This work was supported by internal IU funds. AGO conceived the study. XC and AGO were involved in the design of the described experiments.&#160; FL and AGO analyzed and interpreted the data. KLL, JOB, FL performed all of the ex vivo experiments. FL wrote the MATLAB script. KLL, JOB, and AGO wrote the manuscript.&#160; All authors read and approved the final version of the manuscript.&#160;&#160;

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
<ol>
	<li>Prepare the Krebs Buffer, which contains:&#160; 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 &#176;C using a circulating water bath.</li>
	<li>Prepare Dulbecco&#39;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.</li>
</ol>
2. Animal Preparation
<ol>
	<li>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. &#160; 
	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.</li>
	<li>Place the body on a large weigh boat lined with a paper towel.&#160; 
	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 abortion8,9. CO2 inhalation can be used as a substitute to euthanize mice.&#160;</li>
</ol>
3. Determination of Estrous Cycle Stage
<ol>
	<li>With small forceps, lift the clitoris to gain access to the vaginal ostium and slowly insert a micropipette tip containing 10 &#181;L of DPBS into the vagina.
	<ol>
		<li>Ensure the micropipette tip is inserted through the ostium at an angle of 10 - 30&#176; 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.</li>
		<li>Lightly pull down on the vaginal ostium muscles with the tip of the micropipette to allow air to exit from the vagina.</li>
	</ol>
	</li>
	<li>Slowly flush the vaginal cavity by pipetting up and down 2-3 times with 10 &#181;L of DPBS and place the drawn cell suspension on a glass slide.</li>
	<li>Use an Inverted Phase Contrast Microscope to determine the estrous cycle stage through cytologic analysis. The procedure is done as described elsewhere10,11. Ensure the cell suspension does not dry out before cytologic analysis can be performed. The suspension may be diluted with fresh DPBS, if necessary.</li>
</ol>
4. Mouse Reproductive Tract Ddissection
<ol>
	<li>Arrange the mouse in a supine position and spread its extremities to expose the abdominopelvic region.</li>
	<li>Spray with 70% ethanol to moisten and disinfect the abdominopelvic area.&#160;</li>
	<li>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.</li>
	<li>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&#160;the horns as this can affect uterine motility.</li>
	<li>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).</li>
	<li>To locate the reproductive organs, first identify the urinary bladder (Figure 1C, &#34;4&#34;), which may have a deflated appearance due to voiding after euthanasia. The vagina will be right under the urinary bladder.</li>
	<li>Locate the pubic symphysis at the confluence of the pubic bones (caudally to the bladder).</li>
	<li>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).</li>
	<li>Cut through the perineum located between the anus and lower part of the vulva.</li>
	<li>Using forceps, lift the vagina and slowly excise the rectum.</li>
	<li>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.</li>
	<li>Remove the reproductive tract, which includes the vagina, uterus, oviducts, and ovaries, from the abdominal cavity.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Tissue Imaging
<ol>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wash the entire reproductive tract in 100 mm Petri dish with 10 mL of DPBS to assess the reversibility of treatment.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the video footage to a computer hard drive.</li>
</ol>
6. Data Analysis
<ol>
	<li>Make clips using any video editing software from the original video footage containing the control, treatment, and wash episodes.</li>
	<li>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.
	<ol>
		<li>Open the MATLAB script, go to the Editor tab, and click Run.</li>
		<li>Select the first video file and click Open.</li>
		<li>Enter a label for the video file in the pop-up dialog box and click OK.</li>
		<li>Enter the time interval (s) needed for calculating the rate of horn movement (&#916; Euclidean distance / &#916;s).</li>
		<li>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.</li>
		<li>Monitor the accuracy of the tracking process on the pop-up window.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Use an appropriate software (e.g., Excel or SigmaPlot 13) to perform the statistical analysis.</li>
</ol>

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M., Jafarnejad, F., Kamali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+aerobic+exercise+on+primary+dysmenorrhea:+A+clinical+trial+study.">The Effect of aerobic exercise on primary dysmenorrhea: A clinical trial study.</a> Journal of education and health promotion. 7, 3 (2018).</li><li>Lindner, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+prostaglandins+in+the+regulation+of+cyclic+events+in+the+ovary+and+uterus.">Significance of prostaglandins in the regulation of cyclic events in the ovary and uterus.</a> Advances in prostaglandin and thromboxane research. 8, 1371-1390 (1980).</li><li>Bernardi, M., Lazzeri, L., Perelli, F., Reis, F. M., Petraglia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysmenorrhea+and+related+disorders.">Dysmenorrhea and related disorders.</a> F1000 research. 6, 1645 (2017).</li><li>Marjoribanks, J., Ayeleke, R. O., Farquhar, C., Proctor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsteroidal+anti-inflammatory+drugs+for+dysmenorrhoea.">Nonsteroidal anti-inflammatory drugs for dysmenorrhoea.</a> The Cochrane database of systematic reviews. 7, 001751 (2015).</li><li>Oladosu, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+skeletal+muscle+activity+precedes+spontaneous+menstrual+cramping+pain+in+primary+dysmenorrhea.">Abdominal skeletal muscle activity precedes spontaneous menstrual cramping pain in primary dysmenorrhea.</a> American journal of obstetrics and gynecology. 219 (1), 91 (2018).</li><li>Lawson, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occupational+exposures+among+nurses+and+risk+of+spontaneous+abortion.">Occupational exposures among nurses and risk of spontaneous abortion.</a> American journal of obstetrics and gynecology. 206 (4), 327-328 (2012).</li><li>Mazze, R. I., Wilson, A. I., Rice, S. A., Baden, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+development+in+mice+exposed+to+isoflurane.">Fetal development in mice exposed to isoflurane.</a> Teratology. 32 (3), 339-345 (1985).</li><li>Byers, S. L., Wiles, M. V., Dunn, S. L., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> PLoS One. 7 (4), 35538 (2012).</li><li>Caligioni, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+reproductive+status/stages+in+mice.">Assessing reproductive status/stages in mice.</a> Current protocols in neuroscience. , (2009).</li><li>Segal, S., Csavoy, A. 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The authors have nothing to disclose.

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...

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.&#160; 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.

<table><tbody><tr><td>Epinephrine hydrochloride</td><td>Sigma-Aldrich</td><td>&amp;nbsp;E4642&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Fisher Sceintific&amp;nbsp;</td><td>17-512Q</td><td></td></tr><tr><td>Ethanol 200 PROOF</td><td>Decon Laboratories</td><td>2701</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C5080</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6297</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Patterson Veterinary</td><td>07-893-2374</td><td></td></tr><tr><td>Dissecting Extra-Fine-Pointed Precision Splinter Forceps</td><td>Fisher Sceintific</td><td>13-812-42</td><td></td></tr><tr><td>Curved Hardened Fine Iris Scissors</td><td>Fine Science Tools</td><td>14091-09</td><td></td></tr><tr><td>Dissection High-performance Modular Stereomicroscope</td><td>Leica</td><td>MZ6&amp;nbsp;</td><td></td></tr><tr><td>Digital 5 Megapixel Color Microscope Camera with active cooling system</td><td>Leica</td><td>&amp;nbsp;DFC425 C</td><td></td></tr><tr><td>Stereomaster Microscope Fiber-Optic Light Sources</td><td>Fisher Sceintific&amp;nbsp;</td><td>12-562-21</td><td></td></tr><tr><td>Weigh Boat</td><td>Fisher Sceintific&amp;nbsp;</td><td>WB30304</td><td></td></tr><tr><td>Convertors Astound Standard Surgical Gown&amp;nbsp;</td><td>Cardinal Health&amp;nbsp;</td><td>9515</td><td>Small, Medium or Large</td></tr><tr><td>Gloves</td><td>McKesson Corporation</td><td>20-1080</td><td>Small, Medium, or Large; powder-free sterile latex or nitrile surgical gloves</td></tr><tr><td>&amp;nbsp;Petri Dish</td><td>Corning Falcon</td><td>351029</td><td>100 mm</td></tr><tr><td>&amp;nbsp;Petri Dish</td><td>Corning Falcon</td><td>353001</td><td>35 mm</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;- 5% CO&lt;sub&gt;2&lt;/sub&gt; gas mixture</td><td>Praxair</td><td>&amp;nbsp;MM OXCD5-K</td><td></td></tr><tr><td>Ear-loop Masks</td><td>Valumax International</td><td>5430E-PP</td><td></td></tr><tr><td>DSLR 24.2 MP Camera</td><td>Canon</td><td>EOS Rebel T6i</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>N/A</td><td>version 2019 or later</td></tr></tbody></table>

ex vivo method for assessing the mouse reproductive tract spontaneous motility and a matlab-based uterus motion tracking algorithm for data analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;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 &#181;M 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.

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...

Watch this Scientific Journal Video about Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis at JoVE.c

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

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.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

ex-vivo-method-for-assessing-mouse-reproductive-tract-spontaneous

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.9K Views.  Indiana University School of Medicine. 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.

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

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Developmental Biology

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

Behavior

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Immunology and Infection

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Cancer Research

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Medicine

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after &#945;-catenin depletion in HaCaT keratinocytes.

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To ...

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

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Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

Tracking Mouse Bone Marrow Monocytes In Vivo

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking