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). 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’s health pathologies.
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’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.
Nulliparous 4-6 months female C57BL6J mice (29.4 ± 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
2. Reproductive system dissection
3. Cannulating
4. Pressure myograph set up
5. Basal tone mechanical testing
NOTE: The cervix exhibited a phasic nature during the beginning stages of testing. However, this diminished after preconditioning. Basal tone testing is done utilizing Krebs Ringer Buffer (KRB) in the basin of the DMT device. The buffer is aerated with 95% O2 and 5% CO2. After the basal tone portion is complete, calcium free KRB is utilized.
6. Passive mechanical testing
NOTE: If starting with passive testing start at step 1. If basal tone testing was performed prior to passive start at step 6. If starting with frozen tissue, allow a 30-minute equilibration period at room temperature before cannulating the organ.
7. Clean up
8. Data analysis
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). Failure of either step will result in inability of the vessel to hold pressure. Testing procedure standardization is vital to the success of the protocol in order to yield consistent and repeatable results.
Once the organ is dissected and cannulated properly, power on the pressure myograph system. The setup of the pressure myograph systems involves a controller unit, flow meter, and stage (Figure 4). The pressure myograph system is used to monitor various aspects of the organ as it undergoes mechanical testing (Figure 5). An ultrasound system, or equivalent, is used to measure the thickness of the organs in the unloaded state with and without basal tone (Figure 6). After mechanical testing, the tangent moduli may be calculated for the circumferential and axial directions (Table 2).
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 (Figure 7, Figure 8). Scaling between the organs requires a few adjustments to the protocols (Table 1), as the cervix and vagina experience different loads in vivo46-48. Such variations may be monitored through techniques such as pressure catherization. Pressure catherization is a method used previously to monitor the in vivo conditions within the vagina and uterus49-53. Models in the previous studies range from mice, rabbits, and humans. The same principles would apply similarly to the cervical and vaginal pressure specific for the murine model. Though, regardless which organ is being tested, the same materials are needed for the protocols (Table 3).
Figure 1: Murine dissection diagram. The mouse dissection for the reproductive organs: both uterine horns, cervix, and the vagina. In the figure, the bladder and urethra are removed from the anterior of the vagina. The intestines and abdominal muscles were reflected superiorly. Please click here to view a larger version of this figure.
Figure 2: Size comparison of the two cannula. Size comparison of the two cannulas used for cannulation of the reproductive organs. The larger cannula (D = 3.75 mm) is used for the vaginal tissue (A). The smaller cannula (D = 0.75 mm) is used for cannulating cervical tissue (B). The cervical cannula is smooth while the vaginal cannula has two grooves. Please click here to view a larger version of this figure.
Figure 3: Cannulation method for vagina and cervix. Due to the varying geometry and thickness of the reproductive organs, they are most effectively cannulated in distinct manners. For the vagina, place two sutures in an “X” fashion. When cannulating the cervix, place 3 horizontal sutures on the uterine end and 4 sutures on the external os. Please click here to view a larger version of this figure.
Figure 4: Setup for pressure myograph device. The setup of the DMT device utilized for both basal and passive testing. The DMT is composed of three main hubs: the stage (A), controller unit (B), and flow meter (C). Within the controller unit, there is a reservoir bottle and a waste bottle. The reservoir bottle is initially filled with fluid that empties as the experiment is carried out. The waste bottle, which is initially empty, collects the fluid that runs through the experiment. The controller unit interfaces with the DMT software on the computer and controls the pressure, temperature, and flow. The controller unit reads the outputs from the force and pressure transducers within the stage through a VGA interface cable. The stage component of the system contains an inlet and outlet flow of the system. The inlet and outlet flow have corresponding inlet and outlet pressures measured by the system. Please click here to view a larger version of this figure.
Figure 5: File setup on the pressure myograph program. Display of computer software set-up. A box is drawn around the region of interest and outer diameter of the tissue is optically tracked in real-time (A). Data obtained during mechanical testing is recorded and displayed real-time in the outer diameter, inlet pressure, outlet pressure, mean pressure, force, temperature, pH, and flow tab (B). Within the pressure interface pressure (mmHg), gradient (mmHg/s), and flow is controlled. Further, the axial force (mN) measured by the in-line force transducer is displayed. Flow rate (µL/min) is reported in the flow meter tab (C). Pressure sequencing is shown and controlled in the sequencer tab (D). Data recorded during mechanical testing is recorded and displayed real-time in the outer diameter, inlet pressure, outlet pressure, mean pressure, force, temperature, pH, and flow tab (E). A representative Pressure Diameter test of the vagina is displayed showing outer diameter as a function of time on the outer diameter tab. Please click here to view a larger version of this figure.
Figure 6: Ultrasound Imaging. Ultrasound imaging of the murine reproductive organs. All images were taken using the ultrasound system on the short-axis-B mode. A representative image of the vagina at the unloaded length and pressure (A). Vaginal wall thickness was calculated in ImageJ. A vertical line was drawn along the depth scale (mm) to calibrate the number of pixels per µm. The polygon tool was used to trace the inner and outer diameter. Then transmural lines were drawn to calculate the thickness and averaged (B). This was performed 3 times. A representative image of the cervix at the unloaded length and pressure (C). Wall thickness was then calculated using Image J and the polygon tool in a similar manner to that of the vagina (D). Within the reproductive complex, the outer diameter is tracked at two different locations (E). Throughout the imaging process, the transducer is stabilized by a 3-D printed holder (F). Please click here to view a larger version of this figure.
Figure 7: Representative results for vaginal testing. The representative mechanical testing results of the vaginal basal and passive protocols. With the data obtained by the DMT system, several mechanical relationships can be derived. A) Basal Pressure-Diameter, B) Passive Pressure-Diameter, C) Basal Force-Pressure, D) Passive Force-Pressure, E) Basal circumferential stress-circumferential stretch, F) Passive circumferential stress-circumferential stretch, G) Basal axial stress-circumferential stretch, H) Passive axial stress-circumferential stretch. Please click here to view a larger version of this figure.
Figure 8: Representative results for cervical testing. The representative mechanical testing results of the cervical basal and passive protocols. With the data obtained by the DMT system, several mechanical relationships can be derived. A) Basal Pressure-Diameter, B) Passive Pressure-Diameter, C) Basal Force-Pressure, D) Passive Force-Pressure, E) Basal circumferential stress-circumferential stretch, F) Passive circumferential stress-circumferential stretch, G) Basal axial stress-circumferential stretch, H) Passive axial stress-circumferential stretch. Please click here to view a larger version of this figure.
In Vivo Pressure | Maximum Pressure | 1/3 Max Pressure | 2/3 Max Pressure | Axial Stretch | Cannula Size | Recommended number of sutures |
|
Vagina | 7 mmHg | 15 mmHg | 5 mmHg | 10 mmHg | -2%, in vivo, +2% | 3.75 mm | 2– in an "X" fashion |
Cervix | 10 mmHg | 200 mmHg | 66 mmHg | 133 mmHg | -2%, in vivo, +2% | 0.75 mm for uterine end 3.75 mm for vaginal end |
3 horizontal sutures on the uterine end 4 sutures on the vaginal external os |
Table 1: Summary of information for scaling the mechanical testing methods for each organ. The unloaded pressure values were measured using catherization techniques under anesthesia (4% isoflurane in 100% oxygen). A balloon catheter was utilized for the vaginal measurements and a 2F catheter for the cervix.
Vagina | Cervix | |
Basal Circumferential (kPa) |
127.94 | 188 |
Basal Axial (kPa) |
56.8 | 75.44 |
Passive Circumferential (kPa) |
246.03 | 61.26 |
Passive Axial (kPa) |
112.74 | 19.26 |
Table 2: The representative results for the tangent moduli of the vagina and cervix. The tangent moduli were calculated for both basal and passive conditions as well as for both circumferential and axial directions. All measurements provided are in units of kPa.
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 mechanical contribution within the female reproductive organs54,55. When performing passive mechanical testing, the media does not contain calcium. The lack of calcium inhibits the smooth muscle cells from contracting. This permits elucidation of other ECM components, such as collagen and elastic fibers, which largely dictate the passive mechanical properties. When combined with biochemical and histological analysis, these results permit elucidation of relationships between ECM microstructural composition and mechanical function. This then allows for delineation of the structural and mechanical mechanisms of pathologies relevant to women’s reproductive health.
Previously, the vagina and cervix were tested uniaxially27,28. The vagina and cervix, however, demonstrate anisotropic properties and experience multiaxial loading in vivo29,30 . Hence, pressure myograph systems used herein provide quantitative information on multiaxial loading that may aid in understanding the etiologies of reproductive pathologies, as well as the subsequent design of potential treatments. Further, pressure myography permits assessment of multiaxial properties while preserving the in vivo organ geometry and the native cell-matrix interaction56 . In vivo, the cells actively remodel the surrounding ECM in response to changes in biomechanical and biochemical cues57,58,59. The protocol used herein is advantageous as it permits monitoring of subsequent changes in bulk organ properties under physiologically relevant conditions. This aids in providing a platform to generate systematic datasets of multiaxial active and passive mechanical properties. Further, the data collected in these experiments may be leveraged to formulate and validate microstructurally-motivated nonlinear constitutive models to describe and predict the mechanical response of the female reproductive organs in healthy and pathological states16,60.
An additional system component that was advantageous to the protocol was the use of ultrasound imaging to measure the thickness of the organ walls. The thickness is crucial information for calculating stress experienced while undergoing testing.
With any experimental set up, there are some limitations to this procedure. This protocol currently only considers the elastic response of the vagina and cervix and not the viscoelastic response. A potential method to mitigate this limitation in the future is to modify the existing protocol to include creep and stress relaxation assays61. A second limitation is assuming the organs are incompressible. Within this study, thickness was solely measured at the unloaded configuration, as motivated by prior studies that demonstrate nonpregnant murine tissue exhibits minimal changes in volume during osmotic loading62. Furthermore, additional studies have operated under the same assumption of incompressibility44,60,63. Ideally, an ultrasound would be performed for the entirety of the experiment in order to remove the need for the incompressibility assumption and to better inform finite element models. A final limitation is the lack of quantified in vivo cervical pressure to inform the loading protocols. Literature suggests that cervical pressure in human women is 37 mmHg53. Mice, however, may exhibit different cervical pressure from that of humans. A difference in vaginal pressure was demonstrated between rodent models and human samples64,65. Further studies are needed to quantify pressure in the non-pregnant murine cervix. Towards this end, intra-uterine pressure was recently reported throughout pregnancy49.
The commercially available pressure myograph system utilized in this procedure measures the force properties of elastic, hollow organs. This protocol is easily adaptable to other various organs and tissues by modifying the chemical additives in the bath, cannula size, and suture thickness.
The authors have nothing to disclose.
The work was funded by NSF CAREER award grant #1751050.
2F catheter | Millar | SPR-320 | catheter to measure cervical pressure |
6-0 Suture | Fine Science Tools | 18020-60 | larger suture ties |
CaCl2 (anhydrous) | VWR | 97062-590 | HBSS concentration: 140 mg/ mL |
CaCl2-2H20 | Fischer chemical | BDH9224-1KG | KRB concentration: 3.68 g/L |
Dextrose (D-glucose) | VWR | 101172-434 | HBSS concentration: 1000 mg/mL KRB concentration: 19.8 g/L |
Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | curved forceps |
Dumont SS Forceps | Fine Science Tools | 11203-25 | straight forceps |
Eclipse | Nikon | E200 | microscope used for imaging |
Flow meter | Danish MyoTechnologies | 161FM | flow meter within the testing apparatus |
Force Transducer – 110P | Danish MyoTechnologies | 100079 | force transducer |
ImageJ | SciJava | ImageJ1 | used to measure volume |
Instrument Cases | Fine Science Tools | 20830-00 | casing to hold dissection tools |
KCl | Fisher Chemical | 97061-566 | HBSS concentration: 400 mg/ mL KRB concentration: 3.5 g/L |
KH2PO4 | G-Biosciences | 71003-454 | HBSS concentration: 60 mg/ mL |
MgCl2 | VWR | 97064-150 | KRB concentration: 1.14 g/L |
MgCl2-6H2O | VWR | BDH9244-500G | HBSS concentration: 100 mg/ mL |
MgSO4-7H20 | VWR | 97062-134 | HBSS concentration: 48 mg/ mL |
Mircosoft excel | Microsoft | 6278402 | program used for spreadsheet |
Na2HPO4 (dibasic anhydrous) | VWR | 97061-588 | HBSS concentration: 48 mg/mL KRB concentration: 1.44 g/L |
NaCl | VWR | 97061-274 | HBSS concentration: 8000 mg/mL KRB concentration: 70.1 g/L |
NaHCO3 | VWR | 97062-460 | HBSS concentration: 350 mg/ mL KRB concentration: 21.0 g/L |
Pressure myograph systems | Danish MyoTechnologies | 110P and 120CP | Pressure myograph system: prorgram, cannulation device, and controller unit |
Pressure Transducer | Danish MyoTechnologies | 100106 | pressure transducer |
Student Dumont #5 Forceps | Fine Science Tools | 91150-20 | straight forceps |
Student Vannas Spring Scissors | Fine Science Tools | 91500-09 | micro-scissors |
Tissue dye | Bradley Products | 1101-3 | ink to measure in vivo stretch |
Ultrasound transducer | FujiFilm Visual Sonics | LZ-550 | ultrasound transducer used; 256 elements, 40 MHz center frequency |
VEVO2100 | FujiFilm Visual Sonics | VS-20035 | ultrasound used for imaging |
Wagner Scissors | Fine Science Tools | 14069-12 | larger scissors |