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., 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. 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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.

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	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:208px;">Epinephrine hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:75px;">&#160;E4642&#160;</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:208px;">Dulbecco&#39;s PBS</td>
			<td style="width:161px;">Fisher Sceintific&#160;</td>
			<td style="width:75px;">17-512Q</td>
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			<td height="26" style="height:26px;width:208px;">Ethanol 200 PROOF</td>
			<td style="width:161px;">Decon Laboratories</td>
			<td style="width:75px;">2701</td>
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			<td height="23" style="height:23px;width:208px;">NaCl</td>
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			<td style="width:75px;">P9333</td>
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			<td height="29" style="height:29px;width:208px;">CaCl2 &#183; 2H2O</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:75px;">C5080</td>
			<td style="width:227px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:208px;">NaH2PO4</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:75px;">S0751</td>
			<td style="width:227px;"></td>
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			<td height="29" style="height:29px;width:208px;">MgCl2&#160; &#183; 6H2O</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:75px;">M9272</td>
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			<td height="29" style="height:29px;width:208px;">NaHCO3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:75px;">S6297</td>
			<td style="width:227px;"></td>
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		<tr height="50">
			<td height="50" style="height:50px;width:208px;">Isoflurane, USP</td>
			<td style="width:161px;">Patterson Veterinary</td>
			<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>
			<td style="width:227px;"></td>
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			<td height="39" style="height:39px;width:208px;">Curved Hardened Fine Iris Scissors</td>
			<td style="width:161px;">Fine Science Tools</td>
			<td style="width:75px;">14091-09</td>
			<td style="width:227px;"></td>
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		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Dissection High-performance Modular Stereomicroscope</td>
			<td style="width:161px;">Leica</td>
			<td style="width:75px;">MZ6&#160;</td>
			<td style="width:227px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:208px;">Digital 5 Megapixel Color Microscope Camera with active cooling system</td>
			<td style="width:161px;">Leica</td>
			<td style="width:75px;">&#160;DFC425 C</td>
			<td style="width:227px;"></td>
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		<tr height="45">
			<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>
			<td style="width:227px;"></td>
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		<tr height="45">
			<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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		<tr height="40">
			<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>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Petri Dish</td>
			<td style="width:161px;">Corning Falcon</td>
			<td>351029</td>
			<td style="width:227px;">100 mm</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">&#160;Petri Dish</td>
			<td style="width:161px;">Corning Falcon</td>
			<td style="width:75px;">353001</td>
			<td style="width:227px;">35 mm</td>
		</tr>
		<tr height="42">
			<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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		<tr height="42">
			<td height="42" style="height:42px;">Ear-loop Masks</td>
			<td>Valumax International</td>
			<td style="width:75px;">5430E-PP</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DSLR 24.2 MP Camera</td>
			<td>Canon</td>
			<td style="width:75px;">EOS Rebel T6i</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="40">
			<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...

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

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

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...