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 ...

<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. 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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.

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			<td height="50" style="height:50px;width:208px;">Isoflurane, USP</td>
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			<td style="width:75px;">07-893-2374</td>
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			<td height="50" style="height:50px;width:208px;">Dissecting Extra-Fine-Pointed Precision Splinter Forceps</td>
			<td style="width:161px;">Fisher Sceintific</td>
			<td style="width:75px;">13-812-42</td>
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			<td height="39" style="height:39px;width:208px;">Curved Hardened Fine Iris Scissors</td>
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			<td height="63" style="height:63px;width:208px;">Digital 5 Megapixel Color Microscope Camera with active cooling system</td>
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			<td style="width:75px;">&#160;DFC425 C</td>
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			<td height="45" style="height:45px;width:208px;">Stereomaster Microscope Fiber-Optic Light Sources</td>
			<td style="width:161px;">Fisher Sceintific&#160;</td>
			<td style="width:75px;">12-562-21</td>
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			<td height="45" style="height:45px;width:208px;">Weigh Boat</td>
			<td style="width:161px;">Fisher Sceintific&#160;</td>
			<td style="width:75px;">WB30304</td>
			<td style="width:227px;"></td>
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			<td height="40" style="height:40px;width:208px;">Convertors Astound Standard Surgical Gown&#160;</td>
			<td style="width:161px;">Cardinal Health&#160;</td>
			<td style="width:75px;">9515</td>
			<td style="width:227px;">Small, Medium or Large</td>
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			<td height="63" style="height:63px;width:208px;">Gloves</td>
			<td style="width:161px;">McKesson Corporation</td>
			<td style="width:75px;">20-1080</td>
			<td style="width:227px;">Small, Medium, or Large; powder-free sterile latex or nitrile surgical gloves</td>
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			<td height="40" style="height:40px;">&#160;Petri Dish</td>
			<td style="width:161px;">Corning Falcon</td>
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			<td height="35" style="height:35px;">&#160;Petri Dish</td>
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			<td style="width:75px;">353001</td>
			<td style="width:227px;">35 mm</td>
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			<td height="42" style="height:42px;">95% O2- 5% CO2 gas mixture</td>
			<td>Praxair</td>
			<td style="width:75px;">&#160;MM OXCD5-K</td>
			<td style="width:227px;"></td>
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			<td height="42" style="height:42px;">Ear-loop Masks</td>
			<td>Valumax International</td>
			<td style="width:75px;">5430E-PP</td>
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			<td height="42" style="height:42px;">DSLR 24.2 MP Camera</td>
			<td>Canon</td>
			<td style="width:75px;">EOS Rebel T6i</td>
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			<td height="40" style="height:40px;width:208px;">MATLAB</td>
			<td style="width:161px;">MathWorks</td>
			<td style="width:75px;">N/A</td>
			<td style="width:227px;">version 2019 or later</td>
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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...

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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. ...

Our protocol is significant because it provides the first quantitative ex vivo method for assessing the spontaneous motility of an intact uterus. This novel approach provides an easy and less expensive alternative for testing the relaxant potential of naturally occurring remedies in synthetic compounds. A major advantage of this technique is that all the intra-uterine cellular interactions remain intact throughout the experiment.Over contractility of the uterus is associated with dysmenorrhea, for which uterine relaxants may be useful for treatment. This method provides an easy way to test new relaxants. After confirming the estrous cycle stage, place the mouse in the supine position, and spread the extremities to expose the abdominal pelvic region.Wet the abdomen with 70%ethanol, and use forceps to carefully lift the skin 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. Carefully cut through the peritoneum to expose the gastrointestinal tract without damaging the uterine horns, and use forceps to remove the fascia and adipose tissue covering the gastrointestinal tract.Remove the duodenum, jejunum, ileum, cecum, and the ascending and transverse colon. Locate the urinary bladder to locate the vagina which is found directly under the urinary bladder. Locate the pubic symphysis at the confluence of the pubic bones, caudal to the bladder, and use scissors to carefully make an incision on the lateral sides of the pubic symphysis through the interpubic fibrocartilaginous tissue to gain access and to provide a route for vaginal extraction.Cut through the perineum located between the anus and lower part of the vulva, and use forceps to lift the vagina to allow a slow excision of the rectum. Identify the two uterine horns that bifurcate into a fork rostrally to the vagina and locate a convoluted oviduct and ovary at the end of each horn. Correctly identifying the uterine horns is critical to the procedure to ensure the tissues are not damaged.Use small dissecting scissors to remove any ligaments that connect to and support the horns, oviducts and ovaries within the abdominal cavity, and remove the vagina, uterus, oviducts and ovaries. Transfer the entire isolated reproductive tract into a 100 millimeter Petri dish containing 10 milliliters of Dulbecco's PBS. 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 and wash the tissue two times with fresh Dulbecco's PBS per wash before transferring the isolated reproductive tract into a 35 millimeter Petri dish containing three milliliters of oxygenated Krebs solution. For spontaneous motility imaging, place the Petri dish on a black surface at room temperature, and allow 15 to 30 minutes for spontaneous contractions to begin. When movement can be observed, record the spontaneous uterine motility for 10 minutes from an axial plane using any type of digital video equipment.Next, transfer the preparation into a Petri dish containing oxygenated Krebs buffer supplemented with the test compound and record the spontaneous uterine motility for an additional 10 minutes in the same manner. At the end of the recording period, wash the entire reproductive tract in 10 milliliters of Dulbecco's PBS in a 100 millimeter Petri dish, and transfer the sample into a new Petri dish of freshly oxygenated Krebs buffer to assess the reversibility of the treatment. Record the spontaneous uterine motility for about 10 minutes in the same manner, as for the last two recordings.Then transfer the preparation to another 35 millimeter 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 the spontaneous uterine motility. The reproductive tract preparation exhibited high motility in panels A and C in the absence of epinephrine. But this phenotype is dormant in panel B with the presence of one micro muller epinephrine.Here, a sample data analysis of epinephrine administration and spontaneous motility recovery is shown, illustrating how the effects of the hormone on reproductive tract spontaneous motility can be quantified from the recordings of the tissue taken before, during, and after test compound treatment. It is important to avoid compressing or overstretching the uterine horns otherwise no spontaneous contractility will be observed. Our method may also be used for testing compounds that may increase the contractility of the uterus.This procedure may be applied to pregnant mouse uteri at the earlier stages of pregnancy. This experiment has the potential to be upgraded to a six well plate format for throughput drug screening.

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8.8K visualizações.  Indiana University School of Medicine. Nosso protocolo é significativo porque fornece o primeiro método ex vivo quantitativo para avaliar a motilidade espontânea de um útero intacto. Esta nova abordagem fornece uma alternativa fácil e menos cara para testar o potencial relaxante de remédios que ocorrem naturalmente em compostos sintéticos. Uma grande vantagem dessa técnica é que todas as interações celulares intrauterinas permanecem intactas durante todo o experimento.A sobresemilidade do útero está associada ?...

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