Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Summary

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Abstract

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women's everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 µM epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Introduction

As a major female organ, the uterus is crucial for reproduction and essential for the nourishment of the fetus¹. The uterus consists of three layers: the perimetrium, myometrium and endometrium. The myometrium is the major contractile layer of the uterus and plays a key role in fetus delivery. The endometrium is the innermost layer lining the uterine cavity and is essential for embryo implantation. In non-pregnant females of reproductive age, the endometrial layer is shed monthly at the beginning of the menstrual cycle. The myometrium aids in this shedding process by maintaining the spontaneous myometrial contractions needed for clearing the necrotic endometrial tissue from the uterus¹.

Unfortunately, increased myometrial contractility can result in negative side effects such as dysmenorrhea, or painful menstrual cramps. This is especially seen in young females and nulliparous women². However, dysmenorrhea is different for every woman and depends on the strength of their myometrial contractions; stronger contractions are often associated with the sensation of severe cramping³. Myometrial contractility can be visualized using uterine ultrasound and is often recognized as endometrial waves. Enhanced release of prostaglandins during menstruation⁴ in a uterus undergoing endometrial sloughing is believed to contribute to increased myometrial hypercontractility, resulting in ischemia and hypoxia of the uterine muscle and thus increased pain³.

Severe dysmenorrhea can hinder the day-to-day activity of some women and 3 to 33% of women have very severe pain, which could cause a woman to be bedridden for 1 to 3 days each menstrual cycle⁵. Dysmenorrhea is the leading cause of gynecological morbidity in women of reproductive age regardless of age, nationality, and economic status⁵. The estimated prevalence of dysmenorrhea is both high and variable, ranging from 45% to 93% in women of reproductive age⁵.  Dysmenorrhea-associated pain has an effect on the daily life of women and may result in poor academic performance in adolescents, lower quality of sleep, restriction of daily activities, and mood changes⁵.

Many women who experience severe dysmenorrhea resort to over-the-counter medications to relieve their pain. Such over-the-counter medications contain cyclooxygenase (COX) inhibitors which prevent the formation of prostaglandins⁶. However, COX inhibitors are associated with adverse cardiovascular events, and about 18% of women with dysmenorrhea are unresponsive to these inhibitors⁷. Therefore, there is a need for new medications to reduce menstrual cramps. Since over contractility of the uterus contributes to the pathogenesis of dysmenorrhea, one possible strategy may be the usage of uterine relaxants.

It is beneficial to quantify the effects of potential relaxant drugs in a model of naturally occurring spontaneous myometrial wave-like contractions. However, thus far, no efficient ex vivo method for testing muscle-relaxing drugs in the intact uterus has been described. Currently, isometric tension measurements are used to evaluate relaxant drug effects. During such measurements, a uterine muscle strip is maintained at a constant length under preload in a tissue bath while the force of uterine muscle contractions is recorded before and after oxytocin stimulation in the presence or absence of a relaxant drug. Although this approach is very useful, it requires expensive equipment. Furthermore, isometric contractions do not resemble the spontaneous myometrial wave-like contractions that naturally occur in the intact uterus. Uniquely, the uterine myometrial waves in rodents can be visualized as uterine horn motility when the entire reproductive tract (ovaries, oviducts, uterus, and vagina) is maintained in a buffer solution. Here, we present an ex vivo method for monitoring the spontaneous motility of the intact mouse uterus placed in a Petri dish containing oxygenated Krebs buffer. We also describe a motility quantification algorithm utilizing the MATLAB motion tracker. This novel approach provides an easy and less expensive alternative to test the relaxant potential of naturally occurring remedies and synthetic compounds.

Protocol

All procedures with animals have been approved by the Institutional Animal Care and Use Committee at the Indiana University School of Medicine (Indianapolis, IN). 2-5 month-old F2-129S-C57BL/6 sexually-mature female mice were used in the study.

CAUTION: Ensure safety by wearing a lab coat, mask, and gloves when working with animals and biohazardous materials.

1. Solution Preparation

1. Prepare the Krebs Buffer, which contains:  130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.2 mM NaH₂PO₄, 0.56 mM MgCl₂, 25 mM NaHCO₃, and 5 mM glucose, pH 7.4. Continuously oxygenate the Krebs buffer with a mixture of compressed gases containing 5% CO₂ and 95% O₂ while maintaining the buffer temperature at 37 °C using a circulating water bath.
2. Prepare Dulbecco's Phosphate-Buffered Saline (DPBS), which contains: 2.68 mM KCl, 1.47 mM KH₂PO₄, 136.89 mM NaCl, and 8.1 mM Na₂HPO₄, pH 7.4.

2. Animal Preparation

1. Anesthetize mice using inhalation of isoflurane (3%) with waste gas scavenging. Ensure adequate anesthesia by assessing withdrawal reflexes. Pinch the rear toe to affirm no movements are elicited, indicating loss of reflex responses. After the deep anesthesia is achieved, euthanize the animal by decapitation.  
   NOTE: Isoflurane may cause smooth muscle dilation. Therefore, the reproductive tract preparation should be extensively washed and incubated in the Krebs buffer for at least 15-30 min before beginning the ex vivo experiments. Isoflurane may cause irritation and discomfort when it is in contact with the skin, so proceed with caution.
2. Place the body on a large weigh boat lined with a paper towel. 
   NOTE: Pregnant female laboratory personnel should not be involved in experiments with isoflurane because it can decrease fetal weight, decrease fetal skeletal ossification, and increase the risk of spontaneous abortion^8,9. CO₂ inhalation can be used as a substitute to euthanize mice. 

3. Determination of Estrous Cycle Stage

1. With small forceps, lift the clitoris to gain access to the vaginal ostium and slowly insert a micropipette tip containing 10 µL of DPBS into the vagina.
   1. Ensure the micropipette tip is inserted through the ostium at an angle of 10 - 30° to avoid puncturing the vaginal wall. The liquid should still be visible in the tip after insertion. If the liquid is not visible, the tip was inserted too far into the vagina, and the paracervical area of the vagina might have been perforated.
   2. Lightly pull down on the vaginal ostium muscles with the tip of the micropipette to allow air to exit from the vagina.
2. Slowly flush the vaginal cavity by pipetting up and down 2-3 times with 10 µL of DPBS and place the drawn cell suspension on a glass slide.
3. Use an Inverted Phase Contrast Microscope to determine the estrous cycle stage through cytologic analysis. The procedure is done as described elsewhere^10,11. Ensure the cell suspension does not dry out before cytologic analysis can be performed. The suspension may be diluted with fresh DPBS, if necessary.

4. Mouse Reproductive Tract Ddissection

1. Arrange the mouse in a supine position and spread its extremities to expose the abdominopelvic region.
2. Spray with 70% ethanol to moisten and disinfect the abdominopelvic area. 
3. Using forceps, carefully lift the skin located superior to the clitoris. Make small transverse incisions on the lateral aspects of the lower abdominal area, up to the upper extremities to expose the peritoneum (Figure 1A). During this process, an external flap will form. As small incisions are continually made, the flap will increase in size.
4. Carefully cut through the peritoneum to expose the gastrointestinal tract (Figure 1B). It is important to note that the uterine horns can oftentimes be located directly under the peritoneum, so make incisions cautiously and do not touch the horns as this can affect uterine motility.
5. Using forceps, remove the fascia and adipose tissue covering the gastrointestinal tract. Remove the following gastrointestinal tract segments from the abdominal cavity: the duodenum, jejunum, ileum, cecum, ascending and transverse colon (Figure 1C).
6. To locate the reproductive organs, first identify the urinary bladder (Figure 1C, "4"), which may have a deflated appearance due to voiding after euthanasia. The vagina will be right under the urinary bladder.
7. Locate the pubic symphysis at the confluence of the pubic bones (caudally to the bladder).
8. Using scissors, remove the pubic symphysis by carefully making incisions on its lateral sides through the interpubic fibrocartilaginous tissue to gain access to and provide a route for vagina extraction (Figure 1D).
9. Cut through the perineum located between the anus and lower part of the vulva.
10. Using forceps, lift the vagina and slowly excise the rectum.
11. Identify two uterine horns that bifurcate into a fork, rostrally to the vagina. Locate a convoluted oviduct and ovary at the end of each horn, which may be hidden under the remaining gastrointestinal tract segments. Use small dissecting scissors to remove any ligaments that connect to and support the horns, oviducts, and ovaries within the abdominal cavity.
12. Remove the reproductive tract, which includes the vagina, uterus, oviducts, and ovaries, from the abdominal cavity.
13. Transfer the isolated reproductive tract (Figure 1E) into a 100 mm Petri dish filled with 10 mL of DPBS. Do not compress the uterine horns to avoid damaging the myometrium.
14. Use forceps and surgical scissors to remove any connective and adipose tissue surrounding the uterine horns and vagina as well as any fur in the pubic region that could hinder the image quality. Remove the broad ligament to allow motility of the uterine horns.
15. Wash the isolated reproductive tract with fresh DPBS two times and transfer it into a 35 mm Petri dish filled with 3 mL of oxygenated Krebs solution.

5. Tissue Imaging

1. Place the Petri dish containing the reproductive tract in oxygenated Krebs buffer on a black surface. Maintain the dish at room temperature.
   NOTE: It is possible to use an infrared warming pad to maintain the tissue at 37 °C.
2. Allow 15-30 min for spontaneous contractions to begin. Record spontaneous uterine motility for 10 min from an axial plane using any type of digital video equipment.
3. Transfer the preparation into a Petri dish containing the oxygenated Krebs buffer supplemented with a test compound. Record spontaneous uterine motility for about 10 min from an axial plane using any type of digital video equipment.
4. Wash the entire reproductive tract in 100 mm Petri dish with 10 mL of DPBS to assess the reversibility of treatment.
5. Transfer the preparation into a Petri dish with the freshly oxygenated Krebs buffer. Record spontaneous uterine motility for about 10 min from an axial plane using any type of digital video equipment.
6. Transfer the preparation to another 35 mm Petri dish filled with oxygenated Krebs buffer supplemented with the vehicle for the test compound to ensure that there are no mechanically induced changes in spontaneous uterine motility. This is an important control.
7. Transfer the video footage to a computer hard drive.

6. Data Analysis

1. Make clips using any video editing software from the original video footage containing the control, treatment, and wash episodes.
2. Use the MATLAB software and the provided script (see online Supplemental material) to quantify spontaneous uterine motility.
   NOTE: Computer Vision Toolbox add-on for MATLAB must be installed for the script to be fully functional.
   1. Open the MATLAB script, go to the Editor tab, and click Run.
   2. Select the first video file and click Open.
   3. Enter a label for the video file in the pop-up dialog box and click OK.
   4. Enter the time interval (s) needed for calculating the rate of horn movement (Δ Euclidean distance / Δs).
   5. Use the mouse cursor to select two points on the first frame of the video. A pop-up window will ask to confirm that the selected points should be used for tracking. Click Start to initiate the real time tracking process that will be displayed in the pop-up window. Alternatively, click Reselect Points to reselect the two points.
   6. Monitor the accuracy of the tracking process on the pop-up window.
   7. Observe a rate vs. time scatter plot and distance vs. time scatter plot in a new pop-up window. Save the two figures by selecting File | Save As in the same window to document the data.
   8. Find a folder named PointTrackerData, automatically created by MATLAB, in the same directory where the MATLAB script is located. Identify an Excel file named label_Data containing data points collected from the video in two separate worksheet tabs.
      NOTE: Any alternative motion tracking software can be used to quantify spontaneous uterus motility.
3. Use an appropriate software (e.g., Excel or SigmaPlot 13) to perform the statistical analysis.

Results

Figure 1 shows representative images taken during the entire reproductive tract isolation procedure that is described in this protocol. To avoid contaminating the buffer with fur, which would decrease video quality, we moistened the mouse body with 70% ethanol. The major benchmark for the dissection section of the protocol is to find the urinary bladder. The uterus and vagina will be located inferior to the urinary bladder.

To test the protocol, we treated the ent...

Discussion

Here, we described a method for assessing spontaneous contractility of the entire rodent reproductive tract, which includes the ovaries, oviducts, uterine horns, and the vagina. We used a similar method to demonstrate the relaxant effect of phenylephrine on spontaneous uterine motility¹³, however, in the past we were unable to provide quantitative analysis of the data. In this work, we developed an algorithm for quantitative motility data analysis using the MATLAB motion tracking module. This is a...

Materials

Name	Company	Catalog Number	Comments
Epinephrine hydrochloride	Sigma-Aldrich	E4642
Dulbecco's PBS	Fisher Sceintific	17-512Q
Ethanol 200 PROOF	Decon Laboratories	2701
NaCl	Sigma-Aldrich	S7653
Glucose	Sigma-Aldrich	G7528
KCl	Sigma-Aldrich	P9333
CaCl2 · 2H2O	Sigma-Aldrich	C5080
NaH2PO4	Sigma-Aldrich	S0751
MgCl2  · 6H2O	Sigma-Aldrich	M9272
NaHCO3	Sigma-Aldrich	S6297
Isoflurane, USP	Patterson Veterinary	07-893-2374
Dissecting Extra-Fine-Pointed Precision Splinter Forceps	Fisher Sceintific	13-812-42
Curved Hardened Fine Iris Scissors	Fine Science Tools	14091-09
Dissection High-performance Modular Stereomicroscope	Leica	MZ6
Digital 5 Megapixel Color Microscope Camera with active cooling system	Leica	DFC425 C
Stereomaster Microscope Fiber-Optic Light Sources	Fisher Sceintific	12-562-21
Weigh Boat	Fisher Sceintific	WB30304
Convertors Astound Standard Surgical Gown	Cardinal Health	9515	Small, Medium or Large
Gloves	McKesson Corporation	20-1080	Small, Medium, or Large; powder-free sterile latex or nitrile surgical gloves
Petri Dish	Corning Falcon	351029	100 mm
Petri Dish	Corning Falcon	353001	35 mm
95% O2- 5% CO2 gas mixture	Praxair	MM OXCD5-K
Ear-loop Masks	Valumax International	5430E-PP
DSLR 24.2 MP Camera	Canon	EOS Rebel T6i
MATLAB	MathWorks	N/A	version 2019 or later