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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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

1. Determine the estrous cycle: The estrous cycle was monitored by visual assessment in accordance to previous studies^15,33,34. The estrous cycle consists of four stages: proestrus, ....

Results

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

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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