimentally determined in vivo length and the pressure at zero millimeters of mercury, click start and increase the pressure from zero millimeters of mercury to the maximum pressure and back to zero millimeters of mercury. Hold the zero millimeters of mercury step for 20 seconds and repeat the cycle five times.For force length testing, set the pressure to a nominal pressure and adjust the organ to minus 2%of the in vivo length. Stretch the organ up to plus 2%of the in vivo length and back to minus 2%of the in vivo length at a rate of 10 micrometers per second. After a total of three cycles, save the data and repeat the force length testing for 1/3 of the maximum pressure, 2/3 of the maximum pressure, and at the maximum pressure.For a successful analysis of the mechanical properties of the female reproductive organs, it is imperative to explant the uterine horns to the vagina without any defects. Depending on the organ type, the cannula size will vary. The cannulation must be performed so that the organ cannot move during the experiment but without damaging the wall of the organ during the procedure.The pressure myograph system can be used to monitor various aspects of the organ as it undergoes mechanical testing and the ultrasound system can be used to measure the thickness of the organs in the unloaded state with and without basal tone. After mechanical testing, the tangent moduli may be calculated for the circumferential and axial directions. Both basal tone testing and passive testing yield key mechanical properties of the reproductive tract with and without the contractile contribution of smooth muscle cells.Scaling between the organs requires a few adjustments to the protocols as the cervix and vagina experience different loads in vivo. It is key to perform the dissection quickly to maintain the viability of smooth muscle cells but carefully to not puncture the desired organ. Opening angle experiments which measure the residual strain of the organ may be performed after mechanical testing procedures as well as various histological and biochemical assays.After its development and investigation of the smooth muscle cell basal contribution, this technique also permits evaluating the reproductive system's smooth muscle maximum contractile response under physiologically relevant loads.

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

Abstract