Name: biaxial basal tone and passive testing of the murine reproductive system using a pressure myograph
Uploaded: 2019-08-13T15:00:00
Description: Watch this Scientific Journal Video about Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph at JoVE.com

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). ...

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